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Volume 88-B, Issue SUPP_III October 2006

R.D. Marsh C.N. Emeagi A.E. Goodship M. Amrich G.W. Blunn

Introduction: The use of uncemented arthroplasty in joint replacement surgery requires osseointegration of the prosthesis to maximise function and longevity. It has been demonstrated that osteoblast-like cells will preferentially proliferate, differentiate and produce mineralised matrix in pits and grooves on non-biological surfaces, of similar dimensions to those of Howslip’s lacunae produced by osteoclasts in vitro. The hypotheses of this study were that a photochemically etched titanium alloy surface would 1) induce proliferation and differentiation in osteoblast-like cells; 2) induce osteoblastic differentiation of human mesenchymal stem cells and 3) induce greater bone to implant contact in a caprine model.

Methods: Three microgrooved titanium alloy surfaces (fine, medium & coarse) were created by photochemical etching, with dimensions of 200 to 515 microns. Human Mesenchymal stem cells (MSC) and Human Osteosarcoma (HOS) cells (TE-85) were seeded onto these surfaces and cultured in standard media; in the case of MSC, with and without the addition of osteogenic supplements. At intervals of time each surface and cell type were assessed for proliferation by Alamar blue assay and osteoblastic differentiation by Alkaline Phosphatase expression. A polished titanium surface was used as a control. A plate of each surface dimension was placed into a femoral condyle of ten adult male goats. The animals were euthanased at 6 and 12 weeks post-implantation. The specimens were histologically processed and examined under light and backscattered electron microscopy to establish the percentage of bone to implant contact and the presence of new bone within the grooves.

Results: In vitro, all cells showed an increase in proliferation with time, the greatest occurring on the coarse surface. Alkaline phosphatase expression showed a rise with time on all surfaces, the greatest being on the coarse surface seeded with HOS cells (p< 0.05). MSC could not be induced to differentiate to an osteogenic lineage by these surface textures alone. On addition of osteogenic supplements their results followed the trends of HOS cells. In vivo, histomorphometric analysis showed significantly greater bone implant contact on the coarse surface at both 6 and 12 weeks (p< 0.05). In a number of cases there were signs of osteogenesis occurring deep within the pits and grooves.

Discussion: This study confirms that a photochemically etched surface topography mimicking that created by osteoclasts will increase the proliferation and differentiation of osteoblastic cells in vitro. The rate of differentiation of these cells increased significantly in relation to the size of the grooves. When implanted in vivo these same surfaces were shown to support osseointegration. This surface has the potential to improve the function of uncemented arthroplasties in the future.

P. Pollintine P. Dolan J.H. Tobias D.S. McNally M.A. Adams

Introduction: Age-related hormonal changes and inactivity lead to systemic bone loss and osteoporotic fractures. However, it is not clear why the vertebral body should be affected so often, or why its anterior region should characteristically sustain a “wedge” deformity. We hypothesise that intervertebral disc degeneration in elderly spines leads to altered spinal load-sharing in such a manner that the anterior region of the vertebral body becomes vulnerable to injury.

Methods: Forty thoraco-lumbar “motion segments”, consisting of two vertebrae and the intervening disc and ligaments, were obtained from cadaver spines aged 62–94 yrs. Volumetric bone mineral density (BMD) was measured for various regions of each vertebra using a Lunar Piximus DXA scanner. The distribution of the applied compressive force (1.5 kN) between the anterior and posterior halves of the vertebral body was calculated by pulling a needle-mounted pressure transducer along the sagittal midline of the adjacent disc. Pressure measurements were integrated over area to give force. Anterior and posterior disc forces were subtracted from the applied 1.5 kN to indicate loading of the neural arch. Measurements were repeated with the specimens positioned to simulate various postures in life. The strength of each motion segment was determined by compressing it to failure while positioned in a forward stooped posture. Disc and vertebral morphology were assessed from radiographs, and from digital photographs of tissue sections.

Results: Load-bearing by the neural arch in erect posture increased in the presence of intervertebral disc degeneration, and was inversely proportional to the average height of the disc (P< 0.01). High neural arch load-bearing was associated with relatively low BMD in the anterior vertebral body (P< 0.01), and with low compressive strength (P< 0.0001). BMD in the anterior region of the vertebral body was the best univariate predictor of compressive strength (R2 = 0.78). Stepwise multiple linear regression showed that 86% of the variance in compressive strength could be explained by the following: anterior vertebral body BMD, vertebral body X-sectional area, and neural arch load-bearing (% of applied load). Forcing age, gender and spinal level into the model did little to improve the prediction.

Discussion and Conclusions: Results strongly support our hypothesis. Evidently, intervertebral disc degeneration and narrowing cause the neural arch to “stress shield” the anterior vertebral body whenever the spine is held erect. This leads to reduced BMD in the anterior vertebral body, weakening the spine when it is loaded in a stooped posture. The small age-dependence of results can be attributed to the relatively narrow age range of specimens tested. Vertebral fracture risk can best be assessed from BMD measured in the anterior half of the vertebral body.

B.J.C. Freeman R.D. Fraser C.M.J. Cain D.J. Hall

Introduction: Intra-Discal Electrothermal Therapy (IDET) has been proposed as a treatment for chronic discogenic low back pain. Reports from prospective outcome studies demonstrate statistically significant improvements, but there are no published randomized controlled trials assessing efficacy against a placebo group.

Methods: Ethical committee approval was obtained prior to the study. Patients with chronic low back pain who failed conservative treatment were considered for the study. Inclusion criteria included one or two level symptomatic internal disc disruption as determined by provocative CT/discography. Patients were excluded if there was > 50% loss of disc height or had had previous back surgery. Fifty-seven patients were randomized with a 2:1 (IDET: Placebo) ratio, 38 to the active IDET arm and 19 to the sham (placebo). The IDET catheter was positioned under sedation to cover at least 75% of the annular tear as defined by the CT/discogram. An independent technician connected the catheter to the generator and either delivered electrothermal energy (active group) or did not (sham group). Surgeon, patient and independent outcome assessor were all blinded. All patients followed a standard rehabilitation programme.

Outcome Measures: Low Back Outcome Score (LBOS), Oswestry Disability Index (ODI), SF-36 questionnaire, Zung Depression Index (ZDI) and Modified Somatic Perceptions Questionnaire (MSPQ) were measured at baseline and 6 months. Successful outcome was defined as: No neurological deficit resulting from the procedure, improvement > 7 points in LBOS, improvements > 7 points in SF-36 subsets (pain / disability, physical functioning and bodily pain)

Results: Two subjects withdrew from the study (both IDET). Baseline demographic data, employment and worker’s compensation status, sitting tolerance, initial LBOS, ODI, SF-36, ZDI and MSPQ were similar for both groups. No neurological deficits occurred as a result of either procedure. No subject in either treatment arm showed improvement of > 7 points in LBOS or the specified domains of the SF-36. Mean ODI was 41.4 at baseline and 39.7 at 6 months for the IDET group compared to 40.7 at baseline and 41.5 at six months for the Placebo group. There was no significant change in ZDI or MSPQ scores for either group.

Discussion: No subject in either treatment arm met criteria for successful outcome. There was no significant change in outcome measures in either group at six months. This study demonstrates no significant benefit from IDET over placebo.

J.M. Murnaghan G. Li D.R. Marsh

Introduction: Angiogenesis is essential during bone formation. Many studies have looked at the developing vascular network during normal and abnormal bone growth, using histological, immunohistological and contrast-radiological techniques; however all require sacrifice of animals to obtain tissue samples for examination and consequently chronological investigation of angiogenesis is not possible. We have endeavoured to produce an animal model, whereby quantitative assessment of blood flow, and callus formation across a fracture gap, can be repeatedly assessed.

Methods: The model is an adaptation of a 4-pin externally fixated murine femoral fracture previously developed in this department. Three extra conduits have been drilled onto the fixator cross-bar, such that it now links with an x-ray jig and implantable optical cable. The x-ray jig permits repeated lateral x-rays whereas the optical cable which is implanted adjacent to the fracture gap and connected to a laser, measures blood flow using the principle of the Doppler shift of light. Ten mice underwent surgery. Doppler readings and x-rays were taken on the day of surgery and subsequently at days 1, 2, 4, 8, 12, 16, 24 and 32.

Results: Fracture gap pixel density was seen to rise steadily and plateau at day 24, with significant statistical differences between the day of surgery and early time points, and then again between these early time-points (days 2, 4 and 8) and the late time-point day 24. Blood flow was noted to fall following the day of surgery and then slowly increase, with a rapid rise in flow at day 8 until day 16, when levels began to fall again to resting levels.

Conclusion: The data correlates with previous histo-morphological work performed in this department and also with early results from immunohistochemical studies. The above graph for blood flow conforms to that expected in a murine model of fracture healing, with a short initial drop in flow followed by a large rise as angiogenesis follows chondrocyte hypertrophy at the end of the first week, leading to callus formation. This in vivo model may be used to assess the effects on angiogenesis and callus formation of osteogenic compounds and investigate possible antiangiogenic mechanisms of action of medications such as NSAIDs that are known to be detrimental to fracture repair.

M. Korda J. Sharpe P.A. Rust J. Hua K. Phipps L. Di Silvio M.J. Coathup A.E. Goodship G.W. Blunn

Introduction: Wear particle induced osteolysis is one of the main reasons for revision total hip replacements (THRs). Loss in bone stock as a result of aseptic loosening is responsible for inferior results in revision THRs. Results from impaction grafting to fill osteolytic defects are frequently inconsistent. Our hypothesis is that the combination of autologous mesenchymal stem cells (MSCs) and allograft will enhance bone regeneration. This study asks whether: MSCs with allograft scaffolds survive at a normal impaction force during revision THRs.

Method: MSCs were isolated from a sheep iliac crest aspirate, expanded in culture and seeded onto irradiated sheep allografts (n=9). Viability of MSCs was assayed with alamar blue with absorbance measured on day 4 (before impaction). The constructs were then impacted using forces 3, 6, and 9 kN extrapolated in surgery then assayed daily for 6 days. The control was 0 kN. Samples were resin embedded after 10 days for histology and pieces of graft were taken for scanning electron microscopy (SEM).

Results: The 0KN control shows an MSC growth curve with a lag period and log phase. Compared with the control, the 3 and 6 kN showed initial reduction in cell proliferation measured by alamar blue (^p=0.015, ^p=0.002) but recovered by day 8, while 9kN showed a significant reduction (^p=0.011) over the time (Figure 1).

For cell proliferation over time, 3 and 6 kN showed no differences, but 9 kN showed a significant difference between day 4 and day 8 (^p=0.031). SEM and histological analysis showed a network of cuboidal cells on the allograft surface.

Conclusions: The results showed that MSCs recovered from impaction of 3 and 6 kN after an initial reduction in metabolism and exceeded original cell seeding densities with no significant difference in proliferation. Viability of MSCs were not effected by impaction forces up to 6 kN. This study shows that stem cells mixed with allograft are a potential method for repairing bone defects in revision total hip replacements.

S.E. Aldridge T.W.J. Lennard J.R. Williams M.A. Birch

Introduction: Vascular Endothelial Growth Factor (VEGF) is a proangiogenic cytokine that is expressed highly by many solid tumours often correlating with poor prognosis. VEGF has also been shown to interact with osteoclasts and their precursors in organ cultures to increase differentiation and survival and VEGF receptors have been found on osteoclasts in vitro. In this work we aimed to investigate the expression of VEGF and its receptors in bone metastases from primary breast tumours and further characterise its effects on osteoclasts. We performed immunolocalisation of VEGF in bone metastases and using VEGF and VEGF receptor-specific ligands we assessed their role in osteoclastogenesis in vitro.

Methods: Seventeen specimens of breast cancer metastases to bone were immunohistochemically stained with antibodies to VEGF and its receptors VEGFR1 and 2, and the macrophage marker CD68.

To investigate osteoclastogenesis in vitro Peripheral Blood Mononuclear Cells (PBMC) were isolated from healthy volunteers and cultured over a two-week period under stimulation by cytokines (RANKL, M-CSF, VEGF, PlGF, a specific ligand for VEGFR 1 and VEGF-D, a specific ligand for VEGFR 2). RAW 264.7 cells (a mouse monocyte/macrophage cell line able to differentiate into osteoclast-like cells) were cultured for seven days under stimulation by cytokines (RANKL, VEGF and M-CSF). Osteoclasts were identified by staining for Tartrate Resistant Acid Phophatase (TRAP) and numbers of multinucleated cells counted per treatment. Culture on ivory slices was performed to measure resorption activity of the osteoclasts.

Results: The immunohistochemistry demonstrated that breast cancer metastases express VEGF strongly and that the osteoclasts surrounding metastases express both VEGFR1 (12 of 14 specimens) and VEGFR2 (14 of 14 specimens).

The PBMCs stimulated by VEGF and RANKL together differentiated into multinucleated TRAP positive cells in similar numbers (22±4.7) per field of view to the M-CSF and RANKL (27.3±7.2). Resorption of ivory was identified in these cultures. Stimulation with PlGF and RANKL resulted in increased osteoclastogenesis but VEGF-D with RANKL had little effect. Similar results were seen in triplicate experiments RAW 264.7 cells also differentiated into osteoclast-like cells after stimulation with VEGF and RANKL similar to M-CSF and RANKL.

Discussion and Conclusions: VEGF is able to induce the differentiation of human and mouse osteoclast-like cells from monocyte precursors in the presence of RANKL and this seems to be mediated by VEGFR1. This may lead to an increase in bone resorption in physiological and pathological situations where there is an increase in VEGF, such as in tumours, embryogenesis and fracture repair. VEGF signalling could be a therapeutic target for osteoclast inhibtion in these situations.

C. Pendegrass B. Annand C. Hoare P. Unwin A.E. Goodship G.W. Blunn

Introduction: Normal limb use in amputees with made to measure external prostheses can be impaired by problems at the stump – socket interface. The development of an Intraosseous Transcutaneous Amputation Prosthesis (ITAP) would overcome the problems by protecting the soft tissues, whilst redistributing high stresses to bone. ITAP creates a breach in the skins protective barrier to infection, hence requires a sufficient soft tissue – implant seal to prevent implant failure. Deer antlers are natural analogues of ITAP, and successfully overcome the problems associated with skin penetrating implants such as infection, marsupilisation and avulsion. In this study, an ITAP device has been developed, with a successful soft tissue – implant interface, based on deer antler morphology. It is hypothesised that sub-epithelial dermal fibroblastic, but not epithelial layer adhesion, is directly responsible for the degree of downgrowth observed around ITAP.

Methods: Eleven pairs of deer antler were used to histologically evaluate the interface between the antler and pedicle, and the soft tissue seal around the antler-pedicle structure. The findings were used to develop a titanium alloy (Ti6Al4V) ITAP device in a goat model. Three to five transcutaneous pins were implanted into the medial aspect of the right tibia of skeletally mature female goats. Four implant designs were tested, Machine Finished Straight (MFS), Hydroxyapatite (HA) Coated MFS, Machine Finished Flanged (MFF) and HA Coated MFF. The 70μ thick HA coating was applied to the implant region abutting the sub-epithelium. The implants remained in situ for four weeks after which the histology of the resulting interfaces were analysed qualitatively and quantitatively for degrees of epithelial downgrowth (marsupilisation) and epithelial/sub-epithelial layer attachment to the implant surface.

Results: The histology of the deer antler showed there to be an extremely small area of epithelial attachment, with negligible downgrowth, arrested by soft tissue adhesion to the underlying pedicle surface. There was a significant increase in pore size and frequency in the pedicle structure (abutting the soft tissues), compared to the antler proper. The MFS ITAP implants were associated with significantly greater downgrowth and reduced epithelial and sub-epithelial layer attachment compared to all other implant designs. The HA coating, and porous flange structure significantly reduced downgrowth and increased sub-epithelial layer attachment. Regression correlation showed that there is a significant negative correlation between the extent of downgrowth and the degree of sub-epithelial dermal fibroblastic layer attachment observed around ITAP implants (All p values < 0.05).

Discussion: Deer antlers successfully overcome the potential problems for ITAP. By artificially recreating some of the aspects of the antler, including layering of porous and bioactive surfaces for tissue adhesion, we have successfully developed an ITAP implant that minimises downgrowth and actively encourages epithelial and sub-epithelial soft tissue adhesion.

A. Jafri S.M. Green A.W. McCaskie P.F. Partington S.D. Muller

Introduction: Aseptic loosening is the commonest complication of cemented total hip arthroplasy. Gaseous voids within the cement mantle are thought to act as stress concentrators and points of origin and preferential fracture propagation at the cement stem interface. Assuming a bone tempereature of 37°C, Bishop recommended heating the prosthesis to 44°C, thereby effecting a reduction in cement-prosthesis interface porosity.

The aim of this study was to (I) determine the intra-operative temperature of the femoral cancellous bed prior to insertion of prosthesis, (II) to investigate whether the magnitude of the temperature gradient effects interface porosity (III) to develop clinically relevant recommendations.

Materials and Methods: (I) The intra-operative determination of femoral cancellous boney bed temperature. Sterile, single use thermocouples (Mon-a-therm) were used to record interface temperature in six patients, after canal preparation and lavage. (II) A simulated femoral model was designed consisting of a waterbath, set at temperature determined by (I) with an inner water-tight chamber formed by 19mm diameter polyethylene tubing. Cement (Palacos) was non-vacuum mixed (to exaggerate porosity) for 1 minute and injected in a retrograde manner into the inner tube at 3 minutes. Femoral stems (Exeter) were pre-heated in a second waterbath to 18, 32,35,37,40,44°C, were thoroughly dried and lowered into the inner tube by a Lloyd universal testing machine via a custom jig. The cement was left to polymerise.

The cement mantle was sectioned transversely, then longitudinally to expose the cement-prosthesis interface. This was stained with acrylic dye to facilitate image analysis. Three mantles for each temperature were produced.

Results: (I) The mean femoral canal temperature was 32.3°C, (II) the effect of stem temperature on interface porosity is shown in fig1.

Conclusions: Bone temperature is 32°C after canal preparation using contemporary cementing techniques. Heating to 35°C reduces interface porosity, heating to 40°C is optimal.

M. Shoeb M.J. Coathup J.D. Witt P.S. Walker G.W. Blunn

Introduction: Conservative hip replacements are advantageous because resection of bone in the proximal femur is minimised. This study investigated a new design of conservative hip in the goat model where the femoral head was resected and two hydroxyapatite coated ‘pegs’ were introduced into the femoral neck. The hypothesis was that the ‘pegs’ would provide a direct method of transmitting forces within the femoral neck thus resulting in less adverse bone remodelling and reduced loosening. Bone stock is also preserved should subsequent revision be required.

Methods: Eight unilateral implants were inserted into the right femur of adult female goats for 1 year. Retrieved specimens were analysed radiographically and histologically. Image analysis was used to quantify bone attachment and total bone area adjacent to the implant. Tetracycline bone markers quantified bone turnover. Operated hips were compared with non-operated hips. The students t-test was used for comparative statistical analysis where p< 0.05 were classified as significant.

Results: Radiographic analysis demonstrated bone loss beneath the cup with increased bone density at the distal end of the pins (fig.1). Light microscopy revealed areas of new and mature bone adjacent to the implant. Osseointegration to the HA coating was observed. Bone markers established significantly decreased bone formation rates (p< 0.05) in bone adjacent to the implant in the operated versus control hips.

Image analysis results demonstrated an average bone attachment of 30.94% to the implant surface (fig 2). Greatest bone attachment occurred at the end of the pins (78.99%) contributing 22% of overall attachment to the implant. Least attachment occurred beneath the prosthetic cup (13.82%) and in the medial aspect adjacent to the central pin. Greater total bone area was measured in control hips and no significant correlation between bone attachment to the ‘pegs’ and bone area beneath the prosthetic cup was identified.

Discussion: From this study we have concluded that despite the resorption of bone beneath the prosthetic cup, the conservatve hip design investigated remained well fixed in the femur during the 1 year in vivo period. It appears that an implant design that resurfaces the femoral head with two pins used to transmit forces into the femoral neck is a useful approach in conservative hip design.

S. Alexander M. Hermansson A.L. Wallace J. Saklatvala

Introduction: Osteoarthritis (OA) is a common disease that affects 80% of the population over the age of 65 years. Little is known about the pathogenesis of OA. It is characterised by degradation of articular cartilage. Proteomic studies undertaken at our Institute using 2D gel electrophoresis and mass spectrometry identified about 30 proteins secreted by articular cartilage. Two whose synthesis was upregulated in OA were collagen II and activin A. This study quantified activin A production by human cartilage and investigated factors that may stimulate this.

Methods: Cartilage from normal (n=4) and OA (n=8) specimens were obtained from patients undergoing surgery and explants were cultured. Activin A secretion over five hours was measured in the culture medium by ELISA.

In order to determine factors that stimulate activin A production, chondrocytes were isolated from human cartilage and stimulated with various cytokines. RT-PCR methods were used to measure activin mRNA production and the culture medium was assayed for activin protein. Cartilage explants were also stimulated and activin protein levels were measured.

Results: OA cartilage produced higher amounts of activin A (range 34.9 – 97.1 ng/ml) compared to normal (range 9.4 – 15.6 ng/ml). IL-1, TGF-β and bFGF stimulated activin A mRNA and protein production by isolated chondrocytes. TGF-β and bFGF also stimulated activin production by explants, whereas IL-1 did not. This suggests that environment may determine cellular responses.

Conclusions: Activin A has not previously been described in articular cartilage. It is a homodimer of two inhibin β chains and is a member of the TGF-β superfamily originally purified from ovarian follicular fluid. Activin can induce mesenchymal cell differentiation e.g. chondrogenesis and has been shown to play a role in wound healing. To our knowledge we have shown for the first time that activin is produced by chondrocytes in response to various stimuli and that it may play a regulatory role in osteoarthritis.

A.W. Murray B. Noble A.H.R.W. Simpson

Introduction: It has been suggested that statins may influence bone turnover via an effect on bone morphogenic protein 2 (BMP-2). While the effect on statins in the prevention of osteoporosis remains controversial there is some evidence that they may exert a significant effect on fracture healing.

Using a newly developed fracture model of the proximal tibia of the rat, the effect of simvastatin on osteoporotic and non-osteoporotic fracture healing was investigated. The fracture model was used as it provided a useful model of metaphyseal fracture healing which is particularly relevant to osteoporotic fracture.

Methods: Four groups of 20 3-month-old female Wistar rats were used. Half underwent ovariectomy (ovx) while the remainder had a sham procedure. 8 weeks later a fracture was created in the proximal tibia of each animal by three point bending. The fractures were supported by a narrow intramedullary k-wire. 20 sham and 20 ovx animals were then fed 20mg/kg simvastatin by gavage for 14 days while the rest received placebo. 10 animals from each group were sacrificed at 2 weeks post surgery while the rest were sacrificed at 4 weeks.

X-rays of the healing fractures were taken. Both the intact and fractures tibiae were then taken for mechanical testing by four point bending.

Results: Six animals (7.5%) were excluded because of fracture comminution (5) or loss of stabilisation (1). There was a similar radiological appearance in all 4 groups at each time point. At two weeks: there was no difference in the mechanical properties of the healing bone between the groups. At 4 weeks the fractured and intact tibiae from the sham animals had an equal ultimate load at failure to their intact tibiae. However, the fractured tibiae from the ovx animals remained weaker (ovx & placebo 68%, ovx & statin 60.5% of ultimate load at failure compared with intact tibia). The difference between the fractures ultimate load in ovx and sham animals was statistically significant (p=0.0105). No difference was seen between the statin and placebo group.

Discussion: This work provides evidence that a metaphyseal fracture in the osteoporotic rat model is able to withstand significantly less load at 4 weeks than a fracture from a sham ovx animal suggesting fracture healing is slower in osteoporotic individuals. Simvastatin at 20mg/kg had no effect on the mechanical properties of normal or osteoporotic fracture healing in this study.

R. Milner R. Benson K. Heseltine J. Marotta

Introduction: A major complication associated with external fixation is pin tract infection1. This can occur in as many as 17–57%2 of cases and in severe cases leads to premature removal of the fixator. An antimicrobial coated (AMC) sleeve has been designed to be placed over external fixation pin and wires that delivers an antibiotic, gentamicin, directly into the pin tract. The function of the sleeve is to inhibit bacterial colonisation of the pins and wires, the first step in the development of a clinical infection. This study reports the in vitro testing carried out to establish the effectiveness of the AMC sleeve.

Methods: The prevalence of gentamicin susceptibility amongst bacteria typically associated with pin tract infections was determined by comparing minimum inhibitory concentrations (MIC) of clinical isolates from the SENTRY Antimicrobial Surveillance Programme (1997–2002) to the NCCLS susceptibility breakpoint of < 4 μg/ml. The amount of gentamicin released over time from AMC sleeves into phosphate buffered saline (PBS) was measured using a microbiological zone of inhibition assay against S. epidermidis (NCTC 8853). Three 5 cm long sleeves, fitted over 6 mm diameter pins, were agitated in 5 ml of PBS eluant at 37°C. The eluant was replaced and tested at 2, 24, 48, 72 hours, then weekly until 26 weeks. Concentrations of gentamicin in the pin tract were calculated from these values using an estimated pin-tract volume. The ability of the sleeves to kill bacteria was measured by inoculating single 5 cm long, 5 mm diameter sleeves on pins with 1.5 ml of bacterial suspensions containing approx. 1 x 108 cfu/ml. Surviving numbers of bacteria were counted after contact with the sleeves for 0.5, 1, 2 & 4 hours at 37°C. Effectiveness against clinical isolates of E. coli, S. aureus, S. epidermidis & Ps. aeruginosa was measured.

Results: The SENTRY database showed that of the 1456 individual surgical wound isolates gathered and evaluated, 1210 (83.1%) were found to be susceptible to gentamicin. Estimated concentrations of gentamicin in the pin tract reached 43.3 μg/ml at the end of the first week and exceeded the susceptibility threshold of 4 μg/ml over the next 19 weeks. The sleeves were able to reduce inoculum cell numbers of all organisms tested by 5 logs (99.9999% reduction) in ≤4h.

Discussion and Conclusion: Surveillance data confirms that gentamicin provides high level efficacy against pathogens commonly associated with pin tract infections. The AMC sleeves release gentamicin directly into the pin tract at concentrations above the susceptibility threshold for most clinically-encountered bacteria. These sleeves are also able to reduce significantly bacterial cell numbers when directly in contact with them. Therefore, this study demonstrates that the sleeves will inhibit bacterial colonisation of external fixation pins and emphasises their contribution to reducing the effects of pin tract infection.

S.A. Abusrer A.D. Rowan M.A. Birch

Introduction: The biological processes underlying osteolysis in aseptic loosening are not completely understood, but are believed to include factors such as hydrostatic pressure and wear debris. Characterisation of the pseudosynovial membrane from failed implants has revealed numerous cell types with well characterised roles in osteoclastogenesis and bone resorption. More recent work has demonstrated the presence of immunomodulatory cells, including T cells. IL-17 is a T cell product that is believed to be capable of inducing bone resorption. The aims of our study were to characterise the effects of IL-17 on the expression of RANKL and OPG by synovial fibroblasts and to evaluate its role in supporting osteoclastogenesis in vitro.

Materials and Methods: Synovial fibroblasts (SFB) were isolated from tissue obtained at joint replacement surgery. SFB were expanded in culture and used in experiments between passage 4 and 5. Human SFB, and for comparison the human osteosarcoma cell line MG63, were treated with IL-17 (5 and 50ng/ml) for up to 48 hours. The expression and production of RANKL and OPG at 6, 24 and 48hours was assessed by RT-PCR, quantiative real-time PCR, Northern blot and Western blot analyses. To investigate osteoclastogenesis, peripheral blood mononuclear cells (PBMCs) were cultured with IL-17 (5 and 50ng/ml) either alone or with M-CSF (25 ng/ml). After 14–21 days, cultures were fixed and stained for tartrate-resistant acid phosphatase (TRAP) and multinucleated, TRAP positive cells counted. Experiments were repeated on ivory slices and resorption evaluated.

Results: RT-PCR and QT-PCR analysis demonstrated that RANKL mRNA levels in SFB (4 of 5 patients) are enhanced by IL-17 in a biphasic manner. RANKL expression was elevated at 6 hours, returned to near control values at 24 hours before demonstrating increased levels at 48 hours. The expression of RANKL in MG63 cells was enhanced by IL-17 (5ng/ml) at 6 and 24 hours, and by IL-17 (50ng/ml) at 48 hours. The expression of OPG by SFB was upregulated by IL-17 (5 and 50ng/ml) at 6, 24 and 48 hours. The elevated expression of OPG in MG63 cells by IL-17 was time dependent, and this elevated expression was confirmed by Western blot. In cultures of PBMCs, IL-17 alone increased the numbers of TRAP+ve multinucleate cells dose-dependently. Similar levels of TRAP+ve cells were observed in the cultures treated with RANKL and M-CSF, but numbers of multinucleated cells were further increased when M-CSF was supplemented with IL-17. Resorption of ivory wafers was also observed in cultures treated with IL-17.

Conclusions: These results suggest that IL-17 induced osteoclast formation could contribute to the bone loss associated with a wide range of pathological states involving osteolysis and aseptic loosening.

A.C. Maury C.R.W. Southgate J.H. Kuiper N. Graham

Introduction: The failure rate of cemented hip replacements is about 1% per year, mainly due to aseptic loosening. PMMA acts as a grout, therefore high pressure is needed to ensure fixation. Various plug designs are used to increase pressure. No data is available on their ability to occlude the canal. Factors including canal size, canal shape and cement viscosity may affect performance. The two aims of this study are (I) to determine the effect of cement viscosity, canal shape and canal size on the ability of cement restrictors to withstand cementation pressures, and (II) to determine which of the currently commercially available designs of cement restrictor is able to withstand cementation pressures, regardless of values of other potentially influential factors.

Methods: Artificial femoral canals were drilled in oak blocks. Circular canals had diameters of 12 or 17.5 mm. Oval canals had short axes equal to the diameter of the circular canals and long axes 1.3 times longer. This ellipticity of 1.3 is average for human femoral canals. One of four types of cement plugs (Hardinge, DePuy, UK; Exeter, Stryker, UK; Amber Flex, Summit Medical, UK; and OptiPlug, Scandimed, Sweden) was inserted. A pressure transducer was fitted in the canal just proximal to the plug. Bone cement (Palacos LV-40 low viscosity or Palacos R-20 high viscosity, both Schering Plough, UK) was prepared in a mixing device for 1 min at 21°C, and inserted in the artificial canal after 4 minutes. A materials testing machine was used to generate pressure in the cement. Cement pressure and plug position were measured. All combinations of canal size and shape, plug design and cement viscosity were pre-selected according to a D-optimal experimental design which was optimised to perform a four-way ANOVA to analyse the four main factors plus the interactions between plugs and the other three factors. A total of 23 experiments was performed.

Results: Average cement pressures achieved differed between implants (OptiPlug 448±66 kPa, Hardinge 142±66, Exeter 705±66, Amber Flex 475±72; p=0.002, all mean±SEM). They also differed between canal sizes (12 mm 529±49, 18 mm 356±47; p=0.03), canal shapes (Round 631±45, Oval 254±51; p=0.004) and cement viscosity (High 535±54, Low 350±43; p=0.03). No significant interaction between factors was found.

Discussion and Conclusion: All plugs resisted lower pressures in large canals, oval canals or with low viscosity cement. When comparing plugs, these different circumstances should therefore be taken into account. Of the four tested, the Exeter plug performed best in all adverse circumstances. The OptiPlug and AmberFlex, which are both resorbable, had an intermediate performance. The Hardinge plug performed worse.

G. Datta K.K. Gnanalingham N. Mendoza K. O’Neill D. Peterson J. Van Dellen A. McGregor S.P.F. Hughes

Introduction: Preliminary studies suggest that prolonged retraction of the paraspinal muscle during spinal surgery may produce ischaemic damage. We describe the continuous measurement of intramuscular pressures (IMP) during decompressive lumbar laminectomy and the relationship to back pain and disability.

Methods: In this prospective interventional study, 28 patients undergoing surgery for lumbar canal stenosis were recruited. Back pain and function were assessed using the Visual Analogue Score (VAS), Oswestry Disability Index (ODI) and Short Form 36 (SF36) health survey. During surgery, IMP was continuously recorded from the multifidus muscle using a pressure transducer. The intramuscular perfusion pressure (IPP) was derived as the difference between the patient’s mean arterial pressure (MAP) and IMP (IPP = MAP − IMP). The data was analysed using repeated measures ANOVA (SPSS package).

Results: The mean age was 60.4 ± 3 years and the mean duration of symptoms of 31.0 ± 6 months. The predominant symptoms were neurogenic claudication (14) and/or sciatica (13). Patients underwent 1 (N=3), 2 (N=20) or 3 (N=5) level laminectomies. The muscle retractors used were Norfolk and Norwich (N=16) and McCullock (N=12). The mean duration of deep muscle retraction was 68.5 ± 9 mins (range 19–240). On application of deep muscle retraction, there was a rapid and sustained increase in IMP (F=26.8; p< 0.001; repeated measures ANOVA), and overall the calculated mean IPP approached 0 mmHg or less during this period (F=36.8; p< 0.001). On release of deep muscle retraction there was a rapid decrease in IMP to pre-operative levels. The IPP was greater with Norfolk and Norwich than McCullock retractors (F=12.2; p< 0.001). Compared to pre-operative values, there was a decrease in ODI (F=18.6; p< 0.001) and VAS for back pain (F=9.9; p< 0.001) at discharge, 4–6 weeks and 6 months, post-operatively. Compared to pre-operative values, there was a decrease in SF36 scores at 6 months (F=26.7; p< 0.001). Total duration of muscle retraction over 60 mins was associated with higher VAS scores for back pain at 4–6 weeks and 6 months postoperatively (F=3.7; p< 0.01). There was no relationship between IPP and post-operative ODI or VAS for back pain.

Conclusions: This study demonstrates a simple technique for the continuous monitoring of IMP during spinal surgery, from which the IPP can be derived. Comparison of two muscle retractors has shown that the McCullock retractor generates a higher IMP than Norfolk and Norwich retractor. Decompressive lumbar laminectomy improves the VAS for back pain and ODI and SF36 outcome scores in these patients. The results show that duration of muscle retraction, rather than extent of the pressure generated by the retractor, is related to postoperative back pain.

C. Huber V. Mann H. Simpson B. Noble

Introduction: Oxidative stress occurs when reactive oxygen species (ROS) are produced faster than they can be removed by cellular defence mechanisms contributing to ageing, many chronic diseases, such as atherosclerosis, RA, Parkinson and Alzheimer’s disease and skeletal pathologies. Here we address the impact of ROS on the viability of early osteogenic precursors in the bone marrow and study the influence of estrogen on this interaction. Cells have a number of mechanisms to protect themselves from ROS, which are constantly being formed in the cell through normal metabolic pathways, such as Vitamin E, C and estrogen. Estrogen has been shown to prevent intracellular accumulation of peroxide and to attenuate oxidant-induced death of neuronal and endothelial cells. In addition, it contributes significantly to bone turnover and relieves postmenopausal symptoms. This study has focused on the potential anti-oxidant properties of estrogen against oxidative on bone marrow stromal cells. stress induced by H2O2

Methods: Primary bone marrow stromal cells were pre-treated with several different doses between 10−6M – 10−8M of estrogen prior to H2O2 administration at 0.08–0.4 mM 30% (v/v) for 2–24h. The cellular production of ROS was determined by using the free radical indicator DCFH-DA. Apoptosis was determined by morphological criteria.

Results: H2O2 induced an increase in apoptosis of osteoprogenitor cells (p< 0.05). Determination of apoptosis and cell number by nuclear staining, indicated that pre-treatment of bone marrow stromal cells with 17-beta estradiol reduced the apoptotic response induced by H2O2 (p< 0.05) and restored cell number to control levels. In order to test the anti-oxidant activity of estrogen, the dye DCFH-DA was introduced in a cell free system in the presence or absence of 17-beta estradiol and H2O2. The same experiment was repeated in the presence of bone marrow stromal cells. H2O2 increased both intracellularly and extracellularly oxidant activity and estradiol has the capacity of modifying this activity both inside and outside the cell.

Discussion: These data demonstrate the ability of estrogen, used at physiological doses, to block oxidant-induced apoptosis of osteoprogenitor cells. Estrogen appears to reduce the generation of ROS in these cells. These data could have important implications on the maintenance of osteogenic stem cells during fractures, ageing and disease.

N.L. Shortt B. Noble V. Mann A.H. Simpson

Introduction: The concept of cell senescence has been described as the mechanism responsible for the ageing of tissues, that is a finite ability to replicate and produce new tissue. The senescent cell population is separate and distinct from the cells which are undergoing programmed cell death (apoptosis), and those which are necrosing acutely. Cells reaching the senescent state have an increase in β-galactosidase activity, which is detectable using an established technique for soft tissues including fibroblasts and epithelial tissues. Senescence has not previously been investigated in bone. We have investigated this and hypothesise that new bone formed by distraction osteogenesis will have fewer senescent cells than the adult cortical “old” bone.

Methods: Eight New Zealand white rabbits underwent application of a M100 Orthofix external fixator to the tibia and creation of a mid-diaphyseal osteotomy, using a hand saw. After a seven day latency period, distraction was commenced (0.5mm twice daily) to twenty percent lengthening. After 3 weeks consolidation, the tibae were harvested for histological analysis.Senescent Staining:The sections were stained using a technique described by Faragher, using an X-gal based stain. Sections were incubated for 16 hours at 37 degrees centigrade before counter staining with DAPI. Sections underwent histological analysis and total cell and senescent cell counts performed.

Results: Surprisingly, large numbers of cells within the bone regenerate stained for cell senescence. A mixture of multinucleate and mononucleate cells were present. The location and appearance of the multinucleate cells prompted the use of TRAP staining. This provided support for these cells being osteoclasts.

Discussion: Previously, a high percentage of apoptotic cells and a high rate of cell division has been reported in bone regenerate. The surprisingly high numbers of cells within the newly formed bone staining positively for senescence suggest that there may also be a high senescent cell population. Alternatively, the positive TRAP staining may indicate that the stain is less specific than reported and may be staining osteoclasts and mature macrophages within the bone regenerate.

S.R. Vollans N. Upadhyay B.B. Seedhom

Introduction: Isolated PCL ruptures are most frequently treated non-operatively, although PCL deficiency may ultimately lead to degenerative changes within the patellofemoral compartment. This study investigated, for the first time under physiological loading conditions, the change in patellar tracking as a result of PCL deficiency, hoping to further understand the clinical consequences in situations where such an injury is treated conservatively.

Method: Using eight fresh cadaveric knees, physiological axial tibiofemoral loads and rotatory torques occurring during level walking, were applied to determine tibial rotation angles. These were then used under dynamic Quadriceps femoris loading to determine contact areas and stresses within the patellofemoral joint at 15°, 30°, 60° and 90° of knee flexion. The PCL was then severed, and the procedure repeated under the same loading conditions.

Results: Significant increases in patellofemoral contact stress in the PCL deficient knees were observed at 15° and 30° knee flexion, both in internal and external rotation of the tibia (TABLE I). For these respective rotation positions the increases were 23% and 20% at 15°, and 19% and 28% at 30°, (in all cases p≤0.05). These significantly increased stresses coincided with unchanged contact patterns on the inferior third of the patella, spanning both its medial and lateral facets.

Conclusions: The increased stresses were due to increased patellofemoral joint reaction force, caused by a decreased angle between the quadriceps and patellar tendons due directly to posterior tibial translation in the PCL deficient knees. Significantly increased patellofemoral contact stresses at 15° and 30° of knee flexion, may be implicated in the degeneration of articular cartilage, on both the medial and lateral facets of the inferior third of the patella. These results point out the need for further biomechanical studies to investigate the effects of more strenuous loading conditions. There is also need for clinical studies to investigate focal lesions associated with long-term PCL deficiency.

S.J.A. Kettle M.A. Glasby

Introduction: End-to-side nerve repair is an experimental technique for repairing peripheral nerves when severe injury renders the proximal nerve stump not available for end-to-end repair or for conventional nerve grafting techniques. This study uses a large animal model to compare two variations of end-to-side neurorrhaphy techniques with conventional clinically established methods of nerve repair to assess the feasibility of end-to-side suture as a technique for possible future clinical use.

Methods: 12 age and weight matched sheep underwent end-to-side neurorrhaphy of the distal stump of the transected median nerve to the lateral side of the adjacent intact ulnar nerve through an epineurial window. 12 sheep underwent the same procedure as above but with the proximal stump of the transected median nerve similarly attached 2cm proximal to the first neurorrhaphy site to create a double end-to-side model. 18 sheep underwent conventional methods of nerve repair. All the experiments were randomized and the author performed all the surgery. The nerve repairs were assessed electrophysiologically and histologically and the muscles supplied by the repaired nerves were assessed physiologically at one-year post repair. Normal median nerves and donor ulnar nerves were also tested in the same ways.

Results: There were no significant differences in the outcomes of nerve repair between different conventional techniques. Half the end-to-side repairs failed but the double end-to-side repair consistently supported nerve regeneration. Both end-to-side methods were inferior to conventional techniques of nerve repair in all measures of outcome except twitch and tetanic muscle tensions. The function of the donor ulnar nerves in terms of conduction velocity was compromised in the double end-to-side repair but not the end-to-side repair.

Discussion and Conclusions: End–to-side nerve repair did support nerve regeneration but it was all or nothing. It is likely that the double end-to-side neurorrhaphies regenerated more consistently than the single end-to-side neurorrhaphies due the conduit effect of the donor ulnar nerve bridge supporting axon growth. Donor ulnar nerve damage in the double end-to-side group suggests regeneration may have occurred from terminal sprouts rather than collateral sprouts.

Although end-to-side neurorrhaphy did support nerve regeneration with sometimes good return of muscle function, the use of this technique as a clinical tool at this time cannot be recommended.

N. Upadhyay S.R. Vollans B.B. Seedhom R.W. Soames

Introduction: Anterior cruciate ligament (ACL) rupture impairs knee stability. Reconstruction of the ACL is therefore performed to restore knee stability and avert risk of subsequent ligament and meniscal injury. Bone-patellar tendon-bone autograft is the most commonly employed technique for ACL reconstruction and considered the “gold standard”. Although 10% postoperative patellar tendon shortening has been reported with this technique, there are no systematic studies assessing the effect of this shortening on patellofemoral joint (PFJ) biomechanics under loading conditions simulating normal physiologic activity. The purpose of this study was to determine if 10% shortening of the patellar tendon affected PFJ biomechanics.

Methods: Patellofemoral contact characteristics were evaluated in cadaveric knees before and after patellar tendon shortening. Tendon shortening was performed using a specifically designed device that shortened the tendon without interfering with its anatomic and physiologic integrity. Conditions simulating light physical activity such as level walking were recreated by applying physiological quadriceps loads and corresponding angles of tibial rotation to the PFJ at 15°, 30° and 60° of knee flexion. PFJ contact areas were measured at each position of knee flexion before and after patellar tendon shortening using the silicone oil-carbon black powder suspension squeeze technique (3S technique, Yao & Seedhom, Proc Instn Mech Engrs1991;205:69–72). Differences were compared using the Wilcoxon signed rank t-test, with p< 0.05 required for statistical significance.

Results: Twelve unembalmed cadaveric knees (median age 81.8 years, 8 female: 4 male) were available for study. Five knees had evidence of osteoarthritic changes, and were rejected. The remaining 7 knees were macroscopically intact and were considered adequate for the experimental procedure. The mean patellofemoral contact areas and stresses determined preoperatively were comparable to those reported in normal knees in previous studies. Following patellar tendon shortening, PFJ contact areas were displaced superiorly on the patellar articular surface and distally on the femoral articular surface. Although the PFJ contact area increased by 17% at 15° of knee flexion (p=0.04), no significant change occurred at 30° or 60° of knee flexion (p> 0.05). Patellofemoral contact stress did not differ before and after patellar tendon shortening (p> 0.05) at any angle of knee flexion.

Conclusions: Our results suggest that with light activity such as level walking, a 10% postoperative shortening of the patellar tendon does not alter patellar tracking (in particular contact stresses) and therefore may not impact biomechanics of the patellofemoral joint. Extrapolating these results to the clinical scenario, deleterious consequences on the patellofemoral joint are unlikely after bone-patellar tendon-bone autograft reconstruction of the ACL despite the possibility of postoperative patellar tendon shortening.

H.S. Gill K. Polgar S. Glyn-Jones P. McLardy-Smith D.W. Murray

Introduction: The design philosophy of polished tapered THR stems, such as the Exeter, intend for them to migrate distally within the cement mantle. In addition it is likely that micromotion occurs as a result of functional activity. The pattern of induced stresses will be a function of stem geometry & surface finish, as well as applied loading. Aim: To investigate the stresses induced in the cement mantle of a polished tapered THR stem during functional activity.

Method: Using Roentgen Stereophotogrammetric Analysis (RSA) dynamically induced micro-motion (DIMM) was measured in 21 patients implanted with Exeter stems. DIMM was measured as the difference in stem position in going from double to single leg stance on the operated limb. All subjects were measured 3 months post-operatively. A finite element (FE) model of the femur, including all muscles was used to investigate the stress distribution within the cement; contact was modelled with sliding elements allowing separation. The model was validated by comparison to the DIMM measurements.

Results: The Exeter stem demonstrated significant DIMM(p < 0.017), the average motions are given in the table below. The FE model, with sliding contacts was able to predict similar distal migration of the head. The peak minimum principal stress in the mantle was approx 33MPa and occurred in the proximal medial region. Movements occurred at the stem/cement interface.

Discussion and Conclusion: It is possible to measure DIMM in the Exeter stem and combining this with FE modelling the mechanism of stress transfer between the stem and mantle can be investigated in a manner that can be validated.

J.B. Aderinto G.W. Blunn

Introduction: Bone marrow derived stromal stem cells (BMSSC’s) have the ability to differentiate into a variety of mesenchymal tissues including bone. The objective of this study was to evaluate the use a hydroxyapatite – BMSSC (HA-BMSSC) composite graft for posterior spinal fusion in a rabbit model.

Method: The HA- BMSSC composite graft was prepared by seeding rabbit marrow derived BMSSC’s onto 5 grams of HA granules which were cultured for a further 7 days prior to implantation. Bilateral posterior L4–L5 interlamina spinal fusion was performed using the HA- BMSSC composite graft (4 Rabbits), hydroxyapatite(HA) granules (6 rabbits) or autologous bone graft obtained from the iliac crest (6 rabbits). Rabbits were sacrificed at 5 weeks. Fusion was assessed by manual palpation. Quantitative histological analysis of cartilage, fibrous tissue and bone in the mid portion of the graft was performed using image analysis software.

Results: Three of four of the HA- BMSSC grafts fused successfully compared to 5 of 6 of the autologous bone grafts and 0 of 6 of the HA control grafts. The fusion rate was significantly higher in the iliac crest and HA- BMSSC groups than the HA control group (p< 0.05). In both the HA control and HA stem cell composite grafts there was ingrowth of new bone and encasement of HA granules by new trabecular bone at the graft – host interface. Within the mid region of the grafts there was bone formation in 2 of four fusion masses in the HA- BMSSC group comprising 26% and 45% of tissue in the area examined. In contrast bone formation was seen in the centre of only one of the six 6 HA fusion masses and amounted to only 2% of tissue. There was no significant difference in average percentage area of new bone, cartilage or fibrous tissue within the central region of the HA and HA-BMSSC grafts. There was a higher mean percentage area of new bone formation within the autologous bone graft (27%) than the HA control group (0.3%). p< 0.02.

Discussion: The BMSSC –HA composite was as effective as autologous graft and superior to HA in promoting fusion, but HA when used alone was ineffective. A positive finding to support the osteogenic potential of the stem cell loaded HA granules was the presence of moderate amounts of enchondral new bone isolated within the central regions of the graft away from the graft host interface in 2 of 4 fusion masses. In contrast the HA control grafts only supported significant amounts of bone formation in the periphery, adjacent to the host bed.

S.L. Gouldson M.J. Coathup G.W. Blunn M. Sood

Introduction: One of the most common complications following total joint surgery is aseptic loosening. Improving the bone-cement interlock may increase implant longevity. An ideally prepared bony surface is dry; clean; free from marrow, fat and debris; free from active bleeding; and free from micro-organisms. Lavage removes debris, blood and fat from the interstices of the bone surface so as to allow optimal penetration of the cement. The hypothesis that we investigated in this study was that lavage with a detergent solution obtains a greater depth of cement penetration into bone compared with lavage using 0.9% saline, hydrogen peroxide or an alcohol solution.

Methods: The cancellous bone of ovine femoral condyles were cut into 10×10×13mm blocks. Lavage solutions were delivered via a pulsatile system and directed towards one side of the bone block. All blocks were swabbed dry. A high viscosity cement was manually mixed and applied to the sandblasted surface of titanium alloy plate (10×10mm, weight 0.9g ±0.01g). The titanium plate and cement were placed on the irrigated bone block, and a known weight applied to achieve pressurisation. Time, temperature and method were controlled. The prosthesis-cement-bone composite was sectioned perpendicularly, and image analysis used to quantify penetration depths. 10 readings were recorded per block with 6 blocks per lavage group.

Results: Cancellous bone porosity averaged 75.2% (±4.0) . The mean penetration depth in the saline group averaged 3.39mm (± 0.77); 3.04mm (± 0.59) using a 2% alcohol solution; 3.33mm (±0.79) using a 3% hydrogen peroxide solution; and 5.41mm (± 1.30) when using the detergent lavage. There was no significant difference in cement penetration depth between hydrogen peroxide and saline irrigation (p> 0.05), nor with hydrogen peroxide and alcohol irrigation (p> 0.05). Irrigation with saline however, afforded statistically superior cement penetration than that of alcohol lavage (p < 0.012). Irrigation with detergent solution demonstrated significantly greater depth of penetration than all three other lavage groups (saline p< 0.05; alcohol p< 0.05; hydrogen peroxide p< 0.05).

Discussion: Detergents can physically remove particulate matter and emulsify and remove fats, thereby acting to maximise porosity of the cancellous bone network and optimise space for occupation by intruding cement. This study has proven the ability of a detergent solution to provide a clean, debris free cancellous network, which consequently provides a significantly greater depth of cement penetration than other commonly used irrigating agents. It was noted that cement penetration into cancellous bone followed the line and depth of cleaning from lavage. In conclusion, the hypothesis can be accepted, and lavage with a detergent solution affords a statistically greater depth of cement penetration into bone than that of the universally used 0.9% saline lavage.

S.N. Racey E. Jones M.A. Birch A.W. McCaskie

Introduction: Several recent studies have highlighted the influence of topographical features on the response of cells to biomaterial surfaces, both in terms of their adhesion, morphology and gene expression. Initial cell adhesion events are believed to be pivotal in dictating subsequent host response to implant materials and therefore understanding the mechansims that regulate these events is fundemental to the design and engineering of the next generation of biomaterials. In our studies we evaluated the adhesion associated events of osteoblasts on four orthopaedic metals, each produced to the same surface finnish. Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) were used to determine the nanometre scale topography and immunofluorescence microscopy and image analysis performed to evaluate cell morphology.

Methods: Vitallium, titanium grade 2 (Ti2), Ti6Al4V and TM2F discs were prepared by Stryker, machined and finished to 1 micron. SEM and AFM were then used to analyse surface topography. Rat primary osteoblasts were then seeded at low density onto the metal discs and allowed to adhere and spread for 24 hours. The cells where fixed and focal adhesions stained with an anti-vinculin Mab. The actin cytoskeleton was counterstained with TRITC phalloidin and nuclei stained with DAPI. Images where captured on both a standard epiflourescence microscope and a confocal microscope. Image analysis was performed using ScionImageTM to determine cell area, major X/Y axis lengths and numbers of focal adhesions per cell.

Results: Gross observation of all samples revealed a perfectly smooth and flat surface. SEM and AFM analysis showed that at the nanometre scale each exhibited varying degrees of surface roughness. Vitallium was the smoothest with scratches a few nanometres deep running across the surface. In contrast Ti6Al4V, Ti2 and TM2F had increasing degrees of surface roughness, each with details that measured up to a few microns in height.

We measured 1: the area occupied by a cell and 2: the number of focal adhesions per cell. The largest values of osteoblastic cell area were seen with the smoother vitallium surface. In contrast, samples with more numerous and larger surface features resulted in the osteoblasts covering a smaller area and being confined by topographical elements (Ti2> TM2F> Ti6Al4V). In terms of adhesion, there were generally more focal adhesions per cell on rougher surfaces (Ti6Al4V> TM2F> Vitallium> Ti2).

Conclusions: The different nanometre scale features introduced through the manufacturing process of different orthopaedic implant materials influence the adhesion and cell morphology of osteoblast cells within the first 24 hours of contact. This may have consequences for later differentiation and function of these cells.

P.G. Bush J.S. Huntley M.F. Macnicol A.C. Hall

Introduction: In the growth plate, chondrocyte swelling (hypertrophy) is a crucial event during endochondral ossification and bone lengthening, accounting for ~80% of the increase in bone length (1,3). The swelling is dramatic (~10x) and closely regulated. Failure of chondrocyte hypertrophy may underlie the chondrodysplasias of the vertebrate skeleton (1). However, the mechanisms which control cell swelling are poorly understood although there must be a key role for chondrocyte osmolyte transporters which are sensitive to an increase in cell volume. We have used confocal scanning laser microscopy (CLSM) to study volume regulation by living in situ growth plate chondrocytes at varying degrees of hypertrophy.

Methods: Bovine growth plates were taken from the ends of young (~12d) bovine ribs. In situ growth plate chondrocytes at the proliferative through to hypertrophic stages were fluorescently-labelled (calcein-AM; 5μM), imaged (Zeiss CLSM510) and volumes determined quantitatively as described (2). An acute osmotic challenge (280-140mOsm) was delivered by perfusion to determine volume-regulatory capacity by cells in the various zones.

Results: The resting volumes of proliferative and hypertrophic cells were 550±63μm3 and 5227±1974μm3 respectively. Reducing osmolarity resulted in a rapid (within ~1min) cell swelling, proliferative and hypertrophic chondrocytes increasing in volume by 126±2% and 146±5% (n=5) respectively. Chondrocytes within the proliferative zone then recovered in volume by ~60% over the following 20mins (p=0.04), whereas no volume recovery was detected in hypertrophic cells (p=0.94).

Conclusions: For the increase in growth plate chondrocyte volume to produce hypertrophy it is essential that the membrane transporters which normally prevent cell swelling are suppressed, otherwise the increase in volume will be compromised. These results suggest that chondrocyte hypertrophy is associated with reduced activity of the swelling-stimulated osmolyte transporter whereas the pathway is active in proliferating chondrocytes. Changes in the activity of this pathway are likely to be an important component in the control of chondrocyte hypertrophy. It is clear that the contributions of other membrane transporters in mediating chondrocyte swelling must be identified in order to understand the overall hypertrophic process.

Y. Sasazaki R. Shore B.B. Seedhom

Introduction: Cartilage is an anisotropic material whose structure and tensile properties vary with the depth from the articular surface. Further, ultrastructural changes of articular cartilage under strain are poorly understood. The aim of this study therefore was to visualize the zonal variations in ultrastructural changes of cartilage when subjected to a range of tensile strains to failure.

Materials and Methods: 3 osteochondral plugs were harvested from the femur of a 3 years old bovine with a cylindrical reamer. Cartilage was cut parallel to the articular surface into the superficial, middle and deep layers, 300μm thick each and then each was cut normal to the surface into dumbbell shaped specimen 10 mm long. Each specimen (9 in total) was clamped in an individual mini tension device and subjected to a specific strain, then fixed and processed whilst still under strain within its tension device for observation with SEM.

Results: When specimens were observed in en face view under no strain, a fibrillar meshwork was seen to run parallel to the articular surface in the superficial layer, randomly in the middle layer and perpendicular to the articular surface in the deep layer. Under strain the fibrillar meshwork began to reorient parallel to strain (tangential to the surface) in each layer. At 20% strain the whole fibrillar meshwork was reoriented and formed bundles in the superficial layer. In the middle layer almost whole of the fibrillar meshwork was reoriented at 40% strain. In the deep layer the fibrillar mesh-work was reoriented parallel to the strain in some areas, while in the other areas it was still seen perpendicular to the surface even at 70% strain.

Conclusions: The collagen meshwork of cartilage was reorganised under strain and this appears to play an important role in cartilage extension. Thus the more rapid reorientation in the superficial layer may result in its reduced extensibility compared to that of the deeper layers.

V. Mann C. Towell G. Kogianni H. Simpson B. Noble

Introduction: Evidence exists concerning the anti-oxidant properties of oestrogen in protecting neuronal cells from oxidative stress. The withdrawal of oestrogen after menopause is the major factor determining age related bone loss and apoptotic death of osteocytes. While oestrogen replacement demonstrates clear oestrogen receptor mediated benefits to bone cells little is known regarding oestrogens’ anti-oxidant effects in bone.

Methods: Here we have used MLO-Y4 osteocyte-like cell line to determine whether oestrogen saving effects on osteocytes involves its activities as an anti-oxidant.

MLO-Y4 cells were treated with physiological doses (10−8)M of either 17-beta E2 or the oestrogen receptor inactive stereoisomer 17-alpha E2 with or without the specific oestrogen receptor antagonist ICI 182,780 prior to the addition of 0.4milliM 30% (v/v) H2O2. Cellular apoptosis was determined using morphological and biochemical criteria.

Results: H2O2 induced an increase in apoptosis of MLO-Y4 (14.3 ± 3 SD vs control 1.4 ± 0.9). Pre-treatment of the cells with 17-beta E2 significantly reduced H2O2 induced apoptosis (2.4 ± 0.96). Pre-treatment of cells with 17-alpha E2 or ICI 182,780 also reduced oxidant induced apoptosis to 3.4 ± 1.5 SD and 7.0 ± 2.3 respectively.

The cellular production of reactive oxygen species was determined using the free radical indicator 2′7′- dichlorodihydrofluorescein diacetate. H2O2 induced increases in the number of ROS positive cells (34.6 ± 9.07 SD vs control 0.22 ± 0.39 SD). In contrast pre-treatment with both 17-beta E2 and 17-alpha E2 reduced the number of ROS positive cells associated with H2O2 treatment (Fig 1).

Conclusion: These data suggest that oestrogens ability to save osteocytes from oxidant induced death is independent of the oestrogen receptor and may be related to oestrogens known activity as an anti-oxidant. This raises the possibility that loss of osteocytes during oestrogen insufficiency may occur through a failure to suppress the activity of naturally occurring or disease associated production of oxidant molecules.

J. Blackburn M.J. Coathup T. Smith A.E. Goodship G.W. Blunn

Introduction: The main problem facing the longevity of total hip replacements (THR) is wear particle induced osteolysis, particularly around the acetabular component. The articulating surfaces produce wear particles that migrate in the fibrous tissue membrane along the acetabular implant-bone interface causing osteolysis and subsequent implant loosening. The hypothesis that we investigated was that uncemented acetabular interfaces are more effective than cemented implants at resisting progressive osteolysis through bone attachment and the formation of a biological seal.

Methods: THR surgery was performed in an ovine model. Implants remained in vivo for 1 year. Femoral heads were roughened in order to generate wear debris and aseptic loosening of the acetabular component. Sheep were randomly assigned to one of three experimental groups: cemented polyethylene, grit blasted or plasma sprayed porous acetabular components with a polyethylene insert. Ground Reaction Force (GRF) data was collected pre-op and at 12, 24, 36 and 52 weeks post op. Retrieved specimens were analysed radiographically, histologically and using Scanning Electron Microscopy (SEM). A mould was made of the polyethylene liner and head penetration rates quantified using a shadowgraph technique. Thin sections through the acetabuli were prepared and image analysis used to quantify fibrous tissue (FT) thickness at the bone-implant interface. Mann-Whitney U tests were used for comparative statistical analysis where p< 0.05 were classified as significant.

Results: GRF demonstrated functional hips. A gradual increase was seen until week 36 followed by a decrease until retrieval suggesting the onset of aseptic loosening. 42.86% of control, 60% of grit blasted and 50% of porous coated components were deemed radiographically loose. Mean linear penetration rates demonstrated significantly less penetration within the porous cups (p=0.003, control and p=0.036, grit blasted). SEM established that wear particles generated were < 1μm in size. Light microscopy of thin sections revealed the common mechanism of loosening involving a resorption wedge at the interface with progressive bone loss. In all cases, the FT layer was greatest at the rim of the cup and gradually decreased towards the apex. The grit blasted group had the thickest FT layer adjacent to the cup. Under polarised light, wear debris was seen packed within macrophages in all sections.

Discussion: GRF data demonstrated grit blasted cups to have least function. This was confirmed through histology as they had the thickest FT layer surrounding the acetabular shell suggesting increased aseptic loosening of its component due to wear particles being able to access the interface more easily. Data corroborates radiographic results. In conclusion, porous and control cups performed better than grit blasted cups. Acknowledgments: EPSRC.

K. Mohanty J.N. Powell D. Musso D. Traboulsi I. Belenkie B. Mullen J.V. Tyberg

Introduction: Early stabilization of the skeleton in multiply injured patients has shown to reduce mortality and chest morbidity. Reamed intramedullary nailing is the current method of choice for stablizing femoral and tibial shaft fracture. However several investigators have highlighted the adverse effect of early reamed nailing in polytrauma patients. Intravasation of medullary fat during canal pressurizaton has been suspected to produce a ‘second hit’ and trigger pneumonia and ARDS. The objective of this study is to investigate the effect of a filter placed into the ipsilateral common iliac vein during medullary canal pressurization.

Methods: Using an established model of fat embolization, twelve mongrel dogs were randomized into two groups. Under general anaesthesia, cannulations of carotids and jugular veins and transesophageal echo-cardiography were performed in all animals. Under fluoroscopy control, a special filter was inserted percutaneously into the left common iliac vein in half the animals, where as the other half served as controls. In all dogs, the left knee was exposed; the femor and tiiba were sequentially reamed and then pressurized by injection of bone cement and insertion of intramedullary rods. Hemodynamic measurements and trans-esophageal echocardiography images were recorded continuously during the surgical procedure. After 45 minutes from pressurization, the dogs were sacrificed and the lungs and kidneys were harvested and fixed for histological analysis.

Results: There was significant difference noticed in the right-sided pressures and oxygen tension between the filter and the control groups. The mean pulmonary artery pressure at 3 minutes of pressurization was 12mm of Hg in the filter group and 28mm of Hg in the control group. Transesophageal echocardiography showed less embolic shower in the filter group and also lesser dilatation of right ventricles. Histomorphometry with special staining demonstrated much less proportion of lungs to be occupied by fat in the filter group as compared to the control group.

Discussion and Conclusion: This canine study has demonstrated that mechanical blockade by a venous filter can significantly reduce the emobilic load on the lungs in an established model of fat embolization. A suitable filter with suction system is being designed for possible use in high-risk patients.

J.M. Loughead D. Chesney J.P. Holland A.W. McCaskie

Introduction: Patients following resurfacing frequently remark about the natural feel of the resurfaced hip joint in contrast to those with total hip arthroplasty. Possible reasons for this include the larger femoral head size, conservation of bone and superior biomechanics of the implant with more accurate restoration of femoral offset, leg length and femoral anteversion.

Our aim was to assess femoral offset and leg length following hip resurfacing and hybrid THR (uncemented acetabulum) performed by the same surgeon.

Methods: A consecutive group of patients were identified (35 resurfacing and 25 hybrid). AP pelvis radiographs were evalulated, films with evidence of malrotation or inadequate imaging of the femur were excluded, leaving 21 resurfacing and 15 hybrid. Comparison was made between the pre-op and post-op films together with the contralateral hip on the same film. Patients with hip dysplasia or significant pathology in the contralateral hip were excluded. Magnification of the films was measured by comparison of the templated diameter of the implanted femoral head and the acutal diameter of the implant. To allow comparison between pre-op films a measurement was taken between the obturaror foraminae.

All films were analysed by the same investigator using the technique described by Jolles et al (J Arthroplasty 2002). A horizontal line was drawn between the base of the teardrop on both sides, and perpendicular lines drawn from the back of the teardrops. The anatomical femoral axis was drawn and femoral offset measured from this. The centre of rotation of the femoral head was determined by templating and the acetabular offset obtained.

Distance from tip of the greater trochanter to the centre of the femoral head in the axis of the femur was determined on pre and post-op films, as this shows little variation with rotation of the femur. Leg length was measured from the horizontal line to the tip of the greater trochanter together with the angle between the femoral axis and the horizontal to correct for abduction of the hip.

Results: Mean total femoral offset compared to the contralateral side was −1.3mm (SD 5.3) and −3.2mm (SD 6.5) for the resurfacing and hybrid groups respectively. No significant difference was detected in leg length or other measurements.

Discussion and Conclusion: No significant differences were demonstrated between femoral offset or leg length in the resurfacing and hybrid arthroplasty groups. This study does not support the hypothesis that resurfacing produces more accurate restoration of hip biomechanics than hybrid total hip arthroplasty.

G. Biring J. Meswania C. Wylie S. Muirhead-Allwood J. Hua G. Blunn

Introduction: To investigate the head/neck interface of total hip replacements and to see whether the use of small spigots (minispigots) results in enhanced wear and corrosion of tapers compared to standard spigots and the influence of the surface finish on this.

Methods: In the total hip replacement combinations the heads were made of cobalt-chrome (CoCr) and the stems of titanium alloy (Ti). Firstly wear and corrosion of minisigots were compared with standard spigots (Test 1) and secondly, these minispigots were compared with another minispigot with a smoother taper surface finish (Test 2). The samples were immersed in aerated Ringers solution (37°C) and loaded for 10 million cycles. The specimens surface parameters and profiles were measured before & after the test. Electrochemical static corrosion tests were carried out on the rough & smooth minispigots from Test 2 where the current was measured with constant potential under loaded and non-loaded conditions. A cyclical sinusoidal load of 1500-200 Newtons for 1000 cycles at ~1 Hz was used. Pitting tests measured the current while increasing and then decreasing the potential of non-loaded and loaded specimens. Two newly manufactured rough and smooth minispigots were subjected to the same electrochemical corrosion tests.

Results: In Test 1 the results demonstrated that pre-test the surfaces of the female tapers were similar for all heads. Post-testing the Ra values on the female tapers had become greater for the minispigots compared with standard spigots. An abrupt change was noted on the surface profile of the female taper where it was in contact with the male Ti taper, indicating the the CoCr head had corroded. The Ti male tapers were unchanged. Scanning electron microscopy showed that the coarser profile in the corroded region of the CoCr was similar to the profile on the Ti male taper. Pitting corrosion was evident in the grooves on the CoCr. In Test 2 the smooth spigots were not affected, but in the rough minispigots, Ra values had increased in the female tapers. Static corrosion tests showed evidence of fretting in the rough but not the smooth minispigots. When comparing new rough & smooth minispigots, static corrosion testing with clyclical loading showed that for minispigots with a rough finish the current fluctuated with each cycle. Pitting scans showed a greater hysteresis with the rough minispigot compared with the smooth minispigot indicating potentially greater corrosion in the former.

Conclusion: The cobalt-chrome/titanium alloy combinations where the surface finish on the male taper was coarse, corrosion was increased in minispigots compared with standard spigots. This was due to the smaller area of contact of the minispigot at the interface. This corrosion appears to be mediated through the mechanism of fretting corrosion. Surface finish was crucial and corrosion of the minispigot was reduced if the surface finish was smooth. Manufacturers should investigate the effect of surface finish on the corrosion of their tapers particularly where cobalt-chrome/titanium alloy combinations are used.

T.J. Smith A.E. Goodship H.L. Birch

Introduction: Different tendons and ligaments have a specific elasticity which relates to their role in joint movement and locomotion. To ensure an optimal functional outcome it is essential that this mechanical property is restored following surgical procedures to repair or replace damaged tendons and ligaments. This demands appropriate selection of an autograft or artificial construct aided by an understanding of how molecular composition and morphology determines the stiffness of the material. This study tests the hypothesis that tendons with a higher elastic modulus (stiffer) have larger collagen fibril diameters and lower water and sulphated glycosaminoglycan (GAG) contents.

Methods: The superficial digital flexor tendon (SDFT, 30 pairs), deep digital flexor tendon (DDFT, 6 pairs), suspensory ligament (SL, 6 pairs) and common digital extensor tendon (CDET, 6 pairs) were collected from the forelimbs of horses aged 2–23 years destroyed for reasons other than tendon injuries. Left limb tendons were tested to failure in a hydraulic materials testing machine (Dartec) following measurement of cross sectional area. Collagen fibril diameters, water content and sulphated GAG content were measured in tendon tissue from the right limb. Statistical significance was evaluated using Spearman’s correlation and a general linear model (SPSS software).

Results: The elastic modulus was significantly (p< 0.001) different between the different structures and showed a significant positive correlation with the mass average collagen fibril diameter (MAFD) for the different structures and within the SDFT (FIG. I). The water content showed a significant negative correlation with elastic modulus and significant positive correlation with GAG content.

Discussion and Conclusion: Tendons composed of a stiffer material have larger collagen fibril diameters which are associated with lower water and GAG contents. These characteristics should be considered when choosing suitable replacements in tendon reconstruction procedures. Future work to determine the mechanisms that control collagen fibril diameters and water content will aid in the design of bioengineered constructs.

A Gupta G Marwah J.L. Bassi

Introduction: Road side accidents resulting in polytrauma with an associated fracture of femur is a common pattern of injury in asian countries. We hypothesised that the use of unreamed nailing in the management of such fractures has better outcome than reamed nailing.

Material and Methods: We retrospectively reviewed 116 cases of polytrauma with associated fracture of shaft of femur admitted in our tertiary teaching hospital in North India bewteen Jan 1996 to Dec 2001. The patients were initially resuscitated according to the advanced trauma life support protocol. They were randomally managed by interlocking nail using reamed (n=48) and unreamed (n=68) technique after being haemodynamically stabilized. Five intraoperative parameters were recorded – the surgical time, fluoroscopy time, the intraoperative blood loss, intraoperative oxygen saturation, and any intraoperative complications. The patients were assessed postoperatively for ninety six hours for features of adult respiratory distress syndrome. All patients were clinically and radiologically assessed at 6 weekly intervals till union. The follow-up reassessments were performed by a single surgeon (AG).

Results: There were 80 males, 30 females (6 were bilateral), with an average age of 26 years (range 19 to 64 years). The fractures were closed in 74 and open in 42 (Gustillo Grade 1;n=28, Grade 2;n=9, Grade 3;n=5). 48 were managed by reamed interlock nailing (Group 1) and 68 by unreamed interlock nailing (Group 2). 58 patients had an associated blunt trauma chest, 36 had blunt trauma abdomen, 18 had an associated head injury and 12 had spine injuries. The average surgical time for Group 1 was 118 minutes and for Group 2 was 94 minutes (p=0.014). The average fluorscopy time for Group 1 was 4.30 minutes and for Group 2 was 4.06 minutes. The average intra-operative blood loss for Group 1 was 254 millilitres and for Group 2 was 202 millilitres. The average intraoperative oxygen saturation fall as measured at the time of reaming and nail insertion was 2% in Group 1 and 6% in Group 2. The intraoperative complications were 11 (22.91%) in Group 1 and 18 (26.47%) in Group 2. The features of ARDS were observed in 6 patients in Group 1 (12.5%) and 4 patients in Group 2 (5.88%). The average union time was 25 weeks in Group 1 as compared to 19.4 weeks in Group 2 (p=0.012). The reoperation rate was 6.25% in Group 1 and 11.76% in Group 2.

Discussion: The unreamed interlock nailing is the definitive management of fractures of femur in patients with polytrauma or blunt trauma chest as it requires lesser operative time (and thus exposing the patient to shorter period of anaesthesia), lesser blood loss and lesser fluoroscopy exposure. The incidence of ARDS is significantly lower with unreamed nailing in polytrauma patients. However the union time was significantly longer in unreamed nailing as compared to reamed nailing.

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A.L.N. Tilakawardane J.D. Moorehead

Introduction: A common outcome measure for Total Knee Replacement patients is the measurement of their knee extension angle. In theatre the surgeon usually ensures that the replacement knee can achieve full extension with the patient supine. However patients frequently comment that they are unable to reach full extension while seated. This is due to the flexed hip tightening the hamstrings.

The aim of this study was to deduce the effect of Hip flexion on the knee’s full extension angle (popliteal angle) in a control group of subjects with normal knees.

Method: An electromagnetic motion analysis system (Polhemus Fastrak) was used to assess twenty knees in ten normal subjects. The groups mean age, height and weight were 35 years (SD=7), 1.82 m (SD=0.05) and 83.9 kg (SD=12.9) respectively. Each subject was placed in a supine position on an examination couch with their legs hanging over the end and their knees in 90 degrees of flexion. The electromagnetic source was then positioned 50 cm from the knee joint. Two electromagnetic sensors were then taped to the lateral side of the leg, one over the femoral midpoint and one over the tibial mid point. A recording was then made as the subject extended their knee to full extension. The subjects hip was then flexed to 90 degrees with the knee in 90 degrees flexion. The subject was the asked to extend their knee as far as possible, while keeping their hip flexed. This processed was repeated 3 times for each knee to give average knee extension angles, with the hip straight and flexed.

Results: In the supine hip-straight position the mean extension angle for the fully extended knees was 1.2° (SD=2.7°). In the supine hip-flexed position the mean extension angle for the fully extended knees was 23.8° (SD=12.6°). This gave a mean difference of 22.6°. A paired t test of the extension angles for the two hip positions yielded a significant difference with p=0.0000001.

Conclusion: These results indicate that hip flexion significantly reduces the amount of full knee extension, in normal subjects.

C. Wan D.R. Marsh G. Li

Introduction: Sufficient quantity of osteogenic cells is an essential aspect for a successful cell therapy in the treatment of difficult bone fractures and defects. At present, this was achieved by culturing bone marrow and bone-derived cells in a relatively long duration. A large number of the non-adherent mesenchymal stem cells were discarded during medium change. We hypothesize that collecting the non-adherent cells and re-plating them may result in more osteogenic cells in the same duration of cell culture. The aim of this study was to investigate the possibility of enhancing number of osteogenic cells by collecting non-adherent cells in the pull-off media and to examine their osteognic potentials.

Methods: Mononuclear cells were isolated by density gradient centrifugation method from bone marrow washouts in the bone samples obtained from 5 patients undergone total hip replacement. Mononuclear cells were plated at a density of 1 x 106/cm2 in a T-75 flask with αMEM medium and 15% FCS. The first medium change was made at day 7 and every 3 days thereafter. For the first three times of medium change, the removed media were centrifuged at 250 g for 10 minutes and plated in a separate T-75 (first time change) and T-25 flask (for the 2nd and 3rd times change). The non-adherent cells from the second and the third puff-off flasks were also collected and plated in separate T-25 flasks. Thus, 1xT-75 flask and 4xT-25 flasks of non-adherent cells resulted from the original T-75 flask. The cells in all flasks were harvested at 21 days from the day when the original flask was set up. The total number of cells in all pull off flasks were counted and compared with that of the original T-75 flask. Rate of cell proliferation with or without osteogenic growth medium were also examined by MTT method for passage 1 of both cells types. Osteogenic differentiation was defined with immunocytochemistry of bone markers: ALP, type I collagen, Osteocalcin and cbfa1. It is planed that cells of passage 2 will be mixed with HA powders and to be implanted into the SCID mice to examine the in-vivo osteogenic potential of these cells.

Results: Mesenchymal stem cells (MSCs) derived from the non-adherent population of human bone marrow culture have demonstrated having similar cell proliferation and differentiation potential in vitro, when compared to the MSCs derived from the adherent cell population. These cells expressed bone markers such as: ALP, type I collagen, osteocalcin and cbfa1. When the non-adherent cells were collected and cultured accumulatively, the total number of MSCs was increased to an average of 39.7% (36.6%–42.9%), compared to the number of cells obtained from the original T-75 flask.

Conclusions: Collecting the non-adherent cell population in the bone marrow culture appeared to result in more MSCs. This harvesting method may be used as a non-invasive way for enhancing MSC numbers in a given period of time. Further in vitro and in vivo studies of these MSCs of non-adherent origins may provide information for optimizing cell culture protocols for rapid expanding the osteogenic cells in vitro. This will facilitate the clinical applications of human osteogenic cell therapy.

A. Gray D.C. McMillan C. Wilson C. Williamson D. St J O’Reilly D Talwar

Introduction: The water soluble vitamins B1, B2 and B6 are essential precursors for a wide variety of coenzymes involved in intermediary metabolism. Recent evidence suggests that the systemic inflammatory response associated with disease, injury and infection may lower micronutrient concentrations in plasma independent of tissue stores. Elective knee arthroplasty surgery has been shown to induce a significant and reproducible systemic inflammatory response and therefore provides an ideal model with which to examine the relationship between plasma and erythrocyte (intracellular) concentrations of B-vitamins and the evolution of the systemic inflammatory response.

Methods: The study was approved by the local ethics committee. All subjects were informed of the purpose and procedure of the study and all gave consent. Venous blood samples (EDTA) were withdrawn pre-operatively from 12 primary knee arthroplasty patients and at 12, 24, 48, 72 and 168 hours after the start of surgery. Analysis of plasma and red cell vitamins B1, B2, B6, C-reactive protein and albumin. Data presented as median and range. Data from different time periods were tested for statistical significance using the Freidman test and where appropriate comparisons of data from different time periods were carried out using the Wilcoxon signed rank test.

Results: All patients were over the age of 60 years and had circulating concentrations of B vitamins in the normal range (B1 275–675ng TDP/g Hb; B2 220–410nmol/l; B6 17–135nmol/l). On analysis of serial postoperative values over the study period 0–168hrs there were significant increases in C-reactive protein and significant decreases in albumin concentrations peaking/troughing at 48hrs returning towards normal concentrations at 7 days (p< 0.001). In contrast, during this period plasma albumin (p< 0.001), B2 (p< 0.001) and B6 (p< 0.001) concentrations fell transiently by as much as 50% returning towards normal in parallel with the fall in C-reactive protein concentrations. In contrast, neither red cell B2 nor B6 concentrations fell during the study period.

Conclusions: In this study red blood cell B2 and B6 remained stable over the period of study. In contrast, plasma concentrations of B2 and B6 fell and were outwith the normal range, the trough coinciding with the peak of C-reactive protein before returning to baseline values. These results are consistent with the concept that plasma concentrations of vitamins are unlikely to be a reliable measure of status in patients with evidence of a systemic inflammatory response. Red cell B1, B2 and B6 concentrations more accurately reflect status in patients with evidence of a systemic inflammatory response.

I Papageorgiou E. Ingham J. Fisher E. Jones I. Learmonth C.P. Case

Introduction: Joint replacement surgery is one of the most common operations that take place in United Kingdom. The major problem in total hip arthroplasty is the generation of particulate wear debris and the subsequent biological responses. Wear debris induces osteolysis and a subsequent failure of the implant that lead to the liberation of greater quantities of particulate and soluble debris to bone marrow, blood, lymph nodes, liver and spleen. Recently, it has been suggested that these adverse effects depend not only on the chemical composition but also on the particulate nature of the material (size and shape). Particle size has been shown to influence the inflammatory response of macrophages to wear debris. This study evaluated whether particle size also influences the viability and mutagenic damage.

Methods: Cobalt chrome alloy particles of two sizes (large 2.9±1.1μm, small 0.07±0.04 μm) were generated and characterised by Scanning Electron Microscopy. Different concentrations of particles were added to primary human fibroblasts in tissue culture. The release of cytokines in the medium was assayed by Enzyme-Linked ImunnoSorbent Assay (ELISA). Cell viability was determined by MTT conversion and the degree of DNA damage was quantitatively analysed by the Alkaline Single Cell Gel Electrophoresis (COMET) assay with image analysis.

Results: Small particles initialise DNA damage at much lower volumetric concentrations (0.05 and 0.5 μm3/cell) than larger particles (500 μm3/cell). The difference in the doses was approximately related to the difference in surface area of the particles. DNA damage was related to a delayed decrease in cell viability, which was noted after three days of exposure.

In contrast, the release of the inflammatory cytokine TNF-α and the multifunctional growth factor TGF-β-2 occurred at lower doses (0.0005 to 5 μm3/cell for TNF-α and 0.5 to 50 μm3/cell for TGF-β-2). No release of IL-6 was detected at any dose. Only growth factor FGF-23 was increased in similar pattern to the DNA damage.

Conclusions: This study has demonstrated important differences between the mutagenicity, toxicity and inflammatory potential of small (nanometre sized) and large (micrometer sized) chrome particles.

C.B. Hing L. Shepstone T. Marshall S.T. Donell

Introduction: Patellofemoral instability (PFI) is a disabling condition that occurs in adolescence. Recurrence after patellar dislocation has been reported in 2–50% of cases. This study aimed to compare the shape of the distal femur in PFI to a normal cohort.

Method: 108 CT scans from 54 subjects with PFI were compared to 197 CT scans from 102 normal subjects. Outlines of the trochlear groove and lateral condyle were extracted from CT scans using Interactive Data Language version 5.0 image manipulation software. The shapes were aligned using a modified Procrustes analysis and interpolation performed with a basic cubic spline. A statistical method from the field of functional data analysis was used to quantify shape. A discriminant analysis was then used to provide a tool for deciding which patients had abnormal grooves thus improving patient selection for trochleoplasty.

Results: Mathematical quantification showed a significant difference at the 0.001 level using an independent t-test between the normal and PFI groups for the shape of the trochlear groove with PFI knees having a more complex shape than normal. The second largest source of variation is in the position of the trochlear groove minimum with respect to the coronal plane together with variation in the shape of the medial condyle. The trochlear groove is situated more medially, with a shallower medial condyle, in PFI. In contrast the trochlear groove is situated more laterally, with a more prominent medial condyle in normal knees. Analysis of shape of the lateral condyle showed that in PFI, it is shorter and broader.

Conclusion: Principal components analysis can be used to quantify the variation in shape of the distal femur and allow a discriminant function analysis to be performed comparing PFI knees to normal knees. This quantification of shape can be applied to diagnosis of instability, operative planning for trochleoplasty and implant design.

P.R. Aldinger H.S. Gill C. Rumolo S.J. Breusch D.W. Murray

Introduction: Minimally invasive surgery (MIS) presents challenges in achieving alignment for unicompartmental knee arthroplasty (UKA). Aim: Development and assessment of an image guidance system for MIS implanted Oxford UKA.

Methods: The Surgetics platform which uses intra-operative data acquisition was chosen as the base system. Software was developed to determine height of tibial cut, image guidance of saws, alignment of components and assessment of ligament tension. The accuracy of component placement was assessed in vitro using matched pairs of knees randomised into navigated (NAV n=10) and standard manual (MAN n=10) procedures; standardised postoperative A-P and lateral radiographs were used. Pre and post-operative kinematics were assessed (NAV n=6, MAN n=7). The changes postoperatively over knee flexion and extension were calculated for tibiofemoral rotation (ΔROT) and ab/adduction (ΔABD).

Results: Accurate component placement was achieved with both methods without significant differences. Tibial cut height was more accurately in the NAV group (re-cut rate: NAV 33%, MAN 50%). NAV femoral component placement was as accurate as MAN with intramedullary rod. For the flexing cycle mean ΔROT was −0.06° (range 6.08° to −3.93°) and mean ΔABD was −0.04° (range 3.39° to −5.72°). There were no observable differences between the NAV and MAN kinematics. Overall, no observable differences were found between pre and post-operative kinematics.

Conclusions: Image guidance produces accurate placement through MIS approach and reduces the amount of tibial bone resection.

C.E. Hillier D.J. Beard K.M. Refshauge

Introduction: The factors causing chronic instability, a common sequela of ankle inversion sprains are unclear, despite wide investigation. However, few studies have examined potential factors during the injuring movement. We therefore measured the ability of dancers to control ankle movement during quiet stance and after a perturbation into inversion in a group with chronic instability (N=16) and healthy controls (N=26).

Methods: Control of ankle movement was determined by the magnitude of lateral oscillation at the ankle, measured by a 3SPACE Fastrak. The oscillation was measured during single leg stance (baseline oscillation) for two foot positions, flat and demi-pointe. In both positions, the time taken to return to the baseline oscillation after an inversion perturbation (perturbation time) of 15° for the flat foot and 7.5° for the demi-pointe position was also determined.

Results: The baseline oscillation was significantly smaller (P< 0.005) on the demi-pointe for the sprained group (2.5 ± 0.5 mm) than for controls (4.0 ± 2.3 mm). In addition, the perturbation time for the flat foot was significantly longer (P< 0.05) for the sprained group (2.2 ± 0.4 sec) than for controls (1.8 ± 0.5 sec). However, there was a higher (P< 0.05) failure rate among the sprained group for both the perturbation test with the foot flat and for baseline oscillation on the demi-pointe than among the controls.

Conclusions: Our findings demonstrate altered sensorimotor control in chronically unstable ankles. Those sprainers who successfully completed the tasks “braced” the ankle, allowing a small range of oscillation. The increased perturbation time in the sprained group may reflect a deficit in either detection of inversion movements, peroneal muscle response, or both.

R. Bhatia I. Pallister C. Dent N. Topley

Introduction: Elevated plasma elastase levels have been reported following major trauma and isolated femoral fracture. Reamed femoral nailing has been shown to further increase plasma elastase levels. The aim of this study was to investigate neutrophil (PMN) priming for degranulation following major trauma and isolated long-bone pelvis fracture by assessing the ability of PMN to release elastase in-vitro in response to phorbol myristate acetate (PMA) an analogue of dia-cylglycerol (DAG) a component of the “second messenger” system.

Methods: 11 major trauma (ISS≥18) patients and 18 patients with isolated long-bone/pelvis fracture, were consented to enter the study. Patients in the isolated fracture group were further stratified depending upon the type of fracture stabilization they underwent [reamed nail (n=12), Ex-Fix (n=6)]. Blood samples were obtained on admission, at 24 hours post injury, at day 3 and day 5. 11 healthy volunteers were used as controls. PMN were isolated by dextran sedimentation and ficoll-hypaque density gradient centrifugation. The ability of PMN to degranulate was assessed by an elastase substrate assay.

Results: A significant increase in the capacity of PMN to release elastase in response to a PMA stimulus was seen in major trauma patients on admission as compared with healthy volunteers. However in patients with isolated long-bone/pelvis fracture, there was no difference in levels of elastase release. Further no difference in the ability of PMN to release elastase was seen between the reamed nail and Ex-Fix groups.

Conclusions: In conclusion we show that PMN are primed for increased degranulation (elastase release) following major trauma but not following isolated long-bone/pelvis fracture. These primed PMN are capable of increased tissue damage following major trauma thus increasing the risk of development of multiple organ failure.

D. Kneif R.M. Aspden

Introduction: Aseptic loosening of the acetabular cup is the commonest cause for revision surgery of total hip replacements (THR). Whereas a sound bone cement micro-interlock is believed to contribute most to the stability, most surgeons prefer to enhance their fixation by adding a macro-interlock. Drilling of additional keyholes creates cement pegs intended to resist rotational forces at the acetabular bone-cement interface. Only a few attempts have been made to investigate the effect of number, configuration and shape of these keyholes. Following the limited experiments by Oh (1983) on beechwood blocks, Mburu (1999) systematically optimised diameter, depth and number of keyholes using the same beechwood model. He subsequently developed a novel drill, bell-shaped in cross-section, aiming to minimise the stress concentration observed at the base of cement pegs.

Methods: This study compares the novel drill against a conventional drill using the same beechwood model and the previously optimised number, diameter and depth of keyholes, the shape of the drills being the only difference. The tests were performed on twenty beechwood blocks, half of the blocks allocated to the novel drill and half to the conventional drill. Since the three keyholes were not equidistant, it was also tested whether the direction of torque had an influence on the results. This was done by applying torque clockwise in half of the specimens and anticlockwise in the other half.

Results: The results showed that the static torque to failure was superior in the novel drill (mean: 163 Nm) compared to the conventional drill (mean: 127 Nm), but this was not statistically significant (p= 0.12) due to the wide variation of results. There was no difference for the direction of torque (p= 0.8) and the type of drill and the direction of torque did not show any interaction (p= 0.5).

Conclusions: Results suggest that there is an improvement with the novel drill in static torque to failure. Further testing appears therefore to be worthwhile. Results also suggest that the difference or improvement achieved with the novel drill is less pronounced than it is for number, depth and position of keyholes (Mburu, 1999). However, more variability than expected was encountered. Despite recognition as a possible model for keyhole testing, beechwood blocks testing three keyholes at the same time may not be appropriate when the load to failure for cement pegs is approximating the load to failure for beechwood as encountered in our experiment. Simplification of the system like testing single pegs should be considered.

B. Gargiulo J. Menage H. Evans J.P.G. Urban B. Caterson C. Curtis S.M. Eisenstein S. Roberts

Introduction: Autologous chondrocyte implantation is routinely used for the repair of articular cartilage defects. A similar method may be employed to treat degenerate intervertebral discs or other connective tissues. A system in which cells could not only be delivered, but also retained would offer advantages compared to ACI. Such a vehicle would also allow a homogenous distribution of cells throughout the defect and enhance nutrient penetration to the seeded cells.

Methods: Bovine nucleus cells were isolated via enzyme digestion and expanded in number to passage 3. The cells were resuspended in 0.8% alginate and loaded into poly vinyl alcohol (PVA) cubes. These constructs were placed into a solution of calcium chloride to ‘gel’ the alginate. Constructs were cultured in DMEM+10% FBS within 15ml conical tubes rotated at 37°C for up to 28 days. Cell distribution/morphology and proliferation were assessed on H& E and Ki-67 stained sections, respectively. The re-expression of a disc cell phenotype was assessed using toluidine blue staining and immunohistochemistry (with antibodies to collagen types I, II, IIA, VI and X, and to the glycosaminoglycans, chondroitin-4- and -6-sulphate and keratan sulphate. RT-PCR was performed using oligonucleotide primers to collagen types I, II and X, aggrecan, link protein, and small leucine-rich PGs.

Results: H& E staining of 10μm-thick cryosections revealed an even distribution of loaded cells throughout the scaffold at day 1 being maintained through to day 28. Toluidine blue staining revealed the presence of GAGs, increasing with time. Ki-67 revealed approximately 5% of cells were proliferating at all time points. Immunohistochemistry demonstrated the production of collagen types I, II, IIA, VI and X and the glycosaminoglycans, chondroitin-4-, -6 and keratan sulphate. RT-PCR results showed mRNA expression of fibromodulin throughout the experiment, lumican at days 14, 21 and 28. Types II and X collagen were present at days 21 and 28.

Conclusions: Combining 0.8% alginate with PVA retained 100% of the seeded cells and allowed an even distribution of cells throughout the scaffold. The immunohistochemistry and RT-PCR demonstrated that the system allowed the bovine nucleus cells to express phenotypic markers expressed by disc cells in vivo. These preliminary results indicate that the PVA/alginate system could act as a suitable delivery device for cells during autologous repair of the intervertebral disc or other connective tissues such as meniscus.

K. Phipps J. Pegrum N. Smith G. Blunn

Introduction: ApaPore is a synthetic bone graft extender, made from Hydroxyapatite. It is designed to be used as a 50:50 combination by volume with morcelised bone graft. A typical use for such a product may be in impaction allografting during revision hip surgery. The aim of this study was to compare the mechanical stability of stems inserted using impaction allografting where the graft was composed of a 50:50 mixture of ApaPore and allograft with only allograft.

Methods: Twelve large left sawbones were used in this study, the femoral head was cut off each one and the equivalent cancellous bone was cored out to resemble a femur at revision. Impaction allograft was performed on six of the sawbones, with rinsed human morsellised graft. This procedure was repeated on the remaining six sawbones, with graft produced as a 50:50 mixture by volume of rinsed allograft and blood soaked Apapore. The mechanical test was performed in the red rocket, a six station hydraulic loading machine. The sawbones were mounted in the anatomical position of 7 degrees valgus and 9 degrees posteriorly and the test was run in batches of three. Sinusoidal loading at 2Hz was applied under the following loads: 600N, 1kN, 1.4kN, 1.8kN and 2.2kN, each loading step lasting for 5000 cycles. Migration of the stem during loading was measured using LVDT. Vertical displacement of the prosthesis head was measured using digital height callipers at the beginning and end of each loading step. X-Rays were taken before and after mechanical testing.

Results: There is a significant difference between the groups in the overall displacement of the prosthesis head, measured with the digital height calipers, (Mann-Whitney U Test p=0.01). Total average head movement allograft group: 3.5mm and ApaPore group: 1.8mm. The total average subsidence measured with the LVDT’s was 0.295 mm in the allograft group and 0.119mm in the ApaPore/Allograft group. A sideways displacement of the prosthesis head was observed on the x-rays, which is a direct relationship of head rotation, measurements showed a significantly less rotation in the ApaPore/Allograft group (Mann-Whitney U Test p=0.002).

Conclusions: Rotation of the stem in all planes during loading resulted in a greater observed displacement of the prosthesis head than that measured by the LVDT’s. These results show that ApaPore, when used as a bone graft extender is able to reduce initial rotation and subsidence of the stem. Further research needs to be undertaken to investigate the long-term feasibility of using ApaPore.

J.B. Aderinto G.W. Blunn

Introduction: Human bone marrow stromal stem cells(BMSSC’s) have the ability to differentiate into a variety of mesenchymal cell types including osteoblasts, fibroblasts, adipiocytes and myocytes. These stromal cells are involved in the process of bone formation during the healing of fractures. Collagen lyophilisate is a sterile extract of bovine demineralised bone matrix. This material contains proteins removed from bone that may control the differentiation of osteoblasts from BMSSC’s. These proteins are localised within collagen type 1. The aim of this study was to determine the effects of collagen lyophilisate on the osteogenic differentiation of bone marrow stromal stem cells cultured in vitro.

Methods: Bone marrow was aspirated from the iliac crest of a human donor who was undergoing an unrelated elective orthopadic surgical procedure. Stromal stem cells were isolated from marrow, cultured and then characterised using immunofluorescent antibodies to Stro −1, a stromal stem cell marker. 3x104 BMSSC’s were seeded into each of 3 culture wells and incubated with standard growth medium or standard medium with collagen lyophilisate diluted 1:50 or 1:100. Cells were cultured for a maximum duration of 30 days. At selected time intervals until day 30, osteogenic differentiation was assessed by determination of alkaline phosphatase, osteopontin, pro collagen carboxyterminal (type 1 collagen synthesis) and calcium in cultures using specific assays.

Results: Cells cultured in collagen lyophilisate displayed a polygonal morphology early in the culture period and later formed complex aggregates. Cells in control cultures maintained a fibroblstic morphology until confluence. On day 21 alkaline phosphatase activity was significantly higher in collagen lyophilisate containing cultures than control cultures. Osteopontin levels were not enhanced in the collagen lyophilisate containing cultures. Type 1 collagen synthesis was higher in the collagen lyophilisate 1:50 group than all other groups at day 14. No differences in type 1 collagen synthesis were detected between cultures at other time periods. Calcium was not detected in any of the control cultures for the duration of the culture period. In contrast, calcium was detected in collagen lyophilisate containing cultures on day 15.

Conclusion: Collagen lyophilisate resulted in changes in cellular morphology and arrangement. The ability of collagen lyophilisate to enhance alkaline phosphatase activity, increase collagen type 1 expression and stimulate the deposition of calcium in stromal stem cell cultures provides evidence that it has osteogenic properties.

K. Phipps J. Saksena G. Gie S. Muirhead-Allwood A. Goodship G. Blunn

Introduction: Impaction allografting is a technique that is used at revision where the bone stock in the femur is poor. Femoral heads are ground to create morsellised bone, which is impacted down the femur prior to the cementing a new stem into the canal. The results of this technique are variable and there is a high incidence of stem migration. This variation in results may be due to the degree of bone loss or the techniques used to impact the graft. The aim of this study was to quantify the forces currently used in revision hip surgery with impaction allografting.

Methods: To enable these measurements the Exeter slap hammer (Stryker Howmedica) was altered to include a load washer. The load washer had a special cable welded to it so that sterilisation could still be conducted in an autoclave. During surgery the end of the load washer cable is passed to the operator, who is able to connect it to a laptop computer. A specially written Labview program is then used to store the data and determine the impaction forces. The load washer is mounted within the hammer at the point of impact between the sliding mass and the hammer, consequently it is reading the force transmitted to the hammer, not that transmitted to the graft chips. Calibration was performed in an in vitro experiment with a second load washer, which found that the force in the hammer is three times that in the impactor. The force is so much less because it is taken up in the hammer’s inertia.

Conclusions: The impaction forces have been measured during eight operations performed by three different surgeons. The study shows variability between surgeons, and variability between patients operated on by the same surgeon. These readings show that the forces travelling through the impactor range between three to eleven time body weight.

F. Harrold F. Park-Wesley G. Strugnell S. Whiten R. Abboud C. Wigderowitz

Introduction: Accurate recovery of humeral head geometry in shoulder arthroplasty is an important requirement for a good functional outcome. Despite this, spherical prosthetic components are implanted when the total proximal humerus is described as ovoid1. However, 60 to 80 % of the head is spherical1. If, in the normal glenohumeral joint, only the spherical portion is in contact with the glenoid then recovery of normal mechanics is likely with a spherical prosthetic component.

Contact patterns have been examined ex vivo2 under static conditions but do not reflect the likely in vivo contact pattern under dynamic loading and have not been correlated to changes in sphericity of the articular surface. A recent study of the distal femur found that thickness of normal articular cartilage is positively correlated with loading3 and, thus, contact.

The objective of this study was to determine the feasibility of using a surface laser scanner to determine cartilage thickness and, therefore, likely contact area and to correlate changes in thickness to changes in sphericity of the articular surface.

Methods: A cadaveric arm without bony deformity or evidence of rotator cuff disease was dissected free of soft tissue and mounted on a rigid block within the frame of a surface laser scanner (Kestrel3D Ltd., UK). The articular surface of the humerus was scanned at a resolution of 200 μm. The cartilage was then dissolved away and the humerus re-scanned. The x,y,z coordinate data of the re-scanned bone were used to match the sub-chondral bone with the cartilage from the previous scan using Pointstream™ software (Kestrel3D Ltd., UK).

The cloud point data for the cartilage and bony surfaces were exported into modelling software (McNeal and Assoc., Seattle, WA) and the surface area of the head divided into ten equal sections. For each slice of both the cartilage and bony surface, the radius of curvature was calculated using a least square fit optimisation technique4. The differences in radius of curvature between the cartilage surface and subchondral bone surface were used to calculate the cartilage thickness for each slice. The standard deviation from the radius of curvature was used to calculate the degree of deviation from sphericity.

Results: For the first 60 % of the surface area, the deviation from sphericity was 0.5% of the radius with a cartilage thickness of 0.74 mm. The deviation from sphericity and cartilage thickness for 100% of the articular surface was > 1% and 0.63 mm, respectively.

Conclusions: The experiment proved that the surface laser scanner can be used to elucidate the relationship between contact patterns and articular curvature of the proximal humerus. The changes in sphericity concur with results from previous studies1. Assuming cartilage thickness correlates to contact patterns at the normal glenohumeral joint, the change in cartilage thickness suggests that contact may occur only at the spherical portion of the head. Knowledge of this relationship may aid in future prosthetic design considerations or in modification of the osteotomy technique. To further support these findings, a 50μm laser scanner is being developed and will be used on a larger sample size.

K. Phipps A. Goodship G. Blunn

Introduction: Impaction allografting allows an initial stable function of revision hip replacements and a method of reconstituting the bone stock. A previous in-vivo ovine study has found that the density of impacted morsellised allograft reduces after six weeks but recovers by twelve weeks. This reduction in density during remodelling may also correspond with a reduced mechanical strength. A probable cause of the low density is osteoclastic bone resorption prior to vascular in growth and the formation of new bone by osteoblasts. BoneSave is a 4–6mm porous granules of hydroxyapatite and tricalcium phosphate, and is designed as be used as a 50:50 mix with morsellised allograft. Bonesave takes a long time to be resorbed and replaced with bone compared with allograft. We hypothesised that the inclusion of BoneSave could slow resorption down and hence maintain the mechanical strength of the graft during remodelling. This study investigated the mechanical strength of BoneSave mixed with allograft at six and twelve weeks after insertion into a defect, with pure allograft as a control.

Methods: Twelve yews were used in this study, half were terminated at six weeks the remainder at twelve. The test site was a 15mm diameter hole, approximately 10mm deep, in the medial femoral condyle. Both femurs were operated on consecutively, with allograft on one side and a BoneSave/allograft mix on the other. After euthanasia the distal femurs were removed and CT scans performed to evaluate density. Sixteen millimetre discs were cut from each femur, exposing the test site 4 mm from its proximal end. These graft site was then subjected to non-destructive compression tests in Zwick loading machine. Bone remodelling in the graft was determined using histology.

Results: Wilcoxon paired test were used to compare densities of the allograft group with the BoneSave group at 6 and 12 weeks, at both time points there was a significant difference between the groups (p< 0.05). There was no statistical difference in the density of the allograft groups between 6 and 12 weeks, or the Bonesave groups between 6 and 12 weeks using the Mann-Whitney U test (p> 0.05). There was no significant difference between the stiffness of the two groups at both time points using the Mann Whitney U test (p> 0.05).

Discussion and Conclusion: This result was unexpected in the allograft group because in a previous study looking at different sized allograft chips there was a significant difference between the density at 6 and 12 weeks. The most likely cause for this is that lower forces were used to impact the graft in this experiment compared with the graft size study. This would have resulted in lower density at time zero, so perhaps this lower density didn’t invoke such a large resorption response. Bone-Save is able to maintain mechanical strength during remodelling when used as a bone graft extender.

J.C. Waite H.S. Gill D.J. Beard C.A.F. Dodd D.W. Murray

Introduction: Numerous studies in the orthopaedic literature have reported changes in knee kinematics following rupture of the Anterior Cruciate Ligament (ACL). Gait analysis is currently the preferred method for studying these in vivo kinematics. The accuracy of this method of analysis remains limited due to errors related to skin movement artefact. Most studies have therefore been limited to analysing subjects performing simple tasks such as straight-line walking, since results become increasingly inaccurate as the subject moves faster. Standard skin marker formats allow measurements of knee flexion angle and varus/valgus angles to be recorded relatively accurately during such tasks. Accurate measurements of rotations and translations at the knee joint, however, are not possible with these set-ups.

Aim: To produce a new method for interpretation of kinematic data from gait analysis, to allow accurate measurement of 3-D displacements at the knee joint during dynamic activity.

Method: We employed two different sets of skin markers in an attempt to increase the accuracy of our data, by diminishing the effects of skin movement. The Kabada1 marker set was used with retroreflective spheres of 14.5mm diameter. This marker set was used to establish 3-D femoral and tibial co-ordinate systems. We then established a femoral and tibial co-ordinate centre within the distal femur and proximal tibia respectively. A second set of markers was used similar to the “point-cluster” method described by Andriacchi et al2. This involved groups of eight smaller spheres (9.5mm diameter) placed in a non-uniform distribution on each of the thigh and shank segments. The positions of all these remaining markers, relative to the co-ordinate centres were then established. 15 subjects were then recorded while performing a series of running and cutting tasks. For each trial that was then analysed, we used all visible markers to optimize the recorded position of the tibial and femoral co-ordinate centres, using a method similar to that described by Soderkvist3. The displacements of these co-ordinate centres were then used to calculate the 3-D tibio-femoral kinematics. Reliability and repeatability tests suggest that this method produces results accurate to 3–4mm.

Conclusion: We believe we have developed a practical and accurate method to analyse 3-D joint kinematics from gait laboratory data.

C.E.W. Aylott Y.L. Leung B.J.C. Freeman D. McNally

Introduction: Intra-Discal Electrothermal Therapy (IDET) has been used to treat chronic discogenic low back pain. Proposed mechanisms of action include denervation of the posterior annulus and collagen denaturation. Previous authors have reported on changes in internal disc mechanics following IDET including reduction in stress concentrations possibly leading to a more even distribution of load across the end-plate1. A novel intradiscal decompression catheter has been developed to reduce local disc bulging in cases of contained prolapse. This new catheter is inserted percutaneously into a disc and advanced under radiographic control into a postero-lateral position targeting the herniation. The decompression catheter uses more focused heating and higher temperatures than previous devices and is intended to provide a local decompression of the disc through a thermally-mediated reduction in nuclear volume. The purpose of this study was to investigate changes in internal stress profiles following use of the new catheter.

Methods: Five cadaveric lumbar ‘motion segments’ were dissected from two spines (age 64–84 yrs). Each segment was compressed, normally to 1 kN, while a miniature pressure transducer was withdrawn from posterior to anterior across the mid-sagittal diameter of the disc producing a baseline stress profile. A decompression catheter was inserted into the disc and its position confirmed with plain radiography. The temperature of the catheter was increased to 90°c over a period of 14 minutes. Stress profiles were then repeated.

Results: Stress profiles in three of the five segments showed changes consistent with degenerative change. In these discs stress profiles following ‘treatment’ showed up to a 35% reduction in the magnitude of stress peaks in the posterior annulus. There was very little change in the distribution of stress in the two non-degenerate discs. Stress in the nucleus appeared unchanged in all discs.

Conclusions: Treatment of degenerate discs with the decompression catheter lead to a measurable alteration annular stress peaks that have been associated with degenerative disc disease, while non-degenerate discs were unaffected. These preliminary findings of an ongoing study suggest that the novel decompression catheter has a biomechanical effect in certain classes of disc.

S. Forouzanfar L. Coulton M. Saleh

Introduction: The Sheffield Ring Fixator (SRF) uses wires in the metaphysis and screws in the diaphysis for bone stabilisation. It has four 2mm wires tensioned to 1400N in two parallel groups to stabilise the ring in the metaphysis. For maximum stability, these parallel wires should cross at 60 degrees or greater and the position of the crossing should be in the centre of the bone. Fixation stability and clinical outcome may well depend on the accuracy of surgical application.

Aims: To review the consistency with which the SRF frames were applied by a single surgeon.

Materials and Methods: The fixators of 39 patients aged between 6 and 75 years of age (11.5 years mean age in children and 38.7 years mean age in adults) were examined. 7 patients had proximal and distal metaphyseal wires making a total of 46 recording sites. The angle of the wires was calculated using the number of holes between the wire clamps since each hole subtends an arc of 7.5 degrees. The crossing angles were divided into two groups with crossing angles of greater than 60 degrees and less than 60 degrees. The position of the crossing of the wires was determined by creating a cross section of the metaphyseal rings, reducing the size of the ring to 100mm and transposing a cross section of the tibia of the correct size and at the correct level. Scaling down the distances measured between the inner ring and the patient limb, the position of the cross section and consequently of the wire crossing was determined. Using contour lines the tibia was divided into four zones. Zone 1 was central tibia and zone 4 was the tibial cortex. All crossing within zone 1 and 2 were considered satisfactory, and zone 3 and 4 poor.

Results: 67.5% of patients had crossing angles greater than 60 degrees and 32.5 % had crossing angles of less than 60 degrees. 85% of the rings had zone 1 or 2 crossing positions. 6.5% of the rings had subcortical crossing positions and 8.5% of the rings had cortical crossing positions. 8.5% of rings had crossing angles of less than 60 degrees as well as wire crossing positions in zone 3 and 4.

Discussion: In a carefully controlled situation a surgeon’s surgical technique was consistent in 67.5% of the rings, with satisfactory crossing angles and wire crossing positions. Only 8.5 % of the rings had poor crossing angles and crossing positions. Inability to achieve ideals may be due to technical errors or anatomical variations. There were increased infection rates in patients with reduced crossing angles, however the position of the crossing had no apparent effect on infection rates and patient mobility. A further study would be required to separate the relative importance of these two factors on patient complications.

S.J. Matthews C.R. Gooding M.K. Sood J.A. Skinner G. Bentley

Introduction: Autologous chondrocyte implantation (ACI) is a technique described for treating symptomatic osteochondral defects in the knee. It is contra-indicated, however, in a joint rendered unstable by a ruptured anterior cruciate ligament (ACL). We present our early experience of combined ACL and ACI repair.

Methods: Patients underwent arthroscopic examination and cartilage harvesting of the knee. Chondrocytes were then cultured in plasma and a second operation was undertaken approximately four weeks later to repair the ruptured ACL with hamstring graft and to implant the chondrocytes via formal arthrotomy. Patients then underwent a graduated rehabilitation program and were reviewed at 6 and 12 months. Functional measurements were made using the Bentley functional scale and the modified Cincinnati rating system, with pain measured on a visual analogue scale. All patients also underwent formal clinical examination at each review.

Results: 4 out of the 5 patients reported an improvement in pain as measured on visual analogue scale, with 1 patient reporting no difference. 4 patients had stable knees as determined by negative anterior draw, negative Lachman’s test and negative pivot shift test; one patient showed improvement, but remained pivot shift positive. Improvements in Bentley scores were noted in 3 patients. Cincinnati scores were markedly improved in 3 patients and slightly improved in the remaining 2 patients. The only operative complications were a traction neuropraxia to the saphenous nerve of one patient requiring no treatment and a manipulation under anaesthesia for poor mobilisation in another patient, which was successful in improving range of movement. A further patient required arthroscopic trimming of the cartilage graft which had overgrown; this was also successful.

Conclusion: Symptomatic cartilage defects and ACL deficiency may co-exist in many patients and represent a treatment challenge. Our early results suggest that a combined ACL and ACI repair is a viable option in this group of patients and should reduce the anaesthetic and operative risks of a two-stage repair. More patients and longer follow up will be required to fully assess this technique.

A.A. Smit W.J. Wade

Introduction: Intra-operative visualisation of talonavicular reduction does not exclude the possibility of persistent navicular rotatory subluxation as cause of persistent cavus or adductus deformity. Open perinavicular arthrography accurately defines navicular rotatory status. Similarly, inferior navicular insertion of the tibialis posterior tendon is a reliable predictor of correction of navicular rotation.

Methods: Six operated clubfeet, aged six to ten months and operated on from March 2001 to September 2001, were included in this study. Correction was obtained using a sequential release and reduction was held with talonavicular and calcaneocuboid pinning. Simultaneous perinavicular arthrography was done using contrast soaked surgical patties inserted into the opened talonavicular and naviculocuneiform joints. Naviculocuneiform status and navicular insertion of the tibialis posterior were observed and conventional intra-operative clinical- and radiographic assessment of clubfoot correction was compared with perinavicular arthrographic findings.

Results: Naviculocuneiform displacement was not observed. Visual and arthrographic assessment of talonavicular reduction showed a correlation of 100%, but such reduction often necessitated joint incongruence. Arthrography showed persistent navicular rotatory subluxation after adequate release and talonavicular reduction in 2 cases. Anteroposterior and lateral talar-first metatarsal angles fail to identify inadequate plantar fascia release, failure of talonavicular reduction or persistent navicular rotation as cause of persistent deformity. After adequate plantar fascia release, visual confirmation of talonavicular reduction and arthrographic confirmation of navicular rotatory reduction successfully corrected persistent midfoot deformities. Medial navicular insertion of the tibialis posterior tendon was observed in all cases of navicular rotatory subluxation, while restored inferior navicular insertion of this tendon was confirmed in all cases where navicular rotation was corrected. Recurrent navicular rotation after confirmed correction was observed in one case after single pinning of both talonavicular and calcaneocuboid joints.

Conclusions: The naviculocuneiform joint should not be addressed at clubfoot surgery. Inferior navicular tibialis posterior insertion confirms correction of navicular rotation as cause of persistent midfoot deformity and should be routinely assessed. Recurrent navicular rotatory subluxation suggests double pinning of the talonavicular joint.

A. Patterson C. Curtis B. Caterson D. Edwards S. Roberts L. van Niekerk R. Wade

Introduction: The search continues for ideal markers and methods of monitoring cartilage degeneration. Various cartilage components, whole or fragmented, have been measured in synovial fluids. A common problem in quantitating these markers is often the unknown dilution of synovial fluid which can occur in obtaining the samples. In this study we have used urea (ratio in synovial fluid:serum) as a method to correct for the dilution of synovial fluid, and hence to quantify enzyme levels in patients with a spectrum of cartilage degradation, in addition to identifying aggrecan degradation products, many of them for the first time in such samples.

Methods: Forty synovial fluid samples were obtained from 4 groups of individuals (10 in each):


grade IV chondral damage,

osteochondral defects or

endstage osteoarthritis (OA) of the knee, categorised by the cartilage appearance at arthroscopy.

Levels of matrix metalloproteinases (MMPs) 2 and 3 and the inhibitor, TIMP 1, were measured in the fluids via ELISA assays. Urea levels were measured in blood and synovial fluids and enzymes and their inhibitors were normalized according to the ratio of serum:SF urea, to account for the dilution factor of the SF (Kraus et al 2001). Western blotting was used to identify the presence of aggrecan components (chondroitin-4-sulphate: 2B6 antibody; C-6-S: 3B3 and C-0-S: 1B5; keratan sulphate: BKS-1; the G1 domain: 7D1; interglobular domain: 6B4) and also enzyme degradation products of MMPs (BC14) and aggrecanases (BC3; BC-13).

Results: MMPs 2 and 3 and TIMP 1 were all significantly increased in the synovial fluids from OA patients compared to normals (P< 0.01, 0.001 and 0.01 respectively) and MMP3 was greater in the grade IV chondral and osteochondral defect groups than the normals (P< 0.01). Western blotting demonstrated fragmented aggrecan components with a range of molecular weights. Aggrecanase activity was seen in the OA and grade IV chondral damage groups but not in the osteochondral or normal groups, whereas MMP activity was seen in all 3 groups showing cartilage damage but not in the normals.

Conclusion: Dilution of the synovial fluid, either due to inflammation or joint lavage, is often a problem in quantitating metabolites and markers in joint cavities. This pilot study of a limited number of samples from well characterized patient groups indicates that using urea concentrations in synovial fluid relative to serum provides a mechanism to overcome this. It confirms elevated enzyme activity, both aggrecanase and MMPs, in the joints of patients with degenerate cartilage, compared to normals.

G. Li M. Mushipe H. Rio S. Zhang L. Bonewald J. Q. Feng

Dentin matrix protein (DMP-1), a phosphoprotein highly linked to dentin formation, has recently been reported to have an important role in skeletal development. Previously we reported that adult mice lacking the gene for DMP-1 exhibit the characteristics of chondrodysplasia, osteoarthritis, and showed severe defects in mineralization. DMP-1 knock-out (KO) mice display a profound defect in mineralization, and this is not due to a systemic defect in calcium/phosphate metabolism because serum levels of calcium and phosphate are similar to those in the wild-type mice. Although KO neonates and newborns appear normal, upon closer examination, these animals exhibit skeletal abnormalities, which include delayed secondary ossification and impaired bone remodelling. Heterozygous DMP-1 (H) mice however, show no apparent differences to the wild-type mice. In this study, biomechanical assessment tests of bones from DMP-1 KO mice were performed. Fifteen heterozygous, H, (DMP-1 +/−) and 15 KO, (DMP-1 −/−) male mice were produced and used in this study. At 1, 3 and 7.5 months of age, the mice were sacrificed and 4–5 ulnae from each animal group were harvested and stored in 70% ethanol solution. Volumetric density (BMD) measurements of the intact ulnae were performed using peripheral quantitative computed tomography (XCT960M; Stratec, Pforzheim, Germany) and Norland Stratec software version 5.10. One millimetre thick slices were scanned at a distance of 1 mm under the articular cartilage surface of the elbow as identified by the scout view of the CT scan. BMD of the corticalis and subcortical bone were recorded. Cross-sectional area measurements were also made at the mid-diaphysis of the ulnae. Biomechanical tests were performed in 3-point bending, with supports 3.5 mm apart at a rate of 3 mm/min (Lloyd Instruments Ltd, UK). The ultimate load, yield load and stiffness were determined from the load-displacement curves. All data were analysed using Mann-Whitney U tests (SPSS, Version 9, Chicago, Illinois). Differences were considered significant at p < 0.05. Density studies revealed that H mice had higher BMD than KO mice at all ages (p < 0.001). In the H and KO mice, the cortical BMD peaked at 3 and 7.5 months, respectively. At 1 month, the mean cross-sectional areas of the ulnae were larger in H mice compared to KO mice (0.50 mm2 Vs 0.33 mm2). However at 7.5 months of age, the reverse was observed (H = 0.75 mm2 and KO = 0.98 mm2). Biomechanically, stiffness increased with age at a higher rate in H mice than KO mice. Significant differences were observed at 3 months (p< 0.01) and 7.5 months (p< 0.05) between the two animal groups. There were no significant differences between stiffness values at 1 month. This study has demonstrated that DMP-1 deficiency leads to:

severely compromised bone mineralization;

poor biomechanical properties of the long bone; and

delayed bone development and remodelling. In conjugation with previous findings that DMP-1 plays important roles in the early developmental stage of bone through its effects on osteogenic gene expression of Cbfa1, Col I, and Col II and regulating vascular invasion, the current study may suggest another important role for DMP-1 as a regulator for skeletal mechanostasis.

M. Murnaghan L. McIlmurray M. Mushipe G. Li

The potential importance of bone morphogenic proteins (BMPs) to improve fracture healing is of great interest to orthopaedic surgeons. Although the complex mechanisms leading from the presence of local BMP (either endogenous or exogenous) to form bone is increasingly understood, however most appropriate time to administer exogenous BMP has yet to be elucidated. The purpose of this study was to investigate when BMP may be administered to a fracture arena in order to best improve fracture healing. Forty mice were randomised into 4 groups; (group I) control, treated at day 0 with placebo; (groups II, III and IV) treated with BMP at days 0, 4 and 8, respectively. All animals underwent a previously validated surgical procedure involving the creation of an open femoral fracture which is stabilised using a 4 pin external fixator. Thirty microlitres of bovine serum albumin (BSA) alone was used in group I, and the other groups (II, III and IV) were treated with a combination of the BSA and 2.5 microgrames of rhBMP-2. The BSA and rhBMP were injected through a lateral approach immediately after operation, or at 4, or 8 days postoperatively. At days 0, 8, 16 and 22, sequential radiographs were taken using a digital x-ray machine and at day 22 all animals were sacrificed. Both femora were harvested and assessed biomechanically in 3-point bending prior to fixation for histological evaluation. All data were analysed using Mann-Whitney U tests (SPSS, Version 9, Chicago, Illinois) and differences were considered significant at p < 0.05. X-ray analysis indicated that healing of fractures treated with BMP at day 0(group II) or day 4(group III) was significantly greater than that at both days 16 and 22 (p < 0.05) than those animals in placebo (group I) and BMP day 8(group V) treatment groups. Although the administration of BMP at day 4 seemed to cause more bone formation than treatment at day 0, no significant difference were observed. There were no differences between group IV and group I. Biomechanically, group III exhibited ultimate load values closest to the contralateral unoperated femora followed by group II, then IV and finally the control group I. Significant differences (p < 0.05) were observed between the control animals (group I) and both groups II and III. Qualitative histology suggested that at 22 days after surgery, only groups II and III had healed with woven bone. Group I and group IV had considerable amounts of fibrous tissue and cartilage at the fracture gap. This study suggests that a single percutaneous injection of BMP has a positive effect on fracture healing in this model, when prescribed between the time of injury (day 0) and 4 days. Data suggests that the most effective timing of delivery of BMP may not be at the time of surgery but actually in the early healing phase. The day 4 time point in the mouse model is likely to equate to that of 7–10 days in larger animals or humans. This suggests that current human treatment practices may require further investigation in order to elucidate the most appropriate time of delivery for these important proteins. This work may negate the current requirements for carrier products and large doses of these expensive drugs.

C. Wan G. Li

During bone development and repair, angiogenesis, osteogenesis and bone remodeling (resorption) are closely associated processes with some common mediators involved. BMPs, VEGF and other cytokines are released from bone during bone resorption. Recent study showed that VEGF caused a dose- and time-dependent increase in bone resorption in vitro and in vivo, and BMP-2 markedly enhanced osteoclast differentiation induced by sRANKL and M-CSF in mouse osteoclast culture system. The aim of this study was to further examine the effects of VEGF and BMP-2 on osteoclastogenesis using in vitro human osteoclast culture system. Mononuclear cells were isolated by Lympo-Prep density gradient centrifugation from bone marrow washouts in bone samples from patients undergone total hip replacement. Mononuclear cells were plated at a density of 1 x 106/cm2 in a T-75 flask with aMEM and 15% FCS. The first medium change was made at day 7, when the floating cells were collected from the withdrawn media by centrifugation, and plated in a separate flask. The non-adherent cells in the 2nd flask were harvested again 24 hours later in a similar fashion. The non-adherent cells were then cultured in 24-well plates or calcium phosphate (Ca-P) coated plates, with osteoclast-inducing media (OC media) containing sRANKL 30 ng/ml and M-CSF 30 ng/ml, media were changed every 4 days. After 4 days culture in OC media, rhBMP-2 (3, 30, 300 ng/ml) and VEGF (25 ng/ml) were added respectively or in combination to the cell culture, and the culture was kept for total 16 days. The number of TRAP positive multinuclear cells in each well and the resorptive pit areas on the Ca-P coated plates were calculated and compared. Osteoclastic cell phenotype was defined by expressing tartrate resistant acid phosphatase (TRAP), vitronectin receptor (VNR) and resorptive pit assay. By day 12–14, osteoclastic cells were found in all the experimental groups, they were positive for TRAP and VNR. The number of TRAP+ multinuclear cells were significantly reduced (p< 0.05, t-test) when rhBMP-2 (30 and 300 ng/ml) were present, and this was further reduced (p< 0.01) when rhVEGF was added together with rhBMP-2, comparing to the culture with OC media alone. Extensive lacunar resorption pits in the Ca-P coated plates were found in the culture treated with OC media and OC media with rhVEGF (25 ng/ml). The resorption pit areas were, however, significantly reduced when rhBMP-2 was added at 30 and 300 ng/ml with or without rhVEGF (25 ng/ml, p< 0.05, t-test). The presence of low concentration of rhBMP-2 (3 ng/ml) with VEGF had no effect on osteoclast number or the areas of resorption pit formation. In contrary to previous findings in the mouse osteoclast culture system, the present study had shown that the presence of rhBMP-2 at 30 and 300 ng/ml had strongly inhibited osteoclast differentiation and bone resorptive capability in the human osteoclast culture system, and the inhibition was further enhanced by the presence of rhVEGF. This study implies that VEGF and BMP-2 may be important, yet to be defined regulators, for osteoclastogenesis.

P. Pollintine J.C. Park N. Farooq D.J. Annesley Williams P. Dolan

Introduction: Cement augmentation of osteoporotic vertebral fractures by vertebroplasty can alleviate pain, possibly by restoring normal load-sharing to the affected motion segment. Fracture is known to decrease vertebral compressive stiffness (1), and also affects the compressive stress distribution acting on the vertebral body, causing stress concentrations to appear in the adjoining intervertebral discs (2). We hypothesise that vertebro-plasty can reverse these fracture-induced changes.

Methods: Nineteen cadaver thoraco-lumbar motion segments (64–90 yrs) were used. Each was mounted on a hydraulic materials testing machine and induced to fracture by compressive overload in moderate flexion. Vertebroplasty was performed by injecting 7 cc of poly-methylmethacrylate cement (Simplex P, Stryker Howmedica, NJ) into the fractured vertebral body. Specimens were then creep loaded at 1.5 kN for 1 hour to allow consolidation. Before and after each procedure, profiles of the compressive stress distribution were obtained by pulling a miniature pressure transducer along the mid-sagittal diameter of the intervertebral disc whilst it was compressed at 1.5kN. Using these profiles, stress peaks in the anterior and posterior annulus were measured by subtracting the nucleus pressure from the peak stress in each region (2). Compressive stiffness of the motion segment was also measured before and after vertebroplasty from the tangent of the load-displacement curve at 1 kN. Changes were compared using ANOVA.

Results: Following fracture, motion segment compressive stiffness was reduced by 37% from 2478 N/mm, STD 966N/mm, to 1583 N/mm, STD 585 N/mm (p = 0.0001), stress peaks in the posterior annulus were increased by 139% from 0.24 MPa, STD 0.24 MPa, to 0.57 MPa, STD 0.47 MPa (p = 0.016), and stress peaks in the anterior annulus showed no significant change. The decrease in compressive stiffness was significantly correlated with the increase in the size of the posterior stress peak (Rsq = 0.65, p< 0.001). Following vertebroplasty and subsequent creep loading, compressive stiffness was increased to 2156 N/mm, STD 718 N/mm, and stress peaks in the posterior annulus were reduced to 0.31 MPa, STD 0.43 MPa. These changes were again highly correlated with each other (Rsq = 0.68, p< 0.001). Both compressive stiffness and the size of posterior stress peaks after vertebroplasty showed no significant difference when compared to pre-fracture values.

Discussion: Fracture reduces the ability of vertebrae to resist deformation, thereby decreasing compressive stiffness. These changes impair the disc’s ability to press evenly on the vertebral body, giving rise to increased stress peaks in the posterior annulus. Vertebroplasty can reverse these fracture induced changes by increasing vertebral compressive stiffness which acts to restore pressure in the nucleus. This enables the disc to press more evenly on the vertebral body and thereby reduces the size of stress peaks in the posterior annulus. This restoration of normal load-sharing may possibly contribute to pain relief in patients undergoing this procedure.

M.H.A. Malik B. Rash N. Delcroix P. Day A. Bayat W.E.R. Ollier P.R. Kay

Introduction: In attempting to unravel the complex cellular responses leading to prosthetic loosening investigators have been limited to studying gene expression of extracellular molecules about which most is known whereas new microarray technology allows simultaneous expression profiling of thousands of genes from a complex sample such as the membrane formed around loosened hip prostheses.

Methods: Two groups of 8 patients were recruited who have undergone primary total hip arthroplasty for osteoarthritis and subsequently developed either septic or aseptic loosening +/− osteolysis. The control group consisted of one group of 5 patients with the same initial diagnosis who had undergone identical procedures, developed no clinical or radiological signs of aseptic or septic loosening, but had come to revision surgery for other complications as defined by the Swedish Hip register: fracture without previous osteolysis, dislocation, technical error, implant fracture, polyethylene wear or pain. Periprosthetic membrane was harvested at the time of revision surgery and subjected to RNA extraction. cDNA was then synthesized and hybridised to a Human Genome u95 Genechip ® array which contains a complete set of known human genes. Data normalisation, data filtering and pattern identification was performed using Genechip®3.1 software (Affymetrix, Santa Clara, CA).

Results: This has revealed the involvement of a large number of genes coding for transcriptional regulators upstream from the extracellular and cell-cell signalling molecules already known to be involved in osteolysis and deep infection and which may ultimately control the responses to wear particles and bacterial challenge. Differential expression of genes involved in cell survival and death, cell growth regulation, cell metabolism, inflammation and immune response was found. Most interestingly pathways for control of local bone resorption and inflammatory response have been shown to be highly activated.

Conclusions: The identification of these new pathogenetic mechanisms of total hip replacement failure make new indicators of disease susceptibility and prognosis plus new drug targets direct possibilities.

T.J. Joyce D. Monk P. Thompson P. Chiu A. Unsworth S.M. Green

Introduction: The wear of orthopaedic biopolymers is recognised as a major factor in the failure of total joint replacements. Clinical wear data exists for acetabular cups manufactured from three biopolymers: ultra high molecular weight polyethylene (UHMWPE); poly tetra fluoro ethylene (PTFE); and polyacetal. The aim of this paper was to wear test these biopolymers and compare the results with clinical data.

Method and Materials: The biopolymers were tested using a modified, four-station, pin-on-plate wear rig [1]. In the tests, two of the four stations applied reciprocating motion and two applied multi-directional motion. Biopolymer pins articulated against stainless steel plates under a load of 40N. The lubricant consisted of 25% bovine serum and 75% distilled water. A standardised cleaning and weighing protocol was followed, and the biopolymer wear factors were calculated by dividing the volume lost by the product of the load and the sliding distance.

Discussion and Conclusions: Failed and retrieved UHMWPE acetabular cups have been reported as having a clinical wear factor of 2.1 x 10−6mm3/Nm [2]. However, UHMWPE cups which have been functioning well until removal at post-mortem have been said to show 45 to 69% less wear than revised UHMWPE cups [3]. Combining these values suggests clinical wear factors for functional UHMWPE in the range of 0.95 to 1.45 x 10−6mm3/Nm. This range fits well with the value of 1.1 x 10−6mm3/Nm shown in table 1 for UHMWPE under multi-directional motion. A clinical wear factor of 37 x10−6mm3/Nm has been calculated for PTFE acetabular cups [4]. When compared with the mean wear factor for PTFE pins under multi-directional motion obtained from the pin-on-plate rig, the match is remarkable. For polyacetal cups a mean volumetric wear of 136mm3/ year has been reported [5] and it has been calculated that explanted hip prostheses averaged 1.54 million cycles/year [2]. In polyacetal acetabular cups of 37mm diameter, an average sliding distance of 25mm/cycle can be calculated [6] and it has been said that an equivalent static load of 1000N applies [7]. Taking these four values permits a clinical wear factor for polyacetal cups of 3.5 x 10−6mm3/Nm to be calculated. This number compares well with the value of 3.8 x 10−6mm3/Nm seen for the polyacetal test pins under multi-directional motion. In summary, all three biopolymers subject to multi-directional motion exhibited clinically relevant values of wear.

T. N. Board L. Yang M. Saleh

Fine-wire fixator systems have been used successfully for the treatment of fractures, malunions and for limb lengthening for many years. There has been much research investigating the biomechanical properties of these systems but this has been almost entirely centred on the mechanical properties of the fixator as a whole. Our knowledge of the interactions occurring at the interface between wire and bone remains sparse. To this end we devised an experimental model to analyse the distribution of pressure in cancellous bone surrounding a tensioned wire under loading conditions. The Sawbones cancellous bone material (type 1522-11) was cut into 65x30x40 mm blocks. A 2 mm olive wire was inserted into each block, parallel to the surface and along the 65 mm dimension. The distance from the wire to the surface was variable, from 0.5mm to 5mm in a 0.5mm increment. The wire was mounted on a 150 mm ring and tensioned to 1200 N against a load cell. The ring was rigidly mounted on a material testing machine and a second bone block was incorporated into the testing machine crosshead with a universal joint. Three grades of pressure-sensitive films (Low, Superlow and Ultralow) were sandwiched in turn between the testing block and cross head. The force applied was 175 N for 5 s. The developed film was scanned into a computer and a Matlab program was developed to analyse the pressure image. The results show three phases of pressure distribution. Very close to the wire there is a polar distribution of pressure that is, the pressure is concentrated towards the entry and exit points of the wire. At a depth of 1.5mm away from the wire the pressure becomes evenly distributed along the path of the wire in a beam-loading manner. At a distance of greater than 4mm from the wire there is even distribution of pressure throughout the bone. The peak pressures (6–8 MPa) were found closest to the wire. Most of the pressure measured was less than 1 MPa, which is less than the yield strength of cancellous bone (2–7 MPa, Li and Aspden, 1997). In contrast a similar analysis using threaded half pins under the same conditions showed far higher peak pressures (20 MPa), which were present deeper in the bone specimen. The pressure was concentrated toward the pin entry site and was not well distributed throughout the pin-bone interface. These results allow us to explain why ring fixators are superior to half pin fixators when used in metaphyseal bone.

D.M. Sirkett A.W. Miles G. Mullineux G.E.B. Giddins

Background and Purpose: There is a high incidence of arthritis in the hand, but joint replacement technology in the wrist and other small joints is still in its infancy compared with the larger joints. The wrist is the most complex small joint and so there is a need for fundamental research into the way in which it works. At present there is no generally agreed upon satisfactory explanation for the complex movement patterns of the carpal bones. The purpose of the work was to test a new hypothesis on wrist kinematics. The basis of the hypothesis was that the bones of the wrist move in such a manner as to maximise total contact area in the joint, thereby minimising contact stress. Such a strategy would minimise the bone mass requirements, thereby minimising the biological “cost” of creating and maintaining the joint. This agrees with the minimum energy principle, which governs many natural processes.

Methods: A computer model was created to test the hypothesis. A cadaveric wrist was dissected and 3D faceted models of the carpal bones were created using laser digitisation. The model contained a program to evaluate the closeness of packing of the carpal bones and an optimisation algorithm [1] to maximise this quantity by adjusting the positions of the bones. The evaluation program computed the contact area and level of intersection between nine pairs of interacting bones. Rotation in the radial-ulnar deviation plane was applied in 1.0° increments to four rigidly connected bones defining the overall posture of the wrist, and an optimisation algorithm was used to maximise the contact area by adjusting the positions and orientations of the remaining bones.

Results: The results of the work are encouraging because certain known characteristics of carpal behaviour were clearly predicted by the model. The results for the scaphoid in particular were similar to the characteristic movements of this bone in both radial and ulnar deviation. During 20° of unlar deviation, the bone demonstrated 14.3° of extension, which is near to the 20.4° reported by an experimental study [2]. In 10° of radial deviation, the bone underwent 6.4° of flexion, which again is close to the 8.1° experimental result.

Conclusion: Although the computer model predicted certain aspects of carpal behaviour, the initial hypothesis was not conclusively proved. This is due in part to the computational complexity of the task. Despite some simplifying assumptions, there were still a large number of degrees of freedom, and it is almost certain that the optimisation process was afflicted with local minima problems. If the technical hurdles can be overcome and the hypothesis is proved correct, then we will gain a new explanation of the laws governing the kinematics of the wrist joint, which are not fully understood at present. This will provide invaluable information for surgical applications, where a thorough understanding of normal kinematics is essential for the treatment of joint injury and instability.

F.D Zhao P. Pollintine A.S. Przybyla P. Dolan M.A. Adams

Introduction: Back pain can be associated with erratic and/or excessive movements between adjacent vertebrae. Such movements are normally resisted by intervertebral ligaments, and yet few back pain patients report traumatic rupture of ligaments prior to their onset of symptoms. We suggest that two other mechanisms can lead to ligamentous slack and therefore to spinal instability. The first of these is the age-related dehydration of intervertebral discs, which reduces disc volume and height, bringing the vertebrae closer together. The second mechanism is disc decompression following vertebral endplate fracture, which is a common injury but one which is difficult to detect. Decompression allows the disc to bulge and lose height, increasing ligamentous laxity. In the present experiment, we simulated disc dehydration and endplate injury in cadaveric spines, and compared their effects on spinal (in)stability.

Methods: Cadaveric thoraco-lumbar motion segments were subjected to complex, continuous loading using a hydraulic materials testing machine (Zwick-Roell, Leominster, UK) to simulate full flexion and extension movements in vivo. Vertebral movements were recorded at 50 Hz using the optical “MacReflex” video capture system (Qualisys AB, Sweden). Experiments were repeated following 2 hours of compressive “creep” loading at 1500 N, which reduced disc water content by an amount similar to the aging process, and again following compressive overload sufficient to fracture a vertebral endplate. Bending moment-rotation curves were used to quantify the “neutral-zone” (NZ), range of motion (ROM), and bending stiffness (BS).

Results: Preliminary results (10 motion segments) showed that specimen height was reduced by 1.0 mm (STD 0.3 mm) following creep, and by a further 1.5 mm (STD 0.5 mm) following endplate fracture. Mean ROM in flexion increased from 6.5 deg initially, to 8.9 deg after creep and 12.6 deg after fracture. Corresponding values for NZ in flexion were 4.6 deg, 6.6 deg and 9.5 deg. BS decreased from 28.9 to 23.0 to 15.2 Nm/deg. All changes were statistically significant (p< 0.03). NZ, ROM and BS values in extension were initially 1.6 deg, 4.0 deg and 32.7 Nm, respectively, but no significant changes were noted following creep and endplate fracture. Total ROM (flexion + extension) increased from 10.5 deg to 16.7 deg degrees following both interventions.

Discussion: Results suggest that disc dehydration, which is a normal feature of aging, increases NZ and ROM in flexion, presumably because accompanying disc height loss allows more slack to the posterior intervertebral ligaments. Endplate fracture, which can occur under physiological loads in osteoporotic elderly spines, has an even greater effect. Extension movements were little affected, presumably because loss of disc height also increases the risk of impaction between neural arches.

Conclusion: We conclude that age-related disc dehydration, and relatively minor endplate injury, can increase segmental motion and cause substantial mechanical instability to the thoraco-lumbar spine.

P.L. Lomoro R.K. Wilcox M.C. Levesley R.M. Hall

Percutaneous vertebroplasty (PVP) is an emerging interventional technique for treatment of vertebral compression fractures. Bone cement is introduced to mechanically augment fracture and pain relief is almost immediate. Recent clinical and biomechanical studies have outlined the phenomenon of fractures occurring in adjacent vertebrae following PVP [1,2]. It is widely believed that rigid cement augmentation may cause a shift in the normal loading pattern of the spine thereby resulting in adjacent fractures. However, very few studies have attempted to quantify this effect [3].

Most biomechanical studies adopt a single vertebral body as a model for PVP analysis. With this approach it is not possible to determine the effect of load distribution on adjacent structures. Where multi-segment vertebrae have been used there is little documentation of the fracture characteristics produced or their repeatability. The purpose of this study was to develop a 3-vertebra model for the biomechanical analysis of PVP. The particular focus was on developing a robust technique for generating repeatable level of fracture severity from specimen to specimen.

An alignment device was developed to fit into standard materials testing machine, which allowed constant axial compression without causing lateral bending or flexion-extension of the specimen’s ends. Porcine 3-segment specimens (T8-L2) were mechanically compressed to failure at a rate of 5mm/min applied vertically at a distance of 35% to the anterior edge of the specimen’s anterior-posterior length. During the test load-displacement data was displayed in real time on a PC. In order to generate uniform fractures, a protocol was devised in which the specimens were compressed for a further 6mm after initial yield point. After the initial fracture the segments were augmented with 3ml of PMMA cement injected through each pedicle and then recompressed. The fracture characteristics generated under these conditions were analysed using quantitative microcomputer tomogragy (μCT).

μCT images showed that fractures were generated in the central vertebra, with some propagation towards adjacent vertebra. The results support the use of a 3-segment specimen as a better representation for PVP analysis. The method will enables the load shift and fracture progression on either side of the augmented vertebra to be observed, thereby providing a more complete picture of load-bearing kinetics. Secondly, the middle, augmented motion segment remains unconstrained by platens and cement impressions; hence its anatomical boundary conditions are less compromised. Although longer segments have been shown to be more anatomically appropriate, it is difficult to apply physiologic levels of load without causing the specimen to buckle. We were able to minimise buckling effect by incorporating an alignment device to position the specimen without constraint. Given the preceding observations, the concepts of 3-segment specimen in PVP biomechanical tests provides a suitable compromise in choosing an appropriate clinical setting for in-vitro testing of biological spine specimens.

Y.H. Hsu I.G. Turner A.W. Miles

Introduction: Calcium phosphate based ceramics with a porous configuration are attraction for use as synthetic bone grafts as the porous network allows tissue ingrowth, which further enhances the implant-tissue attachment. The degree of interconnectivity and the nominal pore size are the critical factors that determine the success of the implants. It is generally accepted that a minimum pore size of 100 μm is necessary for the porous implant materials to function well and a pore size greater than 200 μm is an essential requirement for osteo-conduction. However, research has suggested that the degree of interconnectivity is more critical than the pore size. In this study, porous Hydroxyapatite/Tricalcium phosphate (HA/TCP) bioceramics with interconnected porosity and controlled pore sizes were fabricated by a novel technique involving vacuum impregnation of reticulated polymeric foams with ceramic slip. HA/TCP samples with a range of pore sizes and functionally gradient materials (FGM) with porosity gradients were made.

Materials and Methods: Two grades of calcium phosphate powder, TCP 118 and TCP 130, were used. Varying the blend ratios could change the ratios of HA and TCP in the sintered samples. The foams used comprised polyurethane (PU) which had one of three different porosities 20, 30 and 45 pores per inch (ppi). In order to make a FGM with porosity gradients mimicking the bimodal structure of cortical and cancellous bone, two different foams were either joined together by sewing or pressfitting together. The foams were substantially impregnated with slip by vacuum impregnation. The impregnated foams were removed from the vacuum chamber and dried on tissue for at least 24 hours then sintered at temperatures of up to 1280°C.

Results and Discussion: Using a slip with the appropriate viscosity, porous HA/TCP bioceramics having interconnecting pores and a range of pore sizes can be produced successfully. By joining different ppi foams together, it is possible to develop functional gradient materials in which the porosity varies through the thickness of the samples. No weakness could be seen at the interface between the two different structures. This demonstrated that porous HA/TCP with two or more different levels of porosity could be produced in a single block. Image analysis shows the porosity measured for the three different foams was similar. The area equivalent diameters of the pore structure are 197–254 μm with 20ppi foam, 143–183 μm with 30ppi foam and 105–127 μm with 45ppi foam. The compressive strengths of the HA/TCP samples are in the range of 30–170 MPa and the apparent densities were 2.34–2.76 g/cm3. The technique developed for fabricating porous bioceramics can be extended to produce a range of bone substitute materials with properties tailored to specific clinical applications.

A. Anand M. Akmal M. Wiseman A. Goodship G. Bentley

Study Purpose: The cause of intervertebral disc degeneration (IVDD) is multifactorial. One proposed mechanism is that IVDD originates in the nucleus pulposus (NP) and progresses radially to the annulus fibrosis (AF). Failure of current treatment modalities in preventing and treating IVDD and thereby low back pain have led to a growing interest in tissue-engineered solutions where a biological repair is induced. By preventing the abnormality at the NP it may be possible to halt further progression of IVDD. Injection of NP cells into an early degenerative IVD, where the AF is still intact, may retard the degenerative process and is presently under investigation. Using a three-dimensional scaffold that could be successfully introduced into the NP cavity through minimally invasive techniques would prevent the loss of chondrocytic phenotype of the cells and be an improvement over the existing technique by which cells are directly injected into the NP cavity.

Methods: (1) CaSO4 and CaCO3 alginates were injected into the NP cavity of a bovine tail. After 90 minutes the tail was dissected to reveal the gel. (2) NP cells released from pooled bovine NP tissue were dispersed into the CaSO4 and CaCO3 alginate gels (10x106 cells.mL-1) with and without Synvisc® and cultured for 21 days.

Results: (1) Injectable alginate suspensions formed solid viscoelastic gels, filling the exact shape of the NP cavity. (2) NP DNA and ECM synthesis was significantly greater in the CaCO3 alginate gel than in the CaSO4 alginate gel (p< 0.05). (3) Synvisc® significantly increased sulphated GAG (p< 0.01) and collagen (p< 0.05) production. These effects were supported histologically and immunohistologically where cells in the CaCO3 and Synvisc® gels stained more intensely for proteoglycan and collagen type II.

Conclusions: Both CaCO3 alginate gel and CaSO4 alginate gel are injectable and are capable of sustaining NP cells in-vitro. Cells remain viable, maintain their phenotype, proliferate and produce ECM during the culture period. The CaCO3 alginate gel provides a three-dimensional matrix more favourable to NP cellular activity than the CaSO4 alginate gel. Synvisc® behaves as a chondro-stimulant significantly enhancing NP cell metabolic activity. This study demonstrates a successful tissue-engineered approach for replacing the NP and, subject to further studies, may be used for retarding mild-to-moderate IVDD, alleviating lower back pain and restoring a functional NP through a minimally invasive technique.

R.K. Goddard H. Wynn Jones B.I. Singh J.C. Shelton M.A.S. Mowbray

Aims: The aims of this study were to evaluate the biomechanical properties and mode of failure of 4 methods of fixation used for hamstring tendon ACL grafts. The fixation methods investigated included titanium round headed cannulated interference (RCI) screws, bioabsorbable RCI screws, Endobuttons and Bollard fixation. It has been previously shown that a 2 strand tailored equine tendon-Soffix graft has equivalent biomechanical properties to a 4 strand human hamstring tendon-Soffix graft [1,2], therefore this model was used for the graft in the study.

Materials and Method: 32 stifle joints were obtained from skeletally mature pigs, the soft tissues were removed and the ACL and PCL were sacrificed. Tibial tunnel preparation was standardised using the Mayday Rhino horn jig to accurately position a guide wire. An 8 mm cannulated reamer was then used over the guide wire to create the final tibial tunnel. A back radiusing device was then placed into the tibial tunnel to chamfer the posterior margin of the tunnel exit to prevent abrasion and fretting of the graft. A 2 strand equine tendon-Soffix graft was then introduced into the tibial tunnel and secured with one of the four fixation methods. The proximal part of the graft was attached to the cross head of the materials testing machine using the Soffix. Five of each method of fixation were tested mechanically to failure and three of each method were cyclically loaded for 1000 cycles between 5 to 150 N, followed by 2000 cycles at 50 to 450 N.

Results: The mean ultimate tensile loads (UTL) were: titanium RCI screw = 444 N, bioabsorbable RCI screw = 668 N, Endobutton = 999 N and Bollard = 1153 N. The mode of failure for all RCI screws involved tendon slippage past the screw. Two Endobutton failures were encountered and one Bollard pull out occurred. Under cyclic loading conditions the titanium and bioabsorbable RCI screws failed rapidly after several hundred 5 to 150 N cycles due to tendon graft damage and progressive slippage. Both the Bollards and Endobuttons survived 1500 cycles at 50 to 450 N, with less tendon slippage occurring.

Conclusion: Titanium and bioabsorbable RCI screws provide poor initial fixation of tendon grafts used for ACL reconstruction, having significantly lower UTL’s than both Endobutton and Bollard fixation. Under cyclic loading titanium and bioabsorbable RCI screws fail rapidly due to progressive tendon slippage, whereas Bollards and Endobuttons survive cyclic loading. Both Bollard fixation and Endobuttons provide sufficiently high UTL’s and survive cyclic loading to allow early postoperative mobilisation and rehabilitation. Caution must be used in the early postoperative period when using interference screws to secure a hamstring tendon graft because progressive tendon slippage may result in excessive graft elongation and early clinical failure.

R. Balendran A. Sandison J. Moss A.L. Wallace

The purpose of this study was to determine and compare the effects of radiofrequency ablation and mechanical shaving on tendon using histological and ultrastructural techniques. A single cut using a scalpel blade was used to create a standardised reproducible lesion in 12 freshly harvested ovine infraspinatus tendons. Each lesion was then subjected to either bipolar radiofrequency ablation or mechanical shaving. Specimens were either fixed in formalin and processed for light microscopy or fixed in glutaraldehyde and processed for transmission electron microscopy. Samples of normal and untreated cut tendon were analysed as suitable controls. The radiofrequency treated samples showed an area of coagulative necrosis with an average diameter of 2mm around the lesion. Conversely, the shaved samples showed viable cells up to the edges of the lesion. These findings were supported by ultrastructural appearances, which showed preservation of tendon architecture in shaved samples and widespread denaturation of the tendon matrix with loss of fibrillar structure in the radiofrequency treated samples. Radio-frequency electrical energy and mechanical shaving are often used for resection of soft tissues during arthroscopic reconstructive procedures. The effects of these techniques on tendon are not yet clearly understood. The results of this study indicate that thermal resection of tendon causes an immediate additional 2mm area of tissue necrosis which is not present after mechanical shaving. These findings may have implications for the success of arthroscopic debridement and tendon repair procedures.

S.G. Rees A.D. Waggett B. Caterson

Although the function of proteoglycans within the tendon extracellular matrix are not fully understood, changes in their turnover have been associated with tendinopathies. In contrast to cartilage, aggrecanases are constitutively expressed and active in tendon, indicative of a high rate of aggrecan turnover. Clinical trials investigating the use of active site MMP inhibitors have been confounded by side-effects which involve tendonitis and “musculoskeletal syndrome”. Such side effects may relate to non-specific inhibition of tendon aggrecanases required to maintain normal metabolic homeostasis. The purpose of this study, therefore, was to compare the rate turnover of tendon and cartilage proteoglycans derived from the same joint and to determine the effect of MMP inhibitors (actinonin and marimastat) on aggrecan catabolism. Deep digital flexor tendon explants from compressed and tensional regions were dissected from young and mature bovine. Explants were precultured and then cultured for a further 4 days with or without marimastat (0–2 M) or actinonin (0–200 M). Proteoglycan and lactate quantification, Western blot analysis of degradation products and RT-PCR analyses were performed on these samples. In a separate experiment for measurement of proteoglycan turnover, explants were set up as described above then pulse chase labelled with [35S] sulphate. The rate of turnover of 35S-labelled proteoglycans from the matrix of tendon (and articular cartilage obtained from the same animal) was subsequently calculated from the amount of 35S-labelled macromolecules appearing in the medium each day and that remaining in the matrix of explants at the termination of culture. Proteoglycan turnover (presumably predominantly aggrecan) was markedly higher in tendon versus cartilage. This difference was apparent in tendons from all regions and ages. Both marimastat and actinonin inhibited aggrecanase-mediated proteoglycan catabolism in both tendon and cartilage explants. As expected mRNA expression for the aggrecanases, MMPs and TIMPs was unaffected by addition of these inhibitors to the culture medium. Aggrecan turnover in tendon is higher than that of articular cartilage, which may be attributed to distinct physiological properties of this proteoglycan in tendon. Importantly, immunohistochemical staining for aggrecan in tendon indicates its presence in between collagen fibres and fibril bundles and thus aggrecan aggregates may dissipate resultant compressive loads by resisting the flow of water in these locations. In addition, aggrecan may facilitate the sliding of fibrils during the small amount of elongation of the tendon whilst under tension. Thus, the half-life of tendon aggrecan is significantly reduced because it constantly participates in repeated resistance to compression. Our data also demonstrates that both marimastat and actinonin can inhibit aggrecanase-mediated proteoglycan catabolism in tendon cultures. This suggests that the occurrence of “musculoskeletal syndrome” in clinical trial patients may be due to the fact that these inhibitors affect the activity of aggrecanases in tendon, thus preventing them from playing their normal role in tendon aggrecan turnover and consequently perturbing normal physiological function.

R.L. Stanley J.C. Patterson-Kane J.R. Ralphs A.E. Goodship

The energy-storing human Achilles tendon and equine superficial digital flexor tendon (SDFT) show no adaptation to exercise unlike muscle and bone, and are prone to injury. Injury involves microdamage accumulation until there is sufficient weakening for rupture to occur during normal athletic activity. Anatomically opposing positional tendons, such as the common digital extensor tendon (CDET) in the horse rarely suffer exercise–induced injury. Tenocytes maintain the extra-cellular matrix, but in energy-storing tendons they appear unable to adequately repair microdamage as it occurs. Tenocytes have been classified subjectively into 3 subtypes on the basis of histological nuclear morphology. Long, thin type 1 cells are thought to be less synthetically active than cigar-shaped type 2 cells, but their exact morphology and relative proportions in different tendon sites and ages has not been clearly defined. We hypothesised that tenocytes are separable into morphologically distinct subtypes, reflecting differences in age and functional requirements within and between specific tendons. Samples were taken from tensional and compressed regions of the SDFT and CDET of 5 neonates, 5 foals (1–6 m), 5 young adults (2–6 y) and 5 old horses (18–33 y) Cell nuclei were counted and measured in digital images from histological sections by computerised image analysis. Total tenocyte densities and proportions of the 3 subtypes were calculated for each age group, as were nuclear length:width ratios. Length:width ratio distributions for all horses were evaluated using a normality test followed by a paired t-test. There was a significantly higher total cellularity in the SDFT than the CDET, with a higher proportion of type 1 tenocytes in the CDET. With age, total cellularity decreased in all tendon sites and an increase in the proportion of type 1 tenocytes was observed in tensional regions. Foal and neonatal tendons contained significantly higher proportions of type 2 tenocytes than older tendons. The morphology of the two main subtypes in all age groups was significantly different; type 1 tenocytes had a higher nuclear length:width ratio (mean ± SD = 9.6 ± 2.5) than type 2 (mean ± SD =4.7 ±1.1) (p< 0.001). We were able to objectively separate tenocytes into 3 distinct subtypes based on nuclear length:width ratio measurements. There were significant differences in proportions of subtypes with tendon site and age. The positional tendon had significantly lower cellularity and a higher proportion of type 1 tenocytes; these cells may be less functionally active but sufficient to maintain the matrix in a tendon which is not subjected to high levels of strain. The SDFT continues to grow up to 2 years of age and is subjected to high strains, explaining the need for relatively higher proportions of type 2 cells. There is however an age-related increase in type 1 cells in both tendons which may explain an inability of the adult energy-storing tendon to adapt to exercise and to repair microdamage. Understanding the stimulus for age-related changes in tenocyte subtype proportions in tendons with different functions may help us understand the pathogenesis of exercise-induced tendon injury and to develop more appropriate training regimens.

J.C. McGregor-Riley L. Yang M. Saleh

Introduction: Circular fixators are widely utilised in orthopaedic surgery. Their biomechanical characteristics have been studied in some detail and it is known that the widest wire crossing angles yield maximum stability. Unfortunately, due to anatomical constraints, mechanically optimum wire crossing angles are seldom achievable, especially in the tibial diaphysis. Narrowed crossing angles are usually accepted thereby compromising sagittal plane bending stability. With a hybrid circular fixator, narrowed crossing angles exacerbate the problem of fracture site shear. It is hypothesised that by minimising slippage at the wire-bone interface by using threaded wires, stability can be maintained even with narrowed crossing angles. The aim of this study is to examine the effect of threaded wires on fracture site shear with a hybrid fixator.

Method: Bone-fixator models were created from nylon rods and the Orthofix Ring Fixator. Constructs with wire crossing angles of 70, 60, 45 and 30 degrees were loaded axially and in four point bending. Each construct was tested four times; the first test was not analysed. The whole fixator was then rebuilt and all tests repeated. Fracture motion (compression, angulation & shear) was measured using a strain gauge intersegmentary motion device and stiffness calculated by linear regression. Smooth & threaded wires were compared by univariate analysis of variance, which makes allowance for variation between individual frames.

Results: Axial stiffness was comparable to previous studies (85–96N/mm) with no difference between wire types. Threaded wires produced a 29% reduction in shear during axial compression (p=0.02). In four-point-bending, angulation stiffness (in the half-pin plane) was directly related to crossing angle and at all angles threaded wires were associated with a significant improvement. The table shows the effect of wire type on shear (in mm) measured in the plane of the half-pins for a 10Nm bending moment. Shear becomes appreciably higher with narrow crossing angles (almost 2mm) but this is effectively controlled by threaded wires.

Discussion: Of all the factors influencing fracture healing, the mechanical environment is one over which the surgeon has most control. It is generally accepted that excessive shear inhibits fracture healing. This study has shown that by using threaded wires in a circular frame, crossing angles can be narrowed without compromising stability. in particular they control undesirable shear motion seen with hybrid frames and narrow crossing angles. The principle is equally applicable to all-wire frames as they invariably are constructed with compromised crossing angles leading to reduced sagittal plane stability.

M. Day J. Cao S. Li A. Hayes C.E. Hughes R. Evans C. Dent B. Caterson

Introduction: Kashin-Beck disease (KBD) is a special endemic osteoarthropathy whose main pathologic changes occur in growth plate cartilage and articular cartilage of human limbs and joints where it is manifested as cartilage degeneration and necrosis. Past and current research suggests that KBD, and its endemic geographic distribution in China, is due to the combined presence of fungal mycotoxins (on stored food ingested by affected populations) and a regional selenium deficiency in the environment providing local food sources. Thus, we hypothesise that the presence of fungal mycotoxins and the absence of selenium in the diet specifically affects chondrocyte metabolism in the growth plate during limb and joint development and in articular cartilage of adults, which leads to localised tissue necrosis, and the onset of degenerative joint disease. The aim of this study was to examine the effects of mycotoxins; e.g. Nivalenol (NIV), selenium and NIV in the presence of selen! ium in in vitro chondrocyte culture systems to better understand cellular and molecular mechanisms underlying the pathogenesis of KBD.

Methods: Chondrocyte tissue cultures were established using cartilage explant cultures either in the presence or absence of selenium (0.5–1.5 microg/ml) and the mycotoxin nivalenol (0.5–1.5 microg/ml) and culture for 1 to 4 days. Medium was harvested daily at day 1 through 4 and analysed for glycosaminoglycan (GAG) release and the presence of aggrecanase or MMP activity using RT-PCR for gene expression and monoclonal antibodies that detect their respective enzyme-generated neo-epitopes on cartilage aggrecan metabolites.

Results: Our studies to date have shown that NIV exposure induces catabolic changes in chondrocyte metabolism with an increased expression of aggrecanase activity. Addition of selenium did not affect mRNA expression of the aggrecanases ADAMTS-4 & 5. Parallel studies involving immunohistochemical analyses of articular cartilage from KBD showed an increase in aggrecanase activity.

Conclusions: These studies demonstrate that induction of aggrecanase activity as one of the molecular mechanisms involved is the pathogenesis of KBD. However, the addition of selenium does not alter aggrecanase gene expression indicating that its beneficial effects are occurring in other areas of cartilage metabolism.

D. Thyagarajan C. Harris R. Evans C. Dent B. Caterson C. Hughes

Degenerative joint disease (DJD) involves the proteolysis of many extracellular matrix molecules (ECM) present in articular cartilage and other joint tissues such as tendon, meniscus and ligaments. Recent research has identified key enzymes involved in the catabolism of ECM. Two classes of enzyme the Matrix Metalloproteinases (MMP’s) MMP-2, MMP-3, MMP-13 and the ADAMTS family (a disintegrin and metalloproteinase with thrombospondin motifs) of proteinases most notably, ADAMTS-1, -4 and −5, have been shown to be involved in the catabolism of ECM (such as type II collagen and cartilage aggrecan). The presence of several MMPs in the synovial fluid has been reported; however, little data has yet been gathered on the presence of ADAMTS-1, -4 or −5 (the aggrecanases) in synovial fluids. In this study we have used a recombinant artificial substrate and specific neoepitope antibodies that recognise either MMP- generated or aggrecanase -generated degradation products to measure the relative activity of these two enzyme families in the synovial fluid from human patients.

Methods: A recombinant substrate containing the interglobular domain of cartilage aggrecan , flanked by a complement regulator and the Fc region of IgG has been stably transfected into CHO cells. The recombinant protein has been purified from the medium using a Protein A column followed by gel chromatography using a Superose 12 column. Synovial fluid samples were depleted of serum immunoglobulin by pre-absorption with ProSepA. The recombinant substrate was then added to synovial fluid samples and incubated overnight as 37?C. The recombinant substrate was recovered from samples using ProsepA and then separated by SDS-PAGE (10% gels). Gels were transferred to nitrocellulose membranes and immunoblotted with antibodies recognising the undigested substrate and using neoeptiope antibodies specifically recognising MMP or aggrecanase –generated catabolites.

Results: Preliminary analysis by Western blot using the anti IGD neoepitopes BC-14 (detecting cleavage at the major MMP site) and BC-3 (detecting cleavage at the aggrecanase site) demonstrated that enzymes in human synovial fluid collected from patients diagnosed with rheumatoid arthritis cleaved the pro-drug at the MMP site with little or no evidence of aggrecanase catabolism. In contrast, synovial fluid collected from patients diagnosed with osteoarthritis indicted that there was cleavage at the aggrecanase site. In these preliminary studies we have also examined the enzyme activity in a set of clinical samples collected from patients that have undergone knee replacement surgery having been given either n-3 fatty acids or a placebo 10 weeks prior to surgery. Results indicate that aggrecanase generated fragments were found in synovial fluid from placebo patients, and reduced levels of enzyme activity were apparent in fluids tested from patients that had received n-3 fatty acids prior to surgery.

Discussion: This data suggests that the recombinant substrate will aid in the detection of MMP or aggrecanase activities in synovial fluid samples. The ratio of MMP to aggrecanase activity has potential as a biomarker for the severity of cartilage degeneration in degenerative joint diseases.

L. Palmer J. Gidley M. Clare J.R. Sandy J.P. Mansell

Osteoblast growth and differentiation are central to the formation and maintenance of healthy bone tissue. The search for novel mechanisms resulting in osteoblast maturation are highly desirable on several fronts. Firstly they provide potentially important information on the normal development of bone, in addition they may offer alternative therapies for bone diseases like osteoporosis and finally they may facilitate ex-vivo manipulation of cells for the subsequent improvement of oseointegration in transplantation/tissue engineering regimens. Recently we have been addressing how calcitriol, an active metabolite of vitamin D3, integrates with the signalling of epidermal growth factor (EGF) following reports that calcitriol can influence EGF receptor trafficking, expression and ligand binding. We have also extended our studies to investigating how other growth factors known to signal via receptor tyrosine kinases (RTKs) interact with calcitriol in controlling osteoblast growth and differentiation. The co-treatment of human pre-osteoblasts (MG63) with EGF and calcitriol resulted in the synergistic induction of their differentiation as supported by demonstrable increases in alkaline phosphatase activity and osteocalcin. The intracellular components responsible for eliciting the maturation response included protein kinase C and MEK 1/2 since the addition of calphostin C or UO126, respectively, blocked the differentiation response. Other ligands known to signal via RTKs, namely IGF1, VEGF and FGF1 could not induce differentiation in the presence of calcitriol. These findings support the specific integration of calcitriol/EGF signalling in osteoblast maturation. Collectively we have identified a novel, integrated, signalling pathway that drives terminal differentiation of osteoblasts. Our findings support earlier predictions (Yoneda 1996) in identifying novel actions of EGF in bone that will lead to advances in the field. Yoneda, T. 1996. Local regulators of bone: Epidermal growth factor – transforming growth factor-α. In Principles of bone biology (ed. J.P. Bilezikian, L.G. Raisz and G.A. Rodan.), pp. 729–738. Academic press Ltd.

H.I. Roach T. Aigner J.B. Kouri

Evidence has accumulated in recent years that programmed cell death (PCD) is not necessarily synonymous with the classical apoptosis, as defined by Kerr & Wyllie (J Path, 1973, 111:255–261), but that cells use a variety of pathways to undergo cell death, which are reflected by different morphologies. Although chondrocytes with the hallmark features of classical apoptosis have been demonstrated in culture, such cells are extremely rare in vivo. We have examined the morphological differences between dying chondrocytes and classical apoptotic cells in growth plate and osteoarthritic chondrocytes. Unlike classical apoptosis, chondrocyte death involves an increase in the endoplasmic reticulum and Golgi apparatus. This is likely to reflect an increase in protein synthesis with retention of proteins in the ER leading to expansion of the ER lumen, whose membranes surround and compartmentalise organelles and parts of cytoplasm. The final removal of apoptotic remains does not involve phagocytosis, but a combination of three routes: 1) auto-digestion of cellular material within compartments formed by ER membranes; 2) autophagic vacuoles and 3) extrusion of cell remnants into the lacunae. Together these processes lead to complete self-destruction of the chondrocyte as evidenced by the presence of empty lacunae. The involvement of ER suggests that the endoplasmic reticulum pathway of apoptosis may play a greater role in chondroptosis than receptor-mediated and mitochondrial pathways. Lysosomal proteases, present in autophagic digestion, are likely to be as important as caspases in the programmed cell death of chondrocytes in vivo. We propose the term ‘chondroptosis’ to reflect the fact that such cells are undergoing apoptosis, albeit in a non-classical manner, but one that appears to be typical of programmed chondrocyte death in vivo. Chondroptosis may serve to eliminate cells that are not phagocytosed by neighboring cells, which constitutes a crucial advantage for chondrocytes that are typically embedded in an extracellular matrix. Classical apoptosis in that situation is likely to lead to secondary necrosis with all its disadvantages. This may be the reason why most programmed cell death of chondrocytes in vivo appears to follow a chondroptotic pattern and not the classical apoptotic pattern. At present the initiation factors or the molecular pathways involved in chondroptosis remain unclear.

G.D. Smith P. Jones I.K. Ashton J.B. Richardson

Introduction: Autologous Chondrocyte Implantation (ACI) was first described in 1994(1) and has become an increasingly widely used treatment for chondral defects in the knee. The intention of this study was to identify which patient and/or surgical factors affect clinical outcome. In order to do this, a multicentre database of patients treated with ACI was established.

Methods: Four European centres collaborating in the EuroCell project (2) contributed data. These centres have historically used different outcome measures to follow up their patients. In order to analyse this data, a method of z-transformation was used to standardise the clinical scores. This has allowed a large number of patients to be investigated even when different scores have been used. A panel of predictor variables was agreed relating to patient factors and operative technique. Linear multiple regression analysis was performed to determine which predictor variables significantly influenced clinical outcome.

Results: A total of 284 patient datasets from four centres were investigated with 1 to 10 year follow-up. In 213 datasets the Modified Cincinnati (Noyes) clinician evaluation was used (3). The remaining 73 patients had outcome data measured with the modified Lysholm score (4). Outcome was defined as the change in score to latest follow-up. Z-transformation (z-change) was performed for each score type. The regression model was: z-change = − 0.11 − 0.5*z-preop − 0.43*R4 + 0 .30*OC + 0.20*FC (R2=0.30) The regression analysis showed that the factors which affected outcome were one centre (R4), pre-operative score (z-preop), osteochondral defects (OC), and lesions of the femoral condyle (FC). Factors which were found not to affect outcome included the age of the patient, size of the defect treated, number of defects treated and time to follow-up. Variations in operative technique, including the location of the cartilage harvest, the use of fibrin sealant and the timing of patch placement, were not found to have an effect on clinical outcome.

Conclusions: The method of z-transformation is a useful way of compiling multicentre data where different outcome measures have been used. This has allowed a large dataset to be compiled and factors which influence clinical outcome to be identified.

J.H. Kuiper T. Takahashi R. Barker A. Toms

Introduction: Diaphyseal fracture at a cortical perforation is the commonest postoperative complication of hips revised with impacted morsellised bone. To reduce fracture risk, surgeons can apply mesh, augment the bone with plate or strut graft, or bypass the perforation with a longer stem. No biomechanical data exists to choose between these alternatives. The objective of this study was to compare the above methods of cortical repair in terms of (i) bone fracture risk and (ii) stem migration.

Methods: Fourteen large composite femora (Sawbones, Malmö, Sweden) were prepared to simulate cavitary defects. An 18×40 mm lateral cortical perforation was made in 12 diaphyses. These diaphyses were repaired with mesh only, mesh and plate, or mesh and strut graft (n=4 each). Strut graft and plate were fixed with cables. Porcine cancellous bone was morsellised and impacted into each cavitary defect. Simplex P bone cement was injected. In the 12 femora with repaired perforation, a standard or a long Exeter prosthesis, bypassing the perforation 2 cortical diameters, was implanted. Thus, 6 methods of defect repair were created (mesh, plate and strut, combined with either long or short stem, each n=2). Standard stems impaction-grafted in the two femora without perforation served as control (n=2). Femora were placed in a testing machine and loaded at 1 Hz with 100 cycles of joint and abductor force. Peak joint force was 2,500 N. Strain amplitudes at the perforation and stem migration were determined. Statistical analysis was by 2-way and 1-way ANOVA, and the Student-Newman-Keuls (SNK) post-hoc test.

Results: Stem length did not affect average defect strain if used with plate or strut graft (2x2 ANOVA, p=0.62). Four combinations remained for further analysis: standard stem with mesh, long stem with mesh, plate, and strut graft, with defect strains of 5250, 3620, 2940, and 2480 μstrain. In controls, strains were 1750 μstrain. Defect strains differed significantly (ANOVA, p=0.0004), with strains for standard stems with mesh significantly higher than all other groups, those for long stems with mesh significantly higher than controls, and those for plate or strut graft no different from controls (SNK). Maximum permanent subsidence was 0.71 mm and retroversion 1.6°. For repaired perforations, stem length did not affect subsidence (p=0.96), but repair method did (p=0.03, both 2-way ANOVA). For further analysis, subsidence of the three repair methods (mesh, plate and strut graft with subsidence of 0.24, 0.47 and 0.19 mm, resp.) was compared with that of controls (0.52 mm). Subsidence differed significantly (ANOVA, p=0.02), and stems with strut graft subsided significantly less than those with plate or controls (SNK). Permanent retroversion was similar for each group.

Dicussion: Non-reinforced defects with a standard stem generated high defect strain amplitudes. A long stem bypassing the defect reduced these strains by 30%, and might suffice in case of otherwise strong cortex. In other cases, augmentation of the perforated diaphysis with either strut graft or plate needed to minimise fracture risk. Stem migration in reconstructed perforated diaphyses was always less than control cases, suggesting stem migration is no specific problem in reconstruction.

G.C. Cheung J.D. Moorehead C.K. Butcher

Introduction: Pedobarograph systems are used to measure foot pressure characteristics during gait. These measurements help clinicians diagnose pathology and assess treatment outcome. While most patients can walk across the Pedobarograph footplate unaided, some patients ask if they can use their crutch.

Aim: The aim of this study was to assess the effect of using a crutch on pedobarograh measurements.

Materials and Methods: A Musgrave Pedobarograph system was used to measure the foot pressure characteristics of twenty feet in ten normal subjects. The group’s mean age and weight were 31 years (SD=6) and 78 kg (SD=12), respectively. Each subject had each of their feet measured 3 times as they walked across the foot plate unaided. These measurements were then repeated with the subject using a crutch on the contralateral side to that being measured. The force through the crutch was recorded using a set of scales to ensured consistency between measurements.

Results: The main foot pressure characteristics are listed below. The table shows the mean value of the normal parameter, along with the percentage difference of the mean values when the crutch was used. The results of a paired-t test are also given to indicate the statistical significance of the change.

In addition to the above quantitative changes, qualitative assessment of the data showed an alteration in the loading pattern with reduced push off forces. However, eighteen of the twenty feet showed no alteration in the pattern of pressure distribution.

Conclusion: These results show that a crutch affects normal gait by generally increasing the time parameters and reducing the load and pressure parameters. The only parameters not significantly affected by the crutch were “Push off duration” and “Time to peak heel strike pressure”.

P.M. Mayhew C.D.L. Thomas N. Loveridge J.G. Clement J. Reeve

Introduction: Femoral neck (FN) fragility has been attributed to age-related bone loss, with increased loss in women. It has been shown that the mechanical properties of a supporting structure will also change with any alteration to the structure’s dimensions. The purpose of this study was to identify the age-related changes that take place in the morphology of the mid cross-section of the FN, and the implications for its mechanical properties in the different regions around the mid FN cross-section.

Materials and Methods: Measurements were taken from peripheral quantitative computed tomogram (pQCT) images of 81 cadaveric femurs (36 F, 45 M). The mid FN cross-section was segmented radially into eight regions and the cortical bone thickness (CT) and change of the centroid position (CP) of the FN cross-section were measured. The age-related effects of the corresponding changes in the proportion of cortical bone and the “resistance to bending” (section modulus, (Z)) were also measured.

Results: Four femurs were excluded because there were clear signs of OA being present. The maximum difference in regional CT between men and women, was less than 7% (Female: 3.07 ± 0.108mm; Male: 3.28 ± 0.123 mm (mean ± SEM) p =0.21). However, there were regional differences in CT between the young under fifty, (Un50, n=26) and the old, (Abv50), (ANOVAs for young vs old: CT p = 0.001 t 0.01). These effects were attributable to differences in the inferior region, where there was an increase in thickness of the cortical bone of 27% with senior status (Abv50: 3.44 ± 0.09mm; Und50: 2.70 ± 0.12mm. p = 0001) counter balanced by anterior and posterior loss. There was a corresponding change in CP, the distance of the superior, posterior, and superoposterior regions to the FN cross-section’s centroid, 7.6% (Abv50: 20.88 ± 0.28mm; Und50: 19.40 ± 0.47mm; p = 0.005); 6.7% (Abv50: 14.67 ± 0.2mm; Und50: 13.74 ± 0.32mm; p = 0.01); and 8%(Abv50: 17.95 ± 0.24; Und50: 16.61 ± 0.37), respectively. When these two measurements were combined (CP divided by CT) to provide the Local Buckling Ratio (BLR), where the higher the ratio the more unstable the structure, there were significant differences in superoanterior, 30%(Abv50: 15.8 ± 0.52; Und50: 12.1 ± 0.59;p=0.0001); anterior, 20%(Abv50: 10.1 ± 0.32; Und50: 8.3 ± 0.4; p=0.001); inferior, 35%(Abv50: 4.37 ± 0.14; Und50: 5.8 ± 0.34; p=0.0001); inferoposterior 18%(Abv50 8.6 ± 0.27: Und50: 7.36 ± 0.41; p=0.008); posterior, 29%(Abv50: 14.0 ± 0.33; Und50: 10.8 ± 0.5; p=0.0001) and superoposterior, 14%(Abv50: 14.6 ± 0.3; Und50: 12.8 ± 0.4; p=0.001), regions. There was no significant difference in bending resistance nor in the proportion of cortical bone.

Conclusions: A more uniform cortical thickness, seen in the young, would optimise fracture resistance to overloading from unusually loaded directions. Ageing was associated with a thickening of the inferior cortex and thinning of the cortex elsewhere. This effects the location of the area that is least susceptible to the loading forces experienced in stance – that is of the FN mid cross-section’s neutral bending axis – as it will be nearer to the inferior region. Such a change in the morphology will produce deterioration in the FN’s capacity to take a load as shown by the detrimental change in the LBR. This change may indicate that the potential for femoral neck fracture increases with age when load is applied in a direction different to normal stance eg through the greater trochanter.

J.P. Clements S. Gheduzzi J.C.J. Webb H. Schmotzer I.D. Learmonth A.W. Miles

Introduction: Immediate postoperative stability of cementless hip stems is one of the key factors for the long-term success of total hip replacement. The ability to discriminate between stable and unstable stems in the laboratory constitutes a desirable tool for the industry, as it would allow the identification of unsuitable stem designs prior to clinical trials. The use of composite femora for stability investigations is wide spread [1,2] even though their use in this application is yet to be validated. This study is aimed at establishing whether Sawbones composite femora are suitable for the assessment of migration and micromotion of a cementless hip stem. The stability of two SL Plus stems (Precision Implants, CH) implanted into Sawbone was compared to that of two SL Plus stems implanted into cadaveric femora. Ethical approval was obtained for the harvest and use of cadaveric material.

Methods: Stability was assessed in terms of micromotion and migration. Micromotion was defined as the recoverable movement of the implant relative to the bone under cyclic loading. Migration was defined as the non-recoverable movement of the implant with respect to the surrounding bone. Movement of the implant with respect to the surrounding bone was monitored at two locations on the lateral side of the stem by means of two custom made transducers based on the concept described by Berzins et al [3]. Each femur was tested in two different sinusoidal loading configurations: single leg stance (SLS-11° of adduction and 7° of flexion) [4] loaded up to 400N and stair climbing (SC-11° of adduction and 32° of flexion) loaded up to 300N. The effect of the abductor muscles was included in the model [5]. Each test consisted of 200 loading cycles applied at 50 Hz. The captured data was post-processed by a MATLAB routine and converted into translations and rotations of the stem with respect to the bone.

Results: The proximal part of the implant was subject to the highest amplitudes of micromotion in both loading configurations independent of the host. During SLS the largest micromotion was measured in the direction of the axis of the femur, this amplitude was in the order of 20 μm for the stems implanted in sawbones and varied between 13 and 39 μm for the stems implanted in cadaveric femora. The migration of the implants was minimal both in SLS and SC for both hosts with values measured in the sawbones model nearly on order of magnitude smaller than the cadaveric. In the case of SLS the prevalent movement consisted of a translation along the axis of the bone, while during SC the rotations became prevalent.

Discussion: This study has demonstrated that Sawbones provide an effective model to establish micromotion with oscillation patterns and orders of magnitiude similar to cadaveric bone. However the migration is much more dependent on the quality of fit and the internal geometry of the femur and therefore more caution should be placed on interpreting migration data from Sawbones models.

J.H. Kuiper K. Prathapkumar A. New J. Richardson

Introduction: Many designs exist for the femoral component of cemented total hip arthoplasty, but cemented acetabular cups are largely similar. All are essentially hemispheres, made of polyethylene. An important factor determining survival time of cemented implants is cement penetration into the surrounding bone. To ensure sufficient penetration, many surgeons remove the smooth subchondral bone in the acetabulum and drill anchoring holes. This may however weaken the bone. Larger cement pressure during setting will improve penetration. For an acetabular cup, fixation at the rim is most important to prevent loosening, and therefore cement pressure should be high at the rim. A spherical geometry is not ideal to ensure high rim cement pressures. Based on a computer model of cement pressure generation during cup insertion, we designed an improved geometry to ensure higher rim pressures. The aim of this study is to compare the fixation strength of this new design to a conventional design. The effect of the design change will be compared with that of drilling anchoring holes and removing subchondral bone.

Methods: From a larger stock of young bovine acetabula, 14 similarly sized specimens were chosen. Twelve were prepared for a factorial experiment with three factors, based on three cup designs (Ogee either with or without flange, DePuy, Leeds, and the alternative design), preservation or removal of subchondral bone, and presence or absence of anchoring holes. Depth, diameter and position of the anchoring holes were chosen to optimise fixation strength. Two were prepared for replicates of two experiments with the new design, both with sub-chondral bone removed. The order of the experiments was randomised. CMW-3 cement (CMW-DePuy, UK) was hand-mixed for one minute. After four minutes, it was packed in the acetabulum and pressurised for one minute. Then a cup was inserted and manual force applied until setting of the cement. Next, acetabulum and cup were mounted in a materials testing machine and torque applied to the cup until gross failure. Applied force and displacement were sampled into a computer, and used to determine maximum torque.

Results and Discussion: Analysis was done in two steps. First, two-way ANOVA of main effects plus first order interactions was performed. Anchoring holes significantly increased strength (41±8 vs. 114±9 Nm; p=0.004, mean±SEM). No significant effect of reaming or cup design was found. For all experiments, the conventional cups with or without flange behaved almost identical. In step two, these two variations were combined into one “conventional” group, and three-way ANOVA with interactions was performed. Significant interaction between all three factors was found (p=0.02). This indicates that one unique combination (new cup design in acetabula with subchondral bone removed and without anchoring holes) achieved a high average strength. Under these circumstances, the fixation strength of the new design (114±9 Nm) was equal to the overall average achieved with anchoring holes. On average, the new design also had significantly larger fixation strength than a conventional spherical design (95±5 vs. 69±4 Nm; p=0.009). These results justify further studies.

F. Liu Z.M. Jin C. Rieker F. Hirt P. Roberts P. Grigoris

Introduction: Laboratory simulator and clinical retrieval studies of metal-on-metal (MOM) total hip replacements have shown that the metallic alloy, the femoral head radius, the clearance between the acetabular cup and femoral head and the cup thickness can influence the contact mechanics, the lubrication and the wear of the articulation. MOM hip resurfacing procedures have received significant attention recently. The purpose of the present study was to compare the contact mechanics between a MOM hip resurfacing implant and a MOM total hip replacement under identical conditions.

Materials and Methods: A 50mm diameter DUROMTM MOM hip resurfacing prosthesis and a 28mm diameter MetasulTM MOM bearing system (Centerpulse Orthopedics, a Zimmer Company, Winterthur, Switzerland) were investigated. All implants were manufactured from wrought-forged high carbon cobalt chromium alloy (Pro-tasul 21WFTM). The diameters of the DUROMTM femoral head and acetabular cup were 50mm and 50.145mm respectively, and the corresponding wall thickness of the acetabular component was around 4mm. The diameters of the MetasulTM femoral head and acetabular cup were 28mm and 28.12mm. Three-dimensional finite element models were created to simulate the contact between the bearing surfaces of both the femoral head and the acetabular cup fixed to a three dimensional anatomically positioned pelvic and femoral bone consisting of both cortical (with 1mm thickness) and cancellous regions. The load applied to both models was 3200N.

Results: The maximum contact pressure at the bearing surfaces was found to be around 22MPa for the DUROMTM and the contact area between the femoral and acetabular components was predicted to be 237mm2. For the MetasulTM bearing under identical conditions, the maximum contact pressure and the contact area predicted were approximately 47MPa and 74mm2 respectively.

Discussion: A large reduction in the contact pressure, which should improve overall tribological performances, was noted for the DUROMTM hip resurfacing prosthesis, as compared with the MetasulTM bearing. The main reasons for this reduction were the large diameter of the articulation and the small acetabular cup thickness of the DUROMTM system. In contrast, the MetasulTM bearing has a smaller head diameter, and relies on a polyethylene backing underneath the metallic cup inlay to reduce the contact pressure at the articulating surfaces.

C.E. Evans S. Mylchreest A.P. Mee J.L. Berry J.G. Andrew

Aseptic loosening is a growing problem for orthopaedic surgeons and the importance of elevated hydrostatic pressure in its development in vivo is now well documented, but the mechanisms by which pressure could enhance loosening are unclear. We have demonstrated that hydrostatic pressures increased MP synthesis of cytokines, chemokines, PGE2 and M-CSF in vitro, all of which are implicated in bone resorption. 1,25-dihydroxy vitamin D3 (1,25D3) has a pivotal role in bone resorption. It stimulates osteoclastic bone resorption and formation, causes fusion of committed osteoclast precursor cells and activates mature osteoclasts in vitro. Under the correct conditions, macrophages (MP) have the ability to differentiate into osteoclasts. Research has shown that MP can synthesise 1,25D3 and changes in this synthesis occur during MP differentiation. We therefore examined how the application of hydrostatic pressure to MP in vitro influenced their synthesis of 1,25D3. In this study, normal human peripheral blood MP (5x105/ml) were cultured for 7 days then exposed to physiological pressure (34.5x10-3MPa) and/or UHMWPE particles (8mg/ml) and the effect on synthesis of 1,25D3 by endogenous 1a-hydroxylase (1aOHase) was studied. MP were incubated with H3-25, hydroxy vitamin D and 1,25D3 synthesis was analysed by HPLC. 1,25D3 synthesis was increased in cells under pressure by an average of 17% compared to static controls. In situ hybridisation (ISH) was used to demonstrate expression of 1aOHase. Image analysis showed a small increase in 1aOHase mRNA in response to pressure and to particles, and a larger increase to the two stimuli simultaneously. Expressed as % of maximum +Pressure + Particles 100%;+ Particles 59%; +Pressure 37%; No Stimulus < 0.1%. These results suggest that 1,25D3 may be one of the factors which stimulates osteoclastic bone resorption in aseptic loosening. As both these stimuli are likely to be present in vivo, such synthesis could further exacerbate loosening.

E. Edis R. Bayston W. Ashraf

Introduction: One of the most important mechanisms S. epidermidis uses to establish infection on biomaterials is biofilm formation, in which adhesion and the production of polysaccharide intercellular adhesin (PIA) are key factors. Non-steroidal anti-inflammatory drugs (NSAIDs) have been reported to inhibit S. epidermidis biofilm formation and may be useful in prevention or treatment of implant infections (1,2). The potential of these drugs was evaluated by determining the effects of the NSAIDs on bacterial growth, adhesion to bare and conditioned polymethyl-methacrylate (PMMA), on biofilm development, and on established biofilms.

Methods: A PIA-deficient mutant and wild type strain (gift of Prof. D. Mack, Hamburg) and 3 clinical isolates of S. epidermidis were used. The NSAIDs were salicylic acid, acetylsalicylic acid, ibuprofen and phenylbutazone. Their effects on bacterial growth rate and viability were assessed. For adhesion assay, bacteria were exposed to a 1mM concentration of each drug and allowed to adhere for 1h to bare or human plasma – conditioned PMMA before being sonicated and quantified by chemiluminescence and culture. For biofilm assays, bacteria were grown on silicone discs in the presence of various drug concentrations for 4 days before being sonicated and quantified as above. Mature (4 day) biofilms were also exposed to the drugs for a further 4 days and quantified similarly, to assess the effect on established biofilms.

Results: All NSAIDs tested significantly (P< 0.05) reduced the growth rate and viability of each strain, in a concentration – dependent manner. Reduction of adhesion was observed on bare PMMA suggesting interference with either vitronectin – binding protein or charge / hydrophobic interactions. This was independent of the effect on growth. However, adhesion to plasma – conditioned PMMA, presumably mediated by MSCRAMMs, was not significantly affected. Reduction of biofilm formation was observed for all strains and was concentration – dependent, suggesting that inhibition of PIA synthesis was not responsible. There was a significant effect on established biofilms, this was also concentration dependent.


All four NSAIDs reduced S. epidermidis growth rate and viability, but at concentrations above those achievable therapeutically.

The effect on adherence was confined to unconditioned PMMA.

The effect on biofilm formation and on established biofilms appeared to be related to that on growth and viability.

On these grounds, NSAIDs appear to have a limited prospect for use in prevention or treatment of S. epidermidis biomaterial-related infection. However, catheter coating, NSAID-antibiotic combinations, and potential for other types of infection may have greater prospects.

K.C. Wong J. Tong

Interfacial stress distributions in the acetabular region have been studied using plane strain finite element models before and after total hip replacement. The model was adapted from a roentgenogram of a 4 mm slice normal to the acetabulum through the pubic and ilium. The model was divided into 24 regions of different elastic constants with isotropic material properties assumed in each region. The femoral head was modelled as a spherical surface that was mated with a congruent spherical acetabular socket. The implanted hip model was developed by modifying the natural hip model. Contact analyses were carried out between the articulating cartilage layers and between a cobalt chromium head and a cemented ultra-high molecular weight polyethylene (UHMWPE) cup under selected hip contact load cases during normal walking. Local polar coordinates were employed to facilitate the calculation of the interfacial stress components between the cup and cement, cement and subchondral bone as well as between the subchondral and underlying cancellous bones.

The results show that severe reductions in the local stresses in subchondral and cancellous bones were found in the reconstructed case. Both the peak stress and the range of the stress were reduced substantially, suggesting stress shielding in the acetabular region. Load transfer in the reconstructed case was found to occur primarily in the cement layer superior to the cup. Both the peak stress and the stress variation in the cement mantle are substantial, whilst abrupt changes in interfacial stresses occurred between the cement and cup, and cement and subchondral bone. The influence of subchondral bone retention and thickness of the cement (up to 6 mm) on the interfacial stress distribution appears to be insignificant.

The work represents the first stage of research towards developing a numerical tool for pre/post operative assessment of cement/cementless acetabular components.

R. McCann G. Colleary C. Geddis G. Dickson D. Marsh

Introduction: During the development of an externally fixated femoral fracture model in the rat a single dose of Carpofen (Rimadyl) was administered as part of the pre-operative analgesia regime. The negative effect of a NSAID on fracture repair has been well documented.

Materials and Methods: The external fixator was designed and constructed from threaded stainless steel pins and a semi-cylindrical aluminum plate. The pins were passed through the four drill holes made in the plate and were secured by nuts above and below the plate. Forty-five female Sprague-Dawley rats, aged between twelve and eighteen weeks, were used in the model. Twenty-one animals received a single subcutaneous dose of Carpofen (4mg/kg) pre-operatively. Carpofen was then excluded from the pre-operative analgesia regime and the experiment was repeated. All animals received a dose of Buprenorphine hydrochloride (Temgesic, 0.03mg/kg) and a fluid bolus (40–80ml/ kg) both pre and post operatively and antibiotic pre-operatively. Femoral fractures were created after the animals had been anaesthetised. The right femur was then exposed and a mid femoral osteotomy was made prior to the application of the fixator. Post-operative digital x-rays were taken to confirm reduction. A minimum of four animals were assigned to a group for either biomechanical strength testing or histology. Thirty-one animals in total were sacrificed at 4, 6 or 8 weeks for biomechanical strength testing. The fractured limbs were freshly dissected and stored in saline prior to testing. Both the fractured and contralateral limbs were tested mechanically by four point bending. The maximum load to failure was recorded and stiffness was calculated from the load displacement curve obtained. The bending strength of each fractured femur was expressed as a percentage of the strength of the intact contralateral femur. Fourteen fractured limbs were fixed in formaldehyde, decalcified and paraffin embedded for histological analysis. Serial sections were cut and stained with haematoxylin, eosin and Alcin blue at 4, 6 or 8 weeks.

Results: Satisfactory reduction of the fracture was confirmed post-operatively by faxtitron x-ray imaging in all animals. Preliminary data showed that there was a significant difference in stiffness at 8 weeks between the two groups (p= 0.008). Although not a significant difference, stiffness and load to failure were lower in the NSAID group at each of the three time points.

Conclusion: This data suggests that a single pre-operative dose of a NSAID is sufficient to delay fracture repair. The clinical relevance of this finding is that frequently in acute fracture patients a single dose of NSAID is given peri-operatively as it is felt that this will have no effect on fracture repair. This practice may need to be reviewed. On qualitative histology endosteal and periosteal bridging was evident in the group that did not receive NSAID at 1 and 2 weeks. Healing within the NSAID group at 4 weeks was poor.

A. Glaviano C. Mothersill J. Campisi M.A. Rubio V. Navak A. Sood J.S. Clerkin C.P. Case

Joint replacement failure is usually caused by the formation of wear debris resulting in aseptic loosening. Particulate metal and soluble metal ions from orthopaedic alloys (cobalt chromium or vanadium titanium aluminium) that are used in medical prostheses can accumulate in tissues and blood leading to increased chromosome aberrations in bone marrow and peripheral blood lymphocytes. This paper demonstrates that two of the metals used in orthopaedic prostheses, chromium and vanadium can produce delayed as well as immediate effects on the chromosomes of human fibroblasts in vitro. Fibroblasts were exposed to metal ions for only 24 hours and were then expanded over 30 population doublings involving ten passages. The initial increase of chromosomal aberrations, micronuclei formation and cell loss due to lethal mutations persisted over multiple population doublings, thereby demonstrating genomic instability. Differences were seen in the reactions of normal human fibroblasts and those infected with a retrovirus carrying the cDNA encoding hTERT that rendered the normal human fibroblasts telomerase-positive and replicatively immortal. This suggests that chromosomal instability caused by metal ions is influenced by telomere length or telomerase activity. Formerly this syndrome of genomic instability has been demonstrated in two forms following irradiation. One type is non-clonal and involves the appearance of lethal aberrations that cannot have been carried by the surviving cells. The other type is clonal and the aberrations are not lethal. These may arise as a result of complex rearrangements occurring at a high rate post-insult in surviving cells. The consequences of genomic instability are not yet known but it is possible that the increase of chromosomal aberrations that have been previously observed in human patients could be due to immediate and delayed expression of cellular damage after exposure to orthopaedic metals.

Z.R. Yin I. Papageorgiou J.S. Clerkin I.D. Learmonth C.P. Case

Wear debris from worn cobalt chrome joint replacements causes an increase in chromosomal translocations and aneuploidy. In this study the relationship between the amount of DNA damage and the changes in gene expression was investigated in human fibroblasts after exposure to artificial cobalt chrome particles. The comparison was made with different doses of particles, at different time intervals and in fibroblasts of different ages, those that had completed 10 population doublings (10 PD fibroblasts) and those that had completed 35 population doublings (35 PD fibroblasts). The genes (TGF-©¬2, p38 MAPK, Integrin ¥â1, SOD1, Caspase 10, PURA, FRA-1 and VNR) were chosen after a previous screen with cDNA microarrays. The percentage of senescent cells was evaluated using an immunohistochemical assay for ¥â-galactosidase activity. The 35 PD fibroblasts showed significantly more ¥â-galactosidase activity than the 10 PD fibroblasts. The level of DNA damage, as detected with the alkaline comet assay, was greater at higher doses, at longer exposures (up to 24 hours) and in 10 PD fibroblasts. The expression of all the genes listed above was generally lower after exposure to cobalt chrome particles using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The reduction in gene expression, like the increase in DNA damage was greater at higher doses and at longer exposure times. After 24hr exposure the reduction in gene expression was greater in 10 PD fibroblasts compared to 35 PD fibroblasts. After 6hr exposure this was only true at higher doses of particles and the opposite was seen after a lower dose of particles. These results show that levels of gene expression of TGF-©¬2, p38 MAPK, Integrin ¥â1, SOD1, Caspase10, PURA, FRA-1 and VNR may be correlated with the level of DNA damage and that this depends on the dose and length of exposure and the age of the cells. This highlights the potential importance of these genes in the mutagenicity of cobalt chrome particles in human fibroblasts.

G. Colleary R. McCann C. Geddis G. Li G. Dickson D. Marsh

Introduction: The aim of this research project was to establish a simple, reliable and repeatable externally fixed femoral fracture model. The rat was selected, as it was a suitable animal for use in a model of fracture repair and ovariectomy induced osteoporosis, both of which were to be investigated in future experiments. There are femoral fracture models described in the literature based on the insertion of an intramedullary nail prior to inducing a fracture. We felt, based on our experience of the unilateral externally fixed mouse fracture model, that external fixation would allow us to carry out radiographical and histological analysis of fracture healing without any of the tissue trauma caused by the insertion and removal of the intramedullary device.

Materials and Methods: A unilateral external fixator was chosen due to its simplicity. Four threaded stainless steel pins pass through holes in an aluminium plate with nuts placed on the pin above and below the plate. The holes in the plate were 0.1mm bigger than the pins and unthreaded allowing the plate to slide freely over the pins. Tightening of the upper nut compressed the plate against the lower nut holding the pin securely. 41 female Sprague-Dawley rats, aged between 12 and 18 weeks, were used. They were anaesthetised using a standard mixture of hypnorm and midazolam and analgesia, fluids and antibiotic were administered subcutaneously prior to surgery. The femur was exposed through a lateral approach and a standardised osteotomy was made prior to the application of the fixator plate. Accurate reduction was confirmed visually at the time of surgery and also by way of a post-op x-ray. 25 animals were sacrificed at 4 days and 1, 2, 4, 6 and 8 weeks for histology. The fractured limbs were harvested, fixed, decalcified and paraffin embedded as per standard protocol and serial sections were cut. These were stained with H& E and alcian blue and analysed 15 animals were sacrificed at 4,6 or 8 weeks for biomechanical strength testing. Four-point bending was carried out on freshly harvested femurs stored in normal saline between harvest and testing. Both limbs were tested and the fractured limbs were standardised relative to the unfractured limb. Maximum load to failure was recorded and stiffness was calculated from the load-displacement curve.

Results: No post-operative complications of fixation failure or infection occured. On histological assessment at D4 a predominantly lymphocytic inflammatory response was seen within the fracture haematoma. This inflammatory response was replaced with endosteal and periosteal new bone between wks 1 and 2. Bridging of the fracture gap was seen at week 6. Both stiffness and load to failure increased with increasing time. There was a statistically significant improvement in the percentage stiffness and percentage load to failure between 4 and 8 weeks (p=0.03 and p=0.018 respectively). The difference in load to failure between 6 and 8 weeks was also significantly different (p=0.042).

Discussion: A simple, reliable and repeatable externally fixed rat femoral fracture model has been established.

E. Fawzy G. Mandellos S.M. Isaac H. Pandit R. Gundle R. De Steiger D. Murray P. McLardy-Smith

Aim: To investigate the functional and radiological outcome of shelf acetabuloplasty in adults with significantly symptomatic acetabular dysplasia, with a minimum of a 5 year follow-up.

Material and Methods: 77 consecutive shelf procedures (68 patients) with an average follow-up of 10.9 years (range: 6–14) were reviewed. The Oxford hip score (OHS) was used for clinical assessment. Centre-edge angle (CEA) and acetabular angle (AA) were measured as indicators of joint containment. The severity of osteoarthiritis was based primarily on the extent of joint space narrowing. Survivorship analyses using conversion to THR as an endpoint were performed. Logrank tests were used to compare the survivorship of the shelf procedure against the variables of age, preoperative osteoarthiritis, pre and postoperative AA, CEA angles.

Results: The average age at time of surgery was 33 years (range: 17–60). At the time of the last follow-up, the mean OHS was 34.6 (maximum score: 48). Mean postoperative CEA was 55 (Pre-operatively: 13 degrees) while mean postoperative AA was 31 (Pre-operatively: 48 degrees). Thirty percent of hips needed THR at an average duration of 7.3 years. The survival in the 45 patients with only slight or no joint space narrowing was 97% (CI, 93%–100%) at 5 years and 80% (CI, 56%–100%) at 10 years. This was significantly higher (p= 0.0007) than the survival in the 32 patients with moderate or severe osteoarthiritis, which was 72% (CI, 55%–89%) at 5 years and 29% (CI, 13%–45%) at 10 years. There was no significant relationship between survival and age, pre and postoperative AA, CEA angles (p> 0.05).

Conclusion: Shelf-acetabuloplasty offers symptomatic relief to adults with acetabular dysplasia but overall deteriorates with time. About 50% of the patients do not need THR for over 10 years. Best results with shelf-acetabuloplasty were achieved in patients with slight or no joint narrowing.

T. Faram S. Eissa R.K. Smith A.E. Goodship H.L. Birch

Introduction: Energy storing tendons, such as the human Achilles tendon, suffer a much higher incidence of rupture than non- energy storing positional tendons, such as the anterior tibialis tendon. Similarly, in the horse partial rupture of the energy storing superficial digital flexor tendon (SDFT) and suspensory ligament (SL) occurs much more frequently than to the deep digital flexor tendon (DDFT) and common digital extensor tendon (CDET) which are not involved in energy storage. In order to function effectively, energy storing tendons experience strains during high speed locomotion which are much closer to failure strain than non-energy storing tendons. Therefore, these tendons are likely to sustain high levels of microdamage, hence cell metabolism may also be higher in order to repair damage and maintain matrix integrity. Maintenance of the matrix requires not only synthesis of new matrix components but also degradation of matrix macromolecules which is achieved, in part, by a family of matrix metalloproteinase enzymes (MMPs). In this study we test the hypothesis that the energy storing equine SDFT and SL which are prone to degenerative changes have higher levels of MMP2 and MMP9 than the positional DDFT and CDET that are rarely injured.

Methods: Tendons (SDFT, DDFT, SL, CDET) were harvested from the distal part of the forelimbs of 18 month old Thoroughbred horses (n = 12). Tissue from the mid-metacarpal region of each tendon was snap frozen, lyophilised, powdered and MMPs extracted. Gelatin zymography was used to determine levels of the pro and active forms of the gelatinase enzymes, MMP2 and MMP 9. Proteolytic activity (units per mg dry weight tissue) was quantified based on densitometry measurements and standardised between gels using an equine neutrophil MMP extract. Statistical significance was evaluated using a general linear model (SPSS software).

Results: The main activity observed in all tendon samples was that of proMMP2. Quantification showed that the energy storing SDFT (23.4 ± 10.95) and SL (18.9 ± 5.3) had significantly higher levels than the non-energy storing DDFT (2.90 ± 0.99) and CDET (4.06 ± 2.06). Active MMP2 levels were lower than the pro form and were not sufficient to quantify. However, there appeared to be more in the energy storing structures compared with the non energy storing structures. MMP9 activity was detected in some samples. A higher number of the CDET extracts contained MMP9 activity compared to extracts from the other structures.

Discussion: The results of this study show higher levels of MMPs in energy storing structures than in non-energy storing structures. This suggests that there may be an increased demand for repair of micro-damage in these tendons and hence an increased capacity for matrix degradation. Previous studies on energy storing structures in the horse have shown that they do not undergo adaptive hypertrophy or a change in structural architecture in response to mechanical demand, unlike non-energy storing structures. The results of this study indicate that this lack of adaptation in energy storing structures is not due to a general deficiency in cell activity but may be a means of preventing increase in tendon stiffness and a subsequent decrease in efficiency. In order to maintain tendon integrity MMP activity must be matched by mechanisms to inhibit activity and/or to synthesize new matrix components. Degeneration may therefore occur when there is an imbalance between these processes.

H.I. Roach S. Inglis K. Partridge R.O.C. Oreffo N.M.P. Clarke

Clonal chondrocytes of osteoarthritic (OA) cartilage express an aberrant set of genes. We hypothesize that this aberrant gene expression may be due to clonally inherited epigenetic changes, defined as altered gene expression without changes in genetic sequence. The major epigenetic changes are due to altered DNA methylations in crucial parts of the promoter region. If the cytosines of CpG dinucleotides are methylated, the gene will be silenced, even if the right transcription factors are present. Similarly, de-methylations may activate previously silenced genes. Our aims were to provide ‘proof-of-concept’ data by examining the methylation status of genes in OA vs non-OA chondrocytes. Articular cartilage was obtained a) from the cartilage of fracture-neck-of-femur (#NOF) patients and b) from or around the eroded regions of OA samples. The former was full thickness cartilage, the latter was partially degraded cartilage, which contained mostly clonal chondrocytes as confirmed by histology. The cartilage samples were ground in a freezer mill (Glen Creston, UK) and DNA was extracted with a Qiagen DNeasy maxi kit. To assess DNA methylation status, the genomic DNA was treated overnight with methylation-sensitive restriction enzymes. Cleavage of selected sites was detected by PCR amplifications with primer pairs designed to bracket selected promoter regions. Loss of the PCR band after digestion with the enzymes indicated absence of methylations, whereas presence of the band indicated methylated cytosine. We selected MMP-9 as one of genes that is activated in OA. Transcription of mmp-9 is regulated by a 670 bp sequence at the 5′-end flanking region, which contains 6 CpGs and a further 21 CpGs within the 1.5 kb region further upstream. A PCR primer pair was designed to bracket a 350bp sequence upstream from the transcription start site of mmp-9, which contained four of the six potential methylation sites, cleaved by the methylation-sensitive enzymes AciI and HhaI. DNA from 9 OA patients, 5 #NOF patients and 1 rheumatoid arthritic (RA) patient were digested with HhaI or AciI and examined for the presence or absence of PCR bands. In all patients, digestion with HhaI abolished the PCR band, indicating that the HhaI site was never methylated in either #NOF or OA patients. However, a remarkable difference was found after digestion with AciI: in 8/9 OA patients, the PCR band was no longer detectable, while in 4/5 #NOF patients the PCR band was still present. This suggested that all three AciI cleavage sites were methylated in the majority of chondrocytes from #NOF patients, while at least one of the three AciI cleavage sites was unmethylated in OA patients. Interestingly, the PCR band was present in the RA patient, suggesting methylation of the AciI cleavage sites. The present study provides the first ‘proof-of-concept’ data that suggest epigenetic changes may play a role in the etiology of osteoarthritis. Clearly further work is required to establish the generality of the present findings and whether de-methylations are also found in the promoter regions of other genes that are aberrantly expressed in OA.

J.E. Arbuthnot G. Stables J. Hatcher M.J. McNicholas

Introduction: Instrumented arthrometry is a widely used technique for the quantification of cruciate ligament laxity. It is used both before and after surgery. The Rolimeter(Aircast, Europe) is used in such scenarios. It has several advantages over its cousins; it is more compact, lighter, less expensive and amenable to sterilization techniques. The other leading arthrometers have however had over 15 years of clinical use and their reliability has been thoroughly assessed. Muellner et al found no significant difference in the intra-tester and inter-tester results obtained on Rolimeter assessment of the knees of un-injured healthy subjects. Our study assessed the inter-tester and intra-tester variability when the Rolimeter is applied to patients with unilateral ACL-deficient knees. It also examines whether the level of experience of the examiner influences the results in this group of patients.

Materials and Methods: Six examiners each examined thirty-three subjects on two occasions. One examiner was medically qualified but had never performed a Lachman or anterior drawer test. Two examiners were qualified physiotherapists who routinely examined knees, but had never used a Rolimeter. One medically qualified examiner was considered to be of intermediate experience.Two examiners were regarded as expert Rolimeter users.For each examination a Rolimeter reading was taken three times with the knee at 30 degrees of flexion and three times at 90 degrees of flexion for both knees.The interval between examinations was at least thirty minutes. All the readings were acquired on the same day. The examiners were blinded to whether the subject was known to be ACL deficient or not. The results of the examinations were entered onto a data-base.Repeated measures analysis of variance was used to test for the effects of the following factors, difference between examiners, reproduction of results between examinations.

Results: There was no significant difference between each set of measures for each subject between examinations (p=0.767), indicating that the measurement procedure was reliable. Measurements were significantly higher in patients with ACL-deficient knees compared to the control group (p< 0.001) confirming the sensitivity of the Rolimeter to help diagnose ACL-deficient knees. The in-experienced examiner’s measurements were lowest and were more reliable. The examiner with the intermediate experience was the most un-reliable. Both experienced examiners were in close agreement.

Conclusion: We have demonstrated that the rolimeter is reliable in the assesment of ACL deficient patients regardless of the experience of the examiner.

P. Heaton-Adegbile J.G. Hussell J. Tong

Background: It is thought that the forces transmitted across the hip joint produce migration of the prosthesis by failure at either the bone-cement or the prosthesis-cement interface. As symptoms associated with such motions often result from failure at the cement-bone interface, it is this interface and its sub-surfaces that are the critical areas of prosthesis loosening. Our aim is to produce a new and more accurate method of measuring strains at this critical interface.

Objective: To develop in-vitro experiments to measure the strain distributions near the bone-cement interface of the acetabulum region under physiological, quasi-static loading conditions.

Experimental Model: Two hemi-pelvic specimens of saw bones were used. Following careful placement of six protected precision strain gauges (4.6 x 6.4mm, tri-axial EA-13-031RB-120/E). One specimen was prepared to receive a cemented polyethylene cup (Depuy Charnley Ogee LPW 53/22). An uncemented 58mm Duraloc cup was implanted into a second specimen.

Methods: Hip joint force relative to the cup during normal walking (Bergmann, G., 2001. HIP98) was used for quasi-static tests on a Llody LR30K loading machine. The magnitude of the maximum and minimum principal strains, and the orientation of the maximum principal strains were calculated from a 32 channel digital acquisition system.

Results: For both specimens, the maximum principal strains at the maximum loading were highest in the medial wall (dome area) of the acetabulum. The tensile strain from the dome of the uncemented specimen at the maximum loading was twice that of the cemented specimen. In the cemented specimen, the compressive strains in the medial wall were almost twice the tensile strains at the maximum load. Within the acetabular quadrants, the highest strains were recorded in the posterio-inferior quadrant. Compressive strains in the posterio-inferior wall of the acetabulum seem to be comparable to those in the anterior-superior wall.

Conclusion: The critical areas for load transfer in the acetabulum are the medial wall (dome area), the posterio-inferior and the anterior-superior quadrants. The uncemented cup appears to provide a better load transfer mechanism than the cemented cup.

A. Sood R.A. Brooks R. Field E. Jones N. Rushton

Introduction: The Cambridge Acetabular cup is a unique, uncemented prosthesis that has been designed to transmit load to the supporting bone using a flexible material, carbon fibre reinforced polybutyleneterephthalate (CFRPBT). This should significantly reduce bone loss and provide long term stability. The cup consists of a ultra high molecular weight polyethylene liner within a carbon fibre composite backing that was tested with either a plasma sprayed HA coating or with the coating removed. The cup is a horseshoe shaped insert of similar thickness to the cartilage layer and transmits force only to the regions of the acetabulum originally covered with cartilage. The purpose of this study was to evaluate the response of bone and surrounding tissues to the presence of the cup in retrieved human specimens.

Methods: We examined 12 cementless Cambridge acetabular implants that were retrieved at autopsy between 2 and 84 months following surgery. Nine of the implants were coated with HA and three were uncoated. The implant and the surrounding bone were fixed, dehydrated and embedded in polymethylmethacrylate. Sections were cut parallel to the opening of the cup and in two different planes diagonally through the cup. The sections were surface stained with toluidine blue and examined by light microscopy. Image analysis was used to measure the percentage of bone apposition to the implant, the area of bone and fibrous tissue around the implant and the thickness of hydroxyapatite coating.

Results: All 9 HA coated implants showed good bone contact with a mean bone apposition and standard deviation of 50.9% +/− 17.5%. The thickness of the HA coating decreased with time and where this was occurring bone remodelling was seen adjacent to the HA surface. However, even in specimens where the HA coating had been removed completely good bone apposition to the CFRPBT remained. Bone marrow was seen apposed to the implant surface where HA and bone had both been resorbed with little or no fibrous tissue. The uncoated implants showed significantly less bone apposition than the HA coated specimens, mean 11.4% +/− 9.9%(p < 0.01) and significant amounts of fibrous tissue at the interface.

Discussion: The results of this study indicate that the anatomic design of the Cambridge Cup with a flexible CFRPBT backing and HA coating encourages good bone apposition. In the absence of HA the results are generally poor with less bone apposition and often a fibrous membrane at the implant surface. There was a decrease in HA thickness with time in situ and cell mediated bone remodelling seems to be the most likely explanation of the HA loss. However, good bone apposition was seen to the CFRPBT surface even after complete HA resorption in contrast to the uncoated specimens. Though the mean bone apposition percentage to the HA coated implants declined with time, the bone was replaced by marrow apposed to the implant surface. This is in contrast to the uncoated implants where fibrous tissue becomes apposed to the implant surface. We believe this is due to micro-motion occurring at the bone implant interface. The HA coating appears necessary to provide good initial bone bonding to the implant surface that is maintained even after complete loss of HA.

K.L. Chelule B.B. Seedhom M.A. Hafez K.P. Sherman

Aim: To develop a 3-D pre-surgical planner that facilitates selection and placement of correct prosthetic components in the joint, and the design of patient specific templates to use intra-operatively to reproduce the pre-planned implantation procedure, in total knee replacement (TKR) surgery.

Design/Methods: The process begun with loading of pre-operative CT scan data of cadaver knee, onto medical software, followed by reconstructions of 3D models of the joint. Then measurements of anterior-posterior diameter of the femoral condyles of the 3D models of the joint were used to select and import a correct CAD drawing of prostheses from a database of electronic files available in a range of sizes. The selected prosthetic components were positioned and aligned on the 3-d model of the joint, making sure that the anterior flange of the femoral prosthesis component did not violate superior cortical bone of trochlea. Whilst the tibial stem was placed central within the medullar space of the bone, and the plane of the tibial cut was perpendicular to the long axis of the tibia. The planned data were next exported to a CAD environment where template to prepare the bone to receive the prostheses, was designed. A template was designed to press fit on a bone (e.g. femur), via minimum number of cylindrical protrusions with their ends made to conform to the geometry of that bone at the regions of contact. The integrated surgical tools were secured to the bones with pins through each of the protrusions, and were equipped with saw guide slits for cutting the bone, and with drill guides for drilling the fixation holes. Thereafter the files describing templates and prosthetic components selected for cadaveric joint concerned were sent to rapid prototyping machine for manufacturing.

Results: Fourteen procedures were performed on cadaveric knees to date. Visual examination of the joint has revealed the 3-D planning system enabled correct selection of appropriate prosthetic components and alignment, as evidenced by absence of protrusions or overhanging beyond the edges of the bones. The resected bone surfaces were visually smooth and flat. Gaps between the bones and the internal surfaces of the prosthetic components were measured using steel shim gauges, and largest recorded was 0.9mm. Laxity between the femur and tibia was absent and the joint attained full range of flexion. Dimensional deviations of post-operative scans of the prepared bones from the pre-planned ones were between 0.5 and 0.9mm. The templates after their use were shown capable to withstand the rigors of theatre environment.

Conclusion: With the planning software, it has been shown that it is possible to design a simple to use implantation guidance system according to the final position of the restorative prosthesis and the bone pathological condition. Pre-operative planner system relieves the clinician from multiple intra-operative decisions. The system is ideal for critical anatomical situations and eliminates possible manual placement errors such as those from extra and intra-medullary alignment tool. Less inventory required of both implants and instrumentation means reduced complexity of procedure, surgical time and cost.

F. Liu Z.M. Jin C. Rieker F. Hirt P. Roberts P. Grigoris

Introduction: Hip simulator and clinical retrieval studies have shown that metal-on-metal (MOM) hip implants commonly have biphasic wear. An initial high wear or running-in phase is generally followed by a low wear or steady-state phase. A number of hypotheses have been put forward to explain this biphasic phenomenon, including polishing of the metallic bearing surfaces and increasing conformity between the two articulating surfaces. The purpose of the present study was to compare the wear and lubrication of MOM hip implants between the running-in and steady-state periods.

Materials and Methods: A standard 28mm MetasulTM MOM bearing (Centerpulse Orthopedics, a Zimmer Company, Winterthur, Switzerland) was investigated. The wear testing was carried out using a 6-station AMTI hip simulator in the presence of 33% bovine serum and 67% Ringer solution (PH 7.2). The bearing surfaces of both the femoral and acetabular components were measured using a coordinate measurement machine at different stages of wear testing. The dimensional changes of the bearing surfaces due to wear were directly incorporated into the elastohydrodynamic lubrication analysis using an in-house developed code.

Results: The initial running-in period occurred during the first 1 million cycles, and little wear was observed subsequently up to 5 millions cycles. The maximum total wear depth was measured to be around 13 μm at 1 million cycles. The predicted average lubricant film thickness between the two articulating surfaces was increased from 0.024μm at the beginning, to 0.09μm at the end of the first 1 million cycles. For a given composite surface roughness of 0.03μm often quoted for the metallic bearing surfaces, such an increase in the lubricant film thickness represents a transition from a mixed to a fluid film lubrication regime.

Discussion: A large improvement in lubrication was predicted as a direct result of the running-in wear of the bearing surfaces. This was mainly due to the increased conformity between the two articulating surfaces and the decreased diametrical clearance. It was particularly noted that the improvement in lubrication after 1 million cycles was so significant that continuous fluid film lubrication was possible, leading to extremely low wear for up to 5 million cycles, and only material fatigue and start-up and stopping for wear measurements could cause a further increase in wear.

It is possible in theory to optimise the geometry of the metallic bearing surfaces, based on the worn components, to minimise the running-in wear. However, such an improvement in lubrication cannot be readily achieved because of difficulties in surgical techniques and position of the components.

B.D. Cox J.L. Conroy R.K. Wilcox M.C. Levesley R.M. Hall

Introduction: Aseptic loosening is a long-term complication of many cemented arthroplasty procedures. The integrity of the fixation interface, in particular the level of interdigitation between cement and bone, is crucial to sustaining the stability of arthroplasty components[1]. Studies have shown that the viscosity of cement at the time of application is a significant parameter in determining this level of interdigitation[2]. However, the rheological properties of cement at key stages in arthroplasty procedures have not been quantified, and it is unclear if current operative techniques achieve optimum cement delivery properties. Furthermore, because the cure process of bone cement is highly dependent on environmental conditions, it is extremely difficult to accurately predict the time to curing. Oscillatory shear rheometry can be used to characterise the viscoelastic properties of bone cement. However, most commercial rheometers used for this purpose are too large, expensive and delicate for peri-operative use. The aim of this work is to develop a new laboratory method for measuring the viscoelastic properties of bone cement at the time of application and to investigate the relationship between these properties and the level of cement interdigitation.

Methods: A simple, inexpensive electromagnetic rotary actuator has been developed to provide accurate measurements of force, displacement and velocity without the use of sensors. These parameters can be used to continually monitor both viscous and elastic properties of curing bone cement. To consider subjective cementation techniques, a method has been devised where a surgeon indicates early and late doughing stages for a PMMA bone cement within a clinical environment. A computer interface has been developed to plot the real-time properties of the cement that are measured using the self-sensing device concurrently. The range of practical variability of cement delivery properties is then established. In order to investigate the effect of cement viscosity on the level of interdigitation a rig has been developed in which cement is applied to a standardised bone analogue under controlled conditions. The open pore ceramic analogue has been shown through microCT scanning to have a structure that is representative of the trabecular structure in human bone. CMC solution is used to represent back bleeding. Once set, the sample is evaluated using microCT to measure the level of interdigitation.

Results: Preliminary results show that bone cement has largely viscous properties following mixing and largely elastic properties towards setting. Values of dynamic viscosity obtained show the cement to have a low viscosity following mixing, then as polymer beads begin to dissolve in the monomer, the viscosity rapidly increases. The rate of viscosity increase then slows as polymer chains are created, before a final rapid increase in viscosity indicating the onset of setting.

Conclusion: A validated method has been developed to measure the viscoelastic properties of curing bone cement at key stages in arthroplasty procedures and to investigate the effect of these viscoelastic properties using a simple standardised bone model.

S.M. Isaac K.L. Barker I.N.N. Danial D.J. Beard H.S. Gill C.L.M. Gibbons C.A.F. Dodd D.W. Murray

Introduction: Knee joint arthroplasty (total or unicompartmental) is the standard operative treatment for osteoarthritis (OA). Survival rate is good for both types but functional outcome is different. The function of unicompartmental knee arthroplasty (UKA) is substantially better than that of total knee arthroplasty (TKA). As function can be strongly influenced by proprioceptive ability, it is possible that improved outcome seen in patients with UKA results from retaining proprioceptive function associated with the cruciate ligaments. This prospective longitudinal study aimed to evaluate the change in proprioceptive performance after knee replacement; comparing TKA to UKA.

Methods and Materials: Two groups of patients with OA as diagnosed clinically and by X-ray were recruited. Group 1 consisted of 15 patients (mean age 65.8 years range 57–72 years, 10 females and 5 males) listed for TKA with the AGC prosthesis (Biomet, UK). Group 2 consisted of 19 patients (mean age 65.5 years range 52–75 years; 9 females and 10 males) listed for UKA with the Oxford UKA (Biomet, UK) for medial compartment OA. The ACL and PCL were present and preserved in all patients in Group 2, while only the PCL was preserved in Group 1 patients. Joint Position Sense (JPS) and postural sway were used as measures of proprioception performance. Both groups were assessed pre-and 6 months post-operatively in both limbs. JPS was measured using a dynamometer (KinCom, Chatanooga Ltd) as the error in actively and passively reproducing five randomly ordered knee flexion angles (30°, 40°, 50°, 60° and 70°). Postural sway (area, path and velocity) was measured during single leg stance using a Balance Performance Monitor (SMS Medical) for 30 seconds interval. Functional outcome was assessed using the Oxford Knee Score (OKS).

Results: Pre-operatively, no differences in JPS or sway were found between limbs in either group. No differences existed between the two groups. Post-operatively, both groups had significant improvement of JPS in the operated limb only (Mean ± standard deviation for UKA 4.64±1.44° and for TKA 5.18±1.35°). No changes in JPS were seen in the control side. An improvement in sway was found in the UKA group only. UKA patients showed significant improvement in both sway area and path (p< .0001) for both limbs post-operatively. No significant post-operative changes in sway occurred in either limb of TKA patients. The OKS improved postoperatively in both groups, rising from 21.4 to 35.5 for TKA patients and from 23.9 to 38 for UKA patients.

Conclusion: Interestingly, joint position sense improved for both groups but did not seem to show any difference between UKA and TKA. Postural sway was influenced by joint replacement type. Ligament retention may contribute to improved global postural control seen after unicompartmental knee arthroplasty and may explain the higher level of function seen in these patients.

J.P. Gittings I.G. Turner A.W. Miles

Introduction: Joint replacement procedures such as revision impaction grafting and spinal fusion interbody operations are stretching allograft bone stocks to their limits. The need for synthetic alternatives that offer a structural and biological matrix for graft incorporation are paramount for future bone regeneration procedures. Synthetic bone graft alternatives that offer biocompatibility to the host bone (i.e. a biological response) such as hydroxyapatite/tricalcium phosphate (HA/TCP), in addition to possessing an interconnected porosity network have been shown to have a strong influence on the osteoinductive potential of these materials. The current method allows the production of calcium phosphate ceramic components (CPC) that possess an interconnected open porous network in the required size range for osteoid growth and revascularisation.

Materials and Methods: The method can be described as the reticulated foam technique, whereby two grades of calcium phosphate powder are blended together to form a HA/TCP ceramic slip. The slip is then ball milled for 24hrs with zirconia milling media. This slip is used to impregnate polyurethane (PU) foam via a mechanical plunging procedure. The impregnated foam is then held above the slip bath in order for the slip to flow and coat the struts of the foam. The impregnated foam is then dried on tissue paper and treated with high velocity compressed air to avoid the formation of any closed cells. Samples are dried at 120°C for 15hrs. The PU foams are graded as 30 and 45ppi (pores per inch). The slip viscosity ranges from 6000 – 8000 cps (measured with a Brookfield Viscometer, spindle no. 5 and at 10rpm). Samples are sintered slowly until 600°C to ensure PU burnout is complete. Sintering continues up to 1280°C to ensure densification. Image analysis was performed using optical microscopy, digital photography and SEM analysis. Mechanical testing was performed by 3 point bending using an 1122 Instron.

Results: Macroporosity in the samples varied from 40 – 70%. Typical pore sizes far exceeded 300μm (the pore size acknowledged as that needed for osteogenesis). Approx. 79% of all pores were between 150 – 450μm in area equivalent diameter. Typical strut thicknesses ranging from 100 – 500μm were also reported, as was a strut thickness-pore size-mechanical strength relationship. One hundred and twenty samples possessed a breaking stress with a 95% confidence level of 0.30MPa±0.01MPa. The low strengths reported are due to the formation of blow-out holes at triple point junctions on the interconnected struts.

Conclusions: Major requirements for replacement bone materials have been met including a wide range of interconnected porosity from 50 – 1000μm. Bioactivity combined with an excellent porosity size range suggests excellent possibility of osteogenesis. In addition, this fabrication procedure offers consistency and reliability. Future work will focus on improving the strength of these open porous calcium phosphate ceramics.

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M.C. Oliver N. Finan J.A.N. Shepperd

Introduction: This paper reports leg alignment and knee surface measurements taken from 100 young adult limbs using actual physical bone measurements (APB).

Method: The specimens were part of the Spitalfields Collection of 1000 skeletons held in the Natural History Museum, London. Selection criteria included full life documentation, absence of degenerative change and perfect preservation. There were 28 females (45 knees) and 29 males (55 knees). Mean age was 40 years. The femora and tibiae were accurately articulated with the knee extended. Digital images were obtained in neutral, 15 degrees internal rotation and 15 degrees external rotation.

Results: Mean axial alignment of both sexes was valgus (female> male). Using the 2 tailed t-test valgus alignment differed significantly from neutral alignment (p< 0.001) in this population. Internal rotation exaggerated an alignment into valgus and external rotation exaggerated an alignment into varus. The knee joint inclination of both sexes was valgus with respect to the mechanical axis of the tibia (female> male). The physiological valgus angle of the knee more closely resembled the accepted value of 6 degrees in the female.

Discussion: This is the first report of APB measurements as opposed to x-ray analysis in a normal population. It is clear that considerable individual variation occurs in all parameters. Development is likely to be affected by genetic, cultural and occupational factors. Measurements are also affected by limb rotation. We believe that arthroplasty techniques may be more successful if alignment is planned to the individual.

C.E.W. Aylott K.J. McKinlay B.J.C. Freeman D.S. McNally

Introduction: Dynesys is a novel, dynamic stabilization system designed for the treatment of degenerative conditions of the lumbar spine that present with unstable motion segments. This system uses pedicle screws with a modular spacer mounted on a stabilising cord, which controls movement of the instrumented segment in all planes. The purpose of this study was to investigate changes in the biomechanic response of the intervertebral disc (IVD) under normal, flexed and extended loading conditions before and after Dynesys is applied. The IVDs of both the instrumented (bridged) and the adjacent (floating) segment were studied.

Methods: Twelve L3-5 cadaveric segments were dissected and compressed to 1kN in 6° flexion, neutral and 4° extension. The test was done without spacers and with spacers measured to +2mm, neutral and −2mm, where neutral equates to the normal distance between the pedicle screws without an applied load. The stress distribution in the mid-sagittal and posterolateral diameters of both the bridged and floating discs was measured using a miniature pressure transducer. This resulted in greater than 300 stress profiles per specimen. Disc movement and segment motion during loading were recorded using ultrasound imaging and infra-red reflection respectively.

Results: Without stabilization, stress peaks observed in the anterior annulus increased by more than 85% as the specimen was loaded from 4° extension to 6°flexion. With the application of Dynesys, these anterior stress peaks were reduced across the bridged segment. This was most pronounced in 6° flexion where anterior stress peaks of greater than 1 MPa were reduced by 100% in the bridged segment in more than 90% of specimens.

Conclusions: The degree of flexion or extension of the specimen during loading influences the peak stresses generated in the annulus. Dynesys has the potential to relieve peak stresses in the anterior annulus which is most pronounced when the specimen is loaded in flexion.

C.E.W. Aylott A. Tambe G. Taylor

Introduction: The diagnosis of Achilles tendon rupture must be made promptly and reliably to prevent avoidable morbidity. The calf squeeze test (CST) offers a simple clinical test with high sensitivity. However, in our clinical practice we have noticed a lack of clarity in the medical notes. We believe there is confusion regarding what constitutes a positive CST. Movement of the foot being positive or lack of movement of the foot indicating the test is positive. The purpose of this study was to assess the degree of error and to determine whether this is due to lack of knowledge, an inability to perform or correctly interpret and record the result of the CST. We assessed SHO’s , Registrars and Consultants in the Accident & Emergency and Orthopaedic Departments.

Method: Ninety one doctors completed a supervised questionaire. They were asked four questions 1) What tests they chose to diagnose Achilles tendon rupture. 2) How they would perform a CST. 3) What they considered a positive CST to mean . 4) How they would record the diagnosis of a ruptured Achilles tendon.

Results: 92%(84/91) of doctors overall chose to use a CST. 88%(80/91) performed the CST correctly. The CST was interpreted incorrectly by 41%(15/37) and 26%(14/54) of A + E and Orthopaedic doctors respectively. Also 32%(12/37) of A + E and 19%(10/54) of Orthopaedic department doctors mistakenly thought that the diagnosis of an Achilles tendon rupture was consistent with a negative test.

Conclusion: The results suggest that the recording of AT rupture may be inaccurate in as many as 32%(12/37) when patients present to the A + E department. This error is not a result of lack of knowledge or performance of the CST but of interpretation and recording. We would strongly discourage the recording of the CST in terms of a positive or negative result. The result should be described in words, for example ‘No movement of the foot on squeezing the calf muscle.’ We suggest that all SHOs and Registrars who may be called upon to assess patients with suspected Achilles tendon rupture are informed of this source of error.

P. Macnamara

Osteoclasts are cells that resorb bone. They derive from haemopoietic precursors in the presence of Macrophage-Colony Stimulating Factor (M-CSF) and the osteoclast growth factor, Receptor Activator of Nuclear Factor–kB Ligand (RANKL). Tumour Necrosis Factor-a (TNF-a) and M-CSF has been shown to form mature osteoclastic bone resorption in vitro murine cultures in the absence of RANKL. The aim of this study was to investigate the mechanism of action of the pro-inflammatory cytokine Tumour Necrosis Factor-a (TNF-a) with respect to osteoclastic bone resorption. Development of osteoclasts was performed using an in vitro assay of healthy human peripheral blood mononuclear culture (PBMNC) in the presence of M-CSF and RANKL. In the same cultures RANKL was replaced by TNF-a over a wide range of concentrations. Osteoclasts were generated in the presence of M-CSF, TNF-a and RANKL from human PBMNC. However, in the same experiments M-CSF and TNF-a in the absence of RANKL failed to support human osteoclast formation. Aseptic loosening and osteolysis are considered the main long-term complications of hip arthroplasty. Pathogenesis of peri-prosthetic osteolysis is multifactorial and both biological and mechanical factors are important. TNF-a is thought to be involved in orthopaedic implant oste-olysis induced by prosthesis-derived wear particles. The final osteolytic step is undertaken mainly by osteoclasts. This is the first report showing that TNF-a and M-CSF in the absence of RANKL in human PBMNC is not capable of inducing osteoclast formation. TNF-a therefore may increase peri-prosthetic loosening by enhancing the activity of the mature osteoclast.

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Y. Sasazaki R. Shore B.B. Seedhom

Aim: The aim of this study is to visualize the structural changes of both the matrix collagen meshwork and the chondrocyte cytoskeleton of articular cartilage when it is subjected to tensile strain.

Materials and Methods: Dumbbell-shape specimens were harvested from the articular surface of the femur. Specimens were placed with the articular surface uppermost each in individual mini tension device and subjected to a graded series of tensile strains, whilst being observed with phase contrast light microscopy. Thereafter each specimen was fixed in its particular position of strain, and stained with fluorochrome conjugated primary antibodies specific for actin and vimentin and with DAPI for nuclear staining for observation by confocal laser scanning microscopy (CLSM).

Results: Phase contrast microscopy visualized the reorganization of the matrix which became aligned parallel to the direction of strain, resulting in the deformation of the chondrocyte and their nuclei into an elliptical shape. CLSM demonstrated the reorganization of the matrix and chondrocyte cytoskeleton; at no strain, the vimentin meshwork spanned the cytoplasm from plasma membrane to nuclear membrane. At 20% strain, the vimentin meshwork became aligned parallel to the direction of strain and the nucleus was deformed into elliptical shape.

Discussions: There are two possibilities to explain the structural changes in the chondrocyte under tensile strain. 1.The collagen meshwork becomes aligned parallel to the direction of tensile strain, squeezing the chondrocyte into the observed elliptical shape subsequently with the cytoskeleton reorganizing in response to it. 2.The collagen meshwork transfers the tensile strain through the plasma membrane to the vimentin meshwork which reorganizes and subsequently results in the changes in chondrocyte morphology. Further explanation is required to test the above two hypotheses.

R.B. Abu-Rajab I.G. Kelly A.C. Nicol B. Stansfield T. Nunn

The purpose of this study was to evaluate the effect on movement under load of different techniques of reat-tachment of the humeral tuberosities following 4-part proximal humeral fracture. Biomechanical test sawbones were used. 4-part fracture was simulated and a cemented Neer3 prosthesis inserted. Three different techniques of reattachment of the tuberosities were used – 1)tuberosities attached to the shaft, and to each other through the lateral fins in the prosthesis with one cerclage suture through the anterior hole in the prosthesis, 2)as 1 without cerclage suture, and 3)tuberosities attached to the prosthesis and to the shaft. All methods used a number 5 ethibond suture. Both tuberosities and the shaft had multiple markers attached. Two Digital cameras formed an orthogonal photogrammetric system allowing all segments to be tracked in a 3-D axis system. Humeri were incrementally loaded in abduction using an Instron machine, to a minimum 1200N, and sequential photographs taken. Photographic data was analysed to give 3-D linear and angular motions of all segments with respect to the anatomically relevant humeral axis, allowing intertuberosity and tuberosity-shaft displacement to be measured. Techniques 1 and 2 were the most stable constructs with technique 3 allowing greater separation of fragments and angular movement. True intertuberosity separation at the midpoint of the tuberosities was significantly greater using technique 3 (p< 0.05). The cerclage suture used in technique 2 added no further stability to the fixation. In conclusion, our model suggests that the most effective and simplest technique of reattachment involves suturing the tuberosities to each other as well as to the shaft of the humerus. The cerclage suture appears to add little to the fixation in abduction, although the literature would suggest it may have a role in resisting rotatory movements.

R.S. Fish J.R. Ralphs

Introduction: Tendons consist of longitudinally running parallel bundles of collagen with rows of tendon cells between them. The tendon cells are linked to one another via gap junctions1 and cytoskeletally associated adherens junctions. Recent in vitro studies indicate that the two types of gap junction present, made of connexin 32 and connexin 43, regulate tendon cell responses to mechanical load2. In view of the importance of cell-cell interactions in tendon cell behaviour, we describe the behaviour of tendon cells in a novel 3-dimensional culture system designed to allow cells to establish cell-cell contacts and deposit matrix in the absence of scaffolds, which would favour cell-substrate interactions, and without disturbance by medium changes.

Methods: Tendon fibroblasts were isolated from chicken tendons by protease and collagenase digestion3, grown to passage 3 in HEPES buffered DMEM/5% foetal calf serum/1% antibiotic/1% L-glutamine at 370C, in 5% CO2 in air. Cells were suspended at 3x107 cells/ ml and 1 ml placed in a Spectrapor DispoDialyzerTM tube (MW cutoff 300,000). This was then placed into a 60 ml centrifuge tube with 40 ml DMEM containing ascorbate (1mg/ml) on a roller. Medium in the large tube was changed every 2 days. At 24 hours, 7, 14 and 21 days the cell aggregates were fixed in 90% methanol (4°C), frozen on dry ice and cryosections cut at 10–15 μm. Sections were labelled by indirect immunofluorescence with monoclonal type I, II, and III collagen, actin, vimentin and decorin, connexins 32 and 43, vinculin, Pan cadherin and N-cadherin.

Results: Cells in suspension culture formed elongated aggregates up to 3cm long. Immunolabels showed that at 7 days type I and III collagens were present, predominantly in the periphery. At 14 days the collagens were uniformly distributed throughout the aggregates and showed parallel longitudinal organisation. It is also clear from propidium iodide label that the cell nuclei have distinct areas of alignment. The aggregates labelled positively for actin stress fibres, N-cadherin, decorin and connexin 32. Type II collagen and connexin 43 showed no conclusive label.

Discussion: The suspension cultures clearly show that tendon cells can form large scale, organised structures in this culture system, and are capable of assembling an organised extracellular matrix in the absence of a scaffold to support them. The structures formed were similar to tendons in their cell and matrix organization. The amount of collagen deposited by the cells increases over time. Therefore, tendon cells could be used in a tissue engineering context to form well organised tissue in the absence of scaffolds in suitable culture systems. The presence of connexin 32 gap junctions and absence of connexin 43 shows the cell aggregates favouring matrix synthesis pathways – in cell cultures connexin 32 junctions promote collagen sythesis whereas connexin 43 inhibits it2.

N. Heidari M. Korda R. Dattani J. Hua G. Blunn

Introduction: Periprosthetic bone loss, brought about by wear particle induced osteolysis, presents a major challenge and compromises outcome in revision Total Hip Replacement. Poor bone stock at revision hip replacement is the main indication for impaction allografting. There are well documented limitations in the use of bone graft. Autogenous bone graft is osseoinductive, though donor site morbidity and the limited amount available restrict its use. An alternative is allogenic bone graft from cadaveric femoral heads. The drawbacks of using allograft are a limited supply and the risk of disease transmission. An alternative may be the use of bone substitute materials. Usually these are used in conjunction with allograft and therefore a number of drawbacks still apply. This study investigates the use of impaction grafting without bone graft. In this study we tested Apopore, 60% porosity, 2–5 mm hydroxyappatite (HA) granules (ApaTech Ltd) in an animal impaction model with allograft as control. Hypothesis Impaction using porous granular HA induces a similar volume of new bone compared with impaction using allograft.

Methods and Materials: Cylindrical defects of 15mm diameter were created in the medial femoral condyles of 12 sheep (6 sheep in each group) and filled with 3.5 grams of either morselised ovine allograft, washed and defatted according to North London Tissue Bank protocols, or porous HA granules impacted with a specially designed impactor, 20 times with a force of 3 KN. This force was similar to that measured during impaction grafting in clinical cases. After 6 weeks the sheep were euthanized, samples embedded in resin and the amount of bone formation measured by histomorphometric analysis.

Results: Under the impaction forces used the HA graft was more impacted than allograft. In the impacted HA graft the average pore size was smaller than for impacted allograft. After 6 weeks more new bone formation was observed at the host implant interface than the middle of the implant in both groups. At the implant host interface there was 26.64% (± 2.13%) new bone formation in the allograft and 21.13% (± 4.51%) new bone formation in the HA implant. In the middle of the implants allograft produced 11.01% (± 2.07%) new bone whilst the HA produced 7.23% (± 4.05%) new bone. Two tailed t-test showed no significance in either region, p=0.28 at the interface and p=0.40 in the middle. Allograft underwent resorption, from 39.37% at time zero to 5.66% (± 2.04%) at 6 weeks, a total reduction of 85%, where as the volume of HA granules remained the same and was 49% at time zero and 48.59% (± 1.69%) at 6 weeks. Two tailed t-test showed a significant difference (p< 0.0001) between allograft and HA at 6 weeks.

Conclusions: This study shows that granular porous HA induced a similar level of bone formation as compared with allograft. Resorption of allograft in this model allowed greater ingrowth of fibrous tissue. This makes the structural scaffold much more porous, compromising stability of the construct. The HA was not resorbed after 6 weeks and hence may be more stable. HA also has the advantage of being readily available. This study demonstrates that a bone substitute material does not need to be mixed with allograft.

J. Ingram S. Korossis J. Fisher J.E. Ingham

Introduction: The anterior cruciate ligament (ACL) is the most frequently damaged ligament in the knee joint. The patella tendon autograft is the current replacement of choice, however autografts are not always available and grafting often leads to donor site morbidity. Allogeneic implants may cause an adverse immunological reaction [1] The aim of this study was to develop an acellular tendon scaffold with the mechanical and biochemical properties of tissue which could be rapidly recellularised for use in tissue engineering of the anterior cruciate ligament.

Materials and Methods: Porcine patella tendons were dissected less than 24 hours after slaughter and washed in PBS. The tendons were decellularised using 0.1% (w/ v) SDS for 24 hours. Decellularisation was assessed by haematoxylin and eosin staining and light microscopy. The glycosaminoglycan and hydroxyproline (measure of collagen) content of the scaffold were also assessed quantitatively following decellularisation. Following decellularisation the scaffolds were subject to various levels of ultrasonication in order to modify the acellular scaffold prior to reseeding in an attempt to achieve recellularisation of the scaffold. Denaturation of the collagen within the scaffold following ultrasonication was assessed using the ƒÑ-chymotrypsin assay. Decellularised and ultrasonicated scaffolds were subject to uniaxial tensile loading to failure in a Howden tensile testing machine. The sonicated scaffolds were reseeded with human tenocytes (1x105 cells.cm2) and cultured in 5% CO2 in air at 37°C for three weeks. One scaffold was removed every seven days and either fixed in 10% neutral buffered formalin prior to dehydration and H& E staining or was stained with Live/Dead stain (Molecular Probes) and observed using confocal microscopy.

Results: Porcine patella tendons were successfully decellularised using 0.1% (w/v) SDS. Following decellularisation there was no change in the biochemical composition of the scaffold. Ultrasonication of the scaffold at 360W was shown to open up spaces between collagen bundles without damaging the collagen matrix and this was confirmed with the Ą-chymotrypsin assay. Following decellularisation and ultrasonication there was no change in the ultimate force (N) needed to break the tendon scaffold. When cells were seeded onto the sonicated scaffold, the cells were shown to penetrate to the centre of the scaffold within just 3 weeks of culture. Following staining with Live/Dead stain it was shown that after three weeks in static culture approximately 50% of the cells in the centre of the scaffold were viable. In comparison the cells cultured on the acellular non-sonicated scaffold remained on the surface of the scaffold and did not penetrate the matrix during this culture period.

Conclusion: An acellular scaffold with excellent biochemical and mechanical properties has been developed which can be recellularised in an important first step towards tissue engineering of the anterior cruciate ligament. Future work will investigate culture of the reseeded scaffold under appropriate physical stimulation with a view to maintaining tissue homeostasis and increasing cell viability.

A.P. Sprowson A.W. McCaskie M.A. Birch

Introduction: MEPE was identified in patients with tumors and oncogenic hypophosphatemic osteomalacia (OHO), and therefore thought to inhibit osteoblast differentiation and proliferation. However when looking at the structure of MEPE in detail a number of important domains are observed, including a glycosamino-glycan-attachment site, and a RGD cell-attachment motif. The RGD motif is by far the best characterized peptide sequence for stimulating cell adhesion on synthetic surfaces. Glycosaminoglycan attached to MEPE also has the potential to interact with numerous growth factors, proteases and cell surface receptors. MEPE shares molecular similarities with several dentin-bone phosphoglycoproteins which exhibit an ASARM motif shown to potently inhibit calcium crystallization and crystal growth in the salivary duct system. More recently the ASARM peptide sequence has been shown to be a inhibitor of osteoblast mineralization.

Method: To test the hypothesis that MEPE has multiple functional sites, PCR Primers were designed to provide a truncated MEPE protein, which contained pro-osteogenic motifs and had the anti-osteogenic ASARM motif removed. PCR products were cloned using the pBAD TOPO® TA Expression Kit. MEPE was than expressed in E. coli and purified by HIS column chromatography. Expression of truncated MEPE was confirmed by coomassie staining, Western blot with an antibody to an epitope tag and sequence analysis. Truncated MEPE was passively absorbed overnight at 4 oC in a 96 well plate (0.3–50 micrograms) and Fibronectin was laid down (30 micrograms) as a positive control. Primary rat osteoblasts in serum free medium were seeded into the wells (10,000 cells/well) in triplicate and incubated at 37oC for 24 hours. MTT assay was used to estimate cell number, the coloured product absorbance was then determined at 490nm and adhesion was expressed relative to fibronectin. In addition we laid down truncated MEPE into three 8 well chamber slides as above. This was left overnight at 4 oC. Primary rat osteoblasts were then seeded into the wells (10,000 cells/well) in triplicate and incubated at 37oC for 4 hours in serum free medium. Cells were viewed and images captured with a phase contrast microscope.

Results: We have successfully expressed MEPE in E. Coli and devised a purification strategy for obtaining protein. This has been confirmed by coomassie, silver stain and Western blot analysis. The MTT assay showed a significant increase in cell adhesion and proliferation within wells coated with 50 micrograms (70% +/− 0.67(relative to fibronectin)), 30 micrograms (63% +/− 0.81), 3 micrograms (54% +/− 2.4) of MEPE when compared with TCP (32% +/− 0.56). Furthermore we have shown increased osteoblast spreading with increasing dose when compared to tissue culture plastic alone.

Conclusion: The data shows a dose dependent response of osteoblast to increasing concentrations of the novel MEPE protein. This provides evidence that MEPE without the ASARM domain increases osteoblast adhesion, cell anchorage and spreading. Further studies are currently been undertaken to establish its long term effects on osteoblast function and suitability for incorporation into orthopaedic biomaterials.

A. Lakdawala S. Todo G. Scott

Introduction: Aseptic loosening due to polyethylene wear is a mode of failure in knee arthroplasty. No study has evaluated the roughness of the articulating surface of retrieved femoral components & its role in creation of polyethylene wear. AIM The aim of our study was to investigate the in-vivo changes in the surface roughness of retrieved femoral components. Our hypothesis was that the surface finish of the femoral components, articulating with the polyethylene inserts deteriorated in accordance with the duration of implantation.

Materials and Methods: 22 femoral components, all Freeman-Samuelson prostheses, were retrieved from 18 male and 4 female patients at revision knee surgery. The mean age at revision was 68.4 years and the mean period of implantation was 55.64 months. 18 implants were retrieved for aseptic loosening and 4 for infection. Firstly, the surfaces of femoral components & polyethylene inserts were visually inspected for modes of damage in the articulating areas. The surface finish measurements were performed with a contact stylus profilometer with a 2-mm-radius stylus tip and a cut-off length of 0.8mm. The surface roughness was characterised by measuring Ra(mm), which is the arithmetic mean of the absolute values of the measured height deviations taken within the evaluation area and measured from the main line or surface. Both condyles were examined as separate areas articulating with the tibial components from 0° to 60° and 61° to 120° of knee flexion. Surface roughness (Ra) measurements from the sides of the patellar groove at the top of the femoral flange, which do not articulate either with the patella or tibia, were taken as control. The Ewald method of assessing the orientation of the components was applied to derive the coronal angle of the knee (CAK).

Results: The mean CAK was 7.2° ± 1°. Dull edged parallel scratching and burnishing were the main modes of damage identified on the surface in the articulating areas. Visual analysis of polyethylene inserts failed to identify embedded Polymethyl-methacrylate debris or any other damage, which matched the location of the altered surface finish of the femoral components. The mean Ra values recorded were: Control: Mean-0.0230 mm, SD- 0.00821. Medial Femoral condyle (0° – 60°) – 0.0225 mm, SD – 0.00797, P=0.832 Medial Femoral Condyle (61° – 120°) – 0.0244 mm, SD – 0.00532, P= 0.189 Lateral Femoral condyle (0° – 60°) – 0.0263 mm, SD – 0.00694, P= 0.078 Lateral Femoral Condyle (61° – 120°) – 0.0253 mm, SD – 0.00758, P= 0.286 No statistically significant difference was seen in the mean roughness (Ra) of the articulating areas when compared to that of the control (P< 0.05).

Conclusion: This study showed that the surface finish of these implants did not deteriorate during the period of implantation. On this basis we believe that a well-aligned and well-fixed femoral component, without any accumulated wear debris beneath it, does not require mandatory exchange if the revision is carried out for isolated failure of the tibial prosthesis even if the femoral component has fine scratching or burnishing on its surface.

R.M. Barker-Davies B.J.C Freeman R. Bayston W. Ashraf

Introduction: Propionibacterium acnes (P. acnes), a common anaerobic skin commensal, has been implicated in biomaterial-related infections (BRI). Bacteria can adhere to biomaterial surfaces and grow as a bio-film held together by exopolymer, exhibiting increased antimicrobial resistance. To our knowledge, images of P. acnes biofilms have not previously been published. We have demonstrated the ability of P. acnes to adhere to surgical steel and to develop a biofilm on this material. However its ability to adhere to and develop a biofilm on titanium, a commonly used surgical implant material, has not been fully investigated.


To determine the quantitative adherence and biofilm development of P. acnes on titanium compared to surgical steel.

To assess the subsequent effect of penicillin, the therapeutic drug of choice, on mature P. acnes biofilms.

Method: Six clinical isolates of P. acnes were assayed for adherence to materials with and without plasma glycoprotein conditioning film by chemiluminescence and culture. Biofilm development was assessed by chemiluminescence, fluorescence microscopy, environmental (ESEM) and scanning electron microscopy (SEM). Mature biofilms were exposed to plasma concentrations of penicillin and quantified by chemiluminescence and culture. Unpaired student’s t tests and univariate linear regression models were calculated using SPSS software (version 12).

Results: Univariate linear regression showed that P. acnes adherence to titanium was 18% (p=0.001) greater than to steel. Adherence was reduced by the presence of the conditioning film on titanium by 28% (p=0.001), but this made no significant difference to P. acnes adherence to steel. P. acnes biofilms were clearly demonstrated, along with bacterial expolymer, showing an interesting similarity to biofilms of S. epidermidis. P. acnes grows as a thick biofilm on both materials held together by exopolymer and our preliminary results suggest that biofilms on titanium might be less susceptible to antimicrobials after 24 hours of penicillin treatment; a reduction of 94% on steel and 81% on titanium (p=0.057, p=0.39 resp).

Conclusions: P. acnes adheres to steel and titanium, a crucial first step in BRI. Greater numbers of P. acnes adhere to titanium than to steel. The naked surface of titanium is microporous, assisting adhesion. A conditioning film reduces P. acnes adherence to titanium but not to steel. P. acnes develops as a biofilm on steel and titanium. Results indicate that pathogenesis of P. acnes infection on titanium is more successful than on steel. P. acnes biofilms on titanium may be harder to eradicate with antimicrobial agents.

R.T. Wells T.J. Smith A. Galm B. Chatterjee S. Pedersen A.E. Goodship G.W. Blunn

Introduction: External fixation is used widely in the management of fractures, despite a relatively high incidence of complication, arising from pin loosening and infection. Diamond like carbon (DLC) is a low surface energy coating that can be applied to external fixator pins and may reduce biofilm formation and infection resulting in a lower incidence of pin loosening. Hydroxyapatite (HA) is well established as a coating to enhance fixation of external fixator pins. This study tests the hypothesis that HA and DLC coatings on stainless steel (SS) external fixator pin shafts modify integration of the implant with soft/hard tissues.

Materials and Methods: An Orthofix external fixator was used to stabilise a tibial osteotomy with 6 self-drilling/tapping 6mm pins in 32 skeletally mature Friesland ewes. Animals were divided into four groups; SS, DLC, HA partially coated (threads only) and HA fully coated (threads and pin shaft). Pin insertion torque was measured using a torque wrench and extraction torque similarly obtained at 10 weeks when animals underwent euthanasia. Pin performance indices (PPI) were calculated as a ratio of extraction to insertion torque x100%. Pin site 2 was preserved for hard grade resin histology and subsequent pin tissue integration analysis. Pin site 3 was used for analysis of the soft tissue pin shaft interface using transmission electron microscopy. Pin site 5 was examined for the presence of biofilm formation using scanning electron microscopy. Pin site 6 was swabbed for microbiological analysis.

Results: SS and DLC pins achieved significantly higher insertion torques compared to HA partially coated pins (p=0.001, 0.002). Both groups of HA coated pins demonstrated a significantly higher, extraction torque and therefore PPI for all pin site positions compared to SS and DLC (p< 0.001– 0.025). The epithelium was found not to be in contact with the pin shaft in all cases. No significant differences were found between the different pin groups for epidermal down growth and dermal contact. Both groups of HA coated pins showed a significantly higher percentage of new bone in direct contact with the embedded threads compared to SS and DLC pins (p< 0.001, p=0.004). The proportion of soft tissue in contact and within the thread, of fully coated HA pins was significantly lower compared to stainless steel (p=0.003, p=0.017), DLC (p=0.004, p=0.002) and HA partially coated pins (p=0.006, p=0.02). Biofilms were evident on all pins except those coated with DLC. More bacteria were observed on the fully HA coated pins. DLC had significantly lower number of bacterial colonies in culture compared to SS (p=0.028) and fully coated HA pins (p=0.005).

Discussion: Coatings of DLC and HA do have a significant affect on hard/soft tissue reactions. However coatings do not have a significant effect on epidermal down growth or dermal attachment to the pin shaft surface. DLC coated pins had the cleanest surface with no bio-film present and significantly lower numbers of bacteria present. Fully HA coated pins despite evidence of bio-film formation, bacteria and high microbiological counts had significantly higher PPI. In addition fully coated HA pins demonstrated significantly reduced amounts of soft tissue at the pin bone interface. Therefore soft tissue reactions may affect bone integration.

C. Wan Q. He X. Chen G. Li

Introduction: Peripheral blood derived mesenchymal stem cells (PBMSCs) are multipotent cells capable of forming bone, cartilage, fat, and other connective tissues. Bone marrow derived mesenchymal stem cells (BMMSCs) have promoted repair a critical-sized bone defect in several animal models including mouse, rat, rabbit, and dog. The aim of this study was to investigate whether or not the use of allogenic BMMSCs and PBMSCs could regenerate a critical-sized bone defect in rabbit ulnae.

Methods: Rabbit peripheral blood mononuclear cells (PBMNCs) were isolated by density gradient centrifugation method and cultured at a density of 100,000/ cm2 in flasks with DMEM 15% FCS. Colony forming efficiency (CFE) was calculated and their multipotential differentiations into bone, cartilage, and fat were examined under different induction conditions. Specific differentiation markers were examined using cytochemistry and immunocytochemistry methods in the PBMSCs. Critical-sized ulna bone defects, 20 mm in length, were created in the mid-diaphysis of both ulnae in twelve 6 month old NZW rabbits. The ulnar defects were treated as the following 5 groups: empty control (n=4), PBMSCs/Skelite (multi-phase porous calcium phosphate resorbable substitute, EBI Company, USA) (n=5), BMMSCs/Skelite (n=4), PBMNCs/Skelite (n=5), and Skelite alone (n=5). All animals were sacrificed 12 weeks after treatment. The bone regeneration was evaluated by regular radiography, and all samples were subject to peripheral quantitative computed tomography (pQCT) and histological examination at the end point.

Results: The CFE of PBMSCs ranged from 1.2 to 13 per million mononuclear cells. Spindle and polygonal shaped cells were found in the primary PBMSCs colony, showing similar differentiation potential with BMMSCs. Mineralized bone nodules formed under osteogenic media were positive for Alizarin Red S staining in the PBMSCs. Chondrogenic differentiation was identified in serum free media containing TGF-¦Â1 (10 ng/ml), with type II collagen expression and Alcian blue positive nodule formation. Adipocytic differentiation was tested with or without adipogenic media, with positive Oil Red O staining for lipid accumulation and CEBP¦Á expression in the PBMSCs. After twelve weeks implantation, the ulnar defects were not healed in the empty control group; the total bone density in PBMSCs/Skelite and BMMSCs/Skelite treated defects were greater than that of PBMNCs/Skelite and Skelite alone treated groups (p< 0.05), with higher score of X-ray evaluation (p< 0.05). Histologically, there were a greater amount of new bone present in both the PBMSCs/Skelite and BMMSCs/Skelite treated groups compared to the PBMNCs/Skelite and Skelite alone treated groups.

Conclusions: This study demonstrated that PBMSCs were multipotent cells; allogenic PBMSCs loaded onto porous calcium phosphate resorbable substitute had enhanced bone regeneration of a critical-sized segmental defect in the rabbit ulna. PBMSCs may be a new source of osteogenic stem cells for bone regeneration and tissue engineering, and further investigations are undergoing to clarify their functions.

P. Kalia A. Bhalla M.J. Coathup J. Miller A.E. Goodship G.W. Blunn

Introduction: The survival of massive endoprosthesis replacements is not as successful as conventional joint replacements. The main cause of failure of these implants is aseptic loosening. Bone in-growth onto the implant collar on the shaft of the prosthesis adjacent to the transaction site has been correlated with a decrease in radiolucent lines adjacent to the intramedullary stem and reduced implant loosening. We propose that bone contact and in-growth to the collar may be further enhanced with tissue engineering techniques. The hypothesis of this study was that autologous mesenchymal stem cells (MSCs) suspended within fibrin glue and sprayed onto hydroxyapatite (HA)-coated collars of massive prosthesis will augment bone growth and contact to the implant in an ovine model.

Materials and Methods: MSCs were isolated and expanded in vitro from the iliac crest of six adult sheep. Pre-implantation, 2 x 106 autologous MSCS were suspended in thrombin. During surgery, this mixture was combined with fibrinogen and sprayed onto the proximal and distal HA-coated collars of tibial midshaft prostheses using pressurized air. The implants were cemented into the right hind limb of twelve sheep, six of which received MSCs. Radiographs were taken at 2, 4 and 6 months and bone area within defined regions quantified using image analysis software. After six months, specimens were retrieved and processed for undecalcified histology. Transverse thin sections were prepared through the centre of each collar. Image analysis was used to quantify bone area and contact. Mann Whitney U tests were used for comparative statistical analysis, where p< 0.05 was classified as significant.

Results: Anterior-posterior (AP) radiographs taken at 2, 4, and 6 months showed that animals treated with MSCs produced more bone adjacent to the shaft of the implant. Analysis of bone area on both AP and medio-lateral (ML) radiographs taken after sacrifice showed that stem cell-treated implants encouraged significantly more total bone around the implants at 6 months than the control group (171.94 ± 29.04 mm2, and 87.51 ± 9.81 mm2 bone area, respectively, p = 0.016). Analysis of histological sections shows a significant increase in bone area around midshafts treated with MSCs, compared to the implant controls (53.99 ± 10.64 mm2, and 21.07 ± 7.34 mm2, respectively; p = 0.020). The average surface area contact between the midshaft and bone was almost doubled in the MSC-implant group (19.83 ± 8.73 % contact) than in the control group (8.667 ± 8.667 %, p = 0.196). In the MSC group bone was seen deep within the grooves of the HA coated collar whilst a fibrous soft tissue layer separated the newly formed bone in the control group.

Conclusion: Bone contact and in-growth to massive endoprostheses was significantly improved by spraying the implant with autologous MSCs suspended in fibrin glue. Enhanced fixation using stem cells may help prevent aseptic loosening in these massive implants.

Q. He C. Wan G. Li

Introduction: The existence of circulating skeletal stem cells in the peripheral blood from different species including adult mouse and human has been found and documented. The circulating skeletal stem cells may provide a new source of stem cells that may be used for bone regeneration and tissue engineering applications. The aim of this study was to investigate the existence of circulating osteogenic stem cells in the rat peripheral blood, and to compare their osteogenic potentials with bone marrow mesenchymal stem cells (BMMSCs).

Methods: Whole blood from twelve female 3-month old SD rats was harvested by cardiac puncture and bone marrows were also collected. Mononuclear cells from both bone marrow and peripheral blood (PBMNCs) were isolated by Lymphoprep density gradient centrifugation method, and plated at a density of 300000 to 400000/cm2 in flasks with á-MEM medium and 15% FCS. The colony forming efficiency (CFE) was calculated after 10–14 days culture. The osteogenic, adipogenic, and chondrogenic differentiation potential of both BMMSCs and peripheral blood mononuclear cell subset were examined and compared under different specific culture conditions. In addition, both BMMSCs and peripheral blood mononuclear cell subset were seeded into absorbable porous calcium phosphate substitute and implanted subcutaneously into SCID mice for 12 weeks, and the implants were examined histologically.

Results: After 10–14 days in culture, the adherent fibroblast-like colonies were formed in the PBMNCs, with CFE ranging from 1.3 to 3.5 per 10000000 cells. Under osteogenic conditions, both BMMSCs and PBMNCs subset were positive for bone markers such as ALP, type I collagen and osteocalcin; bone nodules were formed in BMMSCs and PBMNCs subset long-term culture with positive Von Kossa and Alizarin Red S staining. Under adipogenic conditions, PBMNCs subset and BMMSCs were positive for Oil Red O and C/EBP á immunostaining. For chondrogenic differentiation studies, PBMNCs subset and BMMSCs were positive for type II collagen and they had Alcian blue positive nodules formation. After implantation with calcium phosphate substitutes in SCID mice for 12 weeks, osteoid and bony tissues were evident in the implants both loaded with PBMNCs subset and BMSCSs.

Conclusions: A subset of mononuclear cells that have multi-differentiation potential similar to BMMSCs exists in the rat peripheral blood. Our present study has shown that these circulating stem cells possess osteogenic potential in vitro and in vivo. Further work is ongoing to investigate the roles of PBMNCs subset in fracture healing and their recruiting and homing mechanisms.

J.S. Huntley I.J. Brenkel J.M. McBirnie A.H. Simpson A.C. Hall

Autologous osteochondral cylinder transfer is a treatment option for small articular defects, especially those arising from trauma or osteochondritis dissecans. There are concerns about graft integration and the nature of tissue forming the cartilage-cartilage bridge. Chondrocyte viability at graft and recipient edges is thought to be an important determinant of quality of repair. The aim was to evaluate cell viability at the graft edge from ex vivo human femoral condyles, after harvest using conventional technique. With ethical approval and patient consent, fresh human tissue was obtained at total knee arthroplasty. Osteochondral plugs were harvested using the commercially available Acufex 4.5mm diameter mosaicplasty osteotome from regions of the lateral femoral condyle (anterior cut) that were macroscopically non-degenerate and microscopically non-fibrillated. Plugs were assessed for chondrocyte viability at the graft edge using confocal laser scanning microscopy (CLSM), fluorescent indicators and image analysis. The central portions of the plugs remained healthy, with > 99% cell viability (n=5). However, there was substantial marginal cell death, of thickness 382 ± 68.2 microm in the superficial zone (SZ). Demi-plugs were created by splitting the mosaicplasty explants with a fresh No. 11 scalpel blade. The margin of SZ cell death was 390.3 ± 18.8 microm at the curved edge of the Acufex, significantly (Mann-Whitney; P= 0.0286; n =4) greater than that at the scalpel cut (34.8 ± 3.2 microm). Findings were similar when the cartilage was breached but the bone left intact. In time-course experiments, the SZ marginal zone of cell death after Acufex harvest showed no increase over the time period 15 minutes to 2 hours. Mathematical modelling of the mosaicplasty surface shows that cell death of this magnitude results in a disturbing 33% of the superficial graft area being non-viable. In conclusion, mosaicplasty, though capable of transposing viable hyaline cartilage, is associated with an extensive margin of cell death that is likely to compromise lateral integration. There would appear to be considerable scope for improvement of osteochondral transplant techniques which may improve graft-recipient healing and clinical outcomes.

C.E. Evans S. Mylchreest J.G. Andrew

Mechanical load is crucial to maintaining skeletal homeostasis, but the pathways involved in mecha-notransduction are still unclear. The OPG/RANK/ RANKL triumvirate has recently been implicated in bone homeostasis. These molecules, which are produced by the osteoblast (OPG and RANKL) and the macrophage/osteoclast (RANK), modulate osteoclastogenesis. We have previously shown that cyclical hydrostatic pressure influenced synthesis of various molecules by cultured human macrophages. These factors are important in osteoclastogenesis and bone resorption and have been linked to the development of aseptic loosening. We have also demonstrated that 1,25-dihydroxyvitamin D3 (1,25D3) influences macrophage response to pressure. For this study human macrophages were co-cultured with osteoblasts and subjected to cyclical hydrostatic pressure (34.5x10–3MPa [5.0 psi]) for up to five days, with or without 1,25D3 supplementation. Cells were immunostained for RANK and culture media were assayed for sRANKL and OPG using specific ELISAs. Immunostaining for RANK showed that macrophages subjected to pressure or 1,25D3 supplementation synthesised more RANK than controls. In addition, when exogenous 1,25D3 and hydrostatic pressure were administered simultaneously, immunostaining for RANK was more intense. There was a reciprocal relationship between OPG and sRANKL in co-cultures subjected to pressure. If pressure increased synthesis of sRANKL, OPG was decreased. In cultures where pressure decreased sRANKL, a corresponding increase in OPG was seen. In addition, samples from different individuals responded differently to pressure. The majority of cell populations responded to pressure by increasing OPG synthesis, compared to non-pressurised controls. These results demonstrate for the first time that the OPG/RANK/RANKL complex is sensitive to hydrostatic pressure and that 1,25-dihydroxyvitamin D3 might be involved in this response. These findings suggest a possible transduction mechanism for mechanical load in the skeleton, which has implications for future therapies for aseptic loosening and for skeletal abnormalities such as osteoporosis.

M. Donnelly M. Timlim P. Kiely C. Condron P. Murray D. Bouchier-Hayes

Introduction: The beneficial effects of insulin in the maintenance of normoglycaemia in non-diabetic myocardial infarct and intensive care patients have recently been reported. Hyperglycaemia and neutrophilia have been shown to be independent prognostic indicators of poor outcome in the traumatised patient. The role of insulin and the maintenance of normoglycaemia in the trauma patient have as yet not been explored. We hypothesised that through the already described anti-inflammatory effects of insulin and the maintenance of normoglycaemia, that the systemic inflammatory response would be attenuated, in the injured patient. This might result in less adult respiratory distress syndrome (ARDS) and multi-organ dysfunction and therefore less morbidity and mortality in trauma patients.

Materials and Methods: We used a previously validated rodent trauma model. There were 3 groups, two groups underwent bilateral femur fracture and 15% blood loss via cannulation and aspiration of the external jugular vein. The third group were anaesthetised only. The treatment group immediately receive subcutaneous insulin according to a recently identified sliding scale, and thereafter subcutaneous boluses, dependent on ½ hourly blood sugar estimations. The control groups received the same volume of normal saline ½ hourly, subcutaneously. The animals were maintained under anaesthetic for 4 hours from injury via inhaled isoflurane and oxygen. Core temperature and O2 saturations were recorded throughout. At 4 hours, each animal underwent midline laparotomy and cannulation of the IVC for blood sampling for full blood counts and lactate levels. Serum was also taken for flow cytometric analysis of neutrophil activation via respiratoy burst and CD11b levels. Broncho-alveolar lavage (BAL) was performed for neutrophil content and total protein estimation. The left lower lobe was harvested for wet-dry lung weight ratios.

Results: While O2 saturations were equal throughout in both groups, respiratory rates were persistently elevated in the controls. Wet:Dry lung weight ratios (p< 0.05) and lactate levels were reduced in the insulin treated animals compared to controls. There were similiarly fewer neutrophils in the BAL specimens of the insuliln treated animals compared to injured controls (p< 0.05).

Conclusions: Insulin reduces leukocyte lung sequestration in the injured animal model. This work confirms that insulin may have a role in reducing ARDS in the trauma patient, be that as an anti-inflammatory agent or anti-hyperglycaemic agent, or both, indicating that outcomes might be improved by treating hyperglycaemic trauma patients with insulin. Further work needs to done to elucidate its exact mechanism of action and role in the injured patient.

C. Brown I. Papageorgiou J. Fisher E. Ingham C.P. Case

Introduction: Cobalt-chrome particles from metal hip implants can accumulate in the liver, spleen, lymph nodes and bone marrow of patients. This is a concern as studies have reported neoplastic changes in cells of patients with metal implants. The aims of this study were to determine the effect of wear particles generated by metal-on-metal and ceramic-on-metal implants from hip simulations upon the viability of L929 cells and to determine their genotoxic potential when cultured with primary human fibroblasts.

Methods: Particles were generated in a 10 station Prosim hip simulator run with water as lubricant under microseparation and standard conditions. Bearings comprised medical grade HIPed ‘BIOLOX Forte’ alumina ceramic femoral heads against Ultima metal CoCr acetabular cups (CoM) and wrought CoCr alloy ASTM F1537 femoral heads and acetabular cups (MoM). Particles were sterilised at 1800C for 4 hours and cultured with L929 fibroblasts at particle volume(μm3):cell number ratios of 500:1, 100:1, 50:1, 5:1, 0.5:1, 0.05:1, 0.005:1 and 0.0005:1. Camptothecin (1 and 2μg.ml-1) and latex beads (100μm3 per cell) were used as positive and negative controls. Cultures were for 0, 1, 2, 3, 4 and 5 days at 37oC in 5%(v/v) CO2 in air. Cell viability was assessed using the ATPlite assay. Sterile particles were cultured with primary human fibroblasts at particle volume (μm3):cell number ratios of 50:1, 5:1 and 0.5:1. Cells were exposed to 30%(v/v) H2O2 (positive control) and latex beads (50μm3 per cell; negative control). Cells were cultured for 24 hours and 5 days at 37oC in 5%(v/v) CO2 in air. Genotoxicity was assessed using the comet assay. Statistical analysis between the cell-only negative controls and the cells with the particles at various concentrations, were determined by ANOVA and calculating the minimum significant difference (MSD;p< 0.05) using the T-method.

Results: Particle volume(μm3):cell ratios of 500:1, 100:1 and 50:1 caused a significant decrease in cell viability over 5 days. Wear particles from MoM implants under microseparation wear conditions were also significantly reduced viability at particle volume(μm3):cell ratios of 5:1 over 5 days. Particles from MoM implants under standard wear conditions and CoM implants under both wear conditions resulted in increases in tail length and tail moment relative to the cells only negative control for all treatment groups after 24 hours. These decreased by day 5. Tail length and tail moment were increased at 24 hours relative to day 5 for each of the three particle types. Particles generated by MoM implants under microseparation conditions had different effects upon cells. Tail lengths increased between days 1and 5 for all particle concentrations. A significant increase in tail moments between days 1 and 5 was recorded.

Discussion: This study has shown that metal particles can cause cytotoxic effects and immediate DNA damage to fibroblasts in vitro. Particles were found to reduce cell viability over 5 days and this may account for the decreases in tail length and moments between 1 and 5 days for three particle types. This is of concern as MoM and CoM implants are designed to be implanted into young patients and, despite their low wear rates generate circa 1013 particles per mm3 of wear.

J. Hua M. Baker S. Muirhead-Allwood P. Mohandas T. Nothall G. Blunn

Introduction: The Resurfacing Hip has been increasingly popular for younger patients. Femoral neck fractures are still the main complication. The problems associated with cement such as thermal necrosis, cement debris and lack of long-term biological fixation, combined with the general use of cementless fixation in young patients invite the question whether a cementless component can be used for resurfacing hip replacement. Given that the cement may reinforce the femoral head preventing collapse, an additional question regarding the effect of bone density in cemented and cementless fixation can be asked. The hypotheses of the study are that:

High bone density will increase the yield point and stiffness of the femoral head and therefore improve the implant fixation.

Cement fixation will increase the yield point and stiffness of the femoral head, especially for the lower density bone compared with cementless fixation.

Materials and Methods: Thirty-six femoral head specimens were obtained from consented patients receiving routine hip arthroplasty. The heads were stored frozen at −20oC until use. pQCT was used to analyse trabecular bone density within each head. Specimens were ranked according to bone density and were assigned to high and low bone density groups. Cemented and cementless fixations were then alternatively assigned to individual heads in each group. Thus the 4 groups included in the study were: High density cemented, high density cementless, low density cemented, and low density cementless. Implantation of Birmingham resurfacing hips was carried out according to recommended surgical procedures. For cementing groups, surgical simplex P bone cement was used. Each sample was potted in a cylindrical polyethylene block for testing. A compressive load up to 5 or 10 KN using a Hounsfield Universal Testing Machine were applied on each sample at a rate of 1 mm min-1. Load versus displacement graphs were plotted for all tests. Yield point and stiffness were measured for each sample.


For yield point, there is no significant difference between cemented or cementless resurfacing (4169 ± 1420 N vs. 3789 ± 1461 N; P = 0.434). However, the high density heads provide a significantly higher yield point than low density heads (4749 ± 1145 N vs. 3208 ± 1287 N; P = 0.01).

The addition of cement significantly contributes to femoral head stiffness compared to cementless resurfacing (5174 ± 1730 N/mm vs. 3678 ± 1630 N/mm; P = 0.012).

Discussion: Bone density plays an important role in resurfacing hip arthroplasty. Higher bone density will reduce the incidence of fractures comparing with lower density. Therefore, resurfacing THR for the older patients and those with sub-optimal bone density should be used with caution. Consequently, it is suggested that a bone density scan should be routinely applied for those patients who are considered for resurfacing hip replacement. There is no difference between the cemented and cementless fixation in reducing femoral head failure, though cement will increase the stiffness of the bone. The study suggests that cementless resurfacing hip could be an alternative design with its clinical advantages of long-term osseointegration if implant is coated with bio-active materials.

K.E. Rudman R.M. Aspden J.R. Meakin

Introduction: Since the mid-1800’s it has been believed that the human femur functions in a similar way to a crane in which the distal end is fixed and body weight is applied to the femoral head (Meyer, 1867, Williams, 1995). This results in tension in the lateral femoral shaft and in the so-called ‘principal tensile system’ of trabeculae while, compression is found in the medial shaft and in the ‘principal compressive system’. Most studies have concentrated on the shaft to find ways of avoiding these tensile stresses and recognised that the inclusion of muscle forces is essential in any realistic modelling. The state of stress in the proximal femur has not been satisfactorily resolved, though a minority view is that muscle forces put all of the trabeculae into compression (Strange, 1965). Our hypothesis is that the majority of the proximal femur is in compression and that the so-called ‘principal tensile system’ functions as an arch, transferring compressive stresses to the diaphysis.

Methods: To begin to test this, we have developed a 2D finite element (FE) model of the femur. The distal end was constrained and a force of half body weight, representing two-legged stance and negligible muscle forces, was applied to a representation of the acetabulum. The material properties used were 17 GPa for cortical bone, and 100–400 MPa for cancellous bone, with a higher modulus assigned to areas of greater apparent density. The model was meshed, using eight-node quadrilateral elements, and solved using ANSYS 8.0 software (ANSYS, Inc., USA). Recognising that the joint capsule is a substantial structure, ligamentous forces were included by spring-like link elements. Contact elements were used between the femoral head and acetabulum.

Results: In the absence of the capsular ligaments, stresses in the proximal femur were similar to those predicted by the crane model, i.e. corresponding to the traditional description of tensile and compressive trabeculae. The inclusion of ligamentous forces resulted in compressive stresses being generated over most of the proximal femur. When the denser trabecular systems were given a higher modulus the stresses become focused along the arch of the horizontal trabeculae.

Discussion: This study shows that inclusion of ligamentous forces results in compressive stresses being generated in the proximal femur and transmitted through the arch-like structure of trabeculae. It is notable that the capsular ligaments are thick and strong and are aligned with the femoral neck. Also, though ignored in this study, some of the major muscle groups have a significant component lying in the same direction. These result in a considerable force pressing the femoral head into the acetabulum and compressive stresses in most of the head and neck. This makes best use of the mechanical properties of bone, which functions better in compression than tension (Cowin, 2001), and avoids tensile forces in the diaphysis.

S.W. Hamilton A.J. Johnstone R.H. Bradley

Introduction: The knee meniscus is prone to injury and has limited intrinsic healing potential despite surgical repair. Methods to enhance fibrochondrocyte function and augment meniscal repair would be invaluable in the treatment of meniscal injuries. Ultraviolet Ozone (UVO) modified surfaces have been characterised chemically and topographically. These surfaces have been shown to promote the function of certain cell types. This study investigated the attachment, proliferation and extracellular matrix production of fibrochondrocytes cultured on UVO modified polystyrene surfaces. Interest was paid to the integrins, a group of transmembrane extracellular matrix attachment glycoproteins. In particular the subunits alpha2 and alpha5, as they specifically bind to the ligands Collagen Type I and Fibronectin, major components in the human meniscus.

Methods: Tissue samples from adult human medial meniscal tears were obtained at knee arthroscopy. Fibrochondrocytes were isolated by standard cell culture techniques and cultured to 100% confluence before seeding onto UVO modified polystyrene surfaces. The untreated polystyrene surfaces of culture dishes were oxidatively modified with an ultraviolet ozone treater. The response of fibrochondrocytes to various surface oxygen concentrations was investigated. Untreated, hydrophobic surfaces acted as controls. Images of cells in culture were obtained with a Leica digital camera mounted on a microscope. Cells were counted at 24, 48, 72 and 96 hours. After 48 and 96 hours of culture standard wet transfer Western Blots were undertaken using antibodies to the alpha2 and aalpha5 integrin subunits (Santa Cruz Biotechnology). To evaluate potential extracellular matrix production total protein assays were undertaken at 1, 2, 3, 4, 6, 8 and 10 days of culture (Bio-Rad Laboratories).

Results: Fibrochondrocytes attached preferentially to the UVO treated surfaces. They proliferated steadily until they reached confluence at 96 hours. Western Blot analysis showed the integrin subunit a5 to be present in the cell lysate after both 48 and 96 hours of culture. The a2 subunit was not detected at these times. There was no increase in total protein concentration on surfaces after fibrochondrocytes had reached confluence. Discussion: UVO modified surfaces promote the attachment and proliferation of human fibrochondrocytes. The alpha2 subunit was not detected in the cell lysate of these surfaces after culture for 96 hours. Whether this is due to defective or absent Collagen Type I at this stage of culture remains to be answered. The presence of the alpha5 subunit suggests that Fibronectin may be involved in the process of fibrochondrocyte attachment to UVO modified polystyrene surfaces. It is suggested that there is little or no extracellular matrix production after 4 days as there is no increase in total surface protein concentration after confluence is reached. The increase in total surface protein concentration up to this point most likely reflects cell proliferation.

Conclusion: Ultraviolet Ozone modified surfaces enhance certain aspects of fibrochondrocyte function and therefore have a potential role in the development of novel therapies for meniscal repair.

J. Page F. Natrass T. Fawcett P. Cook A. Jennings

A phenomenon of methicillin resistance in methicillin sensitive Staphylococcus aureus has been noted in organisms living in biofilm induced by the state of cell wall deficiency. The rate and the amount of biofilm formed by the cell wall deficient organisms far exceeds that of cell wall patent organisms. Once removed from the biofilm the S. aureus had the same sensitivities of the original organism. Cell wall deficient organisms outside the biofilm did not demonstrate the methicillin resistance. A known laboratory strain (ATCC 9144) was induced into a cell wall deficient state and allowed to form biofilm. The rate of formation and amount formed was compared with that formed by cell wall patent organisms. Before inducing cell wall deficiency sensitivity to methicillin was demonstrated using standard microbiological technique. Using an oxacillin containing plate as a culture medium: the biofilm, cell wall deficient organisms and the cell wall competent organisms were inoculated onto separate media. Organisms from the biofilm were isolated and grown free of the biofilm on blood agar. Any growth on the oxacillin containing plate would demonstrate methicillin resistance. There was no growth on the plates containing the cell wall competent or cell wall deficient organisms. There was however growth on the plate inoculated with bio-film, however when organisms were isolated from the biofilm, there was no growth on the media. Antibiotic sensitivities of the original inoculant and the organisms isolated from the biofilm were the same. The biofilm, induced as a result of cell wall deficiency, offers a form of structural protection to the Staphylococcus aureus without altering the resistance pattern of organism. Standard microbiological techniques would therefore report the organism as methicillin sensitive, however clinically the organism may behave as a methicillin resistant organism. The state of cell wall deficiency encourages the formation of biofilm in S. aureus. In-vitro the state of cell wall deficiency is induced using high osmolality media or sub-lethal doses of cell wall active antibiotics. Both these states are found in clinical practice.

H. Kurup P. Rao D. Patro

Use of allograft in orthopaedic surgery is a well-established procedure. Ethylene Oxide sterilization is still controversial in bone banking because of its effect on osteoinductive properties of bone graft. Freeze drying is considered to be the best technique for allograft preparation and storage. High cost of equipment and its maintenance makes this method not feasible option in developing countries like India. This study involved setting up of a bone bank for the first time in JIPMER institute, Pondicherry, India. Cancellous bone was collected from 40 patients (femoral heads removed during joint replacements). They were cleaned thoroughly, chemically processed and sterilized with ethylene oxide gas and stored doubly packed. These were implanted at 11 patients with 14 non-unions, which required cancellous bone grafts. Patients were followed up clinically looking for infection and radiologically for graft incorporation. 85.7 % of grafted sites were united at the end of 12 months. Non-unions took average of 44.8 weeks for the union. Radiological union achieved by 12 months with average time of graft incorporation 44.8 weeks. In 8 cases the allografts were used to pack cavities. Healing occurred at an average of 29 weeks. In 4 patients with arthrodesis following excision of tumor one site failed to unite, one deep infection, which did not resolve with regular chemotherapy had an amputation. The rest of the sites healed at an average 54.8 weeks. This study shows ethylene oxide sterilized cancellous allograft suitable for packing cavities in treatment of benign bone lesions as well as in treatment of non-union. The osteoconductive property of bone allograft may not be affcted by the ethylene oxide sterilization. Achivement of union and a low rate of infection confirms efficacy of ethylene oxide as cost effective and reliable option for bone allograft sterilization.

L.M. Jennings C.J. Bell E. Ingham R. Komistek M.H. Stone J. Fisher

Introduction: In vivo fluoroscopic studies have shown considerable differences in kinematics between different designs of knee prostheses and compared to the natural knee. Most noticeably, lift off of the femoral condyles from the tibial insert has been observed in many patients (Dennis et al, 2003). The aim of this study was to simulate lateral femoral condylar lift off in vitro and to compare the wear of fixed bearing knee prostheses with and without lift off.

Materials and Methods: 12 PFC Sigma cruciate retaining fixed bearing knees (DePuy, Leeds, UK) were tested. The 10 mm thick inserts were manufactured from GUR1020 UHMWPE and gamma irradiated in a vacuum. The inserts snap fitted into titanium alloy tibial trays, and articulated against Co-Cr-Mo alloy femoral components. The testing was carried out on six station simulators (Prosim, Manchester, UK). Femoral axis loading (maximum 2.6 kN) and the flex-ion-extension profile (0–58°) were adopted from ISO 14243 (1999). The internal/external rotation was ± 5° and anterior/ posterior displacement 0–5 mm. Six of the knees were tested under these standard conditions for 4 million cycles. A further six knees were tested under these conditions with the addition of lateral femoral condylar lift off, for 5 million cycles. The lift off was achieved by introducing an adduction moment to the tibial carriage, producing a separation of approximately 1 mm during the swing phase of the simulator cycle. The simulator was run at 1 Hz and the lubricant used was 25% newborn calf serum. Wear was determined gravimetrically, using unloaded soak controls to adjust for moisture uptake. Statistical analysis was performed using Students t-test (p < 0.05).

Results: Under the standard kinematic conditions the mean wear rate with 95% confidence limits was 8.8 ± 4.8 mm3/million cycles. When femoral condylar lift off was simulated the mean wear rate increased to 16.2 ± 2.9 mm3/million cycles, which was statistically significantly higher (p < 0.01). The wear patterns on the femoral articulating surface of all the inserts showed more burnishing wear on the medial condyle than the lateral. However, in the simulation of lift off the medial condyle was even more aggressively worn with evidence of adhesion and surface defects.

Discussion: The presence of lateral femoral condylar lift off resulted in a higher wear rate on the medial compartment of the PFC Sigma fixed bearing knee. This could be due to elevated contact stresses as the lateral lift off produced uneven loading of the bearing. Further, additional medial/lateral sliding of the medial condyle whilst it remained in contact may have accelerated the wear by cross shearing of the polyethylene in the medial/lateral direction. This direction is weakened when the polyethylene is preferentially molecularly orientated by sliding in the flexion-extension axis. The implications of condylar lift off include premature wear of the polyethylene and possible component loosening.

S. Matsuda R. Grogan T. Stewart M. Stone J. Fisher

All polyethylene tibial components (APT) for total knee joint replacement have been recently reintroduced due to their past success and cost savings with respect to knee designs with a metal backed tibial tray (MBT). However, isolated cases of collapse of the medial bone in APT designs have been observed by the authors prompting this investigation. The objective of this study was to investigate the stress/strain distribution within the cancellous bone for the APT and MBT systems, particularly looking at the effects of coverage of the tray over the proximal tibia in each design. A three-dimensional finite element model of the proximal tibia implanted with a tibia tray was generated. An elliptical cylindrical tibia tray with a peg was modeled as being perfectly bonded to a PMMA layer on the superior surface of the cancellous and cortical bone. Gap size between the edge of the tray and outer of the cancellous bone, was introduced in the medial direction. Load was applied on the superior surface of the tibial insert in the medial side. Two lift-off loading cases were used, a low load of 800N (1 body-weight) and a high load of 3200N (4 x BW), both on the medial side. Permanent plastic deformation and collapse was allowed only in the cancellous bone, while all other materials were modeled elastically. Under low load conditions within the elastic limit, introducing a gap between the tray and the cortical bone produced a stress/strain intensity in the cancellous bone beneath the edge of the tray. The strain in the cancellous bone within the APT design was generally 3 times greater than the MBT design, however, peak strain values were similar at the edge of the tray. Whilst the strain increased with the introduction of a gap the resulting strain was not sensitive to the gap size for both designs. Under high load conditions, permanent plastic deformation and bone collapse were observed in the cancellous bone at the edge of the tibial tray in both designs where a gap was introduced. The maximum strain in the cancellous bone was found to be more sensitive to the gap size for the APT design than the MBT design. This can be contributed to the difference in the load transfer through the cancellous bone in the two designs. The MBT design with the more rigid tibial tray transfered higher load through the outer cortical bone than the APT design. The less rigid APT design resulted in progressive collapse of the cancellous bone beneath the tray. Particularly significant was the volume of highly stressed cancellous bone which was 4 times greater in the APT design compared to the MBT design. The results suggest that coverage may be a more important parameter for the APT design than the MBT design. The APT design may, therefore, be more suited to patients with better bone quality.

R. Nanda M. Ahmad A.S. Bajwa S. Green P.J. Gregg A. Port

Aim: To compare of strength of constructs using the newer and part specific nail systems: Polarus and European Humeral Nail with that using PHILOS and Conventional plate systems in a simulated 2-part fracture of proximal humerus, in an osteoporotic bone model.

Materials and Methods: A Biomechanical laboratory study was undertaken. Third generation composite Humerus model was used, with short e-glass epoxy fibres forming cortex and polyurethane cancellous core. Low-density polyurethane core (1.2gm/cc) was used to simulate an osteoporotic model. Osteotomy at surgical neck of humerus was carried out to create 2-part fracture of proximal humerus. Samples were fixed using one of the implants- the Polarus nail, the European Humeral Nail, PHILOS Plate, Clover Leaf Plate or T-Plate. Following fixation samples were placed in a custom made jig to fix proximal and distal ends without interfering with implants and osteotomy site. All samples were subjected to cyclical torque, torque to failure, cyclical compression and Compression loading to failure.

Results: The two Nail systems that are specifically designed for fixation of proximal humerus fracture provided significantly better fixation in all the test modalities. PHILOS construct shows less plastic deformation in cyclical torque and cyclical compression when compared to the other plates but the 2 nail systems were far superior. Locking screws did not ‘back off’ in any of the experiments involving the Polarus, European Humeral Nail and PHILOS construct, however ordinary screws used with the conventional plates did back off both in ‘torque and compression’ testing.

Conclusions: Polarus and European Humeral Nail constructs provide better stability in torque and compression as compared to PHILOS, which in turn is a more stable construct in comparison to conventional plating devices.

S.A. Khan A. Kocialkowski

Back pain is a major cause of disability and absence from work. 80% of the population will experience back pain at some point in their lives. In our study we looked at 2 randomised groups of patients. Group 1 patients had only epidural steroid injections (ESI) and group 2 patients had ESI plus radiofrequency (RF). We hypothesized that there is no difference in outcome between group 1 and 2 patients. The 2 groups were sent out a retrospective questionnaire which had 5 parts to it, including SF-36 health survey, pain drawing chart, visual analogue scale (VAS), oswestry disability score (ODS) and a patient satisfaction questionnaire. The patients had treatment between 2002 and 2003 and the post-treatment questionnaires were sent out in May 2004. The SF-36 was scored giving a physical component score (PCS) and a mental component score (MCS) using an online scoring website. The groups studied were from 2 different referral hospitals. The patients were randomised by GP referral being sent to the 2 different hospitals. 115 questionnaires with stamped addressed envelopes were sent out to group 1 patients, out of which 71 were returned (61.7%) and 113 to group 2 patients out of which 55 were returned (48.7%). Statistical analysis was done using the SPSS software programme. As there was some evidence of non-normality Mann-Whitney test was carried out, and for the patient satisfaction questionnaire, chi-squared and fisher’s exact test was used. We found that there was a significant difference among the 2 groups in the PCS (p< 0.0005) and MCS (p=0.017). There was a statistically significant difference among the 2 groups in their pain draw score, VAS and ODS with p values of < 0.0005. In the patient satisfaction questionnaire, 8 questions were asked. Patients were asked to assess how successful the spinal injection was. 35 (67%) patients from group 2 said it was successful, compared with 25 (37%) patients from group 1. 9 (17%) patients from group 2 said it was not successful compared with 27 (40%) patients from group 1. 8 (15%) patients were not sure from group 2 and 16 (24%) were not sure from group 1. The difference was statistically significant with a p value of 0.003. When asked whether they would recommend this type of injection, more patients from group 2 said they would (p=0.029). When asked about the duration of effectiveness of the injection, group 2 noticed an increased duration of benefit compared with group 1 (p< 0.0005). There was no significant difference between the groups when asked how many injections were required (p=0.089) or when asked whether or not they required painkillers (p=0.062). However, more patients from group 2 said that painkillers controlled their pain (p=0.001). When asked if they were able to return to work and do housework/gardening after injection, there were significantly more patients from group 2 being able to do so (p< 0.0005). We conclude that in the patients studied, the group who had radiofrequency treatment and epidural steroid injection did better as compared with patients who had epidural steroid injection alone.

L. Cardone H. Simpson M. McQueen I. Ekrol A. Muir J. McGeough

Distal radial fractures account for 17% of all fractures treated, with peaks in the bimodal distribution corresponding to young and senior patients. External fixation is one of the best techniques to allow quick patient recovery and is necessary for complex fractures, such as that of the distal radius. However, the safe removal time for these frames remains unclear. A conservative approach commonly leaves the external fixator in place for six weeks, which may be unnecessarily prolonged and lead to increased complications. The aim of this work is to develop a technique to quantify, objectively, a safe removal time for these frames. Studies have been conducted on external fixation of tibial fractures, however there are differences that do not allow transfer of these studies to the external fixation of distal radial fractures. These differences include configuration of the fixation frame, bone and fracture geometries, and the application and transfer of the load to the bone. In this work, the dynamic transfer of the load between the fractured bone and the fixator is investigated. An instrumented grip and a measuring device have been developed to monitor the axial force and displacement when the patient applies a load. Using measurements collected by the instrument and data specifying the frame geometry, a finite element model is used to calculate the load carried by the fixator and by the bone, and the rigidity of the new callus is determined. Plotting the rigidity on semi-logarithmic scale the healing rate can be established. This technique has been successfully verified in a laboratory simplified structure representative of bone fracture. The rigidity of several intra-gap materials has been estimated experimentally using the technique, and the results compared to the real value of the material. These measurements do not interfere in any way with the patient treatment and they can be collected from the first day after the operation. The technique has been tested on 14 volunteer patients and the increase in callus rigidity can be detected by measurements during treatment using the technique described. A randomised prospective study has been initiated to validate this technique and investigate the healing process. A positive outcome would enable the rigidity of the new callus bone and the healing rate to be monitored during clinical assessment. Any healing delay or non-union could be promptly detected, improving the quality of the treatment.

B. Ferris S.P. Ahir G.W. Blunn

Introduction: Fragility of the bone is widely regarded as a cause of Colles’ Fracture particularly in middle aged or elderly women[1]. However not every fall results in fracture of the wrist. The normal volar angle of the distal radius is said to be about 10 degrees although in one study the mean volar angulation was found to be 12 degrees with a range from 4 to 23 degrees[2]. We hypothesised that the volar angle of the distal radius or the position of the wrist at impact could affect where the peak stresses occurred during a fall onto the outstretched arm. We investigated the effect of these two variables on the location and magnitude of the peak stresses using finite element analysis.

Materials and Method: A finite element model of the distal radius was constructed in MARC (MSC software, USA). The model was developed from CT data of the right wrist of a 46 year old male. The data was examined by edge detection software (Materialise, Belgium). The inner and outer boundaries of the cortex were imported as curves into MARC. A surface mesh of the distal radius was constructed, from which a 3D solid mesh of the distal radius was generated automatically. The volar angle was modified to represent between 5 to 25 degrees in 5 degree increments. The wrist position was also changed for each volar angle. This varied in 5 degree increments from 0 to 35 degrees, and then at 45, 75 and 90 degrees. Material properties assigned to cortical and cancellous bone were 20GPa and 6GPa respectively with a Poisson’s Ratio of 0.3. The model consisted of 17660 8 noded hexahedral elements and was fully fixed at the cut end of the proximal radius. For each volar angle a load of 500N and 400N was applied perpendicularly to the articular surface across the scaphoid and lunate fossa respectively. The magnitude and location of peak stresses in the proximal and distal radius were recorded.

Results: Results show that the location and magnitude of peak stresses vary as a result of wrist position. Distally the stress rises with increasing dorsiflexion and at 35 degrees exceeds the load to failure. The volar angle does not influence the stresses unless it is 20 degrees or more. Proximally the volar angle had no effect, but if the wrist is in more than 75 degrees of dorsiflexion then the peak stresses exceeded the load to failure.

Conclusion: Results show that a fall onto the outstretched arm will produce differential stresses in the radius depending on the position of the wrist at impact. The volar angle affected the stresses in the distal radius at greater than 20 degrees but proximally it did not. Proximally stresses above 130MPa (when the wrist is in more than 75 degrees of dorsiflexion) will subject the wrist to fracture[3]. Distally (when the wrist is in more than 35 degrees of dorsiflexion) with high volar angles (greater than 20 degrees) is likely to produce the conditions for a fracture (cancellous bone has been reported to fail as a result of fracture at 50 MPa [4] and for osteoporotic bone at 0.44MPa [4].

A Taylor A. Roques M. Browne

During conventional hip arthroplasties, the diseased femur is rigidified using a metallic stem. The insertion of the stem induces a change in the stress distribution in the surrounding femur, and the bone remodels; this stress distribution is a direct result of the stem stiffness characteristics. Healthy healing of the femur requires that the bone be loaded as naturally as possible. If the bone is not loaded appropriately, it can resorb which may result in stem loosening and revision. Although current rigid metallic femoral stems are very successful, a poor stress distribution may become a critical problem for younger patients as the stem/femoral bone construct will be subjected to higher loads for longer times, and since remodelling is faster, loosening can occur earlier. Reduced stiffness stems have therefore been investigated, but early failures have been reported due to increased movements, poor initial stability and the low proximal stiffness of the stem. A novel biocompatible carbon fibre reinforced plastic (CFRP) stem has been developed in light of these past experiences1. Using a series of analytical models and experimental validation tests1, the fibre type and architecture have been tailored along and across the stem to achieve healthy bone remodelling and proximal strength of the construct. In addition, a biocompatible hydroxyapatite coating was specifically designed to enhance interface strength and stability2. The present study describes the mechanical behaviour of this novel stem with particular emphasis on the stem/bone interface. 4 static and 29 fatigue tests were performed according to ISO7206; these tests were complemented by acoustic emission monitoring to identify failure mechanisms3. A stress versus number of cycles to failure (SN) curve was obtained to describe the fatigue behaviour (i) under constant amplitude cycling at various load levels and (ii) incorporating rest periods and overloads. In addition, a mechanical test was designed to characterise the motions between the bone and the stem during sinusoidal fatigue loading (5000 cycles, 0.2–2kN, 1Hz). Two linear variable differential transformers measured the vertical and horizontal displacements at the stem/ bone interface in the proximal region. 3 tests were performed on CFRP stems and 3 on a metallic stem. The CFRP stem exceeded the standard requirements. The SN curve showed good repeatability across the loading spectrum. The inclusion of overloads/static loads during fatigue had a beneficial effect on the stem endurance. This is attributed to the development of microcracks, which dissipate the load, and to creep of the resin. The amplitude of recoverable motion observed at the interface during each load cycle was similar for both types of stem (20mm and 4mm in the horizontal and vertical directions respectively) and remained below the recommended limit4. Composite materials offer high design flexibility. This has been exploited in the development of a compliant, mechanically tailored biocompatible hip stem for femoral reconstruction, and could provide an answer to hip replacement for younger, more active patients.

B.D. Hawes M. Reeves J.A. McGeough A.H. Simpson

Measuring strain in biological specimens has always been inherently difficult due to their shape and surface properties. Traditional methods such as strain gauges require contact and therefore have reinforcing effects, also the surface preparation can be time consuming and if proper fixation is not achieved the results will be inaccurate. Using a non contact method to measure strain such as photogrammetry has several advantages. The strain over the whole surface of a specimen can be mapped, depending on the field of view of the camera used. It has a large dynamic range, from microns to millimetres which can be decided upon at the post processing stage. Specimens can be tested to destruction without damaging any measurement equipment. Also there is considerably less set up time involved between testing different specimens once the system is in place. We aimed to test speckle photogrammetry, a method used in industry and fluid dynamics as a tool for assessing proximal femur fracture stability and repair techniques. A Zwick Roell materials testing machine was used to axially apply a staircase loading pattern to sawbones femora, simulating the load experienced by the femur when standing. Firstly an intact bone was tested then a set of three identical fractures of each of three common fracture configurations were produced by osteotomy. The first femur of each configuration was loaded un-repaired to failure; the remaining two were repaired using common techniques for that particular fracture type then also loaded to failure. The bone and fixation device were covered with stochastic, high contrast paint speckle prior to testing. This speckle pattern was recorded at regular load intervals by a digital camera which was attached to the materials testing machine via a rigid frame to eliminate any camera movement. These images were then transferred to a computer where they were converted to 8 bit bitmap images. Matlab was used to process the data from subsequent images to produce vector and colour maps of the displacements and strains over the entire visible surface of the proximal femur and to show the comparative displacements and strains experienced by the individual bone fragment and the fixation devices. Non contact optical strain measurement has proved itself to be a useful tool in assessing the stability of fractures and the repair techniques of these fractures. Additionally it can also be used to validate finite element models to compare theoretical and experimental results due to the similar data and graphic visualisation outputs which are produced by both techniques.

A. Galvin E. Ingham J. Fisher

Introduction: Crosslinking has been extensively introduced to reduce the wear of UHMWPE. Zero wear of highly crosslinked UHMWPE has been reported by some groups (1) in hip simulators, clinical studies have reported finite wear rates (2). The aim of this study was to compare the wear rates produced by UHMWPE with different levels of crosslinking.

Materials and Methods: Studies were carried out using 28mm diameter cobalt chrome femoral heads. These were articulated against UHMWPE in the Leeds ProSim hip joint simulator. The acetabular cups were manufactured from UHMWPE GUR 1050. The GUR 1050 was highly crosslinked with 10MRad or 7.5MRad of gamma irradiation in nitrogen followed by re-melting at a temperature above 150°C. Slightly crosslinked GUR 1050 was also tested (gamma irradiated with 2.5MRad in air). Non-crosslinked GUR 1050 UHMWPE was used as a control. Five cups of the materials were tested with one station from each set of five being used for creep data. Wear measurements were taken every million cycles using a coordinate measuring machine and tests were run to 5 million cycles. The tests were carried out in low serum concentrations of 25% (v/v) bovine serum diluted with 0.1% (w/v) sodium azide in water. At each million cycles a 3D measurement was taken of the contact region of the acetabular cups using a Form Talysurf profilometer.

Results and Discussion: The wear rate decreased as crosslinking levels increased. The non-crosslinked material had an overall average wear (mm3/million cycles) determined by volume change of 45.6+/−1.35, the 2.5MRad material 46.9+/−9.4, the 7.5MRad 15.04+/−4.28 and the 10MRad material 8.7+/−3.11. The intentionally cross-linked materials showed a significantly lower volume change than the other two materials, with the 10MRad polyethylene having a slightly lower volume change than the 7.5MRad polyethylene. All four polyethylenes showed greater volume change in the first million cycles than the subsequent four and this was associated with initial creep deformation in the first million cycles. The individual creep deformation cups confirmed this with volume changes in the first million cycles followed by stability. Creep volumes of between 10 and 25 mm3 total were measured with the lowest value being for the 10MRad polyethylene. The steady state wear rates for the PE’s between one and five million cycles were 0MRad 36.9+/−1.92 mm3/million cycles, 2.5MRad 44.12+/−10.09, 7.5MRad 7.89+/−2.32 and 10MRad 4.62+/−2.73. The results of the surface topography of the acetabular cups showed that the highly crosslinked materials became smoother than the other materials as the test progressed. This would benefit the crosslinked materials in aiding lubrication and could have contributed to the lower wear rate seen with these materials.

Conclusion: The highly crosslinked UHMWPE gave lower wear volumes than the noncrosslinked materials. This could have been due to the smoother surfaces of the cups as the study progressed which resulted in better lubrication of the components. Finite wear rates have been recorded for the first time with highly cross-linked polyethylene, that compare with clinical observations.

D. Gordon S. Ng Man Sun C. Pendegrass G. Blunn

Introduction: Transcutaneous Amputation Prosthesis (ITAP) is an alternative for transfemoral amputees to conventional stump-socket prostheses which have many problems. These include: poor fit, stump pressure sores, pain, infections and unnatural gait. ITAP aims to overcome these by being osseointegrated into the femoral medulla with a pin protruding through the skin to which the external prosthesis attaches. Thus, the forces normally encountered by the stump soft tissues are now transferred directly to the skeleton. However, the transcutaneous pin produces a route for infection from the external to internal environment. Therefore, a key feature to the success of the ITAP is to produce a biological seal at the transcutaneous interface. Epithelial cells have been shown to attach to dental transcutaneous titanium devices via hemidesmosomes (HD).2 Focal contacts (FC) are also important in cell adhesion and to the underlying substratum.3 We grew human keratinocytes on different titanium surfaces to assess their morphology, ability to proliferate and produce HD and FC. Hypothesis: Surface topography influences keratinocytes morphology and proliferative capacity and expression of HD and FC.

Materials and Methods: 4 titanium alloy (Ti6Al4V) surface topographies were used (10mm x 4mm discs): polished, machine finished, sandblasted and hydrofluoric acid etched (HF) and a control – plastic thermanox. Surface roughness profiling of titanium discs were measured (Mitutoyo Surftest SV-400). HaCaT keratinocytes were grown on disc surfaces in wells of culture medium at +37oC, 5% CO2 and analysed at 1, 2, 3 and 4 days. Cells were processed to visualise HD with fluorescence microscopy using antibodies to the 6-integrin and plec-tin. Anti-vinculin antibodies were used to visualise FC. Fluorescein isothiocyanate (FITC) secondary antibodies enabled counting of structures (all product: Sigma-Aldrich, UK). Alamar blue (Serotec, UK) measured cell proliferation and SEM (surface morphology, cell area) and TEM were also performed. Cells grown on polished, machined and thermanox discs supported a regular, confluent layer with many cytoplasmic processes and dividing cells. HF and sandblasted discs grew an irregularly layer with fewer cytoplasmic processes and fewer dividing cells (not quantified). Day 3 TEM revealed HD, FC and desmosomes; cells on polished and thermanox were more closely packed and in layers.

Conclusion: Keratinocytes are significantly influenced by titanium surface topography. Smooth polished titanium alloy may be the ideal surface for a transcutaneous pin in the ITAP. Further experiments into isolating favourable biological components needed to encourage keratinocytes to attach onto titanium should be carried out.

Results: No significant difference shown in cell proliferation between titanium discs but cells on thermanox grew significantly more (p< 0.05). FC and HD numbers increased on all surfaces (days 1–3); a negative correlation between surface roughness and HD and FC numbers observed (lower Ra values = more HD and FC expressed).

A.J. McLean C.R. Howie J.A. McGeough A.H. Simpson

Introduction: Tibial component loosening is a common mode of failure in modern total knee arthroplasty and is thus a common cause for revision knee surgery. Direct bone ingrowth of press fit knee prosthesis has been deemed an important prerequisite for long-lasting implant fixation and thus clinical success in both primary and revision TKA whether for cemented or uncemented stems. To achieve good long term biological stabilization, initial secure mechanical stability, (i.e. minimising tibial tray and stem motion with respect to the tibia,) is vital. A lack of initial stability can lead to resorption of bone at the implant-tissue interface and can consequently result in loosening and failure of the prosthesis. Obtaining adequate tibial fixation is difficult in revision patients as often there is insufficient bone stock in the proximal tibia. A longer stem is often recommended with revision surgery as a central stem should guide the migration of the tibial component so that it occurs predominantly along the vertical axis, thus minimising the risk of recurrent malalignment and loosening due to tilting of the tibial tray. It is also thought that the presence of a third rigid peg helps to reduce inducible displacements by anchoring the new implant in robust cancellous bone. However there is no consensus on the length of central stem should be to achieve the best load transfer and fixation and although the use of long stems on the tibial component is advocated, in revision TKA involving bone grafting and augmentation. The effect of the tibial stem length in other cases has received contradictory evaluations. This research deals with an experimentally evaluate the effect that central stem lengths on the initial micromotion of the tibial tray in two revision tibial defects. This is being investigated by measurement of the bone-implant interface motion of the tibial stem.

Method: Composite bones were resected with an extramedullary jig. Three common revision defects were compared 1) no defect requiring no repair(primary); 2) T1 defect requiring bone impaction grafting; 3) T2A requiring augmentation. Three stem configurations were analysed in conjunction with these defects 1) no stem; 2) short 40mm stem; 3) long 80mm stem. Four LVDTs were positioned anteriorly, posteriorly, medially and laterally around the tray and were used to measure the movement of the tibial tray with respect to the tibia. The bones were potted and subjected to axial loading simulating 1– 6 times body weight for 3500 cycles at 1 Hz.

Results: The longer stemmed press fit implants were associated with slightly higher levels of micromotion compared to the “no stem press fit” trays in the primary and T2A defects. This could be due to the fact that cutting errors are accentuated by a longer stem and can cause increased levels of posterior lift off. For bone impaction grafting it seems that a stem sufficiently long to by-pass the defect should be used. The proximal surface cemented trays presented more stable fixation with the inducible displacement between the no stem and stemmed groups being negligible. Subsidence of the tibial tray was reduced marginally by using a longer stem.

J. Caruana K. Mannan A. Sanghrajka D. Higgs G.W. Blunn T.W. Briggs

Introduction: Surgeons in the UK and Europe generally use a thinner cement mantle than their counterparts in the USA for the femoral component in total hip replacement (THR). The aim of this study was to compare the performance of different thicknesses of cement mantle using finite element analysis. The measures by which comparison might be made include cement cracking, subsidence, migration and stress shielding. In this study, we use a linear-elastic model of the implanted femur to give a prediction of the stresses in the cement mantle and in the femoral cortex. These measures give an indication of the relative rates of cement cracking and loss of bone stock due to stress shielding. To assess the reliability of our model in representing patients with different bone densities, we use a range of cancellous bone stiffnesses.

Method: Two cadaveric femora from the same donor were sized, reamed and implanted with identical plastic replica femoral components following standard surgical technique for the Stanmore Hip system. One was prepared using UK rasps, over-reaming by ~2mm, the other using US rasps, over-reaming by ~5mm. Serial CT-scans were used to create three-dimensional geometric models of the implanted femora. Two finite element meshes were hand-built in MSC. Marc finite element software, incorporating cortical and cancellous bone, bone cement and prosthesis. Each model consisted of 10,000 eight-noded brick elements, with a fully bonded stem-cement interface. The thick and thin cement mantles had thicknesses of 2.5mm and 1.0mm respectively, in regions where thickness is affected by rasp size. Models were identical in the distal medullary canal. Cortical bone was modelled as transversely isotropic, with longitudinal and transverse moduli of 17.0 and 11.5 GPa. Bone cement was given a modulus of 2.7 GPa. Loading conditions were chosen to represent the heel-strike phase of gait. In order to assess the impact of variability in patient bone density, cancellous bone modulus was varied between 0.06 and 2.90 GPa.

Results: Equivalent stress was examined on the external surface of the cortex and the internal surface of the cement mantle. The lowest cortical bone stresses were proximal and the highest cement stresses around the distal tip of the prosthesis. In the proximal cortex, higher equivalent stresses were observed medially and laterally with a thick cement mantle. Distally, lower cement stresses were observed in the thick cement mantle. With the highest cancellous modulus, there was little difference between the two models. As this modulus was reduced, stress differences between the models became more apparent. For all cancellous bone moduli, peak distal cement stresses were lower and minimum proximal calcar stresses higher in the thick cement mantle.

Discussion: Proximal stress shielding was greatest in the calcar, in agreement with clinical findings. The thicker cement mantle led to less stress shielding in this region. Cement stresses, highest around the distal tip of the prosthesis, were larger in the thin cement mantle. This suggests a higher rate of both cracking and bone resorption in thin cement mantles. Although observed over a range of cancellous bone stiffness, this finding applies particularly to patients with low bone density.

T. Azzopardi P. McLachlan B. Meadows

Conventional fixed-bearing (FB) knee prostheses have been proved clinically successful. Rotating platform, mobile-bearing (MB) total knee replacements (TKR) have been developed to improve knee kinematics, lower contact stresses on the polyethylene tibial component, minimize constraint, and allow implant self-alignment. The purpose of this study was to characterize and compare the functional outcome of FB- and MB- TKR during gait and deep knee bends, using a motion analysis system. Two groups of five patients with a unilateral FB TKR (PFC) or MB TKR (LCS) underwent a gait analysis study. The normal contralateral limb was used as a control to compare data in the stance phase of gait. Demographic, clinical, and radiographic data were equivalent in the 2 groups. Both MB and FB TKRs gave good functional results in spite of different design rationales. No statistically significant difference was demonstrated between the two groups. However, gait and knee function after TKR was abnormal even though the patients were asymptomatic. A flexional pattern for flex-ion-extension moments at the knee during level walking was present in both types of TKR. Differences in rotational moments between the two groups were observed, with a higher internal rotational moment in the PFC group (PFC, 0.14 Nm/kg; LCS, 0.09 Nm/kg; p=0.094). A stressful weightbearing activity, such as deep knee bends, amplified the functional differences between the different prosthetic designs, indicating that knee kinematics are activity-dependent. Kinetic and kinematic differences noted between the 2 groups reflect different patterns of joint surface motion and loading, with postulated effects on long term failure of the implants through wear, mechanical failure, and loosening. Gait analysis using external skin markers has a limited role in the characterization of the joint surface motion of the prosthetic knee during ambulatory activities because of errors and assumptions inherent in the technique. However, it provides scope for the study of kinetic parameters acting on different knee prostheses during gait.

T. Ibrahim S.M. Ong G.J.S. Taylor

Background: The commonest cause of long term failure of total joint arthroplasty is aseptic loosening. As a result, many patients will require complex revision surgery that is not only technically challenging but associated with poorer results. Revisions procedures are also associated with higher morbidity and costs.

Aim: To quantify osteolysis in a small animal model of aseptic loosening. This model can then be utilised for screening therapeutic agents to inhibit aseptic loosening.

Materials and Methods: 7 time mated female mice were injected with radioactive calcium 45 on day 14 of gestation. The 52 offsprings were divided into 2 equal groups and subjected to either the implantation of clinically relevant ceramic particles or sham surgery into their femora. The non-operated femora were used as control. Animals were killed 4 weeks following surgery. Femora were retrieved, dissolved and radioactivity measured as outcome (CPM/mg = Counts Per Minute per milligram). A Linear mixed effects model was utilised to examine the difference in outcome between the 2 groups.

Results: The mean scintillation count for sham surgery was 388 CPM/mg compared to 449 CPM/mg in the control femora. The mean scintillation count for ceramic particles was 351 CPM/mg compared to 420 CPM/mg in the control femora. The mean effect on outcome of surgery with ceramic particles relative to sham surgery was estimated at 16.7 CPM/mg (95CI%: 0.9 to 32.5 CPM/mg; p = 0.025).

Conclusion: We have successfully shown that this model can quantify osteolysis. However, the difference detected between sham surgery and ceramic particles was biologically small displaying the inert properties of ceramic. Extending the post surgery interval might show a larger difference between sham surgery and ceramic particles and permit quantitative analysis of therapeutic agents to be screened to inhibit aseptic loosening.

Y.S. Lau A. Sabokbar A. Berendt B. Henderson S.P. Nair N. Athanasou

Osteomyelitis commonly causes bone destruction and is most frequently due to infection by Staphylococcus aureus. S. aureus is known to secrete a number of surface-associated proteins which are extremely potent stimulators of bone resorption in the mouse calvarial assay system. The precise cellular and humoral mechanisms whereby this stimulatory effect is mediated, in particular whether osteoclast formation or activity is directly promoted by these factors, have not been determined by this study. Surface-associated material (SAM)(0.001ug/ml)obtained from 24 hour cultures of S. aureus was added to cultures of mouse and human osteoclast precursors (RAW 264.7 cells and human peripheral blood mononuclear cells respectively). These cultures were incubated in the presence and absence of receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony stimulating factor (M-CSF). It was found that independent of RANKL, SAM was capable of inducing osteoclast formation in cultures of RAW cells and human monocytes. This was evidenced by the generation of tartrate-resistant acid phosphatase-positive multinucleated cells, which formed lacunar resorption pits when these cells were cultured on dentine slices. In cultures where M-CSF, RANKL and SAM were added, osteoclast formation was increased, but did not exceed the osteoclast formation in cultures with M-CSF and RANKL. These findings indicate that S. aureus produces a soluble factor which can promote osteoclast formation. Identification of this factor may help to develop therapeutic strategies for treating bone destruction due to Staphylococcal osteomyelitis.

A. Shoaib A. Guha R. Balendran J.H. Kuiper

Introduction: Tension band wiring is a common technique for olecranon fracture fixation. The most commonly used material for the tension band is stainless steel wire. There are however problems associated with stainless steel wire. Ethibond (Ethicon Ltd, Edinburgh) has previously been cited as a suitable alternative material but not FiberWire. The biomechanical properties of FiberWire (Arthrex Ltd, Sheffield) as a tension band material have not been evaluated. This study aimed to investigate the properties of FiberWire and compare them with stainless steel wire and Ethibond.

Methods: Saw-bone olecranons were osteotomised identically to create an olecranon fracture. Identical tension band constructs were produced using stainless steel wire, Ethibond and FiberWire. The construct was tested by cyclical loading with an ESH dynamic testing machine (Brierley Hill, West Mids). A preload of 5N was applied before cyclical loading at levels up to 200N. The fracture gap was measured with a displacement transducer (Tokyo Sokki Kenkyujo Co, Japan).

Results: At loading up to 100N, the stainless steel wire allowed an average fracture gap of 200 micrometers. 5 gauge Ethibond allowed a larger fracture gap of 350 micrometers (p< 0.05). 2 gauge Fiberwire did not allow a significantly different fracture gap to Ethibond.

Discussion: The fracture gap with suture material was greater than with stainless steel wire, but still less then 0.5mm with loading of 100N. Free body diagram calculations determine that in a 70 kg man, this would correspond to the forces expected in extending the elbow against gravity. This means that these alternative materials are mechanically suitable for use in clinical practice for tension bands. This can avoid some of the complications of stainless steel wire.

Conclusion: 5 gauge Ethibond and 2 gauge FiberWire are biomechanically suitable as alternatives to stainless steel wire in tension band wire fixation of olecranon fractures.

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N.M. Everitt S. Rajah D.S. McNally

Bone microhardness has been successfully correlated with important functional parameters such as mineralisation and stiffness. It provides a means of examining the mechanical competence of bone at a micron scale, averaging the effect of osteonal lamellae but sensitive to variation in mineral content within a bone, and, with careful selection of indentation site, able to obtain material characteristics separate from any effects of porosity. However, the effect of bone’s viscoelasticity on such measurements has been largely ignored. This preliminary study investigates the post-indentation size change of Vickers indentations on wet bone. 4 axial slices of bovine femur were harvested from the same shaft, and polished. Each sample was subjected to 4 sets of 10 Vickers indentations with a load of 50 g and holding period of 15 s. The indentation size was measured immediately after the load was removed, and then again at intervals for a period up to 24 hours after the indentation was made. To avoid dehydration, the bone stood in water during the indentation testing and during measurement, and between each measurement period it was fully immersed in water. Measured hardness significantly decreased with time, by approximately 30% in total. The rate of post-indentation recovery is difficult to analyse since the driving force of residual strain decreases as recovery takes place. However a simple exponential fit to the variation of HV with time in the form of H = H(final).(1−exp(−kt)) + H(initial) suggests that the size of the indentation tends towards a constant size between 5 and 24 hours after indentation. Thus we conclude that care should be taken when making “early” measurements given the rapid rate of change in indentation size. Caution should also be employed when interpreting such data.

K. Giesinger M. Reeves H.A. Simpson

Since cementless stem fixation in hip arthroplasty is becoming more and more common, the overall incidence of intraoperative femoral fractures has risen considerably. Depending on primary or revision arthroplasty, literature reports fracture rates between a few percent up to one third of the cases.

In this study, methods commonly applied in the field of structural testing were customized for this specified interference fit situation. A cementless hip system (ABG II, Stryker) was used on animal bones and biomechanical bones.

Transient excitation in the form of regular hammer strokes and sinusoidal excitation using a shaker served as an input. The output of the system under test was measured on the greater trochanter using a piezoelectric accelerometer.

The signals were digitized with a high-speed data acquisition system and analyzed in real-time with spectrum analysis software.

Analysis included threshold detection in the time domain to determine the time delay between the input and output transducer. Spectrum analysis in the frequency domain included FFT analysis and frequency response function analysis to identify shifts of fundamental frequencies and harmonics to describe the vibrational changes with increasing stability.

A digital imaging system was set up to take pictures of the metal-bone site to measure inducible displacement with each hammer impact and correlate it with the vibrometry results. Furthermore a strain gauge circularly mounted around the proximal femur monitored accurately any hairline fracture.

This study shows that changes of the vibrational spectrum are directly related to implant fit. The range of interest is well in the sonic range, which apparently is the reason for many surgeons to listen and ‘feel’ carefully during advancing the broach or the final implant into the femur.

The study is trying to extract critical vibrational parameters correlated with stability and femoral integrity. Due to the different dimensions of the tested animal bones and lack of soft tissue damping, further experiments on cadavers need to be carried out.

Vibrational spectrum analysis could prove to be a useful tool to readily assess implant stability and femoral integrity. It seems to be most beneficial in revision surgery or minimally invasive hip replacement, where the risk of femoral fractures is increased or fissures could easily be missed.

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N.M. Everitt K. Namvari P. Pollintine M.A. Adams D.S. McNally

Cortical porosity is a useful evaluator of bone since it is sensitive to changes in bone turnover. The aim of this study was to evaluate cortical bone porosity of human vertebrae samples using Scanning Acoustic Microscopy (SAM). Currently the common techniques used to determine bone porosity are histomorphometry or scanning electronmicrosopy images. Both methods require extensive preparation of the bone samples. SAM represents a new technique with the great advantage of minimal sample interference since the bone is imaged in water, or saturated, and requires just one flat surface which is scanned (but not contacted) by the transducer. 46 specimens between the ages of 64–90 years were randomly selected and ground before SAM imaging of was carried out using a 400 MHz transducer. For each sample posterior and anterior sections of the cortical bone were scanned several times, and the porosity measured using Scion image software to process the images. It was possible to image the entire anterior or posterior cortex in a single image with 4 mm spatial resolution. Measured porosity was in the region 5 % – 21 %, and showed a significant increase with age for the female specimens but no age dependence in the male specimens. At low porosity (< 6 %) vertebral compressive strength was uncorrelated with porosity. However, at higher porosities strength was highly correlated with porosity. (As would be expected, strength decreased with increasing porosity). High frequency SAM has potential for future bone characterisation, particularly where it is desirable to correlate local measurements of material properties such as nanohardness or microhardness, with microstructure.

F. Harrold M. Apreleva J. Warner C. Wigderowitz A. Gerber

Introduction: Restoration of original humeral head geometry in shoulder arthroplasty is a necessary requirement and may have a bearing on the longevity of the implant. Modern, adaptable, prosthetic components are believed to allow restoration of the individual’s proximal humeral anatomy, provided a precise osteotomy of the humeral head at the level of the anatomical neck is performed. The osteotomy and reconstruction of the humeral head is based on the assumption that the resected articular segment corresponds to a segment of a sphere oriented, identically, in inclination and retroversion to the original humeral head. Resection, along the mid-anterior portion of the cartilage/calcar border, is understood to create a surface that enables a prosthetic component to be mounted, retroverted and inclinated to the same degree as the original head geometry. The objective of this study was to determine the degree of variation in humeral head retroversion relative to the superior and inferior borders of the proximal humeral articular surface.

Methods: Twenty-eight fresh frozen human cadaveric full arms were dissected free of soft tissue to expose the proximal humerus. The distal end of the humeral shaft was potted in PMMA and fixed rigidly in a custom–built jig. The following points and lines were identified and marked on each specimen:

the circumference of the anatomical neck;

(H) as the most superior point of the articular surface at the insertion of the supraspinatus tendon, (L) as the corresponding lowest point of the articular surface at the cartilage/calcar interface;

The medial (MC) and lateral (LC) humeral condyles were exposed and delineated with k-wires.

A Microscribe 3D-X digitizer was used to digitize the points and lines. The data for each humerus were imported into Rhinoceros NURBS modelling software and graphically represented. The constructed graphical model was used to divide the articular portion of the humeral head into six equal sections in the axial plane. The retroversion angle, relative to the epicondyles, was calculated for each section.

Results: A linear decrease in retroversion angle was noted from the most superior to most inferior point on the proximal humeral articular surface. The retroversion angle was greatest at the level of the insertion of the supraspinatus tendon (34.2deg +/−13.7deg) and least at the inferior cartilage/calcar interface (24.3deg +/−10.2deg).

Discussion: Accurate recovery of humeral head geometry is a requirement in order to achieve good function. The variability in retroversion, as it relates to its point of measurement, may effect the accuracy of pre-operative assessment of a patient’s humeral head geometry as well as the osteotomy during shoulder arthroplasty, and, thus, may impact on joint range of motion and stability post-operatively. Further investigation is warranted.

S. Rehman S. Johnson K. McKinlay N.M. Everitt D.S. McNally

Intervertebral disc function and dysfunction is governed by its structural architecture of concentric layers of highly ordered collagen fibres. This architecture is important at the mm scale for overall mechanical performance of the disc; and at the micron scale for mechano-transduction signalling pathways of the disc cells that are responsible for matrix maintenance and therefore disc health. To understand such mechanical behaviour 3-dimensional collagen fibre architecture must be quantified in intact intervertebral discs. Conventional imaging modalities lack either the spatial resolution (e.g. x-ray diffraction) or penetration (e.g. optical, electron or confocal laser microscopy) to yield mechanically important information. Preliminary studies of scanning acoustic microscopy (SAM) at 50 MHz visualises alternating layers of fibre texture, however exactly what is being imaged requires both explanation and validation. Three-dimensional SAM data sets obtained from intact discs were compared to polarised-light and scanning electron micrographs of individual layers of fibres, peeled by micro-dissection from discs. The dimensions of the structural features were measured and recorded. Optical and electron microscopy revealed that each layer consisted of highly oriented collagen fibres of diameter 5 μm with regularly spaced splits between fibres with a spacing of approximately 20–30 μm. The SAM data sets showed layers with a uniform highly oriented fibre texture that reversed between adjacent layers. Resolution of the texture was limited by the acoustic system to approximately 30 μm. It is clear that SAM at 50 MHz cannot resolve and therefore image individual collagen fibres. However, the regular defects in the fibre layers can be visualised and convey complete information about local collagen fibre architecture. SAM therefore provides an effective way of quantifying the fibrous structure of intact, hydrated, unfixed intervertebral discs.

F. Attar R. Shariff D. Selvan D. Machin N. Geary

Background and Aim: It was observed by the senior author over 15 years that if the foot became dependant in the 1st 48 hours after foot surgery, the patient suffered marked swelling and pain. This effect seemed less after about 48 hours. The practice was adopted of keeping the foot elevated for at least 48 hours. Aware of the work of John Tooke and Gerry Rayman with postural effects on laser Doppler skin flow, we set out to see if there was a demonstrable scientific basis for this practice.

Materials and Method: Laser Doppler flow meter was used to assess blood flow in 14 patients, (16 feet), undergoing foot and ankle surgery. Flow was recorded in the big toe, at heart level and on dependency, preoperatively, and then sequentially at 24, 48, 72 and 96 hours post operatively. Postural vasoconstriction was calculated using the formula; Postural Vas.(%)=Blood flow at heart level – Blood flow on depend./ X 100 Blood flow at heart level The time taken for blood flow in the toe to get back to the pre-operative values was assessed. Room temperature, patient temperature and patient position were all kept constant.

Results: Postural vasoconstriction was recorded for all 14 patients at 48 hours, for 7 patients at 72 hours, and for 2 patients at 96 hours post operatively. All patients had an ankle block, except 2 patients who had a popliteal block. The mean postural vasoconstriction preoperatively was 51.31%; mean at 24 hours post op. was 23.05% mean at 48 hours post op. was 36.62% and mean at 72 hours post op. was 44.24%. The mean operative time was 87.25 minutes. There was a significant difference between the pre-op levels and the 24, 48 and 72 hours post-op levels (p< 0.05). At 96 hours post-op, the difference wasn’t significant. Greater operative time was associated with less postural vasoconstriction at the 72 hours postoperatively.

Conclusion: Results showed that it takes longer than 72 hours for microcirculation to get back to normal rather than 48 hours, but the return towards normality was evident by that time. The results emphasised the importance of post-operative foot elevation for at least 48 hours due of this phenomenon. With increasing operative time, it took longer for the microcirculation to get back to normal. The longer the surgery the longer the period of elevation required. We believe that this practice minimises post operative complications; such as oedema, wound breakdown, pain on dependency. No patients suffered DVT’s or PE’s. However, patients did start with active and passive foot and lower limb physiotherapy soon after surgery.

A. Shoaib S. Kili S. Underhill

Introduction: Radial Tunnel Syndrome is characterised by pain over the anterolateral proximal forearm. It is thought to result from compression of the posterior interosseous nerve, and is estimated to be present in 5% of tennis elbow syndrome. This condition has been treated with surgical decompression with varying success. Other nerve compression syndromes are treated with steroid injections and these are reliable in giving relief, even if only temporary. Blind injections have been used as a diagnostic tool in Radial Tunnel Syndrome, but guided steroid injections have not yet been evaluated.

Methods: Patients attending one hospital over three years with unresolved lateral epicondylitis were clinically diagnosed with radial tunnel syndrome. They underwent injection of the radial tunnel guided by a nerve stimulator. The patients were evaluated prior to the injection with a brief pain inventory score produced by the British Pain Society. They were evaluated three months after the injection with a further brief pain inventory score. The scores were compared with Student’s t test.

Results: Ten patients were recruited over three years. The average pain score before and after injection was not significantly different ( P = 0.4386). An interference score reflecting impeding of function as a result of pain was also not significantly different ( P = 0.095).

Discussion: The results of guided injection are worse than a series of blind injections in the literature. We question why this has occurred when the opposite would have been expected. There are several possible explanations. There may have been a double hit phenomenon with the injection only affecting one of two pathological areas. The area of compression may be more proximal to the site of injection. The pathology of radial tunnel syndrome has not been definitively described and the pathology may not be one suitable for treatment with a steroid injection. Post mortem examination of the posterior interosseous nerve in patients with this syndrome may reveal the pathology of the syndrome.

Conclusion: Guided injections for radial tunnel syndrome are not effective. The pathology of this syndrome may be different to conventional nerve compression syndromes. We question indications for surgical decompression and suggest further work to investigate this syndrome.

K. Sokhi S. Whiten C. Wigderowitz

Introduction: The current study investigates the influence of the interosseous portion of the scapholunate and the radioscaphocapitate ligaments in the range of movement of the scaphoid and lunate during flexion and extension of the wrist.

Material and Methods: 10 embalmed cadaveric specimens were studied. A jig was designed to clamp the forearms, holding the wrist in a fixed angle. Metal pointers with 1.5mm diameter were inserted into the scaphoid and the lunate. Digital cameras were aligned from the posterior and radial views and sequential photographs obtained during the full range of motion of the specimens. The SL and the RSC ligaments were then sectioned with new series of photographs obtained between each step. The angles in the photographs were measured with specific software.

Results: Sectioning the SL increased the angle between the lunate and the scaphoid by 12° on average, while sectioning the RSC increased the SL angle by a further 2 ° with the wrist in maximal flexion. With the wrist in extension the angle was also increased by 12° after division of the SL and a further 2° after section of the RSC.

Conclusions: Although volar and dorsal inuries may be associated, our study suggests that the SL has a much greater influence on the relative movement of the scaphoid and lunate than the RSC. The method also suggests that in a number of cases the variation in agulation may be small enough not to be detected as significant on x-rays.

P. Theobald M. Benjamin C. Dent L. Nokes

This study aimed to explore the relationship between the geometry of the tuberosity located superior to the Achilles tendon enthesis and the thickness of its fibro-cartilaginous periosteum. The tuberosity acts as a pulley for the tendon during dorsiflexion of the foot and is thus compressed by the overlying tendon. This can result in pressure-related injuries which account for a significant number of Achilles-related problems among sportsmen or women. We postulated that variations in the contact area between the tendon and the tuberosity (and consequently the pressure exerted by the tendon) affects the periosteum thickness. Here, we report four methods of portraying the two dimensional geometry of the superior tuberosity. Material was obtained from 10 elderly dissecting room cadavers donated to the Cardiff University for anatomical examination and prepared for routine histology. Serial sagittal sections were collected at 1 mm intervals, and stained with Masson’s trichrome, toluidine blue and haematoxylin & eosin. In the first method, the area of the bursal cavity was measured between the deep surface of the tendon and the tuberosity within a 9mm radius of the proximal part of the attachment site. The second technique was similar, though used the long axis of the tendon as a reference, rather than its deep surface. The third technique measured the area of the tuberosity within 20 degrees of the tendon long axis. The final technique measured the cumulative gradient of the first 5 mm of the tuberosity, with reference to the tendon long axis. The periosteum thickness was measured at 500 μm intervals from the proximal part of the enthesis and mean values calculated. A good correlation was seen between all techniques, with the tuberosities having the most localised area of contact with the tendon, showing the thickest periosteum.

S. Kerrigan I.W. Ricketts S.J. McKenna C.A. Wigderowitz

The present study investigates the repeatability of two new methods of measuring acetabular wear with differing levels of automation. Experimental evaluation showed that the more automated method was more repeatable. Both methods segmented the femoral head and acetabular rim with ellipses. The displacement of the ellipse centres was measured and the difference at year 1 and 5 taken as a measure of wear. Measurements were obtained twice for each case. The less automated of the two methods involved the annotation of 9 points on the femoral head and 18 on the acetabular rim to which two least squares ellipse fits[1] were performed. The second and more automated method was active ellipses[2][3]. This method uses iterative robust ellipse fitting and a model of appearance learned from a training set to cause two ellipses to converge on the contours of the femoral head and acetabular rim from a single starting point. Fifty cases with radiographs taken at year 1 and year 5 were measured by both methods. The radiographs contained CPTs with 28mm heads and were digitized at 150 dpi. Fifty postoperative radiographs containing 22.225mm Zimmer CPT heads trained the more automated method. None of the radiographs had metal backed cups or highly eccentric rims. The repeatability coefficient (2 standard deviations) of the active ellipses was 0.23mm and that of the best annotator was 0.40mm while the worst was 2.69mm due to an outlying measurement. Limits of agreement were calculated between the two methods as −0.61mm to 0.91mm and show the active ellipses could replace annotation. Given that the active ellipses are nearly twice as repeatable this is desirable. The range of difference in measurements for the active ellipses is less than that of the annotator.

J. Reilly A. Clift L. Johnston A. Noone G. Philips D. Rowley F. Sullivan

Surgical site infection (SSI) is an important outcome indicator. It is estimated that 70% of post-operative infections present after discharge. A reliable post-discharge surveillance (PDS) method is yet to be described. The aim of this prospective cohort study was to assess the reliability of patient self-diagnosis. Telephone questionnaires were used following hip and knee prosthetic surgery. A trained validation nurse checked the wounds of all patients reporting problems and a sample of those who did not. 376 elective hip and knee arthroplasty procedures from 363 patients were included. In-patient infection rate was 3.1% (13 of 422 procedures) and post-discharge infection rate was 5.2% (22 of 422 procedures). Results suggest that patients can reliably self diagnose SSI. The sensitivity of the procedure (the probability that the telephone surveillance will detect an infection given that the patients has an infection) was 90.9%. The specificity (the probability that the telephone surveillance will report no infection given that no infection is present) was 76.6%. Hence telephone PDS of SSI is a valuable means of identifying accurate rates of hospital acquired infection following surgery. In this study population, 41% of infections were diagnosed post discharge, which is lower than has previously been estimated. PDS of SSI is necessary if accurate rates of hospital acquired infection following surgery are to be available.

T. Ibrahim S.M. Ong G.J.S. Taylor

Background: Aseptic loosening of total joint arthroplasty is characterised by osteolysis caused by osteoclasts and macrophages. Osteolysis occurs by acidification and dissolution of hydroxyapatite crystals then proteolysis of the bone collagen matrix. N-Telopeptide (NTx) and deoxypyridinolone (DPD) represent highly specific markers for bone resorption.

Aim: To investigate whether urinary NTx and DPD generated in-vivo can be used as bone markers in a small animal model of wear debris induced osteolysis.

Materials and Methods: 41 and 38 urinary samples were collected from mice at autopsy four weeks following either the implantation of clinically relevant ceramic particles or sham surgery into their femora and assayed for NTx and DPD respectively. Bone markers were corrected for urinary creatinine.

Results: The mean urinary NTx concentration for mice that underwent the implantation of clinically relevant ceramic particles was 95.0 nM BCE/mM creatinine compared to 85.3 nM BCE/mM creatinine for mice who had sham surgery (p = 0.8, 95%CI: −29.0 to 30.7). The mean urinary DPD concentration for mice that underwent the implantation of clinically relevant ceramic particles was 5.3 nM DPD/mM creatinine compared to 4.0 nM DPD/ mM creatinine for mice who had sham surgery (p = 0.07, 95%CI: −2.8 to 1.4).

Conclusion: The absolute values of NTx and DPD increased in mice that underwent the implantation of clinically relevant ceramic particles compared to sham surgery even though this was not statistically significant. Extending the post operative interval might allow both NTx and DPD to be utilised as bone markers of osteolysis in our small animal model of aseptic loosening.

J.D. Moorehead R.K. Kundra N. Barton-Hanson S.C. Montgomery

Introduction: The Lachman test for anterior cruciate ligament (ACL) deficiency, requires a subjective assessment of joint movement, as the tibia is pulled anteriorly. This study has objectively quantified this movement using a magnetic tracking device.

Materials and Methods: Ten patients aged 21 to 51 years were assessed as having unilateral ACL deficiency with conventional clinical tests. These patients were then reassessed using a magnetic tracking device (Polhemus Fastrak). Patients had magnetic sensors attached around the femoral and tibial mid-shafts using elasticated Velcro straps. The Lachman test was then performed with the patient lying within range of the system’s magnetic source. The test was performed three times on the normal and injured knees of each patient. During the tests, sensor position and orientation data was collected with an accuracy better than 1 mm and 1 degree, respectively. The data was sampled at 10Hz and stored on a computer for post-test analysis. This analysis deduced the tibial displacement resulting from each Lachman pull.

Results: The main Lachman movement is an anterior displacement of the tibia with respect to the femur. The mean anterior movement for the normal knees was 5.6 mm (SD=2.5). By comparison the ACL deficient knees had a mean anterior movement of 10.2 mm (SD=4.2). This is 82 % more. A paired t test of this data showed it to be highly significant with P = 0.005. In addition to the anterior movement, there was also a small proximal tibial movement. In the normal knees the mean movement was 0.7 mm (SD=1.9). In the injured knees the mean movement was 2.1 mm (SD=3.4). However, this difference was not significant (P = 0.12).

Conclusion: This study has quantified the movement produced during the Lachman test for ACL deficiency. The results compare well with reported results from similar arthrometer tests[1]. The main advantage of the magnetic tracker is that its lightweight sensors cause minimal disturbance to the established clinical test. It therefore offers a convenient and non-invasive method of investigation.

M. Ahmad M.J. Trewhella N.C. Bayliss

Aim: A study was done to investigate the range in size and morphological features of a se