Bone has a number of different functions in the skeleton including the physical roles of support, protection and sound wave conduction. The mechanical properties, required for these different functions varies and can be achieved by compositional adaption of the bone material, in addition to changes in shape and architecture. A number of previous studies have demonstrated the relationship between mechanical function and mineral to collagen ratio in bones from different species.

The aim of this study is to test the hypothesis that the mineral to collagen ratio is higher in bone with a mechanically harder matrix within a species.

The red deer (Cervus elaphus) (n=6) was chosen as a model for studying bone with extreme properties. The mechanical properties of the antler, metacarpal bone and tympanic bulla were defined by indentation using a bench-top indentation platform (Biodent). The mineral to collagen ratio was quantified using Raman spectroscopy. The deposition of mineral was studied at macro-level using pQCT.

The results showed that the hardness (Indentation Distance Increase) was lowest in the metacarpal (8.5µm), followed by the bulla bone (9.4µm) and highest in the antler (14.5µm). Raman spectroscopy showed a mineral:collagen ratio of 1:0.10 (bulla), 1:0.13 (metacarpal) and 1:0.15 (antler) for the different bones. This does not follow the more linear trend previously shown between young's modulus and the mineral:collagen ratio. The location of the mineral appeared to differ between bone types with pQCT revealing locations of concentrated density and banding patterns in antler. Interestingly, Raman spectra showed differences in the amide peaks revealing differences in protein structure.

The results reject the hypothesis but also suggest that the organisation of mineral and collagen has an impact on the hardness modulus. We demonstrate that the red deer provides a good model for studying bone specialisation. This work will provide the basis for further investigation into collagen as a controlling factor in mineral deposition.

Summary

Treatment of equine naturally occurring over-strain tendinopathy with mesenchymal stem cells suspended in bone marrow supernatant resulted in significant improvements compared to saline treated tendons in the normalisation of biomechanical, morphological, and compositional parameters with no adverse effects.

Clinical evidence that patients with type 2 diabetes mellitus (T2DM) have increased risk of fractures is reported. Furthermore, thiazolidinediones, used to treat T2DM increases the risk of secondary osteoporosis & subsequent fractures. The osteogenic potency of metformin is reported in vitro, few studies have investigated the effects of metformin on bone mass and fracture healing in vivo. We aimed to investigate the effects of metformin on fracture healing in vivo.

Osteoarthritis is associated with changes to the matrix composition of subchondral bone. Raman spectroscopy has the potential to detect in vivo the molecular changes in osteoarthritic subchondral bone. The objectives were to determine the levels of mineralisation, carbonate accumulation and bone remodelling in osteoarthritic subchondral bone, which we defined as within 3mm of articular cartilage. This was compared to the proximal-compartment (10mm distal to articular cartilage) and the head-neck junction. Five osteoarthritic (average age: 76 years) and five normal cadaveric femoral heads (average age: 72 years) were scanned using peripheral quantitative computed tomography and then sectioned coronally. Raman spectroscopy was then used to scan the femoral heads. All scans were done in the plane of the longitudinal axis of the diaphysis. Cores were subsequently extracted and sodium dodecyl sulphate polyacrylamide gel electrophoresis performed to determine the levels of homotrimeric collagen. The phosphate-to-amide I ratio, from the Raman spectra, in osteoarthritic subchondral bone was significantly greater than controls (p=0.023). Within osteoarthritic specimens, the phosphate-to-amide I ratio increased proximally. The density in osteoarthritic subchondral bone was 89mg/cm3 higher than controls (p=0.022), and 494mg/cm3 higher than the osteoarthritic proximal-compartment (p<0.001). Moreover, carbonate substitution into the apatite crystals decreased in osteoarthritic specimens. The carbonate-to-amide I ratio was highest in osteoarthritic subchondral bone. Furthermore, the median α1-to-α2-chain ratio in osteoarthritic specimens was 2:1. The changes found in subchondral bone are important in the pathogenesis of osteoarthritis. This study shows that Raman spectroscopy can detect differences between osteoarthritic specimens and controls, further supporting its potential use in diagnosing bone disorders.

Osteoarthritis (OA) is a common, debilitating joint disease involving degeneration of cartilage and bone. It has been suggested that subtle changes in the molecular structure of subchondral bone may precede cartilaginous changes in the osteoarthritic joint. To explore these changes Raman spectroscopy was employed as a diagnostic tool. Raman spectroscopy measures inelastic scattered laser light produced when photons interact with chemical materials. Resultant changes in wavelength form spectra relative to the chemical composition of the given sample: with bone this includes the mineral and matrix components, unlike conventional X-rays. The aim of our study is to explore the hypothesis: Changes in matrix composition of osteoarthritic subchondral bone can be detected with Raman spectroscopy. pQCT and Raman spectroscopy were employed to determine the bone mineral density (BMD) and bone quality, respectively. Ten medial compartment OA and five control (non-OA) tibial plateaus were interrogated and analysis performed to compare OA to control, and medial to lateral compartments. The subchondral bone of the medial OA compartments had higher BMD (p=0.05) and thickness compared to lateral and control samples. Spectral analysis revealed there is no difference between the medial and lateral compartments within either cohort. However, there is a statistically significant (p=0.02) spectral difference between the OA and control specimens. The detection of bone matrix changes in osteoarthritis using Raman spectroscopy contributes to the understanding of the biochemical signature of subchondral bone across diseased and control tibial plateaus. This technique has potential to shed light on the role of bone in osteoarthritis.

We investigated the effect of adjuvant and neoadjuvant chemotherapy regimens on the tibial regenerate after removal of the external fixator in a rabbit model of distraction osteogenesis using New Zealand white rabbits.

Forty rabbits were randomly distributed into two groups. In the neoadjuvant group, half of the rabbits received 1mg/kg cisplatinum & 2mg/kg adriamycin at eight weeks of age followed by 1mg/kg cisplatinum & 4mg/kg adriamycin at ten weeks of age. The remaining ten received an identical volume of normal saline using the same regimen. The adjuvant group differed only in the timing of the chemotherapy infusion. Half received the initial infusion ten days prior to the osteotomy, with the second infusion four days following the osteotomy. Again, the remaining ten rabbits received an identical volume of normal saline using the same regimen. This produced an identical interval between infusions and identical age at osteotomy in both groups. All rabbits underwent a tibial osteotomy at 12 weeks of age. Distraction started 24hours after osteotomy at a rate of 0.75mm a day for 10 days, followed by 18 days without correction to allow for consolidation of the regenerate.

At week 16 there was no difference in Bone Mineral Density (BMD), Bone Mineral Content (BMC) or volumetric Bone Mineral Density (vBMD) in the adjuvant group. Neoadjuvant chemotherapy appears to have a significant detrimental effect on BMD, vBMD and BMC. Despite this there were no significant alterations in the mechanical properties of the regenerate. Histologically there was a trend for increased cortical thickness in the control groups compared to intervention however this did not prove statistically significant.

In conclusion, adjuvant chemotherapy may be more beneficial for cases where distraction osteogenesis is being considered to replace segmental bone loss after tumour excision.

Synthetic bone grafts are used in several major dental and orthopaedic procedures. Strontium, in the form of strontium ranelate, has been shown to reduce fracture risk when used to treat osteoporosis. The aim of the study was to compare bone repair in femoral condyle defects filled with either a 10% strontium substituted bioactive glass (StronBoneTM) or a TCP-CaSO4 graft. We hypothesise that strontium substituted bioactive glass increases the rate of bone ingrowth into a bone defect when compared to a TCP-CaSO4 ceramic graft.

A critical size defect was created in the medial femoral condyle of 24 sheep; half were treated with a Sr-bioactive glass (StronBoneTM), and in the other animals defects were filled TCP-CaSO4. Two time points of 90 and 180 days were selected. The samples were examined with regard to: bone mineral density (BMD) from peripheral quantitative CT (pQCT), mechanical properties through indentation testing, and bony ingrowth and graft resorption through histomorphometry.

The radiological density of Sr-bioactive glass in the defect is significantly higher than that of the TCP-CaSO4-filled defect at 90 and 180 days, (p=0.035 and p=0.000). At 90 days, the stiffness of the defect containing Sr-bioactive glass and is higher than that of the TCP-CaSO4 filled defect, (p=0.023). At 6 months there is no significant difference between the two materials. Histomorphometry showed no significant difference in bone ingrowth at any time point, however significantly more of the graft is retained for the StronBoneTM treatment group than the TCP-CaSO4 group at both 0 days (p=0.004) and 180 days (p=0.000). The amount of soft tissue within the defect was significantly less in the StronBoneTM group than for the TCP-CaSO4 group at 90 days (p=0.006) and 180 days (p=0.000)

The data shows the mechanical stability of the defect site is regained at a faster rate with the strontium substituted bioglass than the TCP-CaSO4 alternative. Histomorphmetry shows this is not due to increased bone ingrowth but may be due to the incorporation of stiff graft particles into the trabeculae. Sr-bioactive glass produces a stronger repair of a femoral condyle defect at 3 months compared with TCP-CaSO4.

Unique progenitor cells have been identified recently and successfully cultured in vitro from human articular cartilage. These cells are able to maintain chondrogenic potential upon extensive expansion. In this study, we have developed a sheep, ex-vivo model of cartilage damage and repair, using these progenitor cells. This study addresses the question can such a model be used to determine factors required for progenitor cell proliferation, differentiation and integration of matrix onto bone. The hypothesis was that sheep allogenic cartilage derived progenitor cells could regenerate artificially damaged sheep articular cartilage in an osteochondral culture model. Progenitor cells were derived from ovine articular cartilage using a differential adhesion assay to fibronectin and expanded clonally. These clonal cells were marked with lentiviral vectors derived from the Human Immunodeficiency Virus-1. When a self-inactivating lentiviral vector encoding a ubiquitous phosphoglycerate kinase promoter, driving a Green Fluorescent Protein (GFP) reporter gene, was used to transduce these cells, up to 80% of these progenitor cells expressed GFP. Normal sheep medial femoral condyles containing about 2mm thick sub-condral bone were obtained and 4mm circular defects created on the cartilage surface using a biopsy punch. Condyles were cultured for two weeks in vitro with GFP labelled progenitor cells within a fibrin glue scaffold (Tisseel Lyo) and matrix production (collagen) as determined by spatially offset Raman spectroscopy and immunohistochemistry was demonstrated. Progenitor cells were able to proliferate and differentiate into collagen producing cells. Such an ex-vivo model system is an effective tool for the analysis of cartilage repair from various sources of stem cells. These ex-vivo experiments and variations on defect type, size, titration of scaffold and progenitor cell numbers requirements can further be used as a basis for screening prior to in vivo experiments.

Introduction

Open fractures occur with an annual incidence of 11.5 per 100,000 (6900 pa in UK). Infection rates, even with intravenous broad-spectrum antibiotics, remain as high as 22%. For this reason necessary bone grafting is usually delayed until soft-tissue cover of the bone injury is achieved. A biodegradable bone graft that released sustained high concentrations of antibiotics and encouraged osteogenesis, that could be implanted safely on the day of injury would reduce infection rates and avoid reoperation and secondary grafting. The non –union rate (approx 350 pa in UK) should also be reduced. Such a graft, consisting of a PLA/PGA co –polymer and containing antibiotics, is under development and here we report assessment of spectrum and duration of antimicrobial activity and effect of addition of antibiotics on mechanical properties.

It is thought that metal ions from metal on metal bearing hip replacements cause DNA damage and immune dysfunction in the form of T cell mediated hypersensitivity. To explore the hypothesis that there is a relationship between metal ion levels and DNA damage and immune dysfunction in matched patient groups of hip resurfacings and standard hip replacements reflected in the levels of lymphocyte subtypes (CD3+ T cells, CD4+ T helper cells, CD8 +T cytotoxic/suppressor cells, CD16 +Natural Killer and CD19+ B cells) in peripheral blood samples, we analysed peripheral blood samples from 68 patients: 34 in the hip resurfacing group and 34 in the standard hip arthroplasty group. Samples were analysed for counts of each sub-group of lymphocyte and cytokine production. Whole blood cobalt and chromium ion levels were measured using inductively-coupled mass spectrometry. All hip components were well fixed.

Cobalt and chromium levels were significantly elevated in the resurfacing group compared to the hybrid group (p<0.001). There was a statistically significant decrease in the resurfacing group's level of CD8+ cells (T cytotoxic/suppressor) (p=0.010). No other subgroup of lymphocytes was significantly affected. Gamma interferon levels post antigen challenge were severely depressed in the hip resurfacing group.

A threshold level of blood cobalt and chromium ions for depression of CD8+ T cells was observed. Hip resurfacing patients have levels above this threshold whilst standard hip replacements fall below it. The patients all had normal levels of CD16 +Natural Killer and CD19+ B cells suggesting that this is not a bone marrow toxic effect. Cytokine analysis confirmed that some aspects of T cell function in hip resurfacing patients are severely depressed.

Introduction: Conflicting opinions exist as to whether bone healing is affected by the administration of bisphosphonates for osteoporosis. In an animal model, we assessed the effect of bisphosphonates on osteoporotic fracture healing and whether the timing of administration made a difference.

Methods: 36 female Wistar rats underwent a mid-diaphyseal femoral osteotomy six weeks after ovariectomy. They were then divided into 3 groups:

no treatment (control);

Results: Of the 36 rats, 8 were unable to complete the study. Group 3 differed from control in three respects: higher bone mineral content (BMC) and density (BMD); larger callus; lower torsional stiffness. Group 2 did not differ significantly from control. There was a significant positive correlation between stiffness and change in BMC in group 1 (r=0.85, p< 0.001) but not so for group 2 (r=0.2, p> 0.05) and group 3 (r=0.04, p> 0.05). A similar trend existed for all radiographic parameters in the three groups.

Conclusion: The results suggest that, with early bisphosphonate treatment, although there is an increase in the size of the callus, that callus is biomechanically inferior. Furthermore, administration of bisphosphonates at either stage destroys the relationship between radiographic and mechanical parameters used to assess fracture healing.

Background: 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 3-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:

CaSO4 and CaCO3 3% alginate hydrogels were injected into the NP cavity of a bovine tail. After 90 minutes the tail was dissected to reveal the gel.

Results:

Injectable alginate suspensions formed solid viscoelastic gels, filling the exact shape of the NP cavity.

Discussion: This study demonstrates that slowly polymerising CaCO3 and CaSO4 alginate gels are injectable and capable of sustaining NP cells in-vitro. Cells remain viable, maintain their phenotype, proliferate and produce ECM during the culture period. CaCO3 alginate gel provides a 3-dimensional matrix more favourable to NP cellular activity than the CaSO4 alginate gel. Synvisc® has a chondro-stimulatory effect on NP cells in-vitro. These effects are similar to those observed previously with hyaluronic acid, in that it binds to cell surface CD44 receptors, thereby affecting essential cellular functions and cytoskeleton structure. Synvisc® however has an advantage in that it is highly viscous and can reside longer within an alginate construct thereby having a sustained long-term stimulatory effect. 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.

We previously demonstrated that cartilaginous tissue was induced on a reamed acetabular articulation in an ovine hemiarthroplasty model with three different femoral head sizes. At maximum loading during stance phase, the acetabular peak stresses immediately after reaming could reach approximately 80 MPa under direct implant-bone contact with in-vitro measurements.

We aimed to establish finite element (FE) models of the ovine hip hemiarthroplasty which examine stress distribution on the reamed acetabula by three head sizes. We hypothesized that the stress distribution did not differ between different sizes when the joint is congruent and that the peak stresses in the acetabulum immediately after reaming occurred in the dorsal acetabulum.

Three two-dimensional FE models of ovine hip hemi-arthroplasty were built; each comprised a head component, 25, 28, and 32 mm in diameter, and an acetabular component. The acetabular geometry was acquired from an ovine acetabular histological section. The head was moved to partly intersect with the acetabulum representing the reaming procedure and a congruent contact was confirmed. Cortical bone and cancellous bone were modelled as linear elastic, with moduli of 20 and 1.2 GPa, respectively. Variable moduli were also assessed. The finest mesh for each model consisted of over 100,000 four-node quadrilateral elements. Loading conditions were chosen to represent peak hip joint force developed during the stance phase. Stress distribution in the acetabular area in contact with the head was plotted against the articulating arc length.

The results confirmed that the stress distribution between different prosthetic head sizes in a reamed hemiarthroplasty model did not change when the joint was congruent. The peak compressive stresses occurred in the dorsal acetabulum with the 32 mm model being the highest at approximately 69 MPa, the 28 mm model at 63 MPa, and the 25 mm model at 54 MPa. An increase in the cancellous modulus and a decrease in the cortical modulus increased the peak stresses in the dorsal acetabulum.

This presents an indicative study into the effect of prosthetic femoral head sizes on the stress distribution in the acetabulum. The idealized 2-D models showed reasonable agreement when compared quantitatively with the in vitro study.

Objective: To challenge the validity of using biomarker concentrations in synovial fluid for the assessment of joint pathology.

Hypothesis: Synovial fluid biomarker concentrations are influenced by both cartilage and synovial fluid volumes.

Methods: Synovial fluid volumes were determined from the equine metacarpophalangeal (MCP), proximal inter-phalangeal (PIP) and distal interphalangeal (DIP) joints, which have different disease prevalences.

Chondrocyte density was calculated from a defined site in each joint.

Cartilage volume was measured by novel application of Peripheral Quantitative Computed Tomography (pQCT).

Cartilage oligomeric matrix protein (COMP), glycos-aminoglycans (GAG) and total protein (TP) concentrations were measured and then adjusted for cartilage and synovial fluid volume and compared between joints.

Results: Mean synovial fluid volume was significantly greater in the MCP than the distal joints (p< 0.0001) (3.2 ±0.5ml, 0.5 ±0.1ml and 0.6 ±0.1ml respectively). In contrast, the DIP had the greatest cartilage volume compared to the proximal joints (5360 ±667mm3 2640mm3, 1940 ±331mm3 respectively). There was no significant difference in the cartilage cellularity between all joints.

The DIP had higher TP, COMP and GAG concentrations, however, when values were expressed per unit cartilage volume the opposite was found, with the MCP then exhibiting significantly higher concentrations.

Conclusions: These data show the joint with the highest prevalence to osteoarthritis has the lowest biomarker synovial fluid concentrations but the highest biomarker levels per unit cartilage, suggesting a higher release. These results indicate that meaningful interpretation of biomarkers in synovial fluid require consideration of both fluid and cartilage volume.

Background: As the understanding of bone repair mechanics has advanced the integrity of the bone pin interface has emerged as a key factor in determining the success of external fracture fixation. The benefits of using pins coated with Hydroxyapatite (HA) are well documented however the thickness of the conventional plasma spray coating precludes its use for modification of the surface of fine features in implants. Consequently new electro-chemical techniques for pre-coating implants with a ‘biomimetic’ HA layer using simulated body fluids (SBF) have been pioneered. In this study we test the hypothesis that varying the technique for deposition of HA by electrolysis of SBF alters the morphology of the HA surface which will modify the level of osseointegration. Method: Three alternative methods of HA coating the Barerre, Redepenning and Kumar techniques were compared. Tantalum coated stainless steel pins were coated then used to stabilise a mid-diaphyseal osteotomy in three sheep using an orthofix fixator for a period of ten weeks. Insertion and extraction torques were measured to calculate the pin performance index (PPI). Sections of the bones were then examined using scanning electron microscopy to determine the percentage of bone in contact with the pin surface and the percentage of new bone formation. Results: The different coating protocols resulted in different HA crystal morphologies. The extraction torque exceeded the insertion torque for both the Barerre and Redepenning methods and their PPI exceeds that of plasma spray coatings. The Redepenning technique was shown to perform sig-nificantly better than both the Barerre (p=0,001) and Kumar (p=0,001) techniques with 49.4% of the pin surface in contact with bone. These results were mirrored on analysis of new bone formation with the Redepen-ning technique showing 70.2% of new bone formation compared to the Barerre (55.4%) and Kumar (53.8%) methods. Conclusion: These results indicate that the Redepenning technique is the most effective for creating a bio mimetic HA coating in terms of bonding to bone and promoting new bone formation. This technique holds significant advantages over the conventional plasma spray technique for example the coating thickness can be easily controlled and additional proteins such as bone morphogenic proteins and antibiotics can be incorporated. It may therefore represent a new era in the use of HA coating.

The skeletal system exhibits functional adaptation. For bone the mechanotransduction mechanisms have been well elucidated; in contrast, the response of tendon to its mechanical environment is much more poorly understood despite tendon disorders being commonly encountered in clinical practice. This study presents a novel approach to developing an isolated tendon system in vivo. This model is used to test the hypothesis that stress-shielding, and subsequent restressing, causes significant biomechanical changes. We propose a control mechanism that governs this process.

A custom-built external fixator was used to functionally isolate the ovine patellar tendon(PT). In group 1 animals(n=5) the right PT was stress-shielded for 6 weeks. This was achieved by drawing the patella towards the tibial tubercle, thus slackening the PT. In group 2 (n=5) the PT was stress-shielded for 6 weeks. The external fixator was then removed and the PT physiologically loaded for a further 6 weeks. In each case, the PT subsequently underwent tensile testing and measurement of length(L) and cross-sectional area(CSA). The untreated left PTs acted as controls (n=10).

6 weeks of stress-shielding significantly decreased material and structural properties of tendon compared to controls (elastic modulus(E) 76.2%, ultimate tensile strength(UTS) 69.3%, stiffness(S) 79.2%, ultimate load(UL) 68.5%, strain energy(SE) 60.7%; p< 0.05). Ultimate strain(US), L and CSA were not significantly changed. 6 weeks of subsequent functional loading (Group 2) caused some improvement in material properties, but greater recovery in structural properties (E 79.8%, UTS 91.8%, S 96.7%, UL 92.7%, SE 96.5%). CSA was significantly greater than Group 1 tendons at 114% of control value.

Previous models of tendon remodelling have relied on either joint immobilization or direct surgical procedures. This model allows close control of the tendon’s mechanical environment whilst allowing normal joint movement and avoiding surgical insult to the tendon itself. The hypothesis that stress-shielding, and subsequent restressing, causes significant biomechanical changes has been upheld. We propose that the biomechanical changes observed are governed by a strain homeostasis feedback mechanism.

Introduction: There have been 70,000 hip resurfacings implanted, predictions are for it to become 12% of the US hip replacement market by 2010 (Goldmann Sachs report Oct 2005). There is concern that the cobalt and chromium ions released from metal on polyethylene hip replacements cause immune dysfunction in the form of T cell mediated hypersensitivity (indicated by increased numbers and stimulation of T cells). If metal ions cause significant effects on white blood cells we might reasonably expect to detect this by simply measuring numbers of white blood cells.

Aim : To examine the possibility that raised metal ions may cause an abnormal number of white blood cells, termed a blood dyscrasia.

Method : Peripheral blood samples were analysed from 68 patients: 34 in the hip resurfacing group and 34 in the standard hip arthroplasty group. Samples were analysed for counts of each sub-group of lymphocyte. Functional assessment was also performed using a activation panel of white cell CD markers. Whole blood cobalt and chromium ion levels were measured using inductively-coupled mass spectrometry. All hip components were well fixed.

Results : Cobalt and chromium levels were significantly elevated in the resurfacing group compared to the hybrid group (p< 0.001). There was a statistically significant decrease in the resurfacing groups’ level of CD8⁺ cells (T cytotoxic/suppressor) (p=0.010). There was a characteristic pattern of immune modulation seen on the activation panel.

Conclusions : We found an immune modulation in patients with metal on metal hip resurfacing. This was not a hypersensitivity reaction. This change in T cell function may be detrimental or beneficial to patients.

Current bone grafts include allograft and autografts, both of which have limitations. Tissue engineering biotechnology has shown considerable promise in improving grafts. A competent graft material should ideally have osteoconductive and osteoinductive properties and comprise of bone forming cells and osteoinductive growth factors. In this study, we have evaluated the in vitro formation of bone and have used human demineralised bone matrix [DBM] and human insoluble collagenous matric [ICM] as scaffolds for mesenchymal stem cells [MSCs] and osteogenic protein [OP-1]. The objective was to determine whether combined addition of OP-1 and MSCs resulted in a superior bone graft substitute by improving the inherent osteoinductive property.

DBM and ICM were prepared and combined with rhOP [1.4 mg/0.25 mg of bone] and MSCs [1 x 105/ ml]. Statistically significant differences in MSC proliferation were seen between materials with and without OP-1 [P< 0.05}, n=8] in DBM on day 1, and both DBM and ICM on day 7 and 14. Enhanced osteogenic differentiation was observed in the presence of OP-1 when compared to DBM alone and on DBM and ICM with OP-1. In conclusion MSCs and OP-1 can be seeded together on DBM and ICM and Von Kossa staining and X-ray analysis confirmed in vitro de novo bone formation, with DBM + MSCs + OP-1 being more successful in this regard.

Conclusion: To date, no other study, to the author’s knowledge, has used MSCs and OP-1 together on a graft material; this funding, therefore, has very important clinical implications.

Limitations of allografts and autografts for bone repair have increased the demand for a synthetic bone graft substitute for load-bearing and non-load bearing osseous defects. Tissue engineering of bone has thus been implicated to circumvent and eliminate the limitations of existing therapies, with living cell-scaffold constructs ultimately “integrating” with the patients own tissue. Bone engineering requires cells, growth inducing factors and a scaffold for delivery of cells to the anatomic site, creation of 3-D space for tissue formation and mechanical support. In this study, we investigated whether addition of osteogenic Protein-1 (OP-1) enhanced the osseoinductive properties of hydroxyapatite (HA) loaded with mesenchymal stem cells (MSCs). The study was conducted over a fourteen day period and the two groups HA/MSC and HA/MSC loaded with OP-1 were analysed qualitatively by SEM and quantitatively by assessment of proliferation (Alamar blue assay and total cellular DNA) and differentiation marker alkaline phosphatase activity (ALP). HA/MS/OP-1 showed a statistically significant (p< 0.05) increase in cell proliferation (286.52 ± 58.2) compared to the unloaded samples (175.62 ± 23.51). ALP activity (release) was also significantly enhanced (p < 0.05) in the loaded samples at day 14 (12.63 ± 1.58) compared to the control (2.73 ± 1.07).

Conclusion: the osseoinductive potential of HA was markedly improved by the incorporation of MSC’s and OP-1. This type of graft could provide improved mechanical stability at an earlier time point, and may influence future clinical application of HA for load bearing sites.

Restoration of bone stock is the single greatest challenge facing the revision hip surgeon today. This has been dealt with by means of impaction grafting with morsellised allograft from donor femoral heads.

Alternatives to allograft have been sought. This study investigates the use of a porous biphasic ceramic in impaction grafting of the femur.

Impaction grafting of the femur was performed in four groups of sheep. Group one received pure allograft, group two 50% allograft and 50% BoneSave, group three 50% allograft and 50% BoneSave 2 and group four 10% allograft and 90% BoneSave as the graft material.

Function was assessed by measuring peak vertical reaction forces. Changes in bone mineral density were measured by DEXA scanning. Loosening and subsidence were assessed radiographically and by examination of explanted specimens.

All outcome measures showed no statistically significant difference between the four groups after eighteen months of full function.

Conclusion: When used as allograft expanders, Bone-Save and similar porous biphasic ceramics perform as well as pure allograft in impaction grafting of the femur.

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.

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.

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.

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.

Injury to the core region of energy-storing tendons is a frequent occurrence in both human and equine athletes, the incidence of which increases with age. Such energy-storing tendons include the human Achilles tendon (AT) and the equine superficial digital flexor tendon (SDFT). By definition, energy-storing tendons experience high strains during high-speed athletic activity. In contrast, anatomically opposing tendons (“positional” tendons), such as the common digital extensor tendon (CDET) in the horse and extensor digitorum longus tendon in man act only to transmit muscular force and rarely suffer exercise–induced injury. Functional adaptation of muscle and bone in response to exercise is well – documented, but there has been no convincing evidence to suggest that the energy-storing tendons in adults have the ability to adapt to exercise. We hypothesised that adaptive increases in tenocyte cellularity would occur in the energy-storing and positional tendons of young horses subjected to three specific exercise regimens. Samples were taken from midmeta-carpal regions of the SDFT (periphery and core) and CDET of young Thoroughbred horses from the following groups. Group 1: 6 horses exercised on a high-speed treadmill for 18 months from 21.3 months of age (SD 1.1) with 6 age-matched controls that underwent walking exercise only (long-term); Group 2: 6 horses exercised on a high-speed treadmill for 18 weeks from 19.4 months of age (SD 0.6) with 6 age-matched controls that underwent walking exercise only (short-term) and Group 3: 6 horses trained on pasture in New Zealand for 18 months beginning at 7–10 days of age, with 6 age-matched controls kept at pasture with no additional enforced exercise (Global Equine Research Alliance). Tenocyte nuclei were counted and measured in digital images from histological sections stained with haematoxylin and eosin, by computerised image analysis. Tenocyte densities (per mm2) for exercised and control groups for each study were evaluated using paired t-tests. Tenocyte density was significantly higher in the CDET of exercised horses in Group 3 (mean ± SD =260.4 ± 23.4) compared with the non – exercised controls (mean ± SD =226.9 ± 23.8) (p < 0.01). There was no such difference in the SDFT (core or periphery). There was also no significant exercise-related difference in tenocyte density in either the SDFT (core or periphery) or CDET for Groups 1 or 2. No previous data is available on the effect of exercise on tenocyte populations in equine tendons. The lack of other adaptive changes in previous studies of mature equine tendons had raised the question as to whether immature tendons would be more able to adapt to mechanical stimuli. In this study we were able to show that beginning training of horses shortly after birth (Group 3) stimulated an adaptive response by tenocytes in the positional CDET but not the SDFT. The inability of energy-storing tendons to show functional adaptation to exercise in immature or mature animals may explain the high incidence of strain-induced injury. Understanding the pathway by which exercise-related increases in tenocyte densities occur in immature positional but not energy-storing tendons may increase our understanding of the pathogenesis of strain-induced tendon injury.

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.

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.

Introduction: The ‘gold standard’ currently used to assess bone quality is bone mineral density (BMD) measured by Dual Energy X-ray Absorptiometry (DEXA). However BMD accounts for no more than 60 – 70% of bone strength. X-rays are affected primarily by the mineral phase of bone; the organic phase remains essentially invisible. Yet it is known that the material strength and toughness of bone is critically dependent on its organic phase. A Raman spectroscopic technique was used that permitted visualisation of both phases of bone deep to unbroken skin by successfully removing spectral information from the overlying tissues.

Hypothesis: Spectral features of both the mineral and organic phases of bone from different murine genotypes can be measured objectively through the unbroken skin using time-resolved Raman spectroscopy.

Methods: We used an 800 nm probe laser (1 kHz, 1 ps pulses, focussed to 1 mm diameter) with a synchronised 4 ps optical Kerr gate that had a variable picosecond delay that effectively shuttered out photons from the overlying tissues. We measured bone spectra at a point 2mm above the carpus from two mouse genotypes: wildtype and oim/oim (matched for age, sex and weight) at a typical depth 1.1mm. We then repeated the measurements once the overlying tissues had been carefully removed to expose the bones directly. Oim/oim mice produce only homotrimeric collagen I, (á1(I)3), associated with this change in collagen is a poor mineralisation of the bone tissue, making it an ideal model for a this study.

Results: We recorded the main spectral features in both phases of bone and showed that the ratios of spectral bands from the two phases were similar within each genotype, whether measured through the skin or directly from exposed bone. However, there was a significant difference in the same ratios between genotypes associated with a reduced mineralisation in the oim/oim mice; a significant difference that was apparent both directly from bone and through skin. The band associated with CH2 wag of collagen (organic phase) showed a frequency shift between the genotypes.

Discussion: Measurements of the spectra and their analysis were similar whether made directly on bone or transcutaneously. We were able to detect changes in mineralisation between genotypes and, unlike measurements of BMD, we showed also changes in collagen. Since the material strength of bone is critically dependent on collagen, this indicates an appreciable advantage of this technique over DEXA.

Conclusions: This novel technique allowed objective transcutaneous spectral measurements of bone tissue and was able to distinguish between normal and unhealthy bone tissue. With a laser focussed to 1 mm diameter that was readily moveable, these measurements were specific to that site (2 mm proximal to the carpus). After further optimisation, this technology is likely to improve fracture risk assessments in comparison to the use of DEXA alone, opening opportunities for screening in anticipation of the predicted increase in fragility fractures.

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.

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.

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.

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.

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.

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.

Introduction: Functional outcome following proximal tibial replacement can be impaired by extensor mechanism inefficiency. Current methods used to re-attach the patellar tendon result in varied levels of extensor function. Successful attachment of the patellar tendon requires initial mechanical stability and long-term biological fixation. We have employed a prosthesis, to model patellar tendon re-attachment, to test the hypothesis that biological augmentation of an implant which can provide sufficient mechanical integrity will allow a tendon-implant interface to develop that is similar in function and morphology to a normal tendon-bone interface.

Methods: The right patellar tendon in 24 Skeletally mature Friesland ewes was transfixed between the interlocking spikes of a hydroxyapatite-coated, customized tendon clamp to simulate patellar tendon re-attachment to a proximal tibial replacement. In 12 animals (Autograft group) the clamp attachment was augmented with autologous cancellous bone and marrow graft harvested from the ipsilateral iliac crest at the time of surgery, whilst the remaining animals (HA group) served as un-supplemented controls. Functional outcome was assessed using force plate measurements and two-dimensional optical kinematic gait analysis. Animals were euthanised at 6 and 12 weeks. The specimens were harvested, processed for histology and examined using light microscopy.

Results: The clamp device provided sufficient mechanical fixation of the patellar tendon to allow immediate weight bearing. Gait analysis showed that the range of movement of the stifle (knee) joint was not compromised by the surgical intervention at 6 or 12 weeks post-operation. An extensor lag observed at 6 weeks in both the Autograft and HA group was seen to fully recover by 12 weeks post-operation. There was a significant increase in functional weight bearing through the operated limb of the Autograft group animals between 6 and 12 weeks, which was not observed in the HA group. The tendon-implant interface in the HA group animals showed a fibrous tissue encapsulation of the HA coated surface, with collagen fibrils running parallel to the implant surface. In the Autograft group at 6 weeks post-operation a soft tissue – bone – HA interface had developed, similar in morphology to that of an indirect-type enthesis. Perpendicular orientated Sharpey’s-like fibres were observed spanning the region between the tendon and the HA coated implant and the bone graft material was seen to be undergoing active remodelling. By 12 weeks post-operation the interface was layered with regions of fibrocartilage clearly visible, more closely resembling the morphology of a direct-type enthesis.

Discussion: The clamp device provided sufficient mechanical fixation of the patellar tendon to allow immediate use the operated limb. The incorporation of a bio-active implant coating and biological augmentation encouraged a neo-enthesis to develop with near normal functional properties, and morphology similar to that of a normal patellar tendon-bone direct-type enthesis.

Study Design: Experimental study to assess tissue engineered solutions to disc degeneration.

Objectives: To investigate the use of a novel biodegradable hydrogel which is capable of minimally invasive introduction into an intervertebral disc (IVD) and support of cultured nucleus pulposus (NP) cells for the purpose of developing a tissue-engineered solution to retard progression of IVD degeneration. There were 3 objectives: (1) To introduce a slowly polymerising alginate hydrogel into the NP cavity of a bovine vertebral disc model. (2) To demonstrate the viability and metabolic activity of cultured NP cells in the hydrogel in vitro. (3) To determine the effect of Synvisc (hylan G-F 20) on NP cell proliferation and extracellular matrix (ECM) production.

Summary of Background Data: The cause of intervertebral disc degeneration (IVDD) is multifactorial. One proposed mechanism is that IVDD originates in the NP and progresses radially to the annulus fibrosis (AF). There is 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 progression of IVDD. Injection of NP cells into an early degenerative IVD, where the AF is still intact, may retard the degenerative process.

Subjects/Methods: 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. 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: Injectable alginate suspensions formed solid viscoelastic gels, filling the exact shape of the NP cavity. NP DNA and ECM synthesis was significantly greater in the CaCO₃ alginate gel than in the CaSO₄ alginate gel (p< 0.05). 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.

Introduction: The aim of this study is to develop a novel approach to tissue engineering in vivo, in which the adaptive response of skeletal tissues to the imposed mechanical environment will be utilised to induce a cartilaginous resurfacing of the acetabular articulation in a hemi-arthroplasty model of hip replacement. Our hypothesis was that a cartilaginous resurfacing of subchondral bone can be induced by applying stresses of 0 to 3 MPa to the articular surface of the acetabulum. We used an ovine hemiarthroplasty model where the stresses on the acetabulum were engineered by using different femoral head sizes.

Methods: Three groups of six sheep received unilateral hip hemi-arthroplasties and were sacrificed 24 weeks post-operatively to harvest the acetabula. At operation, acetabular cartilage was removed completely and the subchondral bone was reamed down and left bleeding. Three femoral head sizes, 25, 28, and 32-mm, were used to induce different contact stress levels. Vertical ground reaction force (GRF) data were measured and normalised by body weight for both limbs pre-operatively and every 4 weeks post-operatively. Five specimens from each group and eight unoperated controls were processed and stained with Safranin O and Sirius Red. Cartilage proteoglycans in the regenerated tissues from four specimens in the 25-mm group were detected by immunoblotting using specific monoclonal antibodies.

Results: The operated limbs were subjected to an average of 80 to 90% pre-operative GRF after the eighth post-operative week and maintained till the end of the study. No significant difference was noted during the period between the three groups. A layer of regenerated tissue was noted on all specimens processed and was Sirius positive. Four operated specimens processed in the 25-mm group and three in the 28-mm group were Safranin O positive. The presence of cartilage aggrecan, cartilage link proteins, biglycan, and decorin was confirmed by immunoblotting.

Discussion and Conclusion: We conclude that a cartilaginous resurfacing of acetabulum can be induced in vivo under the mechanical environment imposed by our hemi-arthroplasty model. This approach may be advantageous in clinical practice as a regenerated acetabular cartilaginous surface would avoid the problems associated with wear of the plastic acetabular cup and replacement of the acetabulum.

The purpose of this study was to examine the effects of hyaluronic acid supplementation on chondrocyte metabolism in vitro. The clinical benefits of intra-articular hyaluronic acid injections are thought to occur through improved joint lubrication. Recent findings have shown that exogenous hyaluronic acid is incorporated into articular cartilage where it may have a direct biological effect on chondrocytes through CD44 receptors.

Bovine articular chondrocytes were isolated and seeded into alginate constructs. These were cultured in medium containing hyaluronic acid at varying concentrations. Samples were assayed for biochemical and histological changes.

There was a dose-dependent response to the exposure of hyaluronic acid to bovine articular chondrocytes in vitro. Low concentrations of hyaluronic acid (0.1 mg/mL and 1 mg/mL) significantly increase DNA, sulphated glycosaminoglycan and hydroxyproline synthesis. Immunohistology confirmed the maintenance of cell phenotype with increased matrix deposition of chondroitin-6-sulphate and collagen type II. These findings confirm a stimulatory effect of hyaluronic acid on chondrocyte metabolism.

Aim: The biological activity of demineralised bone matrix (DBM) led to the discovery of bone morphogenetic proteins (BMP). OP-1 (BMP 7) is an osteoinductive protein and has been demonstrated to be capable of inducing new bone formation in rat subcutaneous tissue and in both orthotopic and heterotopic sites in primates. In this study we have investigated whether demineralisation and addition of osteogenic. protein 1 (OP-1) improves osteoinductive properties of allograft. Methods: A randomised controlled blind trial was performed in 16 rats. One group received two pellets of fresh frozen allograft; the other received two pellets of demineralised bone (DBM) intramuscularly. In each rat one pellet was treated with OP-1 (2mg/25mg of graft). The rats were sacriþced at 28 days and tissue þxed and processed for sectioning with haematoxylin and eosin for morphology and Alcian blue and Sirrus red for collagen types I, II. Qualitative observations were made and each specimen graded 0–5 on the degree of new bone formation and integration by two blind observers. Results & Conclusions: DBM with OP-1 yielded optimal results, being signiþcantly superior to allograft alone and allograft with OP-1. DBM alone was shown to be more effective compared to the allograft preparations. Hence we have shown that demineralization and OP-1 signiþcantly improve the osteoinductive properties of allograft

Aim:. The objective of this study was to improve the osseoinductive capacity of human demineralised bone matrix (DBM) and human insoluble collagenous matrix (ICM), following incorporation of recombinant human osteogenic protein 1 (rhOP-1) and human mesenchymal stem cells (MSCs). Ethical Committee approval has been obtained by our Institution. Methods: Recombinant human osteogenic protein Ð 1 (400ng/0.25g of bone) was seeded onto DBM and ICM together with human MSCs (1 x 105). Cellular proliferation was quantitatively evaluated in vitrousing Alamar Blue and 3H-TdR assays. Quantitative cellular differentiation was assessed using the alkaline phosphatase assay. Von Kossa staining, X-ray analysis, and PCR were used for qualitative evaluation of cellular differentiation. Qualitative analysis of proliferation and differentiation was assessed using scanning electron microscopy (SEM). Results: MSC proliferation and differentiation down the osteogenic lineage was observed on DBM and ICM in the presence of OP-1, and also on DBM alone. Alamar blue and 3H-TdR assays conþrmed that MSC proliferation occurred on both DBM and ICM, with the values being signiþcantly greater with addition of OP-1. The ALP activity showed that MSCs differentiated into osteo-blasts on DBM alone, and on DBM and ICM with OP-1. In all cases, OP-1 had a signiþcant effect on MSCs. Discussion: DBM and ICM when seeded with MSCs and OP-1 provide an improved osteoconductive and osteoinductive graft material resulting in de novo-bone formation.

Aim: Mesenchymal stem cells (MSCñs) attach to hydroxyapaptite surfaces (HA) surfaces and given appropriate stimuli from human Bone Morphogenetic Protein 7 (OP -1), will differentiate into osteogenic cells. Our hypothesis is that combining HA/MSC/BMP-7 will provide a superior osseoinductive property compared to HA alone. Methods: Porous hydroxyapatite (74.6% porosity, and 0.65% closed porosity) loaded with MSCñs (2 x 105) were compared to samples loaded with rhBMP-7 (400 ng/0.1g HA,) of the same MSC concentration over a fourteen day period. Quantitative analysis (Cell proliferation, measured by total DNA and the Alamar blue assay and Cell differentiation- alkaline phosphatase activity) and qualitative (Light and Scanning Electron Microscopy) were performed. The Students T-test was performed. Ethical approval for the use of human tissue was obtained prior to experimentation Results: Cell proliferation as indicated by total DNA, and Alamar blue was signiþcantly enhanced (P< 0.05) in the BMP-7 loaded composite at all time points. ALP production and release was enhanced in loaded samples. ALP production per unit DNA was also enhanced in the loaded samples and was signiþ-cant at day fourteen. Conclusion: Results indicate that the loaded composites showed enhanced cell proliferation, and ALP production and release. SEM analysis also demonstrated enhanced cell attachment and an increase number of proliferative cells. Thus the HA/MSC/BMP-7 composite displayed superior osseo-inductive properties in comparison to the HA/MSC composite.

The use of intra-articular hyaluronic acid injections for the treatment of early osteoarthritis is in widespread clinical use. Hyaluronate (HA) is a major component of connective tissue¹ and is available commercially for the intra-articular injective treatment of osteoarthritis of the knee and periarthritis of the shoulder. Although it is known to improve intra-articular lubrication it is also thought to promote articular cartilage structure and prevent catabolism of matrix proteoglycans in osteoarthritis. Clinical studies have shown beneficial effects lasting for many months after cessation of therapy unlike anti-inflammatory drugs that have relatively short term relieving effects^2,3 . Documentation of the true chondroprotective effects of hyaluronic acid (HA) at the cellular level is lacking and therefore this study aimed to identify the effects of HA on chondrocytes cultured in vitro.

Bovine articular chondrocytes were isolated by sequential digestion with pronase and collagenase and seeded in 2% alginate at 1x10⁷ cells/ml. The constructs were cultured for up to 14 days in standard culture medium (DMEM + 20% Fetal calf serum) containing varying concentrations of HA (Sigma), including doses equivalent to those found in vivo. The medium was replaced every 3 days and representative constructs were removed from culture, digested and assayed for DNA, glycosaminoglycans and Collagen. Further constructs were fixed in 4% paraformaldehyde for standard histology and immunolocalisation of collagen types I, II and chondroitin-6-sulphate.

Chondrocytes cultured in the HA system proliferated (increase in DNA) at a faster rate than the controls. There was a 2.2 fold increase in cell concentration at 14 days compared to a 1.2 fold increase in the controls. Total GAG levels at each time point were significantly greater for cells cultured in HA than in controls. Histologically, constructs were characterised by extensive cell cluster formation and intense Safranin-O staining. The newly synthesised matrix also stained positive for type II collagen. By contrast, control constructs exhibited minimal cluster formation, Safranin-O and type II collagen staining.

Cells maintained with HA exhibited a significantly greater rate of proliferation and matrix production. The presence of matrix rich in type II collagen indicates maintenance of chondrocytic phenotype. By contrast, cells cultured without HA did not show these features. These results support the use of intra-articular injections for the treatment of osteoarthritis. The benefits of HA injections may be due to cellular mechanisms as well as mechanical.

The use of intra-articular corticosteroid injections for their anti-inflammatory effects is widespread amongst clinicians. Despite their use in both rheumatoid arthritis and osteoarthritis, the effect of these agents on articular chondrocytes is not fully established. Previous reports suggest a detrimental effect on cartilage explants resulting from inhibition of matrix synthesis¹. However it has also been suggested that the beneficial effects in vivo may be due to prevention of inflamed synovium causing cartilage degradation². Our aim was to assess the effect of a commercially available preparation of methylprednisolone (MP), at clinical doses, on articular chondrocytes cultured in vitro.

Bovine articular chondrocytes were isolated by sequential digestion with pronase and collagenase and seeded in 2% alginate at 1x10⁷ cells/ml. The constructs were cultured for up to 15 days in standard culture medium (DMEM + 20% Fetal calf serum) containing varying concentrations of MP, including doses equivalent to those found in vivo. The medium was replaced every 3 days and representative constructs were removed from culture, digested and assayed for DNA and glycosaminoglycans. Further constructs were fixed in 4% paraformaldehyde for standard histology and immunolocalisation of collagen types I, II and chondroitin-6-sulphate.

Chondrocytes cultured in MP containing medium showed a significant abnormality in cell morphology compared to controls at the day 15 time point. Histologically there was evidence of cell necrosis, reduced amounts of extracellular matrix and loss of collagen type II staining. The effects were dose dependant, with significant damage occurring even at clinical doses. Biochemical analysis revealed a reduction in DNA content and an inhibition of glycosaminoglycan and collagen type II synthesis. In contrast, in the controls, there was cell proliferation with a cell doubling time of 14 days, collagen type II containing extracellular matrix synthesis occurred and the chondrocytes maintained their phenotype throughout the culture period.

Methylprednisolone has a significant detrimental effect on cultured articular chondrocytes in vitro. There was significant cell necrosis associated with inhibition of extracellular matrix synthesis. Based on these results, intra-articular corticosteroid injections should be used with extreme caution.

Nicotine is a constituent of tobacco smoke and is present in the body fluids of smokers^1,2. Numerous studies have confirmed that smoking is a strong risk factor for back pain³. The most widely accepted explanation for the association is that smoking leads to malnutrition of spinal discs due to carboxyhaemoglobin formation. However, other constituents of smoke, such as nicotine, may also be responsible for intervertebral disc (IVD) degeneration by leading to cell necrosis in both the nucleus pulposus and annulus fibrosis. Despite evidence suggesting the detrimental effect on a variety of tissues, the effect of nicotine on IVD cells has not previously been investigated. This study investigated the influence of nicotine on the metabolism and viability of IVD cells cultured in vitro.

Bovine nucleus pulposus (NP) intervertebral disc cells were isolated by sequential digestion of caudal spinal disc nuclei with pronase and collagenase and seeded in 2% alginate at 5x10⁶ cells/ml. The constructs were cultured for 21 days in standard culture medium (DMEM + 20% Fetal calf serum) containing free base nicotine (Sigma) at concentrations ranging from 25nM and 300nM, which reflected the normal physiological concentrations found in the serum of smokers. The medium was replaced every 3 days and representative constructs were removed from culture, digested and assayed for DNA, glycosaminoglycan (GAG) and hydroxyproline content at time points 3, 7, 14 and 21 days. Further constructs were processed for standard histology and immunolocalisation of collagen types I, II and chondroitin-6-sulphate.

The results were analysed statistically using an ANOVA test followed by a non-parametric Dunnit’s test. NP cells demonstrated a dose dependent response. At 25nM dose of nicotine there was a significant increase (p< 0.05) in DNA content, GAG and collagen synthesis in the constructs. At 100nM, 200nM and 300nM doses, there was a significant dose dependent decrease (p< 0.05) in all of these parameters compared to controls cultured under nicotine free conditions. In addition, adverse morphological changes were observed on histology, which included reduced cell proliferation, disrupted cell architecture, disintegration of cells and extracellular matrix. Immunohistochemistry showed the production of type I collagen rather than type II collagen as in the controls.

Nicotine has an overall detrimental effect on cultured nucleus pulposus disc cells in vitro. There was significant inhibition of cell proliferation and extracellular matrix synthesis. Nicotine in tobacco smoke may therefore play a role in the aetiology of disc degeneration that leads to back pain in smokers.

We measured fracture stiffness in 212 patients with tibial fractures treated by external fixation. In the first 117 patients (group 1) the decision to remove the fixator and allow independent weight-bearing was made on clinical grounds. In the other 95 patients (group 2) the frames were removed when the fracture stiffness had reached 15 Nm/degree. In group 1 there were eight refractures and in group 2 there was none (p = 0.02, Fisher's exact test). The time to independent weight-bearing was longer in group 1 (median 24 weeks) than in group 2 (21.7 weeks, p = 0.02). The greater precision of our objective measurement was associated with a reduction in refracture rate and in the time taken to achieve independent weight-bearing. We consider that a stiffness of 15 Nm/degree in the sagittal plane provides a useful definition of union of tibial fractures.

Diaphyseal fractures of the tibia in 80 patients were treated by external skeletal fixation using a unilateral frame, either in a fixed mode or in a mode which allowed the application of a small amount of predominantly axial micromovement. Patients were allocated to each regime by random selection. Fracture healing was assessed clinically, radiologically and by measurement of the mechanical stiffness of the fracture. Both clinical and mechanical healing were enhanced in the group subjected to micromovement, compared to those treated with frames in a fixed mode possessing an overall stiffness similar to that of others in common clinical use. The differences in healing time were statistically significant and independently related to the treatment method. There was no difference in complication rates between treatment groups.

Although it has been well established that fracture healing is influenced by the mechanical environment, the optimal parameters have not yet been established. In two groups of sheep an experimental tibial diaphysial fracture was created, and stabilised using external skeletal fixation. In one group rigid fixation was maintained throughout fracture healing; in the other group controlled axial micromovement, with a loading regime known to be osteogenic in intact bones, was applied for a short period daily. A significant improvement in healing was associated with the application of controlled micromovement. Data from these experiments provide the basis for improving the conditions for fracture healing and may assist in the prevention of delayed union.