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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.
To investigate osteoclastogenesis in vitro Peripheral Blood Mononuclear Cells (PBMC) were isolated from healthy volunteers and cultured over a two-week period under stimulation by cytokines (RANKL, M-CSF, VEGF, PlGF, a specific ligand for VEGFR 1 and VEGF-D, a specific ligand for VEGFR 2). RAW 264.7 cells (a mouse monocyte/macrophage cell line able to differentiate into osteoclast-like cells) were cultured for seven days under stimulation by cytokines (RANKL, VEGF and M-CSF). Osteoclasts were identified by staining for Tartrate Resistant Acid Phophatase (TRAP) and numbers of multinucleated cells counted per treatment. Culture on ivory slices was performed to measure resorption activity of the osteoclasts.
The PBMCs stimulated by VEGF and RANKL together differentiated into multinucleated TRAP positive cells in similar numbers (22±4.7) per field of view to the M-CSF and RANKL (27.3±7.2). Resorption of ivory was identified in these cultures. Stimulation with PlGF and RANKL resulted in increased osteoclastogenesis but VEGF-D with RANKL had little effect. Similar results were seen in triplicate experiments RAW 264.7 cells also differentiated into osteoclast-like cells after stimulation with VEGF and RANKL similar to M-CSF and RANKL.
The aim of this study was to (I) determine the intra-operative temperature of the femoral cancellous bed prior to insertion of prosthesis, (II) to investigate whether the magnitude of the temperature gradient effects interface porosity (III) to develop clinically relevant recommendations.
The cement mantle was sectioned transversely, then longitudinally to expose the cement-prosthesis interface. This was stained with acrylic dye to facilitate image analysis. Three mantles for each temperature were produced.
Image analysis results demonstrated an average bone attachment of 30.94% to the implant surface (fig 2). Greatest bone attachment occurred at the end of the pins (78.99%) contributing 22% of overall attachment to the implant. Least attachment occurred beneath the prosthetic cup (13.82%) and in the medial aspect adjacent to the central pin. Greater total bone area was measured in control hips and no significant correlation between bone attachment to the ‘pegs’ and bone area beneath the prosthetic cup was identified.
In order to determine factors that stimulate activin A production, chondrocytes were isolated from human cartilage and stimulated with various cytokines. RT-PCR methods were used to measure activin mRNA production and the culture medium was assayed for activin protein. Cartilage explants were also stimulated and activin protein levels were measured.
Using a newly developed fracture model of the proximal tibia of the rat, the effect of simvastatin on osteoporotic and non-osteoporotic fracture healing was investigated. The fracture model was used as it provided a useful model of metaphyseal fracture healing which is particularly relevant to osteoporotic fracture.
X-rays of the healing fractures were taken. Both the intact and fractures tibiae were then taken for mechanical testing by four point bending.
Although end-to-side neurorrhaphy did support nerve regeneration with sometimes good return of muscle function, the use of this technique as a clinical tool at this time cannot be recommended.
We measured 1: the area occupied by a cell and 2: the number of focal adhesions per cell. The largest values of osteoblastic cell area were seen with the smoother vitallium surface. In contrast, samples with more numerous and larger surface features resulted in the osteoblasts covering a smaller area and being confined by topographical elements (Ti2> TM2F> Ti6Al4V). In terms of adhesion, there were generally more focal adhesions per cell on rougher surfaces (Ti6Al4V> TM2F> Vitallium> Ti2).
MLO-Y4 cells were treated with physiological doses (10−8)M of either 17-beta E2 or the oestrogen receptor inactive stereoisomer 17-alpha E2 with or without the specific oestrogen receptor antagonist ICI 182,780 prior to the addition of 0.4milliM 30% (v/v) H2O2. Cellular apoptosis was determined using morphological and biochemical criteria.
The cellular production of reactive oxygen species was determined using the free radical indicator 2′7′- dichlorodihydrofluorescein diacetate. H2O2 induced increases in the number of ROS positive cells (34.6 ± 9.07 SD vs control 0.22 ± 0.39 SD). In contrast pre-treatment with both 17-beta E2 and 17-alpha E2 reduced the number of ROS positive cells associated with H2O2 treatment (Fig 1).
Our aim was to assess femoral offset and leg length following hip resurfacing and hybrid THR (uncemented acetabulum) performed by the same surgeon.
All films were analysed by the same investigator using the technique described by Jolles et al (J Arthroplasty 2002). A horizontal line was drawn between the base of the teardrop on both sides, and perpendicular lines drawn from the back of the teardrops. The anatomical femoral axis was drawn and femoral offset measured from this. The centre of rotation of the femoral head was determined by templating and the acetabular offset obtained.
Distance from tip of the greater trochanter to the centre of the femoral head in the axis of the femur was determined on pre and post-op films, as this shows little variation with rotation of the femur. Leg length was measured from the horizontal line to the tip of the greater trochanter together with the angle between the femoral axis and the horizontal to correct for abduction of the hip.
The aim of this study was to deduce the effect of Hip flexion on the knee’s full extension angle (popliteal angle) in a control group of subjects with normal knees.
In contrast, the release of the inflammatory cytokine TNF-α and the multifunctional growth factor TGF-β-2 occurred at lower doses (0.0005 to 5 μm3/cell for TNF-α and 0.5 to 50 μm3/cell for TGF-β-2). No release of IL-6 was detected at any dose. Only growth factor FGF-23 was increased in similar pattern to the DNA damage.
Contact patterns have been examined ex vivo
The objective of this study was to determine the feasibility of using a surface laser scanner to determine cartilage thickness and, therefore, likely contact area and to correlate changes in thickness to changes in sphericity of the articular surface.
The cloud point data for the cartilage and bony surfaces were exported into modelling software (McNeal and Assoc., Seattle, WA) and the surface area of the head divided into ten equal sections. For each slice of both the cartilage and bony surface, the radius of curvature was calculated using a least square fit optimisation technique
normal, grade IV chondral damage, osteochondral defects or endstage osteoarthritis (OA) of the knee, categorised by the cartilage appearance at arthroscopy.
Levels of matrix metalloproteinases (MMPs) 2 and 3 and the inhibitor, TIMP 1, were measured in the fluids via ELISA assays. Urea levels were measured in blood and synovial fluids and enzymes and their inhibitors were normalized according to the ratio of serum:SF urea, to account for the dilution factor of the SF (Kraus et al 2001). Western blotting was used to identify the presence of aggrecan components (chondroitin-4-sulphate: 2B6 antibody; C-6-S: 3B3 and C-0-S: 1B5; keratan sulphate: BKS-1; the G1 domain: 7D1; interglobular domain: 6B4) and also enzyme degradation products of MMPs (BC14) and aggrecanases (BC3; BC-13).
Dentin matrix protein (DMP-1), a phosphoprotein highly linked to dentin formation, has recently been reported to have an important role in skeletal development. Previously we reported that adult mice lacking the gene for DMP-1 exhibit the characteristics of chondrodysplasia, osteoarthritis, and showed severe defects in mineralization. DMP-1 knock-out (KO) mice display a profound defect in mineralization, and this is not due to a systemic defect in calcium/phosphate metabolism because serum levels of calcium and phosphate are similar to those in the wild-type mice. Although KO neonates and newborns appear normal, upon closer examination, these animals exhibit skeletal abnormalities, which include delayed secondary ossification and impaired bone remodelling. Heterozygous DMP-1 (H) mice however, show no apparent differences to the wild-type mice. In this study, biomechanical assessment tests of bones from DMP-1 KO mice were performed. Fifteen heterozygous, H, (DMP-1 +/−) and 15 KO, (DMP-1 −/−) male mice were produced and used in this study. At 1, 3 and 7.5 months of age, the mice were sacrificed and 4–5 ulnae from each animal group were harvested and stored in 70% ethanol solution. Volumetric density (BMD) measurements of the intact ulnae were performed using peripheral quantitative computed tomography (XCT960M; Stratec, Pforzheim, Germany) and Norland Stratec software version 5.10. One millimetre thick slices were scanned at a distance of 1 mm under the articular cartilage surface of the elbow as identified by the scout view of the CT scan. BMD of the corticalis and subcortical bone were recorded. Cross-sectional area measurements were also made at the mid-diaphysis of the ulnae. Biomechanical tests were performed in 3-point bending, with supports 3.5 mm apart at a rate of 3 mm/min (Lloyd Instruments Ltd, UK). The ultimate load, yield load and stiffness were determined from the load-displacement curves. All data were analysed using Mann-Whitney U tests (SPSS, Version 9, Chicago, Illinois). Differences were considered significant at p < 0.05. Density studies revealed that H mice had higher BMD than KO mice at all ages (p < 0.001). In the H and KO mice, the cortical BMD peaked at 3 and 7.5 months, respectively. At 1 month, the mean cross-sectional areas of the ulnae were larger in H mice compared to KO mice (0.50 mm2 Vs 0.33 mm2). However at 7.5 months of age, the reverse was observed (H = 0.75 mm2 and KO = 0.98 mm2). Biomechanically, stiffness increased with age at a higher rate in H mice than KO mice. Significant differences were observed at 3 months (p< 0.01) and 7.5 months (p< 0.05) between the two animal groups. There were no significant differences between stiffness values at 1 month. This study has demonstrated that DMP-1 deficiency leads to:
severely compromised bone mineralization; poor biomechanical properties of the long bone; and delayed bone development and remodelling. In conjugation with previous findings that DMP-1 plays important roles in the early developmental stage of bone through its effects on osteogenic gene expression of Cbfa1, Col I, and Col II and regulating vascular invasion, the current study may suggest another important role for DMP-1 as a regulator for skeletal mechanostasis.
The potential importance of bone morphogenic proteins (BMPs) to improve fracture healing is of great interest to orthopaedic surgeons. Although the complex mechanisms leading from the presence of local BMP (either endogenous or exogenous) to form bone is increasingly understood, however most appropriate time to administer exogenous BMP has yet to be elucidated. The purpose of this study was to investigate when BMP may be administered to a fracture arena in order to best improve fracture healing. Forty mice were randomised into 4 groups; (group I) control, treated at day 0 with placebo; (groups II, III and IV) treated with BMP at days 0, 4 and 8, respectively. All animals underwent a previously validated surgical procedure involving the creation of an open femoral fracture which is stabilised using a 4 pin external fixator. Thirty microlitres of bovine serum albumin (BSA) alone was used in group I, and the other groups (II, III and IV) were treated with a combination of the BSA and 2.5 microgrames of rhBMP-2. The BSA and rhBMP were injected through a lateral approach immediately after operation, or at 4, or 8 days postoperatively. At days 0, 8, 16 and 22, sequential radiographs were taken using a digital x-ray machine and at day 22 all animals were sacrificed. Both femora were harvested and assessed biomechanically in 3-point bending prior to fixation for histological evaluation. All data were analysed using Mann-Whitney U tests (SPSS, Version 9, Chicago, Illinois) and differences were considered significant at p < 0.05. X-ray analysis indicated that healing of fractures treated with BMP at day 0(group II) or day 4(group III) was significantly greater than that at both days 16 and 22 (p < 0.05) than those animals in placebo (group I) and BMP day 8(group V) treatment groups. Although the administration of BMP at day 4 seemed to cause more bone formation than treatment at day 0, no significant difference were observed. There were no differences between group IV and group I. Biomechanically, group III exhibited ultimate load values closest to the contralateral unoperated femora followed by group II, then IV and finally the control group I. Significant differences (p < 0.05) were observed between the control animals (group I) and both groups II and III. Qualitative histology suggested that at 22 days after surgery, only groups II and III had healed with woven bone. Group I and group IV had considerable amounts of fibrous tissue and cartilage at the fracture gap. This study suggests that a single percutaneous injection of BMP has a positive effect on fracture healing in this model, when prescribed between the time of injury (day 0) and 4 days. Data suggests that the most effective timing of delivery of BMP may not be at the time of surgery but actually in the early healing phase. The day 4 time point in the mouse model is likely to equate to that of 7–10 days in larger animals or humans. This suggests that current human treatment practices may require further investigation in order to elucidate the most appropriate time of delivery for these important proteins. This work may negate the current requirements for carrier products and large doses of these expensive drugs.
During bone development and repair, angiogenesis, osteogenesis and bone remodeling (resorption) are closely associated processes with some common mediators involved. BMPs, VEGF and other cytokines are released from bone during bone resorption. Recent study showed that VEGF caused a dose- and time-dependent increase in bone resorption in vitro and in vivo, and BMP-2 markedly enhanced osteoclast differentiation induced by sRANKL and M-CSF in mouse osteoclast culture system. The aim of this study was to further examine the effects of VEGF and BMP-2 on osteoclastogenesis using in vitro human osteoclast culture system. Mononuclear cells were isolated by Lympo-Prep density gradient centrifugation from bone marrow washouts in bone samples from patients undergone total hip replacement. Mononuclear cells were plated at a density of 1 x 106/cm2 in a T-75 flask with aMEM and 15% FCS. The first medium change was made at day 7, when the floating cells were collected from the withdrawn media by centrifugation, and plated in a separate flask. The non-adherent cells in the 2nd flask were harvested again 24 hours later in a similar fashion. The non-adherent cells were then cultured in 24-well plates or calcium phosphate (Ca-P) coated plates, with osteoclast-inducing media (OC media) containing sRANKL 30 ng/ml and M-CSF 30 ng/ml, media were changed every 4 days. After 4 days culture in OC media, rhBMP-2 (3, 30, 300 ng/ml) and VEGF (25 ng/ml) were added respectively or in combination to the cell culture, and the culture was kept for total 16 days. The number of TRAP positive multinuclear cells in each well and the resorptive pit areas on the Ca-P coated plates were calculated and compared. Osteoclastic cell phenotype was defined by expressing tartrate resistant acid phosphatase (TRAP), vitronectin receptor (VNR) and resorptive pit assay. By day 12–14, osteoclastic cells were found in all the experimental groups, they were positive for TRAP and VNR. The number of TRAP+ multinuclear cells were significantly reduced (p< 0.05, t-test) when rhBMP-2 (30 and 300 ng/ml) were present, and this was further reduced (p< 0.01) when rhVEGF was added together with rhBMP-2, comparing to the culture with OC media alone. Extensive lacunar resorption pits in the Ca-P coated plates were found in the culture treated with OC media and OC media with rhVEGF (25 ng/ml). The resorption pit areas were, however, significantly reduced when rhBMP-2 was added at 30 and 300 ng/ml with or without rhVEGF (25 ng/ml, p< 0.05, t-test). The presence of low concentration of rhBMP-2 (3 ng/ml) with VEGF had no effect on osteoclast number or the areas of resorption pit formation. In contrary to previous findings in the mouse osteoclast culture system, the present study had shown that the presence of rhBMP-2 at 30 and 300 ng/ml had strongly inhibited osteoclast differentiation and bone resorptive capability in the human osteoclast culture system, and the inhibition was further enhanced by the presence of rhVEGF. This study implies that VEGF and BMP-2 may be important, yet to be defined regulators, for osteoclastogenesis.
Fine-wire fixator systems have been used successfully for the treatment of fractures, malunions and for limb lengthening for many years. There has been much research investigating the biomechanical properties of these systems but this has been almost entirely centred on the mechanical properties of the fixator as a whole. Our knowledge of the interactions occurring at the interface between wire and bone remains sparse. To this end we devised an experimental model to analyse the distribution of pressure in cancellous bone surrounding a tensioned wire under loading conditions. The Sawbones cancellous bone material (type 1522-11) was cut into 65x30x40 mm blocks. A 2 mm olive wire was inserted into each block, parallel to the surface and along the 65 mm dimension. The distance from the wire to the surface was variable, from 0.5mm to 5mm in a 0.5mm increment. The wire was mounted on a 150 mm ring and tensioned to 1200 N against a load cell. The ring was rigidly mounted on a material testing machine and a second bone block was incorporated into the testing machine crosshead with a universal joint. Three grades of pressure-sensitive films (Low, Superlow and Ultralow) were sandwiched in turn between the testing block and cross head. The force applied was 175 N for 5 s. The developed film was scanned into a computer and a Matlab program was developed to analyse the pressure image. The results show three phases of pressure distribution. Very close to the wire there is a polar distribution of pressure that is, the pressure is concentrated towards the entry and exit points of the wire. At a depth of 1.5mm away from the wire the pressure becomes evenly distributed along the path of the wire in a beam-loading manner. At a distance of greater than 4mm from the wire there is even distribution of pressure throughout the bone. The peak pressures (6–8 MPa) were found closest to the wire. Most of the pressure measured was less than 1 MPa, which is less than the yield strength of cancellous bone (2–7 MPa, Li and Aspden, 1997). In contrast a similar analysis using threaded half pins under the same conditions showed far higher peak pressures (20 MPa), which were present deeper in the bone specimen. The pressure was concentrated toward the pin entry site and was not well distributed throughout the pin-bone interface. These results allow us to explain why ring fixators are superior to half pin fixators when used in metaphyseal bone.
Percutaneous vertebroplasty (PVP) is an emerging interventional technique for treatment of vertebral compression fractures. Bone cement is introduced to mechanically augment fracture and pain relief is almost immediate. Recent clinical and biomechanical studies have outlined the phenomenon of fractures occurring in adjacent vertebrae following PVP [
Most biomechanical studies adopt a single vertebral body as a model for PVP analysis. With this approach it is not possible to determine the effect of load distribution on adjacent structures. Where multi-segment vertebrae have been used there is little documentation of the fracture characteristics produced or their repeatability. The purpose of this study was to develop a 3-vertebra model for the biomechanical analysis of PVP. The particular focus was on developing a robust technique for generating repeatable level of fracture severity from specimen to specimen.
An alignment device was developed to fit into standard materials testing machine, which allowed constant axial compression without causing lateral bending or flexion-extension of the specimen’s ends. Porcine 3-segment specimens (T8-L2) were mechanically compressed to failure at a rate of 5mm/min applied vertically at a distance of 35% to the anterior edge of the specimen’s anterior-posterior length. During the test load-displacement data was displayed in real time on a PC. In order to generate uniform fractures, a protocol was devised in which the specimens were compressed for a further 6mm after initial yield point. After the initial fracture the segments were augmented with 3ml of PMMA cement injected through each pedicle and then recompressed. The fracture characteristics generated under these conditions were analysed using quantitative microcomputer tomogragy (μCT).
μCT images showed that fractures were generated in the central vertebra, with some propagation towards adjacent vertebra. The results support the use of a 3-segment specimen as a better representation for PVP analysis. The method will enables the load shift and fracture progression on either side of the augmented vertebra to be observed, thereby providing a more complete picture of load-bearing kinetics. Secondly, the middle, augmented motion segment remains unconstrained by platens and cement impressions; hence its anatomical boundary conditions are less compromised. Although longer segments have been shown to be more anatomically appropriate, it is difficult to apply physiologic levels of load without causing the specimen to buckle. We were able to minimise buckling effect by incorporating an alignment device to position the specimen without constraint. Given the preceding observations, the concepts of 3-segment specimen in PVP biomechanical tests provides a suitable compromise in choosing an appropriate clinical setting for in-vitro testing of biological spine specimens.
The purpose of this study was to determine and compare the effects of radiofrequency ablation and mechanical shaving on tendon using histological and ultrastructural techniques. A single cut using a scalpel blade was used to create a standardised reproducible lesion in 12 freshly harvested ovine infraspinatus tendons. Each lesion was then subjected to either bipolar radiofrequency ablation or mechanical shaving. Specimens were either fixed in formalin and processed for light microscopy or fixed in glutaraldehyde and processed for transmission electron microscopy. Samples of normal and untreated cut tendon were analysed as suitable controls. The radiofrequency treated samples showed an area of coagulative necrosis with an average diameter of 2mm around the lesion. Conversely, the shaved samples showed viable cells up to the edges of the lesion. These findings were supported by ultrastructural appearances, which showed preservation of tendon architecture in shaved samples and widespread denaturation of the tendon matrix with loss of fibrillar structure in the radiofrequency treated samples. Radio-frequency electrical energy and mechanical shaving are often used for resection of soft tissues during arthroscopic reconstructive procedures. The effects of these techniques on tendon are not yet clearly understood. The results of this study indicate that thermal resection of tendon causes an immediate additional 2mm area of tissue necrosis which is not present after mechanical shaving. These findings may have implications for the success of arthroscopic debridement and tendon repair procedures.
Although the function of proteoglycans within the tendon extracellular matrix are not fully understood, changes in their turnover have been associated with tendinopathies. In contrast to cartilage, aggrecanases are constitutively expressed and active in tendon, indicative of a high rate of aggrecan turnover. Clinical trials investigating the use of active site MMP inhibitors have been confounded by side-effects which involve tendonitis and “musculoskeletal syndrome”. Such side effects may relate to non-specific inhibition of tendon aggrecanases required to maintain normal metabolic homeostasis. The purpose of this study, therefore, was to compare the rate turnover of tendon and cartilage proteoglycans derived from the same joint and to determine the effect of MMP inhibitors (actinonin and marimastat) on aggrecan catabolism. Deep digital flexor tendon explants from compressed and tensional regions were dissected from young and mature bovine. Explants were precultured and then cultured for a further 4 days with or without marimastat (0–2 M) or actinonin (0–200 M). Proteoglycan and lactate quantification, Western blot analysis of degradation products and RT-PCR analyses were performed on these samples. In a separate experiment for measurement of proteoglycan turnover, explants were set up as described above then pulse chase labelled with [35S] sulphate. The rate of turnover of 35S-labelled proteoglycans from the matrix of tendon (and articular cartilage obtained from the same animal) was subsequently calculated from the amount of 35S-labelled macromolecules appearing in the medium each day and that remaining in the matrix of explants at the termination of culture. Proteoglycan turnover (presumably predominantly aggrecan) was markedly higher in tendon versus cartilage. This difference was apparent in tendons from all regions and ages. Both marimastat and actinonin inhibited aggrecanase-mediated proteoglycan catabolism in both tendon and cartilage explants. As expected mRNA expression for the aggrecanases, MMPs and TIMPs was unaffected by addition of these inhibitors to the culture medium. Aggrecan turnover in tendon is higher than that of articular cartilage, which may be attributed to distinct physiological properties of this proteoglycan in tendon. Importantly, immunohistochemical staining for aggrecan in tendon indicates its presence in between collagen fibres and fibril bundles and thus aggrecan aggregates may dissipate resultant compressive loads by resisting the flow of water in these locations. In addition, aggrecan may facilitate the sliding of fibrils during the small amount of elongation of the tendon whilst under tension. Thus, the half-life of tendon aggrecan is significantly reduced because it constantly participates in repeated resistance to compression. Our data also demonstrates that both marimastat and actinonin can inhibit aggrecanase-mediated proteoglycan catabolism in tendon cultures. This suggests that the occurrence of “musculoskeletal syndrome” in clinical trial patients may be due to the fact that these inhibitors affect the activity of aggrecanases in tendon, thus preventing them from playing their normal role in tendon aggrecan turnover and consequently perturbing normal physiological function.
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.
Degenerative joint disease (DJD) involves the proteolysis of many extracellular matrix molecules (ECM) present in articular cartilage and other joint tissues such as tendon, meniscus and ligaments. Recent research has identified key enzymes involved in the catabolism of ECM. Two classes of enzyme the Matrix Metalloproteinases (MMP’s) MMP-2, MMP-3, MMP-13 and the ADAMTS family (a disintegrin and metalloproteinase with thrombospondin motifs) of proteinases most notably, ADAMTS-1, -4 and −5, have been shown to be involved in the catabolism of ECM (such as type II collagen and cartilage aggrecan). The presence of several MMPs in the synovial fluid has been reported; however, little data has yet been gathered on the presence of ADAMTS-1, -4 or −5 (the aggrecanases) in synovial fluids. In this study we have used a recombinant artificial substrate and specific neoepitope antibodies that recognise either MMP- generated or aggrecanase -generated degradation products to measure the relative activity of these two enzyme families in the synovial fluid from human patients.
Osteoblast growth and differentiation are central to the formation and maintenance of healthy bone tissue. The search for novel mechanisms resulting in osteoblast maturation are highly desirable on several fronts. Firstly they provide potentially important information on the normal development of bone, in addition they may offer alternative therapies for bone diseases like osteoporosis and finally they may facilitate ex-vivo manipulation of cells for the subsequent improvement of oseointegration in transplantation/tissue engineering regimens. Recently we have been addressing how calcitriol, an active metabolite of vitamin D3, integrates with the signalling of epidermal growth factor (EGF) following reports that calcitriol can influence EGF receptor trafficking, expression and ligand binding. We have also extended our studies to investigating how other growth factors known to signal via receptor tyrosine kinases (RTKs) interact with calcitriol in controlling osteoblast growth and differentiation. The co-treatment of human pre-osteoblasts (MG63) with EGF and calcitriol resulted in the synergistic induction of their differentiation as supported by demonstrable increases in alkaline phosphatase activity and osteocalcin. The intracellular components responsible for eliciting the maturation response included protein kinase C and MEK 1/2 since the addition of calphostin C or UO126, respectively, blocked the differentiation response. Other ligands known to signal via RTKs, namely IGF1, VEGF and FGF1 could not induce differentiation in the presence of calcitriol. These findings support the specific integration of calcitriol/EGF signalling in osteoblast maturation. Collectively we have identified a novel, integrated, signalling pathway that drives terminal differentiation of osteoblasts. Our findings support earlier predictions (Yoneda 1996) in identifying novel actions of EGF in bone that will lead to advances in the field. Yoneda, T. 1996. Local regulators of bone: Epidermal growth factor – transforming growth factor-α. In Principles of bone biology (ed. J.P. Bilezikian, L.G. Raisz and G.A. Rodan.), pp. 729–738. Academic press Ltd.
Evidence has accumulated in recent years that programmed cell death (PCD) is not necessarily synonymous with the classical apoptosis, as defined by Kerr & Wyllie (J Path, 1973, 111:255–261), but that cells use a variety of pathways to undergo cell death, which are reflected by different morphologies. Although chondrocytes with the hallmark features of classical apoptosis have been demonstrated in culture, such cells are extremely rare in vivo. We have examined the morphological differences between dying chondrocytes and classical apoptotic cells in growth plate and osteoarthritic chondrocytes. Unlike classical apoptosis, chondrocyte death involves an increase in the endoplasmic reticulum and Golgi apparatus. This is likely to reflect an increase in protein synthesis with retention of proteins in the ER leading to expansion of the ER lumen, whose membranes surround and compartmentalise organelles and parts of cytoplasm. The final removal of apoptotic remains does not involve phagocytosis, but a combination of three routes: 1) auto-digestion of cellular material within compartments formed by ER membranes; 2) autophagic vacuoles and 3) extrusion of cell remnants into the lacunae. Together these processes lead to complete self-destruction of the chondrocyte as evidenced by the presence of empty lacunae. The involvement of ER suggests that the endoplasmic reticulum pathway of apoptosis may play a greater role in chondroptosis than receptor-mediated and mitochondrial pathways. Lysosomal proteases, present in autophagic digestion, are likely to be as important as caspases in the programmed cell death of chondrocytes in vivo. We propose the term ‘chondroptosis’ to reflect the fact that such cells are undergoing apoptosis, albeit in a non-classical manner, but one that appears to be typical of programmed chondrocyte death in vivo. Chondroptosis may serve to eliminate cells that are not phagocytosed by neighboring cells, which constitutes a crucial advantage for chondrocytes that are typically embedded in an extracellular matrix. Classical apoptosis in that situation is likely to lead to secondary necrosis with all its disadvantages. This may be the reason why most programmed cell death of chondrocytes in vivo appears to follow a chondroptotic pattern and not the classical apoptotic pattern. At present the initiation factors or the molecular pathways involved in chondroptosis remain unclear.
In addition to the above quantitative changes, qualitative assessment of the data showed an alteration in the loading pattern with reduced push off forces. However, eighteen of the twenty feet showed no alteration in the pattern of pressure distribution.
Aseptic loosening is a growing problem for orthopaedic surgeons and the importance of elevated hydrostatic pressure in its development in vivo is now well documented, but the mechanisms by which pressure could enhance loosening are unclear. We have demonstrated that hydrostatic pressures increased MP synthesis of cytokines, chemokines, PGE2 and M-CSF in vitro, all of which are implicated in bone resorption. 1,25-dihydroxy vitamin D3 (1,25D3) has a pivotal role in bone resorption. It stimulates osteoclastic bone resorption and formation, causes fusion of committed osteoclast precursor cells and activates mature osteoclasts in vitro. Under the correct conditions, macrophages (MP) have the ability to differentiate into osteoclasts. Research has shown that MP can synthesise 1,25D3 and changes in this synthesis occur during MP differentiation. We therefore examined how the application of hydrostatic pressure to MP in vitro influenced their synthesis of 1,25D3. In this study, normal human peripheral blood MP (5x105/ml) were cultured for 7 days then exposed to physiological pressure (34.5x10-3MPa) and/or UHMWPE particles (8mg/ml) and the effect on synthesis of 1,25D3 by endogenous 1a-hydroxylase (1aOHase) was studied. MP were incubated with H3-25, hydroxy vitamin D and 1,25D3 synthesis was analysed by HPLC. 1,25D3 synthesis was increased in cells under pressure by an average of 17% compared to static controls. In situ hybridisation (ISH) was used to demonstrate expression of 1aOHase. Image analysis showed a small increase in 1aOHase mRNA in response to pressure and to particles, and a larger increase to the two stimuli simultaneously. Expressed as % of maximum +Pressure + Particles 100%;+ Particles 59%; +Pressure 37%; No Stimulus < 0.1%. These results suggest that 1,25D3 may be one of the factors which stimulates osteoclastic bone resorption in aseptic loosening. As both these stimuli are likely to be present in vivo, such synthesis could further exacerbate loosening.
All four NSAIDs reduced The effect on adherence was confined to unconditioned PMMA. The effect on biofilm formation and on established biofilms appeared to be related to that on growth and viability. On these grounds, NSAIDs appear to have a limited prospect for use in prevention or treatment of
Interfacial stress distributions in the acetabular region have been studied using plane strain finite element models before and after total hip replacement. The model was adapted from a roentgenogram of a 4 mm slice normal to the acetabulum through the pubic and ilium. The model was divided into 24 regions of different elastic constants with isotropic material properties assumed in each region. The femoral head was modelled as a spherical surface that was mated with a congruent spherical acetabular socket. The implanted hip model was developed by modifying the natural hip model. Contact analyses were carried out between the articulating cartilage layers and between a cobalt chromium head and a cemented ultra-high molecular weight polyethylene (UHMWPE) cup under selected hip contact load cases during normal walking. Local polar coordinates were employed to facilitate the calculation of the interfacial stress components between the cup and cement, cement and subchondral bone as well as between the subchondral and underlying cancellous bones.
The results show that severe reductions in the local stresses in subchondral and cancellous bones were found in the reconstructed case. Both the peak stress and the range of the stress were reduced substantially, suggesting stress shielding in the acetabular region. Load transfer in the reconstructed case was found to occur primarily in the cement layer superior to the cup. Both the peak stress and the stress variation in the cement mantle are substantial, whilst abrupt changes in interfacial stresses occurred between the cement and cup, and cement and subchondral bone. The influence of subchondral bone retention and thickness of the cement (up to 6 mm) on the interfacial stress distribution appears to be insignificant.
The work represents the first stage of research towards developing a numerical tool for pre/post operative assessment of cement/cementless acetabular components.
Joint replacement failure is usually caused by the formation of wear debris resulting in aseptic loosening. Particulate metal and soluble metal ions from orthopaedic alloys (cobalt chromium or vanadium titanium aluminium) that are used in medical prostheses can accumulate in tissues and blood leading to increased chromosome aberrations in bone marrow and peripheral blood lymphocytes. This paper demonstrates that two of the metals used in orthopaedic prostheses, chromium and vanadium can produce delayed as well as immediate effects on the chromosomes of human fibroblasts in vitro. Fibroblasts were exposed to metal ions for only 24 hours and were then expanded over 30 population doublings involving ten passages. The initial increase of chromosomal aberrations, micronuclei formation and cell loss due to lethal mutations persisted over multiple population doublings, thereby demonstrating genomic instability. Differences were seen in the reactions of normal human fibroblasts and those infected with a retrovirus carrying the cDNA encoding hTERT that rendered the normal human fibroblasts telomerase-positive and replicatively immortal. This suggests that chromosomal instability caused by metal ions is influenced by telomere length or telomerase activity. Formerly this syndrome of genomic instability has been demonstrated in two forms following irradiation. One type is non-clonal and involves the appearance of lethal aberrations that cannot have been carried by the surviving cells. The other type is clonal and the aberrations are not lethal. These may arise as a result of complex rearrangements occurring at a high rate post-insult in surviving cells. The consequences of genomic instability are not yet known but it is possible that the increase of chromosomal aberrations that have been previously observed in human patients could be due to immediate and delayed expression of cellular damage after exposure to orthopaedic metals.
Wear debris from worn cobalt chrome joint replacements causes an increase in chromosomal translocations and aneuploidy. In this study the relationship between the amount of DNA damage and the changes in gene expression was investigated in human fibroblasts after exposure to artificial cobalt chrome particles. The comparison was made with different doses of particles, at different time intervals and in fibroblasts of different ages, those that had completed 10 population doublings (10 PD fibroblasts) and those that had completed 35 population doublings (35 PD fibroblasts). The genes (TGF-©¬2, p38 MAPK, Integrin ¥â1, SOD1, Caspase 10, PURA, FRA-1 and VNR) were chosen after a previous screen with cDNA microarrays. The percentage of senescent cells was evaluated using an immunohistochemical assay for ¥â-galactosidase activity. The 35 PD fibroblasts showed significantly more ¥â-galactosidase activity than the 10 PD fibroblasts. The level of DNA damage, as detected with the alkaline comet assay, was greater at higher doses, at longer exposures (up to 24 hours) and in 10 PD fibroblasts. The expression of all the genes listed above was generally lower after exposure to cobalt chrome particles using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The reduction in gene expression, like the increase in DNA damage was greater at higher doses and at longer exposure times. After 24hr exposure the reduction in gene expression was greater in 10 PD fibroblasts compared to 35 PD fibroblasts. After 6hr exposure this was only true at higher doses of particles and the opposite was seen after a lower dose of particles. These results show that levels of gene expression of TGF-©¬2, p38 MAPK, Integrin ¥â1, SOD1, Caspase10, PURA, FRA-1 and VNR may be correlated with the level of DNA damage and that this depends on the dose and length of exposure and the age of the cells. This highlights the potential importance of these genes in the mutagenicity of cobalt chrome particles in human fibroblasts.
Clonal chondrocytes of osteoarthritic (OA) cartilage express an aberrant set of genes. We hypothesize that this aberrant gene expression may be due to clonally inherited epigenetic changes, defined as altered gene expression without changes in genetic sequence. The major epigenetic changes are due to altered DNA methylations in crucial parts of the promoter region. If the cytosines of CpG dinucleotides are methylated, the gene will be silenced, even if the right transcription factors are present. Similarly, de-methylations may activate previously silenced genes. Our aims were to provide ‘proof-of-concept’ data by examining the methylation status of genes in OA vs non-OA chondrocytes. Articular cartilage was obtained a) from the cartilage of fracture-neck-of-femur (#NOF) patients and b) from or around the eroded regions of OA samples. The former was full thickness cartilage, the latter was partially degraded cartilage, which contained mostly clonal chondrocytes as confirmed by histology. The cartilage samples were ground in a freezer mill (Glen Creston, UK) and DNA was extracted with a Qiagen DNeasy maxi kit. To assess DNA methylation status, the genomic DNA was treated overnight with methylation-sensitive restriction enzymes. Cleavage of selected sites was detected by PCR amplifications with primer pairs designed to bracket selected promoter regions. Loss of the PCR band after digestion with the enzymes indicated absence of methylations, whereas presence of the band indicated methylated cytosine. We selected MMP-9 as one of genes that is activated in OA. Transcription of mmp-9 is regulated by a 670 bp sequence at the 5′-end flanking region, which contains 6 CpGs and a further 21 CpGs within the 1.5 kb region further upstream. A PCR primer pair was designed to bracket a 350bp sequence upstream from the transcription start site of mmp-9, which contained four of the six potential methylation sites, cleaved by the methylation-sensitive enzymes AciI and HhaI. DNA from 9 OA patients, 5 #NOF patients and 1 rheumatoid arthritic (RA) patient were digested with HhaI or AciI and examined for the presence or absence of PCR bands. In all patients, digestion with HhaI abolished the PCR band, indicating that the HhaI site was never methylated in either #NOF or OA patients. However, a remarkable difference was found after digestion with AciI: in 8/9 OA patients, the PCR band was no longer detectable, while in 4/5 #NOF patients the PCR band was still present. This suggested that all three AciI cleavage sites were methylated in the majority of chondrocytes from #NOF patients, while at least one of the three AciI cleavage sites was unmethylated in OA patients. Interestingly, the PCR band was present in the RA patient, suggesting methylation of the AciI cleavage sites. The present study provides the first ‘proof-of-concept’ data that suggest epigenetic changes may play a role in the etiology of osteoarthritis. Clearly further work is required to establish the generality of the present findings and whether de-methylations are also found in the promoter regions of other genes that are aberrantly expressed in OA.
It is possible in theory to optimise the geometry of the metallic bearing surfaces, based on the worn components, to minimise the running-in wear. However, such an improvement in lubrication cannot be readily achieved because of difficulties in surgical techniques and position of the components.
Osteoclasts are cells that resorb bone. They derive from haemopoietic precursors in the presence of Macrophage-Colony Stimulating Factor (M-CSF) and the osteoclast growth factor, Receptor Activator of Nuclear Factor–kB Ligand (RANKL). Tumour Necrosis Factor-a (TNF-a) and M-CSF has been shown to form mature osteoclastic bone resorption in vitro murine cultures in the absence of RANKL. The aim of this study was to investigate the mechanism of action of the pro-inflammatory cytokine Tumour Necrosis Factor-a (TNF-a) with respect to osteoclastic bone resorption. Development of osteoclasts was performed using an in vitro assay of healthy human peripheral blood mononuclear culture (PBMNC) in the presence of M-CSF and RANKL. In the same cultures RANKL was replaced by TNF-a over a wide range of concentrations. Osteoclasts were generated in the presence of M-CSF, TNF-a and RANKL from human PBMNC. However, in the same experiments M-CSF and TNF-a in the absence of RANKL failed to support human osteoclast formation. Aseptic loosening and osteolysis are considered the main long-term complications of hip arthroplasty. Pathogenesis of peri-prosthetic osteolysis is multifactorial and both biological and mechanical factors are important. TNF-a is thought to be involved in orthopaedic implant oste-olysis induced by prosthesis-derived wear particles. The final osteolytic step is undertaken mainly by osteoclasts. This is the first report showing that TNF-a and M-CSF in the absence of RANKL in human PBMNC is not capable of inducing osteoclast formation. TNF-a therefore may increase peri-prosthetic loosening by enhancing the activity of the mature osteoclast.
The purpose of this study was to evaluate the effect on movement under load of different techniques of reat-tachment of the humeral tuberosities following 4-part proximal humeral fracture. Biomechanical test sawbones were used. 4-part fracture was simulated and a cemented Neer3 prosthesis inserted. Three different techniques of reattachment of the tuberosities were used – 1)tuberosities attached to the shaft, and to each other through the lateral fins in the prosthesis with one cerclage suture through the anterior hole in the prosthesis, 2)as 1 without cerclage suture, and 3)tuberosities attached to the prosthesis and to the shaft. All methods used a number 5 ethibond suture. Both tuberosities and the shaft had multiple markers attached. Two Digital cameras formed an orthogonal photogrammetric system allowing all segments to be tracked in a 3-D axis system. Humeri were incrementally loaded in abduction using an Instron machine, to a minimum 1200N, and sequential photographs taken. Photographic data was analysed to give 3-D linear and angular motions of all segments with respect to the anatomically relevant humeral axis, allowing intertuberosity and tuberosity-shaft displacement to be measured. Techniques 1 and 2 were the most stable constructs with technique 3 allowing greater separation of fragments and angular movement. True intertuberosity separation at the midpoint of the tuberosities was significantly greater using technique 3 (p< 0.05). The cerclage suture used in technique 2 added no further stability to the fixation. In conclusion, our model suggests that the most effective and simplest technique of reattachment involves suturing the tuberosities to each other as well as to the shaft of the humerus. The cerclage suture appears to add little to the fixation in abduction, although the literature would suggest it may have a role in resisting rotatory movements.
To determine the quantitative adherence and biofilm development of P. acnes on titanium compared to surgical steel. To assess the subsequent effect of penicillin, the therapeutic drug of choice, on mature P. acnes biofilms.
Autologous osteochondral cylinder transfer is a treatment option for small articular defects, especially those arising from trauma or osteochondritis dissecans. There are concerns about graft integration and the nature of tissue forming the cartilage-cartilage bridge. Chondrocyte viability at graft and recipient edges is thought to be an important determinant of quality of repair. The aim was to evaluate cell viability at the graft edge from ex vivo human femoral condyles, after harvest using conventional technique. With ethical approval and patient consent, fresh human tissue was obtained at total knee arthroplasty. Osteochondral plugs were harvested using the commercially available Acufex 4.5mm diameter mosaicplasty osteotome from regions of the lateral femoral condyle (anterior cut) that were macroscopically non-degenerate and microscopically non-fibrillated. Plugs were assessed for chondrocyte viability at the graft edge using confocal laser scanning microscopy (CLSM), fluorescent indicators and image analysis. The central portions of the plugs remained healthy, with > 99% cell viability (n=5). However, there was substantial marginal cell death, of thickness 382 ± 68.2 microm in the superficial zone (SZ). Demi-plugs were created by splitting the mosaicplasty explants with a fresh No. 11 scalpel blade. The margin of SZ cell death was 390.3 ± 18.8 microm at the curved edge of the Acufex, significantly (Mann-Whitney; P= 0.0286; n =4) greater than that at the scalpel cut (34.8 ± 3.2 microm). Findings were similar when the cartilage was breached but the bone left intact. In time-course experiments, the SZ marginal zone of cell death after Acufex harvest showed no increase over the time period 15 minutes to 2 hours. Mathematical modelling of the mosaicplasty surface shows that cell death of this magnitude results in a disturbing 33% of the superficial graft area being non-viable. In conclusion, mosaicplasty, though capable of transposing viable hyaline cartilage, is associated with an extensive margin of cell death that is likely to compromise lateral integration. There would appear to be considerable scope for improvement of osteochondral transplant techniques which may improve graft-recipient healing and clinical outcomes.
Mechanical load is crucial to maintaining skeletal homeostasis, but the pathways involved in mecha-notransduction are still unclear. The OPG/RANK/ RANKL triumvirate has recently been implicated in bone homeostasis. These molecules, which are produced by the osteoblast (OPG and RANKL) and the macrophage/osteoclast (RANK), modulate osteoclastogenesis. We have previously shown that cyclical hydrostatic pressure influenced synthesis of various molecules by cultured human macrophages. These factors are important in osteoclastogenesis and bone resorption and have been linked to the development of aseptic loosening. We have also demonstrated that 1,25-dihydroxyvitamin D3 (1,25D3) influences macrophage response to pressure. For this study human macrophages were co-cultured with osteoblasts and subjected to cyclical hydrostatic pressure (34.5x10–3MPa [5.0 psi]) for up to five days, with or without 1,25D3 supplementation. Cells were immunostained for RANK and culture media were assayed for sRANKL and OPG using specific ELISAs. Immunostaining for RANK showed that macrophages subjected to pressure or 1,25D3 supplementation synthesised more RANK than controls. In addition, when exogenous 1,25D3 and hydrostatic pressure were administered simultaneously, immunostaining for RANK was more intense. There was a reciprocal relationship between OPG and sRANKL in co-cultures subjected to pressure. If pressure increased synthesis of sRANKL, OPG was decreased. In cultures where pressure decreased sRANKL, a corresponding increase in OPG was seen. In addition, samples from different individuals responded differently to pressure. The majority of cell populations responded to pressure by increasing OPG synthesis, compared to non-pressurised controls. These results demonstrate for the first time that the OPG/RANK/RANKL complex is sensitive to hydrostatic pressure and that 1,25-dihydroxyvitamin D3 might be involved in this response. These findings suggest a possible transduction mechanism for mechanical load in the skeleton, which has implications for future therapies for aseptic loosening and for skeletal abnormalities such as osteoporosis.
High bone density will increase the yield point and stiffness of the femoral head and therefore improve the implant fixation. Cement fixation will increase the yield point and stiffness of the femoral head, especially for the lower density bone compared with cementless fixation.
For yield point, there is no significant difference between cemented or cementless resurfacing (4169 ± 1420 N vs. 3789 ± 1461 N; P = 0.434). However, the high density heads provide a significantly higher yield point than low density heads (4749 ± 1145 N vs. 3208 ± 1287 N; P = 0.01). The addition of cement significantly contributes to femoral head stiffness compared to cementless resurfacing (5174 ± 1730 N/mm vs. 3678 ± 1630 N/mm; P = 0.012).
A phenomenon of methicillin resistance in methicillin sensitive Staphylococcus aureus has been noted in organisms living in biofilm induced by the state of cell wall deficiency. The rate and the amount of biofilm formed by the cell wall deficient organisms far exceeds that of cell wall patent organisms. Once removed from the biofilm the S. aureus had the same sensitivities of the original organism. Cell wall deficient organisms outside the biofilm did not demonstrate the methicillin resistance. A known laboratory strain (ATCC 9144) was induced into a cell wall deficient state and allowed to form biofilm. The rate of formation and amount formed was compared with that formed by cell wall patent organisms. Before inducing cell wall deficiency sensitivity to methicillin was demonstrated using standard microbiological technique. Using an oxacillin containing plate as a culture medium: the biofilm, cell wall deficient organisms and the cell wall competent organisms were inoculated onto separate media. Organisms from the biofilm were isolated and grown free of the biofilm on blood agar. Any growth on the oxacillin containing plate would demonstrate methicillin resistance. There was no growth on the plates containing the cell wall competent or cell wall deficient organisms. There was however growth on the plate inoculated with bio-film, however when organisms were isolated from the biofilm, there was no growth on the media. Antibiotic sensitivities of the original inoculant and the organisms isolated from the biofilm were the same. The biofilm, induced as a result of cell wall deficiency, offers a form of structural protection to the Staphylococcus aureus without altering the resistance pattern of organism. Standard microbiological techniques would therefore report the organism as methicillin sensitive, however clinically the organism may behave as a methicillin resistant organism. The state of cell wall deficiency encourages the formation of biofilm in S. aureus. In-vitro the state of cell wall deficiency is induced using high osmolality media or sub-lethal doses of cell wall active antibiotics. Both these states are found in clinical practice.
Use of allograft in orthopaedic surgery is a well-established procedure. Ethylene Oxide sterilization is still controversial in bone banking because of its effect on osteoinductive properties of bone graft. Freeze drying is considered to be the best technique for allograft preparation and storage. High cost of equipment and its maintenance makes this method not feasible option in developing countries like India. This study involved setting up of a bone bank for the first time in JIPMER institute, Pondicherry, India. Cancellous bone was collected from 40 patients (femoral heads removed during joint replacements). They were cleaned thoroughly, chemically processed and sterilized with ethylene oxide gas and stored doubly packed. These were implanted at 11 patients with 14 non-unions, which required cancellous bone grafts. Patients were followed up clinically looking for infection and radiologically for graft incorporation. 85.7 % of grafted sites were united at the end of 12 months. Non-unions took average of 44.8 weeks for the union. Radiological union achieved by 12 months with average time of graft incorporation 44.8 weeks. In 8 cases the allografts were used to pack cavities. Healing occurred at an average of 29 weeks. In 4 patients with arthrodesis following excision of tumor one site failed to unite, one deep infection, which did not resolve with regular chemotherapy had an amputation. The rest of the sites healed at an average 54.8 weeks. This study shows ethylene oxide sterilized cancellous allograft suitable for packing cavities in treatment of benign bone lesions as well as in treatment of non-union. The osteoconductive property of bone allograft may not be affcted by the ethylene oxide sterilization. Achivement of union and a low rate of infection confirms efficacy of ethylene oxide as cost effective and reliable option for bone allograft sterilization.
All polyethylene tibial components (APT) for total knee joint replacement have been recently reintroduced due to their past success and cost savings with respect to knee designs with a metal backed tibial tray (MBT). However, isolated cases of collapse of the medial bone in APT designs have been observed by the authors prompting this investigation. The objective of this study was to investigate the stress/strain distribution within the cancellous bone for the APT and MBT systems, particularly looking at the effects of coverage of the tray over the proximal tibia in each design. A three-dimensional finite element model of the proximal tibia implanted with a tibia tray was generated. An elliptical cylindrical tibia tray with a peg was modeled as being perfectly bonded to a PMMA layer on the superior surface of the cancellous and cortical bone. Gap size between the edge of the tray and outer of the cancellous bone, was introduced in the medial direction. Load was applied on the superior surface of the tibial insert in the medial side. Two lift-off loading cases were used, a low load of 800N (1 body-weight) and a high load of 3200N (4 x BW), both on the medial side. Permanent plastic deformation and collapse was allowed only in the cancellous bone, while all other materials were modeled elastically. Under low load conditions within the elastic limit, introducing a gap between the tray and the cortical bone produced a stress/strain intensity in the cancellous bone beneath the edge of the tray. The strain in the cancellous bone within the APT design was generally 3 times greater than the MBT design, however, peak strain values were similar at the edge of the tray. Whilst the strain increased with the introduction of a gap the resulting strain was not sensitive to the gap size for both designs. Under high load conditions, permanent plastic deformation and bone collapse were observed in the cancellous bone at the edge of the tibial tray in both designs where a gap was introduced. The maximum strain in the cancellous bone was found to be more sensitive to the gap size for the APT design than the MBT design. This can be contributed to the difference in the load transfer through the cancellous bone in the two designs. The MBT design with the more rigid tibial tray transfered higher load through the outer cortical bone than the APT design. The less rigid APT design resulted in progressive collapse of the cancellous bone beneath the tray. Particularly significant was the volume of highly stressed cancellous bone which was 4 times greater in the APT design compared to the MBT design. The results suggest that coverage may be a more important parameter for the APT design than the MBT design. The APT design may, therefore, be more suited to patients with better bone quality.
Back pain is a major cause of disability and absence from work. 80% of the population will experience back pain at some point in their lives. In our study we looked at 2 randomised groups of patients. Group 1 patients had only epidural steroid injections (ESI) and group 2 patients had ESI plus radiofrequency (RF). We hypothesized that there is no difference in outcome between group 1 and 2 patients. The 2 groups were sent out a retrospective questionnaire which had 5 parts to it, including SF-36 health survey, pain drawing chart, visual analogue scale (VAS), oswestry disability score (ODS) and a patient satisfaction questionnaire. The patients had treatment between 2002 and 2003 and the post-treatment questionnaires were sent out in May 2004. The SF-36 was scored giving a physical component score (PCS) and a mental component score (MCS) using an online scoring website. The groups studied were from 2 different referral hospitals. The patients were randomised by GP referral being sent to the 2 different hospitals. 115 questionnaires with stamped addressed envelopes were sent out to group 1 patients, out of which 71 were returned (61.7%) and 113 to group 2 patients out of which 55 were returned (48.7%). Statistical analysis was done using the SPSS software programme. As there was some evidence of non-normality Mann-Whitney test was carried out, and for the patient satisfaction questionnaire, chi-squared and fisher’s exact test was used. We found that there was a significant difference among the 2 groups in the PCS (p< 0.0005) and MCS (p=0.017). There was a statistically significant difference among the 2 groups in their pain draw score, VAS and ODS with p values of < 0.0005. In the patient satisfaction questionnaire, 8 questions were asked. Patients were asked to assess how successful the spinal injection was. 35 (67%) patients from group 2 said it was successful, compared with 25 (37%) patients from group 1. 9 (17%) patients from group 2 said it was not successful compared with 27 (40%) patients from group 1. 8 (15%) patients were not sure from group 2 and 16 (24%) were not sure from group 1. The difference was statistically significant with a p value of 0.003. When asked whether they would recommend this type of injection, more patients from group 2 said they would (p=0.029). When asked about the duration of effectiveness of the injection, group 2 noticed an increased duration of benefit compared with group 1 (p< 0.0005). There was no significant difference between the groups when asked how many injections were required (p=0.089) or when asked whether or not they required painkillers (p=0.062). However, more patients from group 2 said that painkillers controlled their pain (p=0.001). When asked if they were able to return to work and do housework/gardening after injection, there were significantly more patients from group 2 being able to do so (p< 0.0005). We conclude that in the patients studied, the group who had radiofrequency treatment and epidural steroid injection did better as compared with patients who had epidural steroid injection alone.
Distal radial fractures account for 17% of all fractures treated, with peaks in the bimodal distribution corresponding to young and senior patients. External fixation is one of the best techniques to allow quick patient recovery and is necessary for complex fractures, such as that of the distal radius. However, the safe removal time for these frames remains unclear. A conservative approach commonly leaves the external fixator in place for six weeks, which may be unnecessarily prolonged and lead to increased complications. The aim of this work is to develop a technique to quantify, objectively, a safe removal time for these frames. Studies have been conducted on external fixation of tibial fractures, however there are differences that do not allow transfer of these studies to the external fixation of distal radial fractures. These differences include configuration of the fixation frame, bone and fracture geometries, and the application and transfer of the load to the bone. In this work, the dynamic transfer of the load between the fractured bone and the fixator is investigated. An instrumented grip and a measuring device have been developed to monitor the axial force and displacement when the patient applies a load. Using measurements collected by the instrument and data specifying the frame geometry, a finite element model is used to calculate the load carried by the fixator and by the bone, and the rigidity of the new callus is determined. Plotting the rigidity on semi-logarithmic scale the healing rate can be established. This technique has been successfully verified in a laboratory simplified structure representative of bone fracture. The rigidity of several intra-gap materials has been estimated experimentally using the technique, and the results compared to the real value of the material. These measurements do not interfere in any way with the patient treatment and they can be collected from the first day after the operation. The technique has been tested on 14 volunteer patients and the increase in callus rigidity can be detected by measurements during treatment using the technique described. A randomised prospective study has been initiated to validate this technique and investigate the healing process. A positive outcome would enable the rigidity of the new callus bone and the healing rate to be monitored during clinical assessment. Any healing delay or non-union could be promptly detected, improving the quality of the treatment.
During conventional hip arthroplasties, the diseased femur is rigidified using a metallic stem. The insertion of the stem induces a change in the stress distribution in the surrounding femur, and the bone remodels; this stress distribution is a direct result of the stem stiffness characteristics. Healthy healing of the femur requires that the bone be loaded as naturally as possible. If the bone is not loaded appropriately, it can resorb which may result in stem loosening and revision. Although current rigid metallic femoral stems are very successful, a poor stress distribution may become a critical problem for younger patients as the stem/femoral bone construct will be subjected to higher loads for longer times, and since remodelling is faster, loosening can occur earlier. Reduced stiffness stems have therefore been investigated, but early failures have been reported due to increased movements, poor initial stability and the low proximal stiffness of the stem. A novel biocompatible carbon fibre reinforced plastic (CFRP) stem has been developed in light of these past experiences
Measuring strain in biological specimens has always been inherently difficult due to their shape and surface properties. Traditional methods such as strain gauges require contact and therefore have reinforcing effects, also the surface preparation can be time consuming and if proper fixation is not achieved the results will be inaccurate. Using a non contact method to measure strain such as photogrammetry has several advantages. The strain over the whole surface of a specimen can be mapped, depending on the field of view of the camera used. It has a large dynamic range, from microns to millimetres which can be decided upon at the post processing stage. Specimens can be tested to destruction without damaging any measurement equipment. Also there is considerably less set up time involved between testing different specimens once the system is in place. We aimed to test speckle photogrammetry, a method used in industry and fluid dynamics as a tool for assessing proximal femur fracture stability and repair techniques. A Zwick Roell materials testing machine was used to axially apply a staircase loading pattern to sawbones femora, simulating the load experienced by the femur when standing. Firstly an intact bone was tested then a set of three identical fractures of each of three common fracture configurations were produced by osteotomy. The first femur of each configuration was loaded un-repaired to failure; the remaining two were repaired using common techniques for that particular fracture type then also loaded to failure. The bone and fixation device were covered with stochastic, high contrast paint speckle prior to testing. This speckle pattern was recorded at regular load intervals by a digital camera which was attached to the materials testing machine via a rigid frame to eliminate any camera movement. These images were then transferred to a computer where they were converted to 8 bit bitmap images. Matlab was used to process the data from subsequent images to produce vector and colour maps of the displacements and strains over the entire visible surface of the proximal femur and to show the comparative displacements and strains experienced by the individual bone fragment and the fixation devices. Non contact optical strain measurement has proved itself to be a useful tool in assessing the stability of fractures and the repair techniques of these fractures. Additionally it can also be used to validate finite element models to compare theoretical and experimental results due to the similar data and graphic visualisation outputs which are produced by both techniques.
Conventional fixed-bearing (FB) knee prostheses have been proved clinically successful. Rotating platform, mobile-bearing (MB) total knee replacements (TKR) have been developed to improve knee kinematics, lower contact stresses on the polyethylene tibial component, minimize constraint, and allow implant self-alignment. The purpose of this study was to characterize and compare the functional outcome of FB- and MB- TKR during gait and deep knee bends, using a motion analysis system. Two groups of five patients with a unilateral FB TKR (PFC) or MB TKR (LCS) underwent a gait analysis study. The normal contralateral limb was used as a control to compare data in the stance phase of gait. Demographic, clinical, and radiographic data were equivalent in the 2 groups. Both MB and FB TKRs gave good functional results in spite of different design rationales. No statistically significant difference was demonstrated between the two groups. However, gait and knee function after TKR was abnormal even though the patients were asymptomatic. A flexional pattern for flex-ion-extension moments at the knee during level walking was present in both types of TKR. Differences in rotational moments between the two groups were observed, with a higher internal rotational moment in the PFC group (PFC, 0.14 Nm/kg; LCS, 0.09 Nm/kg; p=0.094). A stressful weightbearing activity, such as deep knee bends, amplified the functional differences between the different prosthetic designs, indicating that knee kinematics are activity-dependent. Kinetic and kinematic differences noted between the 2 groups reflect different patterns of joint surface motion and loading, with postulated effects on long term failure of the implants through wear, mechanical failure, and loosening. Gait analysis using external skin markers has a limited role in the characterization of the joint surface motion of the prosthetic knee during ambulatory activities because of errors and assumptions inherent in the technique. However, it provides scope for the study of kinetic parameters acting on different knee prostheses during gait.
Osteomyelitis commonly causes bone destruction and is most frequently due to infection by Staphylococcus aureus.
Bone microhardness has been successfully correlated with important functional parameters such as mineralisation and stiffness. It provides a means of examining the mechanical competence of bone at a micron scale, averaging the effect of osteonal lamellae but sensitive to variation in mineral content within a bone, and, with careful selection of indentation site, able to obtain material characteristics separate from any effects of porosity. However, the effect of bone’s viscoelasticity on such measurements has been largely ignored. This preliminary study investigates the post-indentation size change of Vickers indentations on wet bone. 4 axial slices of bovine femur were harvested from the same shaft, and polished. Each sample was subjected to 4 sets of 10 Vickers indentations with a load of 50 g and holding period of 15 s. The indentation size was measured immediately after the load was removed, and then again at intervals for a period up to 24 hours after the indentation was made. To avoid dehydration, the bone stood in water during the indentation testing and during measurement, and between each measurement period it was fully immersed in water. Measured hardness significantly decreased with time, by approximately 30% in total. The rate of post-indentation recovery is difficult to analyse since the driving force of residual strain decreases as recovery takes place. However a simple exponential fit to the variation of HV with time in the form of H = H(final).(1−exp(−kt)) + H(initial) suggests that the size of the indentation tends towards a constant size between 5 and 24 hours after indentation. Thus we conclude that care should be taken when making “early” measurements given the rapid rate of change in indentation size. Caution should also be employed when interpreting such data.
Since cementless stem fixation in hip arthroplasty is becoming more and more common, the overall incidence of intraoperative femoral fractures has risen considerably. Depending on primary or revision arthroplasty, literature reports fracture rates between a few percent up to one third of the cases.
In this study, methods commonly applied in the field of structural testing were customized for this specified interference fit situation. A cementless hip system (ABG II, Stryker) was used on animal bones and biomechanical bones.
Transient excitation in the form of regular hammer strokes and sinusoidal excitation using a shaker served as an input. The output of the system under test was measured on the greater trochanter using a piezoelectric accelerometer.
The signals were digitized with a high-speed data acquisition system and analyzed in real-time with spectrum analysis software.
Analysis included threshold detection in the time domain to determine the time delay between the input and output transducer. Spectrum analysis in the frequency domain included FFT analysis and frequency response function analysis to identify shifts of fundamental frequencies and harmonics to describe the vibrational changes with increasing stability.
A digital imaging system was set up to take pictures of the metal-bone site to measure inducible displacement with each hammer impact and correlate it with the vibrometry results. Furthermore a strain gauge circularly mounted around the proximal femur monitored accurately any hairline fracture.
This study shows that changes of the vibrational spectrum are directly related to implant fit. The range of interest is well in the sonic range, which apparently is the reason for many surgeons to listen and ‘feel’ carefully during advancing the broach or the final implant into the femur.
The study is trying to extract critical vibrational parameters correlated with stability and femoral integrity. Due to the different dimensions of the tested animal bones and lack of soft tissue damping, further experiments on cadavers need to be carried out.
Vibrational spectrum analysis could prove to be a useful tool to readily assess implant stability and femoral integrity. It seems to be most beneficial in revision surgery or minimally invasive hip replacement, where the risk of femoral fractures is increased or fissures could easily be missed.
Cortical porosity is a useful evaluator of bone since it is sensitive to changes in bone turnover. The aim of this study was to evaluate cortical bone porosity of human vertebrae samples using Scanning Acoustic Microscopy (SAM). Currently the common techniques used to determine bone porosity are histomorphometry or scanning electronmicrosopy images. Both methods require extensive preparation of the bone samples. SAM represents a new technique with the great advantage of minimal sample interference since the bone is imaged in water, or saturated, and requires just one flat surface which is scanned (but not contacted) by the transducer. 46 specimens between the ages of 64–90 years were randomly selected and ground before SAM imaging of was carried out using a 400 MHz transducer. For each sample posterior and anterior sections of the cortical bone were scanned several times, and the porosity measured using Scion image software to process the images. It was possible to image the entire anterior or posterior cortex in a single image with 4 mm spatial resolution. Measured porosity was in the region 5 % – 21 %, and showed a significant increase with age for the female specimens but no age dependence in the male specimens. At low porosity (< 6 %) vertebral compressive strength was uncorrelated with porosity. However, at higher porosities strength was highly correlated with porosity. (As would be expected, strength decreased with increasing porosity). High frequency SAM has potential for future bone characterisation, particularly where it is desirable to correlate local measurements of material properties such as nanohardness or microhardness, with microstructure.
the circumference of the anatomical neck; (H) as the most superior point of the articular surface at the insertion of the supraspinatus tendon, (L) as the corresponding lowest point of the articular surface at the cartilage/calcar interface; The medial (MC) and lateral (LC) humeral condyles were exposed and delineated with k-wires.
A Microscribe 3D-X digitizer was used to digitize the points and lines. The data for each humerus were imported into Rhinoceros NURBS modelling software and graphically represented. The constructed graphical model was used to divide the articular portion of the humeral head into six equal sections in the axial plane. The retroversion angle, relative to the epicondyles, was calculated for each section.
Intervertebral disc function and dysfunction is governed by its structural architecture of concentric layers of highly ordered collagen fibres. This architecture is important at the mm scale for overall mechanical performance of the disc; and at the micron scale for mechano-transduction signalling pathways of the disc cells that are responsible for matrix maintenance and therefore disc health. To understand such mechanical behaviour 3-dimensional collagen fibre architecture must be quantified in intact intervertebral discs. Conventional imaging modalities lack either the spatial resolution (e.g. x-ray diffraction) or penetration (e.g. optical, electron or confocal laser microscopy) to yield mechanically important information. Preliminary studies of scanning acoustic microscopy (SAM) at 50 MHz visualises alternating layers of fibre texture, however exactly what is being imaged requires both explanation and validation. Three-dimensional SAM data sets obtained from intact discs were compared to polarised-light and scanning electron micrographs of individual layers of fibres, peeled by micro-dissection from discs. The dimensions of the structural features were measured and recorded. Optical and electron microscopy revealed that each layer consisted of highly oriented collagen fibres of diameter 5 μm with regularly spaced splits between fibres with a spacing of approximately 20–30 μm. The SAM data sets showed layers with a uniform highly oriented fibre texture that reversed between adjacent layers. Resolution of the texture was limited by the acoustic system to approximately 30 μm. It is clear that SAM at 50 MHz cannot resolve and therefore image individual collagen fibres. However, the regular defects in the fibre layers can be visualised and convey complete information about local collagen fibre architecture. SAM therefore provides an effective way of quantifying the fibrous structure of intact, hydrated, unfixed intervertebral discs.
This study aimed to explore the relationship between the geometry of the tuberosity located superior to the Achilles tendon enthesis and the thickness of its fibro-cartilaginous periosteum. The tuberosity acts as a pulley for the tendon during dorsiflexion of the foot and is thus compressed by the overlying tendon. This can result in pressure-related injuries which account for a significant number of Achilles-related problems among sportsmen or women. We postulated that variations in the contact area between the tendon and the tuberosity (and consequently the pressure exerted by the tendon) affects the periosteum thickness. Here, we report four methods of portraying the two dimensional geometry of the superior tuberosity. Material was obtained from 10 elderly dissecting room cadavers donated to the Cardiff University for anatomical examination and prepared for routine histology. Serial sagittal sections were collected at 1 mm intervals, and stained with Masson’s trichrome, toluidine blue and haematoxylin & eosin. In the first method, the area of the bursal cavity was measured between the deep surface of the tendon and the tuberosity within a 9mm radius of the proximal part of the attachment site. The second technique was similar, though used the long axis of the tendon as a reference, rather than its deep surface. The third technique measured the area of the tuberosity within 20 degrees of the tendon long axis. The final technique measured the cumulative gradient of the first 5 mm of the tuberosity, with reference to the tendon long axis. The periosteum thickness was measured at 500 μm intervals from the proximal part of the enthesis and mean values calculated. A good correlation was seen between all techniques, with the tuberosities having the most localised area of contact with the tendon, showing the thickest periosteum.
Surgical site infection (SSI) is an important outcome indicator. It is estimated that 70% of post-operative infections present after discharge. A reliable post-discharge surveillance (PDS) method is yet to be described. The aim of this prospective cohort study was to assess the reliability of patient self-diagnosis. Telephone questionnaires were used following hip and knee prosthetic surgery. A trained validation nurse checked the wounds of all patients reporting problems and a sample of those who did not. 376 elective hip and knee arthroplasty procedures from 363 patients were included. In-patient infection rate was 3.1% (13 of 422 procedures) and post-discharge infection rate was 5.2% (22 of 422 procedures). Results suggest that patients can reliably self diagnose SSI. The sensitivity of the procedure (the probability that the telephone surveillance will detect an infection given that the patients has an infection) was 90.9%. The specificity (the probability that the telephone surveillance will report no infection given that no infection is present) was 76.6%. Hence telephone PDS of SSI is a valuable means of identifying accurate rates of hospital acquired infection following surgery. In this study population, 41% of infections were diagnosed post discharge, which is lower than has previously been estimated. PDS of SSI is necessary if accurate rates of hospital acquired infection following surgery are to be available.
The present study investigates the repeatability of two new methods of measuring acetabular wear with differing levels of automation. Experimental evaluation showed that the more automated method was more repeatable. Both methods segmented the femoral head and acetabular rim with ellipses. The displacement of the ellipse centres was measured and the difference at year 1 and 5 taken as a measure of wear. Measurements were obtained twice for each case. The less automated of the two methods involved the annotation of 9 points on the femoral head and 18 on the acetabular rim to which two least squares ellipse fits[