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;

Introduction: Fibroblast growth factor receptor 3 (FGFR3) is a tyrosine kinase membrane-spanning protein whose function is to regulate chondrocyte proliferation, differentiation and matrix production during cartilage development. Several mutations in FGFR3 have now been documented to link to human diseases. A number of these mutations result in constitutive activation of the FGFR3, leading to proliferation and premature differentiation of chondrocytes. Depending on the mutation and the resultant level of FGFR3 activation, mild to severe skeletal dysplasias such as achondroplasia (ACH), hypochondroplasia (HCH), thanatophoric dysplasia type I (TDI) and type II (TDII), and severe achondroplasia with developmental delay and acanthosis nigrans (SADDAN) may result. It has been postulated that the signalling pathways downstream of FGFR3 may be responsible for activating transcription factors, leading to up-regulation of cell cycle inhibitors and causing abnormal suppression of chondrocyte cell proliferation. However, the precise signalling pathways involved in FGFR3 mutation have as yet to be elucidated. The aim of this study was to investigate and compare the differences in the downstream signalling pathways between FGFR3 mutants.

Methods and Results: Wild type FGFR3 has been cloned into expression vector pcDNA3 and the construct has been used to generate four different FGFR3 mutants using site-directed mutagenesis. The mutations which have been introduced and the types of dysplasia they correspond to were as follows: K380R (ACH), N540K (HCH) and K650E (TDII). A kinase dead form of the receptor, K504R has also been generated. Wild type and each of the four mutant FGFR3 proteins in pcDNA3 vector have been successfully transfected into 293T cells using the calcium phosphate method. Immunoprecipitation and Western Blot analysis of cell lysates revealed expression of wild type protein in three isoforms of size 135kDa (mature), 120kDa (intermediate) and 98kDa (immature). The mutant proteins all followed a similar pattern of expression with the exception of the TDII mutant that did not express the mature form of the FGFR3. Changes in MAPK, PLCã and STAT 1 signalling pathways in response to FGFs-1, 2, 9 and 18 in the 293-cells of wild type and mutant forms of FGFR3 are now under investigation, in an attempt to define which pathways are mostly responsible for the resultant abnormal phenotypes.

Discussion: Genomics studies have demonstrated that FGFR3 expression is significantly upregulated during the osteoblastic differentiation of mesenchymal stem cells (MSCs) under BMP-2 stimulation in vitro. Subsequent functional studies have demonstrated that a selective ligand for FGFR3, FGF9, is able to induce tyrosine kinase signalling, and the osteoblastic differentiation of MSCs in vitro. Further understanding the signalling mechanisms of FGFR3 activation in normal and mutant forms may lead to discover potential anabolic agents that are based on FGFR3-FGFs pathways.

Introduction: Although distraction osteogenesis techniques have been used clinically for the treatment of many skeletal conditions with great success over the last 2 decades, one-step larger extent tibial lengthening (> 5 cm) still remains a clinical challenge. In which tension unbalance of bone and soft-tissue may occur, and complications such as foot drop, ankle and knee dysfunction, cartilage injure and secondary osteoarthritis were common. We have designed and manufactured a new lengthener, which allows bone and soft tissue to be lengthened in synchronism, and ankle joint remain in functional position and may move freely during lengthening.

Methods: A dynamic cross joint apparatus at ankle level was added to a classic Ilizarov circular four-ring lengthener, the apparatus is consisted of a half ring, two dynamic junctions and an elastic (spring) device. In application pins were inserted into distant and proximal segment of the tibia, also through calcanues, the external fixator with the trans-joint device was then applied. Total 296 patients (age 6–46, average 21), 466 legs, were treated with this new lengthener, among them were 55 cases of infantile paralysis, 38 cases of post-trauma bone defects, 33 cases with congenital dysplasia and 170 cases of chordrodysplasia, rickets, dwarf and short stature (height < 148cm). Unilateral tibia lengthening was performed in 126 legs and bilateral tibia lengthening was performed in 340 legs.

Results: Average lengthening for lower limb discrepancy cases was 6.8 cm (2–8cm), and 8.8 cm (8–18cm) for dwarf and short stature. Patients can stand straight and walk during the lengthening. Average movement of ankle joint remained at 10 degree in all cases and x-ray confirmed that average ankle joint space was 2.2 mm (1–4mm). There was no foot drop and ankle joint deformity seen, and in 98% cases ankle joint function fully recovered within 1.5 years after lengthening (6–8 months). Common complications were pinhole infection (25 cases) and broken pin (8 cases). If total lengthening was over 10cm, 70% cases developed slight ankle joint stiffness that would gradually recover after physiotherapy. Severe complications occurred in 5 cases (1%), including nonunion 1 case, mal-union 1 case, bone deformity 1 case and re-fracture 2 cases. All of those cases were cured with satisfactory clinical outcome.

Discussion: The challenge of larger range tibial lengthening is mainly the soft tissue complications, such as foot drop, varus and valgus deformity of ankle joint and loss of ankle function. Prolonged soft tissue traction around the ankle joint may lead to increasing cartilage compression, cartilage damage and partial or permanent loss of joint function. Our dynamic lengthener would allow synchronized lengthening of triceps, Achilles tendon and prosterior tibia muscle with tibia, maintain ankle joint space and free ankle movement. This device was simple and easy to apply, with no need of additional Achilles tendon lengthening. Our clinical study has demonstrated that this device drastically reduced the rate of soft tissue complication. This device makes larger extent tibial lengthening (> 5cm) safer and realistic in clinical practice.

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.

Introduction: Ten percent of fractures end in delayed or non-union. NSAIDs have been linked to an inhibitory action on fracture repair for three decades yet the mechanism of action remains to be elucidated. Cancer research has identified that NSAIDs impede cell proliferation by inhibiting angiogenesis. It is proposed that a similar mechanism occurs in the induction of NSAID induced non-unions. We have investigated this hypothesis in a randomized placebo control trial of the NSAID rofecoxib using a murine femoral fracture model.

Material and Methods: All animals had an open femoral fracture treated using an external fixator. Outcomes measures included x-ray, histology, and biomechanical testing, with laser Doppler used to assess blood flow across the fracture gap.

Results: Radiology showed similar healing patterns in both groups, however at the later stages (day 32) the NSAID group had significantly poorer healing. Histological analysis showed that controls healed quicker (days 24 and 32), with more callus (day 8) and less fibrous tissue (day 32). Biomechanical testing showed that controls were stronger at day 32. Both groups exhibited a similar pattern of blood flow; however NSAIDs exhibited a lower median flow from day 4 onwards (significant at days 4, 16 and 24).

Discussion: Positive correlations were demonstrated between both histological and radiographic assessments of healing, with increasing blood flow. NSAID animals exhibited lower flows, and poorer healing by all outcomes. Regression analysis demonstrates however that the negative effect of NSAIDs on fracture repair is independent of its inhibitory action on blood flow. In conclusion, COX-2 inhibitors are marketed as having cleaner side effect profiles and are widely used in trauma patients. Following development of a novel method of analyzing functional vascularity across a fracture gap, we have demonstrated that the COX-2 inhibitor rofecoxib has a significant negative effect on blood flow at the fracture gap as well as inhibiting fracture repair.

The thrombin-related peptide, TP508, is a synthetic 23 amino acid peptide, which represents the receptor binding domain of thrombin. TP508 mimics thrombin by interacting with receptors on cells involved in tissue repair. TP508 has been shown to enhance revascularization of injured tissue, and promote soft tissue wound healing, cartilage repair, and fracture repair. The aim of this study is to (1) test the effect of TP508 on bone regeneration during distraction osteogenesis; (2) study the chemotactic effect of TP508 on human osteoblasts.

Unilateral tibial osteoectomies were performed and stabilized with MX100 Orthofix lengthener in 5 male adult NZW rabbits. After 7 days, distraction was initiated at rates of 1.4 mm / day for 6 days. TP508 (100 μg/ml, n=2; 10 μg/ml, n=1) or saline (300 μl, n=2) was injected into the osteotomy / lengthening gap at days 1, 7 and 14 post surgery. Animals were sacrificed at 2 weeks after leg lengthening. Bone formation in the regenerate was assessed by radiography, quantitative computed tomography (pQCT) and histology. For chemotaxis studies, MG63 cells were cultured on glass cover slips for three days, and then inverted onto a Dunn chamber slide and sealed with dental wax. Gradients of TP508 (1, 10, 100 μg/ml) were added to the outer well and plain medium to the inner well. A sequence of images of the cells between the wells was taken via a CCD camera for 9 hours at interval of 10 minutes. Movements of individual cells were tracked and statistically analysed by a specially written Macro program. The Rayleigh test for unimodal clustering was used to determine the directional chemotactic movements.

The radiographic evaluation indicated a significant increase in new bone in the distraction regenerate in the TP508 treated groups at 1 and 2 weeks. pQCT images at 2 weeks demonstrated more advanced bone formation in the TP508 treated animals compared to the control. The mean total bone mineral density (BMD) of the regenerate, obtained from 3 slices was significantly greater (p = 0.019, t-test) in the TP508 treated group (BMD = 479.20 +/− 35.57 mg/ccm) than that in the saline control group (BMD = 355 +/− 2.83 mg/ccm). The histological evaluation supported the radiographic and the pQCT results. For chemotaxis study, no directional movements of the cells were found in the controls, whereas the MG63 cells were strongly chemotactic to TP508 at 1, 10 and 100 μg/ml concentrations.

This preliminary study shows that administration of TP508 enhances bone formation during distraction osteogenesis in the rabbit. The findings also show that TP508 has a chemotactic effect on osteoblasts, consistent with the effect of TP508 on fracture repair. A large animal study is in the process to confirm these findings and explore the underlying mechanisms.

Introduction: Osteoblasts precursors reside in the marrow and small numbers circulate in the blood. Our previous work demonstrated an increase in circulating cells following fracture in humans. Skeletal injury is recognised to stimulate a distant osteogenic response.

We hypothesised that in response to fracture, some integral osteoblasts are recruited via the circulation from remote bone marrow sites.

Method: We established a fracture union model in 3-month-old, male, New Zealand White rabbits and reimplanted labelled autologous osteoblast precursors. At date of submission we have 20 rabbits allocated into 4 groups. Three groups had labelled cells re-implanted, whilst the fourth control group did not receive cells. In groups I, II and III the cells were re-implanted into the fracture gap, into the circulation and into a remote bone marrow cavity respectively. There were six animals in groups I and IV, and four in both II and III.

All animals had bone marrow harvested from their right tibia by saline flush. The mononuclear cells were isolated and culture-expanded in osteogenic medium for 3 weeks. Fluorescent reporter molecules were incorporated into the cell membranes, 24 hours prior to re-implantation of the cells into the fracture model. A 3 mm ulnar defect was preformed in all the animals. In groups I–III this was established 48 hours prior to cell re-implantation.

The animals were sacrificed at least 3 weeks after fracture surgery. Representative samples of the fracture callous, lung, liver, spleen and kidney were harvested from all animals and cryo-sectioned. Using confocal microscopy, the labelled cells were expressed as the average in 5 high power fields for each solid tissue. In addition, cyto-spins were made from blood and marrow and the cell number expressed as a percentage of the total cells.

Results: In group I, labelled cells were identified in the fracture callous, establishing their viability in vivo. Following intravenous re-implantation a smaller number of labelled cells were identified in the callous. When the cells were re-implanted into a remote marrow site, the number of cells in the callous was greater than after venous reimplantation, but less numerous than those in group I.

In all sections, these labelled cells appeared on trabecular surfaces in an osteoblastic fashion, but occasionally they were surrounded by osteoid, corresponding to osteocytes.

A small number of labelled cells were found in the blood, bone marrow, lung, liver and spleen of all animals in groups I–III. No labelled cells were identified in the kidney tissue.

Discussion and Conclusions: We have demonstrated that cells from remote sites are integral in fracture healing. Their presence in callous following venous administration supports recruitment via the circulation. This preliminary data is a proof of concept. This is an exciting new phenomenon, which could provide alternatives for harvesting skeletal progenitor cells and for their delivery in the treatment of bony pathology.

Introduction: The potential of Bone Morphogenic Protein (BMP) to improve fracture healing is of great interest to orthopaedic surgeons. Although the complex mechanisms leading from the presence of local BMP to the fracture scenario has yet to be elucidated. The purpose of this study was to investigate whether introducing rhBMP-2 to the fracture arena, some days after fixation could be more beneficial to fracture repair.

Methods: 40 animals were randomised into 4 groups; namely control treatment at day 0 or rhBMP-2 treatment at days 0, 4 or 8 post-surgery. All animals underwent a previously validated surgical procedure involving creation of an open femoral fracture fixed using a 4-pin external fixator. 30 μl of bovine serum albumin (BSA) alone (control) or mixed with 2.5 μg of rhBMP-2 (treatments) was injected via a lateral approach directly into the fracture gap, either following closure of the wounds (day 0) or at 4 or 8 days postoperatively. Animals were assessed as to the outcome of surgery by digital sequential x-ray at days 0, 8, 16 and 22 using a Faxitron MX-20 camera; and y either biomechanical testing under a 3-point bending technique (Lloyd Instruments Ltd, UK) or histological examination following sacrifice at day 22. Data were analysed using Mann-Whitney U and Wilcoxon Tests for statistical differences (SPSS, Version 9). Differences were considered significant when p< 0.05.

Results: X-ray analysis indicated that healing of fractures treated with rhBMP-2 at day 0 or day 4 was significantly greater than the two other groups at days 16 and 22. BMP given at day 4 tended to a greater effect than when given at day 0, though the range was too great to show a statistical difference. There were no differences between the BMP-8 and the BSA control groups. Mechanical testing showed that only animals that had received rhBMP-2 at day 4 had attained similar peak loads to failure to those of their contralateral unoperated leg. Bones from animals receiving rhBMP-2 at day 0 had attained the next greatest strength, which was followed by rhBMP-2 administration at day 8 animals, whereas the animals receiving BSA attaining the least strength. There was a statistical difference (p< 0.05) between both rhBMP-2 day 4 and day 0 groups compared to the BSA control group. Qualitative histology suggested that the rhBMP-2 day 0 and day 4 groups had almost fully healed with new bone whereas the BSA and rhBMP-2 day 8 groups still had considerable mounts of fibrous tissue and cartilage at the fracture gap 22 days following surgery.

Conclusions: The study demonstrates that a single percutaneous injection of rhBMP-2 has a positive effect on fracture healing, when prescribed at the time of injury or during the early period of fracture repair. Data suggests that the most effective timing of delivery of BMP may not be at the time of surgery but 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. Further investigation as to the most appropriate time for intervention using these proteins is warranted and may negate the current requirement for carried products and large doses of these expensive drugs.

Introduction: Bone mineral density (BMD) is currently the gold standard in predicting osteoporotic fracture, but evidence suggests that over one third of such fractures occur in those with osteopenia or even normal BMD. The level of bone turnover may affect bone quality in these patients independently of BMD. Bone markers have evolved as tools in monitoring anti-resorptive treatment in osteoporosis.

Aims: The aim of this study was to investigate if levels of bone markers in postmenopausal women could be used as an adjunct to BMD measurements in the assessment of fragility fracture risk.

Patients and Methods: 60 postmenopausal women (30 osteoporotic, 30 with normal BDM) were studied. A single BMD measurement by dual energy x-ray absorptiometry (DEXA) enabled categorisation. Serum bone formation markers (bone specific alkaline phosphatase (BSAP) and osteocalcin (OC)), and resorption marker (C-telopetide of type 1 collagen (CTX)), were measured. History of low trauma fracture was documented for each woman.

Results: 36% of the osteoporotic group had experienced at least one fragility fracture. However, the femoral neck and combined spinal BMD in these women was not significantly different from the 64% of osteoporotic women who had no prior fracture. There was also no significant difference in the age of women in both subgroups. Serum bone markers were significantly increased in the osteoporotic fracture subgroup when compared to the non-fracture subgroup and also to the non-osteoporotic controls. The largest increases were seen in the levels of CTX. Smaller increases in all markers were seen when the non-fracture subgroup was compared to the non-osteoporotic control group but these increases did not reach statistical significance.

Conclusions: Bone turnover is significantly increased in postmenopausal osteoporotic women with previous fracture compared to both osteoporotic non-fracture counterparts and non-osteoporotic controls. This suggests higher bone turnover will increase fracture risk in osteoporotic women. It is possible that combining 2 or 3 markers to produce an “index of bone turnover” would be a useful tool when used in addition to BMD to identify those at greatest fracture risk.

Background and Hypothesis: High-energy fractures associated with severe soft tissue injury have a significant incidence of delayed or non-union. The soft tissue envelope may adversely contribute to the healing of a fracture, not only in stripping of the periosteal blood supply, development of compartment syndrome or tissue interposition between the bone ends but also in its ability to generate an intense acute inflammatory response. Inflammation is the initiator of healing; in clinical scenarios of impaired inflammation (immune deficiency, NSAIDs, corticosteroids) healing is delayed; interestingly, in injury with excess inflammation (CVA, MI) healing is also delayed. Would the inflammatory response following high-energy fractures contribute beneficially or adversely to the healing of the underlying fracture? Using an in-house murine femoral fracture model which reliably demonstrated features of delayed fracture healing when associated with a severe overlying muscle crush injury we proposed these hypotheses:

That fracture callus with overlying muscle crush would contain raised expression of acute inflammatory cytokines (IL-1β, IL-6 and TNF-α).

Methods: Total RNA was extracted from normal fracture callus (FO) and muscle crush fracture callus (MC) at day 2, day 4 and day 8. Semi-quantitative RT-PCR was used to compare IL-1β, IL-6 and TNF-α mRNA expression. Histomorpometric analysis of ICC stained sections of the FO and the MC groups was used to estimate IL-1β, IL-6 and TNF-α protein expression within the callus. Positively staining areas for the cytokine within the callus were a semi-quantified and compared between groups. Finally, blocking antibodies to IL-1β and TNF-α were injected into MC fracture callus at day 0, 4 and 8. Control MC group had vehicle only injected. Fracture healing was measured using radiological, histomorphological and biomechanical outcome measures. Following a pilot dosing experiment, the effect of blocking antibodies on fracture healing was compared between MC and MC with antibody groups.

Results: The MC group IL-1β mRNA expression was significantly higher than FO at day 4 and day 8 (p=0.05). ICC for IL-1β protein expression was higher on day 4 and on day 8 in the MC group, significant at day 8 (p=0.03). TNF-α mRNA expression in the MC group at day 8 was significantly higher than the FO group (p=0.05). ICC for TNF-α protein in the MC group peaked at day 8 and was significantly higher than the FO group (p< 0.03). IL-6 mRNA expression was significantly raised in the MC group at day 4 and 8 compared with the FO group (p=0.05). ICC for IL-6 protein showed significantly increased expression at day 8 in the MC group (p=0.05). The patterns of expression of the mRNA and proteins were similar.

Injection of anti-TNF-α antibodies into MC mice caused more new bone formation on day 16 (p=0.03) and day 24 (p=0.06), stiffer calluses at day 24 (p=0.01) and faster fracture gap obliteration at day 16 (p=0.05) and day 24 (p=0.001). IL-1β blockade had slightly less effect, more new bone formationd ay 16 (p=0.01) and day 24 (p=0.03), slightly stiffer (p=0.08), but no significant difference in fracture gap obliteration from controls.

Conclusion: The effect of muscle crush around the fracture callus was to increase and prolong the expression of inflammatory cytokines with the callus. The effect of blocking these excessive inflammatory cytokines in our model was to improve fracture healing. Excessive inflammatory cytokines (IL-1β, IL-6, TNF-α) in bone impair new bone production by osteoblasts, inhibit the recruitment and differentiation of mesenchymal precursors and promote osteoclastogenesis. The mechanism of action of blocking antibodies may be due to inhibition of the antiosteogenic effects of these cytokines.

Introduction: 5–10% of all fractures end in delayed or non-union. It has been reported for 3 decades that NSAIDs have an inhibitory action on fracture repair, yet GPs still prescribe these drugs in up to 50% of fracture patients. Not all fracture patients who are treated with this class of medication go on to develop non-union, yet a strong correlation has been shown in clinical studies between long bone fractures and development of delayed and non-union. The mechanism behind this effect has yet to be elucidated. In cancer research it has been shown that NSAIDs, primarily by a COX-2 pathway, can exert an inhibitory action on cell proliferation by inhibiting angiogenesis. It is proposed that a similar mechanism occurs in the induction of NSAID induced delayed fracture repair. We have investigated this hypothesis using an externally fixated murine model of femoral fracture.

Methods: 158 animals were randomised into either treatment (Rofecoxib 5mg/kg/day in a 0.5% methylcellulose carrier) or control (carrier alone). All had a standard surgical regimen involving creating of an open femoral fracture with treatment using a 4-pin external fixator under the isoflurane inhalational anaesthesia. Outcome measures included standardised x-rays (Faxitron MX-20) and Laser Doppler Flow (Oxford Optronics) measurements taken at days 0, 4, 8, 16, 24 and 32, along with biomechanical testing (Lloyd Instruments Ltd) at days 24 and 32. Data was entered into a spreadsheet and analysed using Mann-Whitney U and Wilcoxon Tests for statistical differences (SPSS, Version 9), with statistical significance being attained when p< 0.05.

Results: A greater number of animals in the NSAID group had a failure of treatment with loss fixation due to pin pullout from poor osseointegration (53% NSAID compared to 26% controls). Of those animals that completed the studies, x-ray analysis showed a change in pixel density at the fracture gap suggesting poorer healing of the NSAID animals that was statistically different at days 16, 24 and 32. Biomechanical testing suggested treatment animals had attained statistically less peak loads and stiffness at day 32. Laser Doppler Flow measurements across the fracture gap showed generally less flow at all time points in the NSAID group. This was statistically significant at days 4 and 24.

Conclusions: The new selective and specific COX-2 inhibitors are marketed as having a cleaner side effect profile and are being widely used by primary care practitioners in trauma patients. Not all animals that are treated with NSAIDs go on to develop a delayed union and some are able to heal with similar mechanical properties to animals in a control group. However, we have illustrated that the highly specific COX-2 inhibitor rofexcoxib has a significant negative effect on maintenance of fracture fixation and fracture repair in this model, both in terms of x-ray and biomechanical analysis. We have also shown that the inhibition of fracture healing is associated with a decrease in blood flow at the fracture site leading to the hypothesis that the mechanism behind the effect is via an inhibition of angiogenesis.

Introduction Posterior cruciate ligament (PCL) reconstruction has been shown to restore the posterior stability of the knee during posterior drawer tests. However, we have previously published a report showing that a PCL deficient knee has abnormal rotation under load. We hypothesise that a PCL reconstruction does not restore rotational stability to the knee.

Methods In-vitro kinematics under simulated muscle loads after PCL reconstruction were measured. Eight fresh-frozen cadaveric knees were tested on a robotic testing system. The system applied a posterior drawer of 130N and a combined quadriceps/hamstrings load (400N/200N) at 0°, 30°, 60°, 90°, and 120° of flexion. Tibial motion with respect to the femur was measured with the PCL intact, resected and reconstructed using an Achilles tendon allograft. Posterior tibial translation (PTT) and internal/external rotation were analyzed using a repeated measures ANOVA.

Results PCL deficiency significantly increased (p< 0.05) PTT under posterior drawer. Reconstruction significantly reduced the increased PTT to the level of the intact knee at all flexion angles. Under the muscle load, the deficiency resulted in significantly higher PTT at 60 to 120, and reconstruction did not significantly reduce the increased PTT. PCL deficiency significantly increased external rotation at 90° and 120°. PCL reconstruction did not significantly reduce the increased external rotation caused by PCL deficiency.

Conclusions Under simulated muscle loading, PCL reconstruction did not restore the translation and rotation of the tibia, despite restoring posterior stability under posterior drawer. Our data may help to identify the biomechanical factors that lead to the long-term development of osteoarthritis following PCL injury and reconstruction.

In relation to the conduct of this study, one or more of the authors is in receipt of a research grant from a non-commercial source.

During the process of distraction osteogenesis new bone is formed rapidly and undergoes remodelling almost immediately. Little is known about the regulatory mechanisms governing the removal of the redundant callus in this process. Tissue homeostasis is achieved by a delicate balance between the processes of cell death (apoptosis) and cell proliferation. The aim of this study was to test the hypothesis that apoptosis is involved during distraction osteogenesis.

Mid-tibial osteotomies were performed in 6 adult male NZW rabbits (age; 24 weeks, weight; 3.0 −3.5 kg), and the tibiae stabilised with unilateral external fixators (Orthofix M-100). 7 days later, twice daily distraction was initiated at rates of 0.7 mm/day for 3 weeks. BrdUrd (40mg/kg) was injected intravenously to the rabbit 1h before killing. The regenerate bone was collected, fixed in 10% buffered formalin and decalcified for paraffin embedding. Some fresh regenerate bone tissues were also prepared for examination under transmission electronic microscopy (TEM). BrdUrd immunohistochemistry has been used to detect proliferating cells and the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-biotin nick end-labelling (TUNEL) method was used to identify cells undergoing apoptosis. To detect bone-resorbing cells, tartrate-resistant acid phosphatase (TRAP) staining was also performed.

BrdUrd positive cells and TUNEL-positive cells were shown to coexist in most of the areas in the regenerates. In the mineralisation front, the majority of the TUNEL-positive cells were present in the transitional region between the fibrous tissue and the new bone. The TUNEL-positive cells were close to or on bone surfaces, and some of the newly formed osteocytes in the new trabeculae were also positive. The TUNEL-positive cells were also seen in the cartilage region of the regenerate. However, the TUNEL labelling was greatly reduced in the new bone close to the osteotomised bone ends, TUNEL-positive labelling were not detected in the cortical bone of the osteotomised bone ends and in the adjacent surrounding periosteum. TRAP staining in the regenerate revealed similar patterns of distribution to those of the TUNEL staining. There were more TRAP-positive cells in the new bone near the mineralisation front than in that of the new bone region, which was close to the osteotomised bone ends. TEM examinations have demonstrated characteristic signs of apoptotic changes in the fibroblast, osteoblast and osteocytes in the specific regions of the distraction regenerate.

The study provided evidence that in the process of rapid bone formation during distraction osteogenesis, superfluous cells are removed by apoptotic mechanisms. The demonstration of a mixture of proliferative and apoptotic cell populations in the regenerating tissue, indicates that apoptosis and cell proliferation may be regulated by local factors. The neovascularisation of the regenerate and withdrawal of growth factors and cytokines may be responsible for apoptosis occurring in some parts of the regenerating tissue. The changes of distribution of apoptotic cells in the different regions of the regenerate, together with the observed patterns of osteoclast activities, suggest that bone cells undergoing apoptosis may initiate rapid bone remodelling seen during distraction osteogenesis.

Fracture repair is a complex physiological process during which bone shows the remarkable ability to mount a repair process, restoring its mechanical integrity and anatomical configuration by original osseous tissue. Programmed cell death, or apoptosis, is a naturally occurring cell suicide pathway with a homeostatic function in the maintenance of continuously renewing tissues. The present study investigated the relation between cell proliferation and cell death (apoptosis) during fracture healing in a mouse femoral model.

Left femoral osteotomies were performed in 20 male CFLP mice (35–45g), immobilised with uniplanar external fixators. 4 animals were sacrificed on days 2, 4, 8, 16 and 24 post-fracture and fracture callus collected for paraffin embedding. Localisation of cell proliferation was examined using immunohistochemistry with proliferating cell nuclear antigen (PCNA) monoclonal antibody. Apoptotic cells were visualised with the terminal deoxynucleotidyl transferase (TdT)–mediated dUTP-biotin nick end-labelling (TUNEL) method. Random images of each time specific specimen were captured via a digital camera and the positive labelling indices of PCNA and TUNEL labelling were calculated and statically compared.

Cell proliferation and apoptosis were found co-existing during the entire period of fracture healing studied. Cell proliferation was predominant in the early phases of fracture healing (days 2–8). PCNA positive labelling index peaked at day 8 (p< 0.01, t-test) and PCNA-positive cells were not limited to the fracture gap mesenchymal tissues but extended in the periosteum along most of the fractured femur. TUNEL positive labelling was minimal in the early stages (days 2–8). In later stages of fracture healing (days 16–24), PCNA expression declined as intramembranous and endochondral ossification spread within the fracture site and apoptosis was the dominant cell activity with the TUNEL positive labelling index peaked at day 16 (p< 0.05, t-test) and then declined sharply at day 24.

The current study indicated that apoptosis was a normal concomitant during fracture repair, confirming programmed cell death in chondrocytes and bone cells, and that cell proliferation and apoptosis were tempero-spatially dependent. These findings support the view that apoptosis is a natural process, genetically programmed and active during fracture repair. The demonstration of a mixture of proliferative and apoptotic cell populations in the regenerating tissues of fracture callus, suggests that apoptosis and cell proliferation may be regulated by local factors during fracture healing.

NSAID’s cycle-oxygenase (COX) inhibitory characteristics are either non-specific, COX-1 preferential or recently COX-2 preferential. NSAID’s have been widely reported to delay fracture repair however the mechanism of this affect remains unclear.

Left femoral osteotomies were performed in 54 male 3 month old CFLP mice immobilised with uniplanar external fixators. 27 externally fixated mice received 4mg/kg meloxicam,b.d., from the day of surgery, by gavage. The control group received the carrier alone. 18 mice had external fixators applied to intact femurs and received no meloxicam as a sham control. Individual mouse movement, was quantified each day by autocounters using an infrared beam motion detection system. Plasma was obtained by right ventricular aspiration under anaesthesia on days 2,4,8 and 16-post surgery.

A validated bioassay and a slot blotting immunoassay were employed to determine the plasma concentration of 11-6 and relative TNF-α levels to normal mouse serum.

TNF-α levels peaked at day 4 and were suppressed by COX-2 inhibition. Both the control and treatment groups had higher levels of TNF-α than the non-fractured controls. The plasma concentration of 11-6 was elevated by COX-2 inhibition at all time points. The levels of TNF-α and 11-6 correlated in fracture control and treatment groups (Spearman’s 0.039 and 0.002 respectively). The 11-6 plasma concentration correlated to the animal motion in the treatment group alone (Spearman’s 0.017).

As it has been shown that TNF-α induces 11-6 production and that this inhibits TNF-α production a possible model for these interaction is shown below.