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.