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.