ASSOCIATION OF CELL PROLIFERATION AND CELL DEATH EVENTS DURING FRACTURE HEALING

Abstract

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.