PROGRAMMED REMOVAL OF CHONDROCYTES DURING ENDOCHONDRAL FRACTURE-HEALING

This investigation tested the hypothesis that the removal of chondrocy tes during endochondral fracture healing involves an ordered process o f programmed cell death. To accomplish this, unilateral closed fractur es were created in the femora of 36 Sprague-Dawley rats. The rats were killed in groups of four on days 1, 3, 7, 14, 21, 28, 42, 49, and 56 after fracture. The femora were embedded in paraffin and tested for ex pression of specific markers of fragmented DNA with use of a terminal deoxyuridyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) technique. To determine the potential for transd ifferentiation of chondrocytes to osteoblasts, calluses were also hybr idized to detect expression of osteocalcin mRNA. Cell proliferation wa s assessed by an immunohistochemical detection method for proliferatin g cell nuclear antigen. A separate group of four rats was killed on da y 28 to represent the later stage of the endochondral ossification, an d the calluses were examined for cellular morphology with transmission electron microscopy. The results showed a coordination in both time a nd space of the activities of cellular proliferation and programmed ce ll death. Cell proliferation was most active in the earlier phases of fracture healing (days 1 through 14), although TUNEL expression was ap parent in hypertrophic chondrocytes on day 14 after fracture and persi sted until day 28. In the later stages of fracture healing (days 14 th rough 28), proliferating cell nuclear antigen was no longer synthesize d in hard callus (intramembranous bone) and cell removal was the domin ant activity in soft callus chondrocytes. Expression of osteocalcin mR NA was detected in osteoblasts but not in hypertrophic chondrocytes or in any other nonosteoblastic cell type. These findings support the hy pothesis that the removal of chondrocytes during endochondral fracture healing is part of an ordered transition of tissue types in which the cellular mechanisms are genetically programmed to involve proliferati on, maturation, and apoptolic cell death.