LOCALIZATION AND QUANTIFICATION OF PROLIFERATING CELLS DURING RAT FRACTURE REPAIR - DETECTION OF PROLIFERATING CELL NUCLEAR ANTIGEN BY IMMUNOHISTOCHEMISTRY

Bilateral femurs of 12-week-old female Sprague-Dawley rats were fractu red, and the fractured femurs were harvested 36 h, 3, 7, 10, and 14 da ys after the fracture. Localization of cell proliferation in the fract ure calluses was investigated using immunohistochemistry with antiprol iferating cell nuclear antigen (PCNA) monoclonal antibodies, Thirty-si x hours after the fracture, many. PCNA-positive cells were observed in the whole callus. The change was not limited to mesenchymal cells at the fracture site where the inflammatory reaction had occurred, but ex tended in the periosteum along almost the entire femoral diaphysis wer e intramembranous ossification was initiated, On day 3, periosteal cel ls or premature osteoblasts in the newly formed trabecular bone during intramembranous ossification still displayed intense staining, On day 7, many premature chondrocytes and proliferating chondrocytes were PC NA positive, Endochondral ossification appeared on days 10 and 14, and the premature osteoblasts and endothelial cells in the endochondral o ssification front were stained with anti-PCNA antibodies, Quantificati on of PCNA-positive cells was carried out using an image analysis comp uter system, obtaining a PCNA score for each cellular event, The highe st score was observed in the periosteum early after the fracture near the fracture site, Immunohistochemistry using anti-PCNA antibodies sho wed that the distribution of proliferating cells and the degree of cel l proliferation varied according to the time lag after the fracture, s uggesting the existence of local regulatory factors such as growth fac tors, and that significant cell proliferation was observed at the begi nning of each cellular event.