Molecular analysis of abnormal fracture healing process

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Impaired fracture healing process was investigated by molecular histological techniques such as in situ hybridization. ODS (Osteogenesis Disorder Shionogi) rat was used for Scurvy model. Membranous ossification process in fracture healing was strongly inhibited by the depletion of vitamin C.Particularly, no expression of osteopontin, extracellular matrix protein located in the bone matrix, was detected. Early stages of endochondral ossification was not strongly inhibited. The unmber of chondrocytes in vitamin C depleted rats was comparable to that of normal rat. However, only a few number of hypertrophic chondrocytes which express osteopontin nRNA was observed. The results suggest the possibility that due to the impaired expression of osteopontin, calcification process in the scurvy was strongly inhibited. Furthermore, transcriptional factors regulating the expression of osteopontin gene was identified in vivo and in vitro. CBFA1, PU.1, MITF were cooperatively interacted with 5'-flanking region of osteopontin gene. No expression of osteopontin gene was detected in the knockout mice of cbfa-1 gene. This knockout mice showed almost no calcification in the bone tissue. The result also suggested the involvement of osteopontin to the calcification process. The biological role of osteopontin for the bone remodeling was also investigated. Enhanced expression of osteopnotin gene was observed by mechanical stress of "press" in the osteocytes. The result indicated that the osteocytes acted as sensory cells respond to echanical stress by espressing osteopontin gene.