Expression of connective tissue growth factor CTGF in the callus during bone regeneration and application for acceleration of regeneration.

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The utility of the endochondral ossification factor CTGF has pointed out in many cases. We investigated to analyze the role of CTGF for the application to accelerate bone regeneration in vivo. The expression of CTGF was found as a band in the hypertrophic chondrocytes during differentiation of proliferative chondrocytes to the hypertrophic chondrocytes. No difference has been found on the angiogenesis in the bone marrow, on the number of monocytes or megakaryocytes in developing liver of CTGF gene targeted mouse embryos to compared with the wild type embryos. However, the morphology of the long bone of CTGF targeted mice was different from that of the wild type. The serious cleft palate, enlarged hypertrophic zone, developmental delay of ossification and the kinks of long bone have observed in the mutants. These morphological abnormalities might be caused by abnormal arrangement of the osteoclasts. The osteoclasts concentrate to the apoptotic zone of the hypertrophic chondrocytes generally, however, the osteoclasts did not concentrate to the apoptotic zone. The femoral bone was fractured to prepare the callus to analyze the behavior of the osteoclasts. The osteoclasts were scattered around the callus for first 1 week. However, the osteoclasts were rearranged rectangular to the axis and then the absorption of callus cartilage by the osteoclasts was occurred actively. This result indicates that the behavior of osteoclasts affects on the ossification. To confirm the relationship between hypertrophic chondrocytes and the osteoclasts, the combination culture of hypertrophic chondrocytes with the osteoclasts was performed to add anti CTGF and anti c-Met antibodies in the medium. After 24 hours, cells were fixed and stained by TRAP method. Interestingly, the osteoclasts behavior and proliferation were activated with addition of anti CTGF. This result is opposite to the observation in vivo.