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

软骨内成骨因子CTGF的应用已经在很多案例中被指出。我们研究分析CTGF在体内应用加速骨再生的作用。在增生性软骨细胞向增生性软骨细胞分化过程中，CTGF在增生性软骨细胞中呈带状表达。CTGF基因靶向小鼠胚胎与野生型胚胎相比，在骨髓血管生成、肝脏单核细胞或巨核细胞数量上均无差异。然而，CTGF靶向小鼠的长骨形态与野生型不同。在突变体中观察到严重的腭裂，肥大带扩大，骨化发育迟缓和长骨扭结。这些形态异常可能是破骨细胞排列异常所致。破骨细胞普遍向肥大软骨细胞的凋亡区集中，但破骨细胞不向凋亡区集中。取股骨骨折制备破骨组织，分析破骨细胞的行为。破骨细胞散布于愈伤组织周围1周。破骨细胞向轴方向重新排列，破骨细胞对骨痂软骨的吸收活跃。这表明破骨细胞的行为对骨化有影响。为了证实增生性软骨细胞与破骨细胞之间的关系，我们将增生性软骨细胞与破骨细胞联合培养，在培养基中加入抗CTGF和抗c-Met抗体。24h后，细胞固定，用TRAP法染色。有趣的是，添加抗CTGF后，破骨细胞的行为和增殖被激活。这一结果与体内观察结果相反。

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.