Elucidation of the mechanism of alveolar bone regeneration by controlled release of recombinant human FGF-2

我们分析了明胶水凝胶控释FGF2诱导小鼠骨再生的生物学机制。牙槽骨再生模型于2005年在JSPS科学研究资助基金的支持下建立。在明胶水凝胶植入后7-14天，FGF-2释放组（试验组）骨膜内PCNA指数显著高于对照组。实验组骨膜增生，碱性磷酸酶活性呈阳性反应，并形成愈伤组织。使用dig标记的碱性磷酸酶、骨钙素、骨桥蛋白、Fgfr-1、Fgfr-2和Runx2的cRNA探针进行原位杂交，检测FGF2诱导的增生性骨膜中Fgfr-1、Fgfr-2和Runx2的成熟和表达分布。成熟的成骨细胞表达骨钙素、骨桥蛋白和碱性磷酸酶，在愈伤组织和原始骨表面可见。仅表达碱性磷酸酶的未成熟成骨细胞广泛分布于骨膜。而成熟成骨细胞中Fgfr-1和Fgfr-2信号高于未成熟成骨细胞，部分骨膜外的梭形细胞也表达Fgfr-1、Fgfr-2。Runx2在骨膜中通过Fgfr-1和Fgfr-2信号共催化。利用实时RT-PCR分析这些基因在激光显微解剖骨膜中的表达水平。实验组Runx2、骨钙素、骨桥蛋白、碱性磷酸酶的表达均显著高于对照组。实验组Fgfr-1和Fgfr-2的表达水平也高于对照组。由此可见，由于FGF2的合成代谢作用不仅促进成骨细胞增殖，而且增强骨基质的生成，骨膜中FGF2的控制释放通过愈伤组织的形成促进了骨再生。此外，在本研究中，我们发现FGF2上调Runx2在组织中的表达，并上调骨钙素、骨桥蛋白和碱性磷酸酶的表达，这与以往体外实验报道的结果相反。少

We analyzed biological mechanism of the bone regeneration induced by controlled release of FGF2 using gelatin hydrogel in mouse. The alveolar bone regeneration model have established in 2005 supported by Grant-in-Aids for Scientific Research from JSPS. Through 7-14 days after implantation of the gelatin hydrogel, PCNA index in the periosteum of the FGF-2 released group (experimental group) was significantly higher than the control group. And hyperplastic periosteum with positive responses of alkaline phosphatase activity was observed with callus formation in the experimental group. In situ hybridization was performed using DIG-labeled cRNA probes for alkaline phosphatase, osteocalcin, osteopontin, Fgfr-1, Fgfr-2 and Runx2 to detect osteoblast maturation and distribution of the Fgfr-1, Fgfr-2 and Runx2 expression in the FGF2 induced hyperplastic periosteum. Mature osteoblast expressing osteocalcin, osteopontin and alkaline phosphatase was observed on a surface of the callus and original … More bone. Beside, immature osteoblast expressing only alkaline phosphatase showed widespread distribution in the periosteum. Whereas, Fgfr-1 and Fgfr-2 signal were greater in mature osteoblast than immature osteoblast, and some spindle shaped Cells out of the periosteum also expressed Fgfr-1, Fgfr-2. Runx2 was colocalyzed in periosteum with the signals of Fgfr-1 and Fgfr-2. Furthermore, expression level of these genes in laser micro-dissected periosteum were analyzed by real time RT-PCR. In experimental group, Runx2, osteocalcin, osteopontin and alkaline phosphatase expression were significantly higher than control group. Fgfr-1 and Fgfr-2 expression level were also higher in the experimental group than those of the control group. It can be concluded that controlled release of FGF2 in periosteum promotes additive bone regeneration through callus formation as a result of anabolic effects of FGF2 not merely promoting osteogenic cell proliferation but also enhancement of bone matrix production. Moreover, in this study we showed that FGF2 up-regulates Runx2 expression revel in the tissue as with osteocalcin, osteopontin and alkaline phosphatase expression contrary to results of previous in vitro experiment reports. Less