Establishment of periodontal tissue engineering by FGF-2

本实验研究了碱性成纤维细胞生长因子对多种细胞外基质生成的调节作用，这些细胞外基质在牙周组织再生中起着重要的调控作用，可作为含碱性成纤维细胞生长因子的再生医学载体材料的候选材料(S)。结果表明，碱性成纤维细胞生长因子优先诱导人牙周膜(HPDL)细胞产生透明质酸和硫酸肝素，且呈剂量依赖关系。高效液相色谱分析显示，在碱性成纤维细胞生长因子处理的HPDL细胞的条件培养液中，HA的相对分子质量比未处理的HPDL细胞高。RT-PCR分析表明，透明质酸合成酶(HAS)1和HAS2的mRNA表达增强，这两种酶都有助于产生高相对分子质量的HA。在硫酸乙酰肝素的作用下，观察到碱性成纤维细胞生长因子处理的HPDL细胞表面Syndecan-2脱落。HPDL细胞的转录组分析表明，牙周膜相关蛋白-1(PLAP-1)是一种新的蛋白多糖样蛋白。我们发现PLAP-1负向调节HPDL细胞向硬组织形成细胞的分化，bFGF下调PLAP-1的表达。在骨相关蛋白方面，碱性成纤维细胞生长因子下调HPDL细胞的骨涎蛋白、骨连接蛋白和骨钙素的表达。然而，有趣的是，碱性成纤维细胞生长因子明显增强了骨桥蛋白在mRNA和蛋白水平的表达。碱性成纤维细胞生长因子处理的HPDL细胞在细胞质和条件培养液中检测到骨桥蛋白，而在细胞表面未检测到骨桥蛋白。此外，研究还发现ERK1/2和PKC参与了碱性成纤维细胞生长因子诱导骨桥蛋白表达的信号通路(S)。我们计划利用Beagle犬重度牙周炎模型，研究碱性成纤维细胞生长因子加支架材料(S)诱导牙周组织再生的效果。

We investigated the regulatory effects of basic fibrovlast growth factor(bFGF) on the production of various extracellular matrices which play important roles in the regulation of periodontal tissue regeneration and can be candidate(s) of the carrier material(s) of bFGF-containing regenerative medicine. It was demonstrated that bFGF preferentially induced hyaluronan and heparan sulfate production by human periodontal ligament (HPDL) cells in a dose dependent manner. Analysis by HPLC revealed that in conditioned medium of bFGF-treated HPDL cells HA had a higher molecular mass, compared to that of untreated HPDL cells. RT-PCR analysis revealed that the enhancement of mRNA expression of hyaluronan synthase (HAS) 1 and HAS2, both of which contribute to the production of HA with a high molecular mass. In terms of heparan sulfate, the shedding of syndecan-2 of the surfaces of bFGF-treated HPDL cells was observed. The transcriptome analysis of HPDL cells revealed the existence of periodontal ligament associated protein-1 (PLAP-1), novel proteoglycan-like protein. We demonstrated that PLAP-1 negatively regulated the cytodifferentiation of HPDL cells into hard-tissue forming cells and that bFGF downregulated the expression of PLAP-1. In terms of bone-related proteins, bFGF downregulated the expressions of bone sialoprotein, osteonectin and osteocalcin by HPDL cells. Interestingly, however, bFGF clearly enhanced the osteopontin expression at both mRNA and protein levels. The osteopontin was detected in the cytoplasm and the conditioned medium but not on the surfaces of bFGF-treated HPDL cells. Furthermore, it was revealed that ERK1/2 and PKC were involved in the signaling pathway(s) of bFGF-induced osteopontin expression. We are now planning to investigate the efficacy of bFGF plus scaffold material(s) to induce periodontal tissue regeneration by utilizing the severe periodontitis model of beagle dogs.