Establishment of periodontal tissue engineering by FGF-2

我们研究了碱性成纤维细胞生长因子（bFGF）对各种细胞外基质的调控作用，这些细胞外基质在牙周组织再生的调控中起重要作用，可以作为含bFGF的再生医学载体材料的候选材料。结果表明，bFGF可诱导人牙周韧带（HPDL）细胞产生透明质酸和硫酸肝素，并呈剂量依赖性。HPLC分析显示，在条件培养基中，bfgf处理的HPDL细胞的HA比未处理的HPDL细胞具有更高的分子质量。RT-PCR分析显示，透明质酸合成酶（HAS） 1和HAS2 mRNA表达增强，两者都有助于高分子量HA的产生。在硫酸肝素方面，观察到bfgf处理的HPDL细胞表面的syndecan-2脱落。HPDL细胞的转录组分析显示，牙周韧带相关蛋白-1 （PLAP-1）是一种新型的蛋白聚糖样蛋白。我们发现PLAP-1负向调控HPDL细胞向硬组织形成细胞的分化，而bFGF下调PLAP-1的表达。在骨相关蛋白方面，bFGF下调HPDL细胞骨涎蛋白、骨连接素和骨钙素的表达。然而，有趣的是，bFGF在mRNA和蛋白水平上都明显增强了骨桥蛋白的表达。经bfgf处理的HPDL细胞在细胞质和条件培养基中检测到骨桥蛋白，但在细胞表面未检测到骨桥蛋白。此外，我们发现ERK1/2和PKC参与bfgf诱导骨桥蛋白表达的信号通路。目前，我们正计划利用比格犬重度牙周炎模型，研究bFGF +支架材料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.