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）对各种细胞外基质产生的调节作用，这些细胞外基质在牙周组织再生的调节中起重要作用，并且可以是含bFGF的再生医学的载体材料的候选者。结果表明，碱性成纤维细胞生长因子优先诱导透明质酸和硫酸乙酰肝素的人牙周膜（HPDL）细胞的生产在剂量依赖性的方式。HPLC分析显示，在bFGF处理的HPDL细胞的条件培养基中，与未处理的HPDL细胞相比，HA具有更高的分子量。RT-PCR分析显示，透明质酸合成酶（HAS）1和HAS 2的mRNA表达增强，这两个基因都有助于产生高分子量的HA。在硫酸乙酰肝素方面，观察到bFGF处理的HPDL细胞表面的多配体蛋白聚糖-2的脱落。HPDL细胞的转录组分析显示存在牙周膜相关蛋白1（PLAP-1），一种新的蛋白多糖样蛋白。我们证明，PLAP-1负调控HPDL细胞分化成硬组织形成细胞和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、骨粘连蛋白和骨钙素的表达。然而，有趣的是，bFGF在mRNA和蛋白水平上明显增强骨桥蛋白的表达。bFGF处理的HPDL细胞的细胞质和条件培养基中检测到骨桥蛋白，但不在细胞表面上。此外，还发现ERK 1/2和PKC参与bFGF诱导骨桥蛋白表达的信号通路。我们目前正计划利用比格犬的重度牙周炎模型来研究bFGF加支架材料诱导牙周组织再生的功效。

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.