狗牙周膜成纤维细胞的体外培养及其生物学活性

Culture of canine periodontal ligament fibroblasts in vitro and their biological characteristics

目的:建立一种简便、可靠的牙周膜成纤维细胞(PDLFs)原代分离培养方法,并探讨PDLFs作为牙周组织工程种子细胞的可行性。方法:采用改良组织块法,全麻下拔狗上颌第3切牙,刮取牙周膜,以玻璃片覆盖组织块贴壁替代传统的干燥贴壁法。细胞培养成功后传代,用四甲基偶氮唑蓝比色、免疫组化染色、VonKossa染色等方法测定其生物学活性。结果:PDLFs原代培养成功率达到89%,免疫组化检测结果为波形丝蛋白阳性,角蛋白阴性,体外培养PDLFs可自然形成矿化结节。结论:采用改良组织块法可以简便、稳定获取原代PDLFs。体外培养PDLFs具有部分成骨样细胞表型特征,可作为牙周组织工程种子细胞来源。

AIM:To establish a simple and reliable approach to primary isolation and culture of periodontal ligament fibroblasts(PDLFs) so as to investigate the feasibility of PDLFs as the seed cells for periodontal tissue engineering. METHODS:With the improved tissue block culture,the periodontal ligament tissues clung to the middle third of the root surface of the totally anesthetized canine incisor teeth were gently curetted,removed,cut into small pieces,and plated onto 6 well plate with cover slip in place of the dry anchoring traditionally.After the successfully cultured cells were subcultured,methyl thiazolyl tetrazolium(MTT) colorimetric assay,immunohistochemical staining and Von Kossa staining were used to determine their biological characteristics. RESULTS:The success rate of primary culture of canine PDLFs was up to about 89%.The PDLFs identified with immunohistochemical staining were positive for vimentin and negative for keratin,and produced mineral like nodules in vitro. CONCLUSION:A simple and stable method to obtain primary PDLFs is established by means of improved tissue block culture.PDLFs cultured in vitro have partial phenotype of osteoblast like cells,indicating that they can be used as seed cells for periodontal tissue engineering.