壳聚糖基温敏水凝胶对牙周膜成纤维细胞相容性的影响

Cytocompatibility of chitosan-based thermosensitive hydrogel to human periodontal ligament cell

目的:体外评价壳聚糖季铵盐/甘油磷酸(CS-HTCC/GP)温敏水凝胶的细胞相容性。方法:体外培养人牙周膜成纤维细胞(HPDLCs),采用四甲基偶氮唑盐(MTT)法测定不同浓度的CS-HTCC/GP温敏凝胶浸提液对1、3、5d细胞增殖和碱性磷酸酶(ALP)活性的影响。采用扫描电镜和透射电镜观察对细胞超微结构的影响。采用SSPS13.0软件包对数据进行统计学分析。结果:免疫组化显示,培养的细胞波形丝蛋白染色阳性,角蛋白染色阴性,证明体外原代培养细胞及传代细胞均为中胚层组织来源,且无上皮来源细胞混杂。不同浓度的CS-HTCC/GP温敏水凝胶浸提液均有促进HPDLCs增殖的作用,在第3天和第5天,差异有高度显著性(P<0.001)。各组均可提高ALP活性。第2组和第3组在第5天与第1天ALP表达的提高有高度显著差异(P<0.001)。扫描电镜和透射电镜观察水凝胶浸提液对HPDLCs的超微结构无影响。结论:CS-HTCC/GP温敏水凝胶具有良好的细胞相容性,具有用于牙周局部药物释放系统载体及组织工程支架的潜力。

PURPOSE： The aim of this investigation was to evaluate the cytocompatibility of an in situ chitosan- quaternized chitosan / , 13-glycerophosphate （CS-HTCC/GP） thermosensitive hydrogel in vitro. METHODS： The primary cells were isolated from human periodontal ligament and cultured. The role of different concentrations of CS-HTCC/GP extract to HPDLCs was evaluated by MTr assay and alkaline phosphatase （ALP） activity. Also, the ultra-architecture of HPDLCs was determined by scanning electron microscopy （SEM） and transmission electron microscopy （TEM） respectively. SPSS13.0 software package was used for statistical analysis. RESULTS： By immunocytochemical method, the cells were stained positively to antibodies against vimentin, and negatively to antibodies against cytokeratin, which indicated that they were external embryo mesenchymal cell without epithelial cell mixure. CS-HTCC/GP thermosensitive hydrogel promoted proliferation of HPDLCs,especially at 3d and 5d, the results was significantly different （P〈0.001）. ALP activity was significantly greater in group 2 and 3 than in group 4 after 5d （P〈0.001）. Also, no negative influence to uhrastructure of HPDLCs was found through SEM and TEM. CONCLUSIONS： The resuhs indicate that CS-HTCC/GP thermosensitive hydrogel exhibits excellent cytocompatibility and has potential to be used as an in situ injectable local periodontal drug delivery vehicle and a tissue-engineering scaffold for periodontal disease therapy. Supported by Award Fund of Excellent Young Scientist of Shandong Province （ BS2010CL015 ）.