不同管径的二氧化钛纳米管对人牙周膜干细胞生物学行为的影响

Effects of titanium dioxide nanotubes with different diameters on the biological behavior of human periodontal ligament stem cells

目的:研究不同管径的二氧化钛(TiO_(2))纳米管对人牙周膜干细胞(PDLSCs)生物学行为的影响。方法:在光滑钛表面通过阳极氧化方法在5、10、20 V电压下分别制备TiO_(2)纳米管形貌,将试样分为光滑钛组(Ti)、5 V纳米管组(NT5)、10 V纳米管组(NT10)和20 V纳米管组(NT20)。分离、培养人PDLSCs,接种至TiO_(2)纳米管表面,扫描电镜观察纳米管表面PDLSCs形态,通过Hoechst染色观察PDLSCs粘附情况,通过碱性磷酸酶(ALP)试剂盒检测PDLSCs的ALP活性,通过天狼星红染色观察PDLSCs的胶原分泌情况,通过茜素红染色观察PDLSCs的胞外基质矿化情况,通过实时定量RT-PCR检测PDLSCs的成骨及牙周相关基因的表达水平。结果:和光滑钛对比,在TiO_(2)纳米管表面的PDLSCs呈纺锤丝样排列,并且伸出大量的丝状伪足和板状伪足;TiO_(2)纳米管能够促进PDLSCs的粘附和胶原分泌;在成骨诱导液环境下,NT5和NT20的ALP染色表面活性优于Ti组;NT10组纳米管形貌表面形成大量矿化结节;纳米管促进了ALP表达,14 d时NT5和NT20在纤维连接蛋白基因和7 d时Runt相关转录因子2基因表达上调。结论:TiO_(2)纳米管能够使PDLSCs展现不同的细胞骨架形态,促进其早期粘附、胶原分泌和胞外基质矿化,并影响了相关基因的表达。

Objective:To evaluate the influence of TiO_(2)nanotubes with different diameters on periodontal ligament stem cells(PDLSCs)biological changes.Methods:TiO_(2)nanotubes with different diameters were prepared by means of anodic oxidation at 5,10 and 20 V voltage on the smooth titanium surface.The samples were divided into smooth titanium group(Ti),5 V nanotube group(NT5),10 V nanotube group(NT10)and 20 V nanotube group(NT20).The PDLSCs were obtained using the limiting-dilution technique,and then cultured on different diameters TiO_(2)nanotubes.The morphology of PDLSCs was observed by using FE-SEM.The cells adhesion was observed by Hoechst staining.The cells'ALP activity was detected by ALP kit.Sirius red staining and Alizarin red staining were used to observe the collagen secretion and the sample surface extracellular matrix mineralization,respectively.Real time quantitative PCR was employed to detect the cells'osteogenic and periodontal related genetic level.Results:The ALP staining experiment showed stronger activity in NT5 and NT20 groups than that in Ti group.A large number of mineralized nodules were formed on the surfaces of NT10 groups.TiO_(2)nanotubes morphology promoted the up-regulation of ALP,FN(NT5 and NT5 at 14 d)and RUNX2(at 7 d)expression.Conclusions:Different diameters TiO_(2)nanotube morphology induced the PDLSCs to form different cell cytoskeletons and promoted the early adhesion,collagen secretion and related genes expression of PDLSCs.