三维培养人牙周膜干细胞的形态、活性及成骨分化能力

Three-dimensional culture affects morphology,activity and osteogenic differentiation of human periodontal ligament stem cells

背景:牙周膜干细胞作为牙源性间充质干细胞一员,是近年来牙周骨组织工程的研究热点。由于干细胞数量不足以及微环境的影响,牙周组织再生能力往往无法满足治疗的需要。近年来,片状培养细胞片和三维培养细胞球体作为单细胞悬浮注射的替代品,能够有效改善数量不足和微环境等问题,成为组织工程的研究趋势。但是目前运用三维培养技术形成牙周膜干细胞球体的研究较少。目的:探究三维培养技术对人牙周膜干细胞形态、活性及成骨分化的影响。方法:采用改良酶消化法培养人牙周膜干细胞并进行细胞鉴定。通过细胞骨架和细胞核荧光标记检测细胞球体的形态;通过Live/Dead染色试剂盒检测细胞球体的活性;通过碱性磷酸酶活性、成骨相关基因表达水平分析三维培养技术对人牙周膜干细胞成骨分化的影响。结果与结论:①人牙周膜干细胞球体形态及活性呈时间依赖性变化。随时间增长,细胞球直径变小,球体核心死亡细胞增加;②与二维培养比较,成骨诱导3 d时三维培养人牙周膜干细胞的碱性磷酸酶活性及成骨诱导3,4,7 d时成骨相关基因的表达增加,β-catenin的表达增加,提示三维培养技术可促进人牙周膜干细胞成骨分化,可能与Wnt/β-catenin通路有关。

BACKGROUND:Periodontal ligament stem cells,as a member of odontogenic mesenchymal stem cells,are the research hotspot of periodontal bone tissue engineering in recent years.Due to the insufficient number of stem cells and the influence of microenvironment,the regeneration ability of periodontal tissue often cannot meet the needs of treatment.In recent years,flaky cultured cell sheets and three-dimensional cultured cell spheres,as substitutes for single cell suspension injection,can effectively improve the problems of insufficient quantity and microenvironment,and become the research trend of tissue engineering.However,there are few studies on the formation of periodontal ligament stem cell spheres by three-dimensional culture technology.OBJECTIVE:To explore the effects of three-dimensional culture technology on the morphology,activity and osteogenic differentiation of human periodontal ligament stem cells.METHODS:Human periodontal ligament stem cells were cultured by modified enzyme digestion method,and then identified.The morphology of cell spheres was detected by cytoskeleton and nuclear fluorescence labeling.The activity of cell spheres was detected by Live/Dead staining kit.The effects of three-dimensional culture on osteogenic differentiation of human periodontal ligament stem cells were analyzed by alkaline phosphatase activity and osteogenic related gene expression.RESULTS AND CONCLUSION:(1)The morphology and activity of human periodontal ligament stem cell spheroid cells changed in a time-dependent manner.With the increase of time,the diameter of cell sphere became smaller and the number of dead cells in the core of sphere increased.(2)Compared with two-dimensional culture,three-dimensional culture of human periodontal ligament stem cells increased alkaline phosphatase activity at 3 days of osteogenic induction and the expression of osteogenic related genes andβ-catenin at 3,4,and 7 days of osteogenic induction,suggesting that three-dimensional culture technology can promote the osteogenic differentiation of human periodontal ligament stem cells,which may be related to Wnt/β-catenin pathway.