细胞外基质PDMS硬度对牙髓干细胞增殖和成骨分化的影响

Effects of extracellular matrix PDMS stiffness on proliferation and osteogenic differentiation of dental pulp stem cells

目的:研究细胞外基质聚二甲基硅氧烷(polydimethylsiloxane,PDMS)硬度对牙髓干细胞(DPSCs)增殖和成骨分化的影响及其机制。方法:收集南京医科大学附属常州市第二人民医院因正畸而拔除的前磨牙作为实验材料,分离、培养DPSCs,制作PDMS基质。根据不同硬度,将PDMS基质分为A组(基料/固化剂=10∶1,硬度135 kPa)、B组(基料/固化剂=20∶1,硬度54 kPa)和C组(基料/固化剂=30∶1,硬度16 kPa),另设不含PDMS基质的对照组,在各组基质上培养DPSCs细胞,采用CCK-8法检测各组DPSCs细胞培养至1、3、5、7天时的增殖率,采用茜素红染色鉴定各组DPSCs细胞成骨效果,采用蛋白免疫印迹法(Western blot)检测各组DPSCs细胞中骨钙素(OCN)、RUNX2、细胞外因子1(Wnt1)、β连环素(β-catenin)蛋白表达量。采用SPSS 22.0软件包对数据进行统计学分析。结果:茜素红染色结果显示,A组DPSCs细胞形态发生明显变化,排列具有明显方向性,由多角形、梭形逐渐变为方形,出现钙化小结节,B组钙化小结节明显少于A组,C组钙化小结节明显少于B组,对照组钙化小结节极少。B组DPSCs细胞各时刻增殖率及OCN、RUNX2、Wnt1、β-catenin蛋白表达量均显著低于A组(P<0.05),C组DPSCs细胞各时刻增殖率及OCN、RUNX2、Wnt1、β-catenin蛋白表达量显著低于B组(P<0.05),对照组DPSCs细胞各时刻增殖率及OCN、RUNX2、Wnt1、β-catenin蛋白表达量显著低于C组(P<0.05)。结论:较硬细胞外基质可能通过激活Wnt/β-catenin信号通路,促进牙髓干细胞增殖和成骨分化,这可为牙周组织工程新材料的研发提供理论基础。

PURPOSE: To investigate the effects of extracellular matrix stiffness on proliferation and osteogenic differentiation of dental pulp stem cells(DPSCs) in polydimethylsiloxane(PDMS)-based cell culture substrate model. METHODS:The premolars removed during orthodontic treatment in Changzhou NO.2 People’s Hospital were collected for DPSCs culture. PDMS matrix membranes were prepared, and divided into three groups according to the different stiffness degrees,group A(binder/hardener: 10∶1;135 kPa), group B(binder/hardener: 20∶1;54 kPa), and group C(binder/hardener: 30∶1;16 k Pa). Group free from PDMS was set as control group. Thereafter, DPSCs cells were cultured on PDMS matrix, and various indexes were detected. The proliferation rate of DPSCs was detected by CCK-8, the osteogenic differentiation of DPSCs was detected by alizarin red staining, and the protein expression levels of osteocalcin(OCN), RUNX2, Wnt1 and β-catenin were detected by Western blot. The data were processed with SPSS 22.0 software package. RESULTS: Alizarin red staining showed that DPSCs cells in group A had obvious morphological changes, and the cell arrangement showed obvious ori-entation, its morphology gradually changed from polygon and spindle shape to square shape, and calcified nodules were also observed. The number of calcified nodules among four groups were the most in the group A, followed by group B and group C, which was the lowest in control group, with significant difference(P<0.05). The cell proliferation rate and the expression of OCN, RUNX2, Wnt1 and β-catenin were the highest in group A, followed by group B and group C, which was the lowest in control group, with significant difference(P <0.05). CONCLUSIONS: The extracellular matrix with high stiffness may promote the proliferation and osteogenic differentiation of DPSCs by activating Wnt/β-catenin signaling pathway, which may provide a theoretical basis for periodontal tissue engineering.