胶原蛋白支架对载杜仲叶提取物处理的人牙周膜干细胞增殖和成骨分化的作用

Effect of collagen scaffold on proliferation and osteogenic differentiation of human periodontal ligament stem cells treated by Eucommia ulmoides Oliver leaf extract

背景:胶原蛋白支架是一种良好的组织工程材料,但目前其对于载杜仲叶提取物的人牙周膜干细胞的生物相容性、增殖及成骨活性的影响尚未有报道。目的:通过对载杜仲叶提取物的人牙周膜干细胞与胶原蛋白支架的体外共培养,研究人牙周膜干细胞在支架材料上的形态特征、黏附情况、增殖及成骨分化功能。方法:将载杜仲叶提取物的人牙周膜干细胞与胶原蛋白支架体外共培养作为实验组,以人牙周膜干细胞与胶原蛋白支架的共培养作为阳性对照组,无支架的人牙周膜干细胞常规培养作为空白对照组。结果与结论:①扫描电镜下可见,实验组支架上有大量人牙周膜干细胞附着,且细胞突触与支架紧密相连,生长状态良好,细胞间相互接触、紧密相连;②共培养4,8和12 h后,实验组和阳性对照组人牙周膜干细胞的黏附率均呈上升趋势,实验组黏附率高于阳性对照组(P<0.05);③MTT结果显示,复合培养3,5和7 d后,实验组的细胞吸光度值高于阳性对照组(P<0.05);④成骨诱导7,14 d后,实验组碱性磷酸酶活性高于阳性对照组(P<0.05);⑤RT-PCR检测结果显示,成骨刺激作用21d后,实验组和阳性对照组中成骨相关基因Runx2、OPN和OCN的表达显著高于空白对照组(P<0.01),实验组上述基因的表达均高于阳性对照组(P<0.05);⑥上述数据说明,胶原蛋白支架对于载杜仲叶提取物的人牙周膜干细胞具有较好的生物相容性,复合培养后可保持细胞形态、促进其增殖及成骨分化功能。

BACKGROUND: Collagen scaffold is a good tissue engineering material. However, there are no reports on the effects of collagen scaffold on the biocompatibility, proliferation, and osteogenic activity of human periodontal ligament stem cells(hPDLSCs) treated by Eucommia ulmoides Oliver leaf extract. OBJECTIVE: hPDLSCs treated by Eucommia ulmoides Oliver leaf extract were co-cultured with collagen scaffolds in vitro to investigate the morphological characteristics, adhesion, proliferation and osteogenic differentiation of hPDLSCs on collagen scaffolds. METHODS: In the experimental group, hPDLSCs treated by Eucommia ulmoides Oliver leaf extract were co-cultured with collagen scaffolds in vitro. In the positive control group, hPDLSCs were co-cultured with collagen scaffolds in vitro. In the blank control group, only hPDLSCs were cultured. RESULTS AND CONCLUSION: Scanning electron microscopy results revealed that in the experimental group, a large number of hPDLSCs adhered to the scaffold, and the synapses of the cells were closely connected with the scaffold. The cells grew well and cont acted closely with each other. After co-culture for 4, 8 and 12 hours, cell adherence rate in both experimental and positive control groups increased and it in the experimental group was significantly higher than that in the positive control group(P < 0.05). MTT results showed that after 3, 5 and 7 days of co-culture, absorbance value in the experimental group was significantly higher than that in the positive control group(P < 0.05). After 7 and 14 days of osteogenic induction, alkaline phosphatase activity in the experimental group was significantly higher than that in the positive control group(P < 0.05). RT-PCR results showed that after 21 days of osteogenic stimulation, the expression of osteogenesis-related genes Runx2, OPN and OCN in the experimental group was significantly higher than that in the blank control group(P < 0.01), and the expression of the above genes in the experimental group was higher than that in the positive control group(P < 0.05). These data indicate that collagen scaffolds have good biocompatibility with hPDLSCs treated by Eucommia ulmoides Oliver leaf extract, can maintain cell morphology, and promote cell proliferation and osteogenic differentiation after co-culture.