纳米羟基磷灰石/细菌纤维素对兔骨髓间充质干细胞成骨分化影响的实验研究

Effects of nano-hydroxyapatite/bacterial cellulose coposite scaffold on the osteoblastic differentiation of rabbit bone marrow mesenchymal stem cell

目的观察纳米羟基磷灰石/细菌纤维素复合支架(nHA/BC)对兔骨髓间充质干细胞(BMSCs)成骨分化的影响。方法将BMSCs与nHA/BC体外复合,活/死实验观察其复合情况,CCK-8检测增殖情况。利用成骨诱导培养基诱导BMSCs向成骨分化,茜素红、碱性磷酸酶染色观察成骨分化情况,免疫荧光检测RUNX2蛋白表达情况,RT-PCR法检测Ⅰ型胶原、骨钙素(OC)、骨桥蛋白(OP)mRNA的表达情况。结果BMSCs在nHA/BC表面生长良好并稳定增殖。诱导后茜素红、碱性磷酸酶染色呈现明显的成骨细胞特征,免疫荧光可检测到RUNX2蛋白表达。RT-PCR结果提示Ⅰ型胶原、OC、OP等成骨标志基因mRNA明显表达。结论nHA/BC支架材料能促进兔BMSCs黏附、增殖、成骨分化,是一种潜在的理想组织工程支架。

Objective To observe the effects of nano-hydroxyapatite/bacterial cellulose(nHA/BC)on the osteoblastic differentiation of rabbit bone marrow mesenchymal stem cells(BMSCs).Methods Rabbit BMSCs were seeded on nHA/BC in vitro,and observed by live and death tests.Their proliferation was detected by CCK-8 assay.Osteogenic induction medium was adopted for inducing the osteogenic differentiation of BMSCs.The osteogenic differentiation was observed by alizarin red staining and alkaline phosphatase staining.The expression of RUNX2 protein was detected by immunofluorescence.The expression of typeⅠcollagen,osteocalcin(OC)and osteopontin(OP)mRNA was detected by RT-PCR.Results BMSCs grew well and steadily proliferated on the surface of nHA/BC.After induction,alizarin red and alkaline phosphatase stainings showed obvious characteristics of osteoblasts,and the expression of RUNX2 protein could be detected by immunofluorescence.RT-PCR results suggested that osteogenic marker genes such as typeⅠcollagen,OC and OP were obviously expressed.Conclusions nHA/BC scaffold can promote the adhesion,proliferation and osteogenic differentiation of rabbit BMSCs,which is a potential ideal scaffold for tissue engineering.