骨髓间充质干细胞在淫羊藿苷/羟基磷灰石/聚乳酸-羟基乙酸共聚物支架上的成骨

Osteogenesis of bone marrow mesenchymal stem cells on hydroxyapatite/icariin/poly(lactic-co-glycolic acid)scaffolds

背景:近年来,骨组织工程技术为临床治疗骨缺损提供了全新的思路和模式。该研究首次将传统中药与组织工程支架的纳米结构结合,以期探索并构建一种可用于骨缺损治疗的新型骨组织替代材料。目的:研究淫羊藿苷(icariin,ICA)/羟基磷灰石(hydroxyapatite,HA)/聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid),PLGA)复合支架的成骨活性。方法:将HA与PLGA通过物理共混的方式制成HA/PLGA复合支架,然后将其浸泡于不同浓度的ICA溶液中,从而得到ICA/HA/PLGA支架。利用兔骨髓间充质干细胞分别对复合支架的细胞黏附、增殖、成骨作用和细胞毒性进行评价。细胞黏附、细胞增殖和细胞毒性采用MTT法进行检测,碱性磷酸酶活性和骨钙素活性采用ELISA法进行检测,成骨相关基因和蛋白表达水平分别用荧光定量PCR和Western blot法进行检测。结果与结论:①PLGA中加入适量HA可以提高支架的力学强度,且在HA含量为10%时效果最佳,拉伸强度为(1.67±0.37)MPa;压缩模量为(4.17±1.62)MPa,且会在支架表面形成纳米结构;该微结构可以促进骨髓间充质干细胞在支架表面的黏附;②ICA不会影响骨髓间充质干细胞在复合支架上的增殖,且1.00 mol/L ICA水溶液浸泡后的ICA/HA/PLGA复合支架具有最优的成骨分化功能,其碱性磷酸酶活性、骨钙素活性、成骨相关基因和蛋白(Runx-2和COLⅠ)的表达水平均最高;③ICA/HA/PLGA复合支架无细胞毒性;④结果表明,HA(10%)/ICA(1.00 mol/L)/PLGA支架具有良好的机械性能、成骨作用和生物相容性,是一种具有良好应用潜力的骨组织工程支架。

BACKGROUND:Bone tissue engineering has provided a novel ideal for treating bone defects in clinic.This study is the first to combine traditional Chinese medicine with the nanostructures of tissue-engineered scaffolds in order to explore and construct a new bone tissue substitute material for the treatment of bone defects.OBJECTIVE:To investigate the osteogenic activity of icariin(ICA)/hydroxyapatite(HA)/poly(lactic-co-glycolic acid)(PLGA)composite scaffolds.METHODS:A HA/PLGA composite scaffold was prepared by physical blending of HA and PLGA,and was then soaked in ICA solution of different concentrations to obtain the HA/ICA/PLGA scaffold.Rabbit bone marrow mesenchymal stem cells were used to evaluate the cell adhesion,proliferation,osteogenesis and cytotoxicity of the composite scaffold.The cell adhesion,proliferation and cytotoxicity were detected by MTT method.The activities of alkaline phosphatase and osteocalcin were detected by ELISA.The expression levels of osteogenic genes and proteins were detected by fluorescence quantitative PCR and western blot assay,respectively.RESULTS AND CONCLUSION:Adding appropriate amount of HA into PLGA could improve the mechanical strength of the scaffold,and 10%HA had the best effect with tensile strength of(1.67±0.37)MPa,and compression modulus of(4.17±1.62)MPa,and nanostructure would be formed on the surface of the scaffold.The nanostructure could promote the adhesion of bone marrow mesenchymal stem cells on the surface of the scaffold.ICA did not affect the proliferation of bone marrow mesenchymal stem cells on the composite scaffold.However,the HA/PLGA composite scaffold soaked in 1.00 mol/L ICA aqueous solution had the optimal osteogenic differentiation function,and the expression levels of alkaline phosphatase,osteocalcin,osteogenic related genes and proteins(Runx-2 and COL I)were increased.The ICA/HA/PLGA scaffold had no cytotoxicity.These results suggest that HA(10%)/ICA(1.00 mol/L)/PLGA scaffold has good mechanical properties,osteogenesis and biocompatibility,which has the potential to be a favorable scaffold for bone tissue engineering.