包埋载辛伐他汀PLGA微球的胶原/壳聚糖支架的制备及其生物相容性研究

摘要

目的 构建一种具有缓释辛伐他汀功能的胶原/壳聚糖复合支架(SIM/COL/CS),并评估其生物相容性.方法 联合应用复乳溶剂挥发法和冻干技术,制备SIM/COL/CS支架.电镜观察支架的表面形态,紫外光分度法测量支架中药物的缓释速率.分别利用CCK-8(Cell Counting Kit 8)法和死活细胞染色,检测接种在支架上的大鼠骨髓间充质干细胞(BMSC)的增殖和活性.利用碱性磷酸酶(ALP)定量分析和茜素红染色,检测支架对BMSCs成骨分化的影响.结果 SIM/COL/CS支架为层状多孔结构,PLGA微球与支架基质紧密结合.支架中的SIM能够以稳定的速率持续释放,释放时间达27 d以上.CCK-8检测显示,在实验的第1、3、5天,COL/CS组和SIM/COL/CS组的细胞数量无差异;在第7天,SIM/COL/CS组的细胞数量显著高于COL/CS组.死活细胞染色显示,COL/CS组和SIM/COL/CS组中的细胞均有良好的活性,较少出现凋亡.此外,SIM/COL/CS组的ALP活性和钙沉积也显著高于COL/CS组.结论 联合应用复乳溶剂挥发法和冻干技术,可成功制备能够缓释SIM的胶原/壳聚糖支架,这种新型支架可显著促进BMSC的增殖和成骨分化,在骨组织工程研究中具有广阔的应用前景.%Objective To prepare a collagen/chitosan scaffold incorporating simvastatin-loaded PLGA microspheres (SIM/COL/CS), which can release simvastatin (SIM) in a sustained manner, and investigate the biocompatibility of the scaffold. Methods Collagen/chitosan scaffolds incorporating SIM-loaded PLGA microspheres were prepared by combining freeze-drying technology and emulsion-solvent evaporation method. The morphology and drug release profile of the scaffolds were characterized by scanning electron microscope and ultraviolet spectrophotometry respectively. The effects of the composite scaffold on the proliferation and viability of the rat bone marrow-derived mesenchymal stem cells (BMSCs) were assessed by Cell Counting Kit 8 (CCK-8) and Live/Dead cell imaging kit separately. The osteoinductive effect of the scaffold on BMSCs was evaluated by alkaline phosphatase (ALP) activity assay and alizarin red S staining. Results The composited scaffolds exhibited an interconnected porous structure and the PLGA microspheres were tightly integrated within the matrix. Besides,the SIM exculpated in the scaffold was released in a sustained manner for up to 27 days. CCK-8 assay showed that although there was no difference in cell number between the COL/CS and SIM/COL/CS groups on days 1, 3 and 5, cell number in the SIM/COL/CS group was significantly higher than that in the COL/CS group on day 7. Live/dead cell staining indicated the viability of the BMSCs on both COL/CS and SIM/COL/CS were good and few dead cells (red) was found. Moreover, both of the ALP activity and calcium deposit in the SIM/COL/CS group were significantly higher than those in the COL/CS group. Conclusion COL/CS scaffolds containing SIM-loaded PLGA microspheres for controlled SIM delivery can be successfully prepared by combining freeze-drying technology and emulsion-solvent evaporation method. The novel SIM-loaded COL/CS scaffold can promote both the proliferation and osteogenic differentiation of BMSCs and has great application potentials in bone repair.