壳聚糖-β-磷酸三钙复合层状支架的制备与研究

Fabrication and research of chitosan-β-TCP composite lamellas scaffold

目的通过合成壳聚糖-β-磷酸三钙(CS-β-TCP)支架,测试其力学性能、生物相容性和体外成骨向分化能力,研究其作为生物支架修复骨缺损的可能性。方法通过双向冻干技术制备CS-β-TCP支架,使用扫描电子显微镜电镜(SEM)、能量分散光谱(EDS)和X线衍射仪(XRD)对该支架进行结构表征、元素分布和组成成分分析,力学万能仪测试其压缩强度。通过CCK-8法和活死染色探究其生物相容性,采用荧光染色法检测复合组(CS-β-TCP)支架的细胞黏附生长情况。qRT-PCR法检测成骨相关基因:骨形态发生蛋白(BMP2),RUNX相关转录因子2(RUNX2)和Ⅰ型胶原(COL1)的表达,ALP染色检测BMSCs的成骨分化效果。结果扫描电镜结果显示CS-β-TCP支架呈平行排列的薄层状结构,疏松多孔,β-TCP颗粒均匀的分布在壳聚糖骨架上,具有良好的机械性能,CS-β-TCP支架的CCK-8结果与对照组无明显差异,活死染色结果表明复合组支架上细胞具有较高的细胞活性。qRT-PCR和ALP结果表明复合组支架体外具有一定的骨诱导能力,能够促进间充质干细胞的成骨向分化。结论复合组支架具备优异的机械性能和生物学性能,能够诱导间充质干细胞成骨方向分化,为骨缺损的修复研究提供了一种新的思路。

Objective To investigate the mechanical property,biocompatibility,and osteogenic differentiation ability of chitosan-β-TCP( CS-β-TCP) scaffold,meanwhile,to study the possibility of the composite as a scaffold to repair bone defect. Methods Briefly,the CS-β-TCP composite scaffold was fabricated utilizing bidirectional lyophilization technique. Then,the scaffold micro-structure was observed by scanning electron microscopy( SEM),X-ray diffraction( XRD) and energy disperse spectroscopy( EDS) were employed to analyze the ingredients and elements distribution of scaffold,respectively. Additionally,the compression strength of the scaffold was tested by mechanical universal testing machine. The biocompatibility of the scaffold and the cell viability research were characterized via CCK-8 assay and Live/Dead staining,respectively,and the cell adhesion was studied by DPAI/Phalloidine fluorescence staining. qRT-PCR was employed to investigate the expression level of osteogenic-related gene such as BMP2,RUNX2 and COL1. ALP staining was carried out to measure the osteogenic differentiation effect of BMSCs. Results The CS-β-TCP scaffold was comprised of bulk parallel,aligned and thin lamellas with many porous structures. β-TCP particles were evenly distributed over CS framework layers and the CS-β-TCP scaffold possess excellent elastic property and biocompatibility,moreover,the cell seeded on scaffold revealed high cell viability and continuous proliferation. qRT-PCR and ALP staining results demonstrated that the CS-β-TCP scaffold could induce osteogenic differentiation of BMSC. Conclusion To sum up,the CS-β-TCP scaffold expressed desired mechanical and biological properties,and could induce BMSC differentiate into osteoblast,the composite scaffold provided a promising strategy for bone defect regeneration.