NUS Graduate School, Integrative Sciences and Engineering Programme, National University of Singapore, Singapore; Department of Biomedical Engineering, National University of Singapore, Singapore.
Department of Biomedical Engineering, National University of Singapore, Singapore.
Mater Sci Eng C Mater Biol Appl. 2021 May;124:112088. doi: 10.1016/j.msec.2021.112088. Epub 2021 Mar 31.
Porous scaffolds have been widely used for bone tissue engineering (BTE), and the pore structure of scaffolds plays an important role in osteogenesis. Silk fibroin (SF) is a favorable biomaterial for BTE due to its excellent mechanical property, biocompatibility, and biodegradation, but the lack of cell attachment sites in SF chemical structure resulted in poor cell-material interactions. In this study, SF scaffolds were coated with fibronectin/gelatin (Fn/G) to improve cell adhesion. Furthermore, the effect of pore size in Fn/G coated SF scaffolds on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) were investigated in vitro. Scaffolds with average pore diameters of 384.52, 275.23, and 173.8 μm were prepared by salt leaching method, labelled as Large, Medium, and Small group. Porcine BMSCs were seeded on scaffolds and cultured in osteogenic medium for 21 days to evaluate cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, gene expression of osteogenic markers, and histological performance. The results showed Fn/G coating effectively improved cell adhesion on SF scaffolds. Cell metabolic rate in each group increased significantly with time, but there was no statistical difference at each time point among the three groups. On day 21, ALP/DNA and calcium/DNA in the Small group were significantly higher than those in the Large group. Among the three pore sizes, the Small group showed higher mRNA expression of COl I on day 7, OPN on day 14, and OCN on day 21. Immunohistochemical staining on day 21 showed that Col I and OCN in Small group were more highly expressed. In conclusion, the Fn/G coated SF scaffolds with a mean pore diameter of 173.8 μm was optimal for osteogenic differentiation of BMSC in vitro.
多孔支架已广泛应用于骨组织工程（BTE），支架的孔结构在成骨中起着重要作用。丝素蛋白（SF）是一种有利于 BTE 的生物材料，具有优异的机械性能、生物相容性和可生物降解性，但 SF 化学结构中缺乏细胞附着位点导致细胞与材料的相互作用较差。在这项研究中，SF 支架涂覆纤维连接蛋白/明胶（Fn/G）以提高细胞黏附性。此外，还研究了 Fn/G 涂覆 SF 支架的孔径对骨髓间充质干细胞（BMSCs）成骨分化的影响。采用盐溶法制备平均孔径为 384.52、275.23 和 173.8 μm 的 SF 支架，分别标记为大孔组、中孔组和小孔组。将猪 BMSCs 接种到支架上，在成骨培养基中培养 21 天，以评估细胞增殖、碱性磷酸酶（ALP）活性、钙沉积、成骨标志物基因表达和组织学性能。结果表明，Fn/G 涂层能有效提高 SF 支架上细胞的黏附性。各实验组细胞代谢率随时间的推移显著增加，但在每个时间点，三组之间均无统计学差异。第 21 天，小孔组的 ALP/DNA 和钙/DNA 显著高于大孔组。在三种孔径中，小孔组在第 7 天的 COl I、第 14 天的 OPN 和第 21 天的 OCN 的 mRNA 表达均较高。第 21 天的免疫组织化学染色显示，小孔组的 Col I 和 OCN 表达较高。综上所述，平均孔径为 173.8 μm 的 Fn/G 涂覆 SF 支架最有利于 BMSC 的体外成骨分化。
