自组装多肽水凝胶支架中MSCs的三维培养及成骨分化

Three-dimensional Culture and Osteogenic Differentiation of MSCs in Self-Assembling Peptide Hydrogel Scaffolds

使用一种新型人工设计自组装多肽(RADA16)水凝胶作为三维培养支架评价MSCs成骨分化情况。将人骨髓MSCs培养增殖后接种到水凝胶中,在成骨分化培养液中进一步培养1～3周。荧光染色法观察细胞形态和存活情况;组织学染色检测MSCs ALP活性;半定量RT-PCR分析成骨特异性基因的表达。绝大多数MSCs在水凝胶支架内能够存活,呈纺锤样形态。诱导培养后蛋白和基因表达水平均检测到ALP活性,在14天时达到峰值。骨晚期分化特异性基因BSP也有表达,且表达量随培养时间延长而增多。自组装多肽水凝胶为MSCs的黏附生长及向成骨细胞分化提供良好的三维微环境,有望成为极具吸引力的骨组织工程支架材料。

The osteogenic differentiation of MSCs was evaluated using a new class of synthetic self - assembling peptide hydrogels, RADA16, as a scaffold for three - dimensional culture. MSCs derived from human bone marrow were culture - expanded and seeded into the hydrogel and further cultured in osteogenic medium for 1 - 3 weeks. The cell viability was visualized and detected under fluorescence microscopy. Alkaline phospharase activity was evaluated by histochemical techniques. Semi - quantitative RT - PCR analyses were preformed to detect the expression of mRNA transcripts. Almost MSCs were living cells and had a typical spindle shape. In osteogenic differentiation, the expression of ALP at the protein and mRNA level was significantly higher than that of control cells. Although the ALP activity peaked at 14 days, the BSP content increased continuously for 21 days. These results showed that the biodegradable/biocompatible hydrogel, RADA16, supports MSCs attachment and differentiation toward the osteogenic lineage, indicating that RADA16 can be considered as an attractive synthetic biomatcrial for using in bone tissue engineering.