新型自组装多肽水凝胶对成骨细胞前体细胞作用的体外研究

Effect of self-assembling peptide hydrogel scaffold on mouse pre-osteoblasts in vitro

目的观察新型自组装肽TRSAWmx水凝胶对在体外小鼠成骨细胞前体细胞MC3T3-E1细胞粘附效果、增殖能力及成骨分化能力的影响。方法通过原子力显微镜观察新型水凝胶的自组装性能;以倒置显微镜对贴壁细胞计数,比较不同材料细胞的粘附;以CCK-8检测和评价不同材料上细胞的增殖;通过激光共聚焦显微镜观察细胞在材料表面的生长;茜素红-S染色观察钙结节形成;检测材料上培养的细胞碱性磷酸酶(alkaline phosphatase,ALP)活性以及细胞ALP、骨钙蛋白(osteocalcin,OCN)、血管内皮生长因子(vascular endothelial growth factor,VEGF)等基因的表达。结果 TRSAWmx能自组装成纳米纤维结构;在接种30、60、90 min时,TRSAWmx组细胞粘附数明显高于空白组(P<0.05);3、5、7 d时TRSAWmx组细胞有着更佳的增殖活力(P<0.05);在成骨诱导培养3、7、14 d时,TRSAWmx组ALP活性明显高于RADA16-Ⅰ组(P<0.05);培养14 d后,TRSAWmx组茜素红-S染色可见钙结节形成多,成骨相关基因ALP、OCN、VEGF表达水平更高(P<0.05)。结论新型自组装多肽纳米水凝胶TRSAWmx在体外能有效提高MC3T3-E1细胞粘附、增殖和成骨分化能力。

Objective To determine the effect of self-assembling peptide hydrogel TRSAWmx on cell adhesion,proliferation and osteogenic differentiation of mouse pre-osteoblasts MC3T3-E1 in vitro. Methods The nanostructure charateristics of TRSAWmx hydrogel were observed with an atomic force microscope（ AFM）. The adhesion of MC3T3-E1 cells to different hydrogels was measured by counting adherent cells under an inverted microscope. The proliferation was investigated by cell counting kit（ CCK）-8 assay. A laser confocal microscope was employed to observe the cell morphology. Alizarin red staining was used to observe calcium nodules. Alkaline phosphatase（ ALP） activity as well as the expressions of ALP,osteocalcin（ OCN）and vascular endothelial growth factor（ VEGF） was detected to evaluate osteogenic differentiation of MC3T3-E1 cells. Results TRSAWmx hydrogel showed nanofiber structure based on self-assembly. The cell adhesion assay showed that TRSAWmx hydrogel had better cell adhesion（ P〈0. 05） than the control. CCK-8 assay showed that the proliferative activity of MC3T3-E1 cells on the TRSAWmx hydrogel was higher than that on the traditional RADA16-Ⅰ gel on day 3,day 5 and day 7（ P〈0. 05）. The activity of ALP was higher in the TRSAWmx hydrogel than in the RADA16-Ⅰ gel on day 3,day 7 and day 14（ P〈0. 05）. Calcium nodules were observed in the TRSAWmx hydrogel on day 14 after osteogenic induction,while the expressions of ALP,OCN and VEGF in the MC3T3-E1 cells were enhanced on day 14（ P〈0. 05）. Conclusion The functionalized self-assembling peptide hydrogel TRSAWmx can enhance the adhesion, proliferation and osteogenic differentiation of MC3T3-E1 cells in vitro.