自组装纳米多肽水凝胶负载干细胞对软骨损伤修复的研究

Repair of cartilage defects with stem cells loaded with silk fibroin degradation products self-assembled nano-peptide hydrogel

目的探讨自组装纳米多肽水凝胶负载干细胞对新西兰兔软骨缺损的修复作用。方法构建自组装纳米多肽水凝胶,利用扫描电镜技术检测丝素蛋白支架的结构,培养兔源性骨髓间充质干细胞(BMSC),流式细胞仪检测第2代BMSC表面蛋白CD34、CD45、CD90和CD105表达,活细胞/死细胞双重染色检测水凝胶与BMSC的生物相容性,CCK-8法检测BMSC增殖情况检测,实时定量PCR检测水凝胶对BMSC的蛋白聚糖(AGN)、Ⅱ型胶原a1(COL2a1)、成软骨分化调控基因Sox-9 mRNA的表达,分析水凝胶对BMSC成软骨分化的影响,并利用甲苯胺蓝染色评估水凝胶对兔软骨缺损的修复效果。结果原子力显微镜观察功能化自组装多肽可以形成纳米纤维网状结构;第2代BMSC表面蛋白结果显示CD105阳性和CD45阴性的比例为91.22%,而CD90阳性和CD34阴性的比例为95.10%。生物相容性检测示兔BMSC在自组装水凝胶中生长状态稳定,以绿染的活细胞为主。CCK-8法检测示BMSC复合水凝胶组在培养3 d时增殖能力高于BMSC组(P<0.001)。PCR检测示空白组、水凝胶、诱导液组和水凝胶与诱导液混合组的AGN、COL2a1、Sox-9 mRNA表达在第2~4周比较差异均有统计学意义(P均<0.001),有水凝胶的环境更有利于BMSC成软骨分化。甲苯胺蓝染色结果显示水凝胶负载干细胞对软骨缺损的修复作用优于BMSC组和缺损对照组(P均<0.05)。结论自组装纳米多肽水凝胶负载BMSC对软骨缺损的修复具有明显的促进作用。

Objective To evaluate the repair of cartilage defects in New Zealand rabbits by selfassembled nano-peptide hydrogel-loaded stem cells loaded with silk fibroin degradation products.Methods The self-assembled nano-peptide hydrogel was constructed.The structure of silk fibroin scaffolds was detected by scanning electron microscopy.The rabbit bone marrow mesenchymal stem cells(BMSCs)were cultured.The expression levels of CD34,CD45,CD90 and CD105 proteins on the surface of second-generation BMSCs were detected by flow cytometry.The biocompatibility between hydrogel and BMSCs was assessed by living/dead cell dual staining.The proliferation of BMSCs was determined by CCK-8 assay.The expression levels of aggrecan(AGN),COL2a1 and Sox-9 mRNA were detected by real-time quantitative PCR.The effect of hydrogel on the cartilage differentiation of BMSCs was evaluated.The repairing effect of hydrogel on cartilage defects was assessed by toluidine blue staining.Results Atomic force microscopy observed that functionalized self-assembled peptides could form nanofiber network structure.The surface proteins of the second-generation BMSCs showed that the proportion of CD105-positive and CD45-negative BMSCs was 91.22%,whereas that of CD90-positive and CD34-negative BMSCs was 95.10%.Biocompatibility test revealed that rabbit BMSCs grew stably in the self-assembled hydrogel,mainly seen as the green-stained live cells.CCK-8 assay showed that the proliferation capability of BMSCs after 3-d culture in the compound hydrogel group was significantly higher than that in the BMSCs group(P<0.001).PCR indicated that the expression levels of AGN,COL2a1 and Sox-9 mRNA at 2-4 weeks significantly differed among the blank,hydrogel,CM and hydrogel combined with CM groups(all P<0.001).The environment containing hydrogen was beneficial to the cartilage differentiation of BMSCs.Toluidine blue staining revealed that hydrogel-loaded stem cells were more effective than the BMSCs and controls in repairing bone defects(both P<0.05).Conclusion The self-assembled nano-peptide hydrogel loaded stem cells can promote the repair of cartilage defects.