功能性纳米自组装多肽KLPP凝胶修复软骨缺损的实验研究

Experimental study of functionalized self-assembling nanofiber peptide KLPP hydrogel in repairing cartilage defects

目的探讨功能性纳米自组装多肽KLPP凝胶对关节软骨缺损的修复作用。方法培养人骨髓间充质干细胞(BMSCs),将BMSCs分为BMSCs-KLD-12组(BMSCs与KLD-12凝胶共培养)和BMSCs-KLPP组(BMSCs与KLPP凝胶共培养),分别行软骨诱导分化培养,MTT法检测细胞的增殖活性。将人软骨组织分为软骨-KLPP组(制作软骨缺损区并填充KLPP凝胶进行培养)、软骨-KLD-12组(制作软骨缺损区并填充KLD-12凝胶进行培养)、软骨组(仅制作软骨缺损区)。Calcein-AM/PI双染法检测BMSCsKLD-12组及BMSCs-KLPP组的细胞毒性。培养49 d后,收集软骨组、软骨-KLD-12组、软骨-KLPP组的软骨组织进行切片,并行阿尔新蓝染色。Western blot法检测各组中Ⅰ型胶原(Col-Ⅰ)、Ⅱ型胶原(Col-Ⅱ)、Ⅹ型胶原(Col-Ⅹ)的表达。结果与BMSCs-KLD-12组比较,BMSCs-KLPP组培养1 d、3 d、7 d的细胞增殖率均明显增加(P<0.05),且Col-Ⅰ、Col-Ⅱ、Col-Ⅹ的相对表达水平明显增加(P<0.05)。Calcein-AM/PI双染色检测表明BMSCs-KLPP组和BMSCs-KLD-12组细胞活性基本一致,KLPP凝胶无细胞毒性。与软骨-KLD-12组比较,软骨-KLPP组软骨修复作用更明显(P<0.05),且Col-Ⅰ、Col-Ⅱ、Col-Ⅹ的相对表达水平更高(P<0.05)。结论功能性纳米自组装多肽KLPP凝胶对关节软骨缺损具有良好的修复作用。

Objective To investigate the repair effect of functionalized self-assembling nanofiber peptide KLPP hydrogel on articular cartilage defects.Methods Human bone marrow mesenchymal stem cells(BMSCs)were cultured and divided into the BMSCs-KLD-12 group(BMSCs co-cultured with KlD-12 hydrogel)and the BMSCs-KLPP group(BMSCs co-cultured with KLPP hydrogel),and chondro⁃genic differentiation of cells were cultured,respectively.The proliferative activity of BMSCs was detected by MTT assay.Human cartilage tissues were divided into the chondro-KLPP group(cartilage defect area was prepared and cultured with KLPP hydrogel),the chondro-KLD-12 group(cartilage defect area was prepared and cultured with KLD-12 hydrogel),and the cartilage group(cartilage defect area was prepared only).The cytotoxicity of the BMSCs-KLD-12 group and the BMSCS-KLPP group was detected by Calcein-AM/PI double staining method.After 49 days of culture,the cartilage tissues of the cartilage group,the chondro-KLD-12 group and the chondro-KLPP group were sliced and stained with Alcian blue.Western blot assay was used to detect the expression of typeⅠcollagen(Col-Ⅰ),typeⅡcollagen(Col-Ⅱ)and typeⅩcollagen(Col-Ⅹ)in all groups.Results Compared with the BMSCs-KLD-12 group,the proliferation rate of the BMSCs-KLPP group was significantly increased at day 1,day 3 and day 7(P<0.05),and the relative expression levels of Col-Ⅰ,Col-Ⅱand Col-Ⅹwere significantly increased(P<0.05).Calcein-AM/PI double staining showed that the cell activity of the BMSCs-KLPP group and the BMSCs-KLD-12 group was basically the same,and KLPP hydrogel had no cytotoxicity.Compared with the chondro-KLD-12 group,the cartilage repair effect of the chondro-KLPP group was significantly increased(P<0.05),and the relative expression levels of Col-Ⅰ,Col-Ⅱand Col-Ⅹwere significantly increased(P<0.05).Conclusion Functional self-assembling nanofiber peptide KLPP hydrogel has a good repair effect on articular cartilage defects.