目标:目前,软骨修复不能提供理想的疗效。幸运的是,最近的研究声称RADA-16肽是修复软骨缺损的一种有吸引力的治疗策略。因此,本研究试图探讨负载转化生长因子-β(TGF-β)1的RADA-16对骨髓间充质干细胞(BMSCs)软骨分化的影响。
方法:首先,通过固相肽合成RADA-16肽,并通过超分子肽自组装形成定义明确的水凝胶。然后,将TGF-β1(加载浓度为10ng/mL)加载到RADA-16中,用扫描电子显微镜观察TGF-β1/RADA-16水凝胶的形态并检测其相关性质。接下来,从骨髓样品中分离并鉴定BMSCs。TGF-β1/RADA-16与L929、BMSCs共培养,和C28/I2细胞,分别,通过活/死细胞染色和MTT法测定细胞的存活和增殖能力。通过Alcian蓝染色和Blyscan测定确定BMSCs的软骨分化和sGAG产生,通过qRT-PCR检测软骨相关基因的表达,ELISA检测炎性因子水平。至于机制调查,Westernblot检测Smad和ERK/MAPK信号通路。
结果:RADA-16水凝胶表现出分布良好且相互连接的多孔表面结构,TGF-β1的加载率为91.9%。TGF-β1/RADA-16水凝胶具有良好的释放和降解性能,对BMSCs的存活和增殖能力无负面影响,L929和C28/I2细胞。重要的是,TGF-β1/RADA-16水凝胶显著促进BMSCs软骨分化和sGAG的生成,和减少促炎因子的产生。此外,水凝胶还显著激活BMSCs的Smad和ERK/MAPK通路。
结论:负载TGF-β1的RADA-16具有良好的生物学特性,可增强BMSCs的软骨分化能力。
OBJECTIVE: At present, cartilage repair does not offer ideal efficacy. Fortunately, recent studies have claimed that RADA-16 peptide is an attractive therapeutic strategy for repairing cartilage defects. Therefore, this study tried to explore the effect of RADA-16 loaded with transforming growth factor-beta (TGF-β) 1 on cartilage differentiation of bone marrow mesenchymal stem cells (BMSCs).
METHODS: First, the RADA-16 peptide was synthesized by solid phase peptide, and a well-defined hydrogel was formed by supramolecular peptide self-assembly. Then, TGF-β1 (loading concentration of 10 ng/mL) was loaded into RADA-16, with scanning electron microscopy to observe the morphology of the TGF-β1/RADA-16 hydrogel and detect its related properties. Next, BMSCs were isolated from bone marrow samples and identified. TGF-β1/RADA-16 was co-cultured with L929, BMSCs, and C28/I2 cells, respectively, and the survival and proliferation ability of the cells was determined by live/dead cell staining and MTT assay. Chondrogenic differentiation and sGAG production of BMSCs were determined by Alcian blue staining and Blyscan assay, the expression of cartilage-associated genes by qRT-PCR, and the levels of inflammatory factors by ELISA. As for mechanism investigation, the Smad and ERK/MAPK signaling pathways were detected by western blot.
RESULTS: RADA-16 hydrogel exhibited a well-distributed and interconnected porous surface structure, with a loading rate of 91.9% for TGF-β1. The TGF-β1/RADA-16 hydrogel had good release and degradation properties, and had no negative effect on the survival and proliferation ability of BMSCs, L929, and C28/I2 cells. Importantly, TGF-β1/RADA-16 hydrogel significantly accelerated chondrogenic differentiation and sGAG generation in BMSCs, and decreased pro-inflammatory factor production. In addition, the hydrogel also significantly activated the Smad and ERK/MAPK pathways of BMSCs.
CONCLUSIONS: RADA-16 loaded with TGF-β1 has good biological properties and can enhance the chondrogenic differentiation ability of BMSCs.