基于 RADA-16 的自组装肽纳米纤维支架负载 TGF-β1 增强 BMSCs 的软骨分化潜能。

RADA-16-based Self-assembled Peptide Nanofiber Scaffolds Loaded with TGF-β1 Enhance the Chondrogenic Differentiation Potential of BMSCs .

机构信息

Department of Joint Surgery, Center for Orthopaedic Surgery, The Third Affiliated Hospital of Southern Medical University, The Third School of Clinical Medicine, Southern Medical University, Guangzhou, 510515, China.

Department of Joint Surgery, The First Affiliated Hospital of Hainan Medical University. Haikou, 570102, China.

出版信息

Curr Stem Cell Res Ther. 2024;19(2):257-266. doi: 10.2174/1574888X18666230316112847.

DOI:10.2174/1574888X18666230316112847

PMID:36927429

Abstract

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.

CONCLUSION

RADA-16 loaded with TGF-β1 has good biological properties and can enhance the chondrogenic differentiation ability of BMSCs.

摘要

目的

目前，软骨修复并不能提供理想的疗效。幸运的是，最近的研究表明，RADA-16 肽是修复软骨缺陷的一种有吸引力的治疗策略。因此，本研究试图探索负载转化生长因子-β（TGF-β）1 的 RADA-16 肽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，通过超分子肽自组装形成明确的水凝胶。然后，将 TGF-β1（负载浓度为 10ng/ml）载入 RADA-16 中，通过扫描电子显微镜观察 TGF-β1/RADA-16 水凝胶的形态，并检测其相关性能。接下来，从骨髓样本中分离出 BMSCs 并进行鉴定。分别将 TGF-β1/RADA-16 与 L929、BMSCs 和 C28/I2 细胞共培养，通过活/死细胞染色和 MTT 测定法来测定细胞的存活和增殖能力。通过 Alcian 蓝染色和 Blyscan 测定法测定 BMSCs 的软骨分化和 sGAG 产生，qRT-PCR 测定软骨相关基因的表达，ELISA 测定炎症因子的水平。至于机制研究，通过 Western blot 检测 Smad 和 ERK/MAPK 信号通路。

结果

RADA-16 水凝胶呈现出分布均匀、相互连通的多孔表面结构，负载 TGF-β1 的载药量为 91.9%。TGF-β1/RADA-16 水凝胶具有良好的释放和降解性能，对 BMSCs、L929 和 C28/I2 细胞的存活和增殖能力没有负面影响。重要的是，TGF-β1/RADA-16 水凝胶显著加速了 BMSCs 的软骨分化和 sGAG 生成，并降低了促炎因子的产生。此外，该水凝胶还显著激活了 BMSCs 的 Smad 和 ERK/MAPK 通路。