Cartilage repair remains a major challenge in clinical trials. These current cartilage repair materials can not effectively promote chondrocyte generation, limiting their practical application in cartilage repair. In this work, we develop an implantable scaffold of RADA-16 peptide hydrogel incorporated with TGF-β1 to provide a microenvironment for stem cell-directed differentiation and chondrocyte adhesion growth. The longest release of growth factor TGF-β1 release can reach up to 600 h under physiological conditions. TGF-β1/RADA-16 hydrogel was demonstrated to be a lamellar porous structure. Based on the cell culture with hBMSCs, TGF-β1/RADA-16 hydrogel showed excellent ability to promote cell proliferation, directed differentiation into chondrocytes, and functional protein secretion. Within 14 days, 80% of hBMSCs were observed to be directed to differentiate into vigorous chondrocytes in the co-culture of TGF-β1/RADA-16 hydrogels with hBMSCs. Specifically, these newly generated chondrocytes can secrete and accumulate large amounts of collagen II within 28 days, which can effectively promote the formation of cartilage tissue. Finally, the exploration of RADA-16 hydrogel-based scaffolds incorporated with TGF-β1 bioactive species would further greatly promote the practical clinical trials of cartilage remediation, which might have excellent potential to promote cartilage regeneration in areas of cartilage damage.

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.