An "EVs-in-ECM" mimicking system orchestrates transcription and translation of RUNX1 for in-situ cartilage regeneration.

The self-repair ability of articular cartilage is limited, which is one of the most difficult diseases to treat clinically. Kartogenin (KGN) induces chondrogenesis by regulating RUNX1 mRNA translation and the small molecule compound TD-198946 (TD) promotes chondrogenic differentiation of mesenchymal stem cells (MSCs) through increasing the transcription of RUNX1 mRNA. GelMA hydrogel and liposomes are respectively similar to the extracellular matrix (ECM) and extracellular vesicles (EVs). So, we developed an "EVs-in-ECM" mimicking system by incorporating GelMA and KGN/TD-loaded liposomes to investigate the repair effects of cartilage defect. First, western-blot, RNA fluorescence in situ hybridization (FISH), cellular immuno-fluorescence, co-immuno-precipitation (CO-IP), and qRT-PCR techniques showed that KGN regulated RUNX1 mRNA expression, and then promote chondrogenic differentiation of MSCs. Second, the role of RUNX1 was amplified by orchestrating RUNX1 transcription and translation through TD-198946 (TD) and KGN respectively, and the synergistic effects of TD and KGN on chondrogenesis of MSCs in vitro were discovered. Finally, an "EVs-in-ECM" mimicking system was designed for in situ cartilage repair. When GelMA loaded with KGN and TD liposomes, the hydrogel (KGN + TD@ GelMA) showed biological functions by the continuously controlled release of KGN and TD while maintaining its porous structure and mechanical strength, which enhanced the chondrogenesis of MSCs in one system. The repair performance of "EVs-in-ECM" in vivo was assessed using the articular osteochondral defect model of rat. The implantation of KGN + TD@ GelMA hydrogels effectively exerted favorable osteochondral repair effects showing structures similar to the native tissue, and prevented chondrocyte hypertrophy. The study indicate that the "EVs-in-ECM" mimicking system can act as a highly efficient and potent scaffold for osteochondral defect regeneration.