Injectable elastin-like recombinamer hydrogels with layered degradation rates for osteochondral regeneration in a rabbit model

• Injectable bioactive elastin-like recombinamer (ELR) hydrogels were designed with distinct degradation kinetics tailored for osteochondral regeneration. • Bilayer hydrogels , containing distinct degradation rates (fast and slow) and cell compositions, effectively promoted long-term regeneration of critical-sized osteochondral defects in rabbits. • Superior cartilage regeneration was achieved using homogeneous hydrogels (uniform degradation rates), outperforming heterogeneous designs. • Mesenchymal stem cells (MSCs) significantly enhanced cartilage regeneration compared to chondrocyte-only or cell-free hydrogels. • The ELR hydrogels demonstrated inherent regenerative potential, promoting cartilage-like tissue formation even in the absence of cells . The regeneration of osteochondral defects remains a significant challenge due to the distinct structural and functional requirements of cartilage and subchondral bone. Current strategies often employ multiphasic scaffolds to mimic the complex osteochondral unit. In this study, we developed an injectable hydrogel system with inner and outer layers exhibiting distinct degradation rates and cell combinations, designed to regenerate critical-sized osteochondral defects in a long-term rabbit model. The hydrogel system is based on elastin-like recombinamers (ELRs), which promote cell adhesion, proliferation, and mechanical integrity. Bioactive sequences, including RGD for cell adhesion and proteolytic motifs with different degradation kinetics, slow-cleaving (DRIR) and fast-cleaving (GTAR), were incorporated into the design. These hydrogels are delivered in liquid form, solidifying in situ to conform to osteochondral defects. To explore the role of cellular interactions, mesenchymal stem cells (MSCs) and chondrocytes were incorporated into the hydrogels. After six months in vivo, outcomes were evaluated via glycosaminoglycan quantification, histological, and immunohistochemistry analyses, revealing cartilage-like tissue production. Statistical analysis using a modified O’Driscoll score demonstrated significant differences (p < 0.05) between homogeneous hydrogels (with either fast or slow degradation rates) and both the untreated control and the chondrocyte-only condition. No significant differences were found among other experimental groups based on the O’Driscoll score; however, histological analysis confirmed promising cartilage-like tissue formation, even in the cell-free hydrogel condition. These findings highlight the potential of in situ crosslinkable ELR hydrogels as a versatile and effective strategy for osteochondral repair, demonstrating substantial cartilage-like tissue formation, even without cellular components. Moreover, the incorporation of MSCs—likely through paracrine signaling—significantly enhanced overall regenerative outcomes, offering a promising stem cell–based approach for treating osteochondral lesions.