3D printed hydrogel scaffold incorporating keratin and melatonin-loaded nanomicelles for cartilage tissue engineering

Articular cartilage possesses limited self-repair capacity due to its avascular nature, necessitating advanced tissue engineering strategies for effective regeneration. This study presents the fabrication and evaluation of a novel 3D-printed hydrogel scaffold composed of chitosan (Cs), carrageenan (Crg), and keratin (Kr), integrated with melatonin (Mel)-loaded nanomicelles to stimulate cartilage repair. The nanomicelles exhibited a mean diameter of 133.36 ± 13.69 nm, and their incorporation into the Cs-Crg/Kr-Mel scaffold enabled a sustained Mel release profile, reaching 88.0 ± 5.5% cumulative release over 264 h. The Cs-Crg/Kr-Mel scaffold demonstrated an equilibrium swelling ratio of 391.6 ± 26.3% after 72 h in distilled water and a degradation rate of 47.0 ± 5.5% after 28 days in PBS. In vitro assays using ATDC5 chondrogenic cells revealed superior cell viability and adhesion on the Mel-containing scaffold. Alcian blue and Safranin O staining confirmed increased proteoglycan synthesis of rBMSCs on the Cs-Crg/Kr-Mel scaffold and gene expression analysis via qRT-PCR indicated significantly elevated levels of COL II, ACAN, and SOX9, confirming enhanced chondrogenic differentiation. The combined presence of Kr and Mel contributed to the scaffold's regenerative efficacy by improving cell adhesion and modulating cellular processes involved in chondrogenic differentiation. These findings suggest that the Cs-Crg/Kr-Mel scaffold holds strong potential for application in cartilage repair and regeneration.