Abstract
Mimicking the structural and functional complexity of native articular cartilage remains a major challenge in osteochondral tissue engineering. In this study, we developed a multilayer hydrogel construct composed of methacrylated chondroitin sulfate (CSMA), gelatin methacryloyl (GelMA), polyethylene glycol dimethacrylate (PEGDMA), and nano-hydroxyapatite (nHA), engineered to replicate the biochemical and biomechanical gradients of the cartilage zones. Each hydrogel layer was systematically tailored to reflect the hydration, stiffness, porosity, and degradation profiles of the superficial, middle, and deep cartilage zones. Mechanical testing revealed a progressive increase in compressive modulus, while SEM imaging demonstrated tunable porosity across layers. PEGDMA incorporation enhanced structural integrity and reduced enzymatic degradation. In vitro chondrocyte encapsulation confirmed high cell viability and zone-specific gene expression, COL2A1 was elevated in GelMA-based middle layers, and COL10A1 was upregulated in nHA-enriched deep layers. Collagen type II immunofluorescence further supported sustained chondrogenic activity. Although whole-construct biological assessment was limited due to post-crosslinking homogenization, characterization of individual layers affirmed their zonal functionality. These findings establish a robust and reproducible platform for stratified hydrogel design and highlight its potential in advancing biomimetic strategies for osteochondral repair. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-025-33222-0.