Stiffness Gradient 3D-Printed Polymeric Scaffolds for Directed Cell Migration and Osteochondral Differentiation

Abstract Multilayered 3D-printed hydrogels with stiffness gradients were developed as platforms for osteochondral tissue engineering, recapitulating native ECM heterogeneity through varying stiffness, porosity, and biochemical cues to regulate cellular migration, behavior, and differentiation. In this study, we created a stiffness gradient scaffold by dual-cross-linked hydrogel scaffolds that were fabricated via initial photo-cross-linking followed by ionic cross-linking with Ca2+, creating stiffness gradients by treating half the scaffold area by chelating Ca2+ from the opposing, using EDTA; Ca2+-cross-linked regions exhibited significantly higher stiffness than EDTA-treated regions. The engineered gradient scaffolds demonstrated cytocompatibility, low protein adsorption, and hemocompatibility, making them suitable for tissue engineering applications. MG-63 cells exhibited stiffness gradient-directed migration from soft to stiff regions via mechanotransduction gene expression. Immunomodulation studies revealed enhanced M0-to-M2 macrophage polarization ratios, promoting osteoimmunomodulation that showed hBMSCs into osteogenic differentiation. Furthermore, hBMSCs seeded on gradient scaffolds underwent spatially controlled differentiation into osteogenic (stiff region) and chondrogenic (soft region) lineages, confirmed by OCN/COL2A marker expression, flow cytometry, alizarin red S mineralization, and Alcian blue staining. Collectively, these findings highlight the gradient hydrogel’s ability to mimic the osteochondral ECM microenvironment, facilitating directed cell migration, lineage-specific differentiation, and functional tissue maturation.