Osteochondral (OC) matrix design poses a significant engineering challenge due to the complexity involved with bone-cartilage interfaces. To better facilitate the regeneration of OC tissue, we developed and evaluated a biodegradable matrix with uniquely arranged bone and cartilage supporting phases: a poly(lactic-co-glycolic) acid (PLGA) template structure with a porosity gradient along its longitudinal axis uniquely integrated with hyaluronic acid hydrogel. Micro-CT scanning and imaging confirmed the formation of an inverse gradient matrix. Hydroxyapatite was added to the PLGA template which was then plasma-treated to increase hydrophilicity and growth factor affinity. An osteogenic growth factor (bone morphogenetic protein 2; BMP-2) was loaded onto the template scaffold via adsorption, while a chondrogenic growth factor (transforming growth factor beta 1; TGF-β1) was incorporated into the hydrogel phase. Confocal microscopy of the growth factor loaded matrix confirmed the spatial distribution of the two growth factors, with chondrogenic factor confined to the cartilaginous portion and osteogenic factor present throughout the scaffold. We observed spatial differentiation of human mesenchymal stem cells (hMSCs) into cartilage and bone cells in the scaffolds in vitro: cartilaginous regions were marked by increased glycosaminoglycan production, and osteogenesis was seen throughout the graft by alizarin red staining. In a dose-dependent study of BMP-2, hMSC pellet cultures with TGF-β1 and BMP-2 showed synergistic effects on chondrogenesis. These results indicate that development of an inverse gradient matrix can spatially distribute two different growth factors to facilitate chondrogenesis and osteogenesis along different portions of a scaffold, which are key steps needed for formation of an osteochondral interface.© 2020 IOP Publishing Ltd.