Knee osteoarthritis (OA) affects about 300 million people worldwide. In its early phases, OA involves the joint cartilage only; however, as it progresses the subchondral bone is more obviously involved, creating an osteochondral (OC) disease pattern. In recent years, 3D tissue engineered substitutes have emerged as promising alternative to traditional strategies (i.e autografts transplantation, subchondral drilling, etc..). Although successful in many aspects, these 3D scaffolds lack in mimicking the complex architecture of the natural stratified OC tissue. To address this hurdle, in this study we investigated the development and optimization of a multi-layered scaffold with incorporated vertical porosity and ceramic content gradients, by using a 3D Bioplotter system.