Articular cartilage is a multi-zonal tissue that coats the epiphysis of long bones and avoids its wear during motion. An unusual friction could micro-fracture this connective membrane and progress into an osteochondral defect (OD), where the affected cartilage suffers inflammation, fibrillation, and forfeiture of its anisotropic structure. Clinical treatment for ODs has been focused on micro-fracture techniques, where the defect area is removed and small incisions are performed in the subchondral bone, which allows the exudation of mesenchymal stem cells (hMSCs) to the abraded zone. However, hMSCs represent less than 0.01% of the total cell population and are not able to self-organise coherently, so the treatments fail in the long term. To select, support and steer hMSCs from the bone marrow into a specific differentiation stage, and recreate the cartilage anisotropic microenvironment, multilayer dual-porosity 3D-printed scaffolds were developed. Dual-porosity scaffolds were printed using prepared inks, containing specific ratios of poly-(d,l)lactide-co-caprolactone copolymer and gelatine microspheres of different diameters, which acted as sacrificial micro-pore templates and were leached after printing. The cell adhesion capability was investigated showing an increased cell number in dual-porosity scaffolds as compared to non-porous ones. To mimic the stiffness of the three cartilage zones, several patterns were designed, printed, and checked by dynamic-mechanical analysis under compression at 37 ºC. Three patterns with specific formulations were chosen as candidates to recreate the mechanical properties of the cartilage layers. Differentiation studies in the selected scaffolds showed the formation of mature cartilage by gene expression, protein deposition and biomolecular analysis. Given the obtained results, designed scaffolds were able to guide hMSC behaviour. In conclusion, biocompatible, multilayer and dual-porosity scaffolds with cell entrapment capability were manufactured. These anisotropic scaffolds were able to recreate the physical microenvironment of the natural cartilage, which in turn stimulated cell differentiation and the formation of mature cartilage.
