Fabrication of a novel bilayered osteochondral scaffold using a bottom up approach

Osteoarthritis and osteochondral injuries, caused by damage to both the articular cartilage and the underlying subchondral bone are suffered by over 40 million people in Europe alone. Consequently, osteochondral injuries and its complications are a serious burden to the health service and society as a whole. Tissue engineered grafts should create a more sustainable and long-term substitutes to clinical interventions because they combine the patientâs own cells with porous biomaterial scaffolds that regenerate cartilage and bone and will restore the function of the joint. The aim of this research was to capture the natural biological properties and mechanical strength of the joint, by developing bi-layered osteochondral plugs composed of superficial cartilaginous layers (coresponding to articular cartilage) and underlying calcified tissue (corresponding to subchondral bone). A novel biomimetic bottom-up approach inspired by the structure of the natural joint, with a different composition, pore size and substrate stiffness in each region of the construct was employed. The materials used for the superficial layer scaffold (chondrogenic region) were collagen type II and polymerised chondroitin sulphate (PCS); and for the underlying scaffold (osteogenic region) were collagen type I and hydroxyapatite. Monomeric type I and type II collagens were used as the starting materials since they mitigate the issue of collagen antigenicity on donor and recipient species. Incorporation of chondroitin sulphate (CS) into collagen scaffolds has been limited in previous research due to a limited number of binding sites available on collagen for the CS attachment. In this study, to increase the incorporation and thus take full advantage of CS presence in scaffolds, CS was successfully polymerised to an 11 time higher molecular weight PCS. Subsequently, scaffolds were developed from crosslinked reconstituted fibrilar collagen II combined with CS or PCS. Rheological properties of collagen dispersions, scaffoldsâ chemical and porous structure, mechanical properties and the scaffolds chondrogenesis potential were examined. PCS significantly increased the complex viscosity, storage and loss modulus of collagen suspensions, while CS reduced them. The greater viscosity of collagen-PCS dispersions, in relation to collagen-CS gives rise to the formation of smaller pores in the scaffolds. PCS significantly increased the Youngâs modulus of scaffold (1.7 time), mainly due to the increase in relative density of scaffold. The higher specific surface area and compressive modulus, as well as a smaller pore size in collagen-PCS scaffolds gives rise to a 1.2 time increase in both (MSCs) cell attachment and proliferation. Most importantly, gene expression results showed 3 times increase in aggrecan and 14 times increase in collagen II expression in collagen-PCS scaffolds. Furthermore, histological analysis showed that cells produced considerably more GAGs and cartilage ECM on collagen-PCS compared to collagen-CS. Osteogenic scaffolds were fabricated using two methods: crosslinked collagen I combined with commercially available HA, and In situ precipitation of HA nano particles on collagen fibrils following by the subsequent crosslinking of suspensions. Rheology of suspensions, scaffolds microstructure and mechanical properties were evaluated. The first method resulted in smaller pores size, significantly higher pore interconnectivity, higher compressive modulus and increased cell differentiation (1.7 times increase in Youngâs modulus and 1.5 times increase in collagen I expression). Lastly, bilayered scaffolds were fabricated based on the optimum outcomes obtained for the composition, micro-architecture and mechanical properties of cartilage and bone scaffolds. It was established that sheep bone marrow mesenchymal stem cells give rise to distinct behavior of chondrogenesis and osteogenesis in the different regions of the bi-layer.