Osteochondral defects present significant challenges for effective tissue regeneration due
to the complex composition of bone and cartilage. To address this challenge, this study presents
the fabrication of hierarchical scaffolds combining chitosan/β-tricalcium phosphate (β-TCP) to
simulate a bone-like layer, interconnected with a silk fibroin layer to mimic cartilage, thus replicating
the cartilage-like layer to mimic the native osteochondral tissue architecture. The scaffolds were
produced by freeze-drying and then crosslinking with genipin. They have a crosslinking degree of up
to 24%, which promotes a structural rearrangement and improved connection between the different
layers. Micro-CT analysis demonstrated that the structures have distinct porosity values on their
top layer (up to 84%), interface (up to 65%), and bottom layer (up to 77%) and are dependent on
the concentration of β-tricalcium phosphate used. Both layers were confirmed to be clearly defined
by the distribution of the components throughout the constructs, showing adequate mechanical
properties for biomedical use. The scaffolds exhibited lower weight loss (up to 7%, 15 days) after
enzymatic degradation due to the combined effects of genipin crosslinking and β-TCP incorporation.
In vitro studies showed that the constructs supported ATDC5 chondrocyte-like cells and MC3T3
osteoblast-like cells in duo culture conditions, providing a suitable environment for cell adhesion and
proliferation for up to 14 days. Overall, the physicochemical properties and biological results of the
developed chitosan/β-tricalcium phosphate/silk fibroin bilayered scaffolds suggest that they may be
potential candidates for osteochondral tissue strategies.
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