Indirect printing of hierarchical patient-specific scaffolds for meniscus tissue engineering

The complex meniscus tissue plays a critical role in the knee. The high susceptibility to injury has led to an intense pursuit for better tissue engineering regenerative strategies, where scaffolds play a major role. In this study, indirect printed hierarchical multilayered scaffolds composed by a silk fibroin (SF) upper layer and an 80/20 (w/w) ratio of SF/ionic-doped beta-tricalcium phosphate (TCP) bottom layer were developed. Furthermore, a comparative analysis between two types of scaffolds produced using different SF concentrations, i.e., 8% (w/v) (Hi8) and 16% (w/v) (Hi16) was performed. In terms of architecture and morphology, the produced scaffolds presented homogeneous porosity in both layers and no differences were observed when comparing both scaffolds. A decrease in terms of mechanical performance of the scaffolds was observed when SF concentration decreased from 16 to 8% (w/v). Hi16 revealed a static compressive modulus of 0.66 +/- 0.05 MPa and dynamical mechanical properties ranging from 2.17 +/- 0.25 to 3.19 +/- 0.38 MPa. By its turn, Hi8 presented a compressive modulus of 0.27 +/- 0.08 MPa and dynamical mechanical properties ranging from 1.03 +/- 0.08 MPa to 1.56 +/- 0.13 MPa. In vitro bioactivity studies showed formation of apatite crystals onto the surface of Hi8 and Hi16 bottom layers. Human meniscus cells (hMCs) and human primary osteoblasts were cultured separately onto the top layer (SF8 and SF16) and bottom layer ( SF8/TCP and SF16/TCP) of the hierarchical scaffolds Hi8 and Hi16, respectively. Both cell types showed good adhesion and proliferation as denoted by the live/dead staining, Alamar Blue assay and DNA quantification analysis. Subcutaneous implantation in mice revealed weak inflammation and scaffold's integrity. The hierarchical indirect printed SF scaffolds can be promising candidate for meniscus TE scaffolding applications due their suitable mechanical properties, good biological performance and possibility of being applied in a patient-specific approach.