Hierarchically porous nagelschmidtite bioceramic-silk scaffolds for bone tissue engineering

Bioactive three-dimensional (3D) scaffolds play a key role in the repair or regeneration of large bone defects. There are many methods to prepare 3D scaffolds, among which the 3D-plotting technique is a promising strategy as the scaffolds prepared by this method possess not only improved mechanical properties and interconnectivity, but also ordered large-pore structure. However, the low cell attachment rate in the interior of the 3D-plotted scaffolds, especially for 3D-plotted bioceramic scaffolds, inhibits the osteogenesis of stem cells in the scaffolds both in vitro and in vivo. The aim of this study is to prepare hierarchically porous composite scaffolds in order to improve the cell attachment, and further stimulate the in vitro and in vivo osteogenesis. We successfully fabricated hierarchically porous bioceramic-silk (BC-silk) composite scaffolds by a combination of the 3D-plotting technique with the freeze-drying method, and further investigated the attachment, proliferation and osteogenic differentiation of bone marrow stromal cells (BMSCs) in the scaffolds as well as the in vivo osteogenesis of the prepared porous scaffolds. The results showed that the hierarchical structure in the composite scaffolds was composed of first-level pores (∼1 mm) of the bioceramic scaffold and second-level pores (∼50-100 μm) of the silk matrix. The prepared BC-silk composite scaffolds possessed excellent apatite-mineralization ability and mechanical properties with compressive strength up to 25 MPa. In addition, hierarchically porous BC-silk scaffolds presented significantly enhanced attachment rate of BMSCs, around 4 times that of pure BC scaffolds without hierarchical pore structures. BC-silk scaffolds with hierarchical pore structures showed distinctively improved cell proliferation, ALP activity and bone-related gene expression as compared to BC scaffolds without hierarchical pore structure. Furthermore, hierarchically porous BC-silk scaffolds significantly enhanced the formation of new bone in vivo as compared to BC scaffolds. Our results suggest that the combination of 3D-plotting with the freeze-drying method is a viable strategy to construct hierarchical pore structures in 3D-plotted scaffolds, and the hierarchical pore structure plays an important role in improving the in vitro and in vivo osteogenesis of 3D-plotted bioceramic scaffolds for bone regeneration application.