Controlled dual delivery of low doses of BMP-2 and VEGF in a silk fibroin–nanohydroxyapatite scaffold for vascularized bone regeneration

The controlled co-release of osteoinductive and angiogenic factors is an efficient approach to promote vascularized bone regeneration, and a suitable controlled release system can largely reduce the usage of these factors to avoid cost and safety problems. In this study, a cell-free vascularized bone tissue engineering system based on a silk fibroin (SF)/nanohydroxyapatite (nHAp) scaffold was developed, in which very low doses of osteoinductive and angiogenic factors, bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF), were embedded and released in a controlled manner to facilitate bone formation and vascularization, respectively. BMP-2 and VEGF were adsorbed onto SF microspheres (diameter of 1.5 ± 0.3 μm) that were prepared using a co-flow capillary device, and these microspheres were subsequently incorporated within the SF/nHAp scaffolds to provide controlled release. BMP-2 and VEGF were incorporated into SF microspheres via chemical covalent bonding and physical adsorption, respectively, leading to their controlled and sustained release from the SF/nHAp scaffolds. The rapid initial release of VEGF mimicked its expression at the early bone healing stage and promoted angiogenesis, and the relatively slow and sustained release of BMP-2 facilitated osteogenic differentiation both in vitro and in vivo, and the bone completely bridged the rat calvarial defects after 12 weeks of implantation. Overall, our findings suggest that the controlled dual release of very low doses of BMP-2 (300 ng per scaffold) and VEGF (20 ng per scaffold) from SF/nHAp scaffolds results in a synergistic effect on vascularized bone regeneration; this controlled release system can largely reduce the usage of BMP-2 as compared to other systems.