Sequential Delivery Of Low Doses Of BMP-2 And VEGF In A Silk Fibroin-nanohydroxyapatite Scaffold For Vascularized Bone Regeneration

Objective: Prepare silk fibroin(SF)/nanohydroxyapatite(nHAp)scaffold in which low doses of BMP-2 and VEGF were released in sequential and sustained manner,and observe its bone formation ability.Methods: SF microspheres were prepared using a co-flow device,and BMP-2 and VEGF were absorbed onto the microspheres in chemical and physical way.Then the SF microspheres were incorporated into the SF/nHAp scaffold to provide sustained release.The morphology and constitute were observed by SEM and FTIR.The release manner of BMP-2 and VEGF were determined by ELISA kits.The influence of cell adhesion and proliferation were observed by SEM and CCK-8.The osteoinductive capacity of composite scaffold was observed by alizarin red staining and alkaline phosphates(ALP).Composite scaffolds were implanted into the rat calvarial defects to test bone formation ability by histological and Immunohistochemistry analysis.Results: At 1,10 and 28 days,6.7 ± 0.1%,13.7 ± 0.4% and 15.5 ± 0.6% of total BMP-2 were released from the scaffold,while 48.7 ± 1.8%,66.4 ± 2.3% and 67.9 ± 2.3% of total VEGF were released from the scaffold.The SEM and CCK-8 proved that composite scaffold had good biocompatibility.The calcium content and ALP activity were improved by the composite scaffold.In vivo study,scaffold with BMP-2 and VEGF showed great bone value(BV/TV),and the newly formed bone could bridge the injury site.Conclusion: BMP-2 and VEGF in composite scaffold were released in sequential way to mimic the process of bone healing.Scaffold with BMP-2 and VEGF had good biocompatibility and could promote bone formation.