Construction Of Bone Microstructure-mimetic Nano-fibrous Scaffolds With Dualdrug Incorporation For Promoting Bone Repair

Bone is a highly vascularized tissue.In the process of bone repair,vascularization and osteogenesis cooperate to promote bone remodeling.The repair of bone defects is a lingering issue due to the poor anastomosis between implants and the host tissues and the delay of early neovascularization.In terms of physical structure,vascular networks are very important in accelerating bone reconstruction.These vascular networks provide appropriate oxygen and nutrients diffusion to the cells affecting cell proliferation and neo-bone formation as well as allow an effective removal of metabolic waste.However,how to quickly and effectively generate well-structured vascular networks in tissue engineered bone scaffolds is a persistent challenge.Besides,natural bone repair is strongly regulated by multiple factors.Endogenous vasculogenic and osteogenic factors are central to the entire spectrum of bone regeneration.Therefore,the fabricated scaffolds exhibiting vascular-like networks and capable of simultaneously releasing vasculogenic and osteogenic factors hold great promise for bone tissue engineering.Herein,inspired by the structural and functional cues of bone remodeling,we developed a microchannels networks-enriched nanofibrous hybrid scaffold(DBM/GP)by the combined utilization of 3D printing and thermally induced phase separation(TIPS)techniques,which can facilitate cells migration and nutrients transportation throughout the scaffold as well as render spatiotemporal release of angiogenic/osteogenic factors.The customizable microchannel structure of polycaprolactone(PCL)within the gelatin–silica nanofibrous scaffold was fabricated based on3 D printed sacrificial caramel templates,while simultaneous delivery of dimethyloxalylglycine(DMOG)and bone forming peptide-1(BFP)was implemented by using mesoporous silica nanoparticles(MSNs).Sequential release of DMOG and BFP,that DMOG released quickly and followed by sustained release of BFP,was monitored by designing the deposition of DMOG-loaded MSNs on the scaffold surface and internal incorporation of BFP-loaded MSNs within the nanofibrous scaffold.The physical and chemical properties of the scaffolds were tested,and the potential of DBM/GP scaffold to promote vascularization and osteogenesis was evaluated both in vitro and in vivo.The main contents are listed as follow:(1)The effect of silica sol content on the pore structure and mechanical properties of gelatinsilica scaffolds were clarified by adjusting the dosage ratio of silica sol.Scanning electron microscopy(SEM)results indicated that an increase in silica sol content loosened the nanofiber network structure.The mechanical testing showed that the compressive modulus of scaffolds increased with an increase in silicon content(10%-50%),and the compressive modulus of nanofiber scaffolds were the highest when the content of silica sol was 30%.Based on sucrose sacrificial template strategy,PCL scaffolds with different specifications(filaments diameter and spacing)could be quickly customized.SEM observation indicated that PCL scaffold possessed perfusable,interconnected microchannel networks and controllable microporous permeable walls.The perfusion experiments further demonstrated the efficiency of this interconnected hollow PCL scaffold to support a long-range functional mass transport.Besides,the micro morphology and in vitro degradation showed that GP scaffold accelerated the flow of materials in the scaffold through microchannel network and improved the degradation performance.The study of drug release and Si ion release in vitro demonstrated that the rapid release of DMOG and the long-term sustained release mode of BFP and Si,which were more conducive to angiogenesis and bone regeneration.(2)In vitro proliferation experiments displayed that the prepared scaffolds had good biocompatibility and were conducive to cell adhesion and proliferation.The results of scratch test,Transwell cell migration and Matrigel angiogenesis test clarified that DBM/GP scaffolds showed chemotactic of human umbilical vein endothelial cells(HUVECs)and angiogenesis.In addition,DBM/GP scaffolds enhanced the osteogenesis of bone marrow mesenchymal cells(BMSCs),upregulated the expression level of osteogenesis-related genes,such as OPN,OCN and COl1 as well as activated the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway mediated angiogenesis.(3)The in vivo angiogenesis of DBM/GP scaffold was evaluated by subcutaneous implantation of scaffolds.Vascular perfusion experiment showed that DBM/GP scaffolds significantly promoted the formation of blood vessels.The bone regeneration ability of DBM/GP scaffold in vivo was evaluated by implantation in a cranial defect model in Sprague-Dawley(SD)rats.Micro-CT analysis suggested that DBM/GP scaffold significantly enhanced the area of new bone formation and promoted bone defect healing.Histological and immunofluorescence staining result further revealed that DBM/GP scaffold had more new bone formation,good vascularization and better bone repair effect.In conclusion,based on the sugar template sacrifice strategy,we designed a DBM/GP scaffold by using 3D printing and TIPS.Physical characterization,in vivo and in vitro experimental results demonstrated that as-prepared DBM/GP scaffold had interconnected microchannel and nanofibrous structure as well as excellent osteogenic and angiogenic activities,which could be a promising scaffold for bone tissue engineering applications.