Research On Vascularized β-TCP/BFP-1/EPCs Construct In Repairing Rabbit Femur Segmental Bone Defect

Objective:1.To explore the effects of bone forming peptide-1(BFP-1)on the proliferation,migration and angiogenic functions of endothelial progenitor cells(EPCs)derived from rabbit peripheral blood,and to investigate the potential molecular mechanisms.2.To study the release kinetics of BFP-1 loaded on the beta-tricalcium phosphate(β-TCP)scaffold,and to probe the viability and proliferation of EPCs seeded on the β-TCP scaffolds loaded with BFP-1.3.To investigate the angiogenesis and osteogenesis of the fabricated tissue engineered bone(TEB)graft by seeding autogenous EPCs on the β-TCP scaffolds loaded with BFP-1(β-TCP/BFP-1/EPCs)in repairing rabbit femur segmental bone defect.Methods:1.Rabbit peripheral blood EPCs were isolated and expanded through the method of density gradient centrifugation combining with adherent culture.The cells were identified with surface markers and functional evaluation.EPCs of passage three were used in the in vitro experiments.Cells were exposed with different concentrations of BFP-1(100,200,300 μg/ml)for 1 to 7 days,and CCK8 test was used to evaluate the proliferation of the cultured cells.The effect of BFP-1 on the migration of EPCs was evaluated using Transwell assay.Matrigel tube formation assay was conducted to assess the role of BFP-1 on the angiogenic function of EPCs.2.The m RNA expressions of angiogenic related genes VEGF,VEGFR2,e NOS,and ALK-1 were detected by quantitative real-time polymerase chain reaction(q PCR)in EPCs treated with BFP-1 for 1 and 3 days.The protein expression of p-Smad 1/5 was evaluated through western blot assay.3.BFP-1 was loaded into the β-TCP through the frozen dry method.The release behavior of BFP-1 released from β-TCP scaffold was detected with an ELISA kit.EPCs were seeded onto the β-TCP scaffolds adsorbed with BFP-1 and cultured for 1 to 10 days,viability of cells was examined under a confocal microscope.4.Vascularized TEB was constructed by loading autogenous EPCs onto the β-TCP scaffolds adsorbed with BFP-1(β-TCP/BFP-1/EPCs),and TEBs of β-TCP/EPCs,β-TCP/BFP-1 and β-TCP were also fabricated and implanted to rabbit femur segmental bone defects.5.Micro-CT based angiography through microfil perfusion and immunohistochemical studies were performed at 4 and 12 weeks post implantation to assess the angiogenesis in the engineered bone grafts.6.Radiography,Micro-CT based bone analysis,and histology studies were used to evaluate bone formation within the engineered grafts.Three-point bending test was carried out 12 weeks after surgery to assess the mechanics of repaired bone.Results:1.Typical cobblestone morphology was observed in the isolated EPCs,and the cells showed positive expression of endothelial markers VEGFR2 and v WF.Di I–Ac-LDL uptake and FITC-UEA-1 lectin binding were also verified.2.BFP-1 of different concentrations had no adverse effect on the proliferation of EPCs.The migration and angiogenic function of EPCs were enhanced after BFP-1 exposure.Elevated expressions of genes VEGF,VEGFR2,e NOS,and ALK-1 were noticed,and increased protein level of phosphorylated Smad 1/5 was also observed.BFP-1 with aconcentration of 200μg/ml exhibited the most significant enhancement in the mentioned effects.3.BFP-1 could be adsorbed to β-TCP scaffold,the loaded BFP-1 showed a relatively sustained release for 28 days after an initial burst release.The EPCs proliferated and retained high viability on the β-TCP/BFP-1 constructs.4.Micro-CT angiography showed that the vascular volume fraction and vascular area value in the β-TCP/BFP-1/EPCs group were significantly greater compared to the β-TCP/EPCs,β-TCP/BFP-1 and β-TCP groups after implantation for 4 weeks and 12 weeks.CD34 staining analysis showed that the newly formed blood vessel density was higher in the β-TCP/BFP-1/EPCs group than that in the β-TCP/EPCs,β-TCP/BFP-1 and β-TCP groups 4 weeks and 12 weeks after surgery.5.The X-ray score and Micro-CT analysis displayed that the formed new bone in the β-TCP/BFP-1/EPCs group were markedly greater compared to the β-TCP/EPCs,β-TCP/BFP-1 and β-TCP groups after implantation for 12 weeks.H&E staining showed that the β-TCP/BFP-1/EPCs group revealed much more continuant lamellar bone formation between the implanted constructs and the host cortical bone.Van-Geison staining demonstrated more bone regeneration in the β-TCP/BFP-1/EPCs group compared to the β-TCP/EPCs,β-TCP/BFP-1 and β-TCP groups after implantation for 4 and 12 weeks.In addition,the β-TCP/BFP-1/EPCs group showed much faster scaffold matrix degradation than that in the β-TCP/EPCs,β-TCP/BFP-1 and β-TCP groups.Biomechanical evaluation results showed that the β-TCP/BFP-1/EPCs group had the highest values of ultimate force and ultimate stress compared to the β-TCP/EPCs,β-TCP/BFP-1 and β-TCP groups 12 weeks post implantation.Conclusions:1.EPCs of high purity with sufficient amounts could be obtained through density gradient centrifugation combining with adherent culture.2.The ALK-1/Smad pathway mediated the enhanced angiogenic activity of EPCs after BFP-1 exposure.3.BFP-1 could be sustainedly released from β-TCP scaffold,the β-TCP/BFP-1 construct has good cell compatibility and supported the proliferation of EPCs.4.The fabricated tissue engineered bone graft β-TCP/BFP-1/EPCs possessed satisfactory biocompatibility,and efficacious in vivo angiogenic and osteogenic capability.The engineered graft is efficiently reliable in repairing large bone defects within the load bearing region.The vascularized tissue engineered bone β-TCP/BFP-1/EPCs construct hold a great potential in clinical application.