Preparation Of A Novel Vascularized Tissue Engineered Scaffold And Evaluation Of Its Biological Properties In Vitro And In Vivo

Objective:With the rapid development of tissue engineering technology,the appearance of tissue engineering bone brings new hope for the repair of large segment of bone defect.However,vascularization is still a key factor restricting the application of tissue engineering bone in clinic.As one of the three elements of bone tissue engineering,scaffold plays an important role in the rapid vascularization and new bone formation for tissue engineered bone.therefore,this study aimed to fabricate a novel vascularized scaffold and evaluate its biological performance in vitro and in vivo.Methods:?1?Preparation and characterization of a novel vascularization scaffold:?1?The calcium-deficient apatite powder was prepared by chemical precipitation method using soluble calcium salt,copper salt and zinc salt,phosphorus salt as original source.Then,the powder was made into paste and further molded into columnsusing calcium alginate as porogen.After calcination,biphasic calcium phosphate scaffolds co-doped with Cu/Zn ions were successfully prepared.When the molar ratio of Cu/Zn was fixed at 1:1,the molar ratio of Cu/?Cu+Zn+Ca?varied from 0 to 0.05,and the corresponding products were named as P0,P1,P2 and P3,respectively.The morphology,chemical composition and phase constitution of the scaffolds were examined by SEM?XRD?FTIR and XPS.The compressive strength of the scaffolds was determined by universal mechanical testing machine.The release profiles of the Ca²⁺,Cu²⁺and Zn²⁺were detected by ICP.?2?PLGA microspheres loaded with GDF-5 were synthesized using emulsion method.Then,the micorspheres were loaded into scaffold P2 by vacuum-drying method and the obtained scaffold was named as P2/GDF-5.The morphology and distribution of microspheres on the pore wall of scaffolds were observed by SEM.The release profile of GDF-5 from scaffolds were determined by BCA assay.?2?In vitro biological performance evaluation:?1?Bone marrow mesenchymal stem cells?BMSCs?and endothelial cells were mixed in a ratio of 1:1 and co-cultured with the scaffolds.The morphology of cells on the scaffold were observed using a SEM and DAPIstaining..The cell proliferation was measured by CCK-8 method and the ALP activity was measured by ALP kit.Also,the expression of bone-related genes were quantitativley measured by RT-PCR.The secretion amount of VEGF was detected by ELISA according to the manufacturer's instruction.?2?The ions extration of scaffold P2 was obtained by immersing the scaffold into DMEM,and co-cultured with BMSCs and endothelial cells.DMEM added with Cu²⁺or Zn²⁺were used as the control group.The cell proliferation and ALP activity were determined as above.The mineral formtion was observed by Alizarin red staining.The angiogenesis capacity of endothelial cells was observed on Matrigel-coated wells.?3?In vivo biological performance evaluation:Firstly,15mm of rabbit radius defect was constructed as a model.The experiment was divided into five groups:Group K:no scaffold was implanted;D:scaffold P0;S1:scaffold P1;S2:scaffold P2;S3 scaffold P2/GDF-5.The X-ray imaging examination was performed at 4,8 and 12 weeks after surgery.At 12 week after the operation,the specimens were fixed,embeded,sliced,and observed by HE and Masson staining.Results:?1?Characterization of the scaffolds:?1?The SEM showed that all scaffolds had diameters ranging from 600⁸00?m,and the pores were interconnected.With the increase of adding ions concentration,the surface of the scaffold varied from smooth grains tomicroparticles,and further to nanoflakesn.XRD,FTIR and XPS analysis showed that the scaffolds were composed of HA and?-TCPforP0?P1 and P2.while P3 was pure?-TCP.The compressive strength of the scaffolds decreased from 1.51MPa to0.91MPa with the ion concentration increase,.The ion release profiles showed that with adding ions concentration increase,the release rates of Cu²⁺,Zn²⁺and Ca²⁺became faster.?2?SEM images showed that the PLGA microsphere loaded with GDF-5-was spherical and smooth-,and the particle size ranged from 8 to 20?m.Moreover,the microspheres were distributed well on the pore wall of the scaffold.In vitro release of GDF-5 showed that GDF-5 had a burst release at the early time,and followed by a steady release for 30 d.?2?In vitro biological performance evaluation:?1?There was no obvious cytotoxicity for all scaffolds,and the cells adhered well onto the pore wall.The number of cells on the scaffold P2/GDF-5 was significantly higher than that of P0,P1,and P2.CCK-8 assay showed that the number of cells gradually increased with the culture time for all scaffolds,especially cells on scaffold P2/GDF-5 was significantly higher than the other three groups?P<0.01?.The ALP activity results showed that on the day 14,the expression of ALP in P2/GDF-5 group was the highest,followed by P2 group.PCR analysis showed that the expression levels of ALP,OCN and OPN in P2/GDF-5 group were significantly higher than those in other groups?P<0.05?;the expression levels of ALP,OCN and OSX in P2 group were higher than those in P0 group?P<0.001?.The VEGF secretion in the P2/GDF-5 group was significantly higher than the other three groups,while that in P2 group was significantly higher than P1 and P0.?2?The cell proliferation in the ions extraction of scaffold P2showed thatcell proliferation was the fastest in theextraction group,Zn²⁺-adding group was more favorable for cell proliferation than Cu²⁺-adding group.The ALP activity for Zn²⁺-adding group-was significantly higher than that of Cu²⁺-adding group and the blank group?P<0.01?.On the day 14,the extraction group was higher than Zn²⁺-adding group.Cells cultured on Matrigel-coated wells showed that tubular structure formed inCu²⁺-adding group and.no tubular structure was formed in Zn²⁺-adding group and blank group.After 21 days of culture,alizarin red staining indicated thatthe number and area of calcium nodules in Zn²⁺-adding group and extraction group were significantly higher than that in blank group and Cu²⁺-adding group.?3?In vivo biological performance evaluation:After the operation,no animals died and all aminals were in good health.At 4weeks after operation,X-ray imaging showed that the gaps between all the defects were clearly visible.A small amount of low-density callus was observed on in S3 group.All the scaffold were clearly visible without shedding or displacement.At 8 weeks after surgery,the scaffolds in D,S1,S2 and S3 group were partially degraded.The degradation amount of S1,S2 and S3 was greater than that of group D.In group K and group D,some new hard bone formed and closed the medullary cavity,and there was no obvious bone connection between the broken ends.S1,S2 and S3 groups all show the broken end part of the osseous connection,and the bony connection area:S3>S2>S1.At 12 weeks after the operation,the broken end of the K group was still closed,and a small amount of bony connections were seen in the K group near the ulnar side.The bone defects of S1,S2 and S3 were almost healed,and the cortical connection was continuous,and the partial pulp cavity was connected.The repair rate of S3 group was significantly higher than that of the other groups.The Lane-Sandhu X score showed thatthe score of S3 group was the highest at each time point,and The differences among the groups were obviously significant?P<0.05?;Histological examination showed thatno inflammation,toxicity and immune rejection were observed in all the implantation sites.At 12 weeks after operation,HE staining showed that a large amount of new bone formed in S1,S2 and S3 groups.Moreover,for S3 group,the pulp cavity showed good plasticity,and the number of new blood vessels was significantly higher than that of S1 and S2.In group D,some osteoid tissues were found,and the new bone grew along the pore wall of the scaffold,while a large amount of fibrous tissue formed in the defect site of K group.At 12 weeks after the operation,Masson staining indicated thatthe number of blood vessels and new bone in the S3 group was significantly higher than those in the other groups.Conclusion:The Cu/Zn co-doped biphasic calcium phosphate scaffolds-derived GDF-5 release system were successfully prepared,using calcium alginate as porogen andPLGA microspheres as carrier for GDF-5,.The scaffold possessed good cytocompatibility,osteoinductivity and vascularization,and could rapidly repair large bone defects.The scaffold showed a promising potential in clinical application for bone tissue engineering.