The Study Of RhBMP2/rhVEGF165 Loaded Hydrogel Combining With Anatomically Biomimetic Artificial Bone For Rabbit Tibia Segemental Defect Repair

Part one:The study of anatomical biomimetic artificial bone for rabbit tibia completely segemental defect repairOBJECTIVE:On the basis of the previous scaffold material study,using 3D rapid prototyping technology manufacture rabbit tibia biomimetic artificial bone scaffold,establishing a simple and convenient plastically anatomical shape bone defect model to simulate the structure properties of rabbit tibia to guide the regeneration and reconstruction of new bone,aiming to improve the anatomical matching,such as stress shielding,linear as well as positional alignment,for artificial bone substitute transplant-And laying a foundation for cells/cytokines-scaffold study and simulate clinical application.METHODS s Mimic software gains 3D reconstruction by CT scan rabbit tibia and establishs 3D electronic model.Manufacture three dimensional biomimetic human bone scaffold under the control of 3D electronic model data by Fused Deposition Modeling equipment(FDM).15 rabbits of six months were randomly divided into blank groups of three,control groups of six,experimental groups of six respectively and made a 1.2cm completely critical-sized defect,among which spacious set,autogenous bone place,anatomic tissue engineered bone place respectively?4?12 weeks later,observed by X-ray,CT,histology,general observation respectively.RESULTS:the defect X-ray show:4,12 weeks after two groups of bone defect transplantation did not see obvious displacement and angulated deformity,autologous control groups manifest better repairment;Biopsy shows new bone filling bone-scaffold in experimental groups after 4 weeks,and new bone formation increased significantly and mineralization after 12 weeks;General observation:two groups of bone defect transplantation did not see obvious displacement and angulated deformity after operation.CONCLUSION:The plastically anatomical biomimetic artificial bone scaffolds can be used as a scaffold model for defect repair of load bearing site,which may simulate the structure function of bone tissue and guide the new bone regeneration specificity,and lay a good foundation for the further development of cell-scaffold or biomolecule-scaffold research.Part two:The study of the biological characteristics of the thermo-induced PLGA-PEG-PLGA hydrogel as a delivery system for rhVEGF165/rhBMP-2OBJECTIVE:This work is to study the thermo-induced PLGA-PEG-PLGA hydrogel as a delivery system of the rhVEGF165 or/and rhBMP-2,and detecting the releasing law of embedding signal factors in vitro and in vivo.In addition,to observe effects of the hydrogel loading cytokines on the biological characteristics of osteoblasts and subcutaneous ectopic ossification.METHODS:The structure and property of thermo-induced PLGA-PEG-PLGA hydrogel was characterized by gel chromatography and rheometer to identify its sol-gel phase-transformation.Mix up the solution with rhVEGF165 or/and rhBMP-2 to dissolve and embed these cytokines,and then subjected to subcutaneous injection for determination of ex vivo degeneration in the gel phase and ectopic ossification.The factors-release was measured by Elesa,and detect effects of the release cytokines on the biological characteristics of osteoblasts was detected osteoblast specific genes by RT-PCR.RESULTS:1.The hydrogel is a kind of homogeneous temperature-sensitive triblock copolymer with reversible sol-gel transition character and the proper phase-transition temperature is 27-30 temperature.2.The thermo-induced hydrogel can maintain structural integrity about 14-21 days in subcutaneous tissues shows a good effect on the drug sustained-release to target site.3.The hydrogel loading rhBMP-2 group and loading rhBMP-2 and rhVEGF165 group promote subcutaneous ectopic ossification in 14 days,and no obvious difference of ectopic ossification between the two groups in 28 days.CONCLUSION:The injectable thermo-induced hydrogel can serve as a localized release rhBMP-2 and/or rhVEGF165 carriers and simulate the key signal factors release in the natural bone healing procedure.And there maybe synergistic effect of rhVEGF165 combined rhBMP-2,the added rhVEGF 165 could induce osteoblasts,osteogenic precursor cells and cytokines migration,improving osteogenesis.The hydrogel can keep rhBMP-2 and rhVEGF165 away from fluid diffusion,enzymolysis,and reduce the minimum effective concentration and complications.Part three:The study of rhBMP2/rhVEGF165 loaded hydrogel combining with anatomically biomimetic artificial bone for rabbit tibia segemental defect repairOBJECTIVE:This part is to build a factors-scaffold strategy by combining 3D printing tissue engineering bone which was printed in the first part with hydrogel loading rhBMP2 or/and rhVEGF 165 which was constructed in the second part to repair bone segemental defect?endowing the scaffold with more intelligence by means of biological activities of signal factors to promote bone regeneration,bone graft osseointegration,and to reconstruct local structural integrity and organ function faster and better to avoid complications such as bone nonunion.METHODS:The hydrogel delivery system was loaded into the 3D printing artificial bone by utilized vacuum techno logy;then transplanting the hydrogel-loaded scaffold to reconstruct and repair the rabbit tibia completely segemental defect.The local situation by general observation,radiological examination were performed 4,12 weeks postoperatively to compare the difference among blank scaffold group,rhBMP2-loaded scaffold group and rhBMP2/rhVEGF165-loaded scaffold group.RESULTS:X-rays show that there is not obvious displacement and angular deformities in all groups 4,12 weeks postoperatively,and the new bone increased after 3 months.The gross specimen were observed without obvious displacement and angular deformities after 3 months;The three group formed varying degrees of bone callus,among which the rhBMP2/rhVEGF 165-loaded scaffold group formed the most callus at the site of bone graft and the scaffold-bone contact surface was osseointegration that couldn't tell the boundary;the rhBMP2-loading scaffold group formed the second-most callus at the site and the scaffold-bone contact surface was still marginally recognizable;the control group formed the least callus at the site and the scaffold-bone contact surface was partial osseointegration and obviously recognizable.CT image reconstruction of the defect site shows the callus was uniform and dense and the contact surface was osseointegration in the rhBMP2/rhVEGF165-loaded scaffold group;the callus was less uniform and dense in the rhBMP2-loaded scaffold group and the contact surface was osseointegration;the control group formed the least new bone and callus and the contact surface was recognizable with partial osseointegration.CONCLUSION:1.The 3D printing biomimetic artificial bone loading rhBMP2 or/and rhVEGF165 hydrogel release system could significantly promote bone repair in the early and bone graft osseointegration in the late,which providing a perfect substitution of bone autograft.2.there maybe synergistic effect of rhVEGF165 combined rhBMP-2,the added rhVEGF 165 could induce osteoblasts,osteogenic precursor cells and cytokines migration,improving osteogenesis.