Fabrication Of Easy To Vascularization Biomaterials And Evaluation Of Their Function On Bone And Soft Tissue Defect Reparation

Diabetes has an impact on metabolism of the bone,leads to osteoporosis in Type I diabetes and increases the fracture risk in patients with Type I/II diabetes,which results in delayed fracture healing,nonunion and pseudarthrosis.Meanwhile,Diabetic patients with open fractures often have to face the skin wounds,especially the foot wounds,which may turn into non-healing chronic wounds,are one of the most dreaded complications of diabetes.As the number of Chinese patients with diabetes has rapidly increased in recent years,the cases which have skin and bone tissue defect are also increasing year by year.How to heal the defect quickly and return the structure to normal becomes a difficult clinical problem.Therefore,we need a safe,effective,easy to popularization way to promote healing process and prevent the occurrence of serious late complications.Currently,the mainstream viewpoint is that vasculopathy and interruption of neovascularization is one of the most important local reasons for defect no-healing.As the study in the mechanism of vascular lesions in diabetes moves towards increasing sophistication,disorder of HIF-1α(Hypoxia Inducible Factor-1α)plays a vital role in this pathological process.DFO(Deferoxamine)is one of the compounds which could stimulate activation of HIF-1α under normoxic conditions and we call them "hypoxia mimetic compound".Thus,this type of compounds may has effect on healing process of diabetic skin and bone defects.At the same time,the scaffolds which is maded by electrospinning,hydrogel and other new material technologies is excellent drug carrier,they could realize fixed-point,quantitative and controlled release.How to combine hypoxia mimetic compounds with biological materials and apply them to repair bone and soft tissue defect is worth of our in-depth and useful exploration.Objectives To fabricate hypoxia mimetic compound loaded scaffolds,verify their easy to vascularizaiton function through experiments in vivo and in vitro and explain its role in the molecular mechanisms and cellular signaling pathways.Methods: 1.Type I diabetes mellitus Sprague Dawley rat(T1DM rats)was induced by intraperitoneal injection of STZ(streptozotocin),and then compared with the normal rats at the same age to demonstrate their osteoporosis by using Micro-CT and three point strength test.Western blot was used to detect impaired pathway of HIF-1α in high glucose.DFO loaded PCL(polycaprolactone)tubular nanofiber scaffold was fabricated by electrospinning.SEM(scanning electron microscope)was used to observe microstructure of the scaffolds.In vitro part,m BMSCs(Murine bone marrow stem cells)was used to verify material’s biocompatibility,angiogenesis and osteogenesis relative m RNA was detected by Realtime-PCR.HUVEC was used to test tube formation.In vivo part,distal femur defect in T1 DM rat model was established,then Micro-CT,X-ray and tissue morphology were used to evaluate the effect of DFO on reparation of diabetic osteoporosis defect.2.DFO loaded hydrophilic PVA(Polyvinyl Alcohol)-CS(Chitosan)composite electrospun fiber membranes were fabricated.Material characterization and sustained drug release rate were detected through SEM,FTIR(Fourier Transform Infrared Spectroscopy)specrometer and other methods,and then their biological properties were verified respectively.In vitro part,HFB(Human dermal fibroblast)was used to verify their biocompatibility and detect the expression of HIF-1α and other angiogenic proteins.The HUVEC was used to test tube formation character.In vivo part,we built full-thickness dorsal skin defects;Tissue morphology,Realtime-PCR,Western blot and other methods was used to evaluate the healing rate and its quality and clarify the changes in HIF-1α signal pathway.3.DFO was mixed with phote-crosslinked Gelma(Gelatin methacrylamide)hydrogel scaffold by using physical blending method.SEM was used to observe microstructure of the hydrogel and drug distribution.The drug release was studied by UV spectrophotometer.HFB and HUVEC were used to verify the biocompatibility of the scaffolds and the expression of relative proteins in vitro.In vivo part,full-thickness dorsal skin defects were built on T1 DM rats,and tissue morphology was used to verify the repair effect of hydrogel scaffold.Results 1.T1 DM rats have obvious osteoporosis and their pathway of HIF-1α was impaired.DFO loaded PCL tubular nanofiber scaffold had good biocompatibility and could up-regulate m RNA of vegf,runx-2 and osteocalcin in m BMSCs and HUVEC tube formation.BMD(Bone mineral density),bone and vessel content were higher than the control group.The expression of HIF-1α,RUNX-2 was higher and osteogenesis was animated in DFO treated group,leading to faster healing process.2.DFO loaded hydrophilic PVA-Chitosan composite electrospun fiber membrane showed excellent water absorption and mechanic property.It could effectively promote HFB adhesion,proliferation and upregulate intracellular expression of HIF-1α,VEGF and other angiogenesis proteins.They could also effectively promote HUVEC tube formation.In vivo,both of the scaffolds could rapidly recruit angiogenesis relative cells and cytokines to the wound area and strengthen the interactivities between HFB and HUVEC,promote angiogenesis,skin regeneration,accelerate type I/III collagen conversion and eventually help the wound totally repaired.3.DFO loaded phote-crosslinked Gelma nanofiber hydrogel scaffold had wonderful biocompatibility,and its shape was freely fixed the wound area in spite of the wound sizes and depths.It could provide relatively closed and stability 3D microstructures for cell ingrowth,proliferation and differentiation and up-regulate HIF-1α in HFB.In vivo,the hydrogel scaffolds supported the reconstruction of microvasculature and induced the vessel formaiton by up-regulating expression of HIF-1α and its downstream protein VEGF.Conclusion DFO loaded porous fiberstructure scaffold could provide excellent three-dimensional microenvironment for cell and tissue adhesion,ingrowth and proliferation and release the drug according to the repair process,and then achieved rapid vascularization and reparation of diabetic skin and bone tissue defects by upregulating Hif-1α signal pathway.