Preparation Of The Tissue Inducing Extracellular Matrix Hydrogel And Characterization Of Osteogenesis And Agiogenesis

Objective Small intestine submucosa(SIS)and demineralized bone matrix(DBM)were used as substrates to prepare injectable extracellular matrix hydrogels.The physical and chemical properties and bioactivities of the ECM hydrogels were studied and their biocompatibility and osteogenesis and angiogenesis activity were also detected.Materials and methods SIS was prepared with three different methods,and the effects of the three methods to the decellularization,composition and microstructure of SIS were detected with HE,Masson and DAPI staining,proteomics analysis and scanning electron microscope(SEM).DBM was prepared by modified Urist method and the degree of decellularization was determined.Then SIS and DBM particles were dissolved in hydrochloric acid with pepsin to produce ECM solution,which was self-assembled to form the appropriate concentration of SIS,DBM and SIS/DBM hydrogel by adjusting the p H and temperature.Gelation kinetics and rheology of ECM hydrogel were detected;and the appearance and microstructure of hydrogel were detected with scanning electron microscopy(SEM).Different types of ECM hydrogels were co-cultured with bone marrow mesenchymal stem cells(BMSCs);living and dead staining and CCK-8 were used to detect the cell compatibility and cell proliferation,alizarin red staining and ALP activity were detected to measure the osteogenic differentiation ability of ECM hydrogel;RT-PCR of HUVEC functinal gene expression were operated to determine angiogenesis of ECM hydrogel with SIS,DBM and SIS/DBM hydrogel.The rats were randomly divided into four groups and received subcutaneous injection with four kinds of injectable hydrogels: SIS,DBM,SIS/DBM and Col I.Five animals were sacrificed at day 7.Biocompatibility and angiogenesis activity of hydrogel were detected by HE,Masson and CD31 immunohistochemical staining.The rat calvarial defects with diameter of 5mm were prepared,which were as follows: SIS,DBM,SIS/DBM and Col I hydogel respectively.Five animals were sacrificed at 4 weeks and 8 weeks postoperatively.HE,Masson,immunohistochemical staining and Micro-CT were used to detect the new bone formation and regeneration.Results SIS prepared by three kinds of methods all showed no visible nucleus of HE and DAPI staining,and the residual DNA content of SIS-1,SIS-2,SIS-3 and DBM were 43.25,21.01 ng/mg,15.69 and 24.53 ng/mg ECM dry weight respectivly.SEM showed that the microstrucure of SIS was destcuted at different extent with randomly messy arangment and fracture of the collagen fiber.Proteomics analysis showed that 644,717,135 and 129 kinds of proteins were detected in SIS-1,SIS-2,SIS-3 and DBM respectively.There were 21 subtypes of collagen,including ?,?,?,?,?,?,?,?,?,? and Collagen type 14 and collagen 16.The relative expression of collagen?was the highest.Among them,the type ?,type ? and type 14 collagen were unique to SIS,and ?,?,?,? type collagen protein is unique to DBM.In addition to collagen,expression of fibronectin,proteoglycan,VEGF,FGF,TGF-?,and BMP-2 was also detected.SIS co-cultured with BMSCs showed that SIS had good cell compatibility.SIS and DBM were digested in HCl with 1mg/ml pepsin with continous stir.After nutrulization with Na OH and PBS,the pre-gel soulution became hydrogel within 1h under 37?.The gelation kinetics of ECM hydrogels showed that all types of hydrogels were able to gel within 1 h,and gelation time of SIS/DBM composite hydrogel was the shortest.Rheological tests showed that the storage modulus of SIS,DBM and SIS/DBM composite hydrogel were 40.33±0.57 Pa,401.67±1.46 Pa and 487±6.08 Pa respectively.The microstructure of hydrogel of ECM hydrogel with SEM showed that the nano-collagen fibers were arranged randomly,with good porosity and three-dimensional scaffold structure.However,SIS hydrogel showed no obvious nano-collagen fiber apprance.Living and death staining by Laser Scanning Confocal Microscope suggested that BMSCs could survive well and keep good mophology and proliferation within ECM hydrogel.CCK-8 suggests that ECM hydrogels could promote the proliferation of BMSCs.RT-PCR showed that the gene expression of agiogenesis relative preoteins KDR,Notch1 and Ang2 of SIS and SIS/DBM hydrogel were significantly higher DBM hydrogel.Alizarin red staining and ALP assay showed that ALP activity was obviously enhanced by DBM and DBM/SIS copmposite hydrogels,which was signifacantly different with SIS hydrogel.RT-PCR showed that the gene expression of osteogenesis relative protein ALP,OCN and OPN of DBM and SIS/DBM hydrogel were significantly higher SIS hydrogel.Subcutaneous injection of ECM hydrogels were operated successfully on the dorsal of the rats.HE,Masson and CD31 immunohistochemical staining showed significant distribution of new blood vessels scattered within SIS and SIS/DBM hydrogel,a small amount of new blood vessels and no obvious angiogenesis within DBM and Col ? hydrogel.There were no significant inflammatory cells within all ECM hydrogels.However,there was no obvious ectopic osteogenesis within all kinds of ECM hydogels.In vivo rat calvarial defect repair showed that both DBM and SIS/DBM hydrogel exhibited satisfied bone defect repair,and SIS hydrogel and Col ? hydrogel exhibited poor bone repair and regeneration,which were significantly different with DBM and SIS/DBM hydrogel.Micro CT demonstrated that bone volume / total volume(BV / TV)of DBM and SIS/DBM composite hydrogel were up to 56.5% and 88.4% in 8 weeks,which were significantly higher than SIS hydrogel and Col ? hydrogel.HE and Masson staining showed that there was obvious new bone formation within DBM hydrogel and SIS/DBM composite hydrogel,which was higher than SIS and Col? hydrogel.And the effect of bone regeneration of 8 week was better than 4 weeks.ALP and OCN immunohistochemistry results showed that ALP and OCN expression of SIS/DBM and DBM hydrogel were higher than the other two kinds of hydrogels.Conclusion Small intestinal submucosa(SIS)and demineralized bone matrix(DBM)prepared by appropriate decellularied method can retain most of bioactive components and microstructure structure the natural extracellular matrix.The injectable ECM hydrogel,which has good physical and chemical properties,can be prepared by adjusting the p H(7.4)and temperature(37?)of acid ECM solution.In vitro experiment showed that the ECM hydrogel had good biocompatibility and increased angiogenesis and osteogenesis differentiation.In vivo experiment demonstrated that ECM hydrogel especially for SIS/DBM composite hydrogel could cause in situ induction of angiogenesis,new bone formation and bone regeneration.,which could be an ideal biomaterial for bone defect repair.