Selecting And Functional Study Of The Seeding Cell And Decellularized Scaffold For Tissue Engineering Blood Vessel

Objective:In consideration of the biological characteristics of perinatal mesenchymal stem cell and the advantages of extracellular matrix(ECM)based biological scaffold.The objective of this study was to explore the feasibility of umbilical cord Wharton's jelly mesenchymal stem cell(WJ-MSC)and amniotic membrane mesenchymal stem cell(AM-MSC)as the seeding cell for construction of tissue engineering blood vessel(TEBV).Porcine carotid artery derived ECM scaffolds were prepared and the physiological and biochemical properties were evaluated.Finally,TEBVs were constructed based on WJ-MSC and ECM scaffold.TEBVs were implanted in vivo and its physiological function was preliminary evaluated.Methods:WJ-MSC and AM-MSC were isolated using explant method and characterized in vitro according to their morphology,immunotype,and multipotent differentiation potential.In order to acquire a suitable MSC for developing TEBV,the proliferation and self-renewal properties were investigated via MTT assay and colony-forming unit fibroblast assay.The hemocompatibility of WJ-MSC and AM-MSC were investigated via platelet adhesion assay,prothrombin time(PT)and activated partial thromboplastin time(APTT)test.Based on cell-sheet technique,the ECM deposition properties and cell viability were further investigated using histological staining,electron microscope oberservation,biochemical assessment,real-time PCR and Live/Dead assessment.Porcine carotid arteries were acquired and randomly divided into 5groups(n=6).Based on sodium dodecyl sulfate(SDS),sodium deoxycholate(SDC)and Triton X-100(Tri),porcine carotid arteries were subjected to decellularize using 4 kinds of methods:1%(m/v)(SDS+ SDC),group B;0.5%(m/v)(SDS + SDC),group C;1%(m/v)SDS and 1%(v/v)Tri,group D;0.5%(m/v)SDS and 0.5%(v/v)Tri,group E;native porcine carotid artery serve as control,group A.For obtaining the optimal ECM scaffold,tissue samples of eachgroup were acquired and comprehensively evaluated using H&E and Movat's Pentachrome staining,scanning electron microscopy,immunohistochemistry,DNA and ECM quantification as well as biomechanical assessment.TEBV was constructed by seeding WJ-MSC on porcine carotid artery derived ECM scaffold,which was processed using 1%(SDS+SDC)method,and analysised using H&E staining.The biological function of TEBV was investigated in vivo using ultrasonography evaluation and H&E staining based on the rabbit model of inferior renal artery intermediategrafting.Results:Fibroblast-like WJ-MSC and AM-MSC were successfully isolated and positively expressed the characteristic markers CD73,CD90 and CD 105 but were negative for CD34,CD45,and HLA-DR.Both MSCs shared tri-lineage differentiation toward the adipogenic,osteogenic and chondrogenic lineages.The proliferative and self-renewal capacity of WJ-MSC was significantly higher than that of AM-MSC(P<0.001).WJ-MSC provided comparable properties of anti-platelet adhesion and did not activation coagulation cascade to endothelial cells.However,aggregated platelets were visualized on the surface of AM-MSC and the intrinsic pathway was activated.Furthermore,WJ-MSCs have a superior property of collagen deposition and higher viability than AM-MSCs during cell sheet formation.The cellular components were completely removed ingroup B andgroup C,however,cellular residues were visualized ingroup D andgroup E.DNA quantification demonstrated thatgroup B with theminimum residual of DNA.Most of the amor:phous ECM was destorized accompanied with decellularization and partial of the xenogeneic antigen a-1,3-Gal was removed.Collagen and elastin fiber bundles were well preserved in all of the decellularizationgroups,however,the morphology and size of porous structure were superior ingroup B than the others.The longitudinal ultimate tensile stress,longitudinal Young's modulus,burst pressure,suture holding capacity and suture retention strength were decreased significantly after decellularization,however,there was no adverse effect on the compliance with decellularization.Elimination of the smooth muscle cell is the major reason for the alteration of biomechanical properties fo porcine carotid artery ECM scaffold.The TEBV was constructed based on ECM scaffold and WJ-MSC,however,histological examination demonstrated that the cell seeding is poor.After implantation in vivo,the physiological function of TEBV is limited.Conclusion:As a seeding cell for construction TEBV,WJ-MSC is superior than AM-MSC.WJ-MSC could serve as an appealing substitute of endothelial cell and fibroblast.More suitable porcine carotid artery ECM scaffold was obtained using 1%(SDS+SDC)based decellularization method.Functional TEBV can be constructed in vitro by cultivating WJ-MSC on porcine carotid artery ECM scaffold.