Fabrication Of Tissue Engineered Artery Using Decellularized Vascular Conduit Seeded With Autologous MSCs Cell Of Goat In Vivo And Vitro

Vascular disease, one of the most common diseases being harmful to thehealth of the human being, is mainly treated by the vascular transplantation.Therefore,the source of vascular grafts becomes the concern of the research.Currently there are three types of grafts: autograft, allograft and artificialmaterials. Autografts have limited sources. Allografts are easy to be attackedby immune rejection. Artificial materials are not fiit for small caliber arteries. The development of tissue engineering provides a new approach for thesource of vascular grafts. Vascular tissue engineering is classified as a fieldthat applies the normal cells from the vascular wall and biologic degradablematerials to produce, reconstruct and regenerate the vessel substitute, makingit possible to reconstruct vascular conduit in vitro which structure andfunction are the same as autogenous ones. In this study, tissue engineered blood vessels (TEBV) were constructedin vitro and in vivo using autologous mensenchymal stem cells, decellularizedvascular conduits as scaffolds. The purpose of our research is to explore thefeasibility of vascular tissue engineering.This article is divided into four parts:Part 1 Seed cells for vascular tissue engineeringPurpose: To establish a method for the isolation and culture of goatmesenchymal stem cells (MSCs) from bone marrow and provide seed cells forthe vascular tissue engineering research.Methods: MSCs of goat was isolated by Percoll and cultured in vitro.Growth curve of cultured cells was drawn. Cell cycle was analyzed bymeasuring DNA content with FAC Scan flow cytometer cell multipotent wasidentified with specific staining.Results: MSCs were both successfully isolated, cultured and passagedand their morphological characteristics were consistent with fibroblast cells ,Immunohistochernical staining results showed that CD44 positive ,whileCD34, negative. The cell cycle analysis showed that 80% of MSCs were inG0?G1 phase.Conclusion : MSCs can be isolated from bone marrow through theiradherent ability. MSCs may be introduced as a valuable model system tostudy the mesenchymal lineages for basic research and tissue engineering.Part 2 Differentiation of MSCs in VitroPurpose: to explore the potentiality that bone marrow mesenchymal stemcells are pluripotent stem cells and can be to induced of differentiating intoendothelial cells (EC), smooth muscle cell and osteoblast in vitro and toprovide the seed cells for study of tissue engineering.Methods : 1The MSCs were incubated for orientation differentiated intoEC in DMEM with 20 % FBS , VEGF , bFGF ,for about 10 days and thedifferentiating cells were evaluated by histology and immunohistochemistry. 2Sufficient dexamethasone, βsodium glycerophosphate and vitamin C wereas inductors to induce MSCs to osteoblasts. The form and specific phenotypeof the cells were detected under alkaline phosphatase activity and type Icollagen secreted by osteoblasts were detected with immnohistochemicalstaining and vonkossa staining was performed. 3 MSCs were with mediumwith TGF-β, Inverted microscopy was used to observe the morphology ofcellsResults : 1 About 80 % -90 %of the differentiating cells from MSCsafter 10days were positively stained for Ⅷ factor (vWF) related antigen byimmunohistochemistry assay to endothelial cell , and show cobblestone-likemorphology;2 to differentiate osteoblast cells , the induced cell expressedtype I collagen , Alkaline phosphatase activity was increased and mineralizednodules formed;3 After induction , MSCs demonstrated smooth muscle Celllike spindle shaped morphology and assumed a hill and valley growth pattern.Immunohistochemical staining revealed positive signals for alpha smoothmuscle actin ,Conclusion: MSCs have the potentiality of differentiation intoosteoblast -like cell, smooth muscle like cell and endothelial cell. MSCs willbe a potential source of seed cells for fabrication of tissue – engineeringPart 3 Preparation of decellularized vascular conduit (DVC) and itscytocompatibility and biocompatibilityPurpose: To prepare DVC for tissue engineering blood vessel and tomeasure its cytocompatibility and biocompatibility.Methods: DVC were prepared by pepsin , Dnase and Rnase digestion andmechanical action. Fresh goat artery was soaked in distilled water in 4 ℃ for24 hours, and then transferred into shaken in 0.125% pepsin for 4hours,and then shaken in Dnase and Rnase for 4 hours,distilled water foranother 24 hours .The production were frzon-dry ing and sterilized usingradiation of CU60. MSCs were harvested and isolated expanded in vitro.The cell were seeded in DVC and specimen were stained with HE,andobserved grossly, underlight microscopy and scanning electromicroscopy. TheDVC were implanted in the back of rats. Results: There were no residues ofcells in the DVC(HE staining). DVC had reticular and porous structure.Poresize was from 100to 200um. The DVC could be seeded with expandedMSCsin vitro. The rat in the implant test had no death, convulsions and otherabnormal response.Conclusion: The enzymatic extraction and mechanical process canremove cells and solvable components effectively and preserve the tissuematrix well and keep the reticular structure. The DVC can be used as an idealscaffold of biological prosthesis for vascular tissue engineering.Part 4 Construction of TEBV in vitro and in vivoPurpose: To explore the construction of TEBV in vitro and in vivo.Methods:MSCs were labeled with Cell Tracker PKH26, were uniformlyseeded onto decellularized tissue matrices Prior to implantation, the SMCs-seeded patches were cultured under static condition for 1 week to allow forcell adherenceand differentiation in Medium supplemented with 20%(v/v)FBS, human VEGF (10 ng/ml), and human bFGF (2 ng/ml) for 1 week .thenBMSCS-seeded vascular conduit were implanted into the Carotid artery ofbone marrow donor goats after anesthetization during the operation. Segments(30mm) of the Carotid artery were resected and replaced by thetissue-engineered vascular conduit using 7-0 polypropylene suture.Result: Mid-portion segments of the vascular conduit retrieved 2 monthssafter implantation .The specimens were stained with HE. collagen werestained using Masson's trichrome method, respectively. The specimens werealso stained immunohistochemically for vWF, SM a-actin. Fluorescentlylabeled BMSCs were examined using fluorescence microscope.The cellseeded blood vessels were all patent at 2 months.No sign of thrombusformation or stenosis was observed in the explanted patches. H&E-stainedsections of the patches retrieved of implantation showed regeneration ofvascular tissues Masson's trichrome stainings showed the presence of asignificant amount of collagen. Immunohistochemical analysis showed thatcells on the luminal sides of the conduits stained positively for vWF,indicating the endothelium regeneration. Cells in the medial parts of thepatches stained positively for SM a-actin, indicating SM regeneration.Next,we investigated if the seeded BMCs survived after implantation. BMCslabeled with fluorescent PKH26 prior to seeding . The labeled cells weredetected in the luminal sides and the medial parts of the explanted patches,suggesting survival of the implanted BMSCs and their contribution toregeneration of vascular tissues. Compared to the fluorescent signals detectedprior to implantation, the signals in the explanted conduits were attenuatedprobably due to migration of the seeded cells to media and signal dilution ofmembrane-incorporated dye by labeled cell division.Conclusion:Tissue-engineered vascular conduit could overcome theproblems of currently available synthetic polymer.