Experimental Research On Construction Of Tissue Engineering Heart Valve With Human Bone Marrow Mesenchymal Stem Cells And Decellularized Porcine Aortic Valve Scaffolds

Partâ… Experimental study on human bone marrow mesenchymal stem cells orientiatedly induced and differentiated into endothelium-like cells and fibroblast-like cells【Objection】To harvest and isolate human bone marrow mesenchymal stem cells(BMMSCs),and to induce and differentiate them into endothelium-like cells and fibroblast-like cells.【Methods】1.Isolation,culture and proliferation of human BMMSCs:BMMSCs were harvested from human ribs and isolated,purified by methods of density gradient centrifugation and adhering to the culture plastic.Cells of P₃ were examined with flow cytometry for the special markers.Cell growth curve of P₀,P₅,P₁₀ passage culture of human BMMSCs was drawn according to cells counting.2.BMMSCs be differentiated into endothelium-like cells and fibroblast-like cells:BMMSCs were treated in medium with 10ng/ml VEGF and 5ng/ml bFGF correspondingly for 21 days. These endothelium-like cells and fibroblast-like cells were observed and identified by morphology and immunohistochemistry.【Results】1.Methods of density gradient centrifugation and adhering to the culture plastic can harvest mononuclear cells with specific character.The cells showed presence of CD 105,CD44 and CD29.Growth curves showed that there was difference of activity periods between primary culture and passage culture.Some cells wound be aging after 10^th passage culture.2.The cells induced by VEGF showed endothelium-like morphology,which immunohistochemistry results showed vWF positive.The level of NO was 166.58±1.91 umol/10⁹ cell/L in medium supernatant on 7^th day after induction.3.The cells induced by bFGF showed fibroblast-like morphology,which immunohistochemistry results showed SMA positive. 【Conclusion】1.Methods of density gradient centrifugation and adhering to the culture plastic can harvest higher purified BMMSCs.2.BMMSCs can be differentiated into cells with characteristics of endothelial cells and fibroblast cells, which suffice the demand of seeds cells to tissue engineering.Partâ…¡Experimental study on porcine aortic valve scaffolds decellularized by different reagents【Objection】To remove cellular components from porcine aortic valve with different reagents,providing acellular tissue matrix scaffolds for tissue engineering heart valve.【Methods】20 porcine aortic valves were decellularized by 4 different reagents. Groupâ…¡:Triton X-100,Groupâ…¢:Trypsin,Groupâ…£:Triton X-100+Trypsin;Groupâ… was control group which valves were fresh.Specimens were observed grossly.And the valve scaffolds were studied respectively by Hematoxylin-Eosin and Mallory-Heidenhain staining,scanning and transmission electron microscope【Results】1.The valve scaffolds in Groupâ…¡were soft and their endothelium is smooth.HE staining showed that the cells were removed from valve,but some nucleolus is left.Mallory-Heidenhain staining showed that the blue collagen and the red elastin were interlaced.Electron microscope showed that fiber were lined wavy and lateral stripe can be observed.2.The valve scaffolds in Groupâ…¢were collapsed, but the endothelium is smooth.HE staining showed that the cells were disappeared and the connective tissue structures are loose.Mallory-Heidenhain staining showed that collagen and elastin were interlaced as a net.Electron microscope showed that some fiber were ruptured,but lateral stripe can be observed.3.The valve scaffolds in Groupâ…£were soft and their,endothelium is smooth.HE staining showed that the cells were removed effectively from valve,Mallory-Heidenhain staining showed that collagen and elastin were lined parallel.Electron microscope showed that fiber were intact and lateral stripe can be observed. 【Conclusion】1.Triton X-100,Trypsin and Triton X-100+Trypsin can remove the valve cells effectively and keep the fiber intact.2.Contrast to other two methods, using Triton X-100+Trypsin can removed the valve cells effectively.Partâ…¢Experimental study on cellular biocompatibility and biomechanical characteristic of decellularized valve scaffolds【Objection】To study the cellular biocompatibility and biomechanical characteristics of porcine aortic valve scaffolds decellularized by different methods.【Methods】The experiment was divided into four groups,Groupâ… was control group, groupâ…¡,â…¢,â…£used the porcine aortic valve scaffolds decellularized respectively by Triton X-100,Trypsin and Triton X-100+Trypsin.Endothelium-like cells derived from BMMSCs were cultured with these scaffolds.After 7 days,morphological characteristics were observed by HE staining and scanning electron microscope.Cells adhesion and proliferation were detected with MTT assay.We also studied physiochemical properties of the four groups,including moisture content, denaturation temperature and wall thickness.Tissue biomechanical characteristic were studied in vitro,which included tension stress of fracture,strain of fracture,stress relaxation rate,area ratio of load curve and unload curve.【Results】1.Cellular biocompatibility:HE staining and scanning electron microscope showed that endothelium-like cells were seeded on these three kinds of decellularized scaffolds and grew well.growth curve from MTT assay showed that the activity periods at 3^rd-6^th day.2.Biomechanical characteristic:there were not obviously different among decellularized scaffolds and fresh valves about denaturation temperature,wall thickness,tension stress of fracture,strain of fracture and area ratio,while had a obviously different about moisture content.【Conclusion】1.Cells grew well on these three kinds of decellularized valve scaffolds.2.The physiochemical properties and biomechanical characteristic of decellularized scaffolds are stable.3.The scaffold decellularized by Triton X-100+Trypsin is the best among the kinds of decellularized scaffolds about tissue structure and biomechanical characteristic.Partâ…£Experimental study on construction of tissue engineering heart valve by layered cell-seeding【Objection】To construct a tissue engineering heart valve exclusively made of endothelium-like cells and fibroblast-like cells,which have a delamination structure as nature valve.【Methods】The porcine aortic valve was decellularized by Triton X-100+Trypsin and coated by fibronectin.Fibroblast-like cells labeled by Brdu were resuspended in a solution of ECMgel,and as a result,a tri-dimensional cell culture system (fibroblast-like cells-ECMgel) was developed.Then the fibroblast-like cells-ECMgel was seeded on the decellularized scaffold.Endothelium-like cells were labeled by DAPI and were seeded as a lining cell.At last this system was incubated for 7 days.The specimen was observed and identified by morphology and immunofluorescence.【Results】Seed cells could be labeled well by Brdu and DAPI.Distinct layered fibroblast-like cells were appeared in ECMgel.The tissue engineering heart valve was observed by HE staining and scanning electron microscope,which showed a typical valve structure,including endothelium,media and fibroblast-like cells.We also could find the cells in the scaffold were those seed cells labeled by Brdu and DAPI.【Conclusion】1.ECMgel is a good matrix of fibroblast-like cells to adhere and grow. 2.Tissue engineering heart valve,which architecture resembled that of natural valve, can be constructed with bone marrow-derived mesenchymal stem cells by layered cell-seeding.