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Experiment Study On Constructing Heart Valve Use Mesenchymal Stem Cells And Decellularized Porcine Valve By Tissue Engineering Approach

Posted on:2006-10-02Degree:DoctorType:Dissertation
Country:ChinaCandidate:X N SunFull Text:PDF
GTID:1104360155460608Subject:Surgery
Abstract/Summary:PDF Full Text Request
Part One1. Tissue-engineered heart valve leaflets: comparison of different decellularization procedures of porcine heart valvesObjective Tissue engineering of heart valves should avoid the disadvantages of conventional prostheses. To determine the most effective method of producing the acellularized porcine heart valve leaflets, we compared pathological findings of the porcine heart valve leaflets produced by three methods: NaCl-SDS, trypsin and Triton -X100, sodium deoxycholate with DNAase 200mg/L, RNAase 20mg/L.treatment and further analyzed the biological character of the best one.Methods Aortic valve leaflet were harvested from pigs in slaughterhouse of Shanghai city. They were immediately immersed in 4℃ PBS solution. After washed at room temperature in povidone-iodine-solution and sterile PBS solution. Leaflets were assigned in one of the three preparation methods for decellularization. And then light and electron microscopic analyses were performed.Results HE stain showed that cells were almost absent in the leaflet treated with trypsin, and Triton-X100, while cells were partly present in the leaflets treated by NaCl-SDS. Scanning electronic microscopic revealed trypsin removed cells completely but caused strong structural alterations. Treatment with Triton-X100 achieved both complete decellularization and preservation of the matrix structure. Conclusion Techniques of decellularization are highly variable in efficiency and matrix preservation and satisfactory results were achieved in our study with Triton-X100, sodium deoxycholate and DNAase, RNAase. 2. Study on the biological properties of decellularized tissue-engineered porcine heart valve leafletsObjective To study the biological properties of decellularized tissue-engineeredporcine heart valve leaflets.Methods The harvesting and immediate treatment of porcine heart valve leafletswas as same as above. Then the valve weight, protein content, stress strain, destroying stress, were examined. Cell toxicity and biocompatibility were also evaluated. Decellularized tissue-engineered porcine heart valve leaflets were implanted in rabbit subcutaneous, and animals were sacrificed after 2,4,8,12 weeks. Results Compared with that before the cell removal, valve weight in the decellularized aortic valve leaflets was increased significantly, but there were no significant difference in stress-strain, destroying stress between normal valve leaflets and decellularized. Cell toxicity was not significant. Biocompatibility study demonstrated that valve leaflets were absorbed gradually, Microscopic inspection of implantation site revealed no pathological inflammatory response. Conclusion The decellularized porcine heart valve leaflets have stable biological properties and can be used as an ideal tissue engineering valve scaffold. Part TwoHuman mesenchymal stem cells —a new cell source for heart valve tissue engineering.Objective Human mesenchymal stem cells (MSC) exhibit the potential to differentiate into multiple cell-lineages and can be easily obtained clinically. This study was designed to assess the feasibility of using human bone marrow-derived mesenchymal stem cells as a kind of source to construct tissue engineering heart valve.Methods Bone marrow was aspirated from the sternum of surgical field. Cells were isolated using Ficoll gradient, cultured, and characterized based on immunofluorescent staining flowcytometry, histology, and immunohistochemistry. Interstitial cells (IC) derived from the human rheumatic aortic heart valves and smooth muscle cells(SMC) from umbilical cord were cultured in vitro as contrast. Results Isolated MSC demonstrated fibroblast-like morphology. Phenotype analysis revealed positive signals for a-smooth muscle actin and vimentin. Flowcytometry of MSC pre-seeding was negative for CD31, CD14, positive for a-smooth muscle actin and vimentin. The mean fluorescence intensity ratio (MFIR) of MSC > IC and SMC was 4.9, 4.5, 4.2 and 2.7, 3.1 and 2.4 respectively. Immunohistology showed production of collagen I and collagen III. Conclusions Mesenchymal stem cells can be isolated noninvasively from thesternum demonstrated myofibroblast-like character, bone marrow may be a potentialsource of cells for tissue engineering heart valves.Part ThreeIn vitro experience with tissue engineered heart valve on deeellularized porcine heart aortic valveObjective To evaluate the feasibility of tissue engineered heart valve on deeellularized porcine aortic valves in vitro.Methods A detergent and enzymatic extraction process has been developed to remove cellular components from porcine aortic valve, glutaraldehyde fixed heart valve was set as control group. MSC were seeded on the deeellularized valves in experiment group, Their growth condition and relationship with deeellularized porcine heart valve leaflets were observed.Results SEM show that a larger amount of cells attached on scaffolds and demonstrated viable secretionally active myofibrolasts and confluent homogenous tissue surface. The cells had covered a larger area of scaffold with a lot of matrix synthesized. The HE stain showed large amount of cells growing on the scaffolds and some cells were found in the central part of scaffolds. There was no cell growth on glutaraldehyde fixed heart valve.Conclusion It is possible to seed MSC on deeellularized porcine heart valve leaflets to create a viable tissue-engineered heart valve leaflets. Part FourExperiment study on constructing heart valve use marrow stromal cells and deeellularized porcine valve by tissue engineering approach Objective The objective of this study was to investigate the feasibility of creating tissue engineered heart valves from human mesenchymal stem cells (MSC) as an alternative cell source and deeellularized porcine heart valve leaflets to obtain viable constructs in nude mice.Methods MSC were isolated, expanded in vitro and labeled with fluorescent tracer DAPI. Porcine heart valves were deeellularized by Triton, RNAse and DNAse, reseeded and cultured under static conditions 7 days. Then cell-scaffold constructs...
Keywords/Search Tags:heart valve, sodium deoxycholate, Triton -X100, trypsin, SDS, tissue engineering, Tissue engineering, decellularized porcine heart valve, mesenchymal stem cells, nude mice, chitosan
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