| Objective:Tissue engineering heart valve ,as an ideal living heart valve prostheses ,has now become a focus of study in the field of heart valve surgery. The definition of tissue engineering valve is to use appropriate cells,deliver those cells onto a3-D supporting scaffold,culture into mature or sub-mature tissue and transplant them into bodies, ultimately form the new functional tissue with the ability to grow and remodel,which would be a major improvement on the current treatment of heart valve disease. Establishing a reliable cell source is a key to effective tissue regeneration. As human MSCs exhibit the potential to differentiate into multiple cell-lineages and can be easily obtained clinically, theoretically speaking ,they will be a promising source of cell populations for the construction of tissue engineering heart valve.In our research we seeded human MSCs which cultured in vitro on acellular porcine heart valve leaflets, observed their growth on the scaffolds in order to investigate the feasibility of creating tissue engineering heart valve from human MSCs as an alternative cell source.Method:1 Preparation of acellular porcine aortic valve leafletsThe porcine aortic valve leaflets were acellularized by using 1% Triton-X 10(K 0.01% trypsin-0.02%EDTA DNase I and RNase I. Biomechanical evaluation was performed using a mechanical tester (Instron), which measured parting intensity and parting intension ratio. Result data were expressed as mean standard error of the mean. An unpaired t test was performed, considering a P<0.05 as statistically significant; Morphological characterization of extracellular matrix (ECM)-formation was analyzed by H-E staining and electron microscopy(EM).2 Study of human MSCsHuman MSCs were isolated from bone marrow aspirate by a simple bone marrow puncture and centrifuged over a Ficoll step gradient.The interface fraction was collected and cultured in DMEM supplemented with 20%FCS. The nonadherent cellsfloated off while MSCs adhered. Flow cytometry analysis of MSCs by CD29, CD34 (HSC) and CDlla (lymphocyte); cultured MSCs were characterized by a -SMA immunohistochemistry staining and western blotting tested the expression of collgen I.3 Study of cells seeding in creation of tissue engineering heart valveCultured and expanded MSCs in vitro were seeded on the acellular porcine aortic valve leaflets,which cultured in the static conditions for seven days. The growth pattern of cells were observed and characterized by H-E staining , a -SMA immunohistochemistry , SEM and TEM. Result:1 The parting intensity and parting intension ratio between acellular valves and fresh valves were similar,and there was no significant difference of biomechanical characteristics between them. After decellularization ,cells could no longer be detected by standard histological analysis. In the same time,the normal structure of extracellular matrix was optimally preserved.2 Flow cytometry demonstrated that expanded MSCs expressed a high lever of CD29 and don not express CD34 and CDlla. Enlogated cells are fibroblast-like morphology .Immunohistochemistry of fixed cells showed the expression of a -SMA. Western blotting test showed cells produced collgen type I.3 H-E staining of TEHV leaflet sections demonstrated cellular tissue organized in a monolayer fashion with a loose outer layer and no cellularity detected in the inner portions. Immunohistochemistry showed cells on the surface positive staining for a -SMA. SEM showed an inhomogenous surface and TEM revealed actin filaments.Conclusion:1 Treatment of 0.01% trypsin -0.02%EDTA with 1% Triton-X 100 RNase I and DNase I was successfully used for complete cell elimination from porcine aortic valvular tissue. The fibrillar structure of decellularized valve leaflet remained well-organized, and its biomechanical characteristics was not evidently affected by accellularization procedure. Acellularied porcine aortic valve can be applied as a scaffold to develop tissue engineering heart valve.2 Human MSCs cultured in 20%FCS DMEM diffe... |
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