| Background:Construction of tissue engineered heart valve is based on planting the seed cells to the valve scaffold which enables cell growth. The interaction between the cells and the scaffold, such as cell migration, differentiation, proliferation etc., depends on the adhesion of cells to the scaffold. Studies found that the adhesive ability of the constructed TEHV in vitro is low, resulting in insufficient physiological functions compared with normal cells. Therefore, it is important to improve the adhesion of the seed cells on the valve scaffold, and thus differentiation and proliferation of THEV constructed in vitro.Platelet lysate contains many growth factors such as the platelet-derived growth factor (PDGF), transforming growth factor-β1(TGFβ1), insulin-like growth factor (IGF) and vascular endothelial growth factor (VEGF). The maximized biological effects of growth factors depend on the regulation of multiple growth factors. Many experiments show that the tissue repair effects of single growth factor are limited.Objective:To construct TEHV in vitro by seeding rat bone marrow stem cells on the acellular porcine aortic valve scaffold. The adhesion and growth of rat BMSCs seeded on the valve scaffold under different static culture conditions and through scouring experiment were examined to evaluate the effect of PL on promoting the adhesive ability of seeded cells.Method:(1) Isolation, culture and identification of rat BMSCs:Whole bone marrow was isolated and cultured in DMEM-F12medium in vitro for growth and expansion. Flowcytometry was performed for phenotyping. Antibodies used include CD34-FITC, HLDR-PE, CD45-PERCT, and CD90-FTC.(2)PL Preparation:According to Song Huiping’s method, PL was prepared by centrifugation for3times followed by repeated freezing and thawing. Intraperitoneal anesthesia was performed by10%chloral hydrate (0.3ml/100g) and6ml of blood was collected from the rat heart. Heparin was added and the blood was centrifuged at150g for10min. Supernatant and platelet layers were taken followed by centrifugation at1500g for10min. Precipitated platelets was taken and resuspended with2mLPBS solution (pH7.2). Platelet membrane and other cell debris were removed by continuously repeated freezing and thawing (-80℃/37℃) for5times and centrifugation at8000g for30min at4℃.(3) The study of PL promoting the division and adhesion of rat BMSCs on the valve scaffold.1. The preparation of the tissue engineered heart valve leaflets:0.25%Trition-X100,025%sodium deoxycholate,0.02%EDTA,0.02%RNA and0.02%DNA,37degrees Celsius were shocked48hours for acellular processing. Decellularization effect were observed by HE method and electron microscopy. Decellularized porcine aortic valve leaflets were used as the stent, They were divided into four groups:group A (10%FBS-DMEMF-12), B(10%FBS-DMEMF-12-2.5%PL),C(10%FBS-DMEMF-12-5%PL)andD(10%FBS-DMEMF-12-5%PL),16valves in each group. TEHV was constructed with BMSCs as seed cells in vitro. The BMSCs on the valve leaflets seven days after the building was counted with light microscope. The BMSCs adhesion, growth and changes on the scaffold were checked by electron microscope, MTT method, HE.2. Vitro erosion experiments of the TEHV:The experiment was divided into2groups, static and experimental groups, each group then divided into four sub-groups, namely Groups A, B, C, D, eight valve leaflets respectively. MTT assay and cell counting of the valve leaflets after building were conducted7days after construction. In experimental group, scouring experiment was done lasting24hours. the remaining BMSCs cells on Valve leaflets were checked with light microscope, MTT assay、tissue sections.Results:1. After primary culture and serial passages, rat bone marrow mesenchymal stem cells appear fusiform, and cell morphology gradually became identical. Flow cytometric analysis showed the negative rate of CD45and HL-DR expression, and positive rate of CD34and CD90expression is consistent with the phenotypic characteristics of bone marrow mesenchymal stem cells.2. HE staining:HE staining Trition-X100me method was able to completely remove the cellular components on the porcine aortic valve leaflets. The decellularized porcine aortic valve leaflets were white translucent, soft texture, and no cells and cell debris was observed; fiber network structure was basically complete; blue deeply stained nuclei and normal valve leaflets structure were visible in fresh porcine aortic valve leaflets; no significant blank area.3. HE staining showed there was continuous cellular multi-layers of BMSCs on the surface of acellular valve leaflets in group B, group C and group D. In group A, though there is cellular layer on the surface of valve leaflets, cell and matrix adhesion is not tight, and some parts do not have cell coverage. Transmission electron microscopy revealed that the fiber structure of the acellular valve leaflets preserved intact. MTT assay and light microscope showed the cells on the valve leaflets in group B, C, D were significantly more than those in group A, while the cells in group C and D were significantly more than those in group B.4. After high-speed fluid erosion, cells counting and MTT showed the cells on the TEHV in group A and B [(2.25±0.28)×104(2.93±0.28)×104]were significantly less than those in groups C, D[(3.56±0.31)×104、(4.68±0.46)×104](p<0.05). The cells in group D was significantly more than those in groups A, B, C (p<0.05). HE staining showed fewer cells remaining in Group A and B after erosion test, while there were significantly more cells on the valvular surface in groups C, D than those in group A and BConclusion:1. Triton method is able to remove the cells on porcine aortic valves.2. PL can promote the proliferation of BMSCs and its adhesion to the valve leaflets. Better effect is obtained with the increase of PL concentration, and the ideal concentration is7.5%. |
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