Effect Comparison Of Myocardial Differentiation For ADMSCs In 2 D And 3 D Cultivation

BackgroudRegeneration ability of Myocardial cells is limited, so the cardiac tissue engineering has brought infinite hopes and new treatment strategies for the restoration of the damaged myocardium in the present study. A wide range of stem and progenitor cells are currently under Investigation, the Adipose Mesenchymal Stem Cells (Adipose Mesenchymal Stem Cells, ADMSCs) has become one of the ideal seed Cells in myocardial tissue engineering due to their optimal safety profile and their abundant sources, conveniently achieve progress (availability via an even less traumatic liposuction) and less ethical limits. Atelocollagen scaffold is one of the most important entities for efficient cardiac tissue engineering, because of its biocompatibility, degradation and mechanical properties which can be made into three-dimensional structure to provide physiological support for cell attachment, proliferation and development. But It has not yet know whether ADMSCs can be trained to be three-dimensional myocardial on Atelocollagen stent. This study tried to plant induced myocardial-ADMSCs on the scaffold in vitro and make them into three-dimensional myocardial.ObjectiveTo explore the effect of myocardial differentiationin of mice Adipose-derived stem cells which cultured under the condition of 2 d and 3 d and induced in vivo and in vitro. and then judge the survival situation of three-dimensional culture ADMSCs within the heart. To provide experimental reference for cardiac tissue engineering.Methods1. ADMSCs were obtained by in vitro culture of the stromal vascular fraction (SVF) isolated from inguinal adipose tissue. The cells were spread to passages 3. Mark the cells with CD29+, CD45- for sorting ADMSCs by flow cytometry.2. Induce ADMSCs with adipogenic, osteogenesis induction medium and detected by oilred O staining and alizarin red staining respectively to test the multiple differentiation potential.3. Adipose-derived stem cells were treated with 5-azacytidine for 24 hours and cultured in cardiac microenvironment respectively to assess the role of 5-zaz and cardiac microenvironment in directing the differentiation of adiposetissue-derived stem cells, after that detect myocardial specific markers cTnT with immunofluorescence technique..4. ADMSCs were labeled with DAPI, PKH26 and GFP slow virus respectively, and then were planted into atelocollagen scaffold for three-dimensional(3D)cultures. Observe the labeling rate of growth cadiocytes in atelocollagen scaffold dynamically by inverted microscopy.5. ADMSCs were planted into atelocollagen three-dimensional scaffold and induced by 5-aza and myocardial micro environment respectively, to observe myocardial differentiation at different time points, and then to detect the expression of myocardial specificity mark cTnT with immunofluorescence technique. To observe the degradation situation of Atelocollagen scaffold in myocardial and ADMSCs survival situation within myocardial. Using immunofluorescence technique to detect expression of gap junction protein Cx43, so as to analysis the functional relationship between cells and the host myocardial cells.Results1. In this study, ADMSCs were extracted from mice inguinal fat, Immune phenotype (CD29+、CD45-)and differentiation potential of its various studies by flow cytometry and immunofluorescence detection suggesting that the cells possess the characteristics of stem cells.2. After induced by adipogenic and osteogenesis for 14 days, ADMSCs can differentiate into adipose cells and bone cells, which showing that the cultured ADMSCs has multiple differentiation potential.3.5-aza in vitro and rat myocardium microenvironment in vivo both can make ADMSCs differentiate into myocardial cells. But the differentiation efficiency of myocardium microenvironment is significantly higher than 5-aza group, and it has the time dependencies(the differentiation efficiency of 5-aza induced group after 3 weeks is (33.33 ±3.79)%, and the differentiation efficiency of myocardium microenvironment induced group after 1 week is (42.93±4.04)%), Compared with 5-aza induced group, myocardium microenvironment induced group after 1 week has a higher efficiency of differentiation (P<0.05)4. DAPI, PKH26 and GFP all can mark ADMSCs greatly, and the labled cells in Atelocollagen collagen scaffold have a strong ability of growth and multiplication. Mark rate are DAPI (92.7%)、PKH26 (89.2%)、GFP (95%)5. In the 3 d after implanted, only very little ADMSCs can be seen, and most of the transplanted cells migrate outside stents. In the 7 d, part of implanted cells express cardiac-specific marker, which prove that implanted cells differentiated into myocardial cells. However,the expression of Cx43 were negative, implied that implanted cells fails to establish contaction with the host cells. In addition, atelocollagen scaffold degrade at the time of 13 days. Adipose-derived stem cell-based three-dimensional scaffold induced by 5-aza and myocardium microenvironment can both differentiate into myocardial cells. Conclusions1. Differentiate efficiency of ADMSCs in rat vivo myocardial microenvironment is higher than 5-aza induced in vitro.2. Atelocollagen scaffold has good biological compatibility, and it can effectively improve ADMSCs adhesion and growth capacity.5-aza and myocardial microenvironment can make ADMSCs based on scaffold differentiate into myocardial cells, but the latter cells ofter migrate can migrate out of scaffold, and differentiate into myocardial cells around the body’s cells.