Construction Of Engineered Myocardial Tissue In Vivo With Cardiomyocyte Cells Derived From Bone Marrow Mesenchymal Stem Cells

BackgroundHeart failure caused by ischemia heart disease was increasing year by year. If heart failure is not treated timely and effectively, there will be a lot of degeneration and necrosis of myocardial cells, leading to more serious consequences and a variety of complications, which seriously threatens human'survival and life qualities. Adult cardiomyocytes are terminally differentiated and can not regenerate after injury.Established pharmacologic treatment can decelerate but not cure and rehabilitate end-stage heart failure once a significant part of the myocardium tissue is damaged. Currently heart transplantation is the only viable treatment option but is not widely used in clinical due to lack of donor heart organs and expensive costs, so it is necessary for researchers to explore a new therapeutic tool. Myocardial cell transplantation is a hot topic in the treatment of ischemic heart disease in recent years and develops rapidly. Since then, the birth and rise of myocardial tissue engineering has brought the dawn for the treatment of ischemic heart disease, brought the new hope for the reconstruction of functional cardiac muscle and become the most challenging goals in the areas of myocardial tissue engineering. Currently, myocardial tissue engineering has made great progress and the reports about this field are also increasing in and abroad, which will surely lay a solid foundation for curing the heart diseases.Objectives1. To investigate the effects and differentiation ratio of bone marrow mesenchymal stem cells (BMMSCs) differentiation into cardiomyocyte-like cells induced by angiotensin (AngⅡ) and 5-azacytidine (5-aza) in vitro.2. To explore the feasibility and effect of constructing engineered myocardial tissues (EMTs) in vivo, using polylactic acid -co-glycolic acid (PLGA) and cardiomyocyte-like cells derived from BMMSCs.Methods1. AngⅡand 5-aza induce BMMSCs to cardiomyocyte-like cells in vitro: BMMSCs were isolated from bone marrow of Sprague-Dawley rats by density gradient centrifugation. The third passage cells were divided into four groups : AngⅡand 5-aza group (0.1μmol/L and 10μmol/L),AngⅡgroup (0. 1μmol/ L),5-aza group (10μmol/ L ),untreated group as control. After 24 h induction, the medium was changed to the complete culture medium without any inductor and the cells were culture for four weeks. The morphological changes were observed under phase contrast microscope. The cell proliferation ability was observed with methyl thiazolyl tetrazolium (MTT) assay. The cardiomyogenic cells were identified by immunofluorescence staining.The induction ratio was examined by flow cytometer. The level of cardiac troponin-I (cTnI) was examined by western blotting. The ultrastructures of the induced cells were viewed with a transmission electron microscope.2. Construction of EMTs in vivo using PLGA and cardiomyocyte-like cells derived from BMMSCs: PLGA polymer was sheared to square pieces, sterilized by 60Co irradiation, and hydrated in PBS and DMEM. After successful induction, the cardiomyocyte-like cells suspension were seeded into PLGA scaffolds to form the cell-scaffold grafts .The grafts were cultured in the incubator for three days and then implanted into the peritoneal cavity of the rats. Four weeks later, routine hematoxylin-eosin (HE) staining, immunohistochemical staining for myocardium-specific cTnI, scanning electron microscopy and transmission electron microscopy were used to analyze the morphology and structure of the EMTs in host rats.Results1. BMMSCs of primary culture formed cell colonies at 14 days. The passaged cells were larger than those of primary culture. After induction, the cells presented long spindle, aligned in parallel and formed muscle island-like structure. MTT assay showed that the absorbance values in the third day in AngⅡand 5-aza group,AngⅡgroup,5-aza group and control group were respectively 0.209±0.044,0.148±0.009,0.129±0.012,0.103±0.008,in the seventh day were respectively 0.284±0.014,0.224±0.010,0.220±0.009,0.169±0.019. The cell proliferation in AngⅡand 5-aza group outweighed that of in AngⅡgroup or 5-aza group(P<0.05). The expression of specific protein cTnI in induced BMMSCs was positive. Flow cytometer showed that the differentiation ratio in AngⅡand 5-aza group,AngⅡgroup,5-aza group and control group were respectively(30.0±1.7) %,(25.3±2.2) %,(24.6±1.9) %, (0.8±0.2)%,demonstrating that the differentiation ratio in AngⅡand 5-aza group was higher than that of in AngⅡgroup or 5-aza group(P<0.05). Transmission electron microscopy showed that the induced cells had myofilaments, Z line-like substances.2. HE staining showed that the cardiomyocyte-like cells distributed equally in the PLGA scaffold, and the nuclei arranged in the spindle shape. Immunohistochemical staining revealed that majority of engrafted cells in the PLGA -Cardiomyocyte-like cells grafts were positive for cTnI. Scanning electron microscopy showed that the inoculated cells well attached to PLGA and grew in 3 dimensions in construct. Transmission electron microscopy showed that the EMTs contained well arranged myofilaments paralleled to the longitudinal cell axis, the cells were rich in endoplasmic reticulum and mitochondria, while desmosomes, gap junction and Z line-like substances were also can be observed as well within the engrafted cells.Conclusion1. AngⅡcombined with 5-aza can induce the differentiation of BMMSCs into cardiomyocyte-like cells, and the differentiation ratio was higher than that of in 5-aza only(P<0.05).2. With PLGA and cardiomyocyte-like cells derived from BMMSCs, the EMTs which resemble native cardiac tissues can be successfully constructed in vivo.