Engineered myocardial tissues constructed in vivo using cardiomyocyte-like cells derived from bone marrow mesenchymal stem cells in rats.

BACKGROUND: To explore the feasibility of constructing engineered myocardial tissues (EMTs) in vivo, using polylactic acid -co-glycolic acid (PLGA) for scaffold and cardiomyocyte-like cells derived from bone marrow mesenchymal stem cells (BMMSCs) for seeded cells. METHODS: BMMSCs were isolated from femur and tibia of Sprague-Dawley (SD) rats by density-gradient centrifugation. The third passage cells were treated with 10 µmol/L 5-azacytidine (5-aza) and 0.1 µmol/L angiotensin II (Ang II) for 24 h, followed by culturing in complete medium for 3 weeks to differentiated into cardiomyocyte-like cells. The cardiomyocyte-like cells were seeded into PLGA scaffolds to form the grafts. The grafts were cultured in the incubator for three days and then implanted into the peritoneal cavity of SD rats. Four weeks later, routine hematoxylin-eosin (HE) staining, immunohistochemical staining for myocardium-specific cardiac troponin I (cTnI), scanning electron microscopy and transmission electron microscopy were used to analyze the morphology and microconstruction of the EMTs in host rats. RESULTS: 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 group 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. CONCLUSION: We have developed an in vivo method to construct engineered myocardial tissue. The in vivo microenvironment helped engrafted cells/tissue survive and share similarities with the native heart tissue.

Construction of engineered myocardial tissues in vitro with cardiomyocyte­like cells and a polylactic­co­glycolic acid polymer.

The aim of the present study was to explore the feasibility of the construction of engineered myocardial tissues in vitro with cardiomyocyte­like cells derived from bone marrow mesenchymal stem cells (BMMSCs) and a polylactic­co­glycolic acid (PLGA) polymer. The PLGA polymer was sheared into square pieces (10x10x1 mm), sterilized by Co60 irradiation, and hydrated in Dulbecco's modified Eagle's medium for 1 h. BMMSCs were isolated from the bone marrow of Sprague­Dawley rats and the third passage cells were induced by 5­azacytidine (5­aza). Following successful induction, the cells were trypsinized and suspended at a density of 1x109/ml. Then, the cell suspension was added to the PLGA scaffold and cultured for 14 days. The morphological changes of BMMSCs were observed using phase contrast microscopy. Immunofluorescence staining was used to identify the cardiomyocyte­like cells. Hematoxylin and eosin (H&E) and immunohistochemical staining were used to observe the morphology of the engineered myocardial tissues. The cell adhesion rates and scanning electron microscopy were used to observe the compatibility of the cardiomyocyte­like cells and PLGA. Transmission electron microscopy was used to view the ultrastructure of the engineered myocardial tissues. BMMSCs in primary culture presented round or short spindle cell morphologies. Following induction by 5­aza, the cells exhibited a long spindle shape and a parallel arrangement. Analysis of the cell adhesion rates demonstrated that the majority of the cardiomyocyte­like cells had adhered to the PLGA scaffolds at 24 h. H&E staining suggested that the cardiomyocyte­like cells with spindle nuclei were evenly distributed in the PLGA scaffold. Immunofluorescence staining revealed that the cardiomyocyte­like cells were positive for cardiac troponin I. Scanning electron microscopy demonstrated that the inoculated cells were well attached to the PLGA scaffold. Transmission electron microscopy indicated that the engineered myocardial tissues contained well­arranged myofilaments, desmosomes, gap junction and Z line­like structures. The present study successfully constructed engineered myocardial tissues in vitro with a PLGA polymer and cardiomyocyte­like cells derived from BMMSCs, which are likely to share various structural similarities with the original heart tissue.