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Construction of Engineered Myocardial Tissues in Vitro with Cardiomyocyte‑Like Cells and a Polylactic‑Co‑Glycolic Acid Polymer

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Construction of Engineered Myocardial Tissues in Vitro with Cardiomyocyte‑Like Cells and a Polylactic‑Co‑Glycolic Acid Polymer MOLECULAR MEDICINE REPORTS 20: 2403-2409, 2019 Construction of engineered myocardial tissues in vitro with cardiomyocyte‑like cells and a polylactic‑co‑glycolic acid polymer YUJIE XING1, SHUANG SHI1, YONG ZHANG1, FUQIANG LIU1, LING ZHU1, BINYA SHI2 and JUNKUI WANG1 1First Department of Cardiology; 2Medical Affairs Department, Shaanxi Provincial People's Hospital, Xi'an, Shaanxi 710068, P.R. China Received May 22, 2018; Accepted February 28, 2019 DOI: 10.3892/mmr.2019.10434 Abstract. The aim of the present study was to explore the PLGA scaffolds at 24 h. H&E staining suggested that the feasibility of the construction of engineered myocardial cardiomyocyte-like cells with spindle nuclei were evenly tissues in vitro with cardiomyocyte-like cells derived from distributed in the PLGA scaffold. Immunofluorescence bone marrow mesenchymal stem cells (BMMSCs) and a staining revealed that the cardiomyocyte-like cells were posi- polylactic-co-glycolic acid (PLGA) polymer. The PLGA tive for cardiac troponin I. Scanning electron microscopy polymer was sheared into square pieces (10x10x1 mm), steril- demonstrated that the inoculated cells were well attached ized by Co60 irradiation, and hydrated in Dulbecco's modified to the PLGA scaffold. Transmission electron microscopy Eagle's medium for 1 h. BMMSCs were isolated from the bone indicated that the engineered myocardial tissues contained marrow of Sprague‑Dawley rats and the third passage cells well-arranged myofilaments, desmosomes, gap junction were induced by 5-azacytidine (5-aza). Following successful and Z line-like structures. The present study successfully induction, the cells were trypsinized and suspended at a constructed engineered myocardial tissues in vitro with density of 1x109/ml. Then, the cell suspension was added to the a PLGA polymer and cardiomyocyte-like cells derived PLGA scaffold and cultured for 14 days. The morphological from BMMSCs, which are likely to share various structural changes of BMMSCs were observed using phase contrast similarities with the original heart tissue. microscopy. Immunofluorescence staining was used to identify the cardiomyocyte-like cells. Hematoxylin and eosin (H&E) Introduction and immunohistochemical staining were used to observe the morphology of the engineered myocardial tissues. The cell The incidence of ischemic heart disease is increasing yearly, adhesion rates and scanning electron microscopy were used and heart failure, as a resulting condition, is a worldwide to observe the compatibility of the cardiomyocyte-like cells problem that seriously threatens the survival and quality of life and PLGA. Transmission electron microscopy was used to of the global population (1). Due to the minimal regenerative view the ultrastructure of the engineered myocardial tissues. capacity of the adult heart, it forms scar tissue to replace the BMMSCs in primary culture presented round or short spindle contractile cardiomyocytes upon injury, eventually leading to cell morphologies. Following induction by 5-aza, the cells heart failure (2). The only feasible treatment for patients with exhibited a long spindle shape and a parallel arrangement. end-stage heart failure is heart transplantation, but this is not Analysis of the cell adhesion rates demonstrated that the widely available due to the limited number of donor organs. majority of the cardiomyocyte-like cells had adhered to the At present, myocardial cell transplantation is a popular topic of study in the treatment of ischemic heart disease. Following myocardial cell transplantation, the development of myocar- dial tissue engineering has generated new possibilities for the Correspondence to: Dr Binya Shi, Medical Affairs Department, treatment of ischemic heart disease. Shaanxi Provincial People's Hospital, 256 Youyi West Road, Xi'an, Myocardial tissue engineering aims to reconstruct ideal Shaanxi 710068, P.R. China myocardial tissue by combining cells with scaffolding poly- E‑mail: [email protected] mers to replace and repair the damaged myocardium (3-8). It involves 3 stages: Isolation of the donor cells; development Professor Junkui Wang, First Department of Cardiology, Shaanxi of the scaffold material; and construction of the engineered Provincial People's Hospital, 256 Youyi West Road, Xi'an, Shaanxi 710068, P.R. China myocardial tissue. The source and species of seed cells E‑mail: [email protected] are the key factors in myocardial tissue engineering. At present, there are a number of data concerning heart autolo- Key words: polylactic-co-glycolic acid polymer, 5-azacytidine, gous stem cells, pluripotent stem cells, embryonic stem bone marrow mesenchymal stem cells, cardiomyocyte-like cells, cells, skeletal myoblasts and bone marrow mononuclear engineered myocardial tissues cells (BMMSCs) (9-11). Of these cell types, BMMSCs are widely used for cardiac repair due to their easy accessibility and availability. For scaffold design, the pore size, distribution 2404 XING et al: CONSTRUCTION OF ENGINEERED MYOCARDIAL TISSUES in vitro and interconnectivity are critical factors that determine mass Induction and differentiation of BMMSCs by 5‑azacytidine transfer of oxygen and nutrients to support effective vascular (5‑aza). BMMSCs at third passage were induced using ingrowth within scaffolds. Polylactic-co-glycolic acid (PLGA) complete DMEM-low glucose medium containing 10 µmol/l scaffolds possess high porosity, and good biocompatibility 5-aza (Sigma‑Aldrich; Merck KGaA). After 24 h, the induc- and biodegradability (12), all of which assist to induce tissue tion medium was removed and the medium was changed to formation through cell migration and nutritional diffusion. complete DMEM-low glucose medium without 5-aza at room At present, there are two predominant methods used for temperature. The medium was changed every 3 days. After myocardial tissue engineering. The first method involves 4 weeks of culture, the cells were prepared for subsequent directly injecting cardiomyocytes or cardiac-like cells into experiments. the heart muscle. A number of studies have confirmed that BMMSCs implantation may induce cardiac regeneration Scaffolds preparation. The PLGA scaffolds consisted of 50% and improve cardiac function through myocardial angiogen- polylactic acid and 50% polyglycolic acid. The porous micro- esis (13-15). However, cell implantation studies are usually structure was prepared by a particulate extraction method with limited by restricted cardiomyogenic potential and low cell particle diameter of 150-200 µm, as described previously (22), survival (16-19). The second method involves transplantation and the porosity was 90%. The PLGA polymer was cut into of three-dimensional heart grafts. Li et al (20) reconstructed squares (10x10x1 mm). Co60 irradiation was used to sterilize myocardial cells in three-dimensional structures using a the PLGA scaffolds prior to cell inoculation, and then the preformed biodegradable gelatin mesh. A previous study has scaffolds were soaked in PBS for 1 h and in DMEM for 1 h, suggested that cell sheet transplantation may treat heart failure and finally for the next step of the experiment after water in animal models (21). The present study aimed to construct drainage. engineered myocardial tissues in vitro using a PLGA scaffold and cardiomyocyte-like cells derived from BMMSCs, which Detection of cell adhesion rate. A total of 12 pieces of spare may support the endogenous ability of induced cells to form a PLGA scaffolds were placed in a 24-well culture plate. Then, cardiac tissue-like structure. 1 ml cardiomyocyte-like cells suspension (1x109 cells/ml) was added to each of the PLGA scaffolds. Following incuba- Materials and methods tion at 37˚C in a CO2 incubator for 4, 12, 24 and 48 h, PBS was added slowly to flush non‑adherent cells. This step was Isolation and culture of BMMSCs. A total of 40 male repeated twice. The cells were digested by 2.5 g/l trypsin and Sprague‑Dawley rats (age, 4-weeks-old;) were purchased then counted using a counting board under optical inverted from the Laboratory Animal Center of Fourth Military phase contrast microscope with x100 magnification. The cell Medical University (Xi'an, China). The housing temperature adhesion rate was calculated as follows: Number of adherent ranged between 18 and 26˚C, the humidity ranged between cells/total number of cells x100%. 40 and 70%. All rats had ad libitum access to food and water, with a 12-h light/dark cycle. All rats were anesthetized and Construction of engineered myocardial tissues in vitro. The sacrificed by cervical dislocation. The femurs and tibias of cardiomyocyte-like cells induced by 5-aza were digested rats were removed under sterile conditions. The marrow cavi- by 2.5 g/l trypsin, and suspended at a concentration of ties were washed with Dulbecco's modified Eagle's medium 1x109 cells/ml. The suspension was slowly instilled onto the (DMEM; HyClone; GE Healthcare Life Sciences, Logan, prepared PLGA scaffold, and moved into a 5% CO2 incubator UT, USA), and the mixed suspension was added slowly to at 37˚C. After 4 h, the initial gelation was observed in the the Percoll® solution (1.073 g/ml; Sigma‑Aldrich; Merck PLGA‑cardiomyocyte-like cells compound, and 2 ml culture KGaA, Darmstadt, Germany) for centrifugation at 2,000 x g medium containing 10% FBS was carefully added to completely for 20 min at room temperature. The enriched cells in the submerge the compound. The PLGA‑cardiomyocyte-like cells middle layer were collected and mixed with incomplete compounds were transferred to a 5% CO2
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