A total of 40 male Sprague-Dawley rats (age, 4-weeks-old;) were purchased from the Laboratory Animal Center of Fourth Military Medical University (Xi'an, China). The housing temperature ranged between 18 and 26°C, the humidity ranged between 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 sacrificed by cervical dislocation. The femurs and tibias of rats were removed under sterile conditions. The marrow cavities were washed with Dulbecco's modified Eagle's medium (DMEM; HyClone; GE Healthcare Life Sciences, Logan, UT, USA), and the mixed suspension was added slowly to the Percoll^® solution (1.073 g/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for centrifugation at 2,000 × g for 20 min at room temperature. The enriched cells in the middle layer were collected and mixed with incomplete culture medium for centrifugation at 1,500 × g for 15 min at room temperature. Following removal of the supernatant, the cells were re-suspended with complete DMEM-low glucose medium supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 µg/ml streptomycin, 100 U/ml penicillin, 10 mmol/l HEPES and 300 mg/l L-glutamine, and then inoculated in a 25 cm² culture flask at 37°C in a humidified atmosphere of 5% CO₂. The non-adherent cells were discarded after 3 days. The culture medium was changed every 3 days. When the cells reached 80% confluence, the cells were digested with 2.5 g/l trypsin and passaged at a 1:2 ratio. The study was approved by the Ethical Review Committee of Shaanxi Provincial People's Hospital (Xi'an, China). All procedures involving animals were in accordance with the ethical standards of the Institutional Research Committee of Shaanxi Provincial People's Hospital.

BMMSCs at third passage were induced using complete DMEM-low glucose medium containing 10 µmol/l 5-aza (Sigma-Aldrich; Merck KGaA). After 24 h, the induction medium was removed and the medium was changed to complete DMEM-low glucose medium without 5-aza at room temperature. The medium was changed every 3 days. After 4 weeks of culture, the cells were prepared for subsequent experiments.

The PLGA scaffolds consisted of 50% polylactic acid and 50% polyglycolic acid. The porous microstructure was prepared by a particulate extraction method with particle diameter of 150–200 µm, as described previously (22), and the porosity was 90%. The PLGA polymer was cut into squares (10×10×1 mm). Co⁶⁰ irradiation was used to sterilize the PLGA scaffolds prior to cell inoculation, and then the scaffolds were soaked in PBS for 1 h and in DMEM for 1 h, and finally for the next step of the experiment after water drainage.

A total of 12 pieces of spare PLGA scaffolds were placed in a 24-well culture plate. Then, 1 ml cardiomyocyte-like cells suspension (1×10⁹ cells/ml) was added to each of the PLGA scaffolds. Following incubation 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 repeated twice. The cells were digested by 2.5 g/l trypsin and then counted using a counting board under optical inverted phase contrast microscope with ×100 magnification. The cell adhesion rate was calculated as follows: Number of adherent cells/total number of cells ×100%.

The cardiomyocyte-like cells induced by 5-aza were digested by 2.5 g/l trypsin, and suspended at a concentration of 1×10⁹ cells/ml. The suspension was slowly instilled onto the prepared PLGA scaffold, and moved into a 5% CO₂ incubator at 37°C. After 4 h, the initial gelation was observed in the PLGA-cardiomyocyte-like cells compound, and 2 ml culture medium containing 10% FBS was carefully added to completely submerge the compound. The PLGA-cardiomyocyte-like cells compounds were transferred to a 5% CO₂ incubator at 37°C for subsequent culture. The medium was changed every 2 days. The total culture time was 14 days.

The immunofluorescence staining of cardiac troponin I (cTnI; cat. no. sc-8118; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was performed to identify whether BMMSCs induced by 5-aza had differentiated into cardiomyocytes. The cells were fixed using 4% polyoxymethylene at 4°C for 20 min, incubated in 3% hydrogen peroxide and methanol for 10 min at room temperature, blocked in normal goat serum (Abcam, Cambridge, UK) at 37°C for 30 min, and incubated with cTnI antibody [polyclonal goat anti-mouse immunoglobulin G (IgG); 1:25 dilution] overnight at 4°C. Subsequent to washing with PBS, the cells were stained by fluorescent isothiocyanate-conjugated rabbit anti-goat IgG (cat. no. sc-2777; Santa Cruz Biotechnology, Inc.; 1:50 dilution) for 40 min at 37°C. Finally, the cells were counterstained with Hoechst 33258 at a 1:1,000 dilution, at room temperature, for 10 min, to visualize the nuclei. Fluorescence microscopy (Olympus BX-51; Olympus Corporation, Tokyo, Japan), at an ×200 magnification, was used to observe the cells and record the results.

After 14 days of culture in vitro, the engineered myocardial tissues were washed with PBS, fixed in 4% paraformaldehyde at 4°C for 24 h, dehydrated on an ascending series of ethanol, embedded in paraffin, sectioned into 10 µm slices and finally were stained with hematoxylin for 10 min and 0.5% eosin for 1 min at room temperature (H&E). For the immunohistochemical staining of cTnI, the sections were incubated with the primary antibodies directed against cTnI (polyclonal goat anti-mouse IgG; cat. no. sc-8118; Santa Cruz Biotechnology, Inc.; 1:25 dilution) at 4°C overnight, and then incubated with secondary antibody (rabbit anti-goat IgG; cat. no. sc-2768; Santa Cruz Biotechnology, Inc.; 1:1,000 dilution concentration) at 37°C for 30 min. A total of 1 mg/ml DAB reagent was added to the cells for 5–10 min at room temperature. The sections were sealed with neutral gum for microscopic examination under optical inverted phase contrast microscope at ×200 magnification. Cells with brown granular DAB reaction product in the cytoplasm were considered positive for the protein.

After 14 days of culture in vitro, the engineered myocardial tissues were fixed in 3% glutaraldehyde at 4°C for 2 h. Samples were then coated with a thin layer of gold for 5 min in a Sputter-coater JFC-1100. The specimen was viewed using a Hitachi S-3400N scanning electron microscope operating at a typical 5 kV accelerating voltage, 20°C and 10⁻⁵ Torr.

After 14 days of culture in vitro, the engineered myocardial tissues were initially fixed in 3% glutaraldehyde at 4°C for 2 h, post-fixed in 1% osmium tetroxide at 4°C for 20 min and embedded in epoxy resin at 45°C for 12 h. Ultrathin sections of 1 µm were prepared and double stained with 2% uranyl acetate at room temperature for 15 min and 6% lead citrate at room temperature for 15 min. The cellular ultrastructure was observed with a JEM-2000EX transmission electron microscope (JEOL, Ltd., Tokyo, Japan).

Data are presented as mean ± standard deviation. Data were compared using standard or repeated measures analysis of variance where appropriate. Pairwise comparisons between groups were performed using the least significant difference test. P<0.05 was considered to indicate a statistically significant difference.

After 3 days of primary culture, the adhered BMMSCs were observed to have round or short spindle morphologies (Fig. 1A). Following subculture, the cells became polygonal or long spindle-shaped (Fig. 1B). Subsequent to induction by 5-aza, a proportion of cells died, but the surviving cells began to proliferate and differentiate. Then, the cells aggregated and gradually increased in size 4 weeks later; the adherent cells were completely in contact with neighboring cells and arranged in a uniform direction (Fig. 1C).

The adhesion rates of cardiomyocyte-like cells in PLGA scaffolds were as demonstrated in Fig. 2. It was identified that the adhesion rates at 12, 24 and 48 h were markedly increased compared with that at 4 h (P<0.01), and the adhesion rates at 24 and 48 h were increased compared with that at 12 h (P<0.05). However, the adhesion rate of 48 h was not statistically significant compared with 24 h (P>0.05). It was indicated that the adhesion rate of the cardiomyocyte-like cells increased gradually with increases in culture time. The majority of the cardiomyocyte-like cells had adhered to PLGA scaffolds at 24 h.

The expression of cTnI was estimated by immunofluorescence staining following 5-aza induction. The results suggested that the cells at 4 weeks after induction by 5-aza exhibited green fluorescence (Fig. 3A). The nuclei of the cells exhibited blue fluorescence (Fig. 3B). Fig. 3C represents the merged data from Fig. 3A and B.

H&E staining demonstrated the presence of an elongated pattern, marked organized striations, and a low nucleus: Cytoplasm ratio (Fig. 4A), which indicated the structural maturation of the engineered myocardial tissue. Immunohistochemical staining revealed that the cells in the engineered myocardial tissues contained a number of differentiated cardiomyocytes that were positive for cTnI (Fig. 4B), revealing the presence of cardiomyocyte-like cells within PLGA scaffolds.

The results of the scanning electron microscopy suggested that the inoculated the cells were well attached to PLGA, and the cells grew and proliferated in 3 dimensions in the engineered myocardial tissues (Fig. 5A). A large number of cell enations in the surface of PLGA were observed, indicating good adhesion of these cells (Fig. 5B). In particular, the cells were able to secrete a large number of extracellular matrix proteins, which are necessary for cell differentiation (Fig. 5C).

The results of the transmission electron microscopy demonstrated the presence uniformly distributed myofilaments (Fig. 6A) and clearly-defined Z line-like structures (Fig. 6B), which are the signs of mature cardiomyocytes. In addition, the presence of specialized junctional structures, including desmosomes and gap junctions, was observed (Fig. 6C and D), which were responsible for electromechanical coupling between neighboring cardiomyocytes.