Introduction: Cardiac tissue engineering has been studied for almost 20 years since the first study about this subject in 1995[1]. Nevertheless, cardiac tissue engineering has not entered into the clinical yet and many patients (adults and children) die each year due to the lack of proper cardiac patches. Myocardial tissue of the heart has advanced muscle fiber architecture along cardiac ECM networks, which facilitates unique cell-to-cell interconnections. The main obstacle in cardiac tissue engineering has been to mimic this unique ECM network. Recently, acellular scaffolds have got so much attention in tissue engineering as scaffolds. Decellularized pericardium scaffold provides optimal characteristics as it preserves natural ultrastructural, mechanical, and compositional cues for cardiac tissue regeneration and bears optimal microenvironments for stem cell reseeding, cardiomyocyte differentiation, and angiogenesis[2]. The aim of this study was to produce a 3D cardiac patch (Bio-Patch) by using acellular bovine pericardium. With this purpose, cardiomyocytes were differentiated from multipotent mesenchymal stem cells and the effect of different stimulators on their cardiomyogenic differentiation was evaluated.
Methods: Bovine pericardium was obtained from slaughter house and put through different decellularization methods by triton x100 or sodium dodecyl sulfate (SDS). The pore size of the acellular structure was optimized by using 1 M acetic acid and 0.5 U/mL collagenase and characterized by SEM. MSCs were isolated from rat bone marrow and characterized by flow cytometry. Also, their multilineage differentiation potential was evaluated. The effect of chemical (5 azacytidine) and physical stimulators like; electrical stimulation and mechanical stimulation on cardiomyogenic differentiation of MSCs were studied. Electrical and mechanical stimulations were applied by using a custom designed bioreactor system. Designed bioreactor system was capable of applying both electrical and mechanical stimulations in a separate or synchronous manner through a software. Acellular pericardia were seeded with MSCs and placed inside the tissue culture chambers. Inside the chambers after adding 5 azacytidine cells were exposed to separate or simultaneous mechanical strain and electrical stimulations during the 2 weeks culture period. Uniaxial mechanical strain of 20% with the frequency of 1 Hz was applied to the scaffold. For the electrical stimulation, voltages of 1V, 3V and 5V with the frequency of 1 Hz and pulse width of 3 ms were applied. Immunohistochemistry and RT-PCR analysis were performed in order to evaluate the cardiac tissue formation and cardiomyogenic differentiation of MSCs.
Results and discussion: Scanning electron microscope images showed that prepared acellular scaffolds have a porous structure with the mean pore size of 54.5 µm. Flow cytometry analysis of isolated cells showed that these cells show 99% MSC positive characteristic. During the differentiation studies it was found that 5 azacytidine in 10 µM concentration stimulate the cardiomyogenic differentiation, however by itself it is not enough for differentiation to cardiomyocytes. On the other hand, it was found that separate mechanical and electrical stimulations can increase the expression of cardiomyocytes specific genes (GATA 4, MEF2c, Nkx2.5, CACNA1C). In the meantime, simultaneous mechanical and electrical stimulation induced a tremendous expression of cardiomyocytes markers.
Conclusion: It was concluded that cardiomyogenic differentiation of MSCs and the expression of cardiomyocyte biomarkers can be increased when synchronous mechanical and electrical stimulations are applied.
This study is supported by The Scientific and Technological Research Council of Turkey, grant number 213M685.
References:
[1] Eschenhagen, T., et al., Faseb J (1997), 11, 683-694.
[2] Wang, B., et al., Journal of Biomedical Materials Research Part A (2010), 94A, 1100-1110.