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Engineering a 3D cardiac patch through cardiomyogenic differentiation of rMSCs by synchronous mechanical and electrical stimulations

机译：通过同步的机械和电刺激通过rMSC的心肌分化来设计3D心脏补丁

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Introduction: Cardiac tissue engineering has been studied for almost 20 years since the first study about this subject in 1995. 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. 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 deceliularization methods by triton ×100 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.
机译：简介：自1995年对该主题进行首次研究以来，心脏组织工程学已进行了近20年的研究。然而，心脏组织工程学尚未进入临床，并且由于缺乏肝组织工程学，每年都有许多患者（成人和儿童）死亡。适当的心脏补丁。心脏的心肌组织沿心脏ECM网络具有先进的肌纤维结构，从而促进了独特的细胞间互连。心脏组织工程学的主要障碍一直是模仿这种独特的ECM网络。近来，无细胞支架在组织工程学中已经引起了人们的广泛关注。去细胞心包支架提供最佳特性，因为它保留了用于心脏组织再生的自然超微结构，机械和组成线索，并具有用于干细胞播种，心肌细胞分化和血管生成的最佳微环境。这项研究的目的是通过使用脱细胞牛心包膜制作3D心脏贴片（Bio-Patch）。为此，将心肌细胞与多能间充质干细胞区分开来，并评估了不同刺激物对其心肌原性分化的影响。方法：从屠宰场获得牛心包，并用Triton×100或十二烷基硫酸钠（SDS）进行不同的去皮方法。通过使用1 M乙酸和0.5 U / mL胶原酶优化无细胞结构的孔径，并通过SEM对其进行表征。从大鼠骨髓中分离出MSC，并通过流式细胞仪进行了表征。此外，他们的多系分化潜力进行了评估。化学作用（5氮杂胞苷）和物理刺激物的作用;研究了电刺激和机械刺激对MSCs心肌分化的影响。通过使用定制设计的生物反应器系统施加电刺激和机械刺激。设计的生物反应器系统能够通过软件以单独或同步的方式施加电刺激和机械刺激。将无细胞心包膜植入MSC，并置于组织培养室内。在添加5个氮杂胞苷后的2分钟内，在2周的培养期间内将细胞暴露于单独的或同时的机械应变和电刺激下。将20％的单轴机械应变（频率为1 Hz）施加到支架上。对于电刺激，施加频率为1 Hz，脉冲宽度为3 ms的1V，3V和5V电压。进行了免疫组织化学和RT-PCR分析，以评估MSCs的心脏组织形成和心肌分化。结果与讨论：扫描电子显微镜图像显示，制备的无细胞支架具有多孔结构，平均孔径为54.5μm。分离细胞的流式细胞术分析表明，这些细胞显示出99％的MSC阳性特征。在分化研究中，发现浓度为10μM的5氮杂胞苷会刺激心肌分化，但是仅靠其自身不足以分化为心肌细胞。另一方面，发现单独的机械和电刺激可以增加心肌细胞特异性基因（GATA 4，MEF2c，Nkx2.5，CACNA1C）的表达。同时，同时进行的机械和电刺激诱导了心肌细胞标志物的大量表达。结论：结论：同步机械和电刺激可增加MSCs的心肌分化和心肌生物标志物的表达。

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