Konjenital Septal Defektlerin Onarımına Yönelik Perikard Bazlı Rejeneratif Kardiyak Yamaların Doku Mühendisliği Yöntemi ile Geliştirilmesi

Abstract

Cardiac patch has been used in the treatment of congenital heart disease and myocardial infarction (MI) while an ideal cardiac patch has not been developed yet. This study aimed to engineer regenerative cardiac patches by cardiac tissue engineering strategies to be used in various cardiac tissue damage such as congenital heart defect and MI repair. In the first part, rat bone marrow-derived mesenchymal stem cells (rt-BMSCs) were seeded into acellular bovine pericardium-based 3-dimensional (3D) tissue scaffolds and differentiated into cardiomyocytes using a biomimetic bioreactor system that mimics excitation-contraction coupling in the heart following 10 µM 5-azacytidine (5-Azc) treatment. The effect of applied biophysical stimulations on cardiomyogenic differentiation was investigated. qPCR analysis showed that cardiac-specific markers (GATA-4, MEF2C, NKX2.5 and CACNA1C) were upregulated in electromechanically stimulated stem cells. The performance of electromechanically stimulated cardiac patches was assessed in a rat MI model and histological stainings were performed after 21 days of post-implantation. Results showed that MI was induced successfully and patch-related calcification was not observed in all tested groups. Moreover, a higher number of BrdU-labelled cells and a low level of CD68 positive cells were observed in the infarct region under electromechanically stimulated conditions compared to static conditions. In addition, cTroT, MHC, SAC, and nCAD positive cells were observed in both infarct region and retrieved patch after 3 weeks. Although promising results were obtained in the first part of the study, it was concluded that biophysical stimulations are not sufficient for cardiomyogenic differentiation of stem cells and novel strategies should be unveiled to enable more effective cardiomyogenic differentiation. Within this scope, we tested several protocols in which chemical (5-Aza and Dimethylsulfoxide (DMSO)) and biological stimulators (TGF-β1, FGF-2 and BMP-2) were used in different combinations to increase cardiomyogenic differentiation of rt-BMSCs in the second part of this study. qPCR results indicated that the highest cardiac-specific marker expressions were measured in the combination of 5-Aza (10 µM) and DMSO (1%) after 7 days. Immunofluorescence analysis results demonstrated that rt-BMSCs expressed cardiac-specific proteins (cTroT, SAC, Cnx43, and α-SMA) and differentiated into cardiomyocytes in 5-Aza and DMSO induction while rt-BMSCs-derived cardiomyocytes showed fetal cardiomyocyte-like proteins expression patterns and disorganized sarcomere structure. Both rt-BMSCs and human bone marrow-derived mesenchymal stem cells (h-BMSCs) were exposed to a variety of small-molecules (valproic acid (Va), 5-Aza, DMSO, IWP-2 (WNT inhibitor) and RepSox (TGF-β inhibitor)] combinations to manipulate epigenetic profile (acetylation activation and methylation inhibition) and signaling pathways (WNT and TGFβ signaling pathway inhibition) to develop adult-like contractile cardiomyocytes with highly organized sarcomere structure. Cell morphology, cell viability, cardiac specific gene and protein expressions were evaluated. Results showed that cell morphology and cardiac-specific gene expression level were highly dependent on small molecule combination and stem cell resources. It was observed that h-BMSCs had a homogeneous morphology (elongated spindle-shaped) with high cell viability and GATA-4, NKX2.5, and MEF2C genes were significantly higher expressed in these cells compared to rt-BMSCs. Immunofluorescence analysis was also performed after 14 days and results clearly showed that protein expression pattern and sarcomere organization were highly dependent on stem cell source and differentiation protocol. Both rt-BMSCs and h-BMSCs were successfully differentiated into cardiomyocytes at the translational level and expressed cardiac-specific proteins including cTroT, SAC, Cnx43, and α-SMA. However, adult-like protein expression patterns and more organized sarcomere structure were observed in h-BMSCs-derived cardiomyocytes compared to rt-BMSCs-derived cardiomyocytes. In conclusion, mesenchymal stem cells showed higher cellular differentiation into cardiomyocytes in the presence of small molecules and h-BMSCs were more convenient cell type to derive adult-like cardiomyocytes for cardiac tissue engineering applications.