Polyvinyl Alcohol - Polyvinyl Pyrrolidone and Polycaprolactone Based Matrices for Cardiac Tissue Engineering Applications

Abstract

Cardiovascular diseases (CVDs) are leading cause of death in the developed and industrialised world and are responsible for approximately ~30% of all global death. In recent years, stem cell-based cardiac tissue engineering (CTE) has emerged as promising technology for the treatment of CVDs in severely damaged heart. The present study deals with the development of polymeric construct for CTE applications. In the first set of experiments, polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) blended film and nanofiber were fabricated and effects of cross-linker and plasticizers on their physico-chemical and biological properties were analyzed. In the second set of experiments, random and aligned polycaprolactone (PCL) nanofibers were spun by electrospinning and were characterized for their application in CTE. Results showed that, PVA-PVP blends could be successfully developed as biodegradable, haemocompatible and cytocompatible cardiac patches with desirable mechanical properties. Optimized molar ratio of PVA, PVP and plasticizers resulted in films, which were dry at room temperature and had desirable folding endurance value (FEV) of at least 300, tensile strength of 5-30 MPa and, percentage elongation at break of more than 250%. Upon contact with PBS, these PVA-PVP films formed hydrogel patches having the tensile strength of 1.3 to 3.0 MPa and supported the attachment, viability and proliferation of H9C2 cardiac cells and primary neonatal cardiomyocytes (CMs). In vivo transplantation of the PVA-PVP patches into subcutaneous pouch and on the heart revealed them to be biodegradable, biocompatible and safe for use. The PVA-PVP blends could also be electrospun to develop nanofibrous matrices (av. dia. 100-200 nm). The study further showed that both random and aligned nanofibrous matrices (av. dia: 350-850 nm) could also be successfully spun from PCL by electrospinning on copper and stainless steel collectors, respectively. Collagen-coated aligned PCL nanofibers showed significantly higher cell adhesion, proliferation and, enhanced cell growth of H9C2 cardiac cells when compared to random PCL nanofibers. Moreover, aligned PCL nanofibers showed an ability to control the orientation of the cells in parallel direction of fibers, thus providing an effective way to control anisotropic nature under in vitro condition. The umbilical cord matrix (UCM)-derived mesenchymal stem cell (MSCs) and adult foreskin-derived induced pluripotent stem cells (iPSCs) were also differentiated into CMs for subsequent construction of cardiac tissue construct. UCM-derived MSCs were isolated successfully by routine enzymatic digestion and a non-enzymatic explant culture method and characterized based on their microscopic observation, differentiation into different lineages such as chondrocytes, osteocytes and adipocytes and surface marker expression of CD34, CD45, CD73, CD14, CD19, HLA-DR, CD90, CD105 by flow cytometry analysis. Treatment of UCM-derived MSCs, with 5-Azacytidine (5 μM) induced their differentiation into putative cardiac cells, as revealed by the expression of Cardiac-specific Troponin T (cTnT), smooth muscles actin (SMA), Myogenin (MYOG), Smoothelin (SMTN), and Cardiac α-actin (ACTC) genes and cTnT and α-actinin proteins by RT-PCR and immunocytochemistry, respectively. However, no beating cells were observed in differentiated MSCs. On the other hand, adult human foreskin-derived iPSCs could be successfully expanded and proliferated on MatrigelTM - coated aligned PCL nanofibrous matrices and was superior to SynthemaxTM - coated PCL nanofiber or tissue culture coated polystyrene (TCP) surfaces. The iPSCs cultured on MatrigelTM - coated aligned PCL nanofibrous matrices showed anisotropic behaviour along the PCL nanofibers and, upon differentiation, expressed cardiac-specific cTnT proteins and showed spontaneous beating. The beating CMs differentiated on MatrigelTM - coated PCL nanofibrous matrices showed significantly higher percentage of cTnT-positive CMs (23.34 vs. 32.55%) and showed more synchronized beating than those differentiated on MatrigelTM - coated TCP. Thus, electrospun aligned PCL nanofibers may be more suitable for proliferation and differentiation of stem cells into beating CMs than random PCL nanofibers or TCP. Results also suggest that the topology of PCL nanofibers, 5-Azacytidine treatment and collagen coating together contributes to the generation of cTnT and α-actinin positive aligned CMs from UCM-MSCs on aligned PCL nanofibers, which was not possible on TCPs. On the other hand, MatrigelTM supported for proliferation of iPSCs whereas PCL topology together with temporal modulation of Wnt signalling has contributed to their differentiation into beating CMs. Furthermore, the aligned PCL fibers not only increased the differentiation of iPSCs into CMs but also resulted in the alignment of the cells to resemble the anisotropic nature of CMs in native heart. In conclusion, the results of the present study suggest that plasticized PVA-PVP may be suitable for developing cardiac patches for CTE. On the other hand, electrospun, aligned PCL nanofibrous matrices, coated with MatrigelTM, may be useful for the development of cardiac tissue construct for culture and differentiation of stem cells into CMs for CTE