Ph.D Thesis


Ph.D StudentYuval Eitan
SubjectAcellular Native Porcine Myocardial Extracellular Matrix as
a Biomaterial Scaffold for Tissue Engineering
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professors Machluf Marcelle
Full Thesis textFull thesis text - English Version


Abstract

We have developed an efficient tissue decellularization process for densely cell populated tissues, and optimized it for the isolation of native extracellular matrix (ECM) from porcine myocardial tissue. The decellularization process is based on a combination of enzymatic digestion with Trypsin and detergent cell removal with Triton-X-100. The isolated ECM maintained a highly intra-connective collageneous macromolecular ultra structure, and significant amounts of glycosaminoglicans (GAGs). It exhibited desirable mechanical properties in terms of elasticity, strength and durability, properties required from scaffolds intended for use in cardiac tissue repair. We further investigated the potential use of the isolated ECM as scaffold for cardiac tissue engineering or repair in terms of interactions with seeded cells and biocompatibility. We used the commonly studied fibroblasts, cardiomyocytes and mesenchymal stem cells, which were isolated and seeded on the scaffold. Cell density and distribution were followed with time by lipophillic membrane staining with DiO. Cell viability was assessed  qualitatively using fluorescin di-acetate and propidium iodide (FDA/PI) staining for live / dead cells, and quantitatively using the metabolic alamarBlue┬« assay. Fibroblast seeded scaffolds shrank to 1-2mm3 spheroids, and their GAG significantly increased by 23%. The expression of ECM remodeling-related mRNAs of collagens I and III, MMP2 and TIMP1 was quantified by real-time PCR, and were significantly elevated in fibroblast-seeded scaffolds, compared to control cells on plates. Fibroblast seeded scaffolds lost some flexibility, yet gained strength compared to acellular scaffolds, as shown by mechanical testing. Scaffold seeded with cardiomyocytes began to beat in concert few days post seeding and the myocytes expressed typical functional cardiac markers such as alpha-actinin, Troponin I and connexin43. The cells revealed aligned elongated morphology, as presented by immunofluorescent staining and SEM. Mesenchymal stem cells seeded scaffolds maintained viability over 24 days in culture. Primary In-vitro assays indicated that the acellular ECM’s immunogenic potential is negligible, as the secretion of nitric oxide and expression of IL-1b and TNF-a by ECM-stimulated macrophage cells remained practically unchanged. In-vivo implantation of the acellular ECM in mice further supported the lack of immunogenic potential, as no inflammation, macrophage infiltration, encapsulation or elevated pro-inflammatory gene expression were observed 1,2,4 and 8 weeks post implantation. To conclude, we suggest that all our findings strengthen the potential of acellular myocardial ECM as a scaffold biomaterial for use in heart tissue engineering and myocardial regeneration.