Ph.D Thesis


Ph.D StudentEvelyne Bao-Vi Nguyen
SubjectEngineering 3D Thick Porcine Extracellular Matrix for
Cardiac Regeneration
(The research and dissertation were con.
at Technion and NTU Singapore)
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professors Machluf Marcelle
Full Thesis textFull thesis text - English Version


Abstract

This project focused on engineering a thick cardiac patch using an acellular scaffold derived from a porcine left ventricle. Whereas most studies demonstrated cardiac constructs with thicknesses up to a few hundred micrometers, this project aims to recellularize an acellular scaffold with comparable thickness to the human native heart (1-1.2 cm).


         The first part of this study focused on the interactions between the chosen cardiomyocyte model cell type, human bone marrow mesenchymal stem cells (bmMSCs), and the porcine cardiac extracellular matrix (pcECM). In initial studies performed in static conditions, pcECMs were treated with different biochemical compounds including addition of functional groups, adhesion proteins and polymers to evaluate possible improvement in cell support ability. Dissolved nitrocellulose treatment demonstrated the most significant enhancement (up to 3 fold, p<0.05) in terms of ultimate cell growth. The second part of this study focused on pcECM functional effects on the seeded cell types. Whereas bmMSCs harvested from pcECMs were found positive for common positive bmMSC markers (CD29, CD44, CD90, CD73 and CD105) by flow cytometry, they started to express CD31 reaching 10% and 55% after 2 and 4 weeks respectively. Further qPCR studies confirmed the conclusions obtained from immunostaining that genes specific for endothelial cells (e.g. PECAM1, VWF, FLT1, FLK1) were upregulated in bmMSCs seeded on pcECMs. In addition, RNA expression of cardiac specific genes and ECM-related genes obtained from bmMSCs and cvFBs respectively seeded on pcECMs were also conducted and demonstrated some interesting gene upregulation. Finally, the third part of this study aimed to recellularize physiologically relevant pcECM slabs. To guarantee oxygen and nutrient supply in such thick constructs, a customized bioreactor system was used. A method combining a static pre-cultivation period prior to dynamic culturing demonstrated to allow up to 12 fold deeper cell infiltration than achieved under static cultures.


          Taken together, those results support the feasibility of obtaining physiologically relevant cardiac constructs for potential scar replacement in the infarcted heart and the suitability of the pcECM for such application. By combining a vasculature-preserved thick pcECM and a customized bioreactor system for sustainable cell viability in such thick construct, significant cell infiltration depth (1,2 mm) was achieved and gives favorable prospects into recellularizing physiologically relevant tissue constructs. It also validates the bioreactor capabilities to sustain cell survival and growth in 3 dimensional environment. In addition, pcECM may provide particular cues towards endothelial transdifferentiation, which could be a substantial advantage in term of angiogenesis.