Date of Award

Spring 2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Biomedical Engineering - (Ph.D.)

Department

Biomedical Engineering

First Advisor

Cheul H. Cho

Second Advisor

George Collins

Third Advisor

William Corson Hunter

Fourth Advisor

Treena Livingston Arinzeh

Fifth Advisor

Diego Fraidenraich

Abstract

The development of an in vitro tissue model that can mimic the 3-dimenisonal (3-D) cellular architecture and mosaic of myocardial tissue holds great value for cardiac tissue engineering, modeling, and cardiovascular drug screening applications. The main objective of this project was to develop a 3-D vascularized cardiac tissue model in vitro for improved survival and function.

The cellular mosaic of the myocardial tissue demands the intricate integration of an extracellular matrix-like scaffold, cellular constituents, and biological factors. The first aim of the research was to fabricate and characterize a biodegradable chitosan nanofiber scaffold that would resemble the extracellular matrix (ECM) physically and chemically. Chitosan, a natural polysaccharide that shares structural homology to the ECM glycosaminoglycans was processed into nanofibers via electrospinning to resemble the physical nano-architecture of the ECM. The second aim was to biologically modify the scaffold using a two step method: (1) Adsorption of fibronectin to improve cellular attachment and migration and (2) Induction of endothelial tubulogenesis to recreate the vascularized architecture of the myocardium. The third aim was to investigate the effect of co-culturing cardiomyocytes with fibroblasts on cardiomyocytes’ survival and contractility in the vascularized 3-D chitosan scaffold. This was based on the fact that 70% of the native myocardial tissue is composed of fibroblasts

The chitosan scaffold was characterized for its physio-chemical properties, including in-vitro structural integrity and bio-degradability. The biomodification of the scaffold via fibronectin adsorption improved cellular attachment, verified by staining of actin (cytoskeletal protein) and vinculin (cell-adhesion protein). The endothelial cells formed a network of interconnected tubes and secreted GAGs that were immobilized onto the chitosan scaffold. The cellular studies showed that cardiomyocyte mono-cultures resulted in islands of isolated contractions and minimal gap junction expression. In addition, cardiomyocyte contractility was lost after four days in the mono-cultures. However, co-culturing the cardiomyocytes with the fibroblasts promoted tissue-like synchronous contractions that were sustained for over three weeks. Gap junction expression in cardiomyocytes-fibroblasts co-cultures was extensive and was expressed along the cardiomyocyte cell membranes.

Finally, to create the cardiac tissue model, the vascularized chitosan nanofibers were impregnated with a co-culture system of cardiomyocytes and fibroblasts. Using real-time intracellular calcium ion staining, the cardiomyocytes were observed to have migrated through the 3-D chitosan scaffold and attained intercellular alignment to form cardiomyocyte tubules which is a characteristic of in vivo cardiomyocytes. The cardiomyocyte tubules were verified to contract in a synchronized and tissue-like rhythmic manner. These results highlight the immense importance of the vascular architecture and fibroblasts co-culture in the development of any cardiac regenerative therapy.

Share

COinS