Date of Award

Winter 2018

Document Type

Dissertation

Degree Name

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

Department

Biomedical Engineering

First Advisor

Lee, Eun Jung

Second Advisor

Del Re, Dominic Pasquale

Third Advisor

Kumar, Vivek A.

Fourth Advisor

Pfister, Bryan J.

Fifth Advisor

Xie, Lai-hua

Abstract

Cardiovascular disease remains the leading cause of mortality in the United States. Current tissue engineering approaches have fallen short of promoting fully functional cardiovascular cells and the post-myocardial infarction microenvironment is still not well understood. These gaps in knowledge are addressed in this dissertation through the development of in vitro engineered cardiac tissues using electroactive materials to enhance the differentiation of pluripotent stem cell derived cardiomyocytes and through the development of in vitro myocardial inflammation models dedicated to understanding cardiomyocytes and macrophages interactions.

Specifically, piezoelectric poly (vinylidene fluoride-trifluoroethylene) (PVDF-TrFE) supports the attachment and survival of mouse embryonic stem cell derived cardiomyocytes (mES-CM) and endothelial cells (mES-EC). Characterization of mES-CM confirms expression of classical cardiac specific marker such as cTnT and Cx43, as well as efficient calcium handling properties when cultured on PVDF-TrFE including response to ryanodine receptor and β-adrenergic stimulation. MES-EC also retain their ability to uptake low density lipoprotein when cultured on PVDF-TrFE scaffolds and express classical endothelial cell specific markers such as eNOS and PECAM-1.

Additionally, a novel graphene composite scaffold (PCL+G) exhibiting even distribution of graphene particles within the matrix allowing miniscule amounts of graphene to increase conductivity is developed and characterized. MES-CM seeded on conductive PCL+G scaffolds attach well and begin migrating into the scaffold matrix. They exhibit well-registered sarcomeres, express cardiac specific markers such as cTnT, MHC and Cx43 and spontaneous beat for up to two weeks. MES-CM on PCL+G scaffolds can be electrically paced and respond to ryanodine receptor and β-adrenergic stimulation. The combination of highly aligned fiber orientation and the presence of graphene promoted significantly improve calcium cycling efficiency by a fractional release of over 40%.

Finally, in vitro myocardial inflammation models are developed to examine both direct and indirect co-culture of mES-CM with polarized macrophage subpopulations present in the post-MI microenvironment. Direct co-culture with macrophage subsets cause significant changes in mES-CM calcium handling function, especially in store operated calcium entry (SOCE), which is accompanied by significant increases in matricellular protein secretion, osteopontin (OPN). A pathway connecting OPN to SOCE response through ERK1/2 activation is analyzed through indirect co-culture with macrophage conditioned media and found to be affected by OPN inhibition, suggesting this pathway is involved with calcium homeostasis in the post-MI microenvironment, specifically in the pro-healing, anti-inflammatory phase.

Taken together, the presented results expand the current state of research in cardiac regenerative medicine by demonstrating the potential of two electroactive biomaterials for the formation of functional cardiac tissues and by illuminating a novel target involved in changes in cardiomyocytes calcium homeostasis during post-MI healing through an in vitro engineered diseased model.

Share

COinS