Development of Novel Bioinks and 3D Printing Methodologies for Cardiac Tissue Engineering Applications

In the modern world, myocardial infarction is one of the most common cardiovascular diseases, which are responsible for around 18 million deaths every year or almost 32% of all deaths. Although there has been some progress in regenerative therapies available for myocardial infarction treatment, translating pre-clinical findings to the clinic remains a major challenge. One reason for this is the lack of reliable and human representative healthy and fibrotic cardiac tissue models that can be used to understand the fundamentals of ischemic/reperfusion injury caused by myocardial infarction and to test new drugs and therapeutic strategies.

With the inability to use human hearts at a rate required for experimental research, the majority of our current knowledge on how the constituents of the cardiovascular system (CVS) function has been obtained through the use of animal models. During the past couple decades, emerging tissue engineering methods allowed researchers to create biomimetic tissue models in vitro, eliminating the need for the interspecies translation of the results, as well as concerns for the ethical controversy in using the animal models. Current in vitro tissue models utilize various methods to combine biological scaffolds, hydrogels, or decellularized matrices with cardiac cells, to provide them with a 3D microenvironment that mimics the native cardiac tissue.

Three dimensional (3D) bioprinting is a layer-by-layer additive manufacturing technology allowing accurate spatial deposition of the biological materials and active cells in a pre-designed pattern and is thus considered a promising technique for fabricating biomimetic cardiac tissue models in vitro.

The scope of this dissertation is to develop novel bioinks and methodologies for extrusion-based 3D bioprinting to engineer more biomimetic post-MI tissue models as well as promising regenerative therapies that can be used to treat cardiac fibrosis replacing the healthy myocardium post-MI. Towards this goal, first I develop a novel two-step crosslinking method to crosslink gelatin methacryloyl (GelMA) hydrogels, which can be used to imitate the relative stiffening of the fibrotic tissue compared to the healthy heart tissue. I investigated the effect of this crosslinking method on mechanical and rheological properties of GelMA hydrogels as well as the pattern fidelity of the 3D bioprinted constructs. Then, I develop novel bioinks by combining decellularized human myocardium extracellular matrix (dhECM) with either GelMA hydrogels or GelMA and methacrylated hyaluronic acid (MeHA) hydrogels to create the softer healthy cardiac tissue or the scar tissue having an order of magnitude higher stiffness compared to the healthy tissue. I then characterize the mechanical, rheological and physical properties of these bioinks as well as their cytotoxicity and printability. As a proof of concept, I combined human induced pluripotent stem cell (hiPSC) derived cardiomyocytes (iCMs) with GelMA-dhECM to represent the healthy tissue and human cardiac fibroblasts(hCFs) with GelMA-MeHA-dhECM to represent the stiffer scar tissue. Then I improve the complexity of the post-MI tissue model by introducing the boundary region between the healthy and the scar regions together with including aged collagen in order to take into consideration the aged microenvironment. Moreover, I increase the cell diversity by using iCMs, hiPSC derived cardiac fibroblasts (iCFs), hiPSC derived endothelial cells (iECs) and hiPSC derived cardiac myofibroblasts (iCMFs). The method I introduce here will allow researchers to create patient-specific, age-specific, benchtop, post-MI tissue models that can be used as a platform to evaluate the efficacy of novel regenerative therapies before animal studies or clinical trials. Finally, we utilize aerosol jet printing to create conductive cardiac patches allowing cell-level resolution enabling the alignment of the iCMs on the hydrogel similar to the native heart tissue. Overall, in this dissertation I develop new bioinks and methods to utilize 3D bioprinting in various cardiac tissue engineering applications, such as post-MI tissue models and cardiac patches, which can be used as a guide for developing new models and treatment strategies.