Treatment of cardiac diseases involves transplantation of donor hearts, since the damaged heart has limited self-regeneration potential. An alternative treatment option has emerged as engineered cardiac tissues, grown in vitro by cultivation of cardiac cells on biomaterials, have comparable properties to native myocardium and can be implanted for cardiac repair. Major current limitations are a viable cell source and adequate vascularization to support cell survival. In this thesis, two proangiogenic biomaterials, a scaffold and a hydrogel, were developed to achieve vascularization in vitro and in vivo for cardiac repair. Scaffold patches are suitable for repairing congestive heart failure or congenital malformations, while injectable biomaterials allow minimally-invasive treatment post-myocardial infarction (MI). In the first aim, a collagen scaffold with covalently immobilized vascular endothelial growth factor (VEGF) was developed, and improved cell mobilization, survival and proliferation when used for free wall repair in adult rats. This increased angiogenesis, which aided in retaining the biomaterial size to allow tissue growth. In the second aim, a collagen-chitosan hydrogel with encapsulated thymosin β4 (Tβ4) was developed to 1) recruit cells from the heart epicardium for repair post-MI in vivo, and 2) guide capillary outgrowths from arteries and veins to form oriented capillary structure for in vitro cardiac tissue engineering. Results showed that the encapsulation of Tβ4 into collagen-chitosan hydrogels led to cell outgrowths from rat or mouse cardiac explants in vitro. A portion of the recruited cells were CD31-positive endothelial cells (ECs) that formed tubes. The hydrogel was injected in vivo to increase vascularization and number of cardiomyocytes within the infarct area post-MI, which improved left ventricular wall thickness. Tβ4-hydrogel also promoted the outgrowth of capillaries from vascular explants that followed the direction of the hydrogel-coated grooves of a micropatterned polydimethylsiloxane (PDMS) substrate. These capillary outgrowths eventually formed a vascular bed for engineering vascularized cardiac tissues. This thesis presents two bioinstructive biomaterials with sustained and localized delivery of angiogenic molecules to be used for in situ cardiac repair based on improved vascularization. The use of cell-free bioactive materials overcomes limitations of cell isolation and expansion as required for cell therapies or implantation of engineered tissues.

Treatment of cardiac diseases involves transplantation of donor hearts, since the damaged heart has limited self-regeneration potential. An alternative treatment option has emerged as engineered cardiac tissues, grown in vitro by cultivation of cardiac cells on biomaterials, have comparable properties to native myocardium and can be implanted for cardiac repair. Major current limitations are a viable cell source and adequate vascularization to support cell survival. In this thesis, two proangiogenic biomaterials, a scaffold and a hydrogel, were developed to achieve vascularization in vitro and in vivo for cardiac repair. Scaffold patches are suitable for repairing congestive heart failure or congenital malformations, while injectable biomaterials allow minimally-invasive treatment post-myocardial infarction (MI). In the first aim, a collagen scaffold with covalently immobilized vascular endothelial growth factor (VEGF) was developed, and improved cell mobilization, survival and proliferation when used for free wall repair in adult rats. This increased angiogenesis, which aided in retaining the biomaterial size to allow tissue growth. In the second aim, a collagen-chitosan hydrogel with encapsulated thymosin β4 (Tβ4) was developed to 1) recruit cells from the heart epicardium for repair post-MI in vivo, and 2) guide capillary outgrowths from arteries and veins to form oriented capillary structure for in vitro cardiac tissue engineering. Results showed that the encapsulation of Tβ4 into collagen-chitosan hydrogels led to cell outgrowths from rat or mouse cardiac explants in vitro. A portion of the recruited cells were CD31-positive endothelial cells (ECs) that formed tubes. The hydrogel was injected in vivo to increase vascularization and number of cardiomyocytes within the infarct area post-MI, which improved left ventricular wall thickness. Tβ4-hydrogel also promoted the outgrowth of capillaries from vascular explants that followed the direction of the hydrogel-coated grooves of a micropatterned polydimethylsiloxane (PDMS) substrate. These capillary outgrowths eventually formed a vascular bed for engineering vascularized cardiac tissues. This thesis presents two bioinstructive biomaterials with sustained and localized delivery of angiogenic molecules to be used for in situ cardiac repair based on improved vascularization. The use of cell-free bioactive materials overcomes limitations of cell isolation and expansion as required for cell therapies or implantation of engineered tissues.

Cardiovascular disease is the leading cause of death in the United States, accounting for approximately 25% of total deaths. Myocardial infarction (MI) is an extreme case of cardiovascular disease where ischemia leads to irreversible tissue necrosis. As the heart lacks the capacity to endogenously regenerate, the infarcted region is negatively remodeled, reducing cardiac function. Current therapies are not able to regenerate cardiac function post-MI, requiring novel approaches such as tissue engineering. However, there are three major pitfalls that are currently limiting the clinical translation of a tissue engineered cardiac patch: lack of proper vascularization within the tissues; biocompatible material; and lack of electrical integration between engineered tissue and host. The research within this dissertation aimed to engineer solutions to overcome these three pitfalls. Plants and animals exploit fundamentally different approaches to transporting fluids, yet there are surprising structural similarities. To take advantage of these similarities, we looked across different kingdoms and investigated whether plants and their innate vasculature could serve as perfusable scaffolds for tissue engineering. Standard perfusion decellularization techniques were adapted and applied to spinach leaves, which were found to be fully devoid of DNA following processing. Leaf vasculature remained patent post-decellularization and supported transport of various sized microparticles. Human cells successfully seeded onto and inside the plant scaffolds. Decellularized leaves were found to be nearly void of any cytotoxic affects. Leaf biocompatibility was then investigated in vivo through subcutaneous implantation in a rat model. Leaf scaffolds were found to be biocompatible after 4 weeks of implantation. Furthermore, leaves that were pre-functionalized with an RGD-dopamine peptide were fully integrated into the host tissue within one week. This shows the leaf scaffold’s potential to be an immuno-modulatory material, depending upon the intended application. Electrically conducting biofibers were engineered through the combination of fibrin microthreads and engineered conductive HEK293 cells. Biofibers could act as a modular platform to allow for electrical integration between the host tissue and any engineered cardiac patch. Biofibers directionally carried electrical current and were found capable of bridging electrical signal between two separate clusters of cardiomyocytes. In vivo investigation bridging a biofiber from the left atria to the left ventricle was accomplished in a rat model. Electrical maps demonstrated a visible accessory pathway that created a feedback electrical signal from the ventricle to the atria through the implanted biofiber. These results demonstrate electrical integration in vivo between host myocardium and the engineered biofiber.

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abstract: Severe cases of congenital heart defect (CHD) require surgeries to fix the structural problem, in which artificial grafts are often used. Although outcome of surgeries has improved over the past decades, there remains to be patients who require re-operations due to graft-related complications and the growth of patients which results in a mismatch in size between the patient’s anatomy and the implanted graft. A graft in which cells of the patient could infiltrate, facilitating transformation of the graft to a native-like tissue, and allow the graft to grow with the patient heart would be ideal. Cardiac tissue engineering (CTE) technologies, including extracellular matrix (ECM)-based hydrogels has emerged as a promising approach for the repair of cardiac damage. However, most of the previous studies have mainly focused on treatments for ischemic heart disease and related heart failure in adults, therefore the potential of CTE for CHD treatment is underexplored. In this study, a hybrid hydrogel was developed by combining the ECM derived from cardiac tissue of pediatric CHD patients and gelatin methacrylate (GelMA). In addition, the influence of incorporating gold nanorods (GNRs) within the hybrid hydrogels was studied. The functionalities of the ECM-GelMA-GNR hydrogels as a CTE scaffold were assessed by culturing neonatal rat cardiomyocytes on the hydrogel. After 8 days of cell culture, highly organized sarcomeric alpha-actinin structures and connexin 43 expression were evident in ECM- and GNR-incorporated hydrogels compared to pristine GelMA hydrogel, indicating cell maturation and formation of cardiac tissue. The findings of this study indicate the promising potential of ECM-GelMA-GNR hybrid hydrogels as a CTE approach for CHD treatment. As another approach to improve CHD treatment, this study sought the possibility of performing a proteomic analysis on cardiac ECM of pediatric CHD patient tissue. As the ECM play important roles in regulating cell signaling, there is an increasing interest in studying the ECM proteome and the influences caused by diseases. Proteomics on ECM is challenging due to the insoluble nature of ECM proteins which makes protein extraction and digestion difficult. In this study, as a first step to perform proteomics, optimization on sample preparation procedure was attempted. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2018

Embryonic stem cells (ESC) constitute a promising source of cells for cardiac transplantation strategies. However, complexities associated with in vivo studies have made it difficult to develop a thorough understanding of cell integration. We have engineered an in vitro system that recapitulates the native cardiac environment using 300μm thick collagen scaffolds seeded with neonatal cardiomyocytes (CM) and electrical field stimulation. The injection of undifferentiated ESC served as a baseline to assess the validity of studying cell transplantation in this model. Yfp-ESC survived and proliferated over several days in model tissue. ESC were not observed to significantly differentiate into the cardiac lineage, and did not integrate with the cardiac cell population. While the injection of ESC improved cardiac cell number, tissue functional properties were hindered. The methods developed herein can be readily adapted to study ESC derived progenitor and differentiated cells, to elucidate the optimal cell state for ESC-mediated cell therapy.

Embryonic stem cells (ESC) constitute a promising source of cells for cardiac transplantation strategies. However, complexities associated with in vivo studies have made it difficult to develop a thorough understanding of cell integration. We have engineered an in vitro system that recapitulates the native cardiac environment using 300μm thick collagen scaffolds seeded with neonatal cardiomyocytes (CM) and electrical field stimulation. The injection of undifferentiated ESC served as a baseline to assess the validity of studying cell transplantation in this model. Yfp-ESC survived and proliferated over several days in model tissue. ESC were not observed to significantly differentiate into the cardiac lineage, and did not integrate with the cardiac cell population. While the injection of ESC improved cardiac cell number, tissue functional properties were hindered. The methods developed herein can be readily adapted to study ESC derived progenitor and differentiated cells, to elucidate the optimal cell state for ESC-mediated cell therapy.

Cardiovascular disease is the leading cause of death in the US, which can result in blockage of a coronary artery, triggering a myocardial infarction (MI). After a MI, hypoxic ventricular myocardial tissue dies, resulting in the deposition of non-contractile scar tissue and remodeling of the ventricle, leading to decreased cardiac output and ultimately heart failure. Currently, the gold-standard solution for total heart failure is a heart transplant. As donor hearts are in short supply, an alternative to total-organ transplantation is surgically remodeling the ventricle with the implantation of a cardiac patch. Acellular cardiac patches have previously been investigated using synthetic or decellularized native materials in effort to improve cardiac function. However, a limitation of this strategy is that acellular cardiac patches only reshape the ventricle and do not increase cardiac contractile function. By incorporating the use of a clinically relevant cell type and by matching native architecture, we propose the use of a highly aligned fibrin scaffold to support the maturation of human induced pluripotent stem cell cardiomyocytes (hiPS-CM) for use as a cell-populated cardiac patch. By micropatterning fibrin hydrogels, hiPS-CM seeded on the surface of this scaffold become highly aligned, which is crucial for increased contractile output. Our lab previously developed a composite fibrin hydrogel and microthread cardiac patch matching mechanical properties of native myocardium. By micropatterning fibrin hydrogel alone, we were able to match cellular alignment of hiPS-CM to that of native myocardium. hiPS-CMs seeded on this surface were found to express distinct sarcomere alignment and circumferential connexin-43 staining at 14 days of culture as well as cellular elongation, which are necessary for mature contractile properties. Constructs were also cultured under electrical stimulation to promote increased contractile properties. After 7 days of stimulation, contractile strains of micropatterned constructs were significantly higher than unpatterned controls. These results suggest that the use of topographical cues on fibrin scaffolds may be a promising strategy for creating engineered myocardial tissue to repair damaged myocardium.

Diseases of the heart have been the most common cause of death in the United States since the middle of the 20th century. The development of engineered cardiac tissue over the last three decades has yielded human induced pluripotent stem cell-derived (hiPSC) cardiomyocytes (CMs), microscale “heart-on-a-chip” platforms, optical interrogation techniques, and more. Having spawned its own scientific field, ongoing research promises lofty goals to address the heart disease burden around the world, such as patient-specific disease models, and clinical trials on chip-based platforms. The greatest academic pursuit for engineered cardiac tissues is to increase their maturity, thereby increasing relevance to native adult tissue. Investigation of cardiomyocyte maturity necessitates the development of 3D-tissue compatible techniques for measuring and perturbing cardiac biology with enhanced precision. This dissertation focuses on the development of biological microelectromechanical systems (BioMEMS) for precision measurement and perturbation of cardiac tissue. We discuss three unique approaches to interfacing MEMS-based tools with cardiac biology. The first is a high resolution magnetic sensor, which directly measures the spatial gradient of a magnetic field. This has an ideal application in magnetocardiography (MCG), as the flux of ions during cardiac contractions produces measurable magnetic signals around the tissue and can be leveraged for noncontact diagnosis. The second is a highly functionalized heart-on-a-chip platform, wherein the mechanical contractions of cardiac microtissues can be simultaneously recorded and actuated. Contractile dynamics are leading indicators of maturity in engineered cardiac tissue and mechanical conditioning has shown recent promise as a critical component of cardiac maturation. The third is the imaging of contractile nanostructures in engineered cardiomyocytes at depth in a 3D microtissue. We use small angle X-ray scattering (SAXS) to discern the periodic arrangement of myofilaments in their native 3D environment. We enable a significant structural analysis to provide insight for functional maturation. Enabling these three thrusts required developing two supporting technologies. The first is the engineered control of dynamic second order systems, a foundational element of all our MEMS and magnetic techniques. We demonstrate numerous algorithms to improve settling time or decrease dead-time such that samples with fast temporal effects can be measured. The second is a microscale gluing technique for integrating myriad of materials with MEMS devices, yielding unique sensors and actuators. / 2022-05-15T00:00:00Z

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