Creating a Functional Cardiac Patch From Decellularized Spinach Leaves

Robbins, Emily

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Creating a Functional Cardiac Patch From Decellularized Spinach Leaves

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Cardiovascular disease (CVD) is the leading cause of death worldwide. Myocardial infarction (MI), one type of CVD, affects over 800,000 Americans each year. MI is a regional disease that generally results from occlusion in a coronary artery leading to cell death, tissue ischemia, inflammation, and fibrotic scar formation. Cardiac scar formation stiffens the heart tissue, results in loss of electromechanical properties, reduced contractile function, and can lead to heart failure. Current treatments for MI and heart failure patients do not regenerate ischemic tissue function. A tissue engineered cardiac patch could be a solution to improve cardiac tissue function in the early months following MI. Decellularized spinach leaves are a promising scaffold for a tissue engineered cardiac patch due to their mature vascular system, biocompatibility, and comparable material properties to that of cardiac tissue. In addition, human induced pluripotent stem cell derived cardiomyocytes (hiPS-CMs) are an encouraging cell type to functionalize a cardiac patch scaffold. hiPS-CMs have been shown to be able to couple with host cardiomyocytes in vivo and improve cardiac function after MI. However, since hiPS-CMs perform functionally like immature cardiomyocytes, inducing maturation to adult cardiomyocyte behavior is a challenge. This dissertation sought to develop a perfusable and functional tissue engineered cardiac patch using a decellularized spinach leaf that can be used to re-functionalize diseased heart tissue of MI patients. We first investigated the leaf’s capability to perfuse cardioactive solutions through its vasculature to hiPS-CMs seeded on the leaf surface. Following perfusion, we found that hiPS-CM beat frequency of perfused leaves significantly increased, supporting our hypothesis. In addition, dye perfusion demonstrated that solutions perfused through the leaf vasculature enter the leaf tissue and cells on its surface. Next, we examined hiPS-CM adherence to and function on various decellularized spinach leaf substrate conditions. After 21 days, hiPS-CMs were found to adhere to protein coated and non-coated leaf scaffolds, with no statistically significant differences between hiPS-CMs’ functional behavior on the coated and non-coated leaf surfaces. These results suggest that coating leaf scaffolds is not necessary to induce or improve cardiomyocyte adhesion and function. Finally, we examined methods to induce cell alignment on the decellularized leaf to mimic cardiac tissue and to mature cell function. We sought to align hiPS-CMs on decellularized spinach leaf scaffolds to promote directional contractile behavior using two different methods: adherent fibrin microthreads and micropatterned fibrin hydrogel rows. Fibrin microthreads induced alignment of human mesenchymal stem cells (hMSCs) on leaves after 7 days; however, hiPS-CMs did not adhere to the leaf using the same techniques. Fibrin hydrogel successfully micropatterned on decellularized spinach leaves, and micropatterned rows provided the appropriate topographical cues for seeded hMSCs and hiPS-CMs to align. Micropatterned hiPS-CMs demonstrated increased contractile strain and synchronized contractile beating across the scaffolds. Overall, we exhibited that decellularized spinach leaves with hiPS-CMs are a viable option for a tissue engineered cardiac patch and aligned hiPS-CMs show enhanced function on the leaf, approaching behavior of cardiac tissue.

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