ORCID Identifier(s)

ORCID 0000-0002-2864-8752

Graduation Semester and Year

Spring 2025

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Biomedical Engineering

Department

Bioengineering

First Advisor

Yi Hong

Second Advisor

Huaxiao Yang

Third Advisor

Jun Liao

Fourth Advisor

Zui Pan

Fifth Advisor

Juhyun Lee

Abstract

Cardiovascular diseases (CVDs) remain the foremost cause of death worldwide, with heart failure (HF), often resulting from myocardial infarction (MI), representing the primary contributor to CVD-related deaths. The adult cardiomyocytes have limited regenerative ability; as a result, MI often leads to the formation of fibrous scar tissue. This fibrotic replacement impairs electrical conduction, sustains inflammation, and promotes adverse left ventricular (LV) remodeling, ultimately compromising cardiac function. Recently, decellularized heart-derived extracellular matrix (ECM) has gained considerable attention as a promising biomimetic scaffold for cardiac repair. Notably, decellularized heart ECM based hydrogels have demonstrated safety and efficacy in preclinical MI models and clinical trials. Ongoing research continues to explore the therapeutic potential of decellularized heart ECM in promoting cardiac regeneration. Despite these advantages, native decellularized ECM is hindered by suboptimal mechanical strength, poor cellular infiltration and integration, and lack of electrical signal propagation, thereby limiting its translational potential. To overcome these barriers and advance clinical translation, engineering strategies to modify and enhance the functionality of decellularized heart ECM are essential. This thesis aims to address these limitations by developing and evaluating engineered ECM-based biomaterials through four distinct strategies to enhance the therapeutic potential of decellularized cardiac ECM for MI repair and to promote the in vitro maturation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).

In Aim 1, we developed a sustained-release system by loading stromal-cell derived factor 1 alpha (SDF-1α) within poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) and incorporating them into an injectable porcine cardiac decellularized extracellular matrix (cdECM) hydrogel. The cdECM was successfully prepared, retaining key matrix components, including collagen, glycosaminoglycans (GAGs), and elastin, while reducing residual DNA to below 50 ng/mg dry weight. SDF-1α-loaded PLGA NPs were fabricated via a double emulsion method, yielding NPs with an average size of 197.86 ± 20.72 nm and the SDF-1α loading content of 102 ± 4 ng/mg. Afterwards, these NPs were blended with neutralized digested cdECM solution, forming a hydrogel at 37 °C within 30 minutes. This composite hydrogel exhibited a sustained release of SDF-1α over four weeks, in contrast to one-week release observed with direct encapsulation in the cdECM hydrogel. Incorporation of PLGA NPs significantly enhanced the mechanical properties of the cdECM hydrogel, without altering the microstructure of the cdECM hydrogel. The nanocomposite hydrogel supported H9C2 cell adhesion and proliferation in vitro, and the released SDF-1α maintained its bioactivity, as confirmed by in vitro chemotactic assays. Furthermore, in vivo studies further demonstrated that intramyocardial injection of the hydrogel promoted angiogenesis in the infarcted myocardium and improved cardiac function. These findings highlight the therapeutic promise of this PLGA NPs-enhanced cdECM hydrogel for MI repair through localized, sustained SDF-1α delivery.

In Aim 2, we engineered a conductive heart-based hydrogel by combining methacrylated cardiac ECM (ECMMA) with hyaluronic acid-doped poly(3,4-ethylenedioxythiophene) nanoparticles (PEDOT:HA) to form an ECMMA/PEDOT:HA (EPH) hydrogel for the study of the effect on maturation of hiPSC-CMs. The ECMMA was synthesized via the reaction between methacrylic anhydride and free amine or hydroxyl groups on heart ECM. PEDOT:HA NPs were synthesized by oxidative polymerization of EDOT, forming PEDOT chains doped with HA. This hydrogel was crosslinked under visible light using a ruthenium/sodium persulfate (Ru/SPS) system. The addition of PEDOT:HA NPs reduced the hydrogel’s pore size and swelling ratio while preserving its mechanical properties. Importantly, the incorporation of conductive PEDOT:HA significantly enhanced electrical conductivity without compromising cytocompatibility. EPH hydrogels supported H9C2 cell proliferation and promoted hiPSC-CMs maturation, as evidenced by synchronized calcium transients and upregulation of key maturation markers, including CX-43 and MYH7/MYH6, indicating improved electrical coupling and structural development. Overall, EPH conductive hydrogels provide a favorable microenvironment for hiPSC-CMs maturation, particularly by enhancing electrical connectivity and structural development.

In Aim 3, we aim to develop the cardiac-derived ECM microspheres to investigate their effects on the maturation of hiPSC-CMs. Electrospray parameters (voltage, flow rate, and needle distance) were optimized to control microsphere size and morphology. In the hydrated state, microspheres exhibited spherical morphology, while freeze-dried microspheres revealed a porous groove/ridge with nonfibrous network structure. Compared to bulk ECM hydrogels, ECM microspheres showed significantly lower swelling ratios and higher Young’s modulus. These microspheres supported the attachment, migration, and growth of multiple cell types (HL-1, C2C12, and H9C2), and enhanced cardiac differentiation in H9C2 cells. In short-term (14 days) hiPSC-CMs culture, ECM microspheres improved the calcium handling of hiPSC-CMs and upregulated cardiac-related genes such as ACTA2, CX-43, and TNNT, although MYH7/MYH6 ratio wasn’t significantly improved. In long-term culture (8 months), ECM microspheres supported sustained hiPSC-CMs viability, consistent beating, enhanced CX-43 expression, and increased binucleation rates, suggesting advanced structural and electrical maturation. Calcium transient analysis showed no significant differences compared to the TCP group. Notably, a decrease in spontaneous beating rate was observed after day 210. Together, these findings highlight the importance of 3D ECM microspheres in supporting sustained cardiomyocyte viability, providing a physiologically relevant microenvironment for hiPSC-CMs culture.

In Aim 4, we developed an injectable, traceable, and conductive ECMMA/GNRs granular hydrogel for MI treatment. Bulk ECMMA/GNRs hydrogels were initially crosslinked using lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), then mechanically fragmented through a needle extrusion process (18G →21G →26G → 30G), and finally re-crosslinked using Ru/SPS to form granular hydrogels. Compared to the bulk hydrogel, these granular hydrogels exhibited an interconnected microporous structure. The incorporation of gold nanorods (GNRs) significantly improved the electrical conductivity of the granular hydrogel and imparted X-ray traceability, enabling non-invasive imaging. In vitro, ECMMA/GNRs granular hydrogels promoted H9C2 cell migration into the hydrogel interior and supported robust cellular proliferation. Intramyocardial injection of this conductive granular hydrogel significantly improved structural remodeling, enhanced systolic function, and facilitated the cardiac repair in a rat MI model.

In summary, this work presents four complementary engineering strategies to overcome the limitations of native cardiac ECM for myocardial repair and hiPSC-CMs in vitro culture. These biomaterial platforms collectively demonstrate improvements in mechanical integrity, bioactivity, electrical functionality, and therapeutic performance. The findings contribute valuable insights into the rational design of multifunctional heart ECM-based biomaterials and highlight their promising potential in advancing cardiac disease treatment.

Keywords

Heart ECM, Decellularization, SDF-1α, Hydrogel, Conductivity, Granular hydrogel, Microspheres, GNRs, Cardiac repair, hiPSC-CMs Maturation

Disciplines

Biochemical and Biomolecular Engineering | Biological Engineering | Biology and Biomimetic Materials | Biomaterials | Molecular, Cellular, and Tissue Engineering

Comments

First, I would like to express my heartfelt gratitude to my advisor and mentor, Dr. Yi Hong, for his exceptional guidance, unwavering support, and constant encouragement. His insight, patience, and leadership have profoundly shaped both my research and my development as a scientist. Next, I would like to acknowledge all my lab members, as well as our collaborators, for their hard work and valuable contributions to my research. Lastly, I am also grateful for the generous support from NIH, NSF, and AHA.

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Available for download on Wednesday, May 12, 2027

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