Graduation Semester and Year

2022

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Biomedical Engineering

Department

Bioengineering

First Advisor

Yi Hong

Second Advisor

Jun Liao

Abstract

In the United States and around the world, cardiovascular disease (CVD) has been one of the main causes of death since 1921, responsible for approximately 1 in 4 deaths domestically. Myocardial infarction (MI), or commonly called a heart attack, is a subset of CVD that often ends in heart failure (HF). Cardiac cells are incapable of regeneration, leading to severe impacts on quality of life. Current treatments for MI fail to target left ventricular remodeling and therefore do little to address the progression towards HF. Cardiac scaffolds for tissue engineering (TE) are a prominent solution in recent research. Developing various biomaterials to promote regaining function has narrowed down critical aspects for a successful scaffold. Trends in development show that biomaterials consisting of bioactive, conductive, elastic, and robust mechanical properties provide good environmental niches for cardiac cells. We aim to develop biomaterial blends that improve the bioactivity and conductivity of biodegradable polyurethane (PU) to study their structural, mechanical, and material characteristics and their interaction with cells. In Aim 1, we integrated digested porcine cardiac extracellular matrix (ECM) into PU at varying concentrations, incorporating biomolecules found natively in cardiac tissue to supply biological cues in cardiac myocyte development. Interaction with the surrounding environment through integrins modulates the development of cardiomyocytes; ECM provides binding sites for integrins not found in synthetic polymers like PU. Aligned electrospinning (ES) of PU-ECM fibers was characterized and compared to the outer wall of a porcine left ventricle (LV). Fabricated fibers displayed highly aligned nanofibrous structures maintained throughout simulated body conditions. The resulting fiber diameter was 650-980 nm after simulated conditions. PU-ECM fibers exhibited anisotropic shrinkage after hydration due to swelling and the release of residual stress from the aligned electrospinning process. The hydrophilicity of fibers was improved through the incorporation of ECM, with PU displaying a contact angle of 107 ± 9° while PU-20%ECM was 70 ± 9°. Improvement of hydrophilicity originated from blending ECM. In vitro degradation of PU-20%ECM fibers showed accelerated degradation retaining a mass of 34.2 ± 2.8% over two weeks in an enzymatic solution; comparatively, PU retained 54.8 ± 7.3% of its mass. Mechanically the LV porcine tissue had a failure stress of 0.36 ± 0.095 MPa, while PU-20%ECM was 15.0 ± 1.3 MPa. The superior mechanical properties of these fibers ensure resistance to failure when bearing forces applied to a heart. Viscoelastic and anisotropic mechanical properties were noted across all fibrous blends. Mouse HL-1 cells and neonatal rat ventricular cardiomyocytes (NNRCM) were seeded on blended fibers. HL-1 cells are organized unidirectionally on aligned fibers, and no significant toxicity to cells. Higher expression of α-actinin and f-actin was noted on PU-20%ECM scaffolds. NNRCM cells displayed significant improvement to viable counted cells in PU-20%ECM. Markers for maturity such as sarcomere and Z-line length were enhanced in PU-20%ECM fibers. In Aim 2, we designed various conductive PU blends with reduced graphene oxide (rGO) to fabricate a potential muscle scaffold to improve electromechanical coupling. Uniformity of contractions is improved by coupling the myocytes, which is critical for practical myocyte function. PU-rGO blends were processed into aligned nanofibrous scaffolds through aligned ES. Initially, PU-rGO blends the highest rGO concentration of 4% w/w. During initial conductivity testing, poor results were obtained. To correct this, we fabricated a PU-10%rGO scaffold. Additionally, fiber size was impacted by rGO blending; PU-10%rGO’s fiber diameter was 689 ± 226 nm, compared to PU, which was 464 ± 87 nm. The contact angle of PU-10%rGO was more hydrophobic than PU, with a contact angle of 132 ± 5° and 120 ± 7°, respectively. The conductivity of fibrous scaffolds was unobtainable, so membranes were made for 4-point probe instrumentation. Dry PU-10%rGO membranes displayed superior conductivity of 0.149 ± 0.035 S/m compared to PU-4%rGO, whose conductivity was 2 magnitudes less, at only 0.006 ± 0.001 S/m. Wet conductance gave erroneous results due to PBS masking bulk conductivity readings. PU-10%rGO displayed vastly reduced failure stresses along and across the fiber preferred direction. However, mechanical properties were still superior along the fiber preferred direction and comparable across the fiber preferred direction to LV tissue. Elasticity curves from cyclic loading revealed no change in trend due to rGO doping; similarly, anisotropic characteristics were consistent with native LV trends. In vitro cell culturing with mouse C2C12 and NNRCM displayed modulation of cell behavior. C2C12 cells were highly aligned on PU-10%rGO fibers, with interconnected cytoskeletal proteins of myosin heavy chain and f-actin. NNRCM displayed highly aligned morphologies, with maturity markers such as sarcomere length, Z-line length, and aspect ratio, which were significantly improved on the PU-10%rGO fibers. In contrast to PU, the expression for connexin-43 (Cx-43) was noted more peripherally, suggesting improved electromechanical coupling. In Aim 3, we built upon the previous two objectives to fabricate a bioactive, conductive, elastic, and robust scaffold for improved biomimicry of native cardiac ECM. Previously found optimal blends of PU-20%ECM and PU-10%rGO were combined to fabricate a composite PU-20%ECM-10%rGO biomaterial blend. PU-20%ECM-10%rGO fibers were produced similarly to the previous iterations. Morphological characteristics of PU-20%ECM-10%rGO were similar to PU-20%ECM and PU-10%rGO, with highly aligned nanofibers, with a fiber diameter of 438 ± 158 nm. Shrinkage of PU-20%ECM-10%rGO (82.4 ± 1.5%) was similar to PU-10%rGO (83.0 ± 2.4%), shrinking less than its PU-20%ECM counterpart (78.0 ± 0.8%). The contact angle of PU-20%ECM-10%rGO was vastly improved compared to PU10%rGO, with the droplet fully absorbing within the first 15 seconds of placement. A unique interaction between ECM and rGO was displayed during conductivity testing. PU-20%ECM-10%rGO was 5-fold more conductive, at 0.92 ± 0.038 S/m compared to PU-10%rGO, at 0.174 ± 0.005 S/m. This improved conductivity may be caused by an improved dispersal of rGO throughout the membrane. Mechanically, PU-20%ECM-10%rGO was superior or comparable to the native LV tissue. A slight increase in failure stress was noticed across the fiber preferred direction in the PU-20%ECM-10%rGO fibers compared to PU-10%rGO. Cyclic loading curves showed similar trends. Biaxial testing revealed PU-20%ECM-10%rGO maintained anisotropic mechanical behavior. NNRCM in vitro studies displayed aligned cell morphologies across all fiber blends. PU-20%ECM-10%rGO sarcomere length was significantly higher than its counterparts. PU-20%ECM-10%rGO Z-line lengths were like PU-20%ECM and were superior to PU and PU-10%rGO. Following the above three aims, future studies should focus on improving various limitations and challenges. Integrating mechanical and electrical stimulation during in vitro studies should promote functionality. Fabrication of a bioreactor capable of mechanical and electrical stimulation has been completed for this purpose. Initial calibrations of mechanical parameters of the bioreactor are ongoing. Secondly, an improvement to diminishing shrink of aligned scaffolds through annealing has also initially been explored. Releasing residual stress before removing the scaffold from the mandrel reduces shrinkage. Thirdly, incorporating a higher concentration of ECM may result in higher bioactivity. Another direction is sourcing and synthesizing monolayer rGO. Issues with aggregation of rGO and the need for high concentrations concern the robustness of mechanical properties and cell viability. Monolayer rGO should reduce the required rGO for improved conductivity while minimizing effects on mechanical properties. Lastly, finite element analysis modeling biomaterial mechanical behavior could reveal scaffold responses in physiological conditions. Computational modeling of the designed biomaterials could offer predictive powers to minimize time and money waste. This dissertation has demonstrated several novel approaches to designing and fabricating new biomaterial blends of biodegradable PU as a potential cardiac scaffold. The approaches for functionalization focused on bioactivity and conductivity separately and combined. Firstly, incorporating bioactive ECM provides environmental cues to modulate cell maturation. Secondly, the incorporation of rGO improves electromechanical coupling by facilitating action potentials. Lastly, we created an improved biomimetic material and scaffold for optimized cell signaling by building upon the previous improvements. These various blends of PU offer a new selection of potential biomaterials for cardiac tissue regeneration and skeletal muscle regeneration.

Keywords

Myocardial infarction, Tissue engineering, Decellularized ECM, Biodegradable polyurethane, Reduced graphene oxide, Cardiomyocytes

Disciplines

Biomedical Engineering and Bioengineering | Engineering

Comments

Degree granted by The University of Texas at Arlington

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