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Ischemic cardiomyopathies, such as myocardial infarction (MI), are a leading cause of heart failure in both men and women throughout the world. Despite timely intervention post-MI, the loss of viable myocardium can lead to global remodeling and loss of function in many patients due to the limited regenerative potential of heart tissue. Thus, there is a critical need to better understand the repair mechanisms involved and to develop new preventative and reparative therapies for treating MI and preventing progression to heart failure. Methylglyoxal (MG) is a highly reactive dicarbonyl metabolite of glycolysis and the main precursor of advanced glycation end-products (AGEs), which can cause oxidative stress and wound healing delay. MG was shown to play an important causative role in the cellular changes, adverse remodeling and functional loss of the infarcted heart. This suggests MG as a target for therapy to restore cell-ECM signaling, inhibit oxidative stress and improve cardiac function post-MI. The aim of this PhD project was to develop new biomaterial therapies that can reduce the effects of MG, decrease oxidative stress, enhance electrical conductivity and improve cardiac contractility and function post-MI. There were three primary objectives: 1) To develop an injectable antioxidant and hydrogel system for minimizing the effects of MG and promoting cardiac repair post-MI; 2) To synthesize a nanoparticle system for targeted delivery of Glyoxalase-1 (Glo1) enzyme to cardiac tissue for reducing the accumulation of MG, limiting adverse remodeling and preserving cardiac function following MI; and 3) To design a sprayable nano-therapeutic that uses surface engineered custom designed multi-armed peptide grafted nanogold for on-the-spot coating of infarcted myocardial surface for increasing contractility of the myocardium post-MI. In the first study, a fisetin-loaded collagen type I hydrogel (fisetin-HG) was injected intramyocardially in mice at 3h post-MI, and compared to fisetin-alone, hydrogel-alone, or saline treatment. The fisetin-HG treatment increased the level of glyoxalase-1 (the main MG-metabolizing enzyme), reduced MG-AGE accumulation, and decreased oxidative stress in the MI heart, which was associated with smaller scar size and improved cardiac function. Treatment with fisetin-HG also promoted neovascularization and increased the number of pro-healing macrophages in the infarct area, while reducing the number of pro-inflammatory macrophages. The second study revealed that when delivered intravenously at 3h post-MI, our Glo1-loaded nanoparticles specifically targeted the damaged cardiac tissue, led to improved cardiac function, protected cell viability and limited infarct expansion by reducing oxidative stress post-MI. Lastly, the third study showed that, when applied at 1-week post-MI, the sprayed nanogold treatment remained at the treatment site for at least 28 days with no significant off-target organ infiltration. Our results demonstrated a remarkable increase in cardiac function, muscle contractility, and myocardial electrical conductivity post-MI. Overall, these findings show that reducing MG levels through both increased activity of Glo1 and direct MG scavenging as well as increasing cardiac contractility may be a promising approach to limit adverse cardiac remodeling, prevent damage, and preserve the function of the infarcted heart

<p>  Peptide biomaterials have drawn great attention in recent decades owing to their tunability and biocompatibility. Coiled-coils specifically have become a well-studied scaffold with a clear sequence-to-structure relationship. As such, the Chmielewski lab has extensively studied peptide assemblies based on the GCN4 leucine zipper. First, we present the peptide <strong>TriCross</strong>, where nitrilotriacetic acid (NTA) and di-histidine ligands are installed at the N- and C-terminus, respectively, and a bipyridine ligand installed at a central, solvent exposed position. Through strategic placement of these metal-binding ligands, TriCross assembled into a three-dimensional (3D) mesh in the presence of zinc ions and dissembled following mild ethylenediaminetetraacetic acid (EDTA) treatment. These properties created a 3D network capable of encapsulating cells for extended periods of time (>1 week) and releasing cells upon metal-chelation. </p> <p>  Next, we describe a stabilized nanotube and enhanced crystal assembly through a heterocoiled-coil assembly. Nanotubes composed of the coiled-coil peptide <strong>TriNL</strong> that assembled likely through ionic interactions rapidly degraded in phosphate buffered saline (PBS). To improve stability, a peptide with metal-binding ligands, <strong>p2L</strong>, was introduced through thermal annealing of the two peptides. Low levels of <strong>p2L </strong>(up to 10:1 <strong>TriNL</strong>:<strong>p2L</strong>) retained nanotube morphology while simultaneously introducing NTA and di-histidine ligands. Upon addition of metal, metal-ligand interactions were established within the nanotube and increased stability of the material. Higher levels of <strong>p2L</strong> (2:1 <strong>TriNL</strong>:<strong>p2L</strong>) led to hexagonal crystals similar to <strong>p2L</strong> but now without the use of metal ions. These crystals expanded the scope of protein inclusion by removing the requirement for His-tags on proteins to be incorporated within the material.</p> <p>  Finally, a self-replicating and self-assembling coiled-coil peptide is reported. The coiled-coil <strong>TriNL</strong> was cysteine modified (N20C) to create a peptide capable of native chemical ligation. At low concentrations, the <strong>N20C FL</strong> peptide acted as a template for the cysteine and thioester fragments while high concentrations led to fibrillar structures. The size of the fibrils was controlled through the addition of preassembled seeds into the native chemical ligation system. </p>

Cell culturing on three-dimensional structures has increased the possibilities in tissue engineering and bioreactor research. These structures enable cells to differentiate, proliferate, mobilize, and function in a conformation that more accurately mimics in vivo conditions. Computer generated models aid in development and rapid alteration of three-dimensional cell substrates, defining their internal structure as well as their external morphology. The rapid transition from substrate design to a viable culture is imperative to quickly advance research in biomedical and tissue engineering applications. The aim of this thesis is to investigate the feasibility of a rapid prototyping process by selectively cross-linking and assembling biocompatible films. This investigation revealed that selectively cross-linking and layering gelatin films could produce a three-dimensional substrate with a defined structure after dissolving uncross-linked gelatin. The study also revealed that freeze-drying aided in the rapid dissolution of uncross-linked gelatin. The line width resolution obtained during tests was .5 mm using a template treatment method and was limited by the template construction resolution. Finally, alteration in treatment time, rinsing agitation, and rinsing temperature yielded stable films that better retained their size and shape compared to films produced in previous processes.

Bone is a largely bipartite viscoelastic composite. Its mechanical behavior is determined by strain rate and the relative proportions of its principal constituent elements, hydroxyapatite and collagen, but is also largely dictated by their geometry and topology. Collagen fibrils include many segments of tropocollagen in staggered, parallel sequences. The physical staggering of this tropocollagen allows for gaps known as hole-zones, which serve as nucleation points for apatite mineral. The distance between adjacent repeat units of tropocollagen is known as D-Spacing and can be measured by Atomic Force Microscopy (AFM). This D-Spacing can vary in length slightly within a bundle, but by an additional order of magnitude within the same specimen, and can significantly alter the proportion of hydroxyapatite. Previous researchers have built and refined a Finite Element Analysis “Complex Model” to capture the consequences of adjusting D-Spacing and the viscoelastic parameters. This will ultimately serve to elucidate and perhaps predict the mechanical consequences of biological events that alter these parameters. This study aims to further refine the model’s precision by accounting for crosslinking between fibrils, the presence of which serves to add mechanical strength. This study also looks to refine the currently used rheological models by way of frequency dependent parameters in the hopes of improving model accuracy over a wider frequency range. Hormonal factors such as estrogen can significantly determine the composition of bone. Menopause marks a significant reduction in circulating estrogen and has been shown to factor heavily in the development of conditions like osteoporosis. Because sheep feature a hormonal cycle and skeletal structure similar to humans, three of six mature Columbia-Rambouillet ewes were randomly selected to undergo an ovariectomy, the remainder serving as sham-operated controls. Twelve months later twenty-five beam samples were harvested from their radius bones for mechanical analysis and other testing, including atomic force microscopy (AFM) and dynamic mechanical analysis (DMA). The data gleaned from these tests provide an experimental basis of comparison with The Complex Model. A 2-D Finite Element Analysis model in Abaqus was first created by Miguel Mendoza, which enforced viscoelasticity and a realistic proportion and placement of hydroxyapatite and collagen. The viscoelasticity was modeled using a Standard Linear Solid involving springs and a dashpot element. Crosslinks of varying number and location were arranged within the former model configuration as node to surface tie-constraints to explore the treatment of the FEA Model as a more realistic assembly of parts. Frequencies utilized for this model included 1, 3, 9 and 12 Hz. This approach is referred to in this research as the Intermolecular Forces (IMF) Scheme. The model was subsequently refined by Christopher Ha and Austin Cummings. The model was characterized by 2x100 unit half-cells, the lengths of which were randomly generated by a Python script. This script ingested the mean and standard deviation D-Spacing length to generate a model geometrically similar to a real specimen bearing those dimensions. A frequency dependent value for the dashpot element in the rheological model used for tropocollagen was developed using this latter FEA model, named the Complex Model. Dashpot values explored for this variable dashpot included 0.0125, 0.125, 0.3125, 0.45, 0.5875, 0.725, 0.8625 and 1.25 GPa-s, some values chosen for their high performance in past studies and others to further narrow the search for the best performing dashpot. All dashpot values were investigated over the previously stated frequencies in addition to 2, 5, 7 and 12 Hz. The best fit dashpot values were plotted against the frequencies in which they best performed and a polynomial trend line was fitted to establish an equation, and that equation was used to modify an existing user material subroutine for tropocollagen to provide an automatic frequency dependent dashpot value to Abaqus. This approach is referred to in this research as the Variable Dashpot (VD) Scheme. Results for the IMF scheme generally performed poorly, with the fully tie-constrained model performing best with 0.77 and 0.024 for R2 and RMSE respectively. Of the randomized crosslink models, that with the lowest number (N=20) of randomly placed non-enzymatic crosslinks performed best with 0.81 and 0.051 for R2 and RMSE respectively. Increasing the number of randomized crosslinks reduced model fit, and the remaining three variants exhibited mean R2 and RMSE values of 0.66-0.67 and 0.052 respectively. For the VD scheme, models running custom modified variable dashpot UMATs yielded R2 and RMSE values of 0.87 and 0.012 for C2207, and 0.89 and 0.008 for C1809. This is a notable fit considering all other material property parameters are held constant throughout each frequency. In the rheological model, this research also found a striking difference between the frequency dependent viscous element values that made each model perform best. This indicates that differences in D-Spacing standard deviations between OVX and control may be associated with distinct strain-rate dependent mechanical responses.

Mammary epithelial cells are highly efficient secreting cells. With genetic engineering, the uses of these cells could be endless. Research is being conducted on these cells to determine their full potential to the biotech industry. This paper investigates whether bovine epithelial mammary cells can survive in glutaraldehyde-treated gelatin tubes in an operating bioreactor. Many bioreactors were developed and tested to suit the needs of the cells. Procedures were created and carried out to ensure sterility of the bioreactors. Bovine mammary epithelial cells were implanted in the bioreactors and samples of their growth were taken over time.

Hundreds of thousands of vascular bypass grafts are implanted in the United States every year, but there has yet to be an ideal graft material to substitute for one’s own autologous vessel. Many synthetic materials have been shown to be successful vessel replacements; however, none have been proven to exhibit the same mechanical properties as native vessels, one of the most important criteria in selecting a vascular graft material. Part of this issue is due to the fact that, currently, there is no “gold standard” for testing the longitudinal and transverse tensile properties of small diameter tubular materials. While there are ASTM and ISO standards that suggest ways to test tubes in their original form, many researchers have published tensile strength data based on cutting the tube and testing it as a flat sample. Thus, it was the aim of this thesis to understand, establish, and implement accurate tensile testing methods of small diameter polymers in their original, tubular state on Cal Poly’s campus. Two test fixtures were created based on specified design criteria in order to test materials in their tubular form in both the longitudinal and transverse directions. Both fixtures were successful in testing PLGA and ePTFE samples, and statistical data was gathered for the transverse test fixture. The new transverse test fixture was tested against the current method of testing, and a significant (α = 0.05) difference between methods was established for ultimate tensile strength. This analysis, however, cannot determine which test method is more accurate, thus more extensive testing is required to verify the design of both fixtures. By developing a method for testing small diameter polymers in tubular form on Cal Poly’s campus, it allows for more testing of various small diameter tubes and more comparative data to validate each design. It also demonstrates a need for a more detailed and widespread standardization of testing for small diameter tubes, especially in vascular substitute applications where the ideal vessel replacement has yet to be found.

“Blood vessel mimics” (BVMs) are tissue-engineered constructs that serve as in vitro preclinical testing models for intravascular devices. The Cal Poly Tissue Engineering lab specifically uses BVMs to test the cellular response to stent implantation. PLGA scaffolds are electrospun in-house using the current “Standard Protocol” and used as the framework for these constructs. The performance of BVMs greatly depends on material and mechanical properties of the scaffolds. It is desirable to create BVMs with reproducible properties so that they can be consistent models that ultimately generate more reliable results for intravascular device testing. Reproducibility stems from the consistency of the scaffolds. Thus, scaffolds with consistent material and mechanical properties are necessary for creating reproducible BVMs. The aim of this thesis was to characterize the reproducibility of the electrospun PLGA scaffolds using fiber diameter measurements and compliance testing. Initial work in this investigation involved designing and testing several experimental electrospinning protocols to obtain smaller fiber diameters, which have been shown to elicit more ideal cellular responses. The most successful protocol in that regard was then analyzed for the reproducibility of fiber diameters and compared to the reproducibility of the Standard Protocol. After determining that the Standard Protocol produced scaffolds with more consistent fibers, a large-scale reproducibility study was performed using this protocol. In this expanded study, both fiber diameter and compliance were analyzed and used to characterize the scaffolds. It was established that the scaffolds demonstrated inconsistent mean fiber diameter and mean compliance. The current standard electrospinning protocol therefore does not create PLGA scaffolds with statistically reproducible properties. Future modifications should be made to the electrospinning parameters in order to reduce variability between the scaffolds and future studies should be performed to determine the acceptable range of properties.

Cardiac disease causes approximately a third of the deaths in the United States. Furthermore, most of these deaths are due to a condition termed atherosclerosis, which is a buildup of plaque in the coronary arteries, leading to occlusion of normal blood flow to the cardiac muscle. Among the methods to treat the condition, stents are devices that are used to restore normal blood flow in the atherosclerotic arteries. Before advancement can be made to these devices and changes can be tested in live models, a reliable testing method that mimics the environment of the native blood vessel is needed. Dr. Kristen Cardinal developed a tissue engineered blood vessel mimic to test intravascular devices. Among the scaffolding material used, electrospun poly (lactide-co-glycolide) (PLGA) has been used as an economic option that can be made in house. PLGA is a biodegradable co-polymer, and when electrospun, creates a porous matrix with tailorable properties. Currently, the standard PLGA electrospinning protocol produces consistent fibrous scaffolds with a mean fiber diameter of 5-6 microns. Research indicates that cell adhesion is more successful in fibrous matrices with a mean fiber diameter at the nanometer level. However, because previous work in the Tissue Engineering Laboratory at Cal Poly sought to ensure a consistent fibrous, there was no model or equation to determine how to change the electrospinning parameter settings to create scaffolds with an optimal mean fiber diameter. To fill this need, biomedical engineering senior Steffi Wong created a design of experiment to systematically approach the electrospinning variables and determine how they interacted with each other, as well as their effect on fiber diameter. The aims of this thesis were to perform the said design of experiments and determine a model to predict the resulting mean fiber diameter of a scaffold based on the electrospinning parameters as well as to determine what combination of parameters would lead to a scaffold with an optimal mean fiber diameter between 100-200 nanometers. The variables tested were solution concentration, gap distance, flow rate, and applied voltage. Each scaffold was imaged and a mean fiber diameter was calculated and used as the predicted variable in a regression analysis, with the variables indicated above as the predictors. The goal of 100-200 nanometer mean fiber diameter was not reached. The smallest mean fiber diameter calculated was 2.74 microns—half of that of the standard protocol. The regression analysis did result in a model to describe how the voltage, gap distance, and flow rate affected the fiber diameter.

Mechanical properties of articular cartilage, including stiffness, biolubrication, and wear-resistance, undergo deterioration during progression of diseases such as osteoarthritis. When the tissue becomes softened and wear-prone, resulting from biochemical alterations within the cartilage matrix, osteoarthritis patients experience painful joint degeneration and erosion of the bone-protective cartilage. Moreover, the synovial fluid bathing the cartilage also experiences a reduction in lubricating capacity as osteoarthritis advances, further hastening wear. An existing treatment paradigm known as viscosupplementation, designed to restore a viscous and lubricating nature to the synovial fluid, involves intraarticular injection of hyaluronic acid into affected joints. While this technique relieves pain for some individuals, the majority of patients experience neither pain relief nor protection of the cartilage from further damage. To address the unmet need of patients requiring chondroprotective thera-pies, this dissertation describes two potential intraarticular strategies based on the application of polymer chemistry principles to bodily tissues and interfaces. One strategy involves the synthesis of a non-hyaluronic-acid synovial fluid sup-plement, based on a phosphorylcholine-containing polyacrylate network, de-signed to functionally mimic the lubricity of the glycoprotein lubricin, phospho-lipid macromolecular assemblies, and high molecular weight hyaluronic acid. The second strategy involves the in situ photopolymerization of a related polyacrylate within cartilage bulk tissue to strengthen, prevent wear, and in-crease the proportion of compressive load supported by the tissue’s interstitial fluid rather than solid matrix. In this strategy, the branched polymer network functionally mimics the glycosaminoglycans that are found in healthy cartilage but depleted in osteoarthritic cartilage. For both potential therapies, chemical and physical properties of the respective fluid and tissue are analyzed, and ex vivo cartilage mechanical testing involving axial and shear deformation reveal the biotribological and compressive reinforcement conferred by the zwitterionic polymer. The synovial fluid supplement significantly decreases cartilage friction through a variety of lubrication mechanisms depending upon tissue fluid flow state and articulation conditions, and the cartilage-reinforcing supplement pro-tects cartilage during accelerated wear testing while also improving synovial flu-id’s ability to lubricate polymer-impregnated cartilage. The fundamental tissue—biomaterial tribological interactions investigated in this dissertation will inform the rational design of therapeutic, friction-reducing polymers for diverse applications. / 2019-04-04T00:00:00Z