Browsing by Author "Whittington, Abby Rebecca"

Now showing 1 - 15 of 15
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    Development and Mechanism of Action of Antimicrobial Coatings
    Behzadinasab, Saeed (Virginia Tech, 2023-07-14)
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    Development of a Nanoparticle Vaccine Delivery System with Polymeric Oral Adjuvants for Poultry
    Cary, Jewel Maria (Virginia Tech, 2019-09-06)
    Development of new vaccination technology has been hindered by a lack of new adjuvants to enable development of protective immunity using different vaccine delivery methods. A vaccine delivery system using oral adjuvants would be applicable across species for both individual and mass vaccination in both the medical and veterinary fields. We sought to create an oral nanoparticle (NP) vaccine delivery system that is easy to produce and uses polymers as oral adjuvants with killed virus. Our hypothesis was gelatin and chitosan would enhance viral uptake and stimulate immune cells to produce protective immunity. This would allow the safer killed form of each virus to be used in place of modified live (MLV) viruses and avoid undesirable side effects like immunosuppression. The research objectives were to 1. Fabricate and characterize gelatin NPs encapsulating inert materials of similar size and shape to the viruses of interest for fabrication proof-of-concept 2. Modify the NP delivery system to minimize immune cell cytoxicity for the vaccine delivery application 3. Fabricate and characterize FPV and HEV viral nanoparticles' stability, cellular uptake/infectivity, and released viruses' ability to replicate 4. Compare the abilities of the killed HEV nanovaccine, killed HEV with loose gelatin and chitosan polymers (no nanoparticle), and a live HEV commercial vaccine to induce textit{in vivo} seroconversion, protective immunity, and side effects during clinical and challenge studies in turkeys We proved our hypothesis to be correct in addition to demonstrating matching the encapsulation material size to empty NP size leads to preferred encapsulated NP formulation parameters, chitosan stabilizes the NP formulation bypassing the need for cytotoxic crosslinkers, and paraformaldehyde is able to kill virus prior to vaccine formulation while still preserving virus morphology sufficiently for immune cell recognition. This development constitutes one of the first novel adjuvants discoveries in 70 years and opens the door for conversion of injectable vaccines to oral delivery across species.
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    Development of an Experimental and Computational Pipeline for Characterizing the Mechanical Properties and Micromechanical Environment within In Vitro 3D Printed Bone Tissue Engineered Scaffolds
    Hunt, Elizabeth Albright (Virginia Tech, 2024-06-10)
    Delayed fracture healing is the improper healing of fractures within a reasonable amount of time and is estimated to impact a sixth of all fractures that occur annually in the United States1. While blood- and imaging-based bone turnover biomarkers have been thoroughly investigated throughout the healing process of bone, there is still a lack of understanding on how well these biomarkers can predict union in individual patients. Although conventional radiography is the most common clinical practice for assessing bone healing progression, this imaging technique—as well as the other imaging methods used—fails to discern the in vivo mechanical environment of bone, and therefore the likeliness of union or nonunion. There is a need to identify mechanical biomarkers that could better differentiate between patients who undergo typical healing progression versus delayed fracture healing. In order to identify these mechanical biomarkers, a 3D in vitro cell culture platform that recapitulates the micromechanical environment must be developed and tested. Success of this in vitro platform relies on the generation of rigorous testing protocols for assessing stiffness and fluid flow within this organoid system. This study aims to develop an experimental and computational pipeline for mechanically characterizing 3D printed (3DP) scaffolds—Voronoi, IsoTruss, and Truncated Octahedron (TO) geometries—that will be the foundation for future studies to explore patient-specific mechanical biomarkers in these bone tissue engineered scaffolds A dynamic mechanical analysis (DMA) strain sweep was performed on the scaffolds (n=6 for 4- and 7-day 3T3 fibroblast seeded Voronoi and TO scaffolds, n=4 for 4- and 7-day seeded IsoTruss scaffolds, n=3 for 4- and 7-day soaked controls for each geometry) to measure storage modulus, loss modulus, and the damping coefficient. The Voronoi geometry increased significantly in storage modulus when seeded for seven days compared to four days (p=0.0293). There was also an overall significant decrease in stiffness when the scaffolds were seeded versus non-seeded (p<<0.001). Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was performed to produce fluid flow experimental validation data, and this provided insights on the micromechanical environment of the IsoTruss scaffold that were consistent with the computational fluid dynamics (CFD) simulation model. The CFD model was used to calculate wall shear stresses (WSS) for various inlet velocities (0.05, 0.10, 0.15, 0.20, and 0.25 mm/s), with 0.15 mm/s producing WSS best within the range of extracellular matrix formation. DMA, DCE-MRI, and CFD all confirmed mechanical characteristics of the IsoTruss geometry that were unique to its specific micromechanical architecture. Out of all scaffolds tested, the IsoTruss geometry achieved the maximum (3.47 MPa) and minimum (0.0631 MPa) storage modulus. The computational analysis pipeline revealed that the patterns observed in the DMA experiments could be caused by buckling due to the fourteen-strut intersections and printing infidelity issue related to the IsoTruss geometry. The protocol developed herein for the experimental and computational analyses done on the scaffolds in this thesis will be used in the future on bone organoids to study individualized fracture healing.
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    Evaluating Microglia Dynamics in Blast and Impact-Induced Neurotrauma and Assessing the Role of Hemostatic Nanoparticles in Microglia Activation
    White, Michelle Renee (Virginia Tech, 2022-10-03)
    Traumatic brain injury (TBI) is a major medical concern that has demonstrated to be particularly challenging to treat because of the disparity amongst injury modes and severities. Increased use of explosive devices during combat has caused blast TBI (bTBI) to become a widespread consequence in military and Veteran populations, and impact-related trauma from contact-related sports or motor vehicle accidents has made mild impact-induced TBIs (concussion) a major health problem. There is a high risk for those who have sustained a TBI to develop behavioral and cognitive disorders following injury, and these symptoms can present as delayed onset, causing diagnosis to be a major feat when planning for treatment and long-term healthcare. Both preclinical and clinical studies report the neuropathological changes following TBI, yet investigating the distinct mechanistic changes in blast and impact trauma that contribute to pathological disparities has yet to be elucidated. Microglia dynamics play a key role in initiating the inflammatory response after injury, as microglia become activated by undergoing morphological changes that influence their function in the injured brain, and unique signaling pathways influence their functional inflammatory states. While previous literature report on the unique responses of microglia, their mediated-inflammatory responses are still not well defined. This work aimed to investigate the acute and subacute responses of microglia to injury through their diverse activation states following blast and impact trauma. The work herein employed rodent models to investigate these changes, finding that microglia activation was spatially and temporally heterogeneous within and across injury paradigms. Three days following bTBI, activated microglia in the cortex displayed morphologies similar to microglia that are known to increase their interactions with dysfunctional synapses, while dystrophic microglia were prevalent in the hippocampus seven days following injury. Moreover, transhemispheric changes in microglia activation were noted following impact TBI, with stressed/primed microglia responding to immune challenges of the cortex at three days, whereas a unique morphological state that was markedly different from those traditionally reported in CNS injury and disease was present within the hippocampus three- and seven-days following injury. State-of-the-art cell sorting techniques were used for in vivo analysis of microglia, which also exhibited that functional changes of microglia vary between injury paradigms, providing insight into how differences in primary insult may elicit distinct signaling pathways involved in microglia-mediated inflammatory responses. These in vivo studies were then crucial in understanding the malleable responses of microglia to complex injuries such as "blast plus impact" TBI, indicating that phenotypic changes in microglia following this injury are also unique and spatially heterogeneous. To date, therapeutic efforts for TBI are limited due to the lack of understanding the underlying mechanisms that influence TBI pathology. This work also investigated novel therapeutic targets, noting that administration of polyester nanoparticles restored microglia to baseline levels following impact. The fundamental research presented in this study is innovative and advantageous as it can provide essential data into targeted and personalized treatments that can improve long-term healthcare and ultimately, the quality of life for those suffering from a TBI.
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    Examining Cellular Interactions and Response to Chemotherapy in The Glioblastoma Perivascular Niche
    Hatlen, Rosalyn Rae (Virginia Tech, 2023-01-17)
    Glioblastoma multiforme (GBM) is the most deadly and common form of brain cancer and is responsible for over 50% of adult brain tumors. A specific region within the GBM environment is known as the perivascular niche (PVN). We have designed a 3D in vitro model of the PVN comprised of either collagen Type 1 or HyStem-C®, human umbilical vein endothelial cells (HUVECs) or human brain microvascular endothelial cells (HBMECs), and LN229 (GBM) cells. A synergistic response between HUVECs and LN229 cells was observed in co-culture, including 10 – 16-fold increased cell proliferation, a decrease in the height of hydrogels of up to 68%, as well as elevated secretion of TGF-β and CXCL12 up to 2.6-fold from Day 8 to 14. These trends correlated with cell colocalization, indicating a chemotactic role for CXCL12 in enabling the migration of LN229 cells towards HUVECs in co-cultures. Von Willebrand factor (vWF) was co-expressed with glial fibrillary acidic protein (GFAP) in up to 40% of LN229 cells after 14 days in co-culture in collagen (2.2 mg/mL) and HyStem-C® gels. The expression of vWF indicates the early stages of trans-differentiation of LN229 cells to an endothelial cell phenotype. We then investigated the effect of chemotherapeutic drugs temozolomide (TMZ) and Avastin® on EC networks, LN229 cell morphology and alignment, cytotoxicity, colocalization, and trans-differentiation. TMZ was observed to primarily affect LN229 cells, with treatment at high concentrations resulting in up to 2.3-fold reduced alignment as well as an increase in cell circularity. Cytotoxicity of up to 94% was also observed up to in LN229 monocultures, and was significantly higher in collagen (1.1 mg/mL) gels. Avastin® treatment resulted in changes to ECs. Network features were significantly reduced and EC cellular proliferation decreased up to 69% with Avastin® treatment. Significant increases in percentages of colocalized and GFAP+/vWF+ cells were also observed when treated with 8 µg/mL Avastin®. This suggests that chemotactic signaling may have been altered. TGF-β secretion was reduced in co-cultures when 150 µM TMZ or 8 µg/mL Avastin® were administered.
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    Low-Input and Single-Cell Transcriptomic Technologies and Their Application to Disease Studies
    Zhou, Zirui (Virginia Tech, 2023-12-19)
    With the rapid progress of next-generation sequencing (NGS) technologies, new tools and methods have emerged to investigate the transcriptomics of various organisms. RNA sequencing (RNA-seq) employs NGS to evaluate the presence and abundance of RNA transcripts in biological samples. This technique offers a comprehensive snapshot of the RNA dynamics within cells. With the ability to profile the entire transcriptome of organisms rapidly and accurately, RNA-seq has become the state-of-the-art method for transcriptome profiling, surpassing the traditional microarray approach. Single-cell RNA sequencing (scRNA-seq) was introduced in 2009 to profile the single-cell gene expression in highly heterogeneous samples such as brain tissue and tumors. The advancement of scRNA-seq technologies enables the in-depth transcriptomic study in each cell subtype. When selecting an scRNA-seq method, researchers must weigh the trade-off between profiling more single cells versus obtaining more comprehensive transcripts per cell, while considering the overall costs. The throughput of full-length scRNA-seq methods is usually lower, as each single cell needs to be processed separately to produce scRNA-seq libraries. However, full-length methods enable the researchers to investigate the splicing variants and allele-specific expression. Non-full-length methods only capture the 3' or 5' ends of transcripts, which limits their application in isoform detection, but as cells are pooled after barcoding for cDNA synthesis, the throughput is 2–3 orders of magnitude higher than full-length methods. We developed a droplet-based platform for full-length single-cell RNA-seq, which enabled the efficient recovery of full-length mRNA from individual cells in a high-throughput manner. The developed platform can process ~8,000 single cells within 2 days and detect ~20% more genes compared to Drop-seq. Besides scRNA-seq technology development, we also applied a low-input RNA-seq method to study the transcriptomics in different biological samples. When handling precious biological samples, a low-input method is necessary to profile the transcriptome of homogeneous cell populations. We first studied the epigenomic and transcriptomic regulations in colorectal cancer (CRC) using MOWChIP-seq, a low-input high-throughput method, in conjunction with our low-input RNA-seq approach. Fusobacterium nucleatum (Fnn) is closely related to the progression of cancers like CRC and pancreatic cancer. However, the molecular mechanisms of how Fnn adjusts the tumor microenvironment (TME) and leads to poor clinical outcomes are still unclear. In this in-vitro study, we characterized how hypoxia, an important TME ignored by previous research, facilitates Fnn infection of CRC and corresponding alterations of global epigenome and transcriptome. We infer that hypoxia has similar effects as Fnn infection alone on the CRC cells. The Fnn infection under hypoxia further boosts the proliferation and progression of CRC. We then applied our low-input RNA-seq method to study brain neuroscience and immunology. Psychedelics like DOI show promising clinical efficacy in patients with psychiatric conditions. Although psychedelics exhibit rapid antidepression action and long-lasting effectiveness compared to conventional treatment, their acute psychotic symptoms and potential for drug abuse discourage their application in clinical practice. In this case, it is important to comprehend the molecular mechanisms responsible for psychedelics' clinical efficacy. This understanding can pave the way for the development of improved treatments that do not rely on psychedelics. After profiling the transcriptome of mouse brain samples exposed to psychedelics with different post-exposure times, we concluded that the psychedelic-induced transcriptomic variations are more transient than epigenomic changes. In the second brain neuroscience project, we first applied 3-color FACS sorting to differentiate four neuron and non-neuron subtypes in human postmortem prefrontal cortex tissues. Then we profiled the gene expression of the four subtypes and validated the FACS sorting by examining the expression of marker genes. Differentially expressed genes between each subtype and the others were extracted and proceeded to gene ontology analysis. We identified unique altered biological pathways related to each subtype. The immunology research focuses on revealing the difference between low-grade inflammation and monocyte exhaustion, as well as the unique biological pathways they regulate. Therefore, we profiled the transcriptome of bone marrow-derived monocytes stimulated by PBS control, a low- or high-dose LPS. In addition to wild-type mice, we also included TRAM-deficient and IRAK-M-deficient mice. We concluded that low-dose LPS specifically regulates the TRAM-dependent pathway of TLR4 signaling, and high-dose LPS exclusively upregulates exhaustion markers by impacting metabolic and proliferative pathways.
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    Method for manufacturing a three-dimensional object
    (United States Patent and Trademark Office, 2020-01-28)
    A method for manufacturing a three dimensional object includes steps of: providing a digital description of the object as a set of voxels; sequentially creating an actual set of voxels corresponding to the digital set of voxels. At least one voxel comprises a polymer derived from: polyol and an ionic monomer. The calculated charge density of the resulting polymer is 0.01 to 0.7 mEq/g.
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    Polymer Nanoparticle Characterization and Applications for Drug Delivery
    Roberts, Rose A. (Virginia Tech, 2019-01-30)
    Nanoparticle usage continues to increase in everyday products, from cosmetics to food preservation coatings, drug delivery to polymer fillers. Their characterization and synthesis is of utmost importance to ensure safety and improved product quality. Nanoparticles can be sourced naturally or synthetically fabricated. Cellulose nanocrystals (CNCs) are rod-like nanoparticles that can be isolated from nature. Reliable methods of characterization are necessary to ensure quality control. However, their physical characteristics cause challenges for imaging under transmission electron microscopy (TEM) with a high enough resolution for dimensional analysis. Heavy metal staining such as radioactive uranyl acetate is often used to increase contrast and TEM sample substrate preparation techniques often use expensive equipment such as glow discharge in order to prevent CNC agglomeration. A method to reliably produce TEM images of CNCs without using radioactive stains or expensive glow discharge equipment was developed, using a vanadium-based stain branded NanoVan® and bovine serum albumin to keep CNCs dispersed while drying on the TEM substrate. Due to their aspect ratio, there is also concern of toxicity to the lungs. The concentration of CNCs in air in production facilities must be monitored, but there is currently no method tailored to CNCs. A method using UV-vis spectroscopy, dynamic light scattering, TEM, and scanning mobility particle sizer in conjunction with impinger collectors was developed for monitoring aerosolized CNC concentration. Synthetic nanoparticles are often used for controlled drug delivery systems. A new peptide drug termed αCT1 has been shown to interact with cell communication in a way that promotes wound healing, reduces inflammation and scarring, and aids in cancer therapy. However, the peptide's half-life in the body is estimated to be less than a day, which is not conducive to long-term treatments. Controlling its release into the body over several weeks can decrease the number of doses required, which is especially useful for glioblastoma treatment. Poly(lactic-co-glycolic acid) (PLGA) is often used for drug encapsulation since it hydrolyzes in the body and is biocompatible. Two methods of αCT1 encapsulation in PLGA were explored. It was found that flash nanoprecipitation increased loading of αCT1 in the particles by 1-2 orders of magnitude compared with the double emulsion method. Particles released αCT1 over three weeks and were non-cytotoxic.
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    Process and Material Modifications to Enable New Material for Material Extrusion Additive Manufacturing
    Zawaski, Callie Elizabeth (Virginia Tech, 2020-07-08)
    The overall goal of this work is to expand the materials library for the fused filament fabrication (FFF) material extrusion additive manufacturing (AM) process through innovations in the FFF process, post-process, and polymer composition. This research was conducted at two opposing ends of the FFF-processing temperature: low processing temperature (<100 °C) for pharmaceutical applications and high processing temperatures (>300 °C) for high-performance structural polymer applications. Both applications lie outside the typical range for FFF (190-260 °C). To achieve these goals, both the material and process were modified. Due to the low processing temperature requirements for pharmaceutical active ingredients, a water-soluble, low melting temperature material (sulfonated poly(ethylene glycol)) series was used to explore how different counterions affect FFF processing. The strong ionic interaction within poly(PEG8k-co-CaSIP) resulted in the best print quality due to the higher viscosity (105 Pa∙s) allowing the material to hold shape in the melt and the high-nucleation producing small spherulites mitigating the layer warping. Fillers were then explored to observe if an ionic filler would produce a similar effect. The ionic filler (calcium chloride) in poly(PEG8k-co-NaSIP) altered the crystallization kinetics, by increasing the nucleation density and viscosity, resulting in improved printability of the semi-crystalline polymer. A methodology for embedding liquids and powders into thin-walled capsules was developed for the incorporation of low-temperature active ingredients into water-soluble materials that uses a higher processing temperature than the actives are compatible with. By tuning the thickness of the printed walls, the time of internal liquid release was controlled during dissolution. This technique was used to enable the release of multiple liquids and powders at different times during dissolution. To enable the printing of high-temperature, high-performance polymers, an inverted desktop-scale heated chamber with the capability of reaching over 300 °C was developed for FFF. The design was integrated onto a FFF machine and was used to successfully print polyphenylsulfone which resulted in a 48% increase in tensile strength (at 200 °C) when compared to printing at room temperature. Finally, the effects of thermal processing conditions for printing ULTEM® 1010 were studied by independently varying the i) nozzle temperature, ii) environment temperature, and iii) post-processing conditions. The nozzle temperature primarily enables flow through the nozzle and needs to be set to at least 360 °C to prevent under extrusion. The environment temperature limits the part warping, as it approaches Tg (217 °C), and improves the layer bonding by decreasing the rate of cooling that allows more time for polymer chain entanglement. Post-processing for a longer time above Tg (18 hrs at 260 °C) promotes further entanglement, which increases the part strength (50% increase in yield strength); however, the part is susceptible to deformation. A post-processing technique was developed to preserve the parts' shape by packing solid parts into powdered salt.
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    Process/Structure/Property Relationships of Semi-Crystalline Polymers in Material Extrusion Additive Manufacturing
    Lin, Yifeng (Virginia Tech, 2024-03-14)
    Material Extrusion additive manufacturing (MEX) represents the most widely implemented form of additive manufacturing due to its high performance-cost ratio and robustness. Being an extrusion process in its essence, this process enables the free form fabrication of a wide range of thermoplastic materials. However, in most typical MEX processes, only amorphous polymers are being used as feedstock material owing to their smaller dimensional shrinkage during cooling and well-stablished process/structure/property (P/S/P) relationship. Semi-crystalline polymers, with their crystalline nature, possess unique properties such as enhanced mechanical properties and improved chemical resistance. However, due to the inherent processing challenges in MEX of semi-crystalline polymers, the P/S/P relationships are much less established, thus limits the application of semi-crystalline polymers in MEX. The overall aim of this thesis is to advance the understanding of P/S/P relationship of semi-crystalline polymers in MEX. This is accomplished through both experimental and simulation-based research. With a typical commodity semi-crystalline polymer, Poly (ethylene terephthalate) (PET), selected as the benchmark material. First, we experimentally explored the MEX printing of both neat and glass fiber (GF) reinforced recycled PET (rPET). Excellent MEX printability were shown for both neat and composite materials, with GF reinforced parts showing a significant improved mechanical property. Notably, a gradient of crystallinity induced by a different toolpathing time was highlighted. In the second project, to further investigate the impact of MEX parameter on crystallinity and mechanical properties, a series of benchmark parts were printed with neat PET and analyzed. The effect of part design and MEX parameter on thermal history during printing was revealed though a comparative analysis of IR thermography. Subsequent Raman spectroscopy and mechanical test indicated that crystallinity developed during the MEX process can adversely affects the interlayer adhesion. In the third project, a 3D heat transfer model was developed to simulate and understand the thermal history of MEX feedstock material during printing, this model is then thoroughly validated against the experimental IR thermography data. While good prediction accuracy was shown for some scenarios, the research identified and discussed several unreported challenges that significantly affect the model's prediction performance in certain conditions. In the fourth project, we employed a non-isothermal crystallization model to directly predict the development of crystallinity based on given temperature profiles, whether monitored experimentally or predicted by the heat transfer model. The research documented notable discrepancies between the model's predictions and actual crystallinity measurements, and the potential source of the error was addressed. In summary, this thesis explored the MEX printing of semi-crystalline polymer and its fiber reinforced composite. The influence of MEX parameters and part designs on the printed part's thermal history, crystallinity and mechanical performance was then thoroughly investigated. A heat transfer model and a non-isothermal crystallization model were constructed and employed. With rigorous validation against experimental data, previously unreported challenges in MEX thermal and crystallization modeling was highlighted. Overall, this thesis deepens the understanding of current semi-crystalline polymer's P/S/P relationship in MEX, and offers insights for the optimization and future research in the field of both experiment and simulation of MEX.
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    Rational Design of Poly(phenylene sulfide) Aerogels Through Precision Processing
    Godshall, Garrett Francis (Virginia Tech, 2024-04-02)
    Poly(phenylene sulfide) (PPS), an engineering thermoplastic with excellent mechanical, thermal, and chemical properties, was gelled for the first time using 1,3-diphenylacetone (DPA) as the gelation solvent in a thermally induced phase separation (TIPS) process. PPS was dissolved in DPA at high temperatures to form a homogeneous solution. The solution was cooled, initiating phase separation and eventually forming a solidified PPS network around DPA-rich domains. Evacuation of DPA from the gel network creates monolithic PPS aerogels, one of few physically crosslinked polymer aerogel systems comprised of a high-performance thermoplastic. In this work, specific properties of PPS aerogels were controlled through the manipulation of various processing parameters, such as polymer concentration, post-process annealing conditions, mode of manufacturing (casting versus additive manufacturing), dissolution temperature, and drying method. The ultimate goal was to elucidate key process-morphology-property relationships in PPS aerogels, to ultimately improve aerogel performance and applicability. The phase diagram of PPS/DPA was first elucidated to determine the phase separation mechanism of the system, which guides all future processing decisions. The phase diagram indicated that the system undergoes solid-liquid phase separation, typical for solutions with relatively favorable polymer-solvent interactions. This assignment was validated by the calculation of the Flory-Huggins interaction parameter through two independent methods - Hansen solubility parameters and fitting melting point depression data. The influence of polymer composition on PPS aerogel properties was then characterized. As polymer concentration increased, aerogel density and mechanical properties increases, and porosity decreased. The particular morphology of PPS aerogels from DPA was that of a fibrillar network, where these axialitic (pre-spherulitic) fibrils are comprised of stacks of PPS crystalline lamellae, as suggested by x-ray scattering and electron microscopy. These interconnected microstructures responded more favorably to compressive load than similar globular PEEK aerogels, highlighting the importance of aerogel microstructure on its mechanical response. Upon solvent extraction, PPS aerogels were annealed in air environments to improve their mechanical behavior. Annealing did not dramatically shrink the aerogels, nor did it appear to affect the micron-scale morphology of PPS aerogels as observed by electron microscopy. The resistance to densification of PPS aerogels was mainly a product of their interconnected fibrillar morphologies, aided by subtle microstructural changes that occurred upon annealing. Exposure to a high temperature oxidative environment (160 – 240 oC) increased the degree of crystallinity of the aerogels, and also promoted chemical crosslinking within the amorphous PPS regions, both of which may have helped to prevent severe densification. With enhanced physical and chemical crosslinking, annealed PPS aerogels displayed improved compressive properties over unannealed analogues. Additionally, the thermal conductivity of both annealed and unannealed aerogel specimens was below that of air (~ 0.026 W/mK) and did not display a dependence on polymer composition nor on annealing condition. Generally, these experiments demonstrate that annealing PPS aerogels improved their mechanical performance without negatively affecting their inherent fibrillar morphology, low density, or low thermal conductivity. To fabricate aerogels with geometric flexibility and hierarchical porosity, PPS/DPA solutions were printed through material extrusion (MEX) and TIPS using a custom-built heated extruder. In this process, solid solvated gels were first re-dissolved in a heated extruder and solutions were deposited in a layer-wise fashion onto a room-temperature substrate. The large temperature gradient between nozzle and substrate rapidly initiated phase separation, solidified the deposited layers and formed a printed part. Subsequent solvent exchange and drying created printed PPS aerogels. The morphology of printed aerogels was compositionally-dependent, where the high extrusion temperature required to dissolve highly-concentrated inks (50 wt % PPS) also destroyed self-nuclei in solution, yielding printed aerogels with spherulitic microstructures. In contrast, aerogels printed from 30 wt % solutions were deposited at lower temperatures and demonstrated fibrillar microstructures, similar to those observed in 30 wt % cast aerogel analogues. Despite these microstructural differences, all printed aerogels demonstrated densities, porosities, and crystallinities similar to their cast aerogel counterparts. However, printed aerogel mechanical properties were microstructurally-dependent, and the spherulitic 50 wt % aerogels were much more brittle compared to the fibrillar cast 50 wt % analogues. This work introduces a widely-applicable framework for printing polymer aerogels using MEX and TIPS. Intrigued by the compositional morphological dependence of the printed PPS aerogels, the dissolution temperature (Tdis), and thus the self-nuclei content, of cast PPS/DPA solutions was systematically varied to understand its influence on aerogel morphology and properties. As Tdis increased, the length and diameter of axialites increased while aerogel density and porosity were relatively unaffected. Thus, the isolated influence of axialite dimensions (analogous to pore size and pore concentration) on aerogel properties could be studied independent of density. At low relative densities (below 0.3, aerogels of 10 – 30 wt %), compressive modulus and offset yield strength tended to decrease with Tdis, due to an increase in axialite length (akin to pore size) and number of axialites (akin to number of pores). At higher relative densities (above 0.3, 40 and 50 wt %), axialitic aerogels were so dense that changes in pore dimensions did not result in systematic changes in mechanical response. All spherulitic aerogels fabricated at the highest Tdis¬ demonstrated reduced mechanical properties due to poor interspherulitic connectivity. The thermal conductivity of all aerogels increased with polymer composition but demonstrated no clear trend with Tdis. A model for thermal conductivity was used to deconvolute calculated conductivity into solid, gaseous, and radiative components to help rationalize the measured conductivity data. This work demonstrates the importance of nucleation density control in TIPS aerogel fabrication, especially at low polymer concentrations. The specific method used to dry an aerogel generally has a great influence on its microstructure and density. Vacuum or ambient drying is the most industrially-attractive technique due to low cost and low energy usage; however, it is typically the most destructive process due to high capillary forces acting on the delicate aerogel microstructure. Three drying methods, vacuum drying, freeze drying, and supercritical CO2 drying, were used to evacuate PPS gels fabricated at three PPS concentrations (10, 15, and 20 wt %). Almost all aerogel specimens displayed excellent resilience against shrinkage as a function of the drying method, besides the 10 wt % vacuum dried sample which shrunk almost 40%. While the micron-scale aerogel morphology captured by electron microscopy appeared to be unaffected by the drying method, other properties such as aerogel surface area, mesoporous volume, and mechanical properties were effectively functions of the degree of aerogel shrinkage. Aerogel thermal conductivity was low for all samples, and in particular, vacuum dried aerogels demonstrated slightly lower conductivities than other ambiently-dried aerogel systems such as silica and carbon. In general, vacuum drying appears to be industrially viable for PPS aerogels at concentrations above 10 wt %.
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    Ruminal Degradation of Polyhydroxyalkanoate and Poly(butylene succinate-co-adipate)
    Galyon, Hailey Roselea (Virginia Tech, 2022-06-21)
    The occurrence of plastic impaction in ruminants is a growing concern. As indiscriminate feeders, cattle may consume plastic foreign materials incorporated into their diets and it is currently estimated that 20% of cattle contain plastic foreign materials in their rumen. These materials are indigestible and accumulate for the lifetime of the animal. As these materials accumulate, they may reduce feed efficiency and production by erosion and ulceration of rumen epithelium, stunting of papillae, blockage of the reticulo-omasal orifice, and leaching of toxic heavy metals. It is necessary to reduce the incidences of plastic impaction in domestic ruminants. Using polyhydroxyalkanoate (PHA) and poly(butylene succinate-co-adipate) (PBSA) biodegradable materials for feed storage products such as bale netting could reduce the incidences and effects of polyethylene-based plastic impaction in ruminants. The objectives of these studies were to evaluate the degradability of PHA and PBSA materials in the reticulorumen via in vitro, in situ, and in vivo methods. Our hypothesis was that these materials would degrade in the rumen and that a melt-blend of PHA and PBSA may degrade faster than its individual components. An in vitro study incubated a proprietary PHA-based polymer, PBSA, and PBSA:PHA melt blend nurdles, and forage controls in rumen fluid for up to 240h in DaisyII Incubators. Mass loss was measured, and digestion kinetic parameters were estimated. Thermogravimetric and differential scanning calorimetry analyses were conducted on incubated samples. Results indicated that the first stage of degradation occurs within 24h and PHA degrades slowly. Degradation kinetics demonstrated that polymer treatments were still in the exponential degradation phase at 240h with a maximum disappearance rate of 0.0031%/h, and mass loss was less than 2% for all polymers. Melting temperature increased and onset thermal degradation temperature decreased with incubation time, indicating structural changes to the polymers starting at 24h. Further in situ degradation, however, indicated these biodegradable materials degrade at more accelerated rates in the rumen. Polyhydroxyalkanote, PBSA, PBSA:PHA blend, and low-density polyethylene (LDPE) films were incubated in the rumens of three cannulated, non-lactating Holsteins for 0, 1, 14, 30, 60, 90, 120, and 150d. In situ disappearance (ISD) and residue length were assessed after every incubation time. Polyhydroxyalkanoate achieved 100% degradation by 30d, with initiation occurring at 14d indicated by ISD and a reduction in residue length. The fractional rate of disappearance of PHA was 7.84%/d. Poly(butylene succinate-co¬-adipate) and Blend did not achieve any significant ISD, yet fragmentation of PBSA occurred at 60d and the blend at just 1d likely due to abiotic hydrolysis. Low-density polyethylene achieved no ISD and residue length did not change over incubation time. From these results, we proposed a PBSA:PHA blend is a valid alternative to polyethylene single-use agricultural plastic products based on its fragmentation within 1d of incubation. Administration of PBSA:PHA film boluses compared to LDPE films and a control further supported this dissemination. Holstein bull calves (n = 12, 62 ± 9d, 74.9 ± 8.0kg) were randomly allocated to one of three daily bolus treatments: 13.6g of PBSA:PHA in 4 gelatin capsules (Blend), 13.6g of LDPE in 4 gelatin capsules (LDPE), or 4 empty gelatin capsules (Control) for 30d. Hemograms were conducted on blood samples collected on d0 and d30. On d31, animals were sacrificed to evaluate gross rumen measurements and pathology, determine papillae length, and characterize polymer residues present in rumen contents. Feed intake, body weight, body temperature, and general health were determined throughout the study. No animals presented any symptoms related to plastic impaction and animal health was not particularly affected by treatment. Daily grain and hay intake, body weight, rectal temperature, hematological parameters, gross rumen measurements and pathology, and rumen pH and temperature were not affected by treatment. There was evidence that degradation of PBSA:PHA may release byproducts that support rumen functionality. Methylene blue reduction time of Blend calves tended to be decreased by 30% compared to LDPE calves, and caudal ventral papillae length of Blend calves were 50% longer than those of Control animals. Though studies are needed to specifically elucidate the production of byproducts due to degradation of PBSA:PHA and their correlations. Polymer accumulation and residue length differed among treatments. Calves dosed with LDPE retained 6.7% of the dosed polymer, undegraded, while Blend calves retained 0.4% of the dosed polymer. The polymer residues in Blend calves were 10% of their original size. Single-use agricultural plastics developed from PBSA:PHA may be a suitable alternative to LDPE-based products in the case of ingestion in ruminants due to no acute health inflictions, fragmentation of polymers with 1d, and improved clearance from the reticulorumen. As such, utilization of these materials may reduce the incidences of plastic impaction in ruminants in commercial operations. Further long-term feeding studies are needed to evaluate specific byproduct production of PBSA:PHA and their potential influences on rumen function and animal health and production in normal commercial conditions.
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    Stimuli-Responsive Peptide-Based Biomaterials: Design, Synthesis, and Applications
    Zhu, Yumeng (Virginia Tech, 2023-05-15)
    Peptide-based biomaterials have gained much interest in various applications in drug delivery and tissue engineering in recent years, in large part due to their typically excellent biocompatibility and biodegradability. Composed of different amino acids, peptides can be designed with numerous sequences, providing flexibility and tunability in biomaterials. Peptides are easy to modify with small molecule drugs, inorganic components, and polymer chains to access multiple functions and tune properties relevant to biology and medicine. Stimuli-responsive peptide-based biomaterials can respond to environmental stimuli, such as light and ultrasound, in addition to local environmental factors, such as temperature, enzyme activity, and pH. Under environmental changes, these materials can be triggered to release therapeutic payloads, change conformations, or induce self-assembly in the target sites. In this work, I introduce the design, synthesis, and potential applications of several stimuli-responsive peptide-based biomaterials. The first half of this dissertation is based on enzyme-responsive, peptide-based biomaterials as extracellular matrix (ECM) mimics in tissue engineering. We synthesized linear and dendritic elastin-like peptides (ELPs) as crosslinkers and conjugated them with hyaluronic acid (HA) to form hydrogels. Trypsin was used as the enzyme trigger for cleaving the C-terminal lysine and to study how crosslinker topology affects enzymatic degradation. Hydrogels with dendritic ELPs degraded more slowly than linear ELPs, providing a novel strategy to tune the degradation rate of hydrogels as ECM mimics by the molecular design of crosslinker topology. Building on this peptide-polysaccharide platform for synthetic ECM design, we subsequently prepared hydrogels embedded with bioactive cryptic sites. These novel polymeric hydrogels mimicked native ECM cryptic sites by using depsipeptides that undergo an enzyme-triggered molecular rearrangement, "switching" from a non-functional epitope to a bioactive sequence. Mass spectrometry, 1H and 13C NMR spectroscopy, and fluorescence studies were applied to track structural changes in the peptide. SEM was used to image these polymer-peptide hybrid hydrogels. Finally, in vitro studies were conducted to evaluate cell interactions with the hydrogels. Switch peptide-modified alginate hydrogels showed increased cell adhesion upon induction of enzymatic activity, which provided a "gain of function" of the synthetic ECM. Critically, enzymes associated with the cells themselves could trigger the peptide switch and change in synthetic ECM behavior. With knowledge of stimuli-responsive peptide-based biomaterials applied in tissue engineering, I then studied how this system could be used in drug delivery by designing peptide-hydrogen sulfide (H2S) donor conjugates (PHDCs). H2S is a gasotransmitter that is produced endogenously, which has been explored in recent years with many potential therapeutical applications. We studied H2S release profiles in dual-enzyme-responsive PHDCs, with a further investigation into PHDC–Fe2+ complexes for potential tumor treatments via chemodynamic therapy. The PHDC–Fe2+ complexes were examined in a C6 glioma cell line, exhibiting an improved cell-killing effect compared with controls, by inducing toxic hydroxyl radical generation (•OH) via a Fenton reaction. To this end, we further discovered how side chains influence self-assembling nanostructures, H2S release profiles, and biological activities via three constitutionally isomeric PHDCs. Different morphologies and varied H2S release rates were observed, paving the way for tuning the properties of PHDCs by simple changes in molecular design. Finally, this dissertation discloses conclusions and future directions on stimuli-responsive peptide-based biomaterials using similar platforms with different designs in the drug delivery and tissue engineering fields.
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    Surface Coatings for Antimicrobial Activity and Fast Evaporation
    Hosseini, Mohsen (Virginia Tech, 2024-05-29)
    Coatings play a pivotal role in everyday life and across various industries. They offer protection, corrosion resistance, insulation, optical improvements, aesthetics, etc. This study investigates the design, fabrication, characterization and evaluation of surface coatings in two areas: antimicrobial activity and fast evaporation. The COVID-19 pandemic underscored the necessity for coatings that mitigate microbial transmission through surfaces, alleviating both contagion and personal fears. The first part of this study presents the design, development, and evaluation of antimicrobial coatings that efficiently inactivate 99.9% of SARS-CoV-2 virus and kill more than 99.9% of pathogenic bacteria such as Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa within one hour. Prioritizing rapid infectivity reduction, we designed and fabricated several coatings using silver oxide (Ag2O), cupric oxide (CuO), and zinc oxide (ZnO) particles as active ingredients. Applying small quantities of micron-sized opaque particles onto a surface yields a transparent film. Although Ag2O particles are inherently opaque, they possess potent antimicrobial properties. Consequently, incorporating small quantities of Ag2O into the coating results in the desired antimicrobial activity while maintaining transparency. Transparent antimicrobial coatings are a necessity for applications such as touchscreens, offering the benefit of reducing disease transmission while maintaining the aesthetic appeal of surfaces. We employed a variant of the Stöber process to bind Ag2O particles to the substrate using a silica matrix. To improve this coating method, we employed room-temperature spin-coating of a suspension of Ag2O/sodium silicate solution on the substrate, eliminating reactions with toxic chemicals in Stöber process and subsequent heat treatment. Two key features of the improved coating are its high robustness and its capability to kill 98.6% of Clostridioides difficile endospores in 60 minutes. On the other hand, CuO and ZnO particles exhibit mild antimicrobial properties; thus, their activity could be enhanced by a porous coating. When an infected droplet lands on such a coating, it is imbibed into the porous structure, where diffusion distances are smaller, and there is a larger active area to inactivate the virus or kill the bacteria. Furthermore, porosity facilitates faster droplet drying, leading to the concentration of cupric and zinc ions in the droplet, which are designed to be toxic to microbes. The second major topic of this thesis is the development, and evaluation of porous coatings for fast evaporation. At low Bond numbers, droplet evaporation is slow on an impermeable surface. We investigated whether application of a thin, porous coating leads to faster droplet evaporation. The droplet will imbibe quickly, but progress normal to the interface will be limited to the thickness of the coating. Therefore, the liquid will spread laterally into a broad disk to expose a large liquid–vapor interface for evaporation. As a result, the evaporation of a droplet is enhanced by a factor of 7–8 on the thin porous coatings. Factors such as coating thickness, pore size and distribution, and the contact angle of the coating, as well as ambient conditions like temperature and relative humidity, could affect the droplet evaporation rates by modifying the droplet's imbibition process and the evaporation driving force. While decreasing the coating thickness and increasing pore size and distribution promoted evaporation, the impact of contact angle is insignificant. Confocal microscopy observations of a coating composed of particles with varying sizes depicted liquid migration along the top of the coating and the edges of the interface. We developed and validated an equation to estimate the rate of evaporation. The rate correlated with the radius of the imbibition area, with higher temperatures and lower humidity further augmenting evaporation.