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Purpose: Within the last 30 years, cancer-nerve interactions have garnered attention for causing patient pain, increasing the risk of recurrence, and decreasing patient prognosis. There are 2 key forms cancer-nerve crosstalk takes: perineural invasion (PNI), in which cancer cells infiltrate nerves as a route for metastasis, and tumor innervation (TI), in which neurites penetrate solid tumors to promote tumor progression. Present in vitro models of cancer-nerve crosstalk primarily include Matrigel® mouse matrix and collagen I hydrogels; whereas collagen I hydrogels neglect tumor extracellular matrix (ECM) complexity, Matrigel® is controversial for its batch-to-batch inconsistencies and inclusion of experiment-altering growth factors. Decellularized ECM (dECM) scaffolds, which utilize clean scaffolds of native tissues, have garnered credibility for their ability to mimic tumors and other diseases. The goal of this dissertation is to fabricate physiomimetic in vitro testbeds for analyzing tumor phenotype and/or neurite outgrowth in PNI and TI. Methodology: A dECM PNI platform to replicate the nerve microenvironment was optimized by examining 3 unique decellularization methods and nerve preparations. Using the selected protocol, hydrogels were fabricated for modeling pancreatic cancer (PDAC) PNI. Tumor and healthy cell-derived extracellular vesicles (EV), which are neglected nanoparticles in cancer-nerve crosstalk research, were harvested, and proteomics and pathway analyses were run. Schwann cells (SC), the support cells of the peripheral nervous system known for perpetrating PNI, were treated with tumor and healthy cell EVs. Invasion assays observed PNI in vitro. Conversely, an adipose dECM hydrogel was created to mimic breast tissue and was combined with collagen I to replicate the desmoplastic breast tumor microenvironment. Using this platform, breast cancer (BC) and healthy cell toxicity, metabolic, and phenotypic responses to Metformin anti-diabetes therapy ± fibroblasts were observed. Neurite outgrowth was measured when PC12 cells were cultured with BC or healthy cells. Results: An SD-based protocol with nerve epineurium removal best removed native cells without compromising ECM composition; the hydrogel digested from these nerves assembled collagen fibrils and maintained mechanical properties similar to nerve tissue. SCs cultured in this hydrogel with tumor EVs were activated in an IL-8-dependent manner, a protein found to be upregulated in tumor EVs over healthy cell EVs, to alter their cytoskeletal protein expression and secretome, including CCL2 upregulation. Invasion studies concluded this signaling pathway facilitated PDAC PNI in vitro. Metabolic cargoes, including those related to glycolysis, gluconeogenesis, and glutathione redox balance, were also altered in tumor EVs; given their association with IL-8 secretion, the exploration of these pathways in regard to PNI was proposed. Additionally, BC cells cultured within the dECM composite scaffold required high doses of Metformin to induce metabolic dysfunction, which also depleted healthy cell health. Fibroblasts cultured with BC cells transformed into cancer-associated fibroblasts with Metformin treatment. Finally, BC cells slightly increased the length of neurite outgrowth. Implications: These findings will help educate future targeted therapies and suggest the negative effects of current treatment options. In summary, the results collected using these platforms will inform future preclinical and clinical work to improve the outcomes of patients with PNI and TI.

This dissertation explores the use of intrinsically disorder proteins (IDPs) in biomedical applications related to the detection and biosensing of nucleic-acid species. We engineer several low critical solution temperature (LCST) nucleic-acid binding elastin-like polypeptides (ELPs) as model IDPs and study their potential in biomedical application via interactions with short single-stranded (ss) DNA, tRNA and viral RNAs. First, we engineer a simple polycationic ELP capable of recruiting DNA on demand in a DNA-protein-rich condensate via LLPS. Moreover, we develop a modified Flory-Huggins theory to quantify the partitioning of DNA and protein upon coacervate formation with and without salts. We then design more robust NA-binding ELPs for use in the detection of NA pathogens such as NA extraction (NAE) or isolation to investigate the potential use of NA-binding ELPs as molecular chaperones of relevant DNA reactions. Our findings suggest that the genetically engineered IDPs we designed can concentrate complementary and structurally complex DNA strands in a biomolecular condensate to accelerate the kinetics of the reaction. Altogether, we show that IDPs can have a major impact on downstream processes involved in the process of NA testing.

This dissertation proposes the GraphGANFed framework, which combines Graph Convolutional Networks (GCN), Generative Adversarial Networks (GAN), and Federated Learning (FL) to generate novel molecules while preserving data privacy. FL allows learning from distributed clients without sharing local datasets, GCN extracts molecular structural properties, and GAN generates new molecules retaining the learned properties. Extensive simulations on three benchmark datasets show GraphGANFed's effectiveness, producing molecules with high novelty (≈ 100) and diversity (> 0.9). Results indicate a trade-off among evaluation metrics, and the right dropout ratio avoids mode collapse. Additionally, Conditional GraphGANFed (cGraphGANFed) incorporates a critic network from Reinforcement Learning (RL) to meet specific client requirements. cGraphGANFed can optimize for particular metrics, producing molecules with higher values in specified metrics or achieving high values across multiple metrics, which may be interdependent. Extensive simulations confirm cGraphGANFed’s efficacy in optimizing specific client objectives and generating molecules with high performance across multiple metrics.

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Bioprinting, akin to 3D printing, employs cell-laden hydrogels such as GelMA (Gelatine Methacrylate) and Alginate instead of plastic or resin to create biological structures. The printing process followed by subsequent tissue maturation is known as 4D bioprinting, where the fourth dimension is time. Ensuring adequate and consistent oxygen, O2, supply within 3D bioprint volume over the entire maturation period process is crucial, as even transient hypoxia can impact cellular behavior and phenotypes. However, printing of fully developed vasculature remains an unresolved technical challenge. Several approaches to chemically (peroxides) or biologically (photosynthesis) generate oxygen during bioprint maturation have been proposed. However, a comprehensive evaluation and optimization of these methods is not currently possible due to the lack of quantitative longitudinal O2 imaging modality. 4D electron paramagnetic resonance imaging (EPRI) is demonstrated to be the method of choice to solve the problem of even bioprint oxygenation in 3D volume and over a prolonged maturation time. EPRI permits longitudinal quantitative noninvasive O2 mapping using incorporate in bioinks probes. Two types of probes have been utilized: water soluble triarylmehyl (trityls) stable radicals and solid lithium octa-nbutoxy-phthalocyanine (LiNc-BuO) particles. Incorporation of these materials in bioinks modifies its chemical and optical properties. For example, LiNc-BuO absorbing light affects photopolymerization. Also, bioprinting process affects the probes. Trityls chemically react with the formed intermediate radicals. Part of the presented work was dedicated to understanding and optimization of the bioprinting process that includes the addition of oxygen probes into the commonly used GelMa and alginate bioinks. Bioprinting was done using locally developed digital light processing (DLP) and extrusion bioprinters. 4D oximetry was performed using a locally built EPRI instrument. Both acellular and cell-laden constructs were successfully printed and imaged. Experiments revealed a previously unreported phenomenon of oxygen depletion due to the presence residual photoinitiator. Oxygen consumption rates by HEK293T cells within printed structures were quantified. 4D EPRI mapping of changing oxygen levels within a murine macrophage-laden alginate constructs have been demonstrated using freeform reversible embedding of suspended hydrogels (FRESH) methodology. Oxygen consumption rates were computed by analyzing consecutive EPR images in the time dimension. Given the incorporation of the EPR probes into the alginate bioink, proper control experiments evaluating the temporal EPR probe stability and EPR signal intensity in acellular bioink were conducted. In conclusion, we've shown the feasibility of integrating 4D EPR with bioprinting, paving the way for diverse applications using various bioinks and molecular spin probes. These findings are foundational for advancing bioprinting technologies and understanding oxygen dynamics in tissue engineering.

Hyperglycemia and dyslipidemia are common comorbidities that often coincide and have a significant impact on the severity of diabetes. This current study investigates the pathology and mechanism behind skeletal muscle cachexia and cardiac dysfunction in diabetic dyslipidemia. Stem cells continue to be critical as a regenerative strategy to restore damaged tissue, however, several drawbacks have been observed with use of stem cells including thrombogenesis, low survival, and tumorigenicity. Therefore, we isolated exosomes from stem cells and assessed their ability to attenuate diabetes-induced sarcopenia and cardiomyopathy. Exosomes are nanosized particles released by cells, containing proteins and nucleic acids that allow it to exhibit similar properties to the cell type of origin. To model diabetic dyslipidemia, we utilized ApoE knockout mice (10±2 weeks) and divided them into 4 groups consisting of control (saline intraperitoneal (IP) injection), diabetic (STZ IP injection), treatment group administered intravenous (IV) exosomes derived from miR-1 ES-Exos (microRNA-1 enriched Embryonic Stem Cells) or MSC-Exos (Mesenchymal Stem Cells), and negative control treatment MEF-Exos (Mouse Embryonic Fibroblasts). Heart and soleus tissue samples were analyzed for inflammation, inflammatory cell death expression, and adverse tissue remodeling using histology, immunohistochemistry, western blotting, RT-PCR, cytokine, and luciferase-based arrays. In summary we found diabetic dyslipidemic mice acquire cardiac and skeletal muscle dysfunction. Administration of miR-1 ES-Exos and MSC-Exos significantly mitigated inflammation and cell death marker expression, resulting in improved cardiac and skeletal muscle function. In conclusion our data shows that miR-1 ES-Exos and MSC-Exos are effective therapeutic agents in attenuating diabetes-induced sarcopenia and cardiomyopathy.

Neuron infiltration is involved in a multitude of diseased states and is critical in the regeneration of physiologic conditions. Modeling of innervation in three-dimensional, heterogenous, environments would allow early investigation in the crosstalk of neurons and native cells in normal and pathophysiologic microenvironments to gain an understanding of mechanistic functions. This would allow for greater insight into the role of metabolism, cell adhesion, microenvironmental alternations, cellular crosstalk, and signaling pathways that play a role in the promotion and prevention of neurite growth. In this thesis, with the construction of collagen-based hydrogels as the foundation for cell culture, we investigate cell behavior in models that mimic native tissue microenvironments where neurite infiltration contributes to pathophysiology.

Mechanical stimulation through physical activity has been shown to play an important role in treating and preventing several non-communicable diseases such as hypertension, lower back pain (LBP), type-2 diabetes mellitus, and several cancers. This is accomplished through the regulation of cellular behavior and tissue remodeling within the body at both the micro- and macro-scale levels. The goal of mechanobiology research is to gain in-depth knowledge and understanding of how cells sense physical forces in conjunction with other biochemical cues and translate those factors into important biological functions that either maintain tissue homeostasis or lead to pathological states. Understanding these processes can lead to the development of medical interventions that influence cells towards a desired outcome. While some of these processes can be observed naturally through thoughtful experimental design, most mechanobiological processes require the development of tools that can enable scientists and researchers to investigate a specific aspect of cell mechanobiology. In this PhD dissertation, we investigated the role that mechanical stimulation plays in regulating cell behavior in different chemical environments by subjecting cells cultured in monolayer to levels of uniaxial tension similar to those experienced in the annulus fibrosus (AF) in the intervertebral disc (IVD). We then developed both an experimental and computational platform that could be used to study the effects of mechanical stimulation on physiological function at both the microscopic and macroscopic levels. The experimental platform was designed to enable long term application of mechanical forces onto cell-seeded tissue scaffolds with the intention of developing a bioreactor to enable in vitro studies focused on investigating how mechanical forces influence cell behavior in healthy and diseased IVDs. The computational platform employed machine learning algorithms and data science strategies to examine how clinical measurements such as blood pressure and blood serum analytes influence patient risk for masked hypertension in a young, apparently healthy population.

Cardiovascular diseases are the leading cause of death worldwide. Heart disease, in particular, accounted for 1 in every 5 deaths in the United States in 2021, representing a substantial public health burden that will require more effective treatment options to alleviate. Heart transplant surgery is currently the only option to completely restore cardiac function, but many patients die while waiting for a transplant due to a longstanding organ donor shortage. Thus, there is a dire need for accessible treatment options that can repair damaged hearts. Recent advances in tissue engineering and regenerative medicine have accelerated the development of therapies for cardiac repair, enabling functional heart cells to be grown in the lab and transplanted directly into injured myocardium. However, cardiac tissue engineering faces several key hurdles, including vascularization of grafted tissue, long-term functional engraftment of transplanted cells, and an unclear understanding of their therapeutic mechanisms. In my doctoral research, I developed novel platforms to address these limitations and advance the translational potential of cardiac tissue engineering. My research focused on leveraging the native microenvironment of cells in vascularized, electrically conductive cardiac tissue to develop bioinspired therapies for cardiovascular disease. Engineering a chemically defined hydrogel matrix inspired by native angiogenesis mechanisms, I developed a bioink for bioprinting modular vascularized tissue engineering constructs that could be applied broadly to cardiovascular disease and wound healing. Next, inspired by the native cardiac conduction system, I developed human cardiac microtissues (organoids) embedded with electrically conductive silicon nanowires (e-SiNWs) to improve the efficiency and efficacy of cardiac cell therapy. Finally, I investigated therapeutic mechanisms of nanowired human cardiac organoids by analyzing the restorative capacity of the extracellular vesicles (EVs) they release. This investigation led to novel insight into the therapeutic nature of cardiac organoid derived EVs and how the nanowired bioelectric interface can be manipulated to augment EV production and function from cardiac tissue engineering constructs. The research established here provides a foundation for developing the next generation of clinically translational therapies for cardiovascular disease.

Heart Failure, sometimes called Congestive Heart Failure, is a collection of pathological disruptions in which the heart is unable to pump blood properly. It affects over 6 million people in the United States, with the cost-per-patient at approximately $30,000 and the total cost estimated to be $160 billion by 2030. There is no single cause of heart failure, as it can manifest as a varying array of diseases, disorders, and syndromes. However, many of these underlying diseases result specifically from dysregulation of mechanically active cells called cardiac fibroblasts, which play a critical role in the remodeling of the extracellular matrix and provide a feedback mechanism to control force distributions within the myocardium via stiffening or softening the tissue. These forces, generated by cardiomyocytes, are then altered into one of many unique disease states. Cardiac fibrosis stimulates excessive collagen production, stiffening the ECM and resulting in Left Ventricle hypertrophy. Myocardial Infarction results in a localized region of dominant and activated fibroblasts, greatly upregulating ECM production to protect against loss of healthy tissue and disrupting electrical signaling propagation. The underlying mechanisms of fibroblast-mediated matrix remodeling and its effects on cardiomyocyte force generation are critical to our understanding of cardiovascular diseases. We have developed an in vitro 3D wound platform that can recreate biologically relevant mechanical conditions utilizing cardiac fibroblasts and cardiomyocytes. It does this by precisely balancing matrix stiffness properties, cell distributions, and electrical/mechanical stimulation. With the ability to create healthy left ventricular myocardial tissue, we also show the ability to produce diseased tissues that either mimic the mode of injury (MI) or upregulate fibroblast signaling pathways (cardiac fibrosis) to recreate an appropriate model of each pathology. This provides a unique platform that can create and characterize, with high precision, the biological and mechanical condition of diseased myocardial tissue for use in clinical or drug development applications.

More than 300,000 women will be diagnosed with breast cancer, and approximately 48,000 women will die this year due to the disease in the United States. HER2+ breast cancer accounts for 20-25% of invasive breast cancer, and it has a worse prognosis and higher early-stage mortality rate than other breast cancer subtypes. Common treatments for HER2+ breast cancer involve surgical removal of the breast or systemic therapy with chemotherapeutic drugs, both of which cause damage to healthy tissue. Due to the lack of a natural ligand for the HER2 receptor, there is a need for improved targeting strategies for the treatment of HER2+ breast cancer. Short interfering RNA, a type of RNA interference, is a promising tool for cancer treatment that works by silencing oncogenes on the mRNA level to reduce the expression of proteins contributing to cancer progression. siRNA requires a carrier to protect it from degradation and to mediate its delivery into a target cell. There are multiple types of carriers for siRNA delivery, including viral vectors and non-viral vectors such as polymeric nanoparticles, liposomes, and peptide-based nanoparticles. Peptides have advantages over other delivery mechanisms, such as control over functionalization, easier synthesis, and better biocompatibility. CD44, an oncogene correlated with HER2+ breast cancer, is directly involved in tumor growth and metastasis. CD44 is expressed in most healthy cells; therefore, silencing must be selective for HER2+ breast cancer. In this study, we investigated a novel strategy for treating HER2+ breast cancer utilizing peptide-mediated delivery of CD44-specific siRNA. We characterized two tandem peptides consisting of HER2 targeting peptides, P25 or P51, and the fusogenic peptide, DIV3W. By linking these two functional peptides, we aim to show that our tandem peptides can target the HER2 receptor and successfully deliver siCD44 to initiate the silencing of CD44 on the gene and protein level in HER2+ breast cancer cells. Characterization assays were performed to determine which tandem peptide possessed more optimal qualities as a carrier for siRNA. We observed that the tandem peptide, P25-DIV3W, protects siRNA from degradation, is cytocompatible, and promotes efficient internalization into HER2+ cell lines. P51-DIV3W showed poor siRNA protection and cytotoxic traits when delivered to HER2+ and HER2-normal cell lines. We then showed that P25-DIV3W loaded with siCD44 initiates the knockdown of CD44 and promotes cell death in HER2+ cell lines while not affecting the viability of normal breast epithelial cells in low concentrations.

Diabetes has become one of the leading causes of lower-limb loss worldwide. Every 30 seconds, a person loses a limb due to diabetic-related vascular complications. About one-third of patients needing lower-limb bypass surgery have debilitated autologous vessels unsuitable for use, and no other good long-term options are available. These detrimental effects on the vasculature are caused mainly by the hyperglycemic and hyperlipidemic conditions derived from diabetes. Under these conditions, an increase in advanced glycation end products (AGEs) and reactive oxygen species leads to irreversible crosslinks of extracellular matrix proteins, accelerating vascular pathology through vascular stiffening, endothelial dysfunction, inflammation, atherosclerosis, fibrosis, and calcification. For many years, scientists have attempted to develop tissue-engineered small-diameter vascular grafts (TEVGs) that can be used as off-the-shelf options for these patients. However, all these attempts have failed so far, leaving thousands of patients waiting. Preliminary studies performed in our lab demonstrated that treating decellularized porcine scaffolds with penta-galloyl glucose (PGG) prior to subdermal implantation in diabetic rats prevents diabetic-induced effects on the implant material, protects them from fast biodegradation, and provides anti-thrombogenic and enhanced mechanical properties. Furthermore, we have shown that our TEVGs can be repopulated with human endothelial cells and adventitial fibroblasts, which are necessary for the long-term integration and patency of the grafts. Our long-term goal is to develop small-diameter TEVGs that provide an optimal off-the-shelf option for diabetic patients in need of lower-limb bypass surgery. To contribute to this goal, we investigated the performance of our decellularized vascular grafts treated with PGG and revitalized with human cells in perfusion models in vitro and in vivo in diabetic conditions. We hypothesized that our TEVGs would perform optimally as perfusion conduits in a diabetic environment by maintaining patency and preventing biodegradation and diabetes-related complications. Decellularized porcine arteries were treated with PGG and seeded with adventitial fibroblasts. For in vitro studies, our TEVGs were placed in vascular bioreactors under normal and diabetic conditions for two weeks. We found that PGG treatment helps prevent detrimental effects on TEVGs under diabetic conditions. PGG treatment helped prevent calcification, impaired mechanical properties, expression of vascular pathology markers such as α-SMA, accumulation of AGEs, imbalance of matrix-metalloproteinases, and overexpression of some pro-inflammatory and apoptotic markers. For the in vivo studies, our TEVGs were implanted as femoral-femoral bypass shunts in diabetic minipigs for 15 weeks. Results showed that PGG treatment provides protection against calcification, biodegradation of elastin, and accumulation of AGEs within the graft’s wall under diabetic conditions. In summary, we found that PGG treatment of decellularized scaffolds provides significant therapeutic benefits to protect scaffolds and vascular cells against several diabetic-related complications. Additionally, our in vitro perfusion models could be considered an alternative to animal models in the early stages of research to study the effects of diabetic conditions on vascular cells and testing of potential therapeutics. Finally, to the best of our knowledge, our diabetic models are the first of their kind and could provide a robust link to clinical translation.

Inherited metabolic diseases (IMDs) affecting the liver are relatively rare but collectively have a prevalence of 1 in 800 live births. These diseases result from autosomal recessive single-gene mutations, leading to organ dysfunction and potentially fatal consequences if left untreated. One potential therapeutic strategy for IMDs of the liver involves using CRISPR-Cas9-induced loss of function mutations. However, translating this approach into the clinic is limited by the need for safe and effective CRISPR delivery methods. Adeno-associated viral vectors (AAVs), commonly used for CRISPR delivery, are associated with significant safety and efficacy concerns, including risks for immunogenicity, off-target mutagenesis, and genotoxicity due to persistent Cas9 expression. Therefore, alternative delivery approaches are needed. In this study, we explore the safety and efficacy of electroporation as an alternative to viral delivery methods for ex vivo gene editing therapy for IMDs of the liver. Our strategy involves isolating hepatocytes from the resected liver of a patient, delivering CRISPR components into the hepatocytes via electroporation ex vivo, and subsequently transplanting the edited hepatocytes back into the patient's liver to replace the native hepatocytes with healthy gene-edited ones. Compared to systemic delivery, this approach offers advantages such as cell-specific editing, the potential to screen for off-target effects, and the opportunity to expand or cryopreserve gene-edited hepatocytes for transplantation when needed. The success of a cell-based therapy will require the edited hepatocytes to engraft and replace approximately 5-10% of the liver mass and persist for the patient's lifetime. iii We develop technical protocols to advance a cell-based therapeutic approach. First, we optimize hepatocyte isolation protocols to achieve high viability in freshly isolated primary hepatocytes for downstream delivery. Second, we established a mouse model for primary hepatocyte isolation and transplantation. After that, we show a successful cell therapy approach to reprogram metabolic pathways by electroporating CRISPR-Cas9 mRNA and RNPs to disrupt the gene encoding 4-hydroxyphenylpyruvate dioxygenase (Hpd) in primary hepatocytes ex vivo, followed by transplantation to treat a mouse model of hereditary tyrosinemia type 1 (HT1). Third, to assess the broader applicability of our strategy, we explore a novel cell-based gene editing approach in a mouse model of familial hypercholesterolemia. Specifically, we demonstrate clonal expansion and liver repopulation by gene-edited hepatocytes after transplantation by transient administration of acetaminophen. This study aims to provide valuable insights into the efficacy and safety of electroporation as a nonviral delivery approach for cell-based gene editing therapies for liver disease. These advancements can be applied to many inherited metabolic liver disorders and are a step toward autologous cell therapy in the liver.

Vascular calcification can be life-threatening, involving calcium phosphate crystal deposits in the blood vessels. The calcification in the intimal layer of the arterial wall is usually associated with atherosclerotic plaque build-up that, in the advanced stage, harbors ‘dispersed’ and ‘spotty’ deposits of calcium phosphate crystals in the necrotic core of the plaque. In contrast, medial arterial calcification (MAC) can form independently of atherosclerosis and appears as ‘Railroad-like’ calcific deposits along degraded elastin lamellae in the medial layer of arteries. The mechanisms of such elastin-associated medial arterial calcification and potential therapies are the key concerns of this dissertation. MAC is common in the aging population and particularly in those with type 2 diabetes and chronic kidney diseases (CKD), which is due to arterial elastin degradation as well as mineral imbalances in the blood, particularly hyperphosphatemia. MAC-associated elastin calcification causes vascular wall stiffening, resulting in increased systolic hypertension and resistance to blood flow. The resultant overload in cardiac work to move blood throughout the body leads to heart failure. Therefore, preventing medial calcification or reversal of existing calcific deposits from the medial layer without imposing any significant side effects is a pressing healthcare challenge. Overall, our study aims to understand the effects of elastase-mediated injury and elastin degradation products to initiate MAC either in the presence or absence of hyperphosphatemia. We test the efficacy of pentagalloyl glucose (PGG) to block elastase- and hyperphosphatemia-induced MAC in an aortic ring culture model and the effectiveness of targeted PGG nanoparticles to prevent systemic MAC in an animal model of CKD. We further developed targeted etidronate-loaded albumin nanoparticles and tested their ability to suppress MAC in aortic ring cultures. We also studied vascular cell behavior upon reversal of heavy aortic calcification using site-specific ethylene diamine tetraacetic acid (EDTA)-loaded albumin nanoparticles in a condition of CKD. We first developed an aortic ring culture model to study the effects of high active elastase on MAC development. High circulating elastase is typical in patients with diabetes, CKD, autoimmune disease, and in aging, either genetic or acquired. This ex vivo study demonstrated that high elastase activity is not only a driving factor for vascular inflammation but also initiates MAC via fragmenting elastin networks and stimulating vascular smooth muscle cells (VSMCs) to produce calcific microvesicles (MVs). Combining high phosphate, another clinical feature of CKD or diabetic nephropathy, exacerbated elastase-induced MAC. However, exogenously added elastin degradation products, such as elastin derived peptides and TGFβ1 alone, do not seem to affect calcium accumulation in the arterial media, though promoted VSMCs phenotypic alterations to osteogenesis. To study if the blockage of damaged sites on elastin or further elastin degradation may prevent high phosphate-induced extensive MAC, PGG was evaluated in an aortic ring culture model. PGG completely abolished MAC development in elastase-injured aortas cultured in high phosphate. By blocking degraded elastin sites, PGG prevented VSMCs-released calcific MVs from depositing onto the arterial media, preventing calcium phosphate crystal growth and development. For in vivo systemic inhibition of calcification with targeted PGG-loaded albumin nanoparticles, we developed a rodent model of progressive CKD combining adenine diet and vitamin D that aggressively caused severe cardiovascular and kidney calcification. Unfortunately, intravenously delivered site-specific PGG nanoparticles were unsuccessful in inhibiting MAC, which could be attributed to the insufficient drug concentration or slow release of PGG from the nanoparticle systems that failed to compete against the severe and aggressive form of calcification in this CKD model. To prevent or halt calcification progression, we developed another nanoparticle system with etidronate-loaded albumin nanoparticles conjugated with anti-elastin antibody for targeting damaged elastin in vasculatures. Herein, a single micro-dose of formulated nanoparticles significantly delayed the hyperphosphatemia-induced calcification in the aortic ring culture model. The aortic rings treated with etidronate nanoparticles showed less LDH release in media supernatants compared to non-treated aortas, indicating no cytotoxicity from etidronate or other components of nanoparticles. Finally, to reverse pre-existing heavy arterial calcification developed with the progressive CKD model, we performed targeted chelation therapy with anti-elastin antibody conjugated ethylene diamine tetraacetic acid (EDTA)-loaded albumin nanoparticles that specifically targeted damaged elastin. EDTA delivery removed calcific deposits from abdominal aortas and returned VSMCs from osteogenic to their natural vascular phenotypes. This chelation therapy also removed calcific deposits from the kidneys. More importantly, targeted chelation therapy significantly improved animals’ survivability than blank therapy. In summary, our research helped us understand MAC in relation to elastin degradation and chronic hyperphosphatemia that mimics CKD. Our nanoparticle targeting system is based on the hypothesis that elastin degradation is the initial step of MAC before calcification; such degraded elastin can be targeted by anti-elastin antibody to deliver drugs to either prevent, or reverse calcification. Here we studied molecular and cellular behavior in response to our targeted nanotherapeutics that have the potential to inhibit/reverse elastin-associated MAC with minimal to no systemic side effects.

Extracellular vesicles (EVs) and exosome-like vesicles (ELVs) are nanoscale lipid particles secreted by cells that mediate intercellular communication by delivering bioactive molecules. Isolation of pure EVs and ELVs are important for downstream analyses and potential therapeutic applications. Isolation of pure actively-loaded engineered ELVs are also important as potential delivery vehicles for future biotherapeutics. Nano-flow cytometry (nFCM) is a recently developed technology that can measure nanoparticle concentration, size, and phenotype individual nanoparticles using suitable antibodies and membrane stains. The objective of this thesis was to use nFCM in combination with other techniques such as nanoparticle tracking analysis (NTA) to characterize EVs secreted by HEK293T cells, which were isolated using various methods including differential ultracentrifugation, polymer-based precipitation, ultrafiltration, and size exclusion chromatography, in order to compare these techniques by quantifying parameters such as EV/ELV yield, size, purity, and concentration. HEK293T cells were transfected with plasmid DNA encoding CD63-GFP to actively load GFP inside ELVs to assess loading efficiency. Using the nFCM, isolation using ultracentrifugation resulted in the highest EV and ELV yield and purity, compared to all other isolation techniques. ELV-enrichment using a commercial CD63-specific immunocapture kit was attempted but was unsuccessful. It was also determined that compared to nFCM, nanoparticle concentrations and sizes were overestimated using NTA highlighting a challenge in the field, as discrepancies can affect downstream applications. We demonstrated that transfection of cells with CD63-GFP can enable high loading of ELVs with GFP. Insights from this thesis will inform future work towards engineering ELVs to deliver biotherapeutics. Advisor: Angela K. Pannier

Chinese hamster ovary (CHO) cell lines are among the most popular expression hosts used in biopharmaceutical manufacturing due to relative ease of culture, capacity to perform human-like post-translational modifications, and non-susceptibility to viruses. However, the intrinsic plasticity of the CHO genome can lead to undesired genetic rearrangements, phenotypic shifts, reduced product quality, and early culture termination that prevents continuous biomanufacturing. A characteristic of plastic and unstable genomes that is poorly understood in CHO cells is extrachromosomal circular DNA (eccDNA). EccDNAs are focal amplifications of the genome that reside in the extranuclear space. These plasmid-like entities are structurally complex and are capable of contributing to a wide variety of biological functions including gene overexpression, regulation of nuclear-encoded genes, immunostimulation, and adaptive stress responses. The objective of this work is to establish the foundational knowledge of eccDNA structure, function, and microevolutionary dynamics in CHO cells under various conditions. This work characterizes eccDNA content in CHO cells grown in bioreactors for two weeks under control and lactate-stressed conditions, two CHO K-1-derived cell lines of different ages, and CHO cells gradually adapted to high extracellular lactate levels. More than 2,000 genes were observed to be encoded on eccDNAs and summaries of gene function are presented using Gene Ontology and KEGG pathway analyses. RNA-seq data is utilized to identify potential changes in gene expression mediated by eccDNAs. Furthermore, the study presents a broad characterization of eccDNA sequence structures and biogenesis sites that may be used as targets in future work.

Glioblastoma (GBM) brain tumors are highly aggressive gliomas due to genetic and cellular heterogeneity. Current GBM treatment consists of surgical resection of the tumor combined with radio- or chemo-therapies. While these treatments have increased the life expectancy for GBM patients up to 20 months, they have had little effect on the 5-year survival rate. The complex cellular and genetic composition of the tumor makes current treatments less effective long term. One approach to developing more effective GBM treatments is to customize nanoparticle-based drug delivery systems that can directly target the aberrant gene expression patterns within a particular GBM tumor. Delivery systems that include oligonucleotide therapies are ideal for this approach due to their ease of synthesis and ability to tailor the oligonucleotide base sequence to allow the targeting of specific gene sequences and proteins. Two therapeutically relevant classes of oligonucleotides, DNA aptamers (e.g., AS1411) and RNA-interfering anti-sense microRNAs (anti-miRs), have exhibited anti-GBM properties. However, their use as standalone therapies is hindered by instability within in vitro and in vivo environments and the inability to cross some biological barriers. The conjugation of individual oligonucleotides to the surface of gold nanoparticles (GNPs) has been shown to help overcome these difficulties to make AS1411 and anti-miRs viable therapeutics for GBM; however, the conjugation of both oligonucleotides to GNPs has not been investigated. This dissertation presents a novel GNP therapy incorporating AS1411 and an anti-miR as a multi-faceted therapy against GBM. Anti-miR-21 (A21) targets miR-21, a microRNA implicated in the increased aggressiveness of GBM tumors. U-87 MG GBM cells treated with GNPs coated with AS1411 and the polymer poly (ethylene glycol) (PEG/AS1411 GNPs) displayed decreased cellular metabolic activity and growth with altered cell morphology. The GNPs were optimized based on the PEG to AS1411 ratio to maximize these bioactive effects against GBM cells. An optimal ratio of 1:3 PEG to AS1411 conjugated GNPs exhibited ~ 72% inhibition of cellular metabolic activity, reduced growth by 75%, and profoundly affected cellular morphology. The effects of AS1411 were enhanced upon conjugation to GNPs. Additionally, using a base-substituted analog of AS1411 showed that the effects on U-87 MG cells were specific to the sequence of AS1411. In addition, this dissertation details the synthesis of AS1411 GNPs coated with A21 (PEG/AS1411/A21 GNPs) and investigates the benefit of A21 addition to PEG/AS1411 GNPs. Treatment of U-87 MG cells with PEG/AS1411/A21 GNPs retained the effects seen from PEG/AS1411 GNPs and significantly lowered the motility rate of U-87 MG cells. PEG/AS1411/A21 GNPs showed additional effects on gene expression patterns of U-87 MG cells due to A21 addition. PEG/AS1411/A21 GNPs reduced miR-21 expression 3-fold in U-87 MG cells. The ability of PEG/AS1411/A21 GNPs to influence the expression of miR-21-associated proteins PTEN and STAT3 within U-87 MG cells was also investigated. The in vivo performance of PEG/AS1411 GNPs, with or without A21, was investigated within a mouse orthotopic xenograft model of GBM. Both GNP types were shown to cross the blood-brain barrier and influence tumor progression. Mice treated with PEG/AS1411 GNPs ultimately survived longer (47 days post-tumor implantation) than untreated mice (37.5 days)

Cell therapy is a technology that relies on replacing diseased or dysfunctional cells with healthy functioning ones. One of the cells used for such advanced therapies are stem cells, owing to their ability to differentiate into specific cells required for repairing damaged or defective tissues or cells. The majority of cell-based products are intended to transiently persist in the patient, secreting factors which then allow the patient’s body to heal; in these products, the cells are subsequently eliminated from the body. Furthermore, unique manufacturing platforms, in addition to novel commercialization strategies, will be required to create a successful, sustainable cell therapy industry. Currently in cell manufacturing companies, need to produce adherent cells, that bind to a solid surface such as tissue culture- treated plastic. In some cases, the growth surface needs to be treated or coated with a matrix (such as natural proteins) to facilitate cell adhesion. Cells can be grown on large flat surfaces or on spherical microcarriers for suspension?based culture in bioreactors. However, those proteins leading to batch-to-batch variability and concerns regarding contamination and affects the cell therapeutic potency. This thesis aims to develop techniques based on the merger of novel materials approaches to manipulate cell microenvironments in culture. For controlling cell-cell interaction and cell-soluble factor interactions, layer-by-layer deposition of ionic biopolymers were developed (Heparin as a negative charge polymer, collagen and Poly-L-Lysin are used as positive charge polymers). In addition, techniques were developed to control the viability of cells under hypoxic conditions within collagen hydrogels by controlling the three-dimensional properties of hydrogels and oxygen delivery (Perfluorocarbon-based oxygen carriers). In addition, to control cell-soluble factor interactions, Metal-Organic-Frameworks (MOFs) nanocarriers application was discussed as a carrier for delivering interferon-gamma (IFN-γ) to cells.

Dissipative particle dynamics (DPD), a coarse grain simulation method, was applied to the membrane formation process of non-solvent induced phase separation (NIPS) to gain further insight on the mechanism of certain variables and how they affect the final morphology. NIPS involves two solutions, an organic polymer dissolved in an organic solvent colloquially called the dope and an aqueous coagulation bath, brought into contact with one another. The solvents then mix, causing the polymer to fall out of solution as an asymmetric membrane with a dense surface layer and a more open subsurface layer in response to the decreasing solubility. Polyethersulfone (PES), a common industrial choice we have previously studied, was utilized as the polymer with N-methyl-2-pyrrolidone (NMP) as the organic solvent and water as the coagulation bath. In this study, our previous model construction was altered in several ways. Firstly, the simulation area was enlarged, allowing for a better sampling of subsurface behavior. Secondly, polymer chain length was increased to bring it more in line with the high molecular weight of industrially common polymers, with our experimental systems ranging from 100 to 200 monomers. Lastly, polyvinylpyrrolidone (PVP) was introduced as an additive to the polymer solution in concentrations varying from 1 to 10% by volume of the dope. PVP is a common polymer additive utilized in industry to produce larger pores, a result that was successfully replicated. We also investigated the effects of adding solvent to the coagulation bath as well as the effects of adding water to the polymer solution.

Volumetric muscle loss (VML) overwhelms muscle’s robust capacity for regeneration. A key event in the etiology of VML injury is the bulk loss of structural cues provided by the underlying extracellular matrix (ECM). While muscle is a highly structured tissue, with cell and ECM alignment in the direction of contractile force production, the impact of scaffold alignment on recovery remains unclear. Bulk human decellularized skeletal muscle (DSM) tissues were sectioned into 10 x 1-2 mm fibers. VML defects were repaired using multi-fiber implants consisting of approximately 8 fibers per defect arranged into two layers. Fibers were oriented 1) to the surrounding tibialis anterior (TA) muscle fibers (aligned), or 2) randomly (unaligned) within the defect and coated with minced muscle paste. At 8 weeks, peak tetanic force was not significantly different between the aligned and unaligned repair groups (73%±14% versus 70%±12%). TA muscle mass was also similar between the repair groups. Collagen and Laminin immuno-stains did not reveal structural differences between the groups. The results suggest it may not be necessary to alter the structure of the scaffold to match the surrounding native muscle. This could suggest that cellular and chemical cues provided by the scaffolds are the key design parameters to be considered. Short-term follow-up studies (1 and 2 weeks following repair) may reveal mechanistic insights.

Glioma is one of the most aggressive forms of brain cancer. It has been shown that the microenvironments differ significantly between the core and edge regions of glioma tumors. This study obtained metabolomic profiles of glioma core and edge regions using paired glioma core and edge tissue samples from 27 human patients. Data was acquired by performing liquid-liquid metabolite extraction and 2DLC-MS/MS on the tissue samples. In addition, a boosted generalized linear machine learning model was employed to predict the metabolomic profiles associated with O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation. A panel of 66 metabolites was found to be statistically significant between the core and edge regions. The machine learning model achieved AUROC values of 0.941 for the core and 0.960 for edge. This proof-of-concept study shows the metabolomic differences are reflected in MGMT promoter methylation status and demonstrates the potential for machine learning to aid as a prognostic and therapeutic tool.

Volumetric muscle loss (VML) remains one of the few skeletal muscle injuries without a reliable and repeatable treatment. In large volume muscle injuries, muscle fibers as well as the surrounding connective tissue are damaged, preventing therapeutic muscle stem cells, called myogenic progenitor cells (MPCs), from reaching the injury site and initiating repair. There is a clinical need to rapidly fabricate in vitro muscle tissue constructs that mimic the native tissue organization, with aligned myotubes, for insertion and integration at the patient’s injury site. In this dissertation, we utilize the MPC’s natural propensity to close gaps across an injury site to guide alignment on collagen I, an extracellular matrix (ECM) protein substrate. Through the binding affinity of biotin and streptavidin molecules, we pre-position MPCs in temporary straight-line patterns separated with small gaps, mimicking small-scale muscle injuries. This temporary positioning initiates the migratory nature of the MPCs to align and form myotubes across the gaps. Additionally, we have developed a novel technique to wash the substrate once it is seeded with cells using centrifugation to maximize the cells adhered in the desired patterned structures, while minimizing the number of cells adhered in the gaps between them. The patterning system we have designed allows for long-range myotube ordering, confirmed through the expression of myosin heavy chain (MyHC). The next part of this dissertation expands our work on cell migration and adhesion to study the properties of 3 pairs of epithelial cancer cell lines (breast, endometrium, tongue) with varying metastatic potential. According to the epithelial-to-mesenchymal transition, cancer cells lose their cell-cell and cell-matrix adhesion junctions as they become more metastatic, contributing to increased detachment from the primary tumor and a migratory response into nearby tissue and vasculature. Current in vitro strategies for characterizing a cell’s metastatic potential heavily favor quantifying the presence of cell adhesion biomarkers; however, little attention is placed on quantitative analysis of metastatic function across well-known cancer cell lines. To place cell lines more accurately within the spectrum of metastasis, we deploy two functional metrics of cancer aggression (wound closure migration velocity and cell detachment from a culture surface) on each cell line. We demonstrate that there is not a relationship between cell migration velocity and metastatic potential. Additionally, we test cell adhesive function through a shear flow assay to confirm that cell lines with higher metastatic potential had greater detachment from a culture surface compared to low metastatic potential cell lines in homogenous populations. Normalizing the data to the highest value within each metric reveals that cell lines with low metastatic potential rank higher wound closure migration velocity while cell lines with high metastatic potential rank higher in detachment from a culture surface. Finally, this dissertation concludes with a study initiated during the COVID-19 pandemic to help local Kentucky distillers transition between hand sanitizer and spirits production. Hand sanitizer introduces the bitter tasting compound denatonium benzoate to production equipment, requiring appropriate cleaning solutions for removal to prevent changes in spirit flavors. Materials compatibility with high proof ethanol dominant in hand sanitizer plays a large role in the effectiveness of the tested cleaning solutions. Metals and rigid polymers do not retain denatonium benzoate due to being compatible with high proof ethanol, so they can be cleaned easily using any of the cleaning solutions. However, elastomers permeate denatonium benzoate into and out of the material due to their poor compatibility with high proof ethanol. Ethanol proves to be a useful cleaner, but can cause irreversible damage to these materials over time.

Currently, there is no standard in vitro model for studying the effects of mechanical stimulation on muscle in type II diabetes. Existing models primarily utilize electrical stimulation, which does not fully recapitulate the effects of exercise. In this thesis, we create a standardized in vitro model of murine muscle that can recapitulate the benefits seen in exercise when mechanically stimulated. Moreover, we show that a type II diabetes environment has similar effects on the muscle in vitro as well as in vivo.

Microvascular disease is hallmarked by pathophysiological conditions such as endothelial senescence, intimal thickening which impairs vasodilation, and regression of the capillary beds causing tissue ischemia in the myocardium or in peripheral vascular networks. Adipose-derived stromal vascular fraction (SVF) has previously demonstrated the ability to revascularize tissue. Increasing evidence shows that regenerative cells elicit their therapeutic benefit by paracrine mechanisms, leaving open extracellular vesicles (EVs) as a potential crux of the cell therapy paradigm. To test this idea, three types of gelatin methacrylate hydrogels were employed: SVF gels, EV gels derived from SVF, and blank control gels, which were used in-vitro and in-vivo to evaluate the effect of cell versus cell-free therapies for revascularizing tissues. Fischer 344 Green Fluorescent Protein positive (GFP+) male and female 6-month-old rats were used as donors for SVF, which was harvested by enzymatic digestion, centrifugation, and filtration steps. Cells were placed directly into gelatin methacrylate (GelMA) hydrogels. EVs were harvested from SVF tissue culture supernatants prior to being loaded in gel constructs. EVs were isolated by ultracentrifugation at 100,000g from cell culture supernatants and detected by spectral flow cytometry (Cytek Aurora) utilizing violet laser side scatter height and characterized by expression of CD9 and CD63 markers. This detection method was also used to estimate the number of particles from a known number of SVF cells. Degradation studies of unloaded GelMA hydrogels were performed using six or eight percent (w/v) concentration over 60 days in PBS. Hydrogels were further characterized by viability assays of the SVF gels to ensure the gel synthesis process, specifically UV exposure, was not cytotoxic to SVF. EV hydrogels were also characterized by their release over 7 days. A plug-in-field assay was used to determine the vasculogenic efficacy of the groups in vitro by staining the constructs with Griffonia Simplicifolia Lectin I (GS1+) and DAPI after seven and fourteen days in culture; plugs were explanted, and images were analyzed for cellular presence and expression of GS1 and αSMA markers. Finally, experimental and control gels were implanted bilaterally into the dorsal subcutaneous space of Rag1 mice for 14 days. Prior to explant, dextran tetramethylrhodamine was infused, gels were explanted and stained for cell markers, and imaged via confocal microscopy. Analysis of images acquired from in vivo experiments were compared for angiogenic metrics such as vascular density, indicated by the dextran infusion.

Heart failure is a broad pathology manifestation categorized by an inability of the heart to successfully pump blood throughout the vast vessel network of the body. Within the United States, heart failure is projected to increase by approximately 46% from 2012 to 2030. Modalities of heart failure are generally related to wall mechanics that are impacted following myocardial infarction events. Interplay exists between the wall mechanics, responding cell populations, and the spatial heterogeneities in the resultant scar. This interplay directs the myocardium towards heart failure modalities governed by overly stiff or compliant states. It is essential to elucidate details underlying the progression, maturation, and remodeling of the collagenous infarct scar to uncover therapeutic avenues and improve outcomes. We developed in vitro platforms that enable these desired insights into infarct progression. Our first platform generates continuous collagen gels possessing spatial fiber alignment heterogeneity utilizing a cost-efficient magnetic microsphere methodology. Spatial heterogeneities in the fiber architecture and cell response were evaluated. The second platform mechanically and electrically stimulates engineered heart tissue constructs and enables the evaluation of spatial heterogeneities in construct mechanics following a simulated infarction. Additionally, to avoid losing sight of the in vivo reality, we evaluated the interplay of the cellular detection of mechanics and spatial strain distributions throughout an infarcted myocardial wall. Histologically evaluated mechanosensor intensities were correlated to in vivo strain data collected via ultrasound imaging of the myocardium of a murine infarction animal model.

Heart failure (HF) currently affects over 6 million Americans, 50% of whom die within 5 years of their initial diagnosis. A major contributor to the onset of HF is cardiac fibrosis in the myocardium, which arises when fibroblasts (FBs) are activated in response to heightened mechanical stress from overload conditions like hypertension. Activated FBs remodel the extracellular matrix (ECM) and secrete ECM proteins including collagen. FB remodeling has been studied in the past by applying forces and/or deformations to three-dimensional, cell-seeded gels and tissue constructs in vitro. Unfortunately, previous stretching platforms have traditionally not enabled mechanical property assessment to be performed with an efficient throughput, thereby limiting the full potential of in vitro mechanobiology studies. We developed a novel in vitro platform to study cell-populated tissue constructs under dynamic mechanical stimulation while also performing repeatable, non-destructive stress-strain tests in living constructs. The multi-well platform is 3D printed out of a Dental LT resin with each well housing a magnetic piston grip. Cell populated tissue constructs were grown within the wells to attach to magnetic grips opposite from stationary grips. Uniaxial mechanical tension was applied to the tissues by a computer-controlled electromagnet placed beside the well plate, which enabled both dynamic stimulation during long-term culture as well as repeatable, non-destructive stress-strain tests for simultaneously comparing tissue stiffnesses across all wells. We have successfully utilized our platform to stretch cell-populated tissue constructs composed of both primary murine and primary human cardiac fibroblasts within 3D fibrin gels. Cyclically stretching cardiac fibroblasts within a 3D fibrin matrix led to collagen accumulation and increased tissue stiffness. Results from these studies validated our novel platform’s ability to enable dynamic mechanical characterization of cell-mediated tissue remodeling in vitro - a capability not possible in standard 3D tissue culture. This multiwell platform offers potential as a high-throughput screening tool for assessing the effect of different treatments on clinically relevant metrics of tissue stiffness using automated measurements.

Cardiovascular Disease(CVD) is the leading cause of mortality in the developed world. CVD is most commonly manifested as atherosclerosis of the coronary arteries leading to Myocardial Infarction(MI). After MI, fibrosis of the ventricular wall leads to heart failure(HF), a pandemic affecting 26 million people globally. While therapies are continuously developed to combat HF, the treatment of choice, whole heart transplant, is limited by the availability of donor hearts. It is clear that there is a need to develop a long-term solution to combat HF and its enormous economic burden. Tissue Engineering and Regenerative Medicine holds promise as a possible solution through the development of cardiac grafts capable of returning the function lost from MI. Current efforts in the development of cardiac tissues have been plagued by the inability to generate tissues of sufficient thickness(>100μm) or contractility of mature tissue, resulting in low engraftment rates or lacking efficacy. This leads to the focus of this research being the generation of a thick vascularized cardiac patch capable of long-term survival. First, we decelled whole porcine hearts for the production of ventricular flap and myocardial scaffolds. Scaffolds showed no evidence of retained cellular content or DNA, but retained key characteristics of the extracellular matrix and maintained the same mechanical properties as native tissue. We then performed a combined cell seeding to reendothelialize the vasculature of the ventricle wall and repopulate the myocardium. Finally, we developed a custom perfusion electromechanical bioreactor for the purpose of conditioning and maintaining the viability of our patch long term. We expect that this research will result in the development of cardiac grafts with long-term implications for therapy to solve MI related HF.

3D bioprinting of biological scaffolds requires control of the physicochemical properties of each unique structures. A promising material for control of properties is hydrogels, which can help create biomimetic scaffolds with controlled spatial arrangement of materials by integrating biological materials directly into layers during the bioprinting process. Nanocellulose offers a unique combination of properties including mechanical, biomimetic, and biocompatibility. These properties offer flexibility over the types, shapes, and applications of their printed hydrogel scaffolds, (i.e., tissue, drug, encapsulation). However, 3D bioprinting of nanocellulose-based hydrogels requires high loading percentages (i.e., >10 wt%) or chemical crosslinkers (i.e., bis(acyl)phosphane oxides (BAPO)). High solid content of nanocellulose results in high printing pressures and stress (i.e., >400kPa, >3kPa respectively) on extruded materials, leading to deviation in desired structures as well as undue stress on active biological materials in inks. Current chemical crosslinking methods require harsh chemicals (i.e., acids or bases) exposure and/or high-intensity light exposure (e.g., photo-crosslinkers), leading to potential for damage to biological components. In this study, solutions were explored to overcome the challenge to 3D bioprinting of nanocelluloses, i.e., the needs of high solid content and/or chemical crosslinking. Specifically, lower solid content inks with biocompatible crosslinking agents were investigated to reduce printing pressure and stress on biological components during bioprinting. Four biocompatible crosslinking agents were selected: methyl cellulose, glycerol, epoxidized soybean oil, and poly-epichlorohydrin. Printability of cellulose nanocrystal (CNC) scaffolds with each crosslinking agent was evaluated by assessing pre-print properties of inks, printing parameters, and printed structure characteristics. Rheological evaluation of crosslinked inks showed potential for 3D printing based on tan(δ) and viscous properties. Consistent printing was observed withing an acceptable range (i.e.,

Porous scaffolds mechanically stimulated with ultrasound are used for the treatment of bone conditions such as osteoporosis or traumatic bone injury. However, one current issue in these treatment techniques is designing a scaffold structure with a high structural integrity that also does not impede tissue growth. A larger ultrasound wave transmission would also allow more of a scaffold to receive mechanical stimulation from ultrasound. Controlling mesenchymal stem cell seeding of tissue scaffolds also has potential for developing optimal seeding patterns to maximize or control tissue growth in the scaffold based on an individual’s physiology. There is also a need for the development of models to evaluate changes in bone pore structure, even when porosity is constant, to predict bone fracture risk more accurately to conditions such as osteoporosis, as well as developing models that can highlight the most important variables that affect the bone growth to tailor the scaffold design to optimize the required amount and rate of bone growth to meet patient’s physiological needs. This study develops experimental and computational models of the ultrasound wave transmission in a bioinspired scaffold structure with high structural integrity using 3D printed scaffolds, immersion ultrasound transducers, finite element simulations and the bioinspired structure is found to have equal amounts of bone tissue growth as structures with uniform pore shapes. A model of bone tissue growth in a bioinspired structure a bone tissue growth algorithm is then developed, and the bioinspired structure is found to have equal amounts of bone tissue growth as the structures with uniform pore shapes. The initial seeding of mesenchymal stem cells into the scaffold is also found to significantly affect the amount of bone tissue growth by as much as 120%. Lastly, unsupervised machine learning models evaluate the effects and presence of unknown patterns on bone tissue growth from different variables using machine learning. The unsupervised machine learning model of ultrasound wave transmission in the scaffolds developed in this study was found to be 81% accurate in classifying between an anisotropic and isotropic scaffold structure. The unsupervised machine learning model in this study classifying data clusters of bone and cartilage growth in the bioinspired and square pore scaffold structures compares the clusters to the effects of features such as pore geometry, scaffold material, ultrasound frequency and wave type, and mesenchymal stem cell distribution. This study thus demonstrates a bioinspired bone tissue scaffold structure with a high structural integrity to oppose structural failure while maintaining a high ultrasound wave transmission to allow more mechanical stimulation to grow bone tissue as well as develops models that can be used to design the optimal bone tissue scaffold design to meet a patient's specific physiological needs.

Microneedle patches are a promising source for transdermal diffusion of macromolecules and are designed to painlessly penetrate the skin. In this study, a biodegradable chitosan microneedle patch to deliver meloxicam for managing pain in cattle was tested. The potential of reuse of the polymeric solution to fabricate the patches, optimization of fabrication, morphological analysis of the microneedle patch and analysis of preservation of the chemical composition after sterilization were evaluated. In-vitro analysis consisted of studying in-vitro penetration mechanical properties, compression testing analysis of microneedle patch, and in-vitro drug release analysis. In-vivo studies were performed to analyze the dissolution capability of the microneedle patch. Results regarding the physical characteristics, chemical composition, and mechanical properties confirmed that rheological properties of the chitosan solution, present significant differences over time, demonstrating that reusing the solution on the fourth day results in failure patches. Morphological characteristics and chemical composition studies revealed that the process of sterilization (ethylene oxide gas) needed for implanting the patches into the skin did not affect the properties of microneedle patches. In-vitro studies showed that approximately 33.02 ± 3.88% of the meloxicam was released over 7 days. A full penetration of the microneedles into the skin can be obtained by applying approximately 3.2 N. In-vivo studies demonstrated that microneedle patches were capable of swelling and dissolving, exhibiting a dissolution percentage of more than 50% of the original height of microneedle after 7 days. No abnormal tissue, swelling, or inflammation was observed in the implanted area. The results of this work show that chitosan biodegradable microneedle patches may be useful to deliver meloxicam to improve pain management of cattle with positive effects for commercial manufacturing.

When a peripheral nerve injury (PNI) occurs, the gold standard for tissue regeneration is the use of autografts. However, due to the secondary effects produced by multiple surgeries involved in the removal and implantation of autografts for very small lesions, it is possible to replace them with the use of Nerve Guide Conduits (NGCs). However, NGCs are limited to short lesions (less than 1 cm). This limitation is caused by the absence of compounds in the extracellular matrix (ECM) that autografts can provide. Since much of the regenerative process takes place on the NGC surface, our work aims to modify its surface using heparin and collagen coatings, two natural polymers present in the ECM. Heparin/collagen bilayers (HEP/COL) will increase biocompatibility, cell adhesion and migration, and allow the incorporation of neurotrophic factors such as Nerve Growth Factor (NGF). This research evaluates the effects on human Schwann cells (hSCs) when cultured on a commercial collagen NGC under four conditions: without coatings and with six heparin/collagen bilayers (HEP/COL)6 with and without NGF supplemented to the culture medium (10 ng/ml). We have exhaustively characterized (HEP/COL)6 coatings. The chemical structures of the polymers, the morphology, and the surface charge of the coatings could be determined. Real-time analysis of hSCs behavior showed three main phases: adhesion, proliferation, and adaptation. The adhesion phase was increased in the conditions with (HEP/COL)6, and the viability studies showed an increase of up to 250% compared to cells grown on polystyrene without coatings. The cell morphology has never been altered by (HEP/COL)6, as well as the morphology of the coated NGC. Cell migration was favored, and (HEP/COL)6 also increased the expression of some proteins such as BDNF, which promotes the duration of the myelination process, or TNF alpha, which is a mediator of inflammatory and immune functions. Concerning the surface modification of a commercial NGC, these studies showed an increase of more than 200% in cell viability when compared to uncoated NGCs. We also determined the presence of heparin and evidenced the cell adhesion on (HEP/COL)6 coated NGCs. In conclusion, (HEP/COL)6 seems to be a promising strategy to improve the performance of NGCs.