Biomaterials are composed of a wide array of macromolecules and have impacted multiple biomedical technologies. Structurally, each of these materials typically only include a small set of monomers that limits their structural complexity, tunability, and functionality. There is a critical need to develop novel biomaterials with greater complexity and tailored functionalities to meet the demands of emerging new technologies in drug delivery, tissue engineering, and regenerative medicine. Inspired by proteins, where complexity and functionality are driven by the organization of thousands of functional domains, we hypothesized that tethering different biomaterial backbones into a domain-structured single-chain polymer (a polymer mosaic) will impart structural complexity with emergent physicochemical properties, novel functionalities, and defined secondary structures. In this thesis, we designed and characterized a series of polymer mosaics designed to test this hypothesis. We first designed and synthesized an alginate-b-polyethylene glycol (PEG)-b-polylactide (PLA) triblock copolymers utilizing a modular click-chemistry strategy. This triblock copolymer was investigated as an amphiphilic material for controlled drug release. The incorporation of a hydrophobic PLA domain and a hydrophilic alginate domain in a single polymer made it an attractive platform for both the encapsulation and release of both hydrophilic and hydrophobic small molecules. Nanoparticles (NPs) formulated from this triblock displayed morphologically discrete compartmentalization of the alginate domains, superior loading efficiencies of both hydrophilic and hydrophobic small molecules, and potential as a drug-combination delivery platform. Next, we evaluated the potential of alginate-b-PLA diblock copolymers to function as degradable hydrogels by combining the hydrogel-forming feature of alginate with the degradation properties of a PLA domain. The fabricated hydrogels had tunable degradation properties from days to weeks by modulating their formulation, and are being evaluated as potential sacrificial scaffolds for tissue engineering. Finally, poly (L-lactide)- poly (amido saccharide) (PLLA-PAS) were synthesized as polymer mosaics amphiphiles with defined secondary structures that formulate into chiral nanoparticles. These chiral particles assembled a unique protein corona of 22 proteins when incubated with mouse serum and analyzed by SDS-PAGE and LC/MS-based proteomics. This result will build a foundation to further our understanding of surface chirality on the protein corona and its potential delivery applications. In summary, we successfully synthesized and developed polymer mosaics with complementary properties, tailored functionalities, and defined secondary structures. These new materials can pave the way for advances in new technologies for drug delivery and tissue engineering.

The primary cilium is an important signaling organelle present on the membrane of almost all cell types during the G0 phase of the cell cycle. In addition to it signaling function, bending of primary cilia allows the cell to sense surrounding fluid flow, important for proper kidney function, as well as left and right direction-- features required during organ development. Cilia are involved in critical signaling functions found within the Hedgehog and PDGFRα pathways. The absence of cilia on the cell surface can significantly alter disease progression and drug response. Many common human ailments, including inflammation, tumorigenesis, and tissue aging, are often associated with changes in the extracellular matrix (ECM) properties, including an increase in ECM stiffness. We wondered whether ECM properties can also affect ciliation. While few past studies have noted that ciliation is affected by a change in substrate stiffness, a deeper insight on the role of mechanical forces from the extracellular matrix (ECM) in ciliogenesis is missing. This study aims to start unveiling the link between mechanical properties of ECM and ciliogenesis, with a focus on cilia signaling and extracellular cues responsible for cilia assembly and disassembly. To address how mechanics of ECM affects cilia biogenesis, we prepared gelatin substrates with various cross-linking degrees and measured ciliation level, loss of cilia, and cilia length. To visualize and measure the cilia, we used immunofluorescent labeling followed by fluorescent and confocal microscopy of the samples. This study displays that increasing the degree of ECM cross-linking promotes ciliogenesis under serum deprivation. Higher cross-linking degree of gelatin substrates resulted in a higher ciliation rate as well as cilia elongation within the cell population. When cilia disassembly is initiated by the addition of serum-enriched medium, cells grown on highly cross-linked gelatin show higher ciliation rate when compared to cells grown on non-cross-linked gelatin. Expression level of cilia-associated protein acetylated α-tubulin was also higher on the highly cross-linked gelatin substrate. The final part of the study describes the action of cilia-targeting drugs in context of ECM cross-linking degree. The performance of ganetespib, a common cancer therapeutic and HSP90 inhibitor, shows a decrease in efficacy on substrate with high degree of cross-linking. / Biomedical Sciences

Soy protein isolate (SPI) has become increasingly attractive for tissue engineering purposes because of its abundance in nature (a plant-derived protein), ease of isolation and processing, customizable biodegradability, inexpensive cost, and minimal immunogenicity. Combining SPI with methacrylic anhydride to form soy-methacryloyl (SoyMA) makes it possible to develop a semi-synthetic bioink that can recapitulate in-vivo tissue constructs when extruded by a three-dimensional (3D) bioprinter. Bioinks offer an ideal biological microenvironment for cells and can be photo-crosslinked easily, ensuring cell encapsulation and form fidelity. The long-term goal of this research is to demonstrate that SoyMA bioinks can be synthesized to form a 3D cell culture material with a particular degree of functionalization (DoF). Specifically, I propose to develop SoyMA bioinks that can be used to fabricate scaffolds mimicking the microenvironment of spinal cord tissue using neuronal progenitor cells (pheochromocytoma (PC12) cells) and endothelial cells (EC). Using 3D bioprinting, we will test the ability of these scaffolds to promote cell adhesion, directed spreading, and proliferation. TO control the mechanical properties of SoyMA, we will parametrically vary polymerization conditions such as concentration, DoF, and photo-crosslinking. We will also evaluate and compare cell viability and morphology of cells grown in various stiffnesses of SoyMA scaffolds. Taken together, we will demonstrate how SoyMA bioinks, coupled with 3D bioprinting, can be used to fabricate dynamic and tunable tissue scaffolds. / Bioengineering

Cartilage tissue engineering is a promising approach for the repair of chondral defects. Engineering of cartilage combines a three-dimensional scaffold with chondrogenic cells and appropriate external stimuli, and ideally results in constructs with properties that resemble, as closely as possible, native cartilage. Once implanted to repair a cartilage defect, the integration of tissue engineered cartilage (TEC) to surrounding native tissue is critical for a successful clinical outcome. However, this depends in part on the initial maturity of the engineered construct, which is challenging to assess a priori. Another challenge relates to the assessment of the longitudinal repair of cartilage defects after tissue engineering approaches are applied. Currently, evaluation is qualitative, based on visual and tactile observations using arthroscopic hook probes which can be a very subjective approach. Furthermore, gold standard techniques (histological, mechanical and biochemical evaluation) to determine compositional and mechanical properties of constructs in vitro and ex vivo are generally destructive.In this thesis, we are proposing the use of a spectroscopic fiber optic probe approach that spans the visible-near infrared (Vis-NIR) regions. This would be a novel, non-destructive technique based on high frequency nonionizing radiation that causes vibrations in the NIR region (750-2500 nm or 12000 to 4000 cm-1), and electronic transitions in the VIS region (400-750 nm) that result in a unique spectrum of the sampled tissues. Previously, we have shown that NIR spectral data collected in a non-destructive manner correlate to compositional and biomechanical properties of tissue engineered cartilage. Additionally, using an arthroscopic fiber optic probe, Vis-NIR spectra can be collected from repairing cartilage tissue in situ. The overarching hypothesis of this thesis is that Vis-NIR fiber optic spectroscopy can be utilized to assess engineered cartilage development in vitro, and in vivo to assess repair in a pre-clinical model of chondral defect. This hypothesis was tested in the following three aims: 1. Assessment of the mid and NIR spectral features of scaffold and extracellular matrix components of cartilage and TECs for in vitro monitoring of TEC development by fiber optic NIR spectroscopy; 2. Assessment of the Vis-NIR spectral features of tissues present in the mini-pig stifle joint during the chondral repair process to facilitate interpretation of in vivo repair; 3. Model-informed design and analysis of an arthroscopic probe for spectral collection during the cartilage repair process in preclinical models and clinical scenarios. Together these studies contribute to an overall approach for spectroscopic assessment of tissue engineered cartilage as a pathway for bench to bedside evaluation of cartilage development and repair. / Bioengineering

In the event of nervous system trauma, there are currently no treatments for functional loss due to thefailure of the mature mammalian central nervous system (CNS) to regenerate. Reduced intrinsic growth ability is believed to be a factor in attributing to the persistent functional deficits in neurological disorders such as spinal cord injury. Our candidate-based genetic screen allows us to examine and reveal several novel targets and pathways that have never been implicated in axon regeneration. However, we still lack a complete understanding of the repertoire of genetic factors that can promote or inhibit axonal regrowth after neural damage. Given that neurons have polarized morphology with distinct cellular compartments, microfluidic platforms had gained considerable impact in neuroscience research. Furthermore, disrupting gene expression is a common approach to understanding the loss-of-function disease mutations. Due to its many advantages, CRISPR technology is an attractive tool to irreversibly remove the gene of interest by targeting its DNA. The overall goal is to identify negative regulators in axon regeneration. Here, we performed candidatebased studies to assess novel candidates in neural regulation (Piezo, Atr, Nup188) and developed several novel axon transection microfluidics platforms that cater to various needs of current research. Results of this integrative approach can either be individually exploited to further neuroscience research or be taken together to result in a list of negative regulators that are potentially suitable as axon disconnection therapy targets. / Bioengineering

The development of engineered and orthogonal biomolecular devices in mammalian cells has granted the ability to customize and tune the way in which cells sense and respond to stimuli. These synthetic signaling pathways have often taken inspiration from endogenous pathways, such as the use of transcription factors or induced nuclear translocation, and have been successful in generating customized cell therapies and advancing tissue engineering. However, one ubiquitous and dominant paradigm for endogenous cellular signaling that has not been fully realized in synthetic contexts is the use of the covalent attachment of small molecules to proteins. These post-translational modifications to proteins often function as an additional regulatory method – one that functions on top of traditional signaling – changing how cells sense and respond to stimuli dependent on their context. Here, we demonstrate the development of a synthetic and orthogonal post-translational modification for mammalian cellular signaling. Using the synthetic Notch receptor as a model pathway, we illustrate the ability for cells to sense and respond to inputs dependent on the modification state of receptors. We further describe the extension of these tools for generalized and inducible signaling of synthetic Notch receptors, as well as their use in generating modification-dependent translocation and transcription. By mimicking the methods in which endogenous pathways control their sensing abilities, engineered modification-dependent pathways are positioned to approach more complex signaling states than with traditional methods alone. / 2024-01-26T00:00:00Z

Elastic and collagen fibers are the two major extracellular matrix (ECM) constituents in large elastic arteries and the primary load-bearing components of the arterial wall. Cardiovascular risk factors, such as age, high blood pressure, obesity, diabetes, and their co-occurrence referred to as metabolic syndrome, directly impact the biomechanical function of arteries. The objective of this study is to provide understandings on the structure and function of elastic arteries under aging, hypertension, and metabolic syndrome using a coupled experimental-modeling approach that integrates multiphoton imaging, mechanical characterization, and constitutive modeling. In this study, we showed that consumption of a high fat high sucrose diet induced metabolic syndrome accelerated arterial remodeling in a mouse model. Aging- and diet-induced arterial remodeling was manifested by a significantly reduced capability of elastic energy storage. Our study further revealed that aging adversely impacted the mechanical homeostasis in elastic arteries with altered structural inhomogeneity in elastic lamellae. Specifically, the uneven thickening of inter-lamellar space and the increased unfolding of inner lamellar layers, resulted in compromised mechanical homeostasis that was manifested by a nonuniform lamellar stretching and stress distribution in the arterial wall. These microstructural changes are likely related to arterial remodeling. Hypertension has long been associated with arterial stiffening and renal sympathetic nervous system plays an important regulatory role in blood pressure. Renal denervation has been emerged as a potential therapeutic approach to resistant hypertension by selectively removing the renal nerves and therefore attenuating sympathetic outflow to the kidney. In this study, we investigated the effects of renal denervation on the biomechanical response and microstructure of elastic arteries using a rat model of spontaneous hypertension. Our results showed that renal denervation effectively reduced blood pressure and reversed the biomechanical properties of carotid arteries under physiological pressure. In the meantime, arteries remained intact after renal denervation without observable changes in the ECM microstructure. Our study provides interesting initial findings on the effect of renal denervation on large elastic arteries mechanical response and microstructure. Future studies are needed to fully reveal the complex interplay between renal sympathetic nervous system and blood pressure regulation. / 2024-09-15T00:00:00Z

Antimicrobial resistance (AMR) is a growing global threat to public health expected to impact 10 million people by 2050, with a disproportionate effect on low- and middle- income countries, that is further exacerbated in communities living in urban informal settlements and refugee camps. As a result, there is a heightened urgency to understand how current antibiotic use is driving the spread of drug resistance in communities with high population density and those that are in proximity to wastewater settings and environmentally contaminated surroundings. Currently, there is a limited quantitative and mechanistic understanding of the evolution and spread of multidrug resistant (MDR) pathogens in these complex settings where there are a multitude of antibiotic residues and bacterial species present. Computational and experimental work in this area can lead to predictive outcomes and more effective strategies to prevent outbreaks of resistant pathogens. The goal of this thesis was to develop and test an integrated mathematical modeling and high-throughput experimental approach to quantitatively analyze AMR evolution in complex environments. The mathematical model captures predicted behavior for systems with multiple antibiotic residues and metal ions, incorporating the effects of both antibiotic-antibiotic interactions and metal-antibiotic interactions. This model is rooted in fundamental principles of biological systems modeling and was continuously integrated with a novel experimental workflow utilizing the eVOLVER for rapid iterative model development and validation. This work has resulted in the development of a robust method of understanding and predicting the development and spread of MDR bacteria in complex environments and has the potential to provide robust strategies to protect the health of vulnerable populations in these environments.

Neuroinflammation is an inflammatory response within the brain or spinal cord that may vary within the context of disease, injury or infection. Several factors can contribute to neuroinflammatory disorders such as cytokine and chemokines that are produced and released from peripherally derived immune cells or from locally activated cells such as microglia in the brain. The primary function of these cells is to clear inflammation, however, following inflammation, circulating monocytes are recruited and enter the CNS and contribute to neuroinflammation. Monocyte-derived macrophages, an important component of CNS inflammation, play a pivotal role in mediating neuroinflammatory responses. Macrophages are heterogeneous both in normal and in pathological conditions due to their plasticity and they are classified in two subsets, classically activated (M1) or alternatively activated (M2). There is accumulating evidence suggesting that extracellular vesicles (EVs) released from activated immune cells may play crucial roles in mediating neurotoxicity in the inflamed brain. EVs may act as antigen-presenting vesicles, carry and transfer cytokines and chemokines between cells, stimulate immune responses, and induce tolerogenic effects to suppress or induce inflammation. However, the possible role of EVs released by activated immune cells such as M1 and M2 macrophages in neurotoxicity seen in the inflamed brain is not known. In order to investigate the molecular and cellular impact of macrophages and EVs released from macrophage subtypes on neuronal functions, we established the conditions for the differentiation of monocytic cell lines into M2-like macrophages and characterize their phenotype in the presence of pro-anti-inflammatory cytokines including PMA, TNF alpha, IFN-gamma, LPS, DEX, and M-CSF. Furthermore, we also isolated and characterized EVs from M1 and M2 macrophages and observed no significant changes in their size and numbers. Furthermore, we treated primary neurons with M2 derived EVs and found a significant reduction in the action potential of neurons when exposed to those EVs. Collectively, these data suggested that M1 and M2 macrophages may possess differential neurotoxic effects mediated by EVs released by monocytic cells in the concept of neuroinflammation. / Bioengineering

Cortical bone fragility increases with age, therapeutic drug use and disease states. Clinically, bone fragility is evaluated by assessment of bone mineral density (BMD); however, studies have shown that other factors such as bone architecture, cell turnover and tissue composition influence bone quality. There is growing evidence that age related changes in bone water associated with collagen and/or mineral have a direct impact on bone mechanical competence and structural integrity. Understanding these compositional changes will aid in improved diagnose and prediction of fractures. Magnetic resonance imaging (MRI) is used to evaluate bone water, but this modality is limited in spatial resolution and is still being developed. Although still in the experimental stage, vibrational spectroscopy in the near infrared region (NIR) also known as near infrared spectroscopy (NIRS) is a nondestructive modality that can spatially evaluate alterations of bone composition. NIRS is a unique nondestructive technique that produces a signature spectrum by penetrating high frequency (4000-12,000 cm-1) non-ionizing radiation into material. NIRS permits a depth of penetration from millimeters to centimeters, dependent on frequency (wavelength). NIRS is very sensitive to water and can be used to provide molecular information of water related to collagen and mineral in bone samples. To date, definitive information on which NIR absorbances are linked to collagen or mineral bound water have not been identified. The overarching hypothesis is that water associated with collagen and/or mineral can be identified using NIRS and will serve as a biomarker for bone fragility in future preclinical studies. This will be achieved with the following three aims: First, to develop a method to image human cortical bone tissue using NIRS; second, to identify NIRS absorbances of water bound with mineral and collagen in bone; and finally, the third aim, to correlate the NIRS-derived water content in human cortical bone to structural properties determined by micro-computed tomography (micro-CT). Together these studies will establish the NIRS technique as a powerful tool to screen and monitor aging and diseased tissues in preclinical studies. / Bioengineering