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<p> Musculoskeletal injuries are one of the leading causes of human disability. Tissue engineers aim to restore damaged musculoskeletal tissues by creating scaffolds that promote cellular adhesion, proliferation, and eventual differentiation into functional tissue. It is known that the extracellular matrix (ECM) regulates cellular behavior and is often used as a basis for biological scaffolds; however, current scaffolds often mimic the ECM of adult, homeostatic tissue and frequently lead to poor tissue restoration. What is rarely taken into consideration is that the ECM undergoes extensive remodeling during development to facilitate growth.</p> <p>In the musculoskeletal system, myogenic progenitors (<em>Pax3</em>+) and connective tissue cells (<em>Prx1</em>+) proliferate and differentiate into muscle, tendon, cartilage, and conjoining interfaces (<em>e.g.</em> myotendinous junction), while depositing and remodeling the ECM. As tissues mature, cells continue to refine ECM networks to withstand the functional demands to facilitate movement. The ECM composition and architecture of adult musculoskeletal tissues have been studied individually and are thought to be distinct; however, there has yet to be a comprehensive comparative analysis of the ECM in adult muscle, tendon, and the myotendinous junction (MTJ) in a single study. Additionally, how the matrisome of adult musculoskeletal system compares to the ECM dynamics during forelimb development, remain largely unknown due to lack of techniques to analyze embryonic matrisome composition and synthesis. </p> <p>To address these research gaps, we (1) used quantitative proteomics to map the matrisome composition in the mature murine MTJ, relative to the tendon and muscle; (2) adapted tissue fractionation and biorthogonal non-canonical amino acid tagging techniques to embryonic tissues as a method to quantify the global and nascent embryonic matrisome; and (3) subsequently used these techniques to establish a baseline of ECM dynamics during forelimb morphogenesis (embryonic day, E11.5-E14.5) and growth (postnatal day, P3 and P35). We hypothesized that proteomic evaluation of ECM composition and synthesis in developing and adolescent limbs would resolve differences between embryonic and growing tissues. Indeed, we saw significant differences in global and nascent matrisome composition between embryonic and adolescent forelimbs. Notably, the relative abundance and ratios of collagens associated with type I fibrillogenesis (I, III, and V) were significantly different as a function of development embryogenesis and across the adult muscle, MTJ, and tendon.</p> <p>Type I collagen fibrils are critical for tissue architecture and function. Using genetic mouse models, the regulatory roles of COL5A1 in the initiation of type I collagen fibrillogenesis, and organization of subsequent fibrils, have been well characterized in tendons and ligaments; however, is it unknown which cell types contribute COL5A1 to the ECM in the forelimb. To identify the functional contribution of COL5A1 by myogenic or connective tissue cell populations, we generated conditional (cre-flox) knock-out mouse models to inactivate <em>Col5a1</em> using <em>Pax3</em>- or <em>Prx1</em>-drivers, respectively. Haploinsufficiency of <em>COL5A1</em> in humans is associated classical Ehlers-Danlos syndrome, characterized by skin fragility and join instability; similar, albeit more severe, phenotypes were present in <em>Prx1Cre/+;Col5a1fl/fl</em> mutants, but not in <em>Pax3Cre/+;Col5a1fl/fl</em> mutants or controls. Interestingly, THBS4+ and COL22A1+ networks at the MTJ were morphologically affected in <em>Prx1Cre/+;Col5a1fl/fl</em> limbs. Additional work needs to be conducted to characterize the systematic phenotypes observed in <em>Prx1Cre/+;Col5a1fl/fl</em> limbs.</p> <p>Together, our results indicate that there are distinct, complex ECM dynamics, originating from distinct cell-types, that drive musculoskeletal morphogenesis in the forelimb. Further, the tools developed here will serve as a foundation for quantitative proteomic analyses of the matrisome composition in embryonic tissues. Collectively, this work provides a baseline of ECM protein dynamics during musculoskeletal morphogenesis, a helpful guide for tissue engineers in designing scaffolds to promote restoration of damaged tissues, with enhanced integration into the host tissue.</p>

Connective tissue extracellular matrix (ECM) consists of an interwoven network of contiguous collagen fibers that inform cell activity, direct biological function, and guide tissue homeostasis throughout life. Recently, ECM analogs have emerged as a unique ex vivo culture platform for studying healthy and diseased tissues and in the latter, enabling the screening for and development of therapeutic regimen. Unfortunately, current commonly used platforms, such as tissue-culture polystyrene (TCPS) or the basement membrane matrix, Matrigel, fail to fully recapitulate the physical and biochemical properties of the ECM. Tissue-culture polystyrene is significantly stiffer than typical ECM tissues and lacks the composition and 3-dimensional architecture that is critical for ECM function. Improving upon TCPS’s shortcomings, Matrigel retains a natural ECM structure and is comprised of native biopolymers. However, it is derived from mouse sarcomas, and thus, has significant batch-to-batch variability and often contains growth factors at non-physiologic concentrations. Moreover, despite being biopolymer based, Matrigel has relatively low amounts of type I collagen and high levels of type IV collagen, and as such, compositionally does not match the predominantly type I collagen matrix intrinsic to connective tissues. Thus, it is clear that new and improved models of the ECM are needed for in vitro culture. In pursuit of developing a highly biomimetic ECM analog, the objectives of this work were three-fold— first, to fabricate collagen-based ECM analogs with nanoscale mimicry, second, to systematically optimize crosslinking protocols in order to produce a stable substrate with continuous fibrous architecture, and third, to evaluate the substrate’s biocompatibility and utility as a platform for studying biomineralization. It was hypothesized that an architecturally and chemically relevant fibrous substrate could be prepared from gelatin and provide an optimal ex vivo platform for cell culture and new therapy screening and development. Thus, the ECM analog will be collagen-like, biocompatible, consist of continuous fibers, demonstrate both viscoelastic and elastic behavior, exhibit relevant mechanical properties, and remain stable for at least 14 days at cell culture conditions. To this end, first, a “green” electrospinning method was developed for preparing fibrous meshes from gelatin, which avoids typical electrospinning solvents that present significant health risks and barriers to large scale production. Next, crosslinking methods were developed using the reactive dialdehyde, glutaraldehyde (GTA), and the naturally derived enzyme, transglutaminase (TGase). These methods stabilized the meshes for over 28 days under cell culture conditions without disrupting its biomimetic architectures and chemical properties. In addition, a third approach to mesh fabrication using gelatin methacryloyl (gelMA) was developed to overcome the shortcomings of GTA and TGase crosslinking. With gelMA, the number of crosslinking sites were customized and, by taking advantage of its ability to undergo free radical polymerization, stable fibrous meshes were prepared with reproducible architecture, chemistry, and tunable mechanical properties. Following fabrication, the biocompatibility of the meshes was evaluated through macrophage, stem cell, and differentiated cell cultures. During culture, the macrophages maintained a naïve, non-polarized state, indicating they were not triggered towards an inflammatory response by the meshes. In addition, fibrochondrocytes, a cell critical for maintaining the collagen-based matrices where ligaments attach to bone, remained viable and maintained phenotypic expression on the meshes, as evident by their enhanced proteoglycan and collagen production relative to TCPS cultures. After demonstrating biocompatibility, the gelatin platform was coupled with a synthetic matrix vesicle (SMV) system and successfully acted as a mineralization platform in the presence of human osteoblast-like cells. Additionally, the platform supported mesenchymal stem cell expansion and mineralization when cultured with an alkaline phosphate conjugated SMV. In this work, three unique methods were developed for preparing ECM analogs. These efforts led to the production of a collagen-like mesh with nano- and micro-scale cues, fibrous continuity with little batch-to-batch variability, and proven stability in both dry and wet conditions. Importantly, these meshes did not instigate any inflammatory responses and supported fibrochondrocyte, osteoblast, and stem cell culture. Furthermore, the mesh successfully functioned as a template for biomineralization using both human osteoblast-like cells and stem cells. Collectively these findings demonstrate the potential of a collagen-like ECM analog with physiological relevance for ex vivo cell culture studies; and furthermore, its potential as a high-fidelity platform for studying cell-mediated biomineralization, cell-matrix interactions, and developing new therapeutic approaches for the treatment of connective tissue disorders.

The in vivo 3D extracellular matrix provides a temporal regulatory environment of chemical cues. Understanding this dynamic environment will be crucial for efficient drug screening, diseases mechanism elucidation, and tissue engineering. Therefore, in vitro 3D cell culture systems with reversible chemical environments are required. To this end, we developed a non-cytotoxic agarose-desthiobiotin hydrogel to sequester streptavidin biomolecule conjugates (KD 10-11 M), which can then be displaced by the addition of biotin (KD 10-15 M). Streptavidin biomolecule conjugates were simultaneously and sequentially immobilized by changing media components. The time required for biochemical environment exchange was minimized by increasing the surface area to volume ratios and pore size of the hydrogels. We temporally controlled the cell adhesive properties of hydrogels with RGD modified streptavidin to influence endothelial cell tube formation. / Thesis / Master of Science (MSc)

The overall objective of this project was to investigate the role of locally derived growth factors and extracellular matrix proteins in regulating prepubertal heifer mammary development. In the first experiment, short-term treatment of heifers with GH or E increased proliferation of mammary epithelial cells. Coinciding with increased epithelial cell proliferation, IGF-I protein increased and IGFBP-3 protein decreased within mammary tissues. Thus, proliferation was associated with an apparent net increase in the biological availability of IGF-I within the mammary gland. In the second experiment, decreased mammary development and epithelial cell proliferation in response to ovariectomy coincided with decreased mammary expression of IGF-I mRNA and decreased binding of IGF-I to mammary microsomes. Taken together, these results imply an important role for locally derived IGF-I in regulating heifer mammary development. However, in contrast to our hypothesis, IGF-I mRNA did not differ between cleared or intact mammary fat pad, suggesting that expression of IGF-I mRNA is not regulated by epithelial:stromal interactions. Neither ovariectomy or epithelial:stromal interactions influenced the mRNA expression of IGFBP-3 or IGFBP-5 within mammary tissues. Ovariectomy increased the proportion of ERa positive mammary epithelial cells. In contrast, GH administration to prepubertal heifers did not influence the proportion of ERa-positive epithelial cells. Interestingly, mammary development was more severely affected in heifers ovariectomized before six weeks of age than heifers ovariectomized at three months of age, implying a critical period of ovarian stimulation during the first six weeks of age. Localization of laminin, fibronectin, and collagen in mammary parenchyma suggested specific roles for extracellular matrix proteins in regulating mammary development and ductal morphogenesis. Laminin was decreased and fibronectin was increased by ovariectomy, suggesting a possible role for interactions between the ovary and extracellular matrix proteins within the heifer mammary gland. Finally, the mitogenic capacities of mammary tissue extracts from control and ovariectomized heifers did not differ in their ability to stimulate in vitro proliferation of MAC-T cells. In conclusion, the overall results support the hypothesis that locally derived IGF-I regulates prepubertal heifer mammary development. However, ERa expression and extracellular matrix proteins also appear to be important regulators of heifer mammary development. / Ph. D.

Radiation therapy has been most commonly used modality in the treatment of brain tumors. About 200,000 patients with brain tumors are treated with either partial large field or whole brain irradiation every year in the United States. The use of radiation therapy for treatment of brain tumor, however, can subsequently lead to devastating functional deficits several months to years after treatment. Unfortunately, there are no known successful treatments and effective strategies for mitigating radiation-induced brain injury. In addition, the specific mechanisms by which irradiation causes brain injury in normal tissues are not fully understood. A deeper understanding of the molecular mechanisms underlying these phenomena could enable the development of more effective therapies to contribute to long-term disease suppression or even cure. Therefore,the primary goal of this research project was to determine the molecular mechanisms responsible for radiation-induced brain injury in normal tissues. In the first study, the effects of whole brain irradiation on pro-inflammatory pathways in the brain were examined. Results demonstrated that brain irradiation induces regionally specific alterations in pro-inflammatory environments through activation of pro-inflammatory transcription factors (e.g., activator protein-1 (AP-1),nuclear factor-κB (NF-κB), and cAMP response element-binding protein (CREB)) and overexpression of pro-inflammatory mediators (e.g., tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1)) in brain. This study provides evidence for a differential induction of pro-inflammatory mediators in specific brain regions that have importance for the neurological/neuropathological consequences of irradiation. In the second study, a mathematical model describing radiation-induced mRNA and protein expression kinetics of TNF-α in hippocampus was reconstructed. This study demonstrated that the reaction kinetic model could predict protein expression levels of TNF-α in cortex, suggesting that this model could be used to predict protein expression levels of pro-inflammatory mediators in other parts of the brain. In the third study, the effects of aging on radiation-mediated impairment of immune responses in brain were examined. Results showed that radiation-induced acute inflammatory responses, such as overexpression of pro-inflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6),adhesion molecules (e.g., intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin), chemokine MCP-1, and matrix metalloproteinase-9 (MMP-9), are significantly impaired in aged brain. This study suggests that reduced production of pro-inflammatory mediators in response to irradiation compromises the normal host defense mechanisms in damaged brain tissue and subsequently leads to impaired repair/remodeling responses in old individuals. In the fourth study, the effects of irradiation on MMPs/tissue inhibitor of metalloproteinases (TIMPs) and extracellular matrix (ECM) degradation in brain were examined. Results demonstrated that whole brain irradiation induces an imbalance between MMPs and TIMPs expression, increases gelatinase activity, and degrades collagen type IV in the brain. This study suggests that a radiation-induced imbalance between MMP-2 and TIMP-2 expression may have an important role in the pathogenesis of brain injury by degrading ECM components of the blood-brain barrier (BBB) basement membrane. In the fifth study, the effects of irradiation on angiogenic factors and vessel rarefaction in brain were examined. Results demonstrated that whole brain irradiation decreases endothelial cell (EC) proliferation, increases EC apoptosis, and differentially regulates the expression of angiogenic factors such as angiopoietin-1 (Ang-1), Ang-2, Tie-2, and vascular endothelial growth factor (VEGF) in brain. This study suggests that radiation-induced differential regulation of angiogenic factors may be responsible for vessel rarefaction. In summary, the results from these studies demonstrated that whole brain irradiation induces brain injury by triggering pro-inflammatory pathways, degrading extracellular matrix, and altering physiologic angiogenesis. Therefore, this work may be beneficial in defining a new cellular and molecular basis responsible for radiation-induced brain injury. Furthermore, it may provide new opportunities for prevention and treatment of brain tumor patients who are undergoing radiotherapy. / Ph. D.

The limited transport of conventional chemotherapy within the tumor microenvironment (TME) is due to irregular vascularization, increased tumor interstitial pressure, and a dense extracellular matrix (ECM). The lack of selectivity of anticancer drugs often leads to systemic toxicity and damage to healthy tissues. Bacteria-based cancer therapy (BBCT) is a promising alternative, as tumor-targeting bacteria have been shown to preferentially colonize primary and metastatic tumors and induce anti-tumor effects. In this dissertation, we focus on several aspects of bacteria-nanoparticle conjugates, wherein BBCT is synergistically combined with nanomedicine to augment the efficacy of both treatment modalities. We explore biofabrication of our bacteria-nanoparticle conjugates called NanoBEADS (Nanoscale Bacteria Enabled Autonomous Drug Delivery Systems) and their interaction with the TME. Specifically, (1) we investigate the effects of two bacteria-NP conjugation chemistry and assembly process parameters of mixing method, volume, and duration, on NP attachment density and repeatability. We evaluate the influence of linkage chemistry and NP size on NP attachment density, viability, growth rate, and motility of NanoBEADS. (2) We investigate the effect of dense stroma and ECM production on the intratumoral penetration of bacteria with a mathematical model of bacterial intratumoral transport and growth. (3) We develop a microfluidic device with multicellular tumor spheroids to study the transport of tumor-targeting bacteria and support real-time imaging and long-term experiments. (4) We develop a new type of bacteria-based bio-hybrid drug delivery system using engineered cell surface display for enhancing the attachment of nanoparticles. / Doctor of Philosophy / Chemotherapy faces challenges in effectively reaching tumors due to factors like irregular blood vessel distribution, increased tumor pressure, and the presence of dense structures such as the extracellular matrix (ECM). This often results in collateral damage to healthy tissues. Bacteria-based cancer therapy (BBCT) offers a promising alternative, utilizing tumor-targeting bacteria to selectively attack tumors. This dissertation focuses on optimizing NanoBEADS (Nanoscale Bacteria Enabled Autonomous Drug Delivery Systems), which are chemotherapy encapsulating nanoparticle-bacteria assemblies to overcome these challenges and characterizing its behavior in tumors. Firstly, we investigated the optimization of bacteria-nanoparticle attachment, exploring various linkage chemistries and assembly processes to enhance attachment density, viability, and motility. Secondly, we examine how dense stroma and ECM affect bacterial penetration providing insights into intratumoral transport dynamics. Thirdly, we develop a microfluidic device integrated with multicellular tumor spheroids to enable real-time imaging and long-term experimentation on bacteria and drug transport. Lastly, we explore the potential of engineered cell surface display to enhance nanoparticle attachment in NanoBEADS, paving the way for self-propelled and highly targeted drug delivery systems. This dissertation strives to contribute to the transformation of current approaches to cancer treatment by refining drug delivery precision and efficacy while minimizing systemic toxicity.

No / Extracellular matrix remodelling and accurate spatio-temporal coordination of growth factor expression are two factors that are believed to regulate mitoses and cell migration in developing and regenerating tissues. The present quantitative videomicroscopical study examined the influence of some of the principal components of extracellular matrix and several growth factors that are known to be expressed in dermal wounds on three important facets of human skin cell behaviour in culture. Keratinocytes, melanocytes and dermal fibroblasts (and myofibroblast controls) exhibited varying degrees of substrate adhesion, division and migration depending on the composition of the culture substrate. Substrates that are recognized components of transitional matrices generally accentuated cell adhesion and proliferation, and were motogenic, when compared with serum-treated control surfaces, whereas components of more stable structures such as basement membrane had less influence. Platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and ¿ fibroblastic growth factor (¿FGF) all promoted cell proliferation and were chemokinetic to dermal fibroblasts, but not keratinocyte growth factor (KGF) or transforming growth factor ß (TGFß). PDGF, EGF and KGF, but not TGFß or ¿FGF, all enhanced proliferation of dermal keratinocytes. The same growth factors, and in addition KGF, all stimulated motility in keratinocytes, but TGFß and ¿FGF again had no effect. Developing a better understanding of the interdependency of factors that control crucial cell behaviour may assist those who are interested in the regulation of histogenesis and also inform the development of rational therapeutic strategies for the management of chronic and poorly healed wounds.

The extracellular matrix is a multi-functional environment that cells inhabit to form living tissue. To maintain the tissue, cells require constant telemetry with the matrix and respond to a variety of cues by remodeling matrix architecture. In this study the physical and biochemical manipulation of the matrix by resident cells is explored to better understand how these are used to remodel tissue. Cell-populated collagen hydrogels are used as a controllable in vitro tissue model. To directly measure mechanical forces involved with gel contraction, a culture force monitor was designed and built. Measuring dimensional changes together with contractile forces presents a method of separating mechanisms that influence tissue remodeling. Together, these techniques revealed a correlation between contractile force and gel deformation, suggesting a novel method for examining the material properties of the matrix. Limiting matrix metalloproteinase (MMP) activity altered the correlation as predicted, indicating a stiffer matrix. Contractile force was found to be regulated independent of MMP activity. In contrast, contractile force was found to be dependent on α2β1 integrin function. Collagen gel contraction correlated with both α2β1 function and MMP activity, and was significantly enhanced when combined. The results of this study indicate cells have the capacity to use multiple mechanisms for remodeling the extracellular matrix and may alternately use them together or independently to vary the rate of matrix contraction.