Vascular grafts are used to repair, replace, or bypass diseased arteries, and there is a growing need for tissue-engineered blood vessels (TEBVs) as replacement grafts. Three-dimensional, self-assembled smooth muscle cell (SMC) rings can be fabricated and fused to create SMC tissue tubes with a structure similar to native vessels; however, this approach is limited by the underdeveloped mechanical integrity of the tissue. Thus, the goal of this research is to design, manufacture, and validate a cyclic circumferential stretch bioreactor to mechanically stimulate SMC tissue rings, with the goal of developing rings that can withstand the physiological forces of the in vivo environment. The bioreactor consists of a closed cam-syringe-tubing system that forces fluid into the tubing with each rotation of the cam, thereby distending and relaxing the tubing. Various sized cams were implemented to modify the distension of the tubing (5%, 7.5%, 10%, and 15% stretch magnitudes). Tissue rings are placed on the tubing, which is housed in a custom culture chamber. The tubing was validated using DVT® imaging technology to distend approximately 5, 7.5, 10, and 15% under static conditions. High density mapping was used to analyze the dynamic distension of the tubing and tissue rings. During bioreactor operation, the tubing distends 1-2% less than expected for the fabricated cams (5, 7.5, 10, 15%), and the tissue ring distends 31-56% less than the tubing on which it is located. To assess the effects of cyclic distension, 7-day-old SMC rings were cultured dynamically for 7 days and exposed to 0%, 5%, 7.5%, 10%, or 15% cyclic stretch (1 Hz, 100% duty cycle). Histology and immunohistochemistry indicate that both stretched and non-stretched rings synthesized collagen and glycosaminoglycans, but the contractile proteins á-smooth muscle actin and calponin were not synthesized. A decrease in cell density was observed as the magnitude of stretch increased, and the 5-15% stretched samples demonstrated more cellular alignment than the 0% stretch control samples. Mechanical testing analysis concluded that the stretched rings exhibited a reduction in ultimate tensile strength, maximum tangent modulus, maximum strain, and maximum load compared to unstretched control samples. It is anticipated that future work, including modifications of the culture medium and mechanical stimulation parameters (eg. reduced duty cycle, reduced frequency), has the potential to achieve the expected outcome of this research - a strong, aligned, contractile vascular smooth muscle cell tissue ring through dynamic culture using a cyclic circumferential stretch bioreactor.

Tissue engineering and regenerative medicine aim to develop materials that mimic some of the characteristics of the tissue they are replacing and control the growth and proliferation of cells. Despite exceptional advances in the range and quality of materials used, much remains to discover about the processes regulating interfaces between cells and their surroundings, or at cell-material interfaces. In order to study and control such interactions, scientists have produced engineered matrices aiming to mimic some of the feature of natural extra-cellular matrix (biochemistry, geometry/topography and mechanical properties). In order to pattern 2D-nanofibers on relatively large areas and throughput, allowing comprehensive biological studies, we developed a nano-fabrication technique based on the deposition of sparse mats of electrospun fibres with different diameters. These mats are used as masks to grow cell resistant polymer brushes from exposed areas. After removal of the fibres, the remaining brushes define a quasi-2D fibrous pattern onto which ECM molecules such as fibronectin can be adsorbed. Chapter 2 includes details of the techniques used to produce and characterize the fibrous nanopattern. Chapter 3 is focused on cell phenotype observed on the different nanofibres sizes. Adhesion assays showed that cell spreading, shape and polarity are regulated by the size of fibres but also the density of the nanofibres, similarly to previous observations made on circular nanopatterns. We then focused on the study of focal adhesion formation and maturations on these nanofibres and the role of key proteins involved in the regulation of the adhesion plaque: integrins and vinculin. Cells expressing different integrins were found to sense the nanoscale geometry differently. Vinculin sensing is the topic of Chapter 4. Although vinculin recruitment dynamics was affected by the nanofibrous patterns and focal adhesions arrange differently on the nanofibres, this protein does not seem to mediate nanoscale sensing. In Chapter 5, we finally focused on the role of the actin cytoskeleton as a direct sensor of nanoscale geometry. A gradual decrease in stress fibre formation was observed as the nanofibres dimensions decrease. Live imaging also demonstrated that the geometry of the extracellular environment strongly affects cytoskeleton rearrangement, stress fibres formation and disassembly. We identify the role of cytoskeleton contractility as an important sensor of the nanoscale geometry. Our study provides a deeper insight in understanding cell adhesion to the extracellular environment and the role of the matrix geometry and topography on such phenomena, but also raises questions regarding the more detailed molecular sensory elements enabling the direct sensing of nanoscale geometry through the actin cytoskeleton.

O avanço da ciência regenerativa está se comprometendo com a busca de novas opções terapêuticas no combate as diversas doenças e disfunções orgânicas. Nessas avançadas linhas de pesquisas, as células-tronco são um símbolo dessa evolução. A variedade da aplicação deste novo e experimental método de tratamento está sendo utilizado, pela bioengenharia, para reparar tecidos e órgãos lesados. Defeitos ósseos extensos ocorrem após diversos tipos de injúrias ao esqueleto facial, como traumas faciais, ressecções por lesões agressivas e malformações congênitas. Essas sequelas são tratadas, preferencialmente, através da reconstrução utilizando enxertos ósseos de origem autógena. Entretanto, às desvantagens proporcionadas pela obtenção do tecido ósseo autógeno, lançaram um dos maiores desafios, da bioengenharia que é a busca pelo aprimoramento dos substitutos ósseos. Nesse caminho do aprimoramento dos biomateriais a adição de células-tronco mesenquimais representam a possibilidade da criação de um sinergismo, entre as células e o arcabouço, para otimizar a osteogênese. Nessa linha, esse estudo propõe-se a avaliar o reparo ósseo em estudo experimental, em modelo animal, através do tratamento de defeito ósseos criados em fêmures de ratos. A amostra dessa pesquisa foi composta de 96 ratos albinos da espécie Rattus novergicus albinus, linhagem SHR (Spontaneously Hypertensive Rats), isogênicos. Os animais foram divididos aleatoriamente em quatro grupos de acordo com o tipo de tratamento (Grupo I: α – TCP + ADSCs; Grupo II: α – TCP + ADSCs/ENDO; Grupo III: α – TCP; Grupo IV). A cultura de células tronco tiveram como tecido de origem o tecido adiposo da região abdominal e as células endoteliais formam coletadas da medula óssea. A peças foram avaliadas em 03 períodos de tempo diferentes (07, 14 e 21 dias). A histomorfometria avaliou a área de neoformação óssea dos defeitos bem como a imunohistoquímica com marcação para a proteína VEGF avaliou a eficácia da adição das células endoteliais. Os resultados demonstraram, através dos testes estatísticos que houve uma diferença estatisticamente significante, no reparo ósseo, favorecendo os tratamentos dos defeitos que utilizaram a terapia celular e a vascularização foi também otimizada no grupo que foi tratado com a adição das células endoteliais. Dessa forma conclui-se que nesse modelo de estudo a utilização das ADSCS foram capazes de otimizar o reparo ósseo. / The regenerative medicine has been searching for a new therapeutic options to manage diseases and also organic dysfunctions. The advanced research fields, has enrolled the stem cells to achieve the upgrade in the new treatment objectives. The bioengineering is application of this new and experimental, treatment method to repair damaged tissues and organs using the stem cells, includes the possibility to acelerating and improving the bone repair process. The autogenous bone graft has been considered the gold standart graft material to reconstruction the bone defects, fundamentally because of the osteogenic potential. However, the harvest disadvantages autogenous bone tissue leads to the search for bone substitutes improvement. In this field a promising alternative has been proposed by tissue engineering. The totipotent cells, also called mesenchymal stem cells, which has the cellular plasticity ability, is associated with biomaterials, creating a synergy, between these cells and the scaffold, to optimize the osteogenesis. The tissue engineering application to tissue repair has been extensively researched with the aim of proposing more reliable and more efficient clinical methods. Although the effects of the use of adult stem cells are well known in bone marrow transplants, in some areas, such as bone repair, there is still lack of scientific data. This research was conducted, in animal model, to assessment bone repair in created femural bone defects treated with mesenchymal stem cells. 96 animals (Rattus novergicus albinus - Spontaneously Hypertensive Rats) were randomly divided into four groups (Group I: α - TCP + ADSCs, Group II: α - TCP + ADSCs/Endo, and Group III: α - TCP; (07, 14 and 21 days). The histological sections were stained in H&E and the histomorphometry was used to evaluated the new bone formation area in the defects and also the immunohistochemical expression of VEGF was analysed. Our results suggest that the combination of ADSCs and the scaffold was able to enhance the bone repair in this study model.

To date, the reconstructive approach addressing chronic non-healing wounds, deep tissue damage, and severe wound defects relies upon avascular dermal grafts and autologous flap techniques. Such flaps are limited by donor site availability and morbidity, while current dermal grafts rely upon host cellular invasion for neovascularization and incorporation. These products fail to include an inherent vascular network and the supporting cells necessary to ensure adequate incorporation and graft survival beyond the most optimal wound beds. Herein, we fabricate a pre-vascularized full-thickness cellularized skin equivalent containing a three-dimensional vascularized network of interconnected macro and microchannels lined with vascular cells, within a collagen neodermis populated with fibroblasts, and an epidermis comprised of human keratinocytes capable of providing whole tissue perfusion. Previously, our lab has employed a sacrificial microfiber technique to develop tissue-engineered scaffolds with an inherent hierarchical network of microvessels, which recapitulates the organization of an arteriole, venule, and capillary bed. Utilizing a type-I collagen hydrogel matrix, vascular cells were seeded within pre-fabricated channels and allowed to proliferate to generate an endothelialized microvasculature. These collagen scaffolds were subsequently anastomosed into rat models to demonstrate the clinical feasibility of such approach. The present study aims to more closely recapitulate the in vivo structure of human skin via the incorporation of vital epidermal and dermal components of native skin into a biocompatible construct containing a complex hierarchical vasculature, which may be anastomosed using standard microsurgical techniques and immediately perfused. Pluronic F127 was used as the sacrificial material: 1.5 mm diameter “U” shaped macrofibers and 100-500 µm-interwoven microfibers were heat extruded and then embedded within type-I collagen into which Cyan Fluorescent Protein (CFP)-tagged human placental pericytes and human foreskin fibroblasts (HFF1) had been encapsulated. Following pluronic sacrifice, resultant channels were intraluminally seeded with Red Fluorescent Protein (RFP)-tagged human aortic smooth muscle cells, Green Fluorescent Protein (GFP)-tagged human umbilical vein endothelial cells, and topically seeded with human epidermal keratinocytes (HEK). Construct microstructure was analyzed using multiphoton microscopy (MPM) after 7, 14 and 28 days of culture. Additionally, after 14 and 28 days of culture, endothelial cells were extracted from the construct using collagenase digestion and Real Time (RT)-qPCR performed to analyze expression of markers of angiogenesis and maturation of the vascular network. MPM demonstrated a hierarchical vascular network containing macro and microvessels lined by endothelial and smooth muscle cells, supported by perivascular pericytes, all in appropriate microanatomic arrangement. Neodermal HFF1 proliferated throughout the observation period and the HEK neoepidermis developed into a stratified epidermis along the superior aspect of the construct. Angiogenic sprouting from the nascent vascular network into neovessel like structures was noted. RT- qPCR revealed relative expression of Jagged1, Dll4, Ve-Cadherin, and CD31. We have successfully fabricated a novel tissue-engineered pre-vascularized full thickness skin flap, which recapitulates the inherent hierarchical vasculature found within human skin and is suitable for in vivo perfusion. We provide the platform for an on- demand, geometrically tunable tissue engineered skin equivalent with an anastomosable vascular network. This tissue-engineered skin flap holds the potential to transform reconstructive surgical practice by eliminating the consequences of donor site morbidity, and enabling rationally designed, patient-specific flaps for each unique wound environment and anatomic location. / 2017-06-16T00:00:00Z

Osteoarthritis is a joint disease associated with the irreversible breakdown of articular cartilage in joints, causing pain, impaired mobility, and reduced quality of life in over 27 million Americans and many more worldwide. The tolls by osteoarthritis (OA) on the workforce and healthcare system represent significant economic burdens. An attractive strategy for treating OA is cartilage tissue engineering (CTE). CTE strategies have been promising at producing cell-scaffold constructs at small sizes (3-5 mm in largest dimension), but OA often does not present symptoms until lesions reach 25 mm in diameter. Using bovine chondrocytes seeded in agarose, our lab has produced small CTE constructs with native cartilage levels of compressive stiffness and proteoglycan content. As construct dimensions are increased, however, the resulting tissue suffers from extreme heterogeneity of deposited matrix due to nutrient transport limitations. The ability to successfully scale up constructs to clinically relevant sizes is a major goal in CTE research. Another major and largely unresolved obstacle is the translation of successes from animal cell models to CTE systems with human cells, which is ultimately necessary for clinical treatment of OA. In this dissertation, experiments are placed forth which seek to address the nutrient limitations in large cartilage constructs and to help bridge the gap from animal cells to human cells for CTE. The growth of CTE constructs is limited by the poor availability of nutrients at construct centers due to consumption by cells at the construct periphery. The first series of studies in this dissertation sought to identify nutrients in culture media that are consumed by cells and are critical for matrix production, and to characterize their transport behavior. Among several candidate nutrients, glucose proved to be the most indispensable; little to no growth transpired in constructs when glucose fell below a critical threshold concentration. A subsequent study provided a system-specific glucose consumption rate. These parameters were informative for computational models of construct growth, which helped predict transport and growth phenomena in constructs and suggest improved culture techniques for later experiments. The cultivation of tissue constructs of increasing size presents logistical challenges, as the constructs’ requirements for nutrients, growth factors, and even sizes of culture vessels increase. The addition of nutrient channels to constructs to improve nutrient transport and tissue growth is a promising strategy, but more sophisticated casting and culture techniques are required for constructs with channels, particularly as construct size is increased. We first designed casting and culture devices for cylindrical 10 mm × 2.3 mm (diameter × height) constructs with 1 mm diameter nutrient channels. With information gleaned from computational models predicting glucose availability in constructs, we refined our culture system and demonstrated beneficial effects of nutrient channels on construct mechanical properties and extracellular matrix contents. This was the most successful instance to date of the use of nutrient channels in CTE, and is highly promising for channels’ ability to mitigate transport limitations in constructs. We next sought to optimize key parameters for culturing channeled constructs. The addition of channels is an optimization problem: greater numbers of closer-packed channels increase nutrient availability within the construct but simultaneously detract from the construct’s initial volume and cell population. Furthermore, we suspected that uneven swelling of 10 mm diameter constructs was a side effect of transient treatment with 10 ng/mL TGF-β, a highly effective and commonly-employed technique for elevating construct functional properties. By increasing channel densities in 10 mm diameter constructs, we identified a channel spacing that yielded optimal construct functional properties. In constructs with this channel spacing, reducing the TGF-β dosage by tenfold resulted in similar or elevated properties by constructs. These experiments supplied us with optimal parameters for further scaling up our constructs to clinically-relevant sizes, a practice that can be adapted for any CTE culture system for large constructs. The ability to treat severe OA by entirely resurfacing diseased joints with CTE would be highly desirable, yet this ability remains elusive, as efforts to grow constructs of such size have thus far been stymied by nutrient transport limitations. We scaled up our culture system for 10 mm diameter constructs, employing previously optimized culture conditions and channel spacing, and cultured articular surface-sized (40 mm diameter, 2.3 mm thick) constructs. These constructs were 100× the size of our small constructs, yet they still attained similar functional properties, reaching native cartilage levels of compressive stiffness and proteoglycan content. These are the largest CTE constructs to ever achieve such favorable properties. These results demonstrate that with nutrient channels, CTE constructs have the potential to replace entire joint surfaces that have been compromised by OA. Finally, we began to explore the feasibility of translating techniques from our bovine and canine model systems into human cells. We harvested adult human chondrocytes from expired osteochondral allografts and cast them in small (3 mm diameter) constructs, culturing the constructs under various conditions that have been previously successful for animal constructs. We observed similarities between human versus bovine and canine constructs, most notably that high initial cell seeding density led to marked increases in functional properties, in some cases approaching mechanical and biochemical properties of native human cartilage. Human constructs also exhibited poor GAG retention and long-term growth relative to animal constructs. By establishing successful techniques for human constructs in addition to identifying new challenges, we provided an in-depth characterization of human chondrocytes in agarose that is promising overall for eventual clinical translation. The body of work presented in this dissertation followed a methodical approach to scaling up CTE constructs to the sizes of entire joint surfaces, through experimentation with nutrient channels in constructs and with the support of predictive computational models. The principle behind nutrient channels is fundamental and therefore can be applied to CTE systems using other scaffold and cell types. By incrementally increasing the scale of bovine chondrocyte-laden constructs and by performing initial studies with small human CTE constructs, we have laid down groundwork for future studies seeking to grow articular surface-sized human engineered cartilage.

The articular cartilage has a limited capacity to regenerate. Cartilage lesions often result in degeneration, leading to osteoarthritis. Current treatments are mostly palliative and reparative, and fail to restore cartilage function in the long term due to the replacement of hyaline cartilage with fibrocartilage. Although a stem-cell based approach towards regenerating the articular cartilage is attractive, cartilage generated from human mesenchymal stem cells (hMSCs) often lack the function, organization and stability of the native cartilage. Thus, there is a need to develop effective methods to engineer physiologic cartilage tissues from hMSCs in vitro and assess their outcomes in vivo. This dissertation focused on three coordinated aims: establish a simple in vivo model for studying the maturation of osteochondral tissues by showing that subcutaneous implantation in a mouse recapitulates native endochondral ossification (Aim 1), (ii) develop a robust method for engineering physiologic cartilage discs from self-assembling hMSCs (Aim 2), and (iii) improve the organization and stability of cartilage discs by implementing spatiotemporal control during induction in vitro (Aim 3). First, the usefulness of subcutaneous implantation in mice for studying the development and maintenance of osteochondral tissues in vivo was determined. By studying juvenile bovine osteochondral tissues, similarities in the profiles of endochondral ossification between the native and ectopic processes were observed. Next, the effects of extracellular matrix (ECM) coating and culture regimen on cartilage formation from self-assembling hMSCs were investigated. Membrane ECM coating and seeding density were important determinants of cartilage disc formation. Cartilage discs were functional and stratified, resembling the native articular cartilage. Comparing cartilage discs and pellets, compositional and organizational differences were identified in vitro and in vivo. Prolonged chondrogenic induction in vitro did not prevent, but expedited endochondral ossification of the discs in vivo. Finally, spatiotemporal regulation during induction of self-assembling hMSCs promoted the formation of functional, organized and stable hyaline cartilage discs. Selective induction regimens in dual compartment culture enabled the maintenance of hyaline cartilage and potentiated deep zone mineralization. Cartilage grown under spatiotemporal regulation retained zonal organization without loss of cartilage markers expression in vivo. Instead, cartilage discs grown under isotropic induction underwent extensive endochondral ossification. Together, the methods established in this dissertation for investigating cartilage maturation in vivo and directing hMSCs towards generating physiologic cartilage in vitro form a basis for guiding the development of new treatment modalities for osteochondral defects.

Approximately 40% of men and women in the United States will be diagnosed with at least one form of cancer in their lifetime, with cancer being implicated in one in four deaths. While great strides have been made in early diagnosis and treatment using standard regimens of chemotherapy and radiation, resulting in an overall decrease in cancer mortality, tumor initiation, growth and metastasis continue to evade control. The continued search for effective and targeted drugs has been hindered by the high failure rate of costly clinical trials, highlighting a need for more accurate preclinical models of disease, not only for pharmaceutical testing, but also biological research and assay development. The dominant role of the tumor microenvironment in regulating tumor initiation, progression, and metastasis has been well documented, driving the application of tissue engineering strategies in cancer biology. In vitro models that recapitulate clinically-relevant features of native tumors with greater fidelity than monolayer tissue cultures have the potential to yield discovery of novel therapeutic targets and regimens while also providing critical insights into mechanisms of tumor resistance. This thesis describes a tissue engineering strategy for generating an in vitro tumor model of human conventional chondrosarcoma using a custom biomimetic scaffold, and characterizes the effect of the biomaterial on cancer cell phenotype. Together with a previously validated and published in vitro model of human Ewing’s sarcoma tumors, we further investigated the effect of microenvironmental factors including matrix stiffness, niche composition, and three-dimensionality on the expression of a key cell cycle regulator and tumor suppressor mutated or lost in a wide variety of cancers, p53. A transcription factor nicknamed the “guardian of the genome,” p53 is activated in normal tissues in response to stress and triggers cellular responses including cell cycle arrest and apoptosis, or induces transcription of DNA repair enzymes to promote cell survival. The unifying hypothesis of this thesis was that the tumor microenvironment does in fact influence expression of tumor suppressors like p53, ultimately contributing to the progression of tumors toward metastasis and chemoresistance, and that these effects can be probed in vitro using disease-specific engineered tumor models to identify novel druggable targets and biomarkers with prognostic significance.

A thin layer of calcified cartilage at the native cartilage-to-bone junction facilitates integration between deep zone articular cartilage and subchondral bone, while maintaining the integrity of the two distinct tissue regions. Regeneration of this interface remains a significant clinical challenge for long-term and functional cartilage repair. The strategy for osteochondral interface formation discussed in this thesis focuses on the design and optimization of a biomimetic scaffold for stable calcified cartilage formation. The ideal interface scaffold supports chondrocyte biosynthesis and the formation of calcified cartilage with physiologically-relevant mechanical properties. Furthermore, the interface scaffold allows for osteointegration and the maintenance of the calcified cartilage matrix. It is hypothesized that ceramic presence and zonal chondrocyte interactions regulate cell biosynthesis and mineralization, and these cell-matrix and cell-cell interactions are essential for calcified cartilage formation and maintenance. Biomimetic design parameters for an interface scaffold were determined by characterizing the native interface in terms of mineral and matrix distribution. A composite hydrogel-hydroxyapatite scaffold was then designed to support formation of a functional calcified cartilage matrix. The hydrogel phase maintains the chondrocyte phenotype and allows for incorporation of ceramic particles, while the biomimetic ceramic phase is osteointegrative and decreases the need for cell-mediated mineralization. This scaffold was optimized <italic>in vitro</italic> based on hydrogel type, chondrocyte population, and ceramic particle size. The collective findings from these cell-ceramic interaction studies determined that hypertrophic chondrocytes, cultured in the presence of micron-sized hydroxyapatite particles, exhibit enhanced hypertrophy and matrix deposition. Scaffold ceramic dose and seeding density were also optimized for promoting calcified cartilage formation <italic>in vitro</italic>. In order to implement the scaffold for integrative cartilage repair, a scaffold was designed to regenerate both uncalcified and calcified cartilage on a bilayered hydrogel scaffold. Furthermore, a polymer-ceramic nanofiber component was added to augment the original design for <italic>in vivo</italic> implementation. The hydrogel-nanofiber composite scaffold was evaluated <italic>in vivo</italic> and found to support mineralization and osteointegration within the bone region while preventing endochondral ossification within the repair tissue. Finally, inspired by the stratified organization of zonal chondrocyte populations above the calcified cartilage interface, the layered hydrogel model was used to determine the role of zonal chondrocyte organization on calcified cartilage stability. This thesis collectively explores cell-ceramic and cell-cell interactions, and their ramifications for calcified cartilage formation and maintenance. Specifically, ceramic presence promotes the deposition of a calcified cartilage matrix by hypertrophic chondrocytes in a dose-dependent manner, and furthermore, communication between surface zone and deep zone chondrocyte populations suppresses mineralization within articular cartilage above the calcified cartilage interface. It is anticipated that the scaffold design strategy developed in this thesis can also be applied to the regeneration of other complex interfaces where there are transitions from soft-to-hard tissue.

Timely reperfusion after a myocardial infarction is necessary to salvage the ischemic region; however, reperfusion itself is a major contributor to the final tissue damage. Currently, there is no clinically relevant therapy available to reduce ischemia-reperfusion injury. While many drugs have shown promise in reducing ischemia-reperfusion injury in preclinical studies, none of these drugs have demonstrated benefit in large clinical trials. Part of the failure to translate therapies can be attributed to the reliance on small animal models for preclinical studies. While animal models encapsulate the complexity of the systemic in vivo environment, they do not fully recapitulate human cardiac physiology. In this thesis, we utilized cardiac tissue engineering methods in conjunction with cardiomyocytes derived from human induced pluripotent stem cells, to establish a biomimetic human tissue-engineered model of ischemia-reperfusion injury. The resulting cardiac constructs were subjected to simulated ischemia or ischemia-reperfusion injury in vitro. We demonstrated that the presence of reperfusion injury can be detected and distinguished from ischemic injury. Furthermore, we demonstrated that we were able to detect changes in reperfusion injury in our model following ischemic preconditioning, modification of reperfusion conditions, and addition of cardioprotective therapeutics. This work establishes the utility of the human tissue model in studying ischemia-reperfusion injury and the potential of the human tissue platform to help translate therapeutic strategies into the clinical setting.

In vitro microphysiological system utilizes engineered tissue constructs from human cells to model functional activity of human tissues or organs in both healthy and diseased state, thereby providing a more accurate drug screening than animal models prior to clinical trials. One essential component of an in vitro microphysiological system is a tissue engineered blood vessel (TEBV) that can accurately recapitulate the functional vasculature in vivo. This thesis first explores two most important considerations to a successful TEBV generation, the cell source and the fabrication method. To engineer a vascular tissue construct, an ideal cell source should demonstrate high availability and accurate vessel functionality. Mesenchymal stem cells (MSC) were explored due to their high availability, proliferation capacity, and capability to deposit adequate extracellular matrix (ECM) for cell sheet formation. Vascular smooth muscle cells (SMC) are the cell components that comprise the medial layer of native blood vessel, and thus optimal for demonstrating equivalent biological functionality. However, SMC are much harder to acquire through biopsy, and they have limited proliferative capacity and quick senescence. Therefore, an alternative cell source for SMC was obtained through direct reprogramming approach involving the induced overexpression of myocardin in more readily available human cell sources. The resulting reprogrammed SMC demonstrated close resemblance to the native SMC in terms of its phenotype, related gene and protein expression levels, and contractile function. Two different fabrication methods, nanopatterned cell sheets and dense collagen hydrogel, were explored to engineer a 1 mm inner diameter blood vessel. The fabricated TEBVs were then compared to that of the native blood vessel and each other in terms of its structure, mechanical properties, and vasoactive function in response to stimuli. After selecting the most optimal cell source and fabrication method for developing a human cell-based TEBV for in vitro microphysiological system, the second part of this thesis assesses the capability of the designed TEBV to model a vascular disease for drug screening purposes. Marfan syndrome was selected as a model vascular disease due to its previous history of contradictory results from the animal models and human clinical trials using losartan, an angiotensin II receptor blocker, in terms of preventing aortic root dilation. TEBV fabricated using reprogrammed SMC from Marfan syndrome patient sample and dense collagen hydrogel showed reduced fibrillin deposition, increased vessel diameter and thickness, and reduced vasoconstriction levels when compared to the wild type TEBV, which is consistent with that observed in native vessels of Marfan syndrome patients. Losartan improved the function of Marfan syndrome TEBV, but still at reduced level when compared to that of the wild type. SB203580, a selective inhibitor of p53 MAPK that has been shown to be a better drug candidate than losartan in recent cell-based studies, showed improved TEBV function comparable to that of the wild type. In overall, this thesis presents a successful development of a highly robust, patient-specific in vitro vascular model. An accurate recapitulation of a drug-induced physiological response in humans can speed up the drug screening process with higher efficiency, and this will eventually increase the chances of successful treatment for patients.