Embryonic stem cells (ESC) constitute a promising source of cells for cardiac transplantation strategies. However, complexities associated with in vivo studies have made it difficult to develop a thorough understanding of cell integration. We have engineered an in vitro system that recapitulates the native cardiac environment using 300μm thick collagen scaffolds seeded with neonatal cardiomyocytes (CM) and electrical field stimulation. The injection of undifferentiated ESC served as a baseline to assess the validity of studying cell transplantation in this model. Yfp-ESC survived and proliferated over several days in model tissue. ESC were not observed to significantly differentiate into the cardiac lineage, and did not integrate with the cardiac cell population. While the injection of ESC improved cardiac cell number, tissue functional properties were hindered. The methods developed herein can be readily adapted to study ESC derived progenitor and differentiated cells, to elucidate the optimal cell state for ESC-mediated cell therapy.

This thesis develops a novel photopatterning strategy for biomimetic scaffolds that enables spatial and biochemical control of engineered cellular architectures, such as the microvasculature. Intricate tools that allow for the three dimensional (3D) manipulation of biomaterial microenvironments will be critical for organizing cellular behavior, directing tissue formation, and ultimately, developing functional therapeutics to treat patients with critical organ failure. Poly(ethylene glycol) (PEG) based hydrogels, which without modification naturally resist protein adsorption and cellular adhesion, were utilized in combination with a two-photon laser patterning approach to covalently immobilize specific biomolecules in custom-designed, three-dimensional (3D) micropatterns. This technique, known as two-photon laser scanning lithography (TP-LSL), was shown in this thesis to possess the capability to micropattern multiple different biomolecules at modular concentrations into a single hydrogel microenvironment over a broad range of size scales with high 3D resolution. 3D cellular adhesion and migration were then explored in detail using time-lapse confocal microscopy to follow cells as they migrated along micropatterned tracks of various 3D size and composition. Further, in a valuable modification of TP-LSL, images from the endogenous microenvironment were converted into instructions to precisely direct the laser patterning of biomolecules within PEG-based hydrogels. 3D images of endogenous microvasculature from various tissues were directly converted into 3D biomolecule patterns within the hydrogel scaffold with precise pattern fidelity. While tissue engineers have previously demonstrated the formation of vessels through the encapsulation of endothelial cells and pericyte precursor cells within PEG-based hydrogels, the vessel structure had been random, uncoordinated, and therefore, ultimately non-functional. This thesis has utilized image guided TP-LSL to pattern biomolecules into a 3D structure that directs the organization of vessels to mimic that of the endogenous tissue vasculature. TP-LSL now stands as a valuable tool to control the microstructure of engineered cellular architectures, thereby providing a critical step in the development of cellularized scaffolds into functional tissues. Ultimately, this thesis develops new technologies that advance the field of regenerative medicine towards the goal of engineering viable organs to therapeutically treat the 18 patients who die every day waiting on the organ transplant list.

Vascularization of tissue-engineered substitutes is imperative for successful implantation into sites of injury. Strategies to promote vascularization within tissue-engineered constructs have focused on incorporating endothelial or endothelial progenitor cells within the construct. However, since endothelial and endothelial progenitor cells are adult cell types and limited in number, acquiring quantities needed for regenerative medicine applications is not feasible. Pluriopotent stem cells have been explored as a cell source for tissue-engineered substitutes because of their inherent ability to differentiate into all somatic cell types, including endothelial cells (ECs). Current EC differentiation strategies require laborious and extensive culture periods, utilize large quantities of expensive growth factors and extracellular matrix, and generally yield heterogenous populations for which only a small percentage of the differentiated cells are ECs. In order to recapitulate in vivo embryonic stem cell (ESC) differentiation, 3D stem cell aggregates or embryoid bodies (EBs) have been employed in vitro. In the developing embryo, fluid shear stress, VEGF, and oxygen are instructive cues for endothelial differentiation and vasculogenesis. Thus, the objective of this work was to study the effects of fluid shear stress pre-conditioning of ESCs on EB endothelial differentiation and vasculogensis. The overall hypothesis is that exposing ESCs to fluid shear stress prior to EB differentiation will promote EB endothelial differentiation and vasculogenesis. Pre-conditioning ESCs with fluid shear stress modulated EB differentiation as well as endothelial cell-like cellular organization and EB morphogenesis. To further promote endothelial differentiation, ESCs pre-conditioned with shear were treated with VEGF. Exposing EBs formed from ESCs pre-conditioned with shear to low oxygen resulted in increased production of VEGF and formation of endothelial networks. The results of this work demonstrate the role that physical forces play in modulating stem cell fate and morphogenesis.

The mature central nervous system has a very limited capacity for self-renewal and repair following injury. Neural stem cells (NSCs), however, provide a promising new therapeutic option and can be readily expanded in vitro . Towards the development of an effective therapy, greater understanding and control is needed over the mechanisms regulating the differentiation of these cells into function-restoring neurons. In vivo, the neural stem cell niche plays a critical role in directing stem cell self-renewal and differentiation. By understanding and harnessing the power of this niche, a tissue engineered system with encapsulated neural stem cells could be designed to encourage neuronal differentiation and ultimately regeneration of damaged neural tissue. Poly(ethylene glycol)-based hydrogels were used here as a platform for isolating and investigating the response of neural stem cells to various matrix, soluble, and cellular components of the niche. When covalently modified with a cyclic RGD peptide, the synthetic scaffold was demonstrated to support attachment and proliferation of a human NSC line under conditions permissive to cell growth. Under differentiating conditions, the scaffold maintained appropriate lineage potential of the cells by permitting the development of both neuronal and glial populations. Expansion and differentiation of NSCs was also observed in a more biomimetic, three dimensional environment following encapsulation within a degradable hydrogel material. To simulate the soluble signals in the niche, fibroblast growth factor and nerve growth factor were tethered to the hydrogel and shown to direct NSC proliferation and neuronal differentiation respectively. Finally, as an example of the cell-cell interactions in the niche, the pro-angiogenic capacity of encapsulated neural stem cells was evaluated both in vitro and in vivo. Ideally, the optimal scaffold design will be applied to guide NSCs in a therapeutic application. Toward this goal, a novel method was developed for encapsulation of the cells within injectable hydrogel microspheres. This technique was optimized for high cell viability and microsphere yield and was demonstrated with successful microencapsulation and delivery of neural stem cells in rodent model of ischemic stroke.

A novel injectable hydrogel system for cell delivery in craniofacial bone tissue engineering was developed in this work. The hydrogel employs a dual solidification mechanism by containing units that gel upon temperature increase to physiological temperature and groups that allow for covalent crosslinking. The successful synthesis of macromers for hydrogel fabrication was demonstrated and structure-property relations were established. The hydrophilic-hydrophobic balance of the macromers was found to be an important design criterion towards their resulting thermal gelation properties. When tested with cells in vitro , macromers with different molecular compositions, molecular weights and transition temperatures were all found to be cytocompatible. The introduction of a chemically crosslinkable group in the macromers resulted in hydrogels with improved stability. The effect of the addition of these highly reactive groups on cell viability was evaluated and parameters that enable viable cell encapsulation in the hydrogels were determined. It was shown that there was a dose- and time-dependent effect of the macromers on cell viability. Increased degrees of modification were found to decrease the thermal transition temperature as well as the cytocompatibility of the macromers. Hydrogels were fabricated at physiological temperature upon physical gelation and chemical crosslinking with the addition of a thermal free radical initiator system. The swelling behavior of the hydrogels was characterized and it was found to be controlled by the chemistry of the macromer end group, the concentration of the initiator system used, the fabrication interval as well as the incubation temperature and medium. In order to evaluate the hydrogels as cell carriers, mesenchymal stems cells were encapsulated in the hydrogels over a 21-day period. Cells retained their viability over the duration of the study and exhibited markers of osteogenic differentiation when cultured with appropriate supplements. These findings hold promise for the use of these hydrogel systems for cell encapsulation in tissue engineering applications.

Damage to synovial joints results in osteochondral defects that only heal with inferior fibrous repair tissue. Since mesenchymal stem cells (MSCs) play a vital role in the natural development, maintenance, and repair of cartilage and bone, tissue engineering strategies to enhance functional regeneration by modulating MSC differentiation are a promising alternative to the limitations and potential complications associated with current conventional therapies. In this work, signals present in the native microenvironment were utilized in fabricating polymer/extracellular matrix composite scaffolds to guide chondrogenic and osteogenic differentiation. In an osteochondral defect environment, interactions exist between bone marrow cell populations. Although MSCs have been extensively utilized for their ability to support hematopoietic stem and progenitor cells (HSPCs), the role of HSPCs in regulating the osteogenic differentiation of MSCs in the bone marrow niche is not well understood, and thus was explored via direct contact co-culture. HSPCs in a low dose with sustained osteogenic induction by dexamethasone accelerated osteogenesis and enhanced mineral deposition, whereas the lack of induction signals affected the spatial distribution of cell populations and minerals. Thus, HSPCs presumably play an active role in modulating the development and maintenance of the osteogenic niche. Since physical signals affect cellular activity, flow perfusion culture was employed to deposit mineralized extracellular matrix (ECM) with different maturity and composition on electrospun poly([varepsilon]-caprolactone) (PCL) microfibers in fabricating mineralized PCL/ECM composite scaffolds. The presence of mineralized matrix induced the osteogenic differentiation of MSCs even in the absence of dexamethasone, and a more mature matrix with higher quantities of collagen and minerals improved osteogenesis by accelerating alkaline phosphatase expression and matrix mineralization. To determine whether PCL/ECM scaffolds can be applied to support the chondrogenic differentiation of MSCs, cartilaginous PCL/ECM composite scaffolds were fabricated. The presence of cartilaginous matrix reduced fibroblastic phenotype and in combination with transforming growth factor-β1 (TGF-β1), further promoted chondrogenesis as evident in elevated levels of glycosaminoglycan synthetic activity. While further investigation is necessary to optimize and test these scaffolds to induce the regeneration of cartilage and bone, this work demonstrates the importance of harnessing signals present in the native microenvironment to modulate chondrogenic and osteogenic differentiation.

<p>Articular cartilage is the connective tissue that lines the ends of long bones in diarthrodial joints, providing a low-friction load-bearing surface that can withstand a lifetime of loading cycles under normal conditions. Despite these unique and advantageous properties, the tissue possesses a limited capacity for self-repair due to its lack of vasculature and innervation. Total joint replacement is a well-established treatment for degenerative joint disease; however, the materials used in these procedures have a limited lifespan in vivo and will likely fail over time, requiring additional - and increasingly complicated - revision surgeries. For younger or more active patients, this risk is unacceptable. Unfortunately, alternative surgical options are not currently available, leaving pain management as the only viable treatment. In seeking to discover a new therapeutic strategy, the goal of this dissertation was to develop a functional tissue-engineered cartilage construct that may be used to resurface an entire diseased or damaged joint.</p><p> A three-dimensional (3-D) woven textile structure, produced on a custom-built miniature weaving loom, was utilized as the basis for producing novel composite scaffolds and cartilage tissue constructs that exhibited initial properties similar to those of native articular cartilage. Using polyglycolic acid (PGA) fibers combined with chondrocyte-loaded agarose or fibrin hydrogels, scaffolds were engineered with anisotropic, inhomogeneous, viscoelastic, and nonlinear characteristics prior to cultivation. However, PGA-based constructs showed a rapid loss of mechanical functionality over a 28 day culture period suggesting that the inclusion of other, less degradable, biomaterial fibers could provide more stable properties. </p><p> Retaining the original 3-D architecture and fiber/hydrogel composite construction, poly (epsilon-caprolactone) (PCL)-based scaffolds demonstrated initial biomechanical properties similar to those of PGA-based scaffolds. Long-term culture of 3-D PCL/fibrin scaffolds seeded with human adipose-derived stem cells (ASCs) showed that scaffolds maintained their baseline properties as new, collagen-rich tissue accumulated within the constructs.</p><p> In an attempt to improve the bioactivity of the PCL scaffold and further induce chondrogenic differentiation of seeded ASCs, we produced a hybrid scaffold system by embedding the 3-D woven structure within a porous matrix derived from native cartilage. We then demonstrated how this multifunctional scaffold could be molded, seeded, and cultured in order to produce an anatomically accurate tissue construct with potential for resurfacing the femoral head of a hip. </p><p>In summary, these findings provide valuable insight into a new approach for the functional tissue engineering of articular cartilage. The results of this work will hopefully lead to the discovery of new strategies for the long-term treatment of cartilage pathology.</p> / Dissertation

<p>Transplantation of a functional engineered skeletal muscle substitute is a promising therapeutic option to repair irreversible muscle damage, and, on the other hand, functional muscle tissue constructs can serve as in vitro 3D tissue models that complement the conventional 2D cell cultures and animal models to advance our limited understanding of intrinsic myogenesis and muscle regeneration process. However, the engineering of skeletal muscle constructs with comparable contractile function to the native muscle is hampered by the lack of 1) effective and reproducible methods to form relatively large muscle constructs composed of viable, dense, aligned and matured myofibers, and 2) beneficial microenvironmental cues as well as physiological stimulations that favor the growth, differentiation and maturation of myogenic cells. Thus, in this thesis, I have developed a mesoscopic hydrogel molding approach to fabricate relatively large engineered muscle tissue networks with controllable thickness, pore dimensions, overall myofiber alignment and regional myofiber orientation. I then investigated the effect of variation in pore length on the force generation and passive properties of engineered muscle networks and the potential to improve the contractile function of engineered muscle networks with the treatment of a soluble neurotrophic factor, agrin.</p><p>Specifically, high aspect-ratio soft lithography was utilized to precisely fabricate elastomeric molds containing an array of staggered hexagonal posts which created elliptical pores in muscle tissue sheets made from a mixture of primary skeletal myoblasts, fibrin and Matrigel. The improved oxygen and nutrient access through the pores increased the viability of the embedded muscle cells and prevented the formation of an acellular core. The differentiated myofibers were locally aligned in tissue bundles surrounding the elliptical pores. The length and direction of the microfabricate posts arbitrarily determined the length of elliptical pores and the mean orientation of myofibers formed around the pores, which enables the control of pore dimensions and regional myofiber orientation. Contractile force analysis revealed that engineered muscle networks with more elongated pores generated larger contractile force due to the increased myonuclear density and degree of overall myofiber alignment, despite the larger porosity and reduced tissue volume. Furthermore, the introduction of elliptical pores resulted in distinct deformational changes in tissue bundles and node regions that connect the ends of bundles with the applied unaxial macroscopic stretch, but such spatial alteration of local strain field resulted in no significant change in macroscopic length- tension relationships among engineered muscle networks with different pore length. </p><p>In addition, supplementing culture medium with soluble recombinant agrin significantly increased contractile force production of engineered muscle networks in the absence of nerve-muscle interaction, primarily or partially due to the agrin-induced upregulation of dystrophin. As expected, alteration in the levels endogenous ACh or ACh-like compound affected the agrin-induced AChR aggregation. Furthermore, increased autocrine AChR stimulation, a novel mechanism underlying survival and maturation of aneural myotubes, attenuated the agrin-induced force increase, while suppressed autocrine AChR stimulation severely comproised the overall force production of engineered muscle networks, of which the underlying mechanisms remains to be elucidated in the future studies. </p><p>In summary, a novel tissue engineering methodology that enables the fabrication of relative large muscle tissue constructs with controllable structure and function has been developed in this thesis. Future studies, such as optimizing cell-matrix interaction, incorporating beneficial regulatory proteins in the fibrin-based matrix, and applying specific patterns of electro-mechanical stimulations are expected to further augment the contractile function of engineered muscle networks. The potential application of this versatile tissue fabrication approach to engineer different types of soft tissue might further advance the development of tissue regeneration therapies as well as deepen our understanding of intrinsic tissue morphogenesis and regeneration process.</p> / Dissertation

The biomaterial component of a tissue engineered device has been shown to enhance the immune response to a co-delivered model shed antigen. The purpose of this research was to investigate in vivo the differential level of the immune response toward different forms of the biomaterial. A model shed antigen, ovalbumin (OVA), was incorporated into polymeric biomaterial carriers made of 50:50 poly(lactic-co-glycolic acid) (PLGA) in the form of microparticles (MP) or scaffolds (SC). These MP and SC biomaterial carrier vehicles with incorporated antigen were then injected or implanted, respectively, into C57BL6 mice to investigate the differential level of the immune response towards OVA controlled release from PLGA MP and PLGA SC. For each polymeric carrier, the resulting time-dependent systemic humoral immune response towards the incorporated OVA, the OVA-specific IgG concentration and isotypes (IgG2a or IgG1, indicating a predominant Th1 or Th2 response, respectively) were determined using ELISA. To assess the differential level of the immune response depending on the form of PLGA, the total amounts of polymer and OVA delivered were kept constant as well as the release rate of OVA. The in vitro protein release kinetics were studied for both PLGA MPs and PLGA scaffolds to examine the release rate of OVA from the polymeric carriers. The level of the humoral immune response was higher and sustained for OVA released from PLGA SC which were implanted with associated tissue damage, and lower and transient when the same amount of polymer and OVA were delivered from PLGA MP, which were minimally invasively delivered by injection. This immune response was primarily Th2 helper T cell-dependent as exemplified by the predominance of IgG1 isotype, although for the strong adjuvant, Complete Freunds adjuvant (CFA), and PLGA SC carriers the anti-OVA IgG2a isotype levels were also significant, potentially indicating both a Th2 and Th1 response. The PLGA SC and PLGA MP exhibited similar protein release kinetics, releasing similar amounts of OVA at each time point. Each carrier incubated contained the same ratio of OVA to polymer. In vitro protein release kinetics experiments suggest that the rate of release of OVA from PLGA SC and PLGA MP was similar, and therefore the enhanced immune response induced by PLGA SC is most likely due to danger signals from implantation which primed the system for an enhanced immune response and not from a difference in concentration of OVA released from the carriers.

Cardiac valves are dynamic, sophisticated structures which interact closely with the surrounding hemodynamic environment. Altered mechanical stresses, including pressure, shear and bending stresses, are believed to cause changes in valve biology, but the cellular and molecular events involved in these processes are not well characterized. Therefore, the overall goal of this project is to determine the effects of pressure and shear stress on porcine aortic valve leaflets biology. Results from the pressure study showed that elevated constant pressure (140 and 170 mmHg) causes significant increases in collagen synthesis. The increases were 37.5% and 90% for 140 and 170 mmHg, respectively. No significant differences in DNA and sGAG synthesis were observed under constant pressure. In the cyclic pressure study, the effects of both pressure magnitude and pulse frequency were studied. With the frequency fixed at 1.167 Hz, collagen and sGAG synthesis increased proportionally with mean pressure level. At a fixed pressure level (80-120 mmHg), collagen and sGAG synthesis were slightly increased by 25% and 14% at 0.5 Hz, respectively. DNA synthesis was significantly increased by 72% at 2 Hz. An experiment combining high magnitude (150-190 mmHg) and high frequency (2 Hz) demonstrated significant increases in collagen and sGAG synthesis (collagen: 74%, sGAG: 56%), but no significant changes in cell proliferation. Shear levels ranging from 1 to 80 dyne/cm2 were studied. Scanning electron microscopy results indicated that 48 hrs exposure to shear stress did not alter the circumferential alignment of endothelial cells. Collagen synthesis was significantly enhanced at 9 and 25 dyne/cm2, but not different from static controls under other shear conditions. Leaflets denuded of the endothelium were exposed to identical shear stress and showed very different responses. Collagen synthesis was not affected at any shear levels, but sGAG content was increased at shear of 9, 25 and 40 dyne/cm2. Further studies showed that the increases in collagen synthesis under pressure or shear stress was concurrent with a decline in the expression and activities of cathepsins L and S. This converse relationship between collagen synthesis and cathepsin activity indicated that cathepsins might be involved in valvular ECM remodeling.