Scientific research is propelled by two objectives: Understanding and recognizing the essential biology of life, and deciphering this to uncover possible therapeutics in order to improve quality of life as well as relieve pain from disease. The aim of the work described in this thesis was to dissect the fundamental requirements of induced pluripotent stem cells both in pluripotency and differentiation with a particular focus on atrial specificity. Drug targeting of atrial-specific ion channels has been difficult because of lack of availability of appropriate cardiac cells, and preclinical testing studies have been carried out in non-cardiac cell lines, heterogeneous cardiac populations or animal models that have been unable to accurately represent human cardiomyocyte physiology. Therefore, we sought out to develop a preparation of cardiomyocytes showing an atrial phenotype with adult characteristics from human induced-pluripotent stem cells. A culture programme involving the use of Gremlin 2 allowed differentiation of cardiomyocytes with an atrial phenotype from human induced-pluripotent stem cells. When these differentiated cultures were dissociated into single myocytes a substantial fraction of cells showed a rod-shaped morphology with a single central nucleus that was broadly similar to that observed in cells isolated from atrial chambers of the heart. Immunolabelling of these myocytes for cardiac proteins (including RyR2 receptors, actinin-2, F-actin) showed striations with a sarcomere spacing of slightly less than 2um. The isolated rod-shaped cells were electrically quiescent unless stimulated to fire action potentials with an amplitude of 100 mV from a resting potential of approximately -70 mV. Proteins expressed included those for IK₁, IK_ur channels. Ca²⁺ Transients recorded from spontaneously beating cultures showed increases in amplitude in response to stimulation of adrenoceptors (both alpha and beta). With the aim of identifying key signaling mechanisms in directing cell fate, our new protocol allowed differentiation of human myocytes with an atrial phenotype and adult characteristics that show functional adrenoceptor signaling pathways and are suitable for investigation of drug effects.

For small diameter (<6 mm) blood vessel replacements, lack of collaterals and vascular disease preclude homografts; while synthetic analogs, ePTFE, expanded polytetrafluoroethylene, and PET, polyethyleneterephathalate, are prone to acute thrombosis and restenosis. It is postulated that the hierarchical assembly of cell populated matrices fabricated from protein analogs provides a new design strategy for generating a structurally viable tissue engineered vascular graft. To this end, synthetic elastin and collagen fiber analogs offer a novel strategy for creating tissue engineered vascular grafts with mechanical and biological properties that match or exceed those of native vessels. This work details techniques developed for the fabrication of prosthetic vascular grafts from a series of extracellular matrix analogs composed of nanofibrous collagen matrices and elastin-mimetic proteins, with and without cells, and subsequent evaluation of their biocompatibility and mechanical properties. The work details the fabrication and mechanical analysis of vascular grafts made from aforementioned protein analogs. Subesequent studies detail seeding and proliferation of rodent mesenchymal stem cells on protein-based composites to recapitulate the media of native vasculature. Finally detailing in vivo biocompatibility and stability of tissue engineered vascular grafts.

Tissue engineering has emerged as a promising alternative to conventional orthopaedic grafting therapies. The general paradigm for this approach, in which phenotype-specific cells and/or bioactive growth factors are integrated into polymeric matrices, has been successfully applied in recent years toward the development of bone, ligament, and cartilage tissues in vitro and in vivo. Despite these advances, an optimal cell source for skeletal tissue repair and regeneration has not been identified. Furthermore, the lack of robust, functional orthopaedic tissue interfaces, such as the bone-ligament enthesis, severely limits the integration and biological performance of engineered tissue substitutes. This works aims to address these limitations by spatially controlling the genetic modification and differentiation of fibroblasts into a mineralizing osteoblastic phenotype within three-dimensional polymeric matrices. The overall objective of this project was to investigate transcription factor-based gene therapy strategies for the differentiation of fibroblasts into a mineralizing cell source for orthopaedic tissue engineering applications. Our central hypothesis was that fibroblasts genetically engineered to express Runx2 via conventional and biomaterial-mediated ex vivo gene transfer approaches will differentiate into a mineralizing osteoblastic phenotype. We have demonstrated that a combination of retroviral Runx2 overexpression and glucocorticoid hormone treatment synergistically induces osteoblastic differentiation and biological mineral deposition in primary dermal fibroblasts cultured in monolayer. We report for the first time that glucocorticoids induce osteoblastic differentiation in this model system by modulating the phosphorylation state of a negative regulatory serine residue (Ser125) on Runx2 through an MKP-1-dependent mechanism. Furthermore, we utilized these Runx2-genetically engineered fibroblasts to create mineralized templates for bone repair in vitro and in vivo. Finally, we engineered a heterogeneous bone-soft tissue interface with a novel biomaterial-mediated gene transfer approach. Overall, these results are significant toward the ultimate goal of regenerating complex, higher-order orthopaedic grafting templates which mimic the cellular and microstructural characteristics of native tissue. Cellular therapies based on primary dermal fibroblasts would be particularly beneficial for patients with a compromised ability to recruit progenitors to the sight of injury as result of traumatic injury, radiation treatment, or osteodegenerative disease.

Biomimetic materials that recapitulate the complex mechanical and biochemical cues in load-bearing tissues are of significant interest in regenerative medicine and tissue engineering applications. Several investigators have endeavored to not only emulate the mechanical properties of the vasculature, but to also mimic the biologic responsiveness of the blood vessel in creating vascular substitutes. Previous studies in our lab generated the elastin-like protein polymer LysB10, which was designed with the capability of physical and chemical crosslinks, and was shown to display a range of elastomeric properties that more closely matched those of the native artery. While extensive validation of the mechanical properties of elastin-mimetic polymers has demonstrated their functionality in a number of tissue engineering applications, limited cell growth on the surfaces of the polymers has motivated further optimization for biological interaction. Recent biologically-inspired surface strategies have focused on functionalizing material surfaces with extracellular matrix molecules and bioactive motifs in order to encourage integrin-mediated cellular responses that trigger precise intracellular signaling processes, while limiting nonspecific biomaterial interactions. Consequently, this dissertation addresses three approaches to modulating cellular behavior on elastin-mimetic analogs with the goal of promoting vascular wall healing and tissue regeneration: genetic engineering of elastin-like protein polymers (ELPs) with cell-binding domains, biofunctionalization of elastin-like protein polymers via chemoselective ligation of bioactive ligands, and incorporation of matrix protein fibronectin for engineering of cell-seeded multilamellar collagen-reinforced elastin-like constructs. The synthesis of recombinant elastin-like protein polymers that integrate biologic functions of the extracellular matrix provides a novel design strategy for generating clinically durable vascular substitutes. Ultimately, the synthesis of model protein networks provides new insights into the relationship between molecular architecture, biomimetic ligand presentation, and associated cellular responses at the cell-material interface. Understanding how each of these design parameters affects cell response will contribute significantly to the rational engineering of bioactive materials. Potential applications for polymer blends with enhanced mechanical and biological properties include surface coatings on vascular grafts and stents, as well as composite materials for tissue engineered scaffolds and vascular substitutes.

The interface between tendon/ligament and bone tissue is a complex transition of biochemical, cellular, and mechanical properties. Investigating computational and tissue engineering models that imitate aspects of this interface may supply critical design parameters for designing future tissue replacements to promote increased biochemical and mechanical integration between tendon/ligament and bone. Strategies for modeling this tissue have typically focused on the development of heterogeneous structures to create gradients or multiphasic materials that mimic aspects of the transition. However, further work is required to elucidate the role of specific mechanical and material stimuli in recapitulating features of the tendon/ligament-bone insertion. In particular, in constructs that exhibit variation in both mechanical and biochemical properties, the interplay of mechanical, material, and chemical signals can complicate understanding of the particular factors at work in interface formation. Thus, the overall goal of this dissertation was to provide insight into the role of mechanical strain and scaffold degradability on cell behavior within heterogeneous biomaterials. Specifically, a method for determining cell vertical position within a degradable gel through a laminated interface was developed. A computational model was created to examine possible variation in local mechanical strain due to heterogeneity in mechanical properties and different interface geometries. Finally, the influence of biomaterial degradability on changes in encapsulated human mesenchymal stem cell morphology under response to cyclic mechanical strain was explored. Together, these studies provide insight into mechanical and material design considerations when devising tissue engineering strategies to regenerate the tendon/ligament-bone interface.

This research centers on the fabrication, characterization, and engineering of inverse opal scaffolds, a novel class of three-dimensional (3D) porous scaffolds made of biocompatible and biodegradable polymers, for applications in tissue engineering and regenerative medicine. The unique features of an inverse opal scaffold include a highly ordered array of pores, uniform and finely tunable pore sizes, high interconnectivity, and great reproducibility. The first part of this work focuses on the fabrication and functionalization of inverse opal scaffolds based on poly(D,L-lactic-co-glycolic acid) (PLGA), a biodegradable material approved by the U.S. Food and Drug Administration (FDA). The advantages of the PLGA inverse opal scaffolds are also demonstrated by comparing with their counterparts with spherical but non-uniform pores and poor interconnectivity. The second part of this work shows two examples where the PLGA inverse opal scaffolds were successfully used as a well-defined system to investigate the effect of pore size of a 3D porous scaffold on the behavior of cell and tissue growth. Specifically, I have demonstrated that i) the differentiation of progenitor cells in vitro was dependent on the pore size of PLGA-based scaffolds and the behavior of the cells was determined by the size of individual pores where the cells resided in, and ii) the neovascularization process in vivo could be directly manipulated by controlling a combination of pore and window sizes when they were applied to a mouse model. The last part of this work deals with the novel application of photoacoustic microscopy (PAM), a volumetric imaging modality recently developed, to tissue engineering and regenerative medicine, in the context of non-invasive imaging and quantification of cells and tissues grown in PLGA inverse opal scaffolds, both in vitro and in vivo. Furthermore, the capability of PAM to monitor and quantitatively analyze the degradation of the scaffolds themselves was also demonstrated.

Severe extremity trauma often involves significant damage to multiple tissue types, including bones, skeletal muscles, peripheral nerves, and blood vessels. Such injuries present unique challenges for reconstruction, and improving structural and functional outcomes of intervention remains a pressing, unmet clinical need. While tissue engineering/regenerative medicine (TE/RM) therapeutics offer promising potential to overcome the status quo limitations of surgical reconstruction, very few products have transitioned to clinical practice. Improving treatment options will likely require advancing our understanding of the biological interactions occurring in the repair of damaged tissues. Bone tissue is known to be innervated and highly vascularized, and both tissue types are involved in normal bone physiology. However, the degree to which these tissue relationships influence the repair of large, multi-tissue defects remains unknown. Accordingly, the goal of this thesis was to investigate tissue regeneration in two novel composite injury models. First, we characterized interactions in a composite bone and nerve injury model where a segmental bone defect was combined with a peripheral nerve gap. Our results indicated that although tissue regeneration was not impaired, the composite injury group experienced a marked functional deficit in the operated limb compared to single-tissue injury. Second, we developed a model of composite bone and vascular extremity trauma by combining a critically-sized segmental bone defect with surgically-induced hind limb ischemia to evaluate the effects on BMP-2-mediated bone repair. Interestingly, our results demonstrated a stimulatory effect of the recovery response to ischemia on bone regeneration. Finally, we investigated early vascular growth and gene expression as potential mechanisms coupling the response to ischemia with bone defect repair. Although the response to ischemia promoted robust vascular growth in the thigh, it did not directly augment vascularization at the site of bone regeneration. In addition, the stimulatory effects of ischemia on bone regeneration could not be explained by gene expression alone based on the genes and time points investigated. Taken together, this thesis presents pioneering work on a new thrust of TE/RM research – tissue regeneration in models of composite injury. This work has provided new insights on the complexity of composite tissue repair, specifically in regard to the relationship between vascular tissue growth and bone healing. Going forward, successful leverage of models of composite tissue injuries will provide valuable test beds for screening new technologies, advance the understanding of tissue repair biology, and ultimately, may produce new therapeutic interventions for limb salvage and reconstruction that improve outcomes for extremity trauma patients.

Our objective was to address current cell source limitations in engineering pancreatic â-cells for the treatment of type 1 diabetes by investigating retroviral genetic modification of murine embryonic stem cells (mESC) with a murine stem cell virus (MSCV) encoding proendocrine transcription factor Neurogenin 3 (Ngn3). We found that expression of Ngn3 and the enhanced green fluorescent protein (eGFP) reporter gene were both significantly silenced in genetically modified mESCs. To overcome this obstacle and enhance the efficiency of retroviral gene transfer to mESCs in general, we employed a virus-polymer complexation method to deliver more transgenes to mESCs. Despite increased transgene delivery and integration in mESCs, transgene expression did not increase. Results suggest mESCs may be restricted in several steps of retrovirus transduction. We then investigated which steps of the virus lifecycle restrict efficient transduction of mESCs by using a recombinant MMuLV-derived retrovirus and a recombinant HIV-1-derived lentivirus to compare three major steps in the transduction of mESCs and NIH 3T3 cells - virus binding, virus integration, and transgene expression. We found that retroviruses and lentiviruses similarly bind 3 or 4-fold less efficiently to R1 mES cells than to NIH 3T3 fibroblasts. We also detected 3-fold fewer integrated retrovirus transgenes and 11-fold lower expression levels in NIH 3T3 cells, suggesting the primary limitation to retrovirus transduction may be low levels of transgene expression. In contrast we detected 10-fold fewer integrated lentivirus transgenes and 8-fold lower expression levels, suggesting lentivirus transduction may be limited by inefficient intracellular post-binding steps of transduction. We then investigated whether depletion of linker histone 1 in mESCs would alleviate silencing of retrovirus transgenes and improve gene transfer by transducing histone H1c, H1d, H1e triple null mESCs with different recombinant vectors. We found this did not improve viral gene transfer. This research is significant for improving protocols for gene transfer to ES cells and facilitating the use of modified ES cells in regenerative medicine.

Thesis (Ph.D)--University of Tennessee Health Science Center, 2007. / Title from title page screen (viewed on October 8, 2007). Research advisor: Joo L. Ong, Ph.D. Document formatted into pages (xiii, 128 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 106-114).

Les premières utilisations du tissu adipeux comme produit de comblement en chirurgie plastique remontent à la fin du 19ème siècle. Depuis quelques décennies, la greffe de tissu adipeux a bénéficié d'un regain d'intérêt utilisant un procédé chirurgical rigoureux. Devant la démonstration de la survie cellulaire et les bons résultats cliniques obtenus, l'utilisation de cette technique s'est élargie à tous les domaines de la chirurgie plastique. Cette technique est simple et efficace et représente actuellement le meilleur moyen de restaurer les défauts de contours et de volume. Récemment, de nouvelles indications utilisant les capacitésrégénératrices du tissu adipeux ont été décrites. Elles concernent la cicatrisation des plaies chroniques et l'amélioration des dystrophies cutanées. Mais la limite de la greffe de tissu adipeux est l'absence de site donneur disponible au prélèvement. Le tissu adipeux est aujourd'hui reconnu comme la source la plus abondante de cellules souches mésenchymateuses multipotentes. Cela a donné un nouvel essor à la médecine régénérative pour réparer, remplacer ou régénérer les tissus et organes endommagés à partirdes cellules souches. Cette régénération se fait soit in-situ après administration des cellules souches, soit après développement in-vitro d'un tissu par ingénierie. Après une présentation du tissu adipeux et ses cellules souches ainsi que leurs applications actuelles en chirurgie plastique, le but de ce travail était de : 1. de clarifier les facteurs influençant les résultats de la greffe adipeuse pour une optimisation de cette technique. 2. d'explorer les possibilités offertes par les cellules souches adipeuses pour la médecine régénérative et l'ingénierie tissulaire, en vue d'une utilisation en chirurgie plastique / The first uses of adipose tissue as filler in plastic surgery started in the late 19th century. In recent decades, the adipose tissue transplantation has received renewed interest using a rigorous surgical procedure. Before the demonstration of cell survival and good clinical results, the use of this technique was extended to all areas of plastic surgery. This technique is simple and effective and is currently the best way to restore the defects of contour and volume. Recently, new indications using the regenerative capacity of adipose tissue have been described. They concern the healing of chronic wounds and the improvement of skin dystrophy. But the limit of the adipose tissue graft is the lack of available donor site for harvesting. Adipose tissue is now recognized as the most abundant source of multipotent mesenchymal stem cells. This gave a boost to regenerative medicine to repair, replace or regenerate damaged tissues and organs from stem cells. This regeneration is done either in-situ after administration of stem cells, after in-vitro development of tissue engineered. After a presentation of adipose tissue and stem cells and their current applications in plastic surgery, the aim of this study was to: 1. clarify the factors influencing the results of fat transplantation to optimize this technique. 2. explore the possibilities offered by ASCs for regenerative medicine and tissue engineering, for use in plastic surgery