The conventional treatments for craniofacial bone defects currently are unsatisfactory due to several drawbacks. Replacement of lost bone by autografts typically causes donor site morbidity while allografts, xenografts, and demineralized bone matrix all have a chance of disease transmission. Current synthetic implants placed within the defect site generally lack osseointegration and biodegradability. There are several methods of generating a hybrid extracellular matrix (ECM) and synthetic material construct. These include coating the synthetic material scaffold with collagen and calcium phosphate, incorporating acellular biological tissue within the scaffold material, and using cells to generate an ECM coating on the synthetic material scaffold. The research performed for this thesis developed and characterized mesenchymal stem cell (MSC)-generated extracellular matrix poly(ε-caprolactone) constructs (PCL/ECM) for the replacement of bone tissue. The osteogenic potential of the PCL/ECM constructs was explored by culturing i) MSCs and ii) whole marrow cells combined with MSCs onto the construct with or without the osteogenic differentiation supplement, dexamethasone. It was established that the osteogenic differentiation of MSCs seeded onto ECM-containing constructs was maintained even in the absence of dexamethasone and that the co-culture of MSCs and whole bone marrow cells without dexamethasone and ECM enhances the proliferation of a cell population (or populations) present in the whole bone marrow. The osteogenicity of the constructs encouraged the characterization of the protein and mineral composition of the ECM coating on the PCL/ECM constructs. Characterization revealed that at short culture durations the MSCs used to generate the ECM deposited cellular adhesion proteins that are a prerequisite protein network for further bone formation. At the later culture durations, it was determined that the ECM was composed of collagen 1, hydroxyapatite, matrix remodeling proteins, and regulatory proteins. The prior studies on the PCL/ECM constructs persuaded exploration of the effect of various devitalization and demineralization processes on the retention of the ECM components within and the osteogenicity of the PCL/ECM constructs. Analysis demonstrated that the freeze-thaw technique is a milder method of devitalization of cell-generated ECM constructs as compared to other methods, but it reduced the osteogenicity of the constructs. In addition, it was elucidated that void spaces in the surface of the constructs are important for allowing access of MSCs into the interior of the constructs.

The traditional view of cancer asserts that a malignant tumour is composed of a population of cells, all of which share the ability to divide without limit. Within the last decade, however, this notion has lost ground to the emerging cancer stem cell hypothesis, which counters that only a (typically small) sub-population of so-called `cancer stem cells' has the capacity to proliferate indefinitely, and hence to drive and maintain tumour growth. Cancer stem cells have been putatively identified in leukemias and, more recently, in a variety of solid tumours including those of the breast and brain. The cancer stem cell hypothesis helps to explain certain clinically-observed phenomena, including the apparent inability of conventional anti-cancer therapies to eradicate the disease despite (transient) reduction of overall tumour bulk -- presumably these treatments fail to kill the underlying cancer stem cells. Herein, we develop stochastic and deterministic temporal models of tumour growth based on the cancer stem cell hypothesis, and apply these models to discussions of the treatment of glioblastoma multiforme, a common type of brain cancer believed to be maintained by cancer stem cells, and to the phenomenon of the epithelial-mesenchymal transition, a process thought to be important in generating cancer cells capable of metastasis.

The traditional view of cancer asserts that a malignant tumour is composed of a population of cells, all of which share the ability to divide without limit. Within the last decade, however, this notion has lost ground to the emerging cancer stem cell hypothesis, which counters that only a (typically small) sub-population of so-called `cancer stem cells' has the capacity to proliferate indefinitely, and hence to drive and maintain tumour growth. Cancer stem cells have been putatively identified in leukemias and, more recently, in a variety of solid tumours including those of the breast and brain. The cancer stem cell hypothesis helps to explain certain clinically-observed phenomena, including the apparent inability of conventional anti-cancer therapies to eradicate the disease despite (transient) reduction of overall tumour bulk -- presumably these treatments fail to kill the underlying cancer stem cells. Herein, we develop stochastic and deterministic temporal models of tumour growth based on the cancer stem cell hypothesis, and apply these models to discussions of the treatment of glioblastoma multiforme, a common type of brain cancer believed to be maintained by cancer stem cells, and to the phenomenon of the epithelial-mesenchymal transition, a process thought to be important in generating cancer cells capable of metastasis.

<p>Vertebrates like urodele and teleost have an enhanced capacity for regeneration, when compared to mammals. Recently, the teleost zebrafish (Danio rerio) has become a popular model for studying regenerative events, due to the ability to regenerate multiple organs such as the fin and the heart, and the diverse genetic approaches available for functional studies. In my thesis studies, I have used the zebrafish caudal fin as a model system to understand molecular and cellular mechanism of appendage regeneration. </p><p>Pharmacological and genetic studies have revealed that Fgf signaling is important for appendage regeneration. To dissect the mechanism of Fgfs during zebrafish fin regeneration, lab colleagues and I have generated and utilized transgenic animals in which Fgf signaling can be experimentally increased or decreased. Through these transgenic studies, I found that position-dependent Fgf signaling directs regenerative growth and blastemal proliferation. Proximally-amputated fin regenerates grow at higher rates than the distally-amputated, owing to position-dependent amounts of Fgf activity. Further studies using new transgenics have provided an understanding of mechanisms by which Fgfs influence epidermal regulation of the blastema. Loss- and gain-of-function studies of Fgfs reveal that Fgf signaling both positively and negatively regulated shh expression in the epidermis to maintain blastemal function.</p><p>During the fin regeneration process, pigmentation pattern is re-established as along with bone structures and connective tissues. While the lineage of the blastema is not precisely clear, pigment cells in the fin regenerates are thought to be derived from melanocyte stem cells. Therefore, melanocyte regeneration is an informative system to understand the mechanism underlying regulation of adult stem cells during regeneration. As part of my thesis studies, we generated transgenic animals in which ectopic Ras expression can be experimentally induced. Transgenic studies, combined with pharmacological approaches, have revealed that Ras controls self-renewal of melanocyte stem cells during fin pigment regeneration.</p> / Dissertation

Bone grafts are used to treat more than 300,000 fracture patients yearly, as well as patients with congenital defects, bone tumors, and those undergoing spinal fusion. Given the established limitations of autograft and allograft bone, there is a substantial need for bone graft substitutes. Tissue engineering strategies employing the addition of osteogenic cells and/or osteoinductive factors to porous scaffolds represent a promising alternative to traditional bone grafts. While many bone defects are in load-bearing sites, very little is known about the response of bone grafts and their substitutes to mechanical loading, despite vast documentation on the ability of normal bone to adapt to its mechanical environment. The goal of this research was to quantify the effects of controlled in vivo mechanical stimulation on bone graft repair and bone graft substitutes and identify the local stress/strain environment associated with load-induced changes in bone formation. The global hypothesis that cyclic in vivo mechanical loading improves mineralized matrix formation within bone grafts and bone graft substitutes was addressed in this work using orthotopic and ectopic models specifically designed to facilitate modeling of local stresses and strains. In the first study, a bone defect repair model utilizing an orthotopic implant capable of supplying a controlled mechanical stimulus to a trabecular allograft showed a significant reduction in new bone formation with controlled in vivo mechanical loading. Although the reason remains unclear, loading conditions may not have been ideal for increased bone formation or potential micromotion may have influenced the results. A second study demonstrated for the first time that controlled in vivo mechanical stimulation enhances mineralized matrix production on a mesenchymal stem cell-seeded polymeric construct using a novel subcutaneous implant system. In addition, the local stresses and strains associated with this adaptive response were predicted. The novel subcutaneous implant represents technology which may be adapted for the preparation of tissue-engineered bone constructs, capitalizing on the benefits of mechanical loading and a vascularized in vivo environment. Such an approach may produce larger, stronger, and more homogeneous constructs than could be developed in a static culture system subject to diffusional limitations.

The growth plate is a cartilaginous tissue responsible for the longitudinal growth of long bones. It is a complex tissue composed of chondrocytes whose maturation and proliferation is tightly regulated by a biochemical feedback loop. Injury to this tissue can result in a limb length discrepancy or angular deformity that may lead to life long disability. Given the recent rise in the number of growth plate injuries and the variability in success of current therapies, there is a significant need for a greater understanding of growth plate injury pathology and the development of improved treatment strategies. Cartilage tissue engineering strategies offer an attractive alternative to regenerating growth plate tissue and restoring growth function. Bone marrow-derived stem cells (BMSCs) have been shown to be able to undergo chondrogenic differentiation and in vitro and in vivo and therefore offers an appealing and abundant cell resource for developing tissue engineering strategies for the treatment of growth plate defects. However, the dependence of chondrogenic differentiation and matrix accumulation on monolayer expansion protocols and three-dimensional (3D) culture environment has received little attention. Prior to developing treatment strategies for growth plate injury repair, it is essential to first understand the interconnection between alterations in growth plate morphology and subsequent limb deformities. To that end, we have established a surgical defect model of growth plate injury in Sprague Dawley rats and developed a novel technique to quantitatively monitor growth plate morphology in health and disease using microcomputed tomography (micro-CT) imaging. In an effort to develop a tissue engineering treatment strategy for growth plate injury, the role of monolayer expansion, 3D scaffold, and growth factor regimen in the chondrogenic differentiation of rat BMSCs was also examined. This research study has demonstrated the utility of micro-CT as a non-invasive imaging modality for assessing growth plate injury and repair. This work has also provided an improved understanding of the interrelationship of monolayer expansion, 3D culture environment, and growth factor regimen in BMSC chondrogenic differentiation. Finally, this work suggests that an injectable in situ gelling hydrogel is a feasible method for decreasing limb length discrepancies, however, neither implantation of agarose alone into the defect nor the inclusion of BMSCs fully corrects growth disruption.

Cartilage tissue engineering represents an exciting potential therapy for providing permanent and functional regeneration of healthy cartilage tissues, but these treatment options have yet to be successfully implemented in a clinical setting. One of the primary obstacles for cartilage engineering is obtaining a sufficient supply of cells capable of regenerating a functional cartilage matrix. Mesenchymal progenitors can easily be isolated from multiple tissues, expanded in vitro, and possess a chondrogenic potential, but it remains unclear what types or combinations of signals are required for lineage-specific differentiation and tissue maturation. The overall goal of this dissertation was to investigate how the coordination of biochemical stimuli with cues from mechanical forces and the extracellular matrix regulate the chondrogenesis of bone marrow stromal cells (BMSCs). These studies explored the potential for cyclic tensile loading and chondrogenic factors, TGF-1 and dexamethsone, to promote fibrochondrocyte-specific differentiation of BMSCs. The application of cyclic tensile displacements to cell-seeded fibrin constructs promoted fibrochondrocyte patterns of gene expression and the development of a fibrocartilage-like matrix. These responses were influenced by the specific loading conditions examined and the differentiation state of the BMSCs. Additionally, the roles of integrin adhesion and cytoskeletal organization in BMSC differentiation were examined within engineered hydrogels presenting controlled densities of biomimetic ligands. Adhesion to the arginine-glycine-aspartic acid (RGD) motif inhibited chondrogenesis in a density-dependent manner and was influenced by interactions with the f-actin cytoskeleton. Together, this research provided fundamental insights into the regulatory mechanisms involved in the chondrogenesis of mesenchymal progenitor cells.

Mesenchymal stem cells are more and more widely used in tissue engineering due to their pluripotency and no relative ethical problems. Traditional characterization techniques to detect mesenchymal stem cell states include flow cytometry, gene expressing profiling and immunohistochemistry. However, these methods can only provide transient and low level information from current RNA or protein levels about mesenchymal stem cells, which may cause problems when predicting the possible downstream lineages they will commit into. We have developed chromatin immunoprecipitation (ChIP)-based epigenetic technique to detect mesenchymal stem cell states. For the systems we tested, this epigenetic assessor successfully characterized cell state changes and gave similar results obtained from gene expression profiling or protein expression assay. This epigenetic technique can provide information about mesenchymal stem cells states from a more fundamental chromatin level, which is promising for predicting future lineages from current states.

Numerous intra and extra cellular factors take role in differentiation of cell towards a given lineage. These factors have crucial role in cell-cell and cell-environment interactions. In this study, the aim is to investigate the effect of mechanical forces on the adipogenic differentiation of preadipocytes and mesenchymal stem cells in an in vitro model. Human preadipocytes and mesenchymal stem cells were embedded in 2 % agarose discs. According to the stress-relaxation test results it was observed that initial mechanical properties of agarose-mesenchymal stem cell (MSC) discs did not change compared to acellular agarose whereas those of preadipocytes decreased significantly. The discs with cells were exposed to compression under different weights (1.4 &plusmn / 0.2 g, 7.5 &plusmn / 0.2 g, and 14.6 &plusmn / 0.3 g.) continuously in differentiation medium for 21 days. The control discs were treated with differentiation medium without any compressive weight on top of them. After 21 days, total ribonucleic acids (RNA) have been isolated. Adipogenic differentiation was investigated via reverse transcription coupled quantitative polymerase chain reaction (PCR). The expression of peroxisome proliferators-activated receptors (PPAR-gamma), CCAAT-enhancer binding protein (C/EBP-Beta), leptin, adiponectin, adipophilin and human stearoyl-CoA desaturase (hSCD) have been assessed as adipogenic markers. Differentiation to adipocytes has been further investigated by histochemical Sudan IV staining and immunochemistry and compared to control group. Decrease in the expression of adipogenic factors, size and number of lipid droplets were observed for both MSCs and preadipocytes subjected to compression in agarose discs. The decreases were correlated with the level of mechanical stress. The highest depletion of gene expression was observed in leptin and C/EBP&amp / #61538 / . From our results, it was shown for the first time that mechanical stress impaired the adipogenic differentiation of MSCs and preadipocytes in agarose discs. However, the differentiation pathways should be further investigated.

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