In the United States, nearly half a million bone graft operations are performed annually to repair defects arising from birth defects, trauma, and disease, making bone the second most transplanted tissue. Autogenous bone is the current gold standard for bone grafts; however it is in limited supply and results in a second injury at the donor site. A promising alternative is a tissue engineered bone graft composed of a biomaterial scaffold, pharmaceutics, and osteoprogenitor cells. One source of osteoprogenitor cells is bone marrow stroma, which can be obtained from the patient - minimizing the risk of an immune response - directed in vitro to proliferate, and differentiate into a bone-like tissue. To date, tissue engineered bone grafts have not been clinically effective; thus, strategies must be developed to improve efficacy. I hypothesize that to facilitate tissue healing in a manner similar to autogenous bone tissue engineering bone must possess a mineralized collagen matrix to support tissue integration, and angiogenic factors to stimulate vascular infiltration, and osteogenic factors to direct normal bone remodeling. I propose that these factors can be synthesized by osteoprogenitor cells in vitro when cultured under the appropriate conditions. Previous work has demonstrated that perfusion culture of osteoprogenitor cells within 3D scaffolds stimulates phenotypic markers of osteoblastic differentiation, but those studies did not determine whether the effects were a consequence of shear stress or increased nutrient availability. Consequently, this work has involved studies in a planar geometry, where nutrient effects are negligible. Three studies that characterize the effect of fluid flow on osteoblastic differentiation of osteoprogenitor cells are presented here. The objective of the first study was to determine the effect of shear stress magnitude on cell density and osteocalcin deposition. In this study, radial flow chambers were used to generate a spatially dependent range of shear stresses (0.36 to 2.7 dynes/cm2) across single substrates, and immunofluorescent techniques were used to assay cell phenotype as a function of shear stress. The objective of the second study was to determine the effect of the duration of fluid flow on cell density and phenotypic markers of differentiation. Here, parallel plate flow chambers were used to generate a single shear stress at the cell surface, and entire cell layers were assayed for expression of osteoblastic genes. The objective of the third study was to compare continuous and intermittent fluid flow strategies. In this study, a microprocessor-controlled actuator was added to the flow loop to periodically halt flow, and markers of mechanosensation and osteoblastic differentiation were measured. These studies demonstrated that shear stresses of 0.36 to 2.7 dynes/cm2 stimulate late phenotypic markers of osteoblastic differentiation but not cell proliferation. In addition, this osteogenic effect is sensitive to duration of fluid flow but insensitive to the magnitude of shear stress. Further, intermittent fluid flow enhances cell retention, biochemical markers of mechanotransduction, and synthesis of the angiogenic factor vascular endothelial growth factor (VEGF). Thus, these studies suggest that intermittent fluid flow may be an attractive component of a biodynamic bioreactor for in vitro manufacture of clinically effective tissue engineered bone grafts. Future studies will further investigate intermittent fluid flow strategies and three-dimensional studies with scaffolds suitable for bone tissue engineering. / Ph. D.

Bone is the second most transplanted tissue after blood and the need for bone graft materials continues to rise at an average annual growth rate of over 18%. An engineered bone substitute consisting of a bone-like extracellular matrix deposited on the internal pores of a resorbable biomaterial scaffold is postulated to stimulate normal bone remodeling when implanted in vivo. Part one of this engineering strategy, the deposition of bone-like extracellular matrix, can be achieved by the directed differentiation of progenitor cells such as bone marrow stromal cells (BMSCs). Part two of the engineering strategy, the biomaterial scaffold, can be fabricated with the appropriate mechanical properties using a synthetic polymer system with tunable properties like polyurethanes. Finally, BMSCs seeded within the biomaterial scaffold can be cultured in a perfusion flow bioreactor to stimulate osteoblastic differentiation and the deposition of bioactive factors. Using the three-part engineering strategy described, I hypothesize that the extracellular matrix produced by BMSCs can be modulated by two stimuli: the stiffness of the scaffold and perfusion flow. First, I propose that culturing BMSCs on polyurethane scaffolds with increasing stiffness will increase markers of osteoblastic differentiation. Secondly, I suggest that mechanically stimulating BMSCs with novel perfusion strategies will also increase markers of osteoblastic differentiation. In aim 1, a family of segmented degradable poly(esterurethane urea)s (PEUURs) were synthesized. The modulus of the PEUUR materials was systematically increased from 0.18 to 0.80 MPa by systematically increasing the molecular weight of the poly(ε-caprolactone) (PCL) soft segment from 1425 to 2700 Da. BMSCs were cultured on both rigid polymer films and on porous foam scaffolds to dissociate the effect of variation in polymer chemistry from the effect of scaffold modulus on cell phenotype. These studies demonstrated changes in osteoblastic differentiation as measured by prostaglandin E2 production, alkaline phosphatase activity (ALP) activity, and osteopontin gene expression. However, the increased levels of these phenotypic markers on the PCL 2700 material could not be attributed to scaffold chemistry or modulus. Instead, the differences may be related to polymer crystallinity or surface topography. In aim 2, novel dynamic perfusion strategies were used to investigate the influence of frequency on osteoblastic differentiation. BMSCs were seeded on porous foam scaffolds and exposed to both steady perfusion and pulsatile perfusion at 0.017, 0.050, and 0.083 Hz frequencies. The data presented here demonstrated that while some markers of osteoblastic phenotype such as ALP activity are enhanced by 0.05 Hz pulsatile flow over continuous flow, they are insensitive to frequency at low frequencies. Therefore, future studies will continue to investigate the effect of a larger range of frequencies. Additionally, fluid flow has also been shown to stimulate the deposition of bioactive factors such as BMP-2 and VEGF-A, and these growth factors are known to significantly enhance healing in bone defect models. Therefore, we plan to investigate the effect of dynamic flow strategies on the deposition of these bioactive factors. We propose that an engineered bone graft material containing a bone-like extracellular matrix and producing these growth factors will show more rapid formation of bone when implanted in vivo. / Ph. D.

This thesis explores the capabilities of cationic polymers modified with lipids of different carbon chain length to deliver DNA molecules to primary cells and transformed cell lines. Our studies focus on two different polymers: polyethylenimine (PEI) and poly(L-lysine) (PLL). Firstly, PEI and PLL were conjugated to palmitic acid (C16). The delivery of plasmid DNA to rat bone marrow stromal cells (rat-BMSC) was evaluated by using a Green Fluorescent Protein gene expressing plasmid (pEGFP-N2) as a reporter system. The rationale for lipid substitution is to give the polymer an amphiphilic character so as to improve the transfection efficiency of native polymers by improving the DNA/polymer translocation through the phospholipid-rich cell membranes. In the case of PLL-C16, transfection efficiency was significantly increased (5 fold) as compared to native PLL, and it was significantly higher than commercially available cationic lipids (LipofectamineTM 2000 and FugeneTM). We further explore the use of other lipids with variable chain lengths (carbon chain length ranging from 8 to 18 saturated and unsaturated) in order to identify other candidates to enhance the gene delivery properties of the PLL. Lipid-modified PLL of high molecular weight (25 vs. 4 kDa) was found to be more effective in delivering plasmid DNA in rat-BMSC. We noted that C14-, C16- and C18-substituted PLL gave the most effective DNA delivery. Moreover, a correlation between the extent of lipid substitution and the plasmid DNA delivery efficiency was found Additionally, transgene expression by BMSC significantly increased when amphiphilic PLLs were used as compared to native PLL. The modified polymers were able to transfect the cells up to 7 days, after which the expression decreased. Encouraged by the successful transgene expression agents obtained by modifying low molecular weight PEI with the same series of lipids described above, we explored the possibility of modifying low molecular weight PEI (2 kDa) with longer lipids; saturated fatty acid (C22), trans fat (C18:1T) and essential fatty acids (C22:1, C22:6 and C18:3). Transfection efficiency proved to be cell dependent. Only the transformed 293T cells were able to express GFP compared to human-derived BMSC. The highest transfection efficiency was found with highly unsaturated lipid-substituted PEI (C18:3 and C22:6) and were able to increase transgene expression overtime (6 days). Furthermore, internalization studies indicated that effective transfection of these carries do not follow any known endocytosis pathway instead the DNA/carrier penetrates the plasma membrane directly. / Pharmaceutical Sciences

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No description available.

Bone is the second most transplanted tissue in the body, with approximately 2.2 million bone graft procedures performed annually worldwide. Currently, autogenous bone is the gold standard for bone grafting due to its ability to achieve functional healing; however, it is limited in supply and results in secondary injury at the donor site. Tissue engineering has emerged as a promising means for the development of new bone graft substitutes in order to overcome the limitations of the current grafts. In this research project, the specific approach for bone tissue engineering involves seeding osteoprogenitor cells within a biomaterial scaffold then culturing this construct in a biodynamic bioreactor. The bioreactor imparts osteoinductive mechanical stimuli on the cells to stimulate the synthesis of an extracellular matrix rich in osteogenic and angiogenic factors that are envisioned to guide bone healing in vivo. Fluid flow, which exerts a hydrodynamic shear stress on adherent cells, has been identified as one of the strongest stimuli on bone cell behavior. It has been shown to enhance the deposition of osteoblastic matrix proteins in vitro, and is particularly important for the delivery of oxygen and nutrients to cells within large scaffolds suitable for bone tissue regeneration. In particular, dynamic flow profiles have been shown to be more efficient at initiating mechanotransductive signaling and enhancing gene expression of osteoblastic cells in vitro relative to steady flow. However, the molecular signaling mechanisms by which bone cells convert hydrodynamic shear stress into biochemical signals and express osteoblastic matrix proteins are not fully understood. Therefore, the overall goal of this research project was to determine the effect of dynamic fluid flow on mechanotransductive signaling and expression of bioactive factors and bone matrix proteins. In the first study, an intermittent flow regimen, in which 5 min rest periods were inserted during fluid flow, was examined. Results showed that signaling molecules, mitogen activated protein kinases (MAPKs) and prostaglandin E2, were modulated with the flow regimen, but that expression of bone matrix proteins, collagen 1α1, osteopontin, bone sialoprotein (BSP), and osteocalcin (OC), were similar under continuous and intermittent flow. Thus, this study suggested that variation of the flow regimen modulates mechanotranductive signaling. In the second study, four flow conditions were examined: continuous flow, 0.074 Hz, 0.044 Hz, and 0.015 Hz pulsatile flow. This study demonstrated that pulsatile flow enhances expression of BSP and OC over steady flow. Similarly, bone morphogenetic protein (BMP)-2 and -7 were enhanced with pulsatile flow, while BMP-4 was suppressed with all flow conditions, suggesting that the mechanism by which fluid flow enhances bone matrix proteins may involve the induction of BMP-2 and -7, but not BMP-4. In the third study, the molecular mechanism by which fluid flow simulates expression of BMPs was examined. Results from this study suggest that this mechanism may involve activation of MAPKs, but BMP-2, -4, and -7 are regulated through multiple different signaling pathways. Overall, the results from this research demonstrate that dynamic flow modulates mechanotransductive signaling and expression of osteoblastic matrix proteins by osteoblast cells. In particular, BMPs, important for formation in vivo, were shown to be induced by fluid flow. Therefore, this work may be beneficial in understanding and developing 3D perfusion culture systems for the creation of a clinically effective engineering bone tissue. / Ph. D.

Rupture of the anterior cruciate ligament (ACL) is one of the most common ligamentous injuries of the knee. Post rupture, the ACL does not heal on itself due to poor vasculature and hence surgical intervention is required to treat the ACL. Current surgical management of ACL rupture consists of reconstruction with autografts or allografts. However, the limitations associated with these grafts have prompted interest in tissue engineered solutions that combine cells, scaffolds and stimuli to facilitate ACL regeneration. This thesis describes a ligament tissue engineering strategy that involves incorporating biomolecules within fibers-based electrospun meshes which mimics the extra-cellular matrix microarchitecture of ligament. However, challenges exist with incorporation of biomolecules. Therefore, the goal of this research project was to develop two techniques to incorporate biomolecules within electrospun meshes: (1) co-axially electrospinning fibers that support surface-grafting of biomolecules, and (2) co-axially electrospinning fibers decorated with biomolecule-loaded microspheres. In the first approach, chitosan was co-axially electrospun on the shell side of poly caprolactone (PCL) and arginine-glycine-aspartate (RGD) was attached to the electrospun meshes. Bone marrow stromal cells (BMSCs) attached, spread and proliferated on these meshes. In the second approach, fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) loaded chitosan-alginate (CS-AL) microspheres were fabricated. The effects of cation to alginate ratio, type of alginate and concentration of CaCl2 on microsphere size, FITC-BSA loading and release were systematically evaluated. The CS-AL microspheres were then incorporated into the sheath phase of co-axially electrospun meshes to achieve microsphere-decorated fiber composite meshes. The results from these model study suggest that both approaches are tractable for incorporating biomolecules within fibers-based electrospun meshes. Both these approaches provide platform for future studies that can focus on ligament-relevant biomolecules such as FGF-2 and GDF-5. / Master of Science

Long-term exposure to pharmacological doses of glucocorticoids has been associated with the development of osteopenia and avascular necrosis. Bone loss may be partially attributed to a steroid-induced decrease in the osteoblastic differentiation of multipotent progenitor cells found in the bone marrow. In order to determine if there is a change in the osteogenic potential of the bone marrow stroma following glucocorticoid treatment, Sprague-Dawley rats were administered methylprednisolone for up to six weeks, then sacrificed at 0, 2, 4, or 6 weeks during treatment or 4 weeks after cessation of treatment. Femurs were collected and analyzed for evidence of steroid-induced osteopenia and bone marrow adipogenesis. Although glucocorticoid treatment did inhibit bone growth, differences in ultimate shear stress and mineral content were not detected. The volume of marrow fat increased with increasing duration of treatment, but returned to near control levels after cessation of treatment. Marrow stromal cells were isolated from tibias, cultured in the presence of osteogenic supplements, and analyzed for their capacity to differentiate into osteoblast-like cells in vitro. Glucocorticoid treatment diminished the absolute number of isolated stromal cells, but did not inhibit the relative levels of bone-like mineral deposition or osteocalcin expression and secretion. Although pharmacological glucocorticoid levels induce bone loss in vivo, physiologically equivalent concentrations have been shown to enhance the formation of bone-like tissue in vitro. However, glucocorticoids have also been reported to inhibit proliferation and type I collagen synthesis in marrow stromal cell cultures. In order to assess the effects of intermittent dexamethasone treatment on the progression of osteogenesis in rat marrow stromal cell culture, this synthetic glucocorticoid was removed from the culture medium after a variable period of initial supplementation. Cell layers were analyzed for total cell number, collagen synthesis, phenotypic marker expression, and matrix mineralization. Prolonged supplementation with dexamethasone decreased proliferation, but did not significantly affect collagen synthesis. Furthermore, increased treatment duration was found to increase bone sialoprotein expression and mineral deposition. The duration of glucocorticoid treatment may be a key factor for controlling the extent of differentiation in vitro. / Master of Science

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Cartilage regeneration is one of the most widely studied areas in tissue-engineering. Despite significant progress, most efforts to date have only focused on generating homogenous tissues whose bulk properties are similar to articular cartilage. However, anatomically and functionally, articular cartilage consists of four spatially distinct regions: the superficial, transitional, deep, and calcified zones. Each zone is characterized by unique extra-cellular matrix (ECM) compositions, mechanical properties, and cellular organization. The ECM is primarily composed of type II collagen and glycosaminoglycans (GAGs), whose relative concentrations vary between zones and therefore lead to distinctive mechanical properties. One of the major unsolved challenges in engineering cartilage has been the inability to regenerate tissue that mimics the zonal architecture of articular cartilage. Recent studies have attempted to imitate this spatial organization using zone-specific chondrocytes isolated from donor animal cartilage. Directed differentiation of a single stem population into zonally organized native-like articular cartilage has not yet been reported. This dissertation reports that hydrogels, incorporating both synthetic and natural polymers as well as cell-induced degradability, are suitable for generating zone-specific chondrogenic phenotypes from a single MSC population. Specifically, cues provided from the unique combinations of chondroitin sulfate (CS), hyaluronic acid (HA), and MMP-sensitive peptide (MMP-pep) within a PEG-based hydrogel, direct the chondrogenic differentiation of MSCs. The findings of this dissertation demonstrate the capability of creating native-like and mechanically relevant articular cartilage consisting of zone specific layers. This ability provides a new direction in cartilage tissue engineering and could be invaluable for cartilage repair if incorporated with current minimally invasive surgical techniques. / text