Transmyocardial revascularization (TMR) has emerged as an additional therapeutic option for patients suffering from diffuse coronary artery disease (CAD), providing immediate angina relief. The current potential of this therapy focuses on the injection of stem cells, in order to create a synergistic angiogenic effect while increasing myocardial repair and regeneration. Although TMR procedures provide increased vascularization within the myocardium, patients suffering from ischemic cardiomyopathy may not benefit from angiogenesis alone. Therefore, the goal of introducing stem cells is to restore the functional state of a failing heart by providing stem cells with a favorable microenvironment that will enhance their engraftment. Since the therapeutic effect of stem cells is dependent on their capacity to survive and retain in the myocardium, laser therapy may provide a strategy for increasing stem cell engraftment. If so, these cells may have the potential to act as mitochondrial donors or as sources of paracrine factors, aiding in the recovery from oxidative stress and providing antioxidant reserves. Furthermore, laser therapy may also play an influential role in regulating cardiac repair and regeneration via epithelial-mesenchymal transition (EMT). By interacting with specific transcription factors TMR may provide another pathway by which it can offer reparative effects. Cumulatively, paracrine release, denervation, and angiogenesis contribute to the therapeutic benefits experienced by TMR patients, including a significant reduction in angina, with increases in myocardial perfusion and survival rates. With the addition of stem cells, these effects may be further augmented, thus providing increased symptomatic relief in patients.

This dissertation provides an in-depth analysis of the practical application and judicial framework pertaining to stem cell research in South Africa. In the realisation of the above-mentioned analysis, and ultimate critique on the current and proposed legal position, focus is placed on aspects of Medical Law, Legal Philosophy and Human Rights. These include concerns on the procurement of informed consent from stem cell donors, ethical and religious influences on the regulation of biomedicine in general as well as the impact of socio-economic indicators in the realisation of the effective implementation of stem cell research. Focus is firstly placed on the medical aspects surrounding the research, whereafter an examination of the current legal position and its practical application is made. Following the discussion of the current legal position, with reference to the array of influences and concerns pertaining thereto, the newly proposed regulative measures are examined within the current international framework. These regulative measures are placed within context of the private and public sector with their different benefits and disadvantages. In a further discussion of the realisation of the private sector’s interests, focus is placed on the role that Intellectual Property Rights play in the protection of monetary incentives to conduct stem cell research. All of the above ultimately leads the author to provide an informed set of recommendations in which the proposed regulative measures can be adapted to ensure the legitimate and practically sound implementation of stem cell research in South Africa. / Dissertation (Magister Legum (Public Law))--University of Pretoria, 2007. / Public Law / unrestricted

La perte de l’organe dentaire entraine une perte de substance de l’os alvéolaire. Les techniques mises en place pour reconstruire l’os alvéolaire préalablement à la pose d’implant utilisent des membranes (seules ou associées à une greffe osseuse), faisant d’une part office de barrière vis-à-vis des tissus mous environnants, et permettant d’autre part le maintien d’un espace nécessaire à l’ostéogénèse. Ce travail de thèse pluridisciplinaire vise à développer des matrices innovantes destinées à la régénération osseuse. Inspirés par la nature hybride du tissu osseux, des revêtements à base de phosphates de calcium et de polymères organiques (chitosane et acide hyaluronique) ont été élaborés par pulvérisation simultanée de solutions d’espèces réactives. La construction de ces revêtements a permis la précipitation d’un composé à base d’apatite carbonatée et de brushite sur des lamelles de verre (preuve de concept), tandis qu’un composé hybride complexe (brushite, phosphate octocalcique et apatite nanocristalline) a été construit sur des membranes de collagène d’origine porcine, utilisées en régénération osseuse. Sur le verre, les revêtements semblent posséder les propriétés intrinsèques (rugosité, élasticité) en faveur d’une différenciation ostéoblastique, ce qui a été confirmé par une différenciation ostéoblastique précoce des cellules souches. Sur la membrane, le revêtement n’induit pas de différenciation mais stimule les propriétés ostéo-immunomodulatrices des cellules souches, nécessaires à la régénération osseuse. De plus, le revêtement a démontré dans les deux cas une activité antibactérienne, le rendant très attractif pour des applications en régénération osseuse. Enfin, le développement d’un dispositif médical de classe II à partir de la gelée de Wharton a été envisagé. Cette source allogénique semble prometteuse en médecine régénératrice osseuse. / Bone loss following tooth extraction requires pre-implantory surgery techniques to regenerate bone. These techniques use an occlusive membrane positioned over a bone graft material or not, providing space maintenance and enabling to seclude soft tissue infiltration to promote bone regeneration. This pluridisciplinary thesis work aims at developing innovative biomaterials for bone regeneration applications. Inspired by bone hybrid composition, coatings made of calcium phosphates and organic polymers (chitosan and hyaluronic acid) were developed using simultaneous spray coating of interacting species process. Coating build-up led to precipitation of a compound made of carbonated apatite and brushite on glass coverslip (proof of concept), whereas a complex hybrid compound (brushite, octacalcium phosphate and nanocrystalline apatite) was formed on collagen membrane. Coating on glass coverslip seems to possess required properties (roughness, elasticity) for osteoblastic differentiation, which was confirmed by stem cells early osteoblast commitment. However, coating on collagen membrane did not induce osteoblastic differentiation, but stimulated stem cells osteo-immunomodulatory properties, required for bone regeneration. Interestingly, coating demonstrated in both cases antibacterial activities, which makes it very attractive for bone regeneration applications. Finally, Wharton’s jelly from umbilical cord was suggested as an innovative source for new biomaterials, to replace xenogeneic membranes.

archives@tulane.edu / Human adipose-derived stem cells (ASCs) efficiently modulate the inflammatory microenvironment, making them ideal for the treatment of inflammatory, autoimmune, and neurodegenerative diseases like multiple sclerosis (MS). Clinical translation of ASC therapies has been limited, making strategies to improve ASC post-transplant immunosuppressive capabilities especially important. Autophagy, a stress-induced degradative pathway, plays a crucial role in the paracrine signaling of ASCs, which drives their therapeutic action. Therefore, I investigated the modulatory effect of autophagy preconditioning in ASC physiology and therapeutic potential using the autophagy inducer Rapamycin (Rapa-ASCs) or the inhibitor 3-methyladenine (3MA-ASCs). Following 4 and 24-hour preconditioning, ASC stemness and immunomodulatory capacity were examined. Results demonstrate that neither Rapa nor 3-MA altered morphology or surface marker expression, indicating preservation of stemness. RT-qPCR analysis revealed that 4-hour preconditioning with Rapa-ASCs and 3MA-ASCs alone upregulated transcription of cyclooxygenase-2 (COX2), but not secretion of its downstream effector molecule prostaglandin E2 (PGE2). Following stimulation with interferon-gamma (IFNγ) to mimic a pathological microenvironment, both 4-hour Rapa-ASCs and 3MA-ASCs upregulated COX2 transcription and PGE2 secretion. 4-hour Rapa-ASCs also upregulated expression of the cytokines transforming growth factor beta (TGF-β) and interleukin-6 (IL-6). As each of these molecules have demonstrated therapeutic effects in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, I hypothesized that 4-hour Rapamycin preconditioning may bestow the greatest improvement to ASC immunomodulatory potential in EAE. To test this, EAE mice were treated at peak disease severity with control ASCs (EAE-ASCs), Rapa-preconditioned ASCs (EAE-Rapa-ASCs), or a vehicle control (EAE). Results revealed that EAE-ASCs enhanced rotarod locomotor activity, improved clinical disease scores, and elevated intact myelin in the spinal cord compared to both EAE and EAE-Rapa-ASC animals. This correlated with augmented CD4+ T helper (Th) and T regulatory (Treg) cells in the spinal cord, and significantly increased interleukin-10 (IL-10) transcripts. Conversely, EAE-Rapa-ASC mice showed no clinical or motor function improvement, reduced myelin levels, and significantly less Th and Treg cells in the spinal cord. These findings suggest that short-term Rapamycin preconditioning diminishes the therapeutic efficacy of ASCs when applied to late-stage EAE and highlights the importance of investigating novel therapeutic strategies in vivo in physiologically relevant disease models. / 1 / Rachel Wise

Cortical injury, such as that following stroke, is one of the leading causes of long-term disabilities world-wide. While some neuroprotective agents given within hours of stroke can reduce damage, there are currently no neurorestorative therapeutics that can enhance long-term recovery. To address this, we tested Mesenchymal Stem Cell (MSC) derived Extracellular Vesicles (EVs) as a treatment for cortical injury in rhesus monkeys (Macaca mulatta). Monkeys treated with EVs 24 hours after injury and again at 14 days after injury recovered more completely and more rapidly than monkeys given a vehicle control. However, the cellular changes associated with enhanced recovery remained unknown. In this dissertation, it was hypothesized that EVs modulated cells within the brain to enhance recovery after cortical injury. To explore this hypothesis, three specific aims were tested. Aim 1: To determine the effects of EVs on microglial reactivity. Since EVs in this study were derived from MSCs, it was hypothesized that they would have an immunomodulatory effect. Using immunohistochemistry, image analyses, and 3-D reconstruction, we showed that microglia shifted from reactive, damaging phenotypes towards homeostatic, surveilling functions in EV-treated monkeys. These effects correlated with reduced time to recovery, suggesting that reduced microglial reactivity enhanced recovery. Aim 2: To assess the effects of EVs on myelination. Because MSCs have regenerative effects, it was hypothesized that these MSC-derived EVs would improve neurorestoration. Using immunohistochemistry, qRT-PCR, Spectral Confocal Reflectance microscopy, and ELISA, we assessed myelination after cortical injury with and without EV treatment. EVs limited oligodendrocyte damage and increased densities of mature oligodendrocytes to enhance myelin maintenance. These effects correlated with improved recovery, suggesting the importance of myelination in recovery after cortical injury. Aim 3: To assess the neuroprotective role of EVs on infarct volumes. While it was hypothesized that EVs would reduce the densities of inflammatory cells (astrocytes, macrophages/microglia, T-cells), hemosiderin accumulation, and infarct volume, we found that EVs did not alter these endpoints. Collectively, our results suggest that EVs modulated microglia and oligodendrocytes to promote neurorestoration. Overall, these findings demonstrate the therapeutic potential of EVs for neurorestoration after cortical injury.

Mesenchymal stem cells (MSCs) are the most widely used source for bone tissue engineering due to their capability of multipotent differentiation. The use of nanotechnology in biomedical applications and therapy has increased in recent years provides an elegant alternative in comparison to current tissue engineering methods. Magnetic nanowires have a high potential in the medical field, as they are biocompatible, are simple to fabricate, possess low cytotoxic effects and can be operated wirelessly via magnetic fields. A nanowire substrate (NW) can provide a surface with tunable elastic properties. Therefore, magnetic nanowires have many promising applications such as in cell therapy, cell separation, cancer treatment, and as a scaffold for cell culture. This thesis explores the effects of alternating magnetic field (AMF) as a biophysical stimulator of osteogenic differentiation of MSCs by culturing the stem cells on a magnetic iron (Fe) NW. To this end, Fe nanowires were fabricated through electrodeposition and interactions between the NW and cells were analysed by electron microscopy. An AMF was applied to the NW in order to induce a vibration. MSCs were exposed to different magnetic field intensities, 250 mT and 50 mT, for different application times, 12 hours on followed by 12 hours off for two days and 24 hours on followed by 12 hours off. Differentiation was determined through the assessment of osteogenic markers at the mRNA level by RT-PCR and at the protein level by flow cytometry and fluorescence microscopy. Different effects were observed on MSCs grown on Fe NWs following exposure to different magnetic field intensities and duration applications. MSC differentiation towards the osteogenic lineage increased with increased field intensities. The most enhanced osteogenic differentiation of MSCs was observed at 250 mT AMF for 12 hours, as evidenced by elevated osteogenic markers at mRNA level compared to that of an AMF free control. Based on these results, we proposed that culturing MSCs on magnetic nanomaterials has the potential to control and promote osteogenesis under magnetic field and without the addition of external differentiation factors. These findings provide a new tool for stem cell research as an effective technology for bone tissue engineering and regenerative medicine.

Osteoporosis is a systemic skeletal disease that affects millions of people worldwide. There are many possible etiologies for osteoporosis, including inherent variables like genetics and sex, and lifestyle variables like diet and exercise. Characterized by low bone mass and increased fracture risk, the disease places a burden on both the patients and the healthcare industry. Therefore, it is vital that research determine the mechanisms by which the risk factors affect BMD so that better diagnosis and treatment options may be developed. The purpose of this study was to examine the relationship between various osteoporosis risk factors and biochemical markers of osteogenic cell activity derived from bone-marrow MSCs. It was hypothesized postmenopausal white women, having the greatest risk for osteoporosis, would have elevated hydroxyproline and decreased ALP, indicative of greater bone resorption. Acetabular reamings were collected from 26 patients (15 males and 11 females) undergoing total hip arthroplasty at Boston Medical Center. MSCs from the reamings were plated and underwent osteoinduction into osteoblasts. The cells were then harvested and assayed for various indicators of cell growth and bone cell activity, such as DNA, ALP, and hydroxyproline. Our hypothesis was generally supported in that postmenopausal white women did have less ALP, an indicator of bone deposition, than premenopausal women and postmenopausal African American women. Additional findings and directions for future studies are further discussed in this paper.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23053号 / 理博第4730号 / 新制||理||1678(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 村山 美穂, 教授 幸島 司郎, 教授 伊谷 原一 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23424号 / 医科博第129号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 林 康紀, 教授 伊佐 正, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23471号 / 医博第4778号 / 新制||医||1053(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 木村 剛, 教授 湊谷 謙司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM