Introduction: Heart failure remains a major public health problem, with recent estimates indicating that end-stage heart failure with two-year mortality rates of 70-80% affects over 60,000 patients in the US each year. Medical management can be used but success declines for patients with end stage heart failure. Although cardiac transplantation is optimal, less than 2500 cardiac transplants are performed annually due to the severely limited supply of donor organs. Mechanical circulatory support (MCS) devices are now routinely used to bridge patients with end-stage heart failure who become critically ill until a donor heart is available. The use of stem cell therapy to treat heart failure has been gaining significant ground in recent years, specifically due to its regenerative properties, and both animal and human models have shown significant improvements in ventricular mass, ejection fraction, vascularization, wall thickness, and infarct size reduction. Using the patients' HeartWare HVAD device diagnostics, we were able to acquire our response variable; pulsatility. Pulsatility is a variable measure of the differential between minimum and maximum flow and is dependent on device motor speed, power, current, and fluid viscosity. This measurement is important as it relates to the contractility of the heart and could potentially be used as an end point in determining when a patient is healthy enough to have their HVAD explanted. We set out to develop a low cost and effective predictive model to determine amniotic mesenchymal stem cell's ability to repair compromised cardiac tissue of patients using the Total Artificial Heart (TAH) and Donovan Mock Circulation Tank (DMC). Methods: Predictive modelling was performed using the TAH and DMC. The system was set to a range from critical heart failure to a normal operating conditions through the variation of preload, afterload, and ventricular drive pressures with the intent of comparing the results to our patient population. Patients (n=7, 3 dilated, 4 ischemic) received intravenous and intra-myocardial injections of a heterogeneous amniotic mesenchymal stem cells mixture and liquid matrix (MSCs+LM) at HVAD implant. Groups were analyzed based on treatment; control (HVAD only, n=7) versus stem cells (HVAD + MSCs+LM). HeartWare log files were acquired from patients' devices and analyzed in SAS and Matlab. Results from the patient study were compared to the predictive model to determine levels of stem cell response. Results: Pulsatility was found to increase with left drive pressure and afterload. Lower drive pressures resulted in a drop off in pulsatility at higher afterloads while higher drive pressures were able to compensate for any afterload. Pulsatility also increased with preload but lower drive pressures were unable to fully eject at the highest preloads, resulting in a reduced pulsatility. We observed the effects of the stem cell injections on pulsatility and found that patients receiving therapy demonstrated statistically significant increases in pulsatility at 15-20 (p=.0487), 25-30 (p=.0131), 35-40 (p=.0333), and 75-80 (p=0.0476) days post implant. At minimum, when comparing the patient results to the in vitro model, the therapy resulted in a progression from end stage HF conditions to medium cardiac function conditions. At maximum, the therapy resulted in a progression from end stage HF to normal healthy operating cardiac function. Conclusions: Stem cells demonstrated a significantly increased rate of change in pulsatility within the first 40 days and at 80 days post implant when compared to control. They also demonstrated progression from end stage HF to normal healthy cardiac function at two time periods (Days 40, 90). These results justify expansion of the study to encompass a larger patient population to verify the results of the in vitro model to predict cardiac regeneration with multiple functional status indicators.

Traumatic injuries to the spinal cord (SCI) and peripheral nerve (PNI) affect several thousand people worldwide every year. At present, there is no effective treatment for SCI and despite continuous improvements in microsurgical reconstructive techniques for PNI, many patients are still left with permanent, devastating neurological dysfunction. This thesis investigates the effects of mesenchymal stem cells (MSC) derived from adipose (ASC) and dental (DSC) tissue and chitosan/microRNA-124 polyplex particles on regeneration after spinal cord and peripheral nerve injury in adult rats. Dental stem cells were obtained from apical papilla, dental pulp, and periodontal ligament. ASC and DSC expressed MSC surface markers (CD73, CD90, CD105 and CD146) and various neurotrophic molecules including BDNF, GDNF, NGF, VEGF-A and angiopoietin-1. Growth factor stimulation of the stem cells resulted in increased secretion of these proteins. Both ASC and DSC supported in vitro neurite outgrowth and in contrast to Schwann cells, ASC did not induce activation of astrocytes. Stimulated ASC also showed an enhanced ability to induce capillary-like tube formation in an in vitro angiogenesis assay. In a peripheral nerve injury model, ASC and DSC were seeded into a fibrin conduit, which was used to bridge a 10 mm rat sciatic nerve gap. After 2 weeks, both ASC and DSC promoted axonal regeneration in the conduit and reduced caspase-3 expression in the dorsal root ganglion (DRG). ASC also enhanced GAP-43 and ATF-3 expression in the spinal cord, reduced c-jun expression in the DRG and increased the vascularity of the implant. After transplantation into injured C3-C4 cervical spinal cord, ASC continued to express neurotrophic factors and laminin and stimulated extensive ingrowth of 5HT-positive raphaespinal axons into the trauma zone. In addition, ASC induced sprouting of raphaespinal terminals in C2 contralateral ventral horn and C6 ventral horn on both sides. Transplanted cells also changed the structure and the density of the astroglial scar. Although the transplanted cells had no effect on the density of capillaries around the lesion site, the reactivity of OX42-positive microglial cells was markedly reduced. However, ASC did not enhance recovery of forelimb function. In order to reduce activation of microglia/macrophages and the secondary tissue damage after SCI, the role of microRNA-124 was investigated. In vitro transfection of chitosan/microRNA-124 polyplex particles into rat microglia resulted in the reduction of reactive oxygen species and TNF-α levels and lowered expression of MHC-II. Upon microinjection into uninjured rat spinal cords, particles formed with Cy3-labeled control sequence RNA, were specifically internalized by OX42 positive macrophages and microglia. Alternatively, particles injected in the peritoneum were transported by macrophages to the site of spinal cord injury. Microinjections of chitosan/microRNA-124 particles significantly reduced the number of ED-1 positive macrophages after SCI. In summary, these results show that human MSC produce functional neurotrophic and angiogenic factors, creating a more desirable microenvironment for neural regeneration after spinal cord and peripheral nerve injury. The data also suggests that chitosan/microRNA-124 particles could be potential treatment technique to reduce neuroinflammation.

The toll skeletal muscle injury, resulting in significant muscle mass loss, has on the patient reaches far more than physical and emotional, as the tolls are financial as well. Approximately more than 3 billion dollars is spent on the initial medical costs and on subsequent disability benefits, following a volumetric muscle loss. Skeletal muscle has a robust capacity for self-repair; this propensity for repair is hindered when skeletal muscle loss is larger than 20% of the total mass of the muscle. Previous work in our lab, has shown functional and morphological improvements following the cellular therapy, with mesenchymal stem cells (MSC), as well as with nerve relocation to the extracellular matrix (ECM). To further observe the regenerative properties of the above treatments, a defect weighing approximately 307 ± 3.7 mg wet weight and measuring approximately 1x 1cm² was removed from the lateral gastrocnemius (LGAS) of male Sprague Dawley rats. Additionally, the medial branch of the plantar nerve was then relocated and implanted to the middle of the ECM. Seven days post injury bone-marrow derived mesenchymal stem cells were injected directly into the implant using a PEGylated Fibrin hydrogel (PEG). Following 56 days of recovery, partial functional restoration was observed in the LGAS ECM seeded with MSC and implanted with the plantar nerve. The LGAS produced 86.3 ± 5.8% of the contralateral LGAS, a value that was significantly higher than ECM implantation alone (p <.05). The implanted ECM seeded with MSC and implanted with the plantar nerve showed significant increases in blood vessel density and myofiber content (p <.05). The data suggest that a volumetric injury can be repaired by neurotization of an implanted muscle-derived ECM seeded with MSCs. / text

Les affections ostéo-articulaires concernent des millions de personnes. L’ingénierietissulaire osseuse, associant cellules souches mésenchymateuses humaines (CSM) etmatériaux synthétiques, pourrait répondre aux besoins cliniques. Pour cela, les différentescomposantes de cette approche et leur association doivent être mieux étudiées pour la rendreutile cliniquement. Durant cette thèse, une première étude animale proche du cas cliniquenous a permis de définir les points à améliorer pour le traitement des pertes osseuses. Nousavons ainsi pu développer un milieu spécifique induisant une différenciation rapide etterminale des CSM en ostéoblastes. Par la suite, l’utilisation de particules de céramiquescomme support cellulaire nous a permis d’obtenir des hybrides riches en matriceextracellulaire. Cet environnement 3D biomimétique permet l’engagement spontané des CSMvers un phénotype ostéoblastique et l’obtention d’une quantité osseuse importante in vivo.L’ensemble de ces résultats met en évidence l’importance de l’environnement et du stade dedifférenciation cellulaire pour la formation osseuse par ingénierie tissulaire osseuse. / Osteo-articular disorders affect millions of people over the world. Bone tissueengineering, an approach combining human mesenchymal stem cells (MSC) and syntheticmaterials, could potentially fulfill clinical needs. However, the different components of thisapproach and their association should be investigated further to make it clinically useful. Inthis thesis, an initial animal study close to clinical situation allowed us to identify areas thatneed improvement for regenerating bone defect. We were then able to develop a specificmedium which induces a rapid and terminal osteoblastic differentiation of MSC.Subsequently, the use of ceramic particles as cell support has allowed us to obtain hybridmainly composed of extracellular matrix. This biomimetic 3D environment allowsspontaneous osteoblastic commitment of MSC and induces a large bone quantity in vivo.Overall, these results highlight the importance of the environment and the cell differentiationstate for bone formation using bone tissue engineering.

This thesis presents deeper insights into bone applicable biomaterials’ design. Poor affinity of BMP-2 towards scaffolds required supra-physiological dose administration. Though molecules containing sulfate could sustain BMP-2 release, side effects occurred due to BMP-2 supra-dose, or these sulfate-containing biomolecules. Improved affinity between BMP-2 and scaffolds was first witnessed by using an acidic carrier (paper I). Hyaluronic acid (HA) hydrazone derived hydrogels having a pH of 4.5-loaded BMP-2 showed sustained release of bioactive BMP-2 in vitro and enhanced bone formation in vivo, while pH 7 HA hydrogels showed Fickian behavior and less bone formation in vivo. Computational evaluation revealed stronger electrostatic interactions between BMP-2, and HA were predominant at pH 4.5, whereas, weaker Van der Waals interactions played a key role at pH 7. During the pre-bone formation phase, endogenous cell responses to pH 4.5 and 7 with or without BMP-2 were investigated. HA hydrogels exhibited extraordinary biocompatibility and recruitment of neutrophils, monocytes, macrophages and stromal cells regardless of hydrogels’ pH and BMP-2 presence.  The different inflammatory responses to HA hydrogels were observed (Appendix). Thiol derivatives can cleave the disulfide bond of BMP-2 to generate inactive monomeric BMP-2. In paper II, thiol-acrylate chemistry-based HA hydrogels (HA-SH) were compared to hydrazone-based HA hydrogels as BMP-2 carriers. Thiol modified HA disrupted BMP-2 integrity and bioactivity. HA-SH hydrogels with BMP-2 exhibited less bioactive BMP-2 release in vitro and induced less bone formation in vivo. Accumulated evidence has shown great osteogenic potential of lithium ions (Li). In paper III, we coordinated Li onto HA-PVA hydrazone hydrogels (Li-gel); Li-gel enhanced 3D cultured hMSCs osteogenic differentiation and induced higher bone formation in CAM defect model. Instead of BMP-2 protein, delivery of BMP-2-coding-plasmid can produce BMP-2 over a long term at a closer physiological level. Yet, efficient gene delivery reagents are needed. In paper IV, two novel gene delivery nanoplexes were developed by post coating DNA-nanoplexes with chondroitin sulfate (CS). To ensure the stability, aldehyde-modified CS (CS-CHO) reacted with free amines of pDNA/PEI complexes. We provided first evidence that CS-CHO coated nanoplexes controlled the release from endosomes, which is essential for higher transfection efficiency.

L’athérosclérose et la pathologie anévrysmale sont principalement caractérisées par un remodelage de laparoi artérielle au cours de leur évolution. Ce travail a examiné un aspect de la destruction de la paroiartérielle à travers l’étude de la métalloprotéase MMP-14 au cours de l’athérome et un aspect dereconstruction artérielle à travers l’étude d’une thérapie cellulaire d’un modèle d’Anévrysme de l’AorteAbdominale (AAA) par Cellules Souches Mésenchymateuses (CSMs).En utilisant un modèle de greffe de Moëlle Osseuse (MO) dans des souris Ldlr-/-, nous avons montré que ladélétion d’expression de MMP-14 dans les cellules issues de la MO provoquait une accumulation decollagène interstitiel dans la plaque athéromateuse sans modification de la composition cellulaire nivariation de taille. Une mesure de l’activité collagénolytique par substrat fluorescent a confirmé que ladélétion en MMP-14 chez les macrophages provoquait une baisse de l’activité collagénolytique. Cetteactivité est indépendante de l’activité MMP-2 et MMP-8 et pourrait être médiée partiellement parl’activation de MMP-13. Nous avons mis en évidence la présence de CSMs à la surface luminale de thrombus de AAA et nous avonsmontré une diminution significative des CSMs circulantes chez des patients porteurs de AAA. Nous avonspu stabiliser la croissance de AAA expérimentaux chez le rat à partir de xénogreffe artérielle par perfusionendoluminale de CSMs. La perfusion de CSMs provoquait une diminution de l’inflammation à court termeet favorisait la reconstruction artérielle par accumulation de collagène et d’élastine à moyen terme.En conclusion, l’activité collagénolytique de MMP-14 est un des mécanismes moléculaires possibles del’évolution de la plaque athéromateuse par rupture de plaque. Elle ouvre la perspective d’une nouvelleapproche thérapeutique et pourrait être une cible comme substrat pour une imagerie fonctionnelle de laplaque athéromateuse. L’évolution de la maladie anévrysmale pourrait être secondaire à une altération dessystèmes de réparation tissulaire dont les CSMs seraient des acteurs clé. La perfusion endoluminale desCSMs dans un modèle expérimental a permis la restauration de ces systèmes de réparation tissulaire etouvre la perspective d’un nouvel outil thérapeutique contre les AAA / Pas de résumé anglais

Osteogenic differentiation of mesenchymal stem cells (MSCs) would be possible to induce by creating of a cell bioactive scaffold that mimic the properties of bone extracellular matrix (ECM). This induction will be not only due to the addition of osteogenic supplements, but also due to the addition of differentiation peptides. These peptides activate signaling pathways leading to cell differentiation. The aim of this study was to evaluate the effect of selected peptides on adhesion, metabolic activity, proliferation and osteogenic differentiation of porcine MSCs. Four peptides with amino acid sequences of DGEA, IAGVGGEKSGGF, GQGFSYPYKAVFSTQ and KIPKASSVPTELSAISTLYL were selected. These peptides were derived from receptor binding sequences of collagen I, collagen III, BMP-7 and BMP-2 respectively. Scaffolds were prepared from a biocompatible and biodegradable poly-ε-caprolactone (PCL) polymer, suitable for cell cultivation. Cells were cultured on scaffolds for three weeks. Various concentration of differentiation peptides were added to the culture medium. As observed in the experiment of cells cultured in basal medium supplemented with differentiation peptides no effect on adhesion, proliferation or metabolic activity of porcine MSCs was observed. In groups treated with peptides derived from BMP-2...

Mesenchymal stem cells (MSC) represent a heterogenous population of nonhematopoietic stem cells with multipotent differential potential. MSC can be isolated from various tissues of organism, the most common tissue are bone marrow or adipose tissue. MSC are good candidates for treatment of autoimmune diseases and possess the ability to prevent graft rejection or graft versus host disease. The immunosuppressive drugs are currently used for inhibition of unwanted immune reaction but they exhibit serious side effects. The use of MSC in therapy can reduce doses of immunosuppressive drugs and eliminate side effects. The study of MSC and immunosuppressant interactions should be detected before MSC can be used for clinical application. We aimed to analyze the interaction between MSC and immunosuppressive drugs and their effect on immune cells. Cyclosporine A and mycophenolate mofetil were used in our research. We demonstrated changes in the expression of surface molecules, production of interleukin 6 and in metabolic activity of MSC after treatment with immunosuppressive drugs. MSC are in organism, in cooperation with the number of other cells. Therefore we studied MSC cocultured with splenocytes in the presence of immunosuppressive drugs. Our results show the effect of MSC and immunosuppressive drugs on different...

Mesenchymal stem cells (MSCs) represent a heterogeneous population of stromal cells with a pluripotent differentiation potential. They can be isolated from multiple tissues of mesodermal origin, such as bone marrow, adipose tissue, umbilical cord blood and peripheral blood and afterwards externally expanded according their adherence to the plastic surfaces. These cells show remarkable immunomodulatory properties, suppressing T-, B- and NK-cell functions, and also modulating dendritic cell activities and influencing immune responses during tissue repair and recovery. MSCs have been shown to possess ability to migrate to sites of inflammation and tissue injury. All these properties make MSCs a promising tool for clinical application. Our primary goal was to identify processes that may influence immunoregulatory effects of MSCs. In order to promote immunossupressive qualities of MSCs we established the scheme comprising MSCs precultivated with various cytokines and Toll-like receptors (TLR) ligands in vitro, with the final aim to improve the therapeutic effect of MSCs on wound healing in vivo. We studied modulation of MSCs properties and consequently the effect of influenced MSCs on cells of the immune system. The immunosuppression is mainly mediated through secreted factors that MSCs produced after...

Electrospinning is widely used technique to produce nanoscale constructs for tissue engineering. This technique can be used to spin wide range of polymers. One of them is polyvinyl alcohol (PVA), which has very good properties for use in this field. PVA is nontoxic, has good mechanical strength and it's degradable and biocompatible. Electrospun PVA nanofibers have limitations because of their -OH side groups. These groups cause solubility of PVA in water. The solubility can be adjusted with crosslinking techniques, but PVA still remains very hydrophilic, which is causing low adhesion of cells. In recent research we decided to reduce the hydrophilicity of PVA using plasma modification. Polymer modification with cold plasma is an economic and quite simple process to change the surface chemistry without side effects that come with conventional chemical treatment. With radical, formed by discharge, we have deposited hydrocarbons on the PVA surface and we rapidly increased hydrophobicity of the polymer surface. The change of surface chemistry has only a little effect on the fiber morphology. The increase of hydrophobicity allowed better adhesion of mesenchymal stem cells on plasma modified PVA as compared to non-modified PVA and a huge change in cell morphology was observed. These changes suggest that we...