The development of functional and oriented microvessels within tissue constructs is a tremendous challenge in tissue engineering and regenerative medicine. It is justified by the need to allow better nutrient and oxygen supply and waste removal within the core of tissue constructs. It is one of the reasons why only few tissue substitutes are available to clinicians. Therefore, the focus of many research studies in the field of tissue engineering has been placed on promoting microvessel ingrowth inside tissue constructs prior to their implantation, as presented in Chapter 1. This process is often referred to as pre-vascularization. As such, Chapter 2 of this thesis is devoted to review the recent development and future challenges related to the microvascularization process of tissue constructs.The experimental work in this thesis is based on the hypothesis that polymer monofilaments precisely distanced from each others can be used to guide endothelial cells when dispersed in a (fibrin) gel and to induce tube-like structures in a directional fashion. In Chapter 3 of this thesis, the use of polymer fibres as contact guidance of endothelial cells to orient microvessel formation and development in a three-dimensional environment was validated.The novel cell attachment method described in Chapter 3 has resulted in an increase in Human Umbilical Vein Endothelial Cell (HUVEC) adhesion and spreading on bare poly(ethylene terephthalate) (PET) fibres. Furthermore, HUVEC-covered fibres were sandwiched between fibrin gels to allow the development of microvessels. Angiogenesis development was characterized and evaluated using a number of imaging techniques, including fluorescence microscopy and confocal microscopy. After 4 days of culture, microvessels formed large tube-like structures (diameter of approximately 100 [micro]m) between adjacent fibres distanced by 0.1mm. This proof-of-concept opens the door to other experiments and to possibility of scaling the system to bioreactor cultures. In Chapter 4, the effect of human fibroblasts and in another set of experiments, the influence of two angiogenic growth factors (i.e., Vascular Endothelial Growth Factor (VEGF) and basic Fibroblast Growth Factor (bFGF)) over the responses of the HUVEC-covered fibres sandwiched in fibrin were investigated.The development and maturation of microvessels as well as the assessment of lumen formation were evaluated using a fluorescent fibrin matrix, confocal microscopy and histology. Histology and fluorescent fibrin experiments confirmed that these microvessel-like structures formed between polymer monofilaments embedded in fibrin contained a lumen.The effects of VEGF and bFGF were dose dependent. Furthermore, the use of fibroblasts significantly improved the maturation of the microvessels compared to control and to samples cultured with VEGF and bFGF. Conclusions and suggested future work are presented in Chapter 5. Appendices A and B present the detailed protocols used to label cells in this study and general information about cell cytoskeleton, respectively.

Toward the objective to create or to replace impaired tissues, it is essential to establish a culture process allowing tissue growth in vitro . The Petri dish culture or the traditional 2D culture has only a limited potential, mainly caused by a poor oxygen mass transfer to feed larger tissue constructs. In this project, an autonomous and complete bioprocess has been built to fullfill these needs. The developed reactor is versatile because many cell culture chamber designs can be connected to it. Two proportional-integral algorithms (PI) can control the dissolved oxygen concentration and the pH. The pressure, the temperature and mass flow rate are recorded in real time. An actuator allows mimicking the cardiac output flow. In this mémoire , it will be demonstrated how the reactor has been optimized to reduce the risk of bioburden. Also, the procedures to develop the control tools of the PI algorithm will be detailed. To characterize the reactor, a RTD study will be presented. To characterize the cell culture chamber, a permeability analysis using fluorescent microscopy and magnetic resonance imaging (MRI) will be exposed. Finally, two cell culture chamber designs to orient microvessel formation have been tested and these results will be presented.

La néphropathie diabétique est l’une des complications les plus fréquentes chez les patients diabétiques à long terme, et est la première cause du besoin de dialyse. Les lésions glomérulaires semblent jouer un rôle clé dans le développement de la néphropathie diabétique. L’épaississement de la membrane basale glomérulaire, l’hypertrophie des cellules glomérulaires et la perte de podocytes font partie des principaux changements pathologiques survenant au cours de la néphropathie diabétique et peuvent conduire à une protéinurie. Il a été suggéré que le dysfonctionnement endothélial joue un rôle important dans la pathogenèse des lésions glomérulaires au cours de la maladie rénale diabétique. En effet, l’altération de la fonction et de l’intégrité des cellules endothéliales glomérulaires est l’une des principales causes de la microalbuminurie observée dans l’insuffisance rénale diabétique précoce. Les lésions des cellules endothéliales glomérulaires peuvent endommager les podocytes et même induire une perte podocitaire ce qui aggrave d’avantage les liaisons des cellules endothéliales glomérulaires et ainsi de suite. Actuellement, les traitements de la néphropathie diabétique visent le contrôle de la glycémie et de la pression artérielle dans le but de maintenir un bon débit de filtration glomérulaire. Cependant, l’étude de traitements pouvant cibler les lésions endothéliales ou les interactions podocyte-cellule endothéliales, qui jouent un rôle essentiel dans la progression de la maladie rénale diabétique, est nécessaire. Les traitements ciblant l’endothélium glomérulaire pourraient offrir des avantages thérapeutiques pour la néphropathie diabétique. En effet, des facteurs anti-angiogéniques tels que les inhibiteurs du VEGF pourraient prévenir les lésions rénales et les altérations glomérulaires sur des modèles de souris diabétiques. Cependant, d’autres données ont montré que des injections d’inhibiteurs du VEGF pouvaient être néfastes pour les cellules endothéliales et podocytaires. Ainsi, de nouvelles molécules ciblant l’endothélium vasculaire pourraient améliorer le pronostic et la qualité de vie chez les patients présentant une insuffisance rénale diabétique à un stade précoce. Nous avons précédemment montré que Alk1, avec son ligand BMP9, joue un rôle important dans le maintien de l’intégrité vasculaire chez les animaux diabétiques. En effet, 4 la perte de signalisation d’Alk1 chez les animaux diabétiques conduit à la dissociation des jonctions vasculaires et à une augmentation des fuites vasculaires dans la rétine. Compte tenu de son rôle dans le maintien de la quiescence et de l’intégrité de l’endothélium, nous avons évalué les effets de la surpression d’Alk1 sur l’intégrité de l’endothélium glomérulaire et la fonction rénale chez la souris diabétique. Nous avons utilisé des souris avec délétion conditionnelle de Alk1 dans l’endothélium (Alk1ΔEC) pour évaluer le rôle de Alk1 dans la filtration glomérulaire chez des souris diabétiques induits par le STZ. Les souris ont été euthanasiées quatre mois après le début du diabète et des analyses sérologiques et urinaires ont été effectuées, ainsi que des études immunohistochimiques. Nous avons démontré que l’haplo-insuffisance d’Alk1 aggrave la microalbuminurie et induit une perte de podocytes chez des souris diabétiques. De plus, une augmentation significative de l’apoptose glomérulaire a été observée chez les souris Alk1ΔEC hétérozygotes diabétiques. L’analyse de souris Alk1ΔEC homozygotes non diabétiques a également révélé une perte importante de cellules endothéliales glomérulaires. Ensemble, ces données suggèrent que la signalisation du récepteur Alk1 joue un rôle essentiel dans le maintien des cellules endothéliales glomérulaires et participe au maintien de l’intégrité glomérulaire à travers un mécanisme de podocyte-endothelial cross-talk. / Diabetic kidney disease one of the most frequent microvascular long-term complications in diabetic patients and is the first cause for the need for dialysis. The glomerular damage seems to play a key role in the development of diabetic nephropathy. Thickening of the glomerular basement membrane, glomerular cell hypertrophy, and podocyte loss is among the main pathological changes occurring during diabetic nephropathy and can lead to proteinuria. Endothelial dysfunction has been suggested to play an important role in the pathogenesis of glomerular damage during diabetic kidney disease. Indeed, alteration of the glomerular endothelial cell function and integrity is a leading cause of microalbuminuria observed in early diabetic kidney disease. Injury to glomerular endothelial cells may lead to podocyte damage, while podocyte loss further exacerbates glomerular endothelial cell injury, forming a vicious cycle. Currently, therapies in diabetic nephropathy are focusing on glycemia control and adequate arterial pressure levels in order to maintain an adequate glomerular filtration rate. However, the study of some treatments that may target endothelial lesions or podocyte-endothelial cell interactions, which play a vital role in the progression of diabetic kidney disease is necessary. It has been suggested that antiangiogenic treatments for diabetic kidney disease could provide therapeutic benefits. Indeed, anti-angiogenic factors such as VEGF inhibitors have been demonstrated to suppress renal damage and glomerular alterations in a diabetic mouse model. However, some other data have shown that anti-VEGF injections could be detrimental for podocytes and endothelial cells. Thus, new molecules targeting the vascular endothelium could possibly improve prognosis and quality of life in patients with early stages of diabetic kidney disease. We have previously shown that Alk1, along with its ligand BMP9, plays an important function to maintain vascular integrity in diabetic animals. Loss of Alk1 signaling in diabetic animals led to dissociation of vascular junctions and increased vascular leakage. Given its role in the maintenance of endothelial quiescence and integrity, we evaluated the effects of Alk1 suppression on kidney integrity and renal function in diabetic mice. 6 We used mice with conditional deletion of Alk1 in the endothelium (Alk1ΔEC) to evaluate the role of Alk1 in glomerular filtration in STZ-induced diabetic mice. Mice were euthanized four months after the onset of diabetes and urine, and serological analyzes were performed, along with immunohistochemical studies. We demonstrated that Alk1 haploinsufficiency worsens microalbuminuria and induces podocyte loss. Furthermore, a significant increase in glomerular apoptosis was observed in Alk1ΔEC mice. Analysis of homozygous Alk1ΔEC mice also revealed a significant loss of glomerular endothelial cells. Together, these data suggest that Alk1/BMP9 signaling plays a critical role in the maintenance of glomerular endothelial cells and has important functions to maintain glomerular integrity through a crosstalk podocyte-endothelial mechanism.