Every year, 400,000 coronary artery bypasses (CABG) are performed in the United States. However, one third of all patients who need a CABG cannot undergo the procedure because of the lack of suitable autologous blood vessels. Both synthetic and tissue engineered vascular grafts have been used clinically for vascular grafts or other surgical applications, but no small- diameter engineered vessels have yet been successfully used for CABG. The success of vascular tissue engineering is strongly dependent on being able to control tissue contractility and extracellular matrix (ECM) production to achieve balance between tissue strength and physiological function. Smooth muscle cells (SMCs), the main contributor of contractility in blood vessels, retain phenotypic plasticity, meaning they possess the ability to switch between a contractile and synthetic phenotype. In 2D culture, a number of biochemical and mechanical cues have been shown to promote the switch to a contractile phenotype in SMCs. However, achieving a stable contractile phenotype in 3D tissue has proven difficult. The work in this dissertation describes an investigation of how passive and dynamic environmental cues influence the smooth muscle phenotype. We studied the effects of substrate modulus in conjunction with changes in cell culture media composition on SMC phenotype in 2D and 3D cultures. Culturing SMCs in a low-serum culture medium resulted in an increase in SMC contractility in 2D cell culture but not in 3D cell-derived tissue. We found that, in SMCs cultured on soft substrates, the ability to modulate SMC phenotype in response to changes in media was diminished. Passively crosslinking the ECM of our cell-derived tissues with genipin resulted in modest increases in elastic modulus, though not enough to observe changes in SMC phenotype. Additionally, we investigated how dynamic cyclic mechanical stretch, in conjunction with cell culture medium, modified SMC contractility in cell and tissue cultures. SMCs increased contractile protein expression when exposed to dynamic stretch in 2D culture, even on soft substrates, which have previously been shown to inhibit phenotypic modulation. In 3D tissue rings, after mechanical stimulation, SMCs became more aligned, the tissue became tougher, and SMCs exhibited a measurable increase in contractile protein expression. In summary, we found that increasing substrate modulus, culturing in low serum cell culture medium, and imparting cyclic mechanical stretch can promote SMC differentiation and cellular alignment, and improve tissue mechanical properties. This information can be used to more accurately recapitulate vascular tissue for use in modeling or in the creation of tissue engineered blood vessels.

La motilité des cellules est un phénomène fondamental en biologie souvent étudié sur des surfaces planes, conditions peu physiologiques. Nous avons analysé la migration cellulaire dans une matrice cellulaire 3D contenant de la fibronectine fluorescente. Nous démontrons que les cellules y sont confinées, et déforment leur environnement de manière cyclique avec une période de ~14 min avec deux centres de contractions à l’avant et à l’arrière de la cellule qui contractent avec un déphasage de ~3.5 min. Une perturbation de ces cycles entraîne une réduction de la motilité. Par l’utilisation d’inhibiteurs spécifiques, nous avons identifié l’acto-myosine comme étant l’acteur principal de ce phénomène. En imposant des contractions-relaxations locales par ablations laser, nous avons déclenché la motilité cellulaire ce qui confirme notre hypothèse. L’ensemble de cette étude met en évidence un nouveau mécanisme fondamental de dynamique cellulaire impliqué dans le mouvement des cellules. / Cell motility is an important process in Biology. It is mainly studied on 2D planar surfaces, whereas cells experience a confining 3D environment in vivo. We prepared a 3D Cell Derived Matrix (CDM) labeled with fluorescently labeled fibronectin, and strikingly cells managed to deform the matrix with specific patterns : contractions occur cyclically with two contraction centers at the front and at the back of the cell, with a period of ~14 min and a phase shift of ~3.5 min. These cycles enable cells to optimally migrate through the CDM, as perturbation of cycles led to reduced motility. Acto-myosin was established to be the driving actor of these cycles, by using specific inhibitors. We were able to trigger cell motility externally with local laser ablations, which supports this framework of two alternating contractions involved in motion. Altogether, this study reveals a new mechanism of dynamic cellular behaviour linked to cell motility.

The purpose of this research was to study the synthesis and remodeling of extracellular matrix (ECM) by fibroblasts with special emphasis on the culture environment (media composition and initial ECM composition) and the resulting mechanical integrity of the ECM. This was investigated by culturing fibroblasts for 3 weeks in a variety of culture conditions consisting of collagen gels, fibrin gels, or media permissive to the self-production of ECM (Cell-Derived Matrix), and quantifying the mechanics of the resulting ECM. The mechanical characteristics were related to the biochemistry of the resulting ECM, notably in terms of collagen accumulation and collagen fibril diameters. The ultimate tensile strength (UTS) of the collagen gels and fibrin gels at the end of the 3-week period was 168.5 ± 43.1 kPa and 133.2 ± 10.6 kPa, respectively. The ultimate tensile strength of the cell-derived matrices was 223.2 ± 9 kPa, and up to 697.1 ± 36.1 kPa when cultured in a chemically-defined medium that was developed for the rapid growth of matrix in a more defined environment. Normalizing the strength to collagen density resulted in a UTS / Collagen Density in these groups of 6.4 ± 1.9 kPa/mg/cm3, 25.9 ± 2.4 kPa/mg/cm3, 14.5 ± 1.1 kPa/mg/cm3, and 40.0 ± 1.9 kPa/mg/cm3, respectively. Cells were synthetically more active when they produced their own matrix than when they were placed within gels. The resulting matrix was also significantly stronger when it was self-produced than when the cells rearranged the matrix within gels that corresponded to a significantly larger fraction of non-acid and pepsin extractable collagen. These studies indicate that cell-derived matrices have potential both as in vitro wound healing models and as soft connective tissue substitutes.