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The overall goal of this research project is to develop methods for producing a tissue engineered ligament. The envisioned tissue engineering strategy involves three steps: seeding bone marrow stromal cells (BMSCs) onto electrospun scaffolds, processing them into cords that allow cell infiltration, and conditioning them with uniaxial cyclic stretch. These steps were addressed in three complimentary studies to establish new methods to engineer a tissue with ligament-like cells depositing organized extracellular matrix (ECM). In the first study scaffold topographies were systematically varied to determine topographies that induce cells to orient and differentiate into ligament-like cells in static culture. Scaffolds — electrospun from poly (ester-urethane urea) (PEUUR) with different fiber diameters degrees of fiber alignments — were biocompatible and supported cell growth. Topographic cues guided cell alignment, and cell elongation increased with increasing fiber alignment. Finally, expression of the ligament-like markers collagen type I and decorin were enhanced on the smallest fiber diameters compared to larger diameters. In the second study BMSCs — seeded onto aligned electrospun PEUUR scaffolds — were cyclically stretched to determine the effect of dynamic mechanical stimulation on BMSC alignment and differentiation. BMSCs remained aligned parallel to the direction of fiber alignment and expressed ligament markers (e.g. collagen type I, decorin, scleraxis, and tenomodulin) on electrospun scaffolds after the application of stretch. However, the cyclic stretch regimen was not able to enhance expression of ECM components. In the third study techniques were developed to produce more clinically relevant constructs with improved cell infiltration. Specifically, a co-electrospun scaffold composed of two well integrated components was developed to create larger pores. The scaffold was also embedding in a photo-crosslinkable hydrogel to prevent the fibers from collapsing. These results demonstrate the feasibility of making a tissue engineered ligament by seeding BMSCs on an aligned, co-electrospun scaffold with submicron diameter fibers and then applying cyclic mechanical stretch. Future work will involve combining these three steps to achieve materials suitable for in vivo testing. / Ph. D.

3D printing technology is rapidly advancing and is being increasingly used for biological applications. The spatial control of 3D printing makes it especially attractive for fabricating 3D tissues and for studying the role of geometry in biology. We utilized two different types of 3D printing to engineer vascularized tissues with complex vascular architectures, to use engineered vasculature to treat ischemia, and to study directional endothelial cell migration on curved wave topography. To engineer 3D tissues, perfusable vascular networks must be embedded within the tissue to supply nutrients and oxygen to cells. 3D-printed sugar filaments have previously been used as a cytocompatible sacrificial template to rapidly cast vascular networks. We improved upon the 3D-printed sugar method and used it to fabricate complex vascular geometries that were not previously possible, such as a branched channel geometry, with controlled fluid flow through the channels. We also integrated an approach utilizing vascular self-assembly to generate thick tissues with dense, capillary-scale vessel networks. The vascularized tissues fabricated using 3D-printed sugar successfully integrated with a host vasculature upon implantation and restored perfusion in two different animal models of ischemia. Cell migration critical to numerous biological processes can be guided by surface topography. However, fabrication limitations constrain topography studies to geometries that may not adequately mimic physiological environments. Direct Laser Writing (DLW) provides the necessary 3D flexibility and control to create well-defined curved waveforms similar to those found in physiological settings, such as the lumen of blood vessels. We found that endothelial cells migrated fastest along square waves, intermediate along triangular waves, and slowest along sine waves and that directional cell migration on sine waves decreased at longer sinusoid wavelengths. Interestingly, inhibition of Rac1 decreased directional migration on 3D sine waves but not on 2D micropatterned lines, suggesting that cells may utilize different molecular pathways to sense curved topographies. Our study demonstrates that DLW can be employed to investigate directional migration on a wide array of surfaces with curvatures that are unattainable using conventional manufacturing techniques. / 2020-10-22T00:00:00Z

In this study, collagen based scaffolds were prepared for cornea tissue engineering. Three different cell carriers (rat tail collagen foam, insoluble collagen foam and patterned collagen film) were produced using two different collagen sources. Scaffolds were designed to mimic the unique topographical features of the corneal stroma. A novel crosslinking method was developed to achieve constant foam thickness. All scaffolds were tested with the primary cells of the native corneal stroma, human keratocytes. Although both foams promoted cell growth and penetration, rat tail foams were found to be superior for keratocyte proliferation. Their degradation rates were high enough but did not compromise their structural integrity during testing. Transparency studies with the foams revealed a progressive improvement. Collagen films degraded significantly over a one month period / however, the presence of cells increased the tensile strength of the films over a 21 day period to close to that of the native cornea and compensated for the loss of strength due to degradation. The micropatterned films proved to have higher transparency than the unpatterned scaffolds. In this study, it was possible to prepare collagen based micropatterned scaffolds using a silicon wafer and then a silicone template, successively, starting from original designs. The resultant collagen films were able to control cell growth through contact guidance, restricted cells and secreted-ECM within the pattern grooves, resulting in a higher transparency in comparison to unpatterned films. Thus, the tissue engineered constructs revealed a significant potential for use as total artificial corneal substitutes.

Metastasis is the leading cause of death among cancer patients. The metastatic cascade, during which cancer cells from the primary tumor reach a distant organ and form multiple secondary tumors, consists of a series of events starting with cancer cells invasion through the surrounding tissue of the primary tumor. Invading cells may perform proteolytic degradation of the surrounding extracellular matrix (ECM) and directed migration in order to disseminate through the tissue. Both of the mentioned processes are profoundly affected by several parameters originating from the tumor microenvironment (extrinsic) and tumor cells themselves (intrinsic). However, due to the complexity of the invasion process and heterogeneity of the tumor tissue, the exact effect of many of these parameters are yet to be elucidated. ECM proteolysis is widely performed by cancer cells to facilitate the invasion process through the dense and highly cross-linked tumor tissue. It has been shown in vivo that the proteolytic activity of the cancer cells correlates with the cross-linking level of their surrounding ECM. Therefore, the first part of this thesis seeks to understand how ECM cross-linking regulates cancer cells proteolytic activity. This chapter first quantitatively characterizes the correlation between ECM cross-linking and the dynamics of cancer cells proteolytic activity and then identifies ß1-integrin subunit as a master regulator of this process. Once cancer cells degrade their immediate ECM, they directionally migrate through it. Bundles of aligned collagen fibers and gradients of soluble growth factors are two well-known cues of directed migration that are abundantly present in tumor tissues stimulating contact guidance and chemotaxis, respectively. While such cues direct the cells towards a specific direction, they are also known to stimulate cell cycle progression. Moreover, due to the complexity of the tumor tissue, cells may be exposed to both cues simultaneously, and this co-stimulation may happen in the same or different directions. Hence, in the next two chapters of this thesis, the effect of cell cycle progression and contact guidance-chemotaxis dual-cue environments on directional migration of invading cells are assessed. First, we show that cell cycle progression affects contact guidance and not random motility of the cells. Next, we show how exposure of cancer cells to contact guidance-chemotaxis dual-cue environments can improve distinctive aspects of cancer invasion depending on the spatial conformation of the two cues. In this dissertation, we strive to achieve the defined milestones by developing novel mathematical and experimental models of cancer invasion as well as utilizing fluorescent time-lapse microscopy and automated image and signal processing techniques. The results of this study improve our knowledge about the role of the studied extrinsic and intrinsic cues in cancer invasion. / Bioengineering / Accompanied by fourteen .avi files.

Grafts currently used for the repair of anterior cruciate ligament (ACL) ruptures integrate poorly with bone due to a significant mismatch in properties between graft and bone. Specifically, conventional grafts (e.g., hamstring tendon) are unable to recapitulate intricate gradients in mechano-chemical properties and extracellular matrix (ECM) architecture found at natural bone-ligament (B-L) transitions, and thus result in stress-concentrations at the graft-bone interface leading to graft failure. In contrast, tissue-engineered scaffolds possessing gradients in properties can potentially guide the establishment of phenotypic gradients in bone marrow stromal cells (BMSCs), and thus aid the regeneration of B-L transitions in the long-term. Towards the eventual goal of regenerating complex tissue transitions, this project employs three complementary strategies to fabricate graded scaffolds. The three strategies involve the presentation of gradients in 1) mineral content, 2) scaffold architecture and 3) growth factor (GF) concentration within scaffolds to control BMSC morphology and phenotype. The first strategy involved co-electrospinning two polymers (one doped with hydroxyapatite) from offset spinnerets onto a rotating drum to produce scaffolds possessing a gradient in mineral content. Post-electrospinning, these graded scaffolds were treated with a simulated body fluid to further enhance the gradient. Analysis of mRNA expression of osteoblastic makers by BMSCs and the deposition of bone-specific ECM proteins indicated that the scaffolds could guide the formation of an osteoblastic phenotypic gradient. The second strategy involved electrospinning two polymer solutions onto a custom-designed dual-drum collector to fabricate scaffolds possessing region-wise differences in fiber alignment, diameter and chemistry. Specifically, electrospinning onto the dual-drum collector resulted in the deposition of aligned fibers from one polymer solution in the gap region between the drums, randomly oriented fibers from the other polymer solution on one of the drums and a mixture of fibers from both polymer solutions in the overlap region in between. The topographical cues within these scaffolds were shown to result in region-dependent BMSC morphology and orientation. Although the long-term goal of the third strategy was to create a co-electrospun scaffold possessing a gradient in GF concentration, a new technique to protect GF activity within electrospun scaffolds via the use of gelatin microspheres was first validated. Preliminary results from these studies indicate that microspheres can protect and deliver a model protein (lysozyme) in active conformation from electrospun scaffolds. These results further suggest that gradients of GF concentration can be achieved in the long-term by protecting GFs within microspheres and co-electrospinning as described in the first strategy. In conclusion, the results from this project suggest that graded scaffolds can help guide the formation of gradients in cell morphology, orientation and phenotype, and thus potentially promote the regeneration of B-L transitions in the long-term. The three strategies described in this project can be employed in concert to create scaffolds intended for the regeneration of complex tissue transitions. / Ph. D.

Dans le cerveau en développement, les interneurones corticaux effectuent une longue migration avant de se positionner dans le cortex et s’intégrer dans les réseaux corticaux dont ils régulent l’activité. Différents facteurs chimiques ont été impliqués dans le guidage de ces cellules, mais l’influence des propriétés physiques de l’environnement dans lequel ils naviguent reste peu connue. Il a été montré que les indices topographiques peuvent guider le mouvement de nombreux types cellulaires, un processus appelé guidage par contact. Mes travaux de thèse ont ainsi cherché à tester et comprendre l’influence de la topographie de l’environnement sur la migration des interneurones corticaux. En utilisant un système expérimental de substrats microstructurés, nous avons mis en évidence pour la première fois l’existence du guidage par contact pour ces cellules. En testant deux types de micro-plots, nous avons établi qu’un changement de forme des structures influence de manière importante l’orientation, la morphologie, l’organisation du cytosquelette et le comportement dynamique des cellules. En particulier, les interneurones en migration entre des plots carrés adoptent majoritairement une morphologie allongée et peu branchée, associée à un mouvement lent et dirigé. A l’inverse, des cellules entre des plots ronds sont plus courtes et montrent un branchement important associé à un mouvement dynamique mais aléatoire. Plus généralement, nous montrons in vitro que la topographie génère des contraintes spatiales globales qui promeuvent la mise en place de différents états cellulaires morphologiques et dynamiques, soulignant ainsi la potentielle importance de ce type d’indices in vivo. / In the developing brain, cortical interneurons undergo a long distance migration to reach the cortex where they integrate into cortical networks and regulate their activity in the adult. Different chemical factors have been involved in the guidance of these cells, but the influence of the physical parameters of the environment in which they navigate remains unclear. It has been shown that topographical cues are able to influence and guide the migration of several cell types, a process called contact guidance. This work therefore aimed at testing and understanding the influence of the topography of the environment in the migration of cortical interneurons. By using an experimental system of microstructured substrates, we demonstrated for the first time the existence of contact guidance for these cells. By testing two types of micron-sized pillars, we showed that a change in the shape of the structures could greatly impact cell orientation, morphology, cytoskeleton organization and dynamic behavior. In particular, most interneurons migrating in between square pillars adopt an elongated, unbranched morphology associated with a slow and directed movement, whereas the majority of cells among round pillars exhibit a short and branched morphology associated with a dynamic but wandering movement. Overall, we show that micron-sized topography provides global spatial constraints promoting the establishment of different morphological and migratory states in vitro, highlighting the potential importance of these types of cues in vivo.