Alignment of Cells and Extracellular Matrix Within Tissue- Engineered Substitutes

Most of the cells in our body are in direct contact with extracellular matrix (ECM) compo‐ nents which constitute a complex network of nano-scale proteins and glycosaminoglycans. Those cells constantly remodel the ECM by different processes. They build it by secreting dif‐ ferent proteins such as collagen, proteoglycans, laminins or degrade it by producing factors such as matrix metalloproteinase (MMP). Cells interact with the ECM via specific receptors, the integrins [1]. They also organize this matrix, guided by different stimuli, to generate pat‐ terns, essential for tissue and organ functions. Reciprocally, cells are guided by the ECM, they modify their morphology and phenotype depending on the protein types and organization via bidirectional integrin signaling [2-4]. In the growing field of tissue engineering [5], control of these aspects are of the utmost importance to create constructs that closely mimic native tis‐ sues. To do so, we must take into account the composition of the scaffold (synthetic, natural, biodegradable or not), its organization and the dimension of the structure. The particular alignment patterns of ECM and cells observed in tissues and organs such as the corneal stroma, vascular smooth muscle cells (SMCs), tendons, bones and skeletal mus‐ cles are crucial for organ function. SMCs express contraction proteins such as alpha-smoothmuscle (SM)-actin, desmin and myosin [6] that are essential for cell contraction [6]. To result in vessel contraction, the cells and ECM need to be organized in such a way that most cells are elongated in the same axis. For tubular vascular constructs, it is suitable that SMCs align in the circumferential direction, as they do in vivo [7, 8]. Another striking example of align‐ ment is skeletal muscle cells that form long polynuclear cells, all elongated in the same axis. Each cell generates a weak and short contraction pulse but collectively, it results in a strong, long and sustained contraction of the muscle and, in term, a displacement of the member. In the corneal stroma, the particular arrangement of the corneal fibroblasts (keratocytes) and ECM is essential to keep the transparency of this tissue [9-13]. Tendons also present a pecu‐ liar matrix alignment relative to the muscle axis. It gives a substantial resistance and excep‐ tional mechanical properties to the tissue in that axis [14, 15]. Intervertebral discs [16], cartilage [17], dental enamel [18], and basement membrane of epithelium are other examples of tissues/organs that present peculiar cell and matrix organization. By reproducing and controlling those alignment patterns within tissue-engineered substitutes, a more physiolog‐ ical representation of human tissues could be achieved. Taking into account the importance of cell microenvironment on the functionality of tissue engineered organ substitutes, one can assume the importance of being able to customise the 3D structure of the biomaterial or scaffold supporting cell growth. To do so, some methods have been developed and most of them rely on topographic or contact guidance. This is the phenomenon by which cells elongate and migrate in the same axis as the ECM. Topographic guidance was so termed by Curtis and Clark [19] to include cell shape, orientation and movement in the concept of contact guidance described by Harrison [20] and implemented by Weiss [21, 22]. Therefore, if one can achieve ECM alignment, cells will follow the same pattern. Inversely, if cells are aligned on a patterned culture plate, the end result would be aligned ECM deposition [23]. The specific property of tissues or materials that present a variation in their mechanical and structural properties in different axis is called anisotropy. This property can be evaluated ei‐ ther by birefringence measurements [24, 25], mechanical testing in different axis [26], immu‐ nological staining of collagen or actin filaments [23] or direct visualisation of collagen fibrils using their self-fluorescence around 488 nm [27, 28]. Several techniques have been recently developed to mimic the specific alignment of cells within tissues to produce more physiologically relevant constructs. In this chapter, we will describe five different techniques, collagen gel compaction, electromagnetic field, electro‐ spinning of nanofibers, mechanical stimulation and microstructured culture plates

Rôle de la microcirculation et du microenvironnement sur la fonctionnalité de substituts vasculaires reconstruits par génie tissulaire

Les cellules et la matrice extracellulaire composant les tissus humains ont une architecture tridimensionnelle organisée leur conférant des propriétés propres à leur physiologie. La plupart des substituts créés par génie tissulaire ne possèdent pas cette organisation architecturale leur permettant d'être physiologiquement comparable aux tissus à remplacer. Cela s'avère particulièrement crucial lors de la conception de substituts vasculaires où les propriétés mécaniques ont une importance primordiale afin de résister à la pression artérielle, mais aussi la compliance nécessaire pour ne pas induire une athérosclérose postimplantation due à une trop grande rigidité. De plus, les présents modèles vasculaires conçus par génie tissulaire n'intègrent pas la microcirculation dans les parois vasculaires. Les vasa vasorum, capillaires de l'adventice, servent à nourrir et oxygéner les parois des vaisseaux sanguins et ont probablement un rôle clé dans l'intégration dans les tissus avoisinants des substituts vasculaires. À ces fins, nous avons conçu un biomatériau pouvant être microstructuré afin de reproduire une topographie similaire à celle composant le micro environnement des tissus. Les polymères thermoplastiques permettent la production à grande échelle de substrats avec une nanotopographie de surface permettant l'alignement des cellules et de la matrice extracellulaire. Nous avons fait la démonstration que ces substrats permettent l'auto-assemblage tridimensionnel des cellules et de la matrice extracellulaire suivant un angle précis, lequel correspond à la même organisation physiologique intrinsèque du tissu et varie en fonction du type cellulaire en culture. Pour les cellules musculaires lisses vasculaires composant la média, nous avons montré que cette organisation se traduit par une augmentation de plus de 100% en résistance mécanique. D'autre part, une nouvelle technique de culture cellulaire pour les adventices vasculaires a permis de créer un réseau de capillaires in vitro s'apparentant au vasa vasorum. Ces substituts vasculaires ont été implantés en sous-cutané dans un modèle animal permettant de démontrer la rapidité d'intégration des tissus pré-vascularisés (48h) en comparaison avec des tissus non-vascularisés (141). Ces améliorations aux substituts vasculaires leurs confèrent une importante ressemblance anatomique et physiologique avec les vaisseaux utilisés notamment pour les pontages des artères soit coronaires ou périphériques, les rapprochant ainsi d'une utilisation en clinique

Ultrashort-pulsed laser ablation of poly-L-lactide (PLLA) for cell and tissue engineering applications

El contenido de los capítulos 3 y 4 están sujetos a confidencialidad 167 p.La tecnología de ablación láser es una herramienta bien establecida para la modificación superficial de materiales de distinta naturaleza (metales, polímeros, cerámicas, vidrio¿). La ablación de material mediante láseres de pulso ultracorto (menor que 10 picosegundos) es capaz de generar motivos topográficos micrométricos con una alta precisión debido a un proceso de ablación ¿frío¿ minimizando los efectos térmicos en el material sin producir cambios químicos en el mismo. Es por tanto una tecnología versátil para la fabricación de superficies microestructuradas en un proceso directo y sin contacto y aplicable a una gran variedad de materiales para generar motivos con distintas geometrías sobre superficies no planas. En este trabajo de tesis se aplica la tecnología de ablación mediante láser pulsado de picosegundos para la creación de micro-patrones topográficos en planchas de ácido poli-L-láctico (PLLA), para investigar el mecanismo de ablación del mismo y el efecto de los micro-patrones en el comportamiento de varios tipos de células mediante ensayos in vitro, con el objetivo final de elucidar el alcance de la influencia de estos micro-patrones en el comportamiento celular y evaluar la tecnología como método de fabricación de soportes en la ingeniería de tejidos

Supported Engineered Extracellular Matrices for 3D Cell Culture

In the current shift away from 2D tissue culture polystyrene and towards 3D cell culture models, several important design criteria have yet to be considered: (1) provision of large open areas where cells can create their own niche (2) fabrication of a scaffold that is chemically and mechanically tunable and (3) presentation of proteins that mimic native extracellular matrix (ECM). Polymer scaffolds fabricated by 3D jet writing provide extensive void space for maximum cell-cell and cell-ECM interactions. This work expands on such electrospinning technologies to establish a micromanufacturing process that modulates the flow of various polymer solutions through a manifold. The resulting scaffolds contain spatially distinct domains that can be customized to exhibit specific bulk or surface properties. Such tunability is not limited to the synthetic design space. We have discovered that hydrodynamically induced fibrillogenesis can yield remarkably stable networks of protein fibrils suspended across a support or scaffold that recapitulate important structural and functional hallmarks of cell-secreted ECM. These engineered networks of fibronectin serve as a breast cancer microenvironment, making it possible to culture an unfractionated patient sample (n=14), where less than 5% are cancer cells, into a self-selected composition of differentiated cancer cells, stem-like cancer cells, and various stromal cells. An average of 40% increase in the tumor-initiating population and at least a 7-fold increase in the cancer cell population was observed after six days (n=3). This user-defined 3D cell culture platform will enable investigation into the bidirectional relationship between cells and the ECM, not just for breast cancer but a variety of diseased or healthy tissue types.PHDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/140892/1/stacyram_1.pd

Cell Shape and Forces in Elastic and Structured Environments: From Single Cells to Organoids

With the advent of mechanobiology, cell shape and forces have emerged as essential elements of cell behavior and fate, in addition to biochemical factors such as growth factors. Cell shape and forces are intrinsically linked to the physical properties of the environment. Extracellular stiffness guides migration of single cells and collectives as well as differentiation and developmental processes. In confined environments, cell division patterns are altered, cell death or extrusion might be initiated, and other modes of cell migration become possible. Tools from materials science such as adhesive micropatterning of soft elastic substrates or direct laser writing of 3D scaffolds have been established to control and quantify cell shape and forces in structured environments. Herein, a review is given on recent experimental and modeling advances in this field, which currently moves from single cells to cell collectives and tissue. A very exciting avenue is the combination of organoids with structured environments, because this will allow one to achieve organotypic function in a controlled setting well suited for long-term and high-throughput culture

Ultrashort-pulsed laser ablation of poly-L-lactide (PLLA) for cell and tissue engineering applications

El contenido de los capítulos 3 y 4 están sujetos a confidencialidad 167 p.La tecnología de ablación láser es una herramienta bien establecida para la modificación superficial de materiales de distinta naturaleza (metales, polímeros, cerámicas, vidrio¿). La ablación de material mediante láseres de pulso ultracorto (menor que 10 picosegundos) es capaz de generar motivos topográficos micrométricos con una alta precisión debido a un proceso de ablación ¿frío¿ minimizando los efectos térmicos en el material sin producir cambios químicos en el mismo. Es por tanto una tecnología versátil para la fabricación de superficies microestructuradas en un proceso directo y sin contacto y aplicable a una gran variedad de materiales para generar motivos con distintas geometrías sobre superficies no planas. En este trabajo de tesis se aplica la tecnología de ablación mediante láser pulsado de picosegundos para la creación de micro-patrones topográficos en planchas de ácido poli-L-láctico (PLLA), para investigar el mecanismo de ablación del mismo y el efecto de los micro-patrones en el comportamiento de varios tipos de células mediante ensayos in vitro, con el objetivo final de elucidar el alcance de la influencia de estos micro-patrones en el comportamiento celular y evaluar la tecnología como método de fabricación de soportes en la ingeniería de tejidos

Engineered environments for biomedical applications: anisotropic nanotopographies and microfluidic devices

During the last two decades micro- and nano-fabrication techniques originally developed for electronic engineering have directed their attention towards life sciences. The increase of analytical power of diagnostic devices and the creation of more biomimetic scaffolds have been strongly desired by these fields, in order to have a better insight into the complexity of physiological systems, while improving the ability to model them in vitro. Technological innovations worked to fill such a gap, but the integration of these fields of science is not progressing fast enough to satisfy the expectations. In this thesis I present novel devices which exploit the unique features of the micro- and nanoscale and, at the same time, match the requirements for successful application in biomedical research. Such biochips were used for optical detection of water-dispersed nanoparticles in microchannels, for highly controlled cell-patterning in closed microreactors, and for topography-mediated regulation of cell morphology and migration. Moreover, pilot experiments on the pre-clinical translation of micropatterned scaffolds in a rat model of peripheral nerve transaction were initiated and are ongoing. Given these results, the devices presented here have the potential to achieve clinical translation in a short/medium time, contributing to the improvement of biomedical technologies

Fabrication and Applications of Micro/Nanostructured Devices for Tissue Engineering

Nanotechnology allows the realization of new materials and devices with basic structural unit in the range of 1–100 nm and characterized by gaining control at the atomic, molecular, and supramolecular level. Reducing the dimensions of a material into the nanoscale range usually results in the change of its physiochemical properties such as reactivity, crystallinity, and solubility. This review treats the convergence of last research news at the interface of nanostructured biomaterials and tissue engineering for emerging biomedical technologies such as scaffolding and tissue regeneration. The present review is organized into three main sections. The introduction concerns an overview of the increasing utility of nanostructured materials in the field of tissue engineering. It elucidates how nanotechnology, by working in the submicron length scale, assures the realization of a biocompatible interface that is able to reproduce the physiological cell–matrix interaction. The second, more technical section, concerns the design and fabrication of biocompatible surface characterized by micro- and submicroscale features, using microfabrication, nanolithography, and miscellaneous nanolithographic techniques. In the last part, we review the ongoing tissue engineering application of nanostructured materials and scaffolds in different fields such as neurology, cardiology, orthopedics, and skin tissue regeneration