acase@tulane.edu / The ability of humans to regenerate complex tissue structures after amputation is not completely absent. Clinical reports have described random spontaneous cases of digit tip regeneration in young adults. Regeneration of a structure such as a limb or a digit requires tight orchestration of environmental cues and cells that come together and coordinate the regeneration of the missing body part. Studies on animal models have been crucial to have a better understanding on relevant components and mechanisms that are involved in epimorphic regeneration. Mechanistic studies however, are difficult to perform due to the lack of spatial and temporal control of microenvironmental factors. The overall approach of this proposal is to develop a blastema-like in vitro model to conduct comparative studies between connective tissue cells from regeneration-competent (P3) and incompetent (P2) regions of the mouse digit tip, and to control the cellular microenvironment to modulate P2 cells regeneration-incompetent behavior. A 3D spheroid culture model was identified to serve as a 3D biomimetic blastema model that preserves the inherent regenerative properties of regeneration-incompetent and regeneration-competent phenotypes. Relevant factors associated with either wound healing or a regenerative response, were evaluated in both P2 and P3 cells cultured as spheroids. It was found that the expression of wound healing markers, particularly known to be involved in scar tissue formation, were significantly higher in P2 spheroids. Conversely, the expression of markers indicative of a regeneration-permissive microenvironment was significantly higher in P3 spheroids. We evaluated the effects of oxygen modulation in P2 during 2D expansion and/or during 3D spheroid culture and found that preconditioning of P2 cells in 2D increases P2 cell number and promotes spontaneous aggregation. We also found that modulation of oxygen concentration during 3D culture significantly decreases expression of both, wound healing and regenerative markers. This physiologically relevant in vitro model provides a platform to characterize cellular processes involved in the wound healing and regenerative responses. Additionally, it allows for the incorporation of environmental cues, such as oxygen concentration, to better understand the key target mechanisms to shift the default wound response from scar formation to epimorphic regeneration. / 1 / Lina M. Quijano

Cells in multicellular organisms are surrounded by a complex three-dimensional macromolecular extracellular matrix (ECM). This matrix, traditionally thought to uniquely serve a structural function providing support and strength to cells within tissues, is increasingly being recognized to have pleiotropic effects in neurogenesis and regeneration processes such as neocortex folding, stem cell niche maintenance, peripheral nerve regeneration, axonal growth, and many more. ECM mediates these processes via cell-ECM interactions which provide the cells with a wealth of signals including biophysical and mechanical cues in a spatiotemporal manner. Owing to the importance of the surrounding microenvironment, modern neural tissue engineering strategies have focused on the development of engineered biomaterials capable of finely instructing the neuronal response according to their physicochemical characteristics. Neurons and neural stem cells are in fact sensitive to their mechanical and topographical environment, and cell–substrate binding contributes to this sensitivity by activating specific signaling pathways for basic cell function. In addition, the advances in nanotechnology have opened the possibility of introducing decorative nano-motifs that interact with cells at the molecular level. Successful strategies in tissue engineering are driven by not only advances in the synthesis of highly instructive biomaterials but also greatly depend on the right selection of cell sources. As a matter of fact, advances in neural tissue engineering have been strongly hampered by the poor availability of cell sources, considering that primary neurons are the only type of cells that do not proliferate. The discovery of induced pluripotent stem cells (iPSCs) has addressed many of the cell-related limitations in neural tissue engineering, offering the possibility to consistently produce a wide range of neural cell lines. Advances in cell biology have led to the development of iPSCs-derived brain spheroid, which surely represent the most promising tools for several neural tissue engineering applications ranging from in vitro modelling of neurodegenerative diseases (i.e., Parkinson's, Huntington's and Alzheimer's), biomaterials testing and drug screening platforms. The overarching goal of my doctoral work was to engineer biomaterials with instructive physicochemical properties to elicit beneficial cellular responses that are suitable for different neural tissue engineering applications such as nerve regeneration and 3D in vitro modelling. In the first study (Chapter 2), I evaluated the compounded effects of surface stiffness and micro-topography on dorsal root ganglion and human bone-marrow mesenchymal stem cells behavior. To this end, arrays of parallel microchannels of different geometries were introduced on the surface of chitosan films by electrophoretic replica deposition. In addition, a novel chemical crosslinking with citric acid was performed to both enhance the long-term stability of the chitosan films and fine-tune the surface stiffness for the investigation of its role in cell behavior. In the second study (Chapter 3), I developed a novel nanocomposite consisting of a collagen hydrogel decorated with glycine-derived carbon nanodots (Gly-CNDs). After a comprehensive physicochemical characterization of the resulting nanocomposite, I evaluated the effects exerted on neuronal differentiation and electrophysiological maturation of mouse iPSCs-derived brain spheroid. In the third study (Chapter 4), I optimized an alignable collagen-based hydrogel characterized by anisotropically oriented fibers with potential applications in both peripheral and central nervous system repair. I established a protocol that encompasses the introduction in the collagen solution of biodegradable laminin-functionalized magnetic microbeads and the time-controlled application of an external magnetic field. The regenerative potential of the hydrogel was unveiled using mouse iPSCs-derived neural stem cells.

Two-dimensional (2D) in vitro models of tissues and organs have long been used as one of the main tools to understand human physiology and for applications such as drug discovery. But there is a huge disparity between in vivo conditions and these models which has created the need for better models. It has been shown that making three-dimensional models with dynamic environments that provide proper physical and chemical cues for cells, can bridge this gap between 2D models and in vivo conditions but the toolbox for creating such models has been imperfect and rudimentary. Introduction of tissue engineering concept and advent of biofabrication tools to meet its demands has provided new possible avenues for in vitro modeling but many of these tools are specifically designed to create tissue and organ replacements and lack features such as the ability to investigate cellular behavior with ease that are necessary for in vitro modeling purposes. The objective of this doctoral thesis was to introduce a novel toolbox of biofabrication techniques, based on bioprinting and bioassembly, that together are capable of recapitulating anatomical and physiological requirements of different tissue in in vitro setups in a more relevant way while creating the possibility of investigating cellular behavior. A bioprinting technique was developed that allowed formation of large constructs with proper mechanical stability, perfusion, and direct access to cells in different locations. The second technique was based on bioassembly of collagenous grafts in micro-molds and cells from different tissues with the ability to control cell positioning and create tissue-relevant cell densities with higher degree of similarity to human tissues compared to previous techniques. The third technique was based on bioassembled stand alone and dense cell-sheets for cells capable of fusion. These techniques were subsequently used for modeling a few chosen biological phenomenon to showcase the advantages of the techniques over previously developed ones and to further shed light on possible shortcomings of each of the techniques in their application for those specific tissues. In conclusion, our techniques may serve as valuable and easy to use tools for researchers, specifically biologists to investigate different aspects of human biology and disease mechanism in more details. / Thesis / Doctor of Philosophy (PhD) / Experimentation on humans is unethical, therefore in order to understand how human body works and test new therapeutic drugs researchers have used animals and cells isolated from animals or humans. Animals are inherently different from humans and isolated cells are culture in conditions different than human body, therefore a huge gap exists between the knowledge derived from these models and what happens in human body. Since there is no one-size-fits-all technique to model all of the human tissues, the objective of current study was set to build a toolbox of techniques that each could create better environment in the lab for cells isolated from different tissues and organs with more similarity to original tissues, to bridge the gap and eliminate the need to use animal models entirely. During the course of this PhD studies, three different techniques that can be used to make such models for different tissues and organs, as well as different diseases, were developed and characterized. These techniques were also used to shed light on some of the cellular behavior that are already observed in human body but either are not explained or aren’t re-created in the lab for mechanistic studies. Certain questions regarding selected tissues were chosen and the technique most compatible with that tissue was used for the modeling purposes. For example, one investigated niche was the origin of the bone sensory cells which could be important to heal damaged bones or prevent osteoporosis. The first technique was deemed most suitable for this question while for the next question, how the fat and muscle cells are affecting each other that can be useful to better understand conditions such as diabetes and obesity, the second technique was the best option. Overall, a variety of tools were developed that can be used by biologists to create better models of human tissues in the lab as platforms to study human physiology and as media for developing treatments for different diseases.

Le glioblastome est un cancer du cerveau très agressif dont les thérapies actuelles n’augmentent que très peu la durée de vie. Dans cette thèse, nous étudions un nouveau traitement par effet magnéto-mécanique de particules (TEMMP). Un champ magnétique rotatif à faible fréquence (20 Hz) est appliqué pour faire vibrer des particules magnétiques en contact avec les cellules cancéreuses. Les particules développées sont produites par une approche top-down en salle blanche. Les disques de permalloy utilisés présentent une configuration en vortex avec une faible rémanence et une bonne dispersion en suspension. Des particules multicouches de Co/Pt avec une anisotropie perpendiculaire et des vortex de permalloy en forme d’ellipses sont aussi étudiés. L’efficacité du TEMMP est évaluée in-vitro sur des cellules de glioblastome et les différents paramètres sont optimisés. Une forte diminution du nombre de cellules après traitement est alors observée et le comportement des cellules restantes est affecté. Le TEMMP est ensuite adapté pour une étude in-vivo dans un modèle orthotopique de glioblastome chez la souris nude. L’injection des particules en intra-tumoral est mise au point. Les tissus sont peu affectés par le TEMMP comparé à une injection de particules, et une faible augmentation de la survie est observée. Pour mimer les propriétés mécaniques du cerveau de manière plus pertinente, un modèle in-vitro 3D est alors développé et validé. Conçu avec des sphéroïdes de cellules pris dans un gel d’agarose, ce modèle apporte des pistes d’optimisation. / Glioblastoma is a brain cancer with a very poor prognosis. Existing therapies improve only slightly the median survival. In this work, we study a new treatment by magneto-mechanical actuation of particles (TMMAP). A low frequency (20 Hz) rotating magnetic field is applied to stimulate magnetic particles localized near cancer cells. Magnetic particles are produced by a top-down approach in clean room. Permalloy disks with a vortex configuration have a low remanence and are well dispersed in suspension. Multilayers of Co/Pt with a perpendicular anisotropy and permalloy vortex particles with an ellipse shape are also studied. TMMAP efficiency is tested in-vitro on glioblastoma cell line and the parameters are optimized. A huge diminution of living cells and an affected behavior of the remaining cells are observed after treatment. TMMAP is then adapted to an in-vivo study on glioblastoma orthotopic model on nude mice and the intratumoral injection of the particles is developed. Few differences are observed between tissues submitted to TMMAP or injected with particles, and survival is slightly increased. To mimic mechanical properties of the brain in a more relevant model, an in-vitro 3D model is proposed and validated. Based on cells grown as a spheroid and encapsulated in an agarose gel, this model brings optimization tracks.

Skin is an organ with a complex structure which plays a crucial role in thebody’s defence against external threats and in maintaining major homeostatic functions. The need for in vitro models that mimic the in vivo milieu is therefore high and relevant with various applications including, among others, penetration, absorption, and toxicity studies. In this context, the choice of the biomaterial that will provide a 3D scaffold to the cultured cells is defining the model’s success. The FN-4RepCT silk is here suggested as a potent biomaterial for skin tissue engineering applications. This recombinantly produced spider silk protein (FN-4RepCT), which can self-assemble into fibrils, creates a robust and elastic matrice with high bioactivity, due to its functionalization with the fibronectin derived RGD-containing peptide. Hence it overcomes the drawbacks of other available biomaterials either synthetic or based on animal derived proteins. Additionally, the FN-4RepCT silk protein can be cast in various 3D formats, two of which are utilized within this project. We herein present a bilayered skin tissue equivalent supported by the FN-4RepCT silk. This is constructed by the combination of a foam format, integrated with dermal fibroblasts and endothelial cells, and a membrane format supporting epidermal keratinocytes. As a result, a vascularized dermal layer that contains ECM components (Collagen I, Collagen III, and Elastin) is constructed and attached to an epidermal layer of differentiated keratinocytes.The protocol presented in this project offers a successful method of evenly integrating cells in the FN-4RepCT silk scaffold, while preserving their ability to resume some of their major in vivo functions like proliferation, ECM secretion, construction of vascular networks, and differentiation. The obtained results were evaluated with immunofluorescence stainings of various markers of interest and further analysed, when necessary, with image processing tools. The results that ensued from the herein presented protocol strongly suggest that the FN-4RepCT silk is a promising biomaterial for skin tissue engineering applications.