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

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.