Using a Quasi-3D Ex Vivo Skin Dermis Model to Investigate the Potential of Biomaterials to Reprogram Gene Expression in Human Dermal (Myo)Fibroblasts

<b>Introduction</b><br/>Myofibroblasts are specialized cells that play a key role during the inflammatory phase of normal wound healing. They deposit extracellular matrix (ECM) components to restore the mechanical properties of the skin and physically contract to encourage wound closure. However, dysregulation of myofibroblast activity can lead to development of chronic inflammatory conditions such as delayed healing¹. To date, many studies have explored how material cues, including physicochemical and biological signals, influence cell behaviors such as differentiation, reprogramming, and proliferation². These findings raise the possibility of reprogramming gene expression of inflamed tissues via biomaterial contact to promote regeneration. In this study, we use a bio-inspired quasi-3D <i>ex vivo </i>skin dermis model³ that is comprised of human dermal (myo)fibroblasts seeded on top of a silk/collagen substrate to evaluate the influence of various material cues on gene expression that may delay or reverse pathological development.<br/><b>Materials and Methods</b><br/>Collagen and silk fibroin was crosslinked using HRP-mediated catalysis at 37°C³. To characterize the silk/collagen matrix, Texas Red^TM-X Succinimidyl Ester was conjugated to amine groups present on collagen. To compare cell morphology in 2D and in quasi-3D conditions, immunofluorescence imaging was performed with Hoechst 33342 dye and an anti-vimentin antibody to visualize nuclei and cytoskeletal vimentin, respectively.<br/><b>Results and Discussion</b><br/>It is now well appreciated that cells are sensitive to the geometry they are cultured in. Several cell types exhibit a flattened morphology in 2D culture, which leads to changes in chromatin topology that in turn, alters gene expression⁴. We address these issues using a quasi-3D model that allows for physiological recapitulation of the extracellular matrix to conserve typical baseline gene expression, as well as preservation of mechanical integrity, offered by collagen and silk components, respectively³. Originally, we elected to seed the cells within a 3D silk/collagen gel, but the dimensions were such that the majority of encapsulated cells would not be in contact with the material. To reduce the thickness of the model, we sought to dip glass slides in the reactive solution in a serial manner to obtain a thin coating of gel. However, only the first layer successfully adhered to the slides. We propose depositing the solution directly on top of the glass slides is optimal to achieve a homogeneous coating that preserves elongated cell morphology observed <i>in vivo</i>.<br/><b>Conclusions</b><br/>This skin model enables a biologically relevant yet accessible platform that more accurately recapitulates the mechanics of the 3D <i>in vivo</i> microenvironment for the purpose of identifying biomaterials that have the potential to delay the onset of and/or reverse pathological inflammatory phenotypes.<br/><b>References</b><br/>1. Darby, I. A., Laverdet, B., Bonté, F. & Desmoulière, A. Fibroblasts and myofibroblasts in wound healing. <i>Clin. Cosmet. Investig. Dermatol.</i> <b>7</b>, 301–311 (2014).<br/>2. Crowder, S. W., Leonardo, V., Whittaker, T., Papathanasiou, P. & Stevens, M. M. Material Cues as Potent Regulators of Epigenetics and Stem Cell Function. <i>Cell Stem Cell</i> <b>18</b>, 39–52 (2016).<br/>3. Vidal, S. E. L. <i>et al.</i> 3D biomaterial matrix to support long term, full thickness, immuno-competent human skin equivalents with nervous system components. <i>Biomaterials</i> <b>198</b>, 194–203 (2019).<br/>4. Smithmyer, M. E., Cassel, S. E. & Kloxin, A. M. Bridging 2D and 3D culture: Probing impact of extracellular environment on fibroblast activation in layered hydrogels. <i>AIChE J.</i> <b>65</b>, e16837 (2019).<br/><b>Acknowledgements</b><br/>This work was supported by the Department of Bioengineering at Imperial College London.