Design and fabrication of a physiologically relevant human skin tissue model for efficacy testing using 3d bioprinting

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Motter Catarino, Carolina
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Electronic thesis
Chemical engineering
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A variety of human skin models have been developed for applications in regenerative medicine and in vitro efficacy studies. Typically, these reconstructed skin models employ matrix proteins and scaffolds that are derived from non-human sources along with human skin cells: fibroblasts (dermis) and keratinocytes (epidermis). A key limitation of these models is that they still fail in recapitulating the cellular and microenvironmental complexity, such as presence of vasculature, multiple cell types (e.g., melanocytes, neural and immune skin cell) and adnexal structures (e.g., hair follicles, sebaceous and sweat glands), that are representative of human physiology. The use of recombinant extracellular matrix proteins, as well as the introduction of other cell types, can overcome these limitations. In parallel, there is a growing interest in employing three-dimensional (3D) bioprinting platforms for tissue engineering given the possibility for precise cell positioning, flexibility, reproducibility, and high-throughput production. We have been working on the evaluation of animal and non-animal derived scaffold proteins and glycosaminoglycans for the design of bioinks for skin reconstruction using 3D bioprinting. At the dermal-epidermal junction (DEJ), proteins, glycoproteins, and proteoglycans form a thin bilayer membrane known as the basement membrane. The screening of proteins from the DEJ (collagen IV, laminin and fibronectin) demonstrates that certain protein combinations increase the proliferation of keratinocytes compared to the control (no protein coating). In the investigation of the effect of components from the dermal layer (collagen I and III, elastin, hyaluronic acid, and chondroitin sulfate B), the primary influence on the viability of fibroblast was attributed to the source of the collagen type I (rat tail, human and bovine) used as scaffold materials. Furthermore, the incorporation of chondroitin sulfate B in the dermal bioink and the increase in collagen I concentration has led to a reduction of the skin samples contraction compared to the control samples whose dermal scaffold is composed primary of collagen type I. This approach highlights the relevance of the bioinks composition to the development of complex reconstructed skin models. In parallel to the design of bioinks, we have evaluated and optimized the printing parameters for skin reconstruction using two platforms: a custom model and a commercial 3D bioprinter. We have been able to successfully demonstrate the feasibility of, using both platforms, to generate skin samples with characteristics equivalent to the tissues obtained by manual deposition of the bioinks and similar to the human skin. We further explored the advantages of the 3D bioprinting technology in the development of three-dimensional hair follicle models. For the reconstructed skin model, a bioink containing dermal papilla cell (DPCs) and human umbilical vein cells (HUVECs) was precisely printed within the gelled dermis. Keratinocytes and melanocytes migrated from the epidermis into the vertical opening left by the nozzle and enveloped the spheroid of DPCs and HUVECs. The resulting model contained hair follicle-like structures whose morphology and biomarker pattern mimics that of the native tissue. Additionally, we have developed a hair follicle spheroid model formed by a core of DPCs and HUVECs (step 1) enveloped by a sheath of epithelial cells (step 2). The resulting spheroid, generated in an automated, precise and reproducible manner by 3D bioprinting, resembled the structure of the native hair follicle and could potentially be used for high-throughput screening of substances. The development of reconstructed skin models with increased complexity that better mimic the native tissue can have an important impact both in regenerative medicine and in the diversification of in vitro models available for safety and efficacy assessment of chemical compounds.
School of Engineering
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Rensselaer Polytechnic Institute, Troy, NY
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