Digital Light Processing Bioprinting Full-Thickness Human Skin for Modelling Infected Chronic Wounds in Vitro

Date

2022-08-08

Authors

Stefanek, Evan

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Abstract

Chronic wounds have a detrimental impact on patient quality of life, a significant economic cost, and often lead to severe outcomes such as amputation, sepsis or death. The elaborate interplay between bacteria, cutaneous cells, immune cells, growth factors, and proteases in chronic wounds has complicated the development of new therapies that could improve outcomes for chronic wound patients. Existing in vitro models of chronic wounds do not appreciably mimic the complexity of the wound environment. In this work, tissue-engineered skin was developed with the goal of creating an in vitro platform appropriate for testing potential clinical therapies for chronic wounds. The Lumen-X, a digital light processing bioprinter, was used to create tissue-engineered skin from a 7.5% (w/v) gelatin methacryloyl hydrogel laden with primary dermal fibroblasts. This dermal layer was developed with an emphasis on providing a favourable microenvironment for the fibroblasts in order to mimic their in vivo phenotype. An epidermal layer of human keratinocytes was formed on the hydrogel surface and stratified through culture at the air-liquid-interface. The maturation of the epidermis was thoroughly characterized with histology, immunohistochemistry, and trans-epithelial electrical resistance analyses which showed a degree of maturation suitable for wound healing studies. To verify the suitability of this tissue-engineered skin for studying healing in vitro, sharp tweezers were used to create physical wounds in the epidermis which were then infected with Pseudomonas aeruginosa. Reepithelialisation, the production of the pro- inflammatory cytokine TNF-α, and the presence of bacteria were monitored over time, showing healing in wounds without infection and those treated with antibiotics, and potential biofilm formation in infected wounds. The tissue-engineered skin developed here is suitable for use as an in vitro model of the infected chronic wound environment. Future work includes developing better methods for creating the physical wound and characterizing the bacterial biofilm in order to improve the reproducibility and clarity of results. Such a model will then be well-poised to begin testing potential chronic wound therapies in vitro.

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Keywords

Bioprinting, Tissue engineering, Skin, Wound healing, Hydrogels, Photo-crosslinking

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