AccScience Publishing / IJB / Online First / DOI: 10.36922/ijb.3925
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RESEARCH ARTICLE

Bioprinted keratinocyte and stem cell-laden constructs for skin tissue engineering

Eva Bettendorf1 ,  Rafael Schmid1 ,  Raymund E. Horch1 ,  Annika Kengelbach-Weigand1 ,  Yvonne Kulike1 ,  Stefan Schrüfer2 ,  Dirk W. Schubert2 ,  Zan Lamberger3 ,  Philipp Stahlhut3 ,  Gregor Lang3 ,  Celena A. Sörgel1*
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1 Department of Plastic and Hand Surgery and Laboratory for Tissue Engineering and Regenerative Medicine, University Hospital of Erlangen, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany
2 Department of Materials Science and Engineering, Institute of Polymer Materials, Friedrich- Alexander University Erlangen-Nürnberg, Erlangen, Germany
3 Department for Functional Materials in Medicine and Dentistry, University Hospital of Würzburg, Julius-Maximilians University Würzburg, Würzburg, Germany
IJB, 3925 https://doi.org/10.36922/ijb.3925
Submitted: 12 June 2024 | Accepted: 6 August 2024 | Published: 12 August 2024
© 2024 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
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Abstract

Treating large-scale skin wounds remains a significant therapeutic challenge, often due to insufficient autologous material for complete coverage. Recent advances in biofabrication offer a solution with reproducible and precise large-scale production. Herein, this study aims to evaluate the feasibility of biofabrication and develop a customized three-dimensional (3D) bioprinted skin construct containing immortalized HaCaT keratinocytes and adipose-derived stem cells (ADSCs). Keratinocytes were cultured in various hydrogels (e.g., containing alginate [Alg], fibrin [Fib], collagen, gelatin [Gel], gelatin methacryloyl [GelMA], hyaluronic acid [HA], and a pre-fabricated collagen-elastin-matrix) for 7 days. The metabolic activity of cultured keratinocytes was then evaluated during the co-cultivation of HaCaT and ADSCs in a transwell model. The metabolic activity in all groups increased over the experimental period. Alg/HA/Gel and GelMA hydrogels demonstrated good printability and high diffusion rates. There was no significant difference in pore size between all hydrogels. Based on the results of printability and diffusion assays, as well as scanning electron microscopy (SEM) and rheological measurements, Alg/HA/Gel and GelMA hydrogels were selected for the bioprinted 3D model. Fib hydrogel was integrated into the biofabricated constructs for its excellent metabolic activity in the transwell model. Hydrogel stability, cell survival, and metabolic activity in bioprinted 3D models containing keratinocytes and ADSCs were evaluated over 14 days. On day 14, metabolic activity and live cell count within the bioprinted constructs of the co-cultured groups were significantly higher compared to day 1. The biofabricated GelMA constructs displayed higher cell viability than Alg/HA/Gel constructs. Additionally, to evaluate cell migration out of the constructs, the metabolic activity and viability of the cells on the well bottom were examined. After 14 days, an average of 50% of the well bottom was covered by HaCaT cells, which were initially printed in co-culture into the constructs. These findings indicate that GelMA constructs containing keratinocytes and ADSCs may offer a promising therapeutic option in the treatment of large chronic wounds.

Keywords
Keratinocytes
Stem cells
Bioprinting
Skin substitution
Wound healing
Hydrogel
Funding
The work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation; project number 326998133, TRR 225; subprojects C03, A07, and C04).
Conflict of interest
The authors declare no conflict of interest.
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International Journal of Bioprinting, Electronic ISSN: 2424-8002 Print ISSN: 2424-7723, Published by AccScience Publishing
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