AccScience Publishing / IJB / Volume 10 / Issue 6 / 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 2024, 10(6), 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.

Graphical abstract
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.
References
  1. Tottoli EM, Dorati R, Genta I, Chiesa E, Pisani S, Conti B. Skin wound healing process and new emerging technologies for skin wound care and regeneration. Pharmaceutics. 2020;12(8):735. doi: 10.3390/pharmaceutics12080735
  2. Sörgel CA, Cai A, Schmid R, Horch RE. Perspectives on the current state of bioprinted skin substitutes for wound healing. Biomedicines. 2023;11(10):2678. doi: 10.3390/biomedicines11102678
  3. Zuo KJ, Medina A, Tredget EE. Important developments in burn care. Plast Reconstr Surg. 2017;139(1):120e-138e. doi: 10.1097/prs.0000000000002908
  4. Wang Y, Beekman J, Hew J, et al. Burn injury: challenges and advances in burn wound healing, infection, pain and scarring. Adv Drug Deliv Rev. 2018;123:3-17. doi: 10.1016/j.addr.2017.09.018
  5. Jeschke MG, Shahrokhi S, Finnerty CC, Branski LK, Dibildox M. Wound coverage technologies in burn care: established techniques. J Burn Care Res. 2018;39(3):313-318. doi: 10.1097/BCR.0b013e3182920d29
  6. Varkey M, Visscher DO, van Zuijlen PPM, Atala A, Yoo JJ. Skin bioprinting: the future of burn wound reconstruction? Burns Trauma. 2019;7:4. doi: 10.1186/s41038-019-0142-7
  7. Sorgel CA, Schmid R, Kengelbach-Weigand A, Promny T, Horch RE. Air-pressure-supported application of cultured human keratinocytes in a fibrin sealant suspension as a potential clinical tool for large-scale wounds. J Clin Med. 2022;11(17):5032. doi: 10.3390/jcm11175032
  8. Tan SH, Ngo ZH, Sci DB, Leavesley D, Liang K. Recent advances in the design of three-dimensional and bioprinted scaffolds for full-thickness wound healing. Tissue Eng Part B Rev. 2022;28(1):160-181. doi: 10.1089/ten.TEB.2020.0339
  9. Weng T, Zhang W, Xia Y, et al. 3D bioprinting for skin tissue engineering: current status and perspectives. J Tissue Eng. 2021;12:20417314211028574. doi: 10.1177/20417314211028574
  10. Park J, Lee SJ, Chung S, et al. Cell-laden 3D bioprinting hydrogel matrix depending on different compositions for soft tissue engineering: characterization and evaluation. Mater Sci Eng C. 2017;71:678-684.
  11. Catanzano O, D’Esposito V, Acierno S, et al. Alginate– hyaluronan composite hydrogels accelerate wound healing process. Carbohydr Polym. 2015;131:407-414. doi: 10.1016/j.carbpol.2015.05.081
  12. Wang S, Xiong Y, Chen J, et al. Three dimensional printing bilayer membrane scaffold promotes wound healing. Front Bioeng Biotechnol. 2019;7:348. doi: 10.3389/fbioe.2019.00348
  13. Yue K, Trujillo-de Santiago G, Alvarez MM, Tamayol A, Annabi N, Khademhosseini A. Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels. Biomaterials. 2015;73:254-271. doi: 10.1016/j.biomaterials.2015.08.045
  14. Cohen B, Pinkas O, Foox M, Zilberman M. Gelatin–alginate novel tissue adhesives and their formulation–strength effects. Acta Biomater. 2013;9(11):9004-9011. doi: 10.1016/j.actbio.2013.07.002
  15. Schmid R, Schmidt SK, Detsch R, et al. A new printable alginate/hyaluronic acid/gelatin hydrogel suitable for biofabrication of in vitro and in vivo metastatic melanoma models. Adv Funct Mater. 2022;32(2):2107993. doi: 10.1002/adfm.202107993
  16. Kim BS, Gao G, Kim JY, Cho D-W. 3D cell printing of perfusable vascularized human skin equivalent composed of epidermis, dermis, and hypodermis for better structural recapitulation of native skin. Adv Healthc Mater. 2019;8(7):1801019. doi: 10.1002/adhm.201801019
  17. Zhao X, Lang Q, Yildirimer L, et al. Photocrosslinkable gelatin hydrogel for epidermal tissue engineering. Adv Healthc Mater. 2016;5(1):108-118. doi: 10.1002/adhm.201500005
  18. Billiet T, Gevaert E, De Schryver T, Cornelissen M, Dubruel P. The 3D printing of gelatin methacrylamide cell-laden tissue-engineered constructs with high cell viability. Biomaterials. 2014;35(1):49-62. doi: 10.1016/j.biomaterials.2013.09.078
  19. Heltmann-Meyer S, Steiner D, Müller C, et al. Gelatin methacryloyl is a slow degrading material allowing vascularization and long-term usein vivo. Biomed Mater. 2021;16(6). doi: 10.1088/1748-605X/ac1e9d
  20. Gopinathan J, Noh I. Recent trends in bioinks for 3D printing. Biomater Res. 2018;22(1):11. doi: 10.1186/s40824-018-0122-1
  21. Horch RE, Bannasch H, Kopp J, Andree C, Stark GB. Single-cell suspensions of cultured human keratinocytes in fibrin–glue reconstitute the epidermis. Cell Transplant. 1998;7(3):309-317. doi: 10.1177/096368979800700309
  22. Bannasch H, Unterberg T, Föhn M, Weyand B, Horch RE, Stark GB. Cultured keratinocytes in fibrin with decellularised dermis close porcine full-thickness wounds in a single step. Burns. 2008;34(7):1015-1021. doi: 10.1016/j.burns.2007.12.009
  23. Hunyadi J, Farkas B, Bertényi C, Oláh J, Dobozy A. Keratinocyte grafting: a new means of transplantation for full-thickness wounds. J Dermatol Surg Oncol. 1988;14(1):75-78. doi: 10.1111/j.1524-4725.1988.tb03343.x
  24. Thamm OC, Theodorou P, Stuermer E, et al. Adipose-derived stem cells and keratinocytes in a chronic wound cell culture model: the role of hydroxyectoine. Int Wound J. 2015;12(4):387-396. doi: 10.1111/iwj.12120
  25. Jorgensen AM, Varkey M, Gorkun A, et al. Bioprinted skin recapitulates normal collagen remodeling in full-thickness wounds. Tissue Eng Part A. 2020;26(9-10):512-526. doi: 10.1089/ten.TEA.2019.0319
  26. Schulik J, Salehi S, Boccaccini AR, et al. Comparison of the behavior of 3D-printed endothelial cells in different bioinks. Bioengineering (Basel). 2023;10(7):751. doi: 10.3390/bioengineering10070751
  27. Safikhani MM, Asefnejad A, Aghdam RM, Rahmati S. Fabrication, and characterization of crosslinked sodium alginate/hyaluronic acid/gelatin 3Dprinted heparin-loaded scaffold. J Polym Res. 2024;31(4):121. doi: 10.1007/s10965-024-03942-4
  28. Christensen K, Xu C, Chai W, Zhang Z, Fu J, Huang Y. Freeform inkjet printing of cellular structures with bifurcations. Biotechnol Bioeng. 2015;112(5):1047-1055.
  29. Sakai S, Ohi H, Hotta T, Kamei H, Taya M. Differentiation potential of human adipose stem cells bioprinted with hyaluronic acid/gelatin-based bioink through microextrusion and visible light-initiated crosslinking. Biopolymers. 2018;109(2). doi: 10.1002/bip.23080
  30. Law N, Doney B, Glover H, et al. Characterisation of hyaluronic acid methylcellulose hydrogels for 3D bioprinting. J Mech Behav Biomed Mater. 2018;77:389-399. doi: 10.1016/j.jmbbm.2017.09.031
  31. Marfia G, Navone SE, Di Vito C, et al. Mesenchymal stem cells: potential for therapy and treatment of chronic non-healing skin wounds. Organogenesis. 2015;11(4):183-206. doi: 10.1080/15476278.2015.1126018
  32. Mazini L, Rochette L, Admou B, Amal S, Malka G. Hopes and limits of adipose-derived stem cells (ADSCs) and mesenchymal stem cells (MSCs) in wound healing. Int J Mol Sci. 2020;21(4):1306. doi: 10.3390/ijms21041306
  33. Seo BF, Kim KJ, Kim MK, Rhie JW. The effects of human keratinocyte coculture on human adipose-derived stem cells. Int Wound J. 2016;13(5):630-635. doi: 10.1111/iwj.12335
  34. Lee SH, Jin SY, Song JS, Seo KK, Cho KH. Paracrine effects of adipose-derived stem cells on keratinocytes and dermal fibroblasts. Ann Dermatol. 2012;24(2):136-143. doi: 10.5021/ad.2012.24.2.136
  35. Hermann M, Peddi A, Gerhards A, et al. Secretome of adipose-derived stem cells cultured in platelet lysate improves migration and viability of keratinocytes. Int J Mol Sci. 2023;24(4):3522. doi: 10.3390/ijms24043522
  36. Ong HT, Redmond SL, Marano RJ, et al. Paracrine activity from adipose-derived stem cells on in vitro wound healing in human tympanic membrane keratinocytes. Stem Cells Dev. 2017;26(6):405-418. doi: 10.1089/scd.2016.0204
  37. Li S, Sun J, Yang J, et al. Gelatin methacryloyl (GelMA) loaded with concentrated hypoxic pretreated adipose-derived mesenchymal stem cells(ADSCs) conditioned medium promotes wound healing and vascular regeneration in aged skin. Biomater Res. 2023;27(1):11. doi: 10.1186/s40824-023-00352-3
  38. Müller SJ, Fabry B, Gekle S. Predicting cell stress and strain during extrusion bioprinting. Phys Rev Appl. 2023;19(6):064061. doi: 10.1103/PhysRevApplied.19.064061
  39. Schmid R, Schmidt SK, Schrüfer S, et al. A vascularized in vivo melanoma model suitable for metastasis research of different tumor stages using fundamentally different bioinks. Mater Today Bio. 2024;26:101071. doi: 10.1016/j.mtbio.2024.101071
  40. Chand R, Muhire BS, Vijayavenkataraman S. Computational fluid dynamics assessment of the effect of bioprinting parameters in extrusion bioprinting. Int J Bioprint. 2022;8(2):545. doi: 10.18063/ijb.v8i2.545
  41. Joodaki H, Panzer MB. Skin mechanical properties and modeling: a review. Proc Inst Mech Eng H. 2018;232(4):323-343. doi: 10.1177/0954411918759801
  42. Martin P. Wound healing--aiming for perfect skin regeneration. Science. 1997;276(5309):75-81. doi: 10.1126/science.276.5309.75
  43. Zhang Y, Han F, Gu L, et al. Adipose mesenchymal stem cell exosomes promote wound healing through accelerated keratinocyte migration and proliferation by activating the AKT/HIF-1α axis. J Mol Histol. 2020;51(4):375-383. doi: 10.1007/s10735-020-09887-4

 

 

 



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International Journal of Bioprinting, Electronic ISSN: 2424-8002 Print ISSN: 2424-7723, Published by AccScience Publishing
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