Mimicking human skin constructs using norbornene-pullulan-based hydrogels
There has been a huge demand for engineered skin tissues in the realms of both in vitro and in vivo applications. Selecting the right material scaffold is a critical consideration in making engineered skin tissues, since it should possess a good balance between elasticity and mechanical stability while promoting an adequate cell microenvironment to support both the dermal and the epidermal compartments of skin tissue. In this study, 3D-bioprinted norbornene-pullulan photocrosslinkable hydrogels were utilized as alternative scaffolds to produce epithelized dermal skin models. By employing visible light, 2.5 mm3 cell-laden hydrogels could be printed in 10 s. The thiol-ene photocrosslinking chemistry employed in this work enabled the formation of a well-defined extracellular matrix with orthogonal crosslinks, where encapsulated fibroblasts maintained high cellular viability rates. Through this method, an epidermal layer could be grown on top of the fibroblasts. The coexistence and interaction of human fibroblasts and keratinocytes were visualized by determining the expression of specific markers. This approach represents a promising starting point for the development of photocrosslinkable hydrogel-based human skin constructs by using thiol-ene norbornene chemistry, paving the way toward manufacture of complex in vitro models of human tissues.
- Moniz T, Costa Lima SA, Reis S. Human skin models: from healthy to disease-mimetic systems; characteristics and applications. Br J Pharmacol. 2020;177(19):4314-4329. doi: 10.1111/bph.15184
- Dearman BL, Boyce ST, Greenwood JE. Advances in skin tissue bioengineering and the challenges of clinical translation. Front Surg. 2021;8:640879. doi: 10.3389/fsurg.2021.640879
- Shores JT, Gabriel A, Gupta S. Skin substitutes and alternatives. Adv Skin Wound Care. 2007;20(9):493-508. doi: 10.1097/01.ASW.0000288217.83128.f3
- Manon-Jensen T, Kjeld NG, Karsdal MA. Collagen-mediated hemostasis. J Thromb Haemost. 2016;14(3):438-448. doi: 10.1111/jth.13249
- Sorushanova A, Delgado LM, Wu Z, et al. The collagen suprafamily: from biosynthesis to advanced biomaterial development. Adv Mater. 2019;31(1):e1801651. doi: 10.1002/adma.201801651
- Eming SA, Martin P, Tomic-Canic M. Wound repair and regeneration: Mechanisms, signaling, and translation. Sci Transl Med. 2014;6(265):265sr6. doi: 10.1126/scitranslmed.3009337
- Silvipriya KS, Krishna Kumar K, Bhat AR, Dinesh Kumar B, John A, Lakshmanan P. Collagen: animal sources and biomedical application. J Appl Pharm Sci. 2015;5(3): 123-127. doi: 10.7324/JAPS.2015.50322
- Schmidt FF, Nowakowski S, Kluger PJ. Improvement of a three-layered in vitro skin model for topical application of irritating substances. Front Bioeng Biotechnol. 2020;8(May):1-11. doi: 10.3389/fbioe.2020.00388
- Wang H. A review of the effects of collagen treatment in clinical studies. Polymers (Basel). 2021;13(22):3868. doi: 10.3390/polym13223868
- Keefe J, Wauk L, Chu S, DeLustro F. Clinical use of injectable bovine collagen: a decade of experience. Clin Mater. 1992;9(3-4):155-162. doi: 10.1016/0267-6605(92)90095-b
- Bacakova M, Pajorova J, Broz A, et al. A two-layer skin construct consisting of a collagen hydrogel reinforced by a fibrin-coated polylactide nanofibrous membrane. Int J Nanomedicine. 2019;14:5033-5050. doi: 10.2147/IJN.S200782
- Ackermann K, Lombardi Borgia S, Korting HC, Mewes KR, Schäfer-Korting M. The Phenion® full-thickness skin model for percutaneous absorption testing. Skin Pharmacol Physiol. 2010;23(2):105-112. doi: 10.1159/000265681
- Zhang Y, Wang Y, Li Y, et al. Application of collagen-based hydrogel in skin wound healing. Gels. 2023;9(3):185. doi: 10.3390/gels9030185
- Zhu J, Marchant RE. Design properties of hydrogel tissue-engineering scaffolds. Expert Rev Med Devices. 2011;8(5):607-626. doi: 10.1586/erd.11.27
- Sun M, Sun X, Wang Z, Guo S, Yu G, Yang H. Synthesis and properties of gelatin methacryloyl (GelMA) hydrogels and their recent applications in load-bearing tissue. Polymers (Basel). 2018;10(11):1290. doi: 10.3390/polym10111290
- Yegappan R, Selvaprithiviraj V, Amirthalingam S, Jayakumar R. Carrageenan based hydrogels for drug delivery, tissue engineering and wound healing. Carbohydr Polym. 2018;198:385-400. doi: 10.1016/j.carbpol.2018.06.086
- Park D, Kim Y, Kim H, et al. Hyaluronic acid promotes angiogenesis by inducing RHAMM-TGFβ receptor interaction via CD44-PKCδ. Mol Cells. 2012;33(6):563-574. doi: 10.1007/s10059-012-2294-1
- Toole BP. Hyaluronan: from extracellular glue to pericellular cue. Nat Rev Cancer. 2004;4(7):528-539. doi: 10.1038/nrc1391
- Gobi R, Ravichandiran P, Babu RS, Yoo DJ. Biopolymer and synthetic polymer-based nanocomposites in wound dressing applications: a review. Polymers (Basel). 2021;13(12):1962. doi: 10.3390/polym13121962
- Li R, Tomasula P, de Sousa AMM, et al. Electrospinning pullulan fibers from salt solutions. Polymers (Basel). 2017;9(1):32. doi: 10.3390/polym9010032
- Leathers TD. Biotechnological production and applications of pullulan. Appl Microbiol Biotechnol. 2003;62(5-6): 468-473. doi: 10.1007/s00253-003-1386-4
- Cheng N, Jeschke MG, Sheikholeslam M, Datu A, Oh HH, Amini‐Nik S. Promotion of dermal regeneration using pullulan/gelatin porous skin substitute. J Tissue Eng Regen Med. 2019;13(11):1965-1977. doi: 10.1002/term.2946
- Wang Y, Yuan X, Yao B, Zhu S, Zhu P, Huang S. Tailoring bioinks of extrusion-based bioprinting for cutaneous wound healing. Bioact Mater. 2022;17:178-194. doi: 10.1016/j.bioactmat.2022.01.024
- Antezana PE, Municoy S, Álvarez-Echazú MI, et al. The 3D bioprinted scaffolds for wound healing. Pharmaceutics. 2022;14(2):464. doi: 10.3390/pharmaceutics14020464
- Zhang Q, Bei HP, Zhao M, Dong Z, Zhao X. Shedding light on 3D printing: Printing photo-crosslinkable constructs for tissue engineering. Biomaterials. 2022;286:121566. doi: 10.1016/j.biomaterials.2022.121566
- Yu C, Schimelman J, Wang P, et al. Photopolymerizable biomaterials and light-based 3D printing strategies for biomedical applications. Chem Rev. 2020;120(19): 10695-10743. doi: 10.1021/acs.chemrev.9b00810
- Torras N, Zabalo J, Abril E, Carré A, García-Díaz M, Martínez E. A bioprinted 3D gut model with crypt-villus structures to mimic the intestinal epithelial-stromal microenvironment. Biomater Adv. 2023;153:213534. doi: 10.1016/j.bioadv.2023.213534
- Lin CC, Raza A, Shih H. PEG hydrogels formed by thiol-ene photo-click chemistry and their effect on the formation and recovery of insulin-secreting cell spheroids. Biomaterials. 2011;32(36):9685-9695. doi: 10.1016/j.biomaterials.2011.08.083
- McCall JD, Anseth KS. Thiol–ene photopolymerizations provide a facile method to encapsulate proteins and maintain their bioactivity. Biomacromolecules. 2012;13(8):2410-2417. doi: 10.1021/bm300671s
- Fairbanks BD, Schwartz MP, Halevi AE. A Versatile Synthetic Extracellular Matrix Mimic via Thio-Norbornene Photopolymerization. Adv Mater. 2009;21(48):5005-5010. doi: 10.1002/adma.200901808
- Lin CC, Ki CS, Shih H. Thiol-norbornene photoclick hydrogels for tissue engineering applications. J Appl Polym Sci. 2015;132(8):1-11. doi: 10.1002/app.41563
- Van Hoorick J, Dobos A, Markovic M, et al. Thiol-norbornene gelatin hydrogels: Influence of thiolated crosslinker on network properties and high definition 3D printing. Biofabrication. 2020;13(1): 1-22. doi: 10.1088/1758-5090/abc95f
- Anseth KS, Bowman CN, Brannon-Peppas L. Mechanical properties of hydrogels and their experimental determination. Biomaterials. 1996;17(17):1647-1657. doi: 10.1016/0142-9612(96)87644-7
- Vila A, Torras N, Castaño AG, et al. Hydrogel co-networks of gelatine methacrylate and poly(ethylene glycol) diacrylate sustain 3D functional in vitro models of intestinal mucosa. Biofabrication. 2020;12(2):025008. doi: 10.1088/1758-5090/ab5f50
- Nguyen AL, Grothe S, Luong JHT. Applications of pullulan in aqueous two-phase systems for enzyme production, purification and utilization. Appl Microbiol Biotechnol. 1988:27;341-346. doi: 10.1007/BF00251765
- Singh RS, Kaur N, Singh D, Kennedy JF. Investigating aqueous phase separation of pullulan from Aureobasidium pullulans and its characterization. Carbohydr Polym. 2019;223:115103. doi: 10.1016/j.carbpol.2019.115103
- Knight CG, Willenbrock F, Murphy G. A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases. FEBS Lett. 1992;296(3):263-266. doi: 10.1016/0014-5793(92)80300-6
- Holback H, Yeo Y, Park K. Hydrogel swelling behavior and its biomedical applications. Biomed Hydrogels. 2011;1:3-24. doi: 10.1533/9780857091383.1.3
- Chyzy A, Plonska-Brzezinska ME. Hydrogel properties and their impact on regenerative medicine and tissue engineering. Molecules. 2020;25(24):5795. doi: 10.3390/molecules25245795
- Suhaeri M, Noh MH, Moon JH, et al. Novel skin patch combining human fibroblast-derived matrix and ciprofloxacin for infected wound healing. Theranostics. 2018;8(18):5025-5038. doi: 10.7150/thno.26837
- Wu DQ, Zhu J, Han H, et al. Synthesis and characterization of arginine-NIPAAm hybrid hydrogel as wound dressing: in vitro and in vivo study. Acta Biomater. 2018;65:305-316. doi: 10.1016/j.actbio.2017.08.048
- Peppas NA, Bures P, Leobandung W, Ichikawa H. Hydrogels in pharmaceutical formulations. Eur J Pharm Biopharm. 2000;50(1):27-46. doi: 10.1016/S0939-6411(00)00090-4
- Lai VK, Nedrelow DS, Lake SP, et al. Swelling of collagen-hyaluronic acid co-gels: an in vitro residual stress model. Ann Biomed Eng. 2016;44(10):2984-2993. doi: 10.1007/s10439-016-1636-0
- Bachmann B, Spitz S, Schädl B, et al. Stiffness matters: fine-tuned hydrogel elasticity alters chondrogenic redifferentiation. Front Bioeng Biotechnol. 2020;8:373. doi: 10.3389/fbioe.2020.00373
- Ahearne M, Yang Y, El Haj AJ, Then KY, Liu KK. Characterizing the viscoelastic properties of thin hydrogel-based constructs for tissue engineering applications. J R Soc Interface. 2005;2(5):455-463. doi: 10.1098/rsif.2005.0065
- Orwin EJ, Borene ML, Hubel A. Biomechanical and optical characteristics of a corneal stromal equivalent. J Biomech Eng. 2003;125(4):439-444. doi: 10.1115/1.1589773
- Awad HA, Quinn Wickham M, Leddy HA, Gimble JM, Guilak F. Chondrogenic differentiation of adipose-derived adult stem cells in agarose, alginate, and gelatin scaffolds. Biomaterials. 2004;25(16):3211-3222. doi: 10.1016/j.biomaterials.2003.10.045
- R. Ibañez RI, do Amaral RJFC, Reis RL, Marques AP, Murphy CM, O’Brien FJ. 3D-printed gelatin methacrylate scaffolds with controlled architecture and stiffness modulate the fibroblast phenotype towards dermal regeneration. Polymers (Basel). 2021;13(15):2510. doi: 10.3390/polym13152510
- Raub CB, Putnam AJ, Tromberg BJ, George SC. Predicting bulk mechanical properties of cellularized collagen gels using multiphoton microscopy. Acta Biomater. 2010;6(12): 4657-4665. doi: 10.1016/j.actbio.2010.07.004
- Weng T, Zhang W, Xia Y, et al. 3D bioprinting for skin tissue engineering: current status and perspectives. J Tissue Eng. 2021;12:204173142110285. doi: 10.1177/20417314211028574
- Tahri S, Maarof M, Masri S, Che Man R, Masmoudi H, Fauzi MB. Human epidermal keratinocytes and human dermal fibroblasts interactions seeded on gelatin hydrogel for future application in skin in vitro 3-dimensional model. Front Bioeng Biotechnol. 2023;11:1200618. doi: 10.3389/fbioe.2023.1200618
- Bott K, Upton Z, Schrobback K, et al. The effect of matrix characteristics on fibroblast proliferation in 3D gels. Biomaterials. 2010;31(32):8454-8464. doi: 10.1016/j.biomaterials.2010.07.046
- Feng Z, Li J, Zhou D, Song H, Lv J, Bai W. A novel photocurable pullulan-based bioink for digital light processing 3D printing. Int J Bioprint. 2022;9(2):104-117. doi: 10.18063/ijb.v9i2.657
- Boelsma E, Verhoeven MCH, Ponec M. Reconstruction of a human skin equivalent using a spontaneously transformed keratinocyte cell line (HaCaT). J Invest Dermatol. 1999;112(4):489-498. doi: 10.1046/j.1523-1747.1999.00545.x
- Schoop VM, Fusenig NE, Mirancea N. Epidermal organization and differentiation of HaCaT keratinocytes in organotypic coculture with human dermal fibroblasts. J Invest Dermatol. 1999;112(3):343-353. doi: 10.1046/j.1523-1747.1999.00524.x
- Maas-Szabowski N, Stärker A, Fusenig NE. Epidermal tissue regeneration and stromal interaction in HaCaT cells is initiated by TGF-α. J Cell Sci. 2003;116(Pt 14):2937-2948. doi: 10.1242/jcs.00474
- Wilson VG. Growth and differentiation of HaCaT keratinocytes. Methods Mol Biol. 2014;1195:33-41. doi: 10.1007/7651_2013_42
- 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
- Meyle J, Guttig K, Rascher G, Wolburg H. Transepithelial electrical resistance and tight junctions of human gingival keratinocvtes. J Periodontal Res. 1999;34(4):214-222. doi: 10.1111/j.1600-0765.1999.tb02244.x
- Le Ferrec E, Chesne C, Artusson P, et al. In vitro models of the intestinal barrier: the report and recommendations of ECVAM workshop 46. Altern Lab Anim. 2001;29(6): 649-668. doi: 10.1177/026119290102900604
- Lenselink EA. Role of fibronectin in normal wound healing. Int Wound J. 2015;12(3):313-316. doi: 10.1111/iwj.12109
- Flaten GE, Palac Z, Engesland A, Filipović-Grčić J, Vanić Ž, Škalko-Basnet N. In vitro skin models as a tool in optimization of drug formulation. Eur J Pharm Sci. 2015;75:10-24. doi: 10.1016/j.ejps.2015.02.018
- Randall MJ, Jüngel A, Rimann M, Wuertz-Kozak K. Advances in the biofabrication of 3D skin in vitro: healthy and pathological models. Front Bioeng Biotechnol. 2018;6:154. doi: 10.3389/fbioe.2018.00154
- Yun YE, Jung YJ, Choi YJ, Choi JS, Cho YW. Artificial skin models for animal-free testing. J Pharm Investig. 2018;48(2):215-223. doi: 10.1007/s40005-018-0389-1
- Abd E, Yousef SA, Pastore MN, et al. Skin models for the testing of transdermal drugs. Clin Pharmacol. 2016;8: 163-176. doi: 10.2147/CPAA.S64788
- Cirulli A, Neves Borgheti-Cardoso L, Torras N, García-Díaz M, Martínez E. Hydrogels as tissue barriers. In: Oliveira JM, Silva-Correia J, Reis RL, eds. Hydrogels for Tissue Engineering and Regenerative Medicine. Elsevier; 2024:433-466. doi: 10.1016/B978-0-12-823948-3.00017-8
- Mawazi SM, Kumar M, Ahmad N, Ge Y, Mahmood S. Recent applications of chitosan and its derivatives in antibacterial, anticancer, wound healing, and tissue engineering fields. Polymers (Basel). 2024;16(10):1351. doi: 10.3390/polym16101351