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RESEARCH ARTICLE

Physicochemical properties of gelatin/ hyaluronic acid-printed hydrogel incorporating thymoquinone: In vitro evaluation of biocompatibility for wound healing

Nur Izzah Md Fadilah1,2 ,  Yasuhiko Tabata3 ,  Manira Maarof1,2 ,  Mh Busra Fauzi1,2*
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1 Department of Tissue Engineering and Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, 56000 Cheras, Kuala Lumpur, Malaysia
2 Advance Bioactive Materials-Cells UKM Research Group, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
3 Cell Biotechnology Group, Department of Plastic and Reconstructive Surgery, Graduate School of Medicine, Kyoto University, South Research Bldg. No.1 (Institute for Life and Medical Sciences) 53 Kawara-cho Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
IJB 2025, 11(2), 387–411; https://doi.org/10.36922/ijb.8551
Submitted: 15 January 2025 | Accepted: 20 February 2025 | Published: 20 February 2025
(This article belongs to the Special Issue Advances in 3D Bioprinting)
© 2025 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

Wound healing is a multifaceted biological process that necessitates the development of advanced materials that can effectively support tissue regeneration and repair. The fabrication of bioengineered wound dressings has evolved significantly, with three-dimensional (3D) bioprinting emerging as a promising method to produce personalized, structurally stable hydrogels. In this study, we leveraged extrusion-based 3D bioprinting technology to develop gelatin-hyaluronic acid (GEL-HA) hydrogels, incorporating thymoquinone (TQ), a bioactive compound known for its regenerative properties. The use of 3D printing allowed for precise control over the scaffold’s architecture, optimizing its compressive strength and resilience while creating a bioactive, biocompatible platform for wound healing applications. This enables precise control over their architecture and mechanical properties to enhance wound healing where it offers promising potential as biocompatible scaffolds for wound healing applications due to their favorable physicochemical properties and ability to promote cell proliferation and migration. GEL-HA hydrogels were fabricated with varying HA concentrations (0.1–1.0 wt%), and the effects on the gelation process and physical characteristics were evaluated. Results showed that the ideal gelation temperature for the GEL-HA hydrogel was 22 °C, with the inclusion of HA reducing polymerization time. The printed hydrogels exhibited high water retention (>1000%) and satisfactory mechanical properties, with a degree of crosslinking of up to 40.21%. Furthermore, the hydrogels demonstrated a low biodegradation rate (less than 0.300 mg/h) and favorable water vapor transmission rate (WVTR) in a range of 2000– 3000 gm–2day–1, which are crucial for maintaining a moist environment for wound healing. The incorporation of TQ further enhanced the biocompatibility and cellular proliferation of human dermal fibroblasts (HDFs). Cell viability assays indicated that TQ promoted HDFs growth at concentrations of 0.005–0.1 μg/mL without toxicity. Moreover, the wound scratch assay demonstrated that TQ facilitated cell migration, with the optimum concentration of 0.1 μg/mL showing the most significant effect. The GEL-HA-TQ hydrogel also supported HDFs attachment and proliferation, as confirmed by live and dead cell staining, also with Ki67 level assessment, and collagen type-I immunocytochemistry. These findings suggest that GEL-HA hydrogels, combined with TQ, provide a promising and biocompatible platform for wound healing. It effectively promotes cell viability, migration, and extracellular matrix synthesis, which could be beneficial in regenerative medicine and tissue engineering applications.

Graphical abstract
Keywords
3D bioprinting
Biocompatibility
Gelatin
Hyaluronic acid
Physicochemical properties
Thymoquinone
Wound healing
Funding
This study was supported by grants from the Geran Fundamental Fakulti Perubatan (GFFP), Universiti Kebangsaan Malaysia with a code project (Grant Code: FF-2022-310).
Conflict of interest
The authors declare that they have no conflict of interest.
References
  1. Almadani YH, Vorstenbosch J, Davison PG, Murphy AM. Wound healing: a comprehensive review. Semin Plast Surg. 2021;35(3):141-144. doi: 10.1055/s-0041-1731791
  2. Baron JM, Glatz M, Proksch E. Optimal support of wound healing: new insights. Dermatology. 2020;236(6):593-600. doi: 10.1159/000505291
  3. Kaur G, Narayanan G, Garg D, et al. Biomaterials-based regenerative strategies for skin tissue wound healing. ACS Applied Bio Mater. 2022;5(5):2069-2106. doi: 10.1021/acsabm.2c00035
  4. Ansari M, Darvishi A. A review of the current state of natural biomaterials in wound healing applications. Front Bioeng Biotechnol. 2024;12:1309541. doi: 10.3389/fbioe.2024.1309541
  5. Fadilah NIM, Isa ILM, Zaman WSWK, Tabata Y, Fauzi MB. The effect of nanoparticle-incorporated natural-based biomaterials towards cells on activated pathways: a systematic review. Polymers (Basel). 2022;14(3):476. doi: 10.3390/polym14030476
  6. Md Fadilah NI, Shahabudin NA, Mohd Razif RA, et al. Discovery of bioactive peptides as therapeutic agents for skin wound repair. J Tissue Eng. 2024;15: 20417314241280359. doi: 10.1177/20417314241280359
  7. Zarei F, Soleimaninejad M. Role of growth factors and biomaterials in wound healing. Artif Cells Nanomed Biotechnol. 2018;46(suppl 1):906-911. doi: 10.1080/21691401.2018.1439836
  8. Fadilah NIM, Fauzi MB, Maarof M. Effect of multiple-cycle collections of conditioned media from different cell sources towards fibroblasts in in vitro wound healing model. Pharmaceutics. 2024 ;16(6):767. doi: 10.3390/pharmaceutics16060767
  9. Md Fadilah NI, Mohd Abdul Kader Jailani MS, Badrul Hisham MAI, et al. Cell secretomes for wound healing and tissue regeneration: next generation acellular based tissue engineered products. J Tissue Eng. 2022;13:20417314221114273. doi: 10.1177/20417314221114273
  10. Zhong Y, Wei E-T, Wu L, et al. Novel biomaterials for wound healing and tissue regeneration. ACS Omega. 2024; 9(30):32268-32286. doi: 10.1021/acsomega.4c02775
  11. Fadilah NIM, Nizam NAAK, Fauzi MB. Antibacterial compounds-incorporated functional biomaterials for chronic wound healing application via 3D bioprinting: the mechanism of action. IJB. 2024;10(4):3372. doi: 10.36922/ijb.3372
  12. Fang H, Xu J, Ma H, et al. Functional materials of 3D bioprinting for wound dressings and skin tissue engineering applications: a review. Int J Bioprint. 2023; 9(5):757. doi: 10.18063/ijb.757
  13. Abuhamad AY, Masri S, Fadilah NIM, Alamassi MN, Maarof M, Fauzi MB. Application of 3D-printed bioinks in chronic wound healing: a scoping review. Polymers (Basel). 2024;16(17):2456. doi: 10.3390/polym16172456
  14. 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
  15. Khanna A. The new era of tissue engineering-3D printing of organs and tissue constructs. BME Horizon. 2024;2(1):112. doi: 10.37155/2972-449X-vol2(1)-112
  16. Masri S, Fadilah NIM, Hao LQ, et al. Multifunctionalised skin substitute of hybrid gelatin-polyvinyl alcohol bioinks for chronic wound: injectable vs. 3D bioprinting. Drug Deliv Transl Res. 2024;14(4):1005-1027. doi: 10.1007/s13346-023-01447-z
  17. Nizam AAK, Md Fadilah NI, Ahmad H, Maarof M, Fauzi MB. Injectable gelatin-palmitoyl-GDPH hydrogels as bioinks for future cutaneous regeneration: physicochemical characterization and cytotoxicity assessment. Polymers (Basel). 2024;17(1):41. doi: 10.3390/polym17010041
  18. Camci-Unal G, Cuttica D, Annabi N, Demarchi D, Khademhosseini A. Synthesis and characterization of hybrid hyaluronic acid-gelatin hydrogels. Biomacromolecules. 2013;14(4):1085-1092. doi: 10.1021/bm3019856
  19. Cao H, Wang J, Hao Z, Zhao D. Gelatin-based biomaterials and gelatin as an additive for chronic wound repair. Front Pharmacol. 2024;15:1398939. doi: 10.3389/fphar.2024.1398939
  20. Salih ARC, Farooqi HMU, Amin H, et al., Hyaluronic acid: comprehensive review of a multifunctional biopolymer. Future J Pharm Sci. 2024;10(1):63. doi: 10.1186/s43094-024-00636-y
  21. Lee SJ, Kim H-J, Choi EJ, et al. Immediately injectable modified gelatin and hyaluronic acid-based hydrogel encapsulating nano-hydroxyapatite and human adipose-derived MSCs for use as a bone filler in situ therapy. Carbohydr Polym Technol Appl. 2024;8:100625. doi: 10.1016/j.carpta.2024.100625
  22. Yang D, Chen H, Wei H, et al. Hydrogel wound dressings containing bioactive compounds originated from traditional Chinese herbs: a review. Smart Mater Med. 2024;5(1): 153-165. doi: 10.1016/j.smaim.2023.10.004
  23. López-Gutierrez J, Ramos-Payan R, Ayala-Hum A, et al. Biofunctionalization of hydrogel-based scaffolds for vascular tissue regeneration. Front Mater. 2023; 10. doi: 10.3389/fmats.2023.1168616
  24. Gu Q, Tomaskovic-Crook E, Wallace GG, Crook JM. Engineering human neural tissue by 3D bioprinting. Methods Mol Biol. 2018;1758:129-138. doi: 10.1007/978-1-4939-7741-3_10
  25. Abbas M, Gururani MA, Ali A, et al. Antimicrobial properties and therapeutic potential of bioactive compounds in Nigella sativa: a review. Molecules. 2024; 29(20):4914. doi: 10.3390/molecules29204914
  26. Sallehuddin N, Hao LQ, Wen APY, Fadilah NIM, Maarof M, Fauzi MB. Thymoquinone-incorporated collagee biomatrix: a promising approach for full-thickness wound healing. Pharmaceutics. 2024;16(11):1440. doi: 10.3390/pharmaceutics16111440
  27. Fan F, Saha S, Hanjaya-Putra D. Biomimetic hydrogels to promote wound healing. Front Bioeng Biotechnol. 2021;9:718377. doi: 10.3389/fbioe.2021.718377
  28. Masri S, Maarof M, Aziz IA, Idrus R, Fauzi MB. Performance of hybrid gelatin-PVA bioinks integrated with genipin through extrusion-based 3D bioprinting: An in vitro evaluation using human dermal fibroblasts. Int J Bioprint. 2023;9(3):677. doi: 10.18063/ijb.677
  29. Ng WC, Lokanathan Y, Fauzi MB, et al. In vitro evaluation of genipin-crosslinked gelatin hydrogels for vocal fold injection. Sci Rep. 2023;13(1):5128. doi: 10.1038/s41598-023-32080-y
  30. Phang SJ, Teh HX, Looi ML, et al. PVA/PVP nanofibres incorporated with Ecklonia cava phlorotannins exhibit excellent cytocompatibility and accelerate hyperglycaemic wound healing. Tissue Eng Regen Med. 2024; 21(2):243-260. doi: 10.1007/s13770-023-00590-5
  31. Amirrah IN, Zulkiflee I, Mohd Razip Wee MF, et al. Plasma-polymerised antibacterial coating of ovine tendon collagen type I (OTC) crosslinked with genipin (GNP) and dehydrothermal-crosslinked (DHT) as a cutaneous substitute for wound healing. Materials (Basel). 2023;16(7):2739. doi: 10.3390/ma16072739
  32. Salleh A, Mustafa N, Teow YH, et al. Dual-layered approach of ovine collagen-gelatin/cellulose hybrid biomatrix containing graphene oxide-silver nanoparticles for cutaneous wound healing: fabrication, physicochemical, cytotoxicity and antibacterial characterisation. Biomedicines. 2022;10(4):816. doi: 10.3390/biomedicines10040816
  33. Samadian H, Zamiri S, Ehterami A, et al., Electrospun cellulose acetate/gelatin nanofibrous wound dressing containing berberine for diabetic foot ulcer healing: in vitro and in vivo studies. Sci Rep. 2020;10(1):8312. doi: 10.1038/s41598-020-65268-7
  34. Maarof M, Chowdhury SR, Saim A, Bt Hj Idrus R, Lokanathan Y. Concentration dependent effect of human dermal fibroblast conditioned medium (DFCM) from three various origins on keratinocytes wound healing. Int J Mol Sci. 2020;21(8):2929. doi: 10.3390/ijms21082929
  35. Khaleghi M, Ahmadi E, Khodabandeh Shahraki M, Aliakbari F, Morshedi D. Temperature-dependent formulation of a hydrogel based on Hyaluronic acid-polydimethylsiloxane for biomedical applications. Heliyon. 2020;6(3):e03494. doi: 10.1016/j.heliyon.2020.e03494
  36. Wei P, Chen W, Song Q, et al. Superabsorbent hydrogels enhanced by quaternized tunicate cellulose nanocrystals with adjustable strength and swelling ratio. Cellulose. 2021;28(6):3723-3732. doi: 10.1007/s10570-021-03776-z
  37. Agubata CO, Mbah MA, Akpa PA, Ugwu G. Application of self-healing, swellable and biodegradable polymers for wound treatment. J Wound Care. 2021;30(Sup9a):IVi-IVx. doi: 10.12968/jowc.2021.30.Sup9a.IV
  38. Baseer A, Koenneke A, Zapp J, et al. Design and characterization of surface-crosslinked gelatin nanoparticles for the delivery of hydrophilic macromolecular drugs. Macromol Chem Phys. 2019;220(18):1900260. doi: 10.1002/macp.201900260
  39. Chang KC, Lin DJ, Wu YR, et al. Characterization of genipin-crosslinked gelatin/hyaluronic acid-based hydrogel membranes and loaded with hinokitiol: In vitro evaluation of antibacterial activity and biocompatibility. Mater Sci Eng C Mater Biol Appl. 2019;105:110074. doi: 10.1016/j.msec.2019.110074
  40. Zawani M, Maarof M, Tabata Y, Motta A, Fauzi MB. Quercetin-embedded gelastin injectable hydrogel as provisional biotemplate for future cutaneous application: optimization and in vitro evaluation. Gels. 2022;8(10):623. doi: 10.3390/gels8100623
  41. Liu L, Li X, Nagao M, Elias AL, Narain R, Chung HJ. A pH-indicating colorimetric tough hydrogel patch towards applications in a substrate for smart wound dressings. Polymers (Basel). 2017;9(11):558. doi: 10.3390/polym9110558
  42. Hazrati R, Davaran S, and Omidi Y. Bioactive functional scaffolds for stem cells delivery in wound healing and skin regeneration. React Funct Polym. 2022;174:105233. doi: 10.1016/j.reactfunctpolym.2022.105233
  43. Zhang M, Chu L, Chen J, et al. Asymmetric wettability fibrous membranes: Preparation and biologic applications. Compos Part B: Eng. 2023;269:111095. doi: 10.1016/j.compositesb.2023.111095
  44. Radakisnin R, Abdul Majid MS, Jamir MRM, Tahir MFM. Physical, thermal, and mechanical properties of highly porous polylactic acid/cellulose nanofibre scaffolds prepared by salt leaching technique. Nanotechnol Rev. 2021;10(1): 1469-1483. doi: 10.1515/ntrev-2021-0098
  45. Sallehuddin N, Md Fadilah NI, Hwei NM, et al. Characterization and cytocompatibility of collagen-gelatin-elastin (collagee) acellular skin substitute towards human dermal fibroblasts: in vitro assessment. Biomedicines. 2022;10(6):1327. doi: 10.3390/biomedicines10061327
  46. Gholamali I, Vu TT, Jo SH, Park SH, Lim KT. Exploring the progress of hyaluronic acid hydrogels: synthesis, characteristics, and wide-ranging applications. Materials (Basel). 2024;17(10):2439. doi: 10.3390/ma17102439
  47. Zhang S, Kang L, Hu S, et al. Carboxymethyl chitosan microspheres loaded hyaluronic acid/gelatin hydrogels for controlled drug delivery and the treatment of inflammatory bowel disease. Int J Biol Macromol. 2021;167:1598-1612. doi: 10.1016/j.ijbiomac.2020.11.117
  48. Song M, Furuya K. Fabrication and characterization of nanostructures on insulator substrates by electron-beam-induced deposition. Sci Technol Adv Mater. 2008;9(2):023002. doi: 10.1088/1468-6996/9/2/023002
  49. Miranda DG, Malmonge SM, Campos DM, Attik NG, Grosgogeat B, Gritsch K. A chitosan-hyaluronic acid hydrogel scaffold for periodontal tissue engineering. J Biomed Mater Res B Appl Biomater. 2016;104(8): 1691-1702. doi: 10.1002/jbm.b.33516
  50. Algahtani MS, Ahmad MZ, Shaikh IA, Abdel-Wahab BA, Nourein IH, Ahmad J. Thymoquinone loaded topical nanoemulgel for wound healing: formulation design and in-vivo evaluation. Molecules. 2021;26(13):3863. doi: 10.3390/molecules26133863.
  51. Trinh XT, Long NV, Van Anh LT, et al. A comprehensive review of natural compounds for wound healing: targeting bioactivity perspective. Int J Mol Sci. 2022;23(17): 9573. doi: 10.3390/ijms23179573
  52. Masri S, Fadilah NIM, Hao LQ, et al. Multifunctionalised skin substitute of hybrid gelatin-polyvinyl alcohol bioinks for chronic wound: injectable vs. 3D bioprinting. Drug Deliv Transl Res. 2024;14(4):1005-1027. doi: 10.1007/s13346-023-01447-z
  53. Thambirajoo M, Md Fadilah NI, Maarof M, et al. Functionalised sodium-carboxymethylcellulose-collagen bioactive bilayer as an acellular skin substitute for future use in diabetic wound management: the evaluation of physicochemical, cell viability, and antibacterial effects. Polymers (Basel). 2024;16(16):2252. doi: 10.3390/polym16162252
  54. Fadilah NIM, Phang SJ, Kamaruzaman N, et al. Antioxidant biomaterials in cutaneous wound healing and tissue regeneration: a critical review. Antioxidants (Basel). 2023;12(4):787. doi: 10.3390/antiox12040787
  55. Mohanto S, Narayana S, Merai KP, et al. Advancements in gelatin-based hydrogel systems for biomedical applications: a state-of-the-art review. Int J Biol Macromol. 2023; 253(Pt 5):127143. doi: 10.1016/j.ijbiomac.2023.127143
  56. Barrulas RV, Corvo MC. Rheology in product development: an insight into 3D printing of hydrogels and aerogels. Gels. 2023;9(12):986. doi: 10.3390/gels9120986
  57. Liu S, Li D, Wang Y, Zhou G, Ge K, Jiang L. Adhesive, antibacterial and double crosslinked carboxylated polyvinyl alcohol/chitosan hydrogel to enhance dynamic skin wound healing. Int J Biol Macromol. 2023; 228:744-753. doi: 10.1016/j.ijbiomac.2022.12.169
  58. Nguyen HM, Le TTN, Nguyen AT, et al. Biomedical materials for wound dressing: recent advances and applications. RSC Adv. 2023;13(8):5509-5528. doi: 10.1039/D2RA07673J
  59. Griveau L, Lafont M, le Goff H, et al. Design and characterization of an in vivo injectable hydrogel with effervescently generated porosity for regenerative medicine applications. Acta Biomater. 2022;140:324-337. doi: 10.1016/j.actbio.2021.11.036
  60. Balion Z, Sipailaite E, Stasyte G, et al. Investigation of cancer cell migration and proliferation on synthetic extracellular matrix peptide hydrogels. Front Bioeng Biotechnol. 2020;8:773. doi: 10.3389/fbioe.2020.00773
  61. Zhang Y, Xu Y, Gao J. The engineering and application of extracellular matrix hydrogels: a review. Biomaterials Sci. 2023; 11(11):3784-3799. doi: 10.1039/D3BM00183K

 

 

 

 

 

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