Preparation and characterization of acellular membrane from yellowfin tuna (Thunnus albacares) skin for skin regeneration

Abstract

Full-thickness skin wounds pose a considerable clinical challenge because of the limited capacity for self-regeneration. Acellular materials derived from animals offer a promising solution to this issue. In the present investigation, an acellular scaffold is prepared from yellowfin tuna skin (Thunnus albacares) for skin regeneration application by comparing the efficacy of three chemical decellularization agents: sodium hydroxide (NaOH), Triton X-100 (TT), and sodium dodecyl sulfate (SDS). The impact of these agents on the resulting acellular dermal matrices was evaluated by assessing collagen preservation, DNA removal, residual fat and ash content, and structural integrity using hydroxyproline assay and chemical composition analysis. Mechanical properties, swelling behavior, degradation rate, water vapor transmission rate, moisture loss, and biocompatibility of the acellular membrane were also characterized. Furthermore, the regenerative potential of these samples was assessed in a porcine full-thickness skin defect model. The results demonstrated that all three decellularization methods effectively removed cellular components, with varying degrees of collagen preservation and ECM structural alteration. TT treatment yielded the highest collagen retention and a relatively intact fibrous structure, while NaOH caused significant structural damage. Mechanical testing revealed that hydration significantly improved the elasticity of TT- and SDS-treated samples. In vitro biocompatibility assays showed no significant cytotoxicity or hemolysis. These findings suggest that the acellular membrane holds promise as a biomaterial for skin regeneration applications due to its effective decellularization, preserved collagen structure, and favorable biocompatibility.

Keywords

Thunnus albacares skin / Yellowfin tuna / Decellularization / Acellular membrane / Skin regeneration / Wound healing

References

[1] VeerurajA, LiuL, ZhengJ, WuJ, ArumugamM. Evaluation of astaxanthin incorporated collagen film developed from the outer skin waste of squid Doryteuthis singhalensis for wound healing and tissue regenerative applications. Mater Sci Eng C, 2019, 95: 29-42.

[2] LvK, WangL, HeX, LiW, HanL, QinS. Application of tilapia skin acellular dermal matrix to induce acute skin wound repair in rats. Front Bioeng Biotechnol, 2022, 9: 1-10.

[3] StoneR, et al.. Accelerated wound closure of deep partial thickness burns with acellular fish skin graft. Int J Mol Sci, 2021, 224. 1590

[4] PhamDT, ThaoNTP, ThuyBTP, TranVD, NguyenTQC, NguyenNNT. Silk fibroin hydrogel containing Sesbania sesban L. extract for rheumatoid arthritis treatment. Drug Deliv, 2022, 29(1): 882-888.

[5] TranBLT, et al.. Antibacterial efficacy of low-dosage silver nanoparticle-sodium alginate-chitosan nanocomposite films against pure and clinical acne strains. RSC Adv, 2024, 14(45): 33267-33280.

[6] BlancoM, VázquezJA, Pérez-MartínRI, SoteloCG. Hydrolysates of fish skin collagen: an opportunity for valorizing fish industry byproducts. Mar Drugs, 2017.

[7] LiD, et al.. Evaluation of a novel tilapia-skin acellular dermis matrix rationally processed for enhanced wound healing. Mater Sci Eng C, 2021, 127112202.

[8] MaP, WangY, LiB, HouH. Cross-linking effects of carbodiimide, oxidized chitosan oligosaccharide and glutaraldehyde on acellular dermal matrix of basa fish (Pangasius bocourti). Int J Biol Macromol, 2020, 164: 677-686.

[9] BiazarE, Heidari KeshelS, Rezaei TaviraniM, KamalvandM. Healing effect of acellular fish skin with plasma rich in growth factor on full-thickness skin defects. Int Wound J, 2022, 19(no. 8): 2154-2162.

[10] PatelM, LantisJCII. Fish skin acellular dermal matrix: potential in the treatment of chronic wounds. Chronic Wound Care Manag Res, 2019, 6: 59-70.

[11] LauCS, et al.. Evaluation of decellularized tilapia skin as a tissue engineering scaffold. J Tissue Eng Regen Med, 2019, 13(10): 1779-1791.

[12] AbbasnezhadS, BiazarE, AavaniF, KamalvandM, Heidari KeshelS, PourjabbarB. Chemical modification of acellular fish skin as a promising biological scaffold by carbodiimide cross-linker for wound healing. Int Wound J, 2023, 20(5): 1566-1577.

[13] ZhaoJ, et al.. Structural and biofunctional evaluation of decellularized jellyfish matrices. J Mater Chem B, 2023, 11(16): 3740-3751.

[14] HuangW, et al.. Application of decellularized tilapia skin in rabbit urethral reconstruction: an experimental study. Transl Androl Urol, 2025, 14(2): 266-279.

[15] ChenW, et al.. Decellularization of fish tissues for tissue engineering and regenerative medicine applications. Regen Biomater, 2025.

[16] LiuZ, et al.. Decellularized tilapia fish skin: a novel candidate for tendon tissue engineering. Mater. Today Bio, 2022, 17100488.

[17] WuLC, et al.. Optimized decellularization protocol including α-Gal epitope reduction for fabrication of an acellular porcine annulus fibrosus scaffold. Cell Tissue Bank, 2017, 18(3): 383-396.

[18] MorrisAH, StamerDK, KyriakidesTR. The host response to naturally-derived extracellular matrix biomaterials. Semin Immunol, 2017, 29: 72-91.

[19] WongML, GriffithsLG. Immunogenicity in xenogeneic scaffold generation: antigen removal vs. decellularization. Acta Biomater, 2014, 10(5): 1806-1816.

[20] LeKM, et al.. Effect of freeze-thaw cycles on the acellular scaffold fabricated from tuna fish skin. IFMBE Proc, 2025, 123: 267-274.

[21] BualR, et al.. Characterization of decellularized extracellular matrix from milkfish (Chanos chanos) skin. Biomimetics, 2022, 74. 213

[22] YangB, et al.. Development of a porcine bladder acellular matrix with well-preserved extracellular bioactive factors for tissue engineering. Tissue Eng Part C Methods, 2010, 16(5): 1201-1211.

[23] ZhangQ, LvS, LuJ, JiangS, LinL. Characterization of polycaprolactone/collagen fibrous scaffolds by electrospinning and their bioactivity. Int J Biol Macromol, 2015, 76: 94-101.

[24] LeM-AT, et al.. Investigation of fast in situ injectable carboxymethylcellulose-xanthan gum hydrogel via acyl hydrazone linkages for tissue regeneration. Cellulose, 2025.

[25] AnhT, LamT, MinhK, LeMT. Fabrication and characterization of hydrophobic montmorillonite-modified PCL / COS nanofibrous electrospun membrane for hemostatic dressing. Colloids Surf A Physicochem Eng Asp, 2024, 708135975.

[26] ISO 29943, “International Standard International Standard,” 61010–1 © Iec2001, vol. 2003, p. 13, 2003.

[27] CosteaR, EneI, PavelR. Pig sedation and anesthesia for medical research. Animals, 2023.

[28] FengJ, FuS, LuanJ. Regenerated fat induced by a decellularized adipose matrix can survive long-term in vivo. Acta Biomater, 2025, 191: 233-243.

[29] FelicianFF, et al.. The wound healing potential of collagen peptides derived from the jellyfish Rhopilema esculentum. Chin J Traumatol, 2019, 22(1): 12-20.

[30] BarzidehZ, LatiffAA, GanCY, BenjakulS, KarimAA. Isolation and characterisation of collagen from the ribbon jellyfish (Chrysaora sp.). Int J Food Sci Technol, 2014, 49(6): 1490-1499.

[31] NagaiT, SuzukiN, NagashimaT. Collagen from common minke whale (Balaenoptera acutorostrata) unesu. Food Chem, 2008, 111(2): 296-301.

[32] LiJ, et al.. Carbodiimide crosslinked collagen from porcine dermal matrix for high-strength tissue engineering scaffold. Int J Biol Macromol, 2013, 61: 69-74.

[33] VavkenP, JoshiS, MurrayMM. Triton-X is most effective among three decellularization agents for ACL tissue engineering. J Orthop Res, 2009, 27(12): 1612-1618.

[34] TsengHT, et al.. Advances and challenges of tissue vascular scaffolds and supercritical carbon dioxide technology in cardiovascular diseases. Tissue Eng Regen Med, 2025, 22(3): 273-284.

[35] WoodsT, GratzerPF. Effectiveness of three extraction techniques in the development of a decellularized bone-anterior cruciate ligament-bone graft. Biomaterials, 2005, 26(35): 7339-7349.

[36] KimBS, DasS, JangJ, ChoDW. Decellularized extracellular matrix-based bioinks for engineering tissue- and organ-specific microenvironments. Chem Rev, 2020, 120(19): 10608-10661.

[37] PoornejadN, et al.. The impact of decellularization agents on renal tissue extracellular matrix. J Biomater Appl, 2016, 31(4): 521-533.

[38] EivazkhaniF, et al.. Evaluating two ovarian decellularization methods in three species. Mater Sci Eng C, 2019, 102: 670-682.

[39] ZhaoJ, et al.. Decellularization techniques pave the way for tissue engineering and regenerative medicine: a narrative review. Regener Med Rep, 2024.

[40] HeM, CallananA, LagarasK, SteeleJAM, StevensMM. Optimization of SDS exposure on preservation of ECM characteristics in whole organ decellularization of rat kidneys. J Biomed Mater Res-Part B Appl Biomater, 2017, 105(6): 1352-1360.

[41] GroverCN, CameronRE, BestSM. Investigating the morphological, mechanical and degradation properties of scaffolds comprising collagen, gelatin and elastin for use in soft tissue engineering. J Mech Behav Biomed Mater, 2012, 10: 62-74.

[42] BoseS, LiS, MeleE, SilberschmidtVV. Exploring the mechanical properties and performance of type-I collagen at various length scales: a progress report. Materials (Basel), 2022, 15(8): 1-17.

[43] BlairMJ, JonesJD, WoessnerAE, QuinnKP. Skin structure-function relationships and the wound healing response to intrinsic aging. Adv Wound Care, 2020, 9(3): 127-143.

[44] ZhuJ, et al.. Smart bioadhesives for wound healing and closure. Bioact. Mater., 2023, 19: 360-375.

[45] YuHP, ZhuYJ. Guidelines derived from biomineralized tissues for design and construction of high-performance biomimetic materials: from weak to strong. Chem Soc Rev, 2024, 53(9): 4490-4606.

[46] KamalvandM, BiazarE, Daliri-JoupariM, MontazerF, Rezaei-TaviraniM, Heidari-KeshelS. Design of a decellularized fish skin as a biological scaffold for skin tissue regeneration. Tissue Cell, 2021, 71101509.

[47] NuutilaK, ErikssonE. Moist wound healing with commonly available dressings. Adv Wound Care, 2021, 10(12): 685-698.

[48] SoodA, GranickMS, TomaselliNL. Wound dressings and comparative effectiveness data. Adv Wound Care, 2014, 3(8): 511-529.

[49] ZhouL, ZhangY, YiX, ChenY, LiY. Advances in proteins, polysaccharides, and composite biomaterials for enhanced wound healing via microenvironment management: a review. Int J Biol Macromol, 2024.

[50] YaşayanG, AlarçinE, Bal-ÖztürkA, Avci-AdaliM. Natural polymers for wound dressing applications. Stud Nat Prod Chem, 2022, 74: 367-441.

[51] Bal-ÖztürkA, et al.. Innovative approaches in skin therapy: bionanocomposites for skin tissue repair and regeneration. Mater Adv, 2024, 5(12): 4996-5024.

[52] XuR, et al.. Controlled water vapor transmission rate promotes wound-healing via wound re-epithelialization and contraction enhancement. Sci Rep, 2016, 6: 1-12.

[53] WinogradoffD, JohnS, AksimentievA. Protein unfolding by SDS: the microscopic mechanisms and the properties of the SDS-protein assembly. Nanoscale, 2020, 12(9): 5422-5434.

[54] MaquartFX, MonboisseJC. Extracellular matrix and wound healing. Pathol Biol, 2014, 62(2): 91-95.

[55] OlczykP, MencnerŁ, Komosinska-VassevK. The role of the extracellular matrix components in cutaneous wound healing. Biomed Res Int, 2014, 2014: 12-14.

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