Fish collagen is gaining attention as a sustainable biomaterial for three-dimensional (3D) scaffold fabrication in tissue engineering. In this study, a biomimetic, one-pot crosslinking strategy for native fish skin collagen was developed and compared with a conventional periodate oxidation–Schiff base approach using oxidized maltose. Both approaches increased viscosity and thermal stability while preserving native structural features. The oxidized maltose-crosslinked collagen demonstrated Schiff-base crosslinking, and the one-pot crosslinking method produced a covalent bond that was not a Schiff base. Precrosslinked collagens were processed into microgels and incorporated into calcium alginate to yield a shear-recovering, extrudable ink suitable for 3D extrusion printing. The printed scaffolds maintained structural resilience under physiological conditions, exhibited shear-recovery behavior confirmed by rheological analysis, and supported high cell viability. To enhance biofunctionality, vascular endothelial growth factor was conjugated to the 3D scaffolds, which were subsequently seeded with human bone marrow-derived mesenchymal stem cells. Immunofluorescence staining indicated endothelial lineage differentiation, suggesting that this platform may support the development of vascularized 3D tissue constructs.
Impact Statement
This study presents a one-pot crosslinking approach to enable three-dimensional extrusion printing of a shear-recovery, precrosslinked fish collagen ink design, eliminating the need for postprinting treatments. Functionalization with vascular endothelial growth factor further enhanced the bioactivity of the printed scaffolds by promoting angiogenic response. Collectively, these findings demonstrate a sustainable and biocompatible strategy that broadens the applicability of fish collagen-based inks for vascularized tissue engineering applications.
San AntonioJD, JacenkoO, FertalaA, et al.Collagen structure-function mapping informs applications for regenerative medicine. Bioengineering (Basel), 2020; 8(1):3; doi: 10.3390/bioengineering8010003
2.
ShouldersMD, RainesRT. Collagen structure and stability. Annu Rev Biochem, 2009; 78:929–958; doi: 10.1146/annurev.biochem.77.032207.120833
3.
DavidenkoN, SchusterCF, BaxDV, et al.Evaluation of cell binding to collagen and gelatin: A study of the effect of 2D and 3D architecture and surface chemistry. J Mater Sci Mater Med, 2018; 29(4):39; doi: 10.1007/s10856-016-5763-9
4.
TarafdarA, GaurVK, RawatN, et al.Advances in biomaterial production from animal derived waste. Bioengineered, 2021; 12(1):8247–8258; doi: 10.1080/21655979.2021.1982321
5.
BenwoodC, ChrenekJ, KirschRL, et al.Natural biomaterials and their use as bioinks for printing tissues. Bioengineering (Basel), 2021; 8(2):27; doi: 10.3390/bioengineering8020027
6.
PawelecKM, BestSM, CameronRE. Collagen: A network for regenerative medicine. J Mater Chem B, 2016; 4(40):6484–6496; doi: 10.1039/C6TB00807K
7.
SchrieberR, GareisH. Gelatine Handbook: Theory and Industrial Practice. John Wiley & Sons; 2007.
8.
PienN, PezzoliD, Van HoorickJ, et al.Development of photo-crosslinkable collagen hydrogel building blocks for vascular tissue engineering applications: A superior alternative to methacrylated gelatin? Mater Sci Eng C Mater Biol Appl, 2021; 130:112460; doi: 10.1016/j.msec.2021.112460
9.
SubhanF, IkramM, ShehzadA, et al.Marine collagen: An emerging player in biomedical applications. J Food Sci Technol, 2015; 52(8):4703–4707; doi: 10.1007/s13197-014-1652-8
10.
FelicianFF, XiaC, QiW, et al.Collagen from marine biological sources and medical applications. Chem Biodivers, 2018; 15(5):e1700557; doi: 10.1002/cbdv.201700557
11.
XuX, SuiB, LiuX, et al.Superior low-immunogenicity of tilapia type I collagen based on unique secondary structure with single calcium binding motif over terrestrial mammals by inhibiting activation of DC intracellular Ca2+-mediated STIM1-Orai1/NF-кB pathway. Mater Sci Eng C Mater Biol Appl, 2021; 131:112503; doi: 10.1016/j.msec.2021.112503
12.
de SouzaA, de Almeida CruzM, de AraújoTAT, et al.Fish collagen for skin wound healing: A systematic review in experimental animal studies. Cell Tissue Res, 2022; 388(3):489–502; doi: 10.1007/s00441-022-03625-w
13.
YamadaS, YamamotoK, IkedaT, et al.Potency of fish collagen as a scaffold for regenerative medicine. Biomed Res Int, 2014; 2014:e302932; doi: 10.1155/2014/302932
14.
GraysonWL, MartensTP, EngGM, et al.Biomimetic approach to tissue engineering. Semin Cell Dev Biol, 2009; 20(6):665–673; doi: 10.1016/j.semcdb.2008.12.008
15.
HsuH-H, UemuraT, YamaguchiI, et al.Chondrogenic differentiation of human mesenchymal stem cells on fish scale collagen. J Biosci Bioeng, 2016; 122(2):219–225; doi: 10.1016/j.jbiosc.2016.01.001
16.
FangZ, LvB, ZhanJ, et al.Flexible conductive decellularized fish skin matrix as a functional scaffold for myocardial infarction repair. Macromol Biosci, 2023; 23(12):e2300207; doi: 10.1002/mabi.202300207
17.
LeonardAR, CummingMH, AliMA, et al.Fish collagen cross-linking atrategies to improve mechanical and bioactive capabilities for tissue engineering and regenerative medicine. Adv Funct Materials, 2024; 34(45):2405335; doi: 10.1002/adfm.202405335
18.
ChenF, LeP, Fernandes-CunhaGM, et al.Bio-orthogonally crosslinked hyaluronate-collagen hydrogel for suture-free corneal defect repair. Biomaterials, 2020; 255:120176; doi: 10.1016/j.biomaterials.2020.120176
19.
PatiF, DattaP, AdhikariB, et al.Collagen scaffolds derived from fresh water fish origin and their biocompatibility. J Biomed Mater Res A, 2012; 100(4):1068–1079; doi: 10.1002/jbm.a.33280
20.
Mh BusraMF, LokanathanY. Recent development in the fabrication of collagen scaffolds for tissue engineering applications: A review. Curr Pharm Biotechnol, 2019; 20(12):992–1003; doi: 10.2174/1389201020666190731121016
21.
ChengK, ChenX, YiY, et al.Novel biomimetic collagen-based corneal repair material achieved via a “Killing two birds with one stone” strategy using carboxymethyl-β-cyclodextrin. ACS Biomater Sci Eng, 2025; 11(4):2263–2273; doi: 10.1021/acsbiomaterials.4c02203
22.
YamauchiM, SricholpechM, TerajimaM, et al.Glycosylation of Type I collagen. In: Post-Translational Modification of Proteins: Tools for Functional Proteomics. Methods in Molecular Biology. (KannichtC ed). Springer: New York, NY; 2019; pp. 127–144; doi: 10.1007/978-1-4939-9055-9_9
23.
XuZ, YuanL, LiuQ, et al.Crosslinking effect of dialdehyde cholesterol modified starch nanoparticles on collagen hydrogel. Carbohydr Polym, 2022; 285:119237; doi: 10.1016/j.carbpol.2022.119237
24.
BiderF, MiolaM, ClejanuC-E, et al.3D bioprinting of multifunctional alginate dialdehyde (ADA)–gelatin (GEL) (ADA-GEL) hydrogels incorporating ferulic acid. Int J Biol Macromol, 2024; 257(Pt 2):128449; doi: 10.1016/j.ijbiomac.2023.128449
25.
DingW, ZhouJ, ZengY, et al.Preparation of oxidized sodium alginate with different molecular weights and its application for crosslinking collagen fiber. Carbohydr Polym, 2017; 157:1650–1656; doi: 10.1016/j.carbpol.2016.11.045
26.
XuJ, LiuY, HsuS. Hydrogels based on Schiff base linkages for biomedical applications. Molecules, 2019; 24(16):3005; doi: 10.3390/molecules24163005
27.
FengR, DanN, ChenY, et al.Crosslinking of dialdehyde heparin: A new strategy for improving the anticoagulant properties of porcine acellular dermal matrix. RSC Adv, 2022; 12(11):6811–6820; doi: 10.1039/D1RA08982J
28.
YiY, ZhangY, ManselB, et al.Effect of dialdehyde carboxymethyl cellulose cross-linking on the porous structure of the collagen matrix. Biomacromolecules, 2022; 23(4):1723–1732; doi: 10.1021/acs.biomac.1c01641
29.
KamimuraW, KoyamaH, MiyataT, et al.Sugar-based crosslinker forms a stable atelocollagen hydrogel that is a favorable microenvironment for 3D cell culture. J Biomed Mater Res A, 2014; 102(12):4309–4316; doi: 10.1002/jbm.a.35106
30.
GomezCG, RinaudoM, VillarMA. Oxidation of sodium alginate and characterization of the oxidized derivatives. Carbohydr Polym, 2007; 67(3):296–304; doi: 10.1016/j.carbpol.2006.05.025
31.
SunF, ShanP, LiuB, et al.Gelatin-based multifunctional composite films integrated with dialdehyde carboxymethyl cellulose and coffee leaf extract for active food packaging. Int J Biol Macromol, 2024; 263(Pt 1):130302; doi: 10.1016/j.ijbiomac.2024.130302
32.
KwakHW, LeeH, ParkS, et al.Chemical and physical reinforcement of hydrophilic gelatin film with di-aldehyde nanocellulose. Int J Biol Macromol, 2020; 146:332–342; doi: 10.1016/j.ijbiomac.2019.12.254
33.
CorreiaC, SousaRO, ValeAC, et al.Adhesive and biodegradable membranes made of sustainable catechol-functionalized marine collagen and chitosan. Colloids Surf B Biointerfaces, 2022; 213:112409; doi: 10.1016/j.colsurfb.2022.112409
34.
BrudzyńskaP, Kulka-KamińskaK, PiwowarskiŁ, et al.Dialdehyde starch as a cross-linking agent modifying fish collagen film properties. Materials (Basel), 2024; 17(7):1475; doi: 10.3390/ma17071475
35.
PietruchaK, SafandowskaM. Dialdehyde cellulose-crosslinked collagen and its physicochemical properties. Process Biochemistry, 2015; 50(12):2105–2111; doi: 10.1016/j.procbio.2015.09.025
36.
HuangS, KongX, XiongY, et al.An overview of dynamic covalent bonds in polymer material and their applications. Eur Polym J, 2020; 141:110094; doi: 10.1016/j.eurpolymj.2020.110094
37.
Devi V. KA, ShyamR, PalaniappanA, et al.Self-healing hydrogels: Preparation, mechanism and advancement in biomedical applications. Polymers (Basel), 2021; 13(21):3782; doi: 10.3390/polym13213782
SinclairA, O’KellyMB, BaiT, et al.Self-healing zwitterionic microgels as a versatile platform for malleable cell constructs and injectable therapies. Adv Mater, 2018; 30(39):e1803087; doi: 10.1002/adma.201803087
40.
HighleyCB, SongKH, DalyAC, et al.Jammed microgel inks for 3D printing applications. Adv Sci (Weinh), 2019; 6(1):1801076; doi: 10.1002/advs.201801076
41.
DalyAC, RileyL, SeguraT, et al.Hydrogel microparticles for biomedical applications. Nat Rev Mater, 2020; 5(1):20–43; doi: 10.1038/s41578-019-0148-6
MuirVG, PrendergastME, BurdickJA. Fragmenting bulk hydrogels and processing into granular hydrogels for biomedical applications. J Vis Exp, 2022(183):e63867; doi: 10.3791/63867
44.
CarvalhoD, DaniS, SoteloC, et al.Assessing non-synthetic crosslinkers in biomaterial inks based on polymers of marine origin to increase the shape fidelity in 3D extrusion printing. Biomed Mater, 2023; 18(5); doi: 10.1088/1748-605X/acecec
45.
GovindharajM, RoopavathUK, RathSN. Valorization of discarded Marine Eel fish skin for collagen extraction as a 3D printable blue biomaterial for tissue engineering. J Clean Prod, 2019; 230:412–419; doi: 10.1016/j.jclepro.2019.05.082
46.
WierzbickaA, BartniakM, WaśkoJ, et al.The impact of gelatin and fish collagen on alginate hydrogel properties: A comparative study. Gels, 2024; 10(8):491; doi: 10.3390/gels10080491
47.
MilazzoM, RovelliR, RicciC, et al.Rheology and printability of hydroxyapatite/sodium alginate bioinks added with bovine or fish collagen peptides. Gels, 2025; 11(3):209; doi: 10.3390/gels11030209
48.
TanJ, LuoY, GuoY, et al.Development of alginate-based hydrogels: Crosslinking strategies and biomedical applications. Int J Biol Macromol, 2023; 239:124275; doi: 10.1016/j.ijbiomac.2023.124275
49.
MaihemutiA, ZhangH, LinX, et al.3D-printed fish gelatin scaffolds for cartilage tissue engineering. Bioact Mater, 2023; 26:77–87; doi: 10.1016/j.bioactmat.2023.02.007
50.
CurtiF, DrăgușinD-M, SerafimA, et al.Development of thick paste-like inks based on superconcentrated gelatin/alginate for 3D printing of scaffolds with shape fidelity and stability. Mater Sci Eng C Mater Biol Appl, 2021; 122:111866; doi: 10.1016/j.msec.2021.111866
51.
GovindharajM, Al HashimiN, SomanSS, et al.3D-bioprinted tri-layered cellulose/collagen-based drug-eluting fillers for the treatment of deep tunneling wounds. Bio-Des Manuf, 2024; 7(6):938–954; doi: 10.1007/s42242-024-00305-2
52.
ShenYH, ShoichetMS, RadisicM. Vascular endothelial growth factor immobilized in collagen scaffold promotes penetration and proliferation of endothelial cells. Acta Biomater, 2008; 4(3):477–489; doi: 10.1016/j.actbio.2007.12.011
53.
SizelandKH, HofmanKA, HallettIC, et al.Nanostructure of electrospun collagen: Do electrospun collagen fibers form native structures? Materialia (Oxf), 2018; 3:90–96; doi: 10.1016/j.mtla.2018.10.001
54.
SimonJ, FliriL, DrexlerF, et al.Debugging periodate oxidation of cellulose: Why following the common protocol of quenching excess periodate with glycol is a bad idea. Carbohydr Polym, 2023; 310:120691; doi: 10.1016/j.carbpol.2023.120691
55.
HermansonGT. Functional targets for bioconjugation. In: Bioconjugate Techniques. Elsevier; 2013; pp. 127–228; doi: 10.1016/B978-0-12-382239-0.00002-9
56.
SunL, ShenY, LiM, et al.Preparation and modification of collagen/sodium alginate-based biomedical materials and their characteristics. Polymers (Basel), 2024; 16(2):171; doi: 10.3390/polym16020171
57.
ZhaoH, HeindelND. Determination of degree of substitution of formyl groups in polyaldehyde dextran by the hydroxylamine hydrochloride method. Pharm Res, 1991; 8(3):400–402; doi: 10.1023/A:1015866104055
58.
McKinneyW. Pandas: A foundational Python library for data analysis and statistics. Python for High Performance and Scientific Computing, 2011; 14(9):1–9.
59.
HarrisCR, MillmanKJ, van der WaltSJ, et al.Array programming with NumPy. Nature, 2020; 585(7825):357–362; doi: 10.1038/s41586-020-2649-2
60.
PedregosaF, VaroquauxG, GramfortA, et al.Scikit-learn: Machine learning in Python. Journal of Machine Learning Research, 2011; 12:2825–2830.
61.
Plotly Technologies Inc. Plotly. Available from: https://plotly.com/python/ [Last accessed: Novmber2, 2025].
62.
VirtanenP, GommersR, OliphantTE, et al.; SciPy 1.0 Contributors. SciPy 1.0: Fundamental algorithms for scientific computing in Python. Nat Methods, 2020; 17(3):261–272; doi: 10.1038/s41592-019-0686-2
63.
SchindelinJ, Arganda-CarrerasI, FriseE, et al.Fiji: An open-source platform for biological-image analysis. Nat Methods, 2012; 9(7):676–682; doi: 10.1038/nmeth.2019
64.
TurnerPR, McConnellM, YoungSL, et al.3D living dressing improves healing and modulates immune response in a thermal injury model. Tissue Eng Part C Methods, 2022; 28(8):431–439; doi: 10.1089/ten.tec.2022.0088
GautieriA, PassiniFS, SilvánU, et al.Advanced glycation end-products: Mechanics of aged collagen from molecule to tissue. Matrix Biol, 2017; 59:95–108; doi: 10.1016/j.matbio.2016.09.001
67.
NakamuraA, KawaharadaR, NakamuraA, et al.Advanced glycation end products and oxidative stress in a hyperglycaemic environment. In: Fundamentals of Glycosylation. IntechOpen; 2021; doi: 10.5772/intechopen.97234
68.
SellDR, BiemelKM, ReihlO, et al.Glucosepane is a major protein cross-link of the senescent human extracellular matrix: Relationship with diabetes*. J Biol Chem, 2005; 280(13):12310–12315; doi: 10.1074/jbc.M500733200
69.
GirtonTS, OegemaTR, TranquilloRT. Exploiting glycation to stiffen and strengthen tissue equivalents for tissue engineering. J Biomed Mater Res, 1999; 46(1):87–92; doi: 10.1002/(sici)1097-4636(199907)46:1<87::aid-jbm10>3.0.co;2-k
LeeHJ, Fernandes-CunhaGM, PutraI, et al.Tethering growth factors to collagen surfaces using copper-free click chemistry: Surface characterization and in vitro biological response. ACS Appl Mater Interfaces, 2017; 9(28):23389–23399; doi: 10.1021/acsami.7b05262
72.
ThantAA, RuangpornvisutiV, SangvanichP, et al.Characterization of a bioscaffold containing polysaccharide acemannan and native collagen for pulp tissue regeneration. Int J Biol Macromol, 2023; 225:286–297; doi: 10.1016/j.ijbiomac.2022.11.015
73.
DavidenkoN, SchusterCF, BaxDV, et al.Control of crosslinking for tailoring collagen-based scaffolds stability and mechanics. Acta Biomater, 2015; 25:131–142; doi: 10.1016/j.actbio.2015.07.034
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