Collagen hydrogels serve as biomimetic scaffolds that closely resemble the natural extracellular matrix, thus providing an ideal 3D biocompatible environment for cells. However, based on our previous experience, not all collagen isolates are capable of gelling, which appears to depend on the type, origin, species, age and sex of the source animal and the collagen isolation method applied. We therefore decided to evaluate porcine collagen-rich materials isolated from two different porcine genotypes applying two different specific isolation methods, and to analyse other main components, i.e., lipids and glycosaminoglycans, as well as amino acid composition and structural and morphological properties. While all the collagen isolates obtained were subjected to the gelling process, only one of them successfully gelled. In addition, the gelling ability of this isolate was confirmed repeatedly on collagens that were isolated from other pigs of the same porcine genotype. The results revealed that the gelling process proceeds via cooperation between the composition and the structure of the collagen isolate. With respect to the composition, one of the most important factors in terms of the success of the gelation process of collagen isolates concerns elevated glycosaminoglycan contents. The structural factors that characterise collagen isolates, i.e., cross-links (immature and mature) and their mutual ratio, as well as the presence of telopeptides, strongly impact the progress of the gelling process and the resulting character of the hydrogel structure. All these factors are influenced by the isolation procedure.

Department of Composites and Carbon Materials Institute of Rock Structure and Mechanics Czech Academy of Sciences 182 09 Prague Czech Republic

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Taghipour Y.D., Hokmabad V.R., Bakhshayesh A.R.D., Asadi N., Salehi R., Nasrabadi H.T. The Application of Hydrogels Based on Natural Polymers for Tissue Engineering. Curr. Med. Chem. 2020;27:2658–2680. doi: 10.2174/0929867326666190711103956. PubMed DOI

Yang J., Sun X., Zhang Y., Chen Y. Chapter 10—The application of natural polymer–based hydrogels in tissue engineering. In: Chen Y., editor. Hydrogels Based on Natural Polymers. 1st ed. Elsevier; Amsterdam, The Netherlands: 2019. [(accessed on 1 September 2024)]. pp. 273–307. Available online: https://shop.elsevier.com/books/hydrogels-based-on-natural-polymers/chen/978-0-12-816421-1.

Badylak S.F., Freytes D.O., Gilbert T.W. Extracellular matrix as a biological scaffold material: Structure and function. Acta Biomater. 2015;23:S17–S26. doi: 10.1016/j.actbio.2015.07.016. PubMed DOI

Syedain Z., Reimer J., Lahti M., Berry J., Johnson S., Bianco R., Tranquillo R.T. Tissue engineering of acellular vascular grafts capable of somatic growth inyoung lambs. Nat. Commun. 2016;7:12951. doi: 10.1038/ncomms12951. PubMed DOI PMC

Antoine E.E., Vlachos P.P., Rylander M.N. Tunable Collagen I Hydrogels for Engineered Physiological Tissue Micro-Environments. PLoS ONE. 2015;10:e0122500. doi: 10.1371/journal.pone.0122500. PubMed DOI PMC

Flanagan T.C., Wilkins B., Black A., Jockenhoevel S., Smith T.J., Pandit A.S. A collagen-glycosaminoglycan co-culture model for heart valve tissue engineering applications. Biomaterials. 2006;27:2233–2246. doi: 10.1016/j.biomaterials.2005.10.031. PubMed DOI

Duan B., Hockaday L.A., Kang K.H., Butcher J.T. 3D Bioprinting of heterogeneous aortic valve conduits with alginate/gelatin hydrogels. J. Biomed. Mater. Res. Part A. 2013;101A:1255–1264. doi: 10.1002/jbm.a.34420. PubMed DOI PMC

Duconseille A., Astruc T., Quintana N., Meersman F., Sante-Lhoutellier V. Gelatin structure and composition linked to hard capsule dissolution: A review. Food Hydrocoll. 2015;43:360–376. doi: 10.1016/j.foodhyd.2014.06.006. DOI

Fratzl P. Collagen: Structure and Mechanics. Springer; New York, NY, USA: 2008.

Sarrigiannidis S.O., Rey J.M., Dobre O., González-García C., Dalby M.J., Salmeron-Sanchez M. A tough act to follow: Collagen hydrogel modifications to improve mechanical and growth factor loading capabilities. Mater. Today Bio. 2021;10:100098. doi: 10.1016/j.mtbio.2021.100098. PubMed DOI PMC

Silver F.H., Freeman J.W., Seehra G.P. Collagen self-assembly and the development of tendon mechanical properties. J. Biomech. 2003;36:1529–1553. doi: 10.1016/S0021-9290(03)00135-0. PubMed DOI

Yang Y., Motte S., Kaufman L.J. Pore size variable type I collagen gels and their interaction with glioma cells. Biomaterials. 2010;31:5678–5688. doi: 10.1016/j.biomaterials.2010.03.039. PubMed DOI

Yang Y.L., Kaufman L.J. Rheology and confocal reflectance microscopy as probes of mechanical properties and structure during collagen and collagen/hyaluronan self-assembly. Biophys. J. 2009;96:1566–1585. doi: 10.1016/j.bpj.2008.10.063. PubMed DOI PMC

Prockop D.J., Fertala A. Inhibition of the Self-assembly of Collagen I into Fibrils with Synthetic Peptides: Demonstration that Assembly is Driven by Specific Binding Sites on the Monomers. J. Biolog. Chem. 1998;273:15598–15604. doi: 10.1074/jbc.273.25.15598. PubMed DOI

Zhang X., Xu S., Shen L., Li G. Factors affecting thermal stability of collagen from the aspects of extraction, processing and modification. J. Leather Sci. Eng. 2020;2:19. doi: 10.1186/s42825-020-00033-0. DOI

Matinong A.M.E., Chisti Y., Pickering K.L., Haverkamp R.G. Collagen Extraction from Animal Skin. Biology. 2022;11:905. doi: 10.3390/biology11060905. PubMed DOI PMC

Gobeaux F., Mosser G., Anglo A., Panine P., Davidson P., Giraud-Guille M.-M., Belamie E. Fibrillogenesis in dense collagen solutions: A physicochemical study. J. Mol. Biol. 2008;376:1509–1522. doi: 10.1016/j.jmb.2007.12.047. PubMed DOI

Forgacs G., Newman S.A., Hinner B., Maier C.W., Sackmann E. Assembly of collagen matrices as a phase transition revealed by structural and rheologic studies. Biophys. J. 2003;84:1272–1280. doi: 10.1016/S0006-3495(03)74942-X. PubMed DOI PMC

Wood G.C., Keech M.K. The formation of fibrils from collagen solutions 1. The effect of experimental conditions: Kinetic and electron-microscope studies. Biochem. J. 1960;75:588–598. doi: 10.1042/bj0750588. PubMed DOI PMC

Joseph A. Bachelor’s Thesis. Saint Mary’s University; Halifax, Nova Scotia: 2020. [(accessed on 1 October 2023)]. Assessment of pH and Temperature Effects on Collagen Gel Microstructure Using Second Harmonic Generation and Scanning Electron Microscopies. Available online: https://library2.smu.ca/bitstream/handle/01/29355/Joseph_Ariana_Honours_2020.pdf?sequence=1&isAllowed=y.

Furusawa K., Sato S., Masumoto J., Hanazaki Y., Maki Y., Dobashi T., Yamamoto T., Fukui A., Sasaki N. Studies on the Formation Mechanism and the Structure of the Anisotropic Collagen Gel Prepared by Dialysis-Induced Anisotropic Gelation. Biomacromolecules. 2012;13:29–39. doi: 10.1021/bm200869p. PubMed DOI

Wood G.C. The formation of fibrils from collagen solutions. 2. A mechanism of collagen-fibril formation. Biochem. J. 1960;75:598–605. doi: 10.1042/bj0750598. PubMed DOI PMC

Kar K., Amin P., Bryan M.A., Persikov A.V., Mohs A., Wang Y.-H., Brodsky B. Self-association of Collagen Triple Helic Peptides into Higher Order Structures. J. Biol. Chem. 2006;281:33283–33290. doi: 10.1074/jbc.M605747200. PubMed DOI

Holmes D.F., Capaldi M.J., Chapman J.A. Reconstitution of collagen fibrils in vitro; the assembly proces depends on the initiating procedure. Inter. J. Biol. Macromol. 1986;8:161–166. doi: 10.1016/0141-8130(86)90020-6. DOI

Gómez-Guillén M.C., Giménez B., López-Caballero M.E., Montero M.P. Functional and bioactive properties of collagen and gelatin from alternative sources: A review. Food Hydrocoll. 2011;25:1813–1827. doi: 10.1016/j.foodhyd.2011.02.007. DOI

Shu F., Dai C., Wang H., Xu C., Wie B., Zhang J., Xu Y., He L., Li S. Formation, Stability and Self-Assembly Behaviour of the Collagen-Like Triple Helix Confirmation: The Role of Ser, Ala and Arg/Glu. ChemistrySelect. 2019;4:13370–13379. doi: 10.1002/slct.201903500. DOI

Stepanovska J., Supova M., Hanzalek K., Broz A., Matejka R. Collagen Bioinks for Bioprinting: A Systematic Review of Hydrogel Properties, Bioprinting Parameters, Protocols, and Bioprinted Structure Characteristics. Biomedicines. 2021;9:1137. doi: 10.3390/biomedicines9091137. PubMed DOI PMC

Holder A.J., Badiei N., Hawkins K., Wright C., Williams P.R., Curtis D.J. Control of collagen gel mechanical properties through manipulation of gelation conditions near the sol–gel transition. Soft Matter. 2018;14:574. doi: 10.1039/C7SM01933E. PubMed DOI

Correa de Moraes M., Lopes Cunha R. Gelation property and water holding capacity of heat-treated collagen at different temperature and pH values. Food Res. Inter. 2013;50:213–223. doi: 10.1016/j.foodres.2012.10.016. DOI

Kim J., Bonassar L. Controlling collagen gelation pH to enhance biochemical, structural, and biomechanical properties of tissue-engineered menisci. J. Biomed. Mater. Res. A. 2023;111:478–487. doi: 10.1002/jbm.a.37464. PubMed DOI

Wang H. A Review of the Effects of Collagen Treatment in Clinical Studies. Polymers. 2021;13:3868. doi: 10.3390/polym13223868. PubMed DOI PMC

Stepanovska J., Otahal M., Hanzalek K., Supova M., Matejka R. pH Modification of High-Concentrated Collagen Bioinks as a Factor Affecting Cell Viability, Mechanical Properties, and Printability. Gels. 2021;7:252. doi: 10.3390/gels7040252. PubMed DOI PMC

Kanokova D., Matejka R., Zaloudkova M., Zigmond J., Supova M., Matejkova J. Active Media Perfusion in Bioprinted Highly Concentrated Collagen Bioink Enhances the Viability of Cell Culture and Substrate Remodeling. Gels. 2024;10:316. doi: 10.3390/gels10050316. PubMed DOI PMC

Jackson M., Choo L.P., Watson P.H., Halliday W.C. Beware of connective tissue proteins: Assignment and implications of collagen absorptions in infrared spectra of human tissues. Biochim. Biophys. Acta. 1995;1270:1–6. doi: 10.1016/0925-4439(94)00056-V. PubMed DOI

Rýglová Š., Braun M., Suchý T., Hříbal M., Žaloudková M., Vištejnová L. The investigation of batch-to-batch variabilities in the composition of isolates from fish and mammalian species using different protocols. Food Res. Int. 2023;169:112798. doi: 10.1016/j.foodres.2023.112798. PubMed DOI

Veeruraj A., Arumugam M., Ajithkumar T., Balasubramanian T. Isolation and characterization of collagen from the outer skin of squid (Doryteuthis singhalensis) Food Hydrocoll. 2015;43:708–716. doi: 10.1016/j.foodhyd.2014.07.025. DOI

Payne K.J., Veis A. Fourier transform IR spectroscopy of collagen and gelatin solutions: Deconvolution of the Amide I band for conformational studies. Biopolymers. 1988;27:1749–1760. doi: 10.1002/bip.360271105. PubMed DOI

Rabotyagova O.S., Cebe P., Kaplan D.L. Collagen Structural Hierarchy and Susceptibility to Degradation by Ultraviolet Radiation. Mater. Sci. Eng. C. 2008;28:1420–1429. doi: 10.1016/j.msec.2008.03.012. PubMed DOI PMC

PRYSTUPA D.A., DONALD A.M. Infrared study of gelatin conformations in gel and sol states. Polym. Gels Netw. 1996;4:87–110. doi: 10.1016/0966-7822(96)00003-2. DOI

Gaar J., Naffa R., Brimble M. Enzymatic and non-enzymatic crosslinks found in collagen and elastin and their chemical synthesis. Org. Chem. Front. 2020;7:2789–2814. doi: 10.1039/D0QO00624F. DOI

Sanden K.W., Böcker U., Ofstad R., Pedersen M.E., Høst V., Afseth N.K., Rønning S.B., Pleshko N. Characterization of Collagen Structure in Normal, Wooden Breast and Spaghetti Meat Chicken Fillets by FTIR Microspectroscopy and Histology. Foods. 2021;10:548. doi: 10.3390/foods10030548. PubMed DOI PMC

Rýglová Š., Braun M., Hříbal M., Suchý T., Vörös D. The proportion of the key components analysed in collagen-based isolates from fish and mammalian tissues processed by different protocols. J. Food Comp. Anal. 2021;103:104059. doi: 10.1016/j.jfca.2021.104059. DOI

Stenzel K.H., Miyata T., Rubin A.L. Collagen as a biomaterial. Ann. Rev. Biophysic. Bioeng. 1974;3:231–253. doi: 10.1146/annurev.bb.03.060174.001311. PubMed DOI

Bailey A.J., Paul R.G., Knott L. Mechanisms of maturation and ageing of collagen. Mech. Ageing Dev. 1998;106:1–56. doi: 10.1016/S0047-6374(98)00119-5. PubMed DOI

Pešáková V., Štol M., Gillery P., Maquart F.X., Borel J.P., Adam M. The effect of different collagens and of proteoglycan on the retraction of collagen lattice. Biomed. Pharmacother. 1994;48:261–266. doi: 10.1016/0753-3322(94)90142-2. PubMed DOI

Bell E., Ivarsson B., Merrill C. Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc. Natl. Acad. Sci. USA. 1979;76:1274–1278. doi: 10.1073/pnas.76.3.1274. PubMed DOI PMC

Blidi O.E., Omari N.E., Balahbib A., Ghchime R., Menyiy N.E., Ibrahimi A., Kaddour K.B., Bouyahya A., Chokairi O., Barkiyou M. Extraction Methods, Characterization and Biomedical Applications of Collagen: A Review. Biointerf. Res. Appl. Chem. 2021;11:13587–13613. doi: 10.33263/BRIAC115.1358713613. DOI

Salim N.V., Madhan B., Glattauer V., Ramshaw J.A.M. Comprehensive review on collagen extraction from food by-products and waste as a value-added material. Int. J. Biol. Macromol. 2024;278:134374. doi: 10.1016/j.ijbiomac.2024.134374. PubMed DOI

Schmidt M.M., Dornelles R.C.P., Mello R.O., Kubota E.H., Mazutti M.A., Kempka A.P., Demiate I.M. Collagen extraction process. Int. Food Res. J. 2015;23:913–922.

Davison P.F., Cannon D.J., Andersson L.P. The effects of acetic acid on collagen cross-links. Connect. Tissue Res. 1972;1:205–216. doi: 10.3109/03008207209152076. DOI

Skierka E., Sadowska M. The influence of different acids and pepsin on the extractability of collagen from the skin of Baltic cod (Gadus morhua) Food Chem. 2007;105:1302–1306. doi: 10.1016/j.foodchem.2007.04.030. DOI

Bella J., Brodsky B., Berman H.M. Hydration structure of a collagen peptide. Structure. 1995;3:893–906. doi: 10.1016/S0969-2126(01)00224-6. PubMed DOI

Sionkowska S.A., Skopinska-Wisniewska J., Gawron M., Kozlowska J., Planecka A. Chemical and thermal cross-linking of collagen and elastin hydrolysates. Int. J. Biol. Macromol. 2010;47:570–577. doi: 10.1016/j.ijbiomac.2010.08.004. PubMed DOI

Neves M.I., Araújo M., Moroni L., da Silva R.M.P., Barrias C.C. Glycosaminoglycan-Inspired Biomaterials for the Development of Bioactive Hydrogel Networks. Molecules. 2020;25:978. doi: 10.3390/molecules25040978. PubMed DOI PMC

Chen X., Chen D., Ban E., Toussaint K.C., Janmeya P.A., Wells R.G., Shenoy V.B. Glycosaminoglycans modulate long-range mechanical communication between cells in collagen networks. Proc. Natl. Acad. Sci. USA. 2022;119:e2116718119. doi: 10.1073/pnas.2116718119. PubMed DOI PMC

Kaczmarek B., Sionkowska A., Osyczka A.M. Collagen-based scaffolds enriched with glycosaminoglycans isolated from skin of Salmo salar fish. Polym. Test. 2017;62:132–136. doi: 10.1016/j.polymertesting.2017.06.022. DOI

Badylak S.F., Brown B.N., Gilbert T.W. Biomaterials Science. 3rd ed. Academic Press; Cambridge, MA, USA: 2013. Chapter II.6.16.—Tissue Engineering with Decellularized Tissues; pp. 1316–1331. DOI

Bi Y., Patra P., Faezipour M. Structure of Collagen-Glycosaminoglycan Matrix and the Influence to its Integrity and Stability. Annu. Int. Conf. IEEE Eng. Med. Biol. Soc. 2014;2014:3949–3952. doi: 10.1109/EMBC.2014.6944488. PubMed DOI

Sato K., Ebihara T., Adachi E., Kawashima S., Hattori S., Irie S. Possible involvement of aminotelopeptide in self-assembly and thermal stability of collagen I as revealed by its removal with proteases. J. Biol. Chem. 2000;275:25870–25875. doi: 10.1074/jbc.M003700200. PubMed DOI

Walton R.S., Brand D.D., Czernuszka J.T. Influence of telopeptides, fibrils and crosslinking on physicochemical properties of type I collagen films. J. Mater. Sci. Mater. Med. 2010;21:451–461. doi: 10.1007/s10856-009-3910-2. PubMed DOI

Piez K.A., Trus B.L. Microfibrillar structure and packing of collagen: Hydrophobic interactions. J. Mol. Biol. 1977;110:701–704. doi: 10.1016/S0022-2836(77)80086-7. PubMed DOI

Suchý T., Horný L., Šupová M., Adámek T., Blanková A., Žaloudková M., Grajciarová M., Yakushko O., Blassová T., Braun M. Age-related changes in the biochemical composition of the human aorta and their correlation with the delamination strength. Acta Biomater. 2024;190:344–361. doi: 10.1016/j.actbio.2024.11.002. PubMed DOI

Amirrah I.N., Lokanathan Y., Zulkiflee I., Wee M.F.M.R., Motta A., Fauzi M.B. A Comprehensive Review on Collagen Type I Development of Biomaterials for Tissue Engineering: From Biosynthesis to Bioscaffold. Biomedicines. 2022;10:2307. doi: 10.3390/biomedicines10092307. PubMed DOI PMC

Sloseris D., Forde N.R. AGEing of collagen: The effects of glycation on collagen’s stability, mechanics and assembly. Matrix Biol. 2025;135:153–160. doi: 10.1016/j.matbio.2024.12.007. PubMed DOI

Gandhi M., Elfeky O., Ertugrul H., Chela H.K., Daglilar E. Scurvy: Rediscovering a Forgotten Disease. Diseases. 2023;11:78. doi: 10.3390/diseases11020078. PubMed DOI PMC

ČSN 46 7092-25 (467092)Methods of Testing for Feedingstuffs—Part 25: Determination of Amino Acid Content. [(accessed on 18 May 2025)]; Available online: https://www.technicke-normy-csn.cz/csn-46-7092-25-467092-207000.html#.

Meat and Meat Products—Determination of Hydroxyproline Content. ISO; Geneva, Switzerland: 2022. [(accessed on 18 May 2025)]. Available online: https://www.iso.org/standard/8848.html.

Lee D.K. Data transformation: A focus on the interpretation. Korean J. Anesthesiol. 2020;73:503–508. doi: 10.4097/kja.20137. PubMed DOI PMC