{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T20:59:44Z","timestamp":1790369984441,"version":"4.1.0"},"reference-count":28,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2022,5,14]],"date-time":"2022-05-14T00:00:00Z","timestamp":1652486400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Chemical Faculty of the Gdansk University of Technology","award":["032408T103"],"award-info":[{"award-number":["032408T103"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The article presents a new approach in the purification of chitosan (CS) hydrogel in order to remove a significant amount of endotoxins without changing its molecular weight and viscosity. Two variants of the method used to purify CS hydrogels from endotoxins were investigated using the PyroGene rFC Enzymatic Cascade assay kit. The effect of the CS purification method was assessed in terms of changes in the dynamic viscosity of its hydrogels, the molecular weight of the polymer, microbiological purity after refrigerated storage and cytotoxicity against L929 cells based on the ISO 10993-5:2009(E) standard. The proposed purification method 1 (M1) allows for the removal of significant amounts of endotoxins: 87.9\u201397.6% in relation to their initial concentration in the CS hydrogel without affecting the solution viscosity. Moreover, the final solutions were sterile and microbiologically stable during storage. The M1 purification method did not change the morphology of the L929 cells.<\/jats:p>","DOI":"10.3390\/ijms23105505","type":"journal-article","created":{"date-parts":[[2022,5,14]],"date-time":"2022-05-14T09:27:56Z","timestamp":1652520476000},"page":"5505","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["A Novel Method of Endotoxins Removal from Chitosan Hydrogel as a Potential Bioink Component Obtained by CO2 Saturation"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2992-7611","authenticated-orcid":false,"given":"Adrianna","family":"Banach-Kope\u0107","sequence":"first","affiliation":[{"name":"Department of Chemistry, Technology, and Biotechnology of Food, Faculty of Chemistry, Gda\u0144sk University of Technology, 11\/12 G.Narutowicza Str., 80-233 Gdansk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2748-0393","authenticated-orcid":false,"given":"Szymon","family":"Mania","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Technology, and Biotechnology of Food, Faculty of Chemistry, Gda\u0144sk University of Technology, 11\/12 G.Narutowicza Str., 80-233 Gdansk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3476-2540","authenticated-orcid":false,"given":"Joanna","family":"Pilch","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gda\u0144sk University of Technology, 11\/12 G.Narutowicza Str., 80-233 Gdansk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0807-021X","authenticated-orcid":false,"given":"Ewa","family":"Augustin","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gda\u0144sk University of Technology, 11\/12 G.Narutowicza Str., 80-233 Gdansk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0833-5596","authenticated-orcid":false,"given":"Iwona","family":"Gabriel","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gda\u0144sk University of Technology, 11\/12 G.Narutowicza Str., 80-233 Gdansk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7643-8664","authenticated-orcid":false,"given":"Robert","family":"Tylingo","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Technology, and Biotechnology of Food, Faculty of Chemistry, Gda\u0144sk University of Technology, 11\/12 G.Narutowicza Str., 80-233 Gdansk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,5,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"119028","DOI":"10.1016\/j.carbpol.2021.119028","article-title":"A novel method of creating thermoplastic chitosan blends to produce cell scaffolds by FDM additive manufacturing","volume":"280","author":"Tylingo","year":"2022","journal-title":"Carbohydr. 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Test."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"5156","DOI":"10.3390\/md13085156","article-title":"Chitosan: An update on potential biomedical and pharmaceutical applications","volume":"13","author":"Cheung","year":"2015","journal-title":"Mar. Drugs"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2589","DOI":"10.1016\/S0142-9612(00)00126-5","article-title":"Application of Chitosan-Based polysaccharide biomaterials in cartilage tissue engineering: A review","volume":"21","author":"Matthew","year":"2000","journal-title":"Biomaterials"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"901","DOI":"10.1016\/j.carbpol.2013.10.060","article-title":"Preparation and characterization of genipin cross-linked porous chitosan\u2013collagen\u2013gelatin scaffolds using chitosan\u2013CO2 solution","volume":"102","author":"Gorczyca","year":"2014","journal-title":"Carbohydr. Polym."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Ravindranathan, S., Koppolu, B.P., Smith, S.G., and Zaharoff, D.A. (2016). Effect of chitosan properties on immunoreactivity. Mar. Drugs, 14.","DOI":"10.3390\/md14050091"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1385\/MB:19:3:279","article-title":"The biology of endotoxin","volume":"19","author":"Heine","year":"2001","journal-title":"Mol. Biotechnol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"5058","DOI":"10.1128\/AEM.00122-09","article-title":"Inactivation of Escherichia coli endotoxin by soft hydrothermal processing","volume":"75","author":"Miyamoto","year":"2009","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_11","first-page":"1","article-title":"Endotoxin limits for parenteral drug products","volume":"1","author":"Dawson","year":"2017","journal-title":"BET White Pap."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1509","DOI":"10.1002\/biot.201200220","article-title":"Endotoxin removal and prevention for pre-clinical biologics production","volume":"7","author":"Kerins","year":"2012","journal-title":"Biotechnol. J."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1089\/ten.teb.2012.0636","article-title":"Endotoxins-the invisible companion in biomaterials research","volume":"19","author":"Lieder","year":"2013","journal-title":"Tissue Eng. Part B Rev."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Piehler, M., Roeder, R., Blessing, S., and Reich, J. (2020). Comparison of LAL and RFC assays\u2014Participation in a proficiency test program between 2014 and 2019. Microorganisms, 8.","DOI":"10.3390\/microorganisms8030418"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1016\/S0167-7799(01)01694-8","article-title":"A new era in pyrogen testing","volume":"19","author":"Ding","year":"2001","journal-title":"Trends Biotechnol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Rodr\u00edguez-V\u00e1zquez, M., Vega-Ruiz, B., Ramos-Z\u00fa\u00f1iga, R., Salda\u00f1a-Koppel, D.A., and Qui\u00f1ones-Olvera, L.F. (2015). Chitosan and its potential use as a scaffold for tissue engineering in regenerative medicine. BioMed Res. Int., 1\u201315.","DOI":"10.1155\/2015\/821279"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1007\/s40204-018-0094-1","article-title":"Tailoring the Gelatin\/Chitosan electrospun scaffold for application in skin tissue engineering: An in vitro study","volume":"7","author":"Zandi","year":"2018","journal-title":"Prog. Biomater."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"920","DOI":"10.1111\/jphp.13082","article-title":"Easy and effective method to generate endotoxin-free chitosan particles for immunotoxicology and immunopharmacology studies","volume":"71","author":"Lebre","year":"2019","journal-title":"J. Pharm. Pharmacol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"646","DOI":"10.3389\/fbioe.2021.699418","article-title":"Advances in the development of biomaterials for endotoxin adsorption in sepsis","volume":"9","author":"Yang","year":"2021","journal-title":"Front. Bioeng. Biotechnol."},{"key":"ref_20","first-page":"1082","article-title":"Interaction of bacterial endotoxins with chitosan. Effect of endotoxin structure, chitosan molecular mass, and ionic strength of the solution on the formation of the complex","volume":"65","author":"Davydova","year":"2000","journal-title":"Biochem. Biokhimiia"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"728","DOI":"10.1016\/j.rinp.2016.10.012","article-title":"Study on the degradation of chitosan slurries","volume":"6","author":"Martini","year":"2016","journal-title":"Results Phys."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Sikorski, D., Gzyra-Jagie\u0142a, K., and Draczy\u0144ski, Z. (2021). The kinetics of chitosan degradation in organic acid solutions. Mar. Drugs, 19.","DOI":"10.3390\/md19050236"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1819","DOI":"10.3390\/md13041819","article-title":"Stability of chitosan-A challenge for pharmaceutical and biomedical applications","volume":"13","author":"Winnicka","year":"2015","journal-title":"Mar. Drugs"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Ke, C.-L., Deng, F.-S., Chuang, C.-Y., Lin, C.-H., and Bardosova, M. (2021). Polymers antimicrobial actions and applications of chitosan. Polymers, 13.","DOI":"10.3390\/polym13060904"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"San Juan, A., Montembault, A., Gillet, D., Say, J.P., Rouif, S., Bouet, T., Royaud, I., and David, L. (2012). Degradation of chitosan-based materials after different sterilization treatments. IOP Conference Series: Materials Science and Engineering, IOP Publishing.","DOI":"10.1088\/1757-899X\/31\/1\/012007"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"344","DOI":"10.1023\/B:PHAM.0000016249.52831.a5","article-title":"Uptake and cytotoxicity of chitosan molecules and nanoparticles: Effects of Molecular weight and degree of deacetylation","volume":"21","author":"Huang","year":"2004","journal-title":"Pharm. Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1080\/09540105.2011.569883","article-title":"Food and Agricultural immunology determination of in vivo toxicity and in vitro cytotoxicity of lipopolysaccharide isolated from salmonella enteritidis and its potential use for production of polyclonal antibody","volume":"22","author":"Nalbantsoy","year":"2011","journal-title":"Food Agric. Immunol."},{"key":"ref_28","first-page":"39","article-title":"Determination of viscosity-average molecular weight of chitosan using intrinsic viscosity measurement","volume":"10","author":"Yacob","year":"2013","journal-title":"J. Nucl. Relat. Technol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/10\/5505\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:10:52Z","timestamp":1760137852000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/10\/5505"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,5,14]]},"references-count":28,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2022,5]]}},"alternative-id":["ijms23105505"],"URL":"https:\/\/doi.org\/10.3390\/ijms23105505","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,5,14]]}}}