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Lookup NU author(s): Sophie Reay, Emma Jackson, Dan Salthouse, Dr Ana Ferreira-DuarteORCiD, Professor Catharien Hilkens, Professor Katarina NovakovicORCiD
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Introduction Chitosan-based biomaterials are becoming increasingly popular for biomedical applications including wound healing, drug delivery and tissue regeneration. As chitosan is naturally derived, it is susceptible to endotoxin contamination. Bacterial endotoxins elicit potent proinflammatory responses in vivo, therefore, it is critical that endotoxin is quantified and removed from any biomaterial intended for in vivo use. In the presented work, heat-treatment (180°C for 1.5 hours) and sodium hydroxide-treatment (1M for 2 hours) are investigated as two endotoxin removal methods from chitosan. While endotoxin removal is the prime task, it is important to note that preservation of chitosan structure is vital for synthesis and in vivo lysozyme degradation of chitosan-based hydrogels. Methods The limulus amebocyte lysate (LAL) assay was used to quantify the endotoxin content of chitosan samples. Endotoxin removal was also assessed by measuring TNF-alpha production from PBMCs cultured with treated or native chitosan. FTIR was employed to determine the effect of endotoxin removal methods on chitosan structure. Chitosan-genipin hydrogels were synthesized and lysozyme degradation was investigated gravimetrically. Hydrogels were co-cultured separately with immature monocyte-derived DCs (moDCs) and cell viability and expression of maturation markers (CD83, CD86 and PDL-1) expression was assessed by flow cytometry. Results Both heat and NaOH treatment significantly reduced the endotoxin level compared to native chitosan, to concentrations below the FDA limit for medical devices (0.5 EU/ml). NaOH-treatment significantly reduced TNF-alpha secretion by PBMCs compared to native chitosan; however, the difference was non-significant for the heat-treated chitosan, which was excluded from further testing. Although the FTIR spectra of native and NaOH-treated chitosan were extremely similar, NaOH treatment reduced the degree of acetylation of native chitosan by 6%. As lysozyme only interacts with the acetylated units of chitosan, it was important to determine if NaOH-treated chitosan is susceptible to lysozyme degradation. Chitosan-genipin hydrogels were synthesized and gravimetric degradation studies using lysozyme showed that hydrogels composed of native and NaOH-treated chitosan had comparable degradation rates. NaOH-treated chitosan did not negatively affect viability of immature DCs, furthermore the expression of maturation markers was non-significant between NaOH chitosan-genipin hydrogel condition and the immature DC control. Conclusion NaOH treatment is a cheap, effective endotoxin removal method that preserves chitosan structure. Resultant chitosan-genipin hydrogels synthesised with NaOH-treated chitosan are biocompatible with moDCs, as they do not induce cell death or maturation.
Author(s): Reay S, Jackson E, Salthouse D, Ferreira AM, Hilkens C, Novakovic K
Publication type: Conference Proceedings (inc. Abstract)
Publication status: Published
Conference Name: Tissue Engineering and Regenerative Medicine International Society Americas Europe Chapter Conference (TERMIS-EU 2023)
Year of Conference: 2023
Print publication date: 28/03/2023
Online publication date: 28/03/2023
Acceptance date: 30/01/2023
Publisher: TERMIS-EU
URL: https://eu2023.termis.org/
