Crosslinking of genipin and autoclaving in chitosan-based nanofibrous scaffolds: structural and physiochemical properties
- 179 Downloads
Chitosan-based electrospun nanofibrous scaffolds have been selected as wound healing/tissue scaffolds because of their extracellular matrix nature and biocompatible properties. However, crosslinking of scaffolds is necessary to avoid lysozyme degradation in an aqueous environment, as a stable scaffold is crucial for the activities of fibroblasts, including adhesion and proliferation during wound healing. Autoclaving (physical) and genipin crosslinking (chemical) methods have been employed to stabilize chitosan-based scaffolds individually. However, the differences in scaffold microstructure induced by the individual or combined crosslinking methods have yet to be investigated systematically. In this study, autoclaving crosslinking improved mainly the structural properties (tensile strength and crystallinity), but it also expanded the chitosan and PEO network by hydrolysis, which enlarged the fiber diameter and caused chitosan chain degradation. Meanwhile, genipin crosslinking improved the physiochemical properties, primarily hydrophilicity. On the other hand, the combined crosslinking significantly improved both the structural and physiochemical properties through the unique reorganization of the polymeric network. The confined geometry of the nanofiber as well as the genipin crosslinks resulted in maximal crystallization of chitosan and amorphization of PEO chains. Unfortunately, the combined crosslinking resulted in the lowest antibacterial activity because of the consumption of amino and protonated amino groups in the crosslinking process. Despite this, the combined crosslinking scaffold achieved the best stability under lysozyme degradation and therefore it is preferred over autoclaving or genipin crosslinking alone. In conclusion, the results show that chemical and physical crosslinking methods induce different changes in crystallinity and hydrophilicity that affect the physicochemical properties. Therefore, crystallinity and hydrophilicity are significant considerations when designing a tissue scaffold.
Yi Wah Mak acknowledges the funding support from the Hong Kong Research Grant Council PhD fellowship for three years. She also acknowledges additional studentship for 1 year from the Department of Mechanical Engineering, the Hong Kong Polytechnic University (HKPolyU). The authors are grateful to Mr. Kenneth K.S. Lo from the Dept. of Mechanical Engineering (ME), HKPolyU, for comments, Dr. Kit Ying Choy from the Dept. of Applied Biology and Chemical Technology (ABCT), HKPolyU, for bacterial culture technology, Prof. Thomas Leung from the Dept. of ABCT, HKPolyU, for the gift of S. aureus, and Dr. Y.S. Szeto from the Department of ITC, HKPolyU, for his comments.
Yi Wah Mak received the PhD fellowship funding from the Hong Kong Research Grant Council for 3 years. She also received additional studentship for 1 year from the Department of Mechanical Engineering, the Hong Kong Polytechnic University.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- 5.Paul W, Sharma CP (2004) Chitosan and alginate wound dressings: a short review. Trends Biomater Artif Organs 18:18–23Google Scholar
- 11.Bhattarai N, Edmondson D, Veiseh O, Matsen FA, Zhang M (2005) Electrospun chitosan-based nanofibers and their cellular compatibility. Biomaterials 26:6176–6184. https://doi.org/10.1016/j.biomaterials.2005.03.027 CrossRefGoogle Scholar
- 13.Lim LY, Khor E, Ling CE (1999) Effects of dry heat and saturated steam on the physical properties of chitosan. J Biomed Mater Res 48:111–116. https://doi.org/10.1002/(SICI)1097-4636(1999)48:2%3c111:AID-JBM3%3e3.0.CO;2-W CrossRefGoogle Scholar
- 15.Mayol L, De Stefano D, Campani V, De Falco F, Ferrari E, Cencetti C, Matricardi P, Maiuri L, Carnuccio R, Gallo A, Maiuri M, De Rosa G (2014) Design and characterization of a chitosan physical gel promoting wound healing in mice. Eur Soc Biomater 25:1483–1493. https://doi.org/10.1007/s10856-014-5175-7 Google Scholar
- 20.Pauliukaite R, Ghica ME, Fatibello-Filho O, Brett CMA (2009) Comparative study of different cross-linking agents for the immobilization of functionalized carbon nanotubes within a chitosan film supported on a graphite–epoxy composite electrode. Anal Chem 81:5364–5372. https://doi.org/10.1021/ac900464z CrossRefGoogle Scholar
- 21.Huang LLH, Sung HW, Tsai CC, Huang DM (1998) Biocompatibility study of a biological tissue fixed with a naturally occurring crosslinking reagent. J Biomed Mater Res 42:568–576. https://doi.org/10.1002/(sici)1097-4636(19981215)42:4%3c568:aid-jbm13%3e3.0.co;2-7 CrossRefGoogle Scholar
- 22.Norowski PA, Fujiwara T, Clem WC, Adatrow PC, Eckstein EC, Haggard WO, Bumgardner JD (2012) Novel naturally crosslinked electrospun nanofibrous chitosan mats for guided bone regeneration membranes: material characterization and cytocompatibility. J Tissue Eng Regen Med 9:577–583. https://doi.org/10.1002/term.1648 CrossRefGoogle Scholar
- 31.Norowski PA, Mishra S, Adatrow PC, Haggard WO, Bumgardner JD (2012) Suture pullout strength and in vitro fibroblast and RAW 264.7 monocyte biocompatibility of genipin crosslinked nanofibrous chitosan mats for guided tissue regeneration. J Biomed Mater Res Part A 100:2890–2896. https://doi.org/10.1002/jbm.a.34224 CrossRefGoogle Scholar
- 33.Fessel G, Cadby J, Wunderli S, van Weeren R, Snedeker JG (2014) Dose- and time-dependent effects of genipin crosslinking on cell viability and tissue mechanics—toward clinical application for tendon repair. Acta Biomater 10:1897–1906. https://doi.org/10.1016/j.actbio.2013.12.048 CrossRefGoogle Scholar
- 56.Toffey A, Samaranayake G, Frazier CE, Glasser WG (1996) Chitin derivatives. I. Kinetics of the heat-induced conversion of chitosan to chitin. J Appl Polym Sci 60:75–85. https://doi.org/10.1002/(SICI)1097-4628(19960404)60:1%3c75:AID-APP9%3e3.0.CO;2-S CrossRefGoogle Scholar
- 68.Mejía A, García N, Guzmán J, Tiemblo P (2013) Confinement and nucleation effects in poly(ethylene oxide) melt-compounded with neat and coated sepiolite nanofibers: modulation of the structure and semicrystalline morphology. Eur Polym J 49:118–129. https://doi.org/10.1016/j.eurpolymj.2012.09.014 CrossRefGoogle Scholar
- 72.Klein MP, Hackenhaar CR, Lorenzoni ASG, Rodrigues RC, Costa TMH, Ninow JL, Hertz PF (2016) Chitosan crosslinked with genipin as support matrix for application in food process: support characterization and β-d-galactosidase immobilization. Carbohydr Polym 137:184–190. https://doi.org/10.1016/j.carbpol.2015.10.069 CrossRefGoogle Scholar
- 80.Hasmann A, Wehrschuetz-Sigl E, Kanzler G, Gewessler U, Hulla E, Schneider KP, Binder B, Schintler M, Guebitz GM (2011) Novel peptidoglycan-based diagnostic devices for detection of wound infection. Diagn Microbiol Infect Dis 71:12–23. https://doi.org/10.1016/j.diagmicrobio.2010.09.009 CrossRefGoogle Scholar