Genipin cross-linked electrospun chitosan-based nanofibrous mat as tissue engineering scaffold


1 Department of Medical Nanotechnology, School of Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran, Iran

2 National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran


To improve water stability of electrospun chitosan/ Polyethylene oxide (PEO) nanofibers, genipin, a biocompatible and nontoxic agent, was used to crosslink chitosan based nanofibers.
Materials and Methods:
Different amounts of genipin were added to the chitosan/PEO solutions, chitosan/PEO weight ratio 90/10 in 80 % acetic acid, and the solutions were then electrospun to form nanofibers. The spun nanofibers were exposed to water vapor to complete crosslinking. The nanofibrous membranes were subjected to detailed analysis by scanning electron microscopy (SEM), Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy, swelling test, MTT cytotoxicity, and cell attachment.  
SEM images of electrospun mats showed that genipin-crosslinked nanofibers retained their fibrous structure after immerging in PBS (pH=7.4) for 24 hours, while the uncrosslinked samples lost their fibrous structure, indicating the water stability of genipin-crosslinked nanofibers. The genipin-crosslinked mats also showed no significant change in swelling ratio in comparison with uncrosslinked ones. FTIR-ATR spectrum of uncrosslinked and genipin-crosslinked chitosan nanofibers revealed the reaction between genipin and amino groups of chitosan. Cytotoxicity of genipin-crosslinked nanofibers was examined by MTT assay on human fibroblast cells in the presence of nanofibers extraction media. The genipin-crosslinked nanofibers did not show any toxic effects on fibroblast cells at the lowest and moderate amount of genipin. The fibroblast cells also showed a good adhesion on genipin-crosslinked nanofibers.  
This electrospun matrix would be used for biomedical applications such as wound dressing and scaffold for tissue engineering without the concern of toxicity.


1. Bhardwaj N, Kundu SC. Electrospinning: a fascinating fiber fabrication technique. Biotechnol Adv. 2010; 28(3): 325-347.
2. Lala NL, Ramaseshan R, Bojun L, Sundarrajan S, Barhate RS, Ying-jun L, Ramakrishna S. Fabrication of nanofibers with antimicrobial functionality used as filters: protection against bacterial contaminants. Biotechnol Bioeng. 2007; 97(6): 1357-1365.
3. Piperno S, Passacantando M, Santucci S, Lozzi L, La Rosa S. WO nanofibers for gas sensing applications. J Appl Phys. 2007; 101: 124504. Available from URL: doi: 10.1063/1.2748627.
4. Kim K, Luu YK, Chang C, Fang D, Hsiao BS, Chu B , Hadjiargyrou M. Incorporation and controlled release of a hydrophilic antibiotic using poly(lactide-co-glycolide)-based electrospun nanofibrous scaffolds. J Control Release. 2004; 98(1): 47-56.
5. Rho KS, Jeong L, Lee G, Seo B-M, Park YJ, Hong S-D, Roh S, Cho JJ, Park WH, Min B-M. Electrospinning of collagen nanofibers: Effects on the behavior of normal human keratinocytes and early-stage wound healing. Biomaterials. 2006; 27(8): 1452-1461.
6. Inoguchi H, Kwon IK, Inoue E, Takamizawa K, Maehara Y, Matsuda T. Mechanical responses of a compliant electrospun poly(l-lactide-co-[epsilon]-caprolactone) small-diameter vascular graft. Biomaterials. 2006; 27(8): 1470-1478.
7. Wang Z-G, Wan L-S, Liu Z-M, Huang X-J , Xu Z-K. Enzyme immobilization on electrospun polymer nanofibers: An overview. J Mol Catal B: Enzym. 2009; 56(4): 189-195.
8. Lee KY, Jeong L, Kang YO, Lee SJ, Park WH. Electrospinning of polysaccharides for regenerative medicine. Adv Drug Del Rev. 2009; 61(12): 1020-1032.
9. Jayakumar R, Prabaharan M, Nair S , Tamura H. Novel chitin and chitosan nanofibers in biomedical applications. Biotechnol Adv. 2010; 28(1): 142-150.
10. Vondran JL, Sun W, Schauer CL. Crosslinked, electrospun chitosan–poly(ethylene oxide) nanofiber mats. J Appl Polym Sci. 2008; 109(2): 968-975.
11. Speer DP, Chvapil M, Eskelson CD, Ulreich J. Biological effects of residual glutaraldehyde in glutaraldehyde-tanned collagen biomaterials. J Biomed Mater Res. 1980; 14(6): 753-764.
12. Mi F-L, Sung H-W, Shyu S-S. Synthesis and characterization of a novel chitosan-based network prepared using naturally occurring crosslinker. J Polym Sci Part A: Polym Chem. 2000; 38(15): 2804-2814.
13. Ko C-S, Huang J-P, Huang C-W , Chu I M. Type II collagen-chondroitin sulfate-hyaluronan scaffold cross-linked by genipin for cartilage tissue engineering. J Biosci Bioeng. 2009; 107(2): 177-182.
14. Sung HW, Huang RN, Huang LLH , Tsai CC. In vitro evaluation of cytotoxicity of a naturally occurring cross-linking reagent for biological tissue fixation. J Biomater Sci Polym Ed. 1999; 10(1):63-78.
15. Sung H-W, Huang R-N, Huang LLH, Tsai C-C, Chiu C-T. Feasibility study of a natural crosslinking reagent for biological tissue fixation. J Biomed Mater Res. 1998; 42(4): 560-567.
16. Muzzarelli RAA. Genipin-crosslinked chitosan hydrogels as biomedical and pharmaceutical aids. Carbohydr Polym. 2009; 77(1): 1-9.
17. Yuan Y, Chesnutt BM, Utturkar G, Haggard WO, Yang Y, Ong JL , Bumgardner JD. The effect of cross-linking of chitosan microspheres with genipin on protein release. Carbohydr Polym. 2007; 68(3): 561-567.
18. Ko J, Yin H, An J, Chung D, Kim J-H, Lee S, Pyun D. Characterization of cross-linked gelatin nanofibers through electrospinning. Macromol Res. 2010; 18(2): 137-143.
19. Mekhail M, Wong KKH, Padavan DT, Wu Y, O'Gorman DB , Wan W. Genipin-cross-linked electrospun collagen fibers. J. Biomater Sci Polym Ed. 2011; 22(17): 2241-2259.
20. Zhang K, Qian Y, Wang H, Fan L, Huang C, Yin A, Mo X. Genipin-crosslinked silk fibroin/hydroxybutyl chitosan nanofibrous scaffolds for tissue-engineering application. J Biomed Mater Res A. 2010; 95A(3): 870-881.
21. Austero MS, Donius AE, Wegst UGK , Schauer CL. New crosslinkers for electrospun chitosan fibre mats. I. Chemical analysis. J R Soc Interface. 2012; 9(75): 2551-2562.
22. Frohbergh ME, Katsman A, Botta GP, Lazarovici P, Schauer CL, Wegst UGK, Lelkes PI. Electrospun hydroxyapatite-containing chitosan nanofibers crosslinked with genipin for bone tissue engineering. Biomaterials. 2012; 33(36): 9167–9178.
23. Pakravan M, Heuzey MC, Ajji A. A fundamental study of chitosan/PEO electrospinning. Polymer. 2011; 52(21): 4813-4824.
24. Jin J, Song M. Chitosan and chitosan–PEO blend membranes crosslinked by genipin for drug release. J. Appl. Polym. Sci. 2006; 102(1):436-444.
25. Schiffman JD , Schauer CL. Cross-Linking Chitosan Nanofibers. Biomacromolecules. 2006; 8(2): 594-601.
26. Chen ZG, Wang PW, Wei B, Mo XM, Cui FZ. Electrospun collagen–chitosan nanofiber: A biomimetic extracellular matrix for endothelial cell and smooth muscle cell. Acta Biomater. 2010; 6(2): 372-382.
27. Silva SS, Motta A, Rodrigues MT, Pinheiro AFM, Gomes ME, Mano JF, Reis RL, Migliaresi C. Novel genipin-cross-linked chitosan/silk fibroin sponges for cartilage engineering strategies. Biomacromolecules. 2008; 9(10): 2764-2774.
28. Harish Prashanth K, Kittur F, Tharanathan R. Solid state structure of chitosan prepared under different N-deacetylating conditions. Carbohydr Polym. 2002; 50(1): 27-33.
29. Schiffman JD, Schauer CL. One-step electrospinning of cross-linked chitosan fibers. Biomacromolecules. 2007; 8(9): 2665-2667.