Fabrication of macroporous soft hydrogels of Chitosan scaffolds by hydrothermal reaction and cytotoxicity to 3T3 L1 cells


A novel method of fabricating a macroporous chitosan (CH) hydrogel scaffold that is partly crosslinked physically and chemically is introduced. This involved the hydrothermal reaction of chitosan in the presence of succinic acid (SA) and urea (UR) in addition to chemical crosslinking in the presence of genipin (G). The physical crosslinks could be removed by extraction with dilute sodium hydroxide, leaving a macroporous and lightly crosslinked CH that could function as a biocompatible macroporous scaffold. The structure of the product was characterized by 13C solid-state NMR, FTIR, TGA, and PXRD. The porosity of the gel was assessed by micro CT x-ray imaging, while the rheological properties were evaluated by rheometry. The gels were observed to absorb a significant quantity of water (~ 500 g/g maximum), but their rheological properties were not improved significantly as a result of the additional mild chemical crosslinking. The scaffold of the desired shape can be prepared in this method through the variation in the shape of the reacting vessel as the gel takes the shape of the container. The gels were non-toxic to 3T3 L1 (mouse fibroblast) cells and thus offer scope for both haemostatic and drug delivery applications. The constraint of the existing system, at this phase, is the lack of ability to fabricate microporous CH with higher porosity and higher surface area.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12





Cuccinic acid






Product of hydrothermal reaction between chitosan, succinic acid and urea


G product of hydrothermal reaction between chitosan, succinic acid urea and genipin


  1. 1.

    Bhatnagar A, Sillanpää M (2009) Applications of chitin-and chitosan-derivatives for the detoxification of water and wastewater—a short review. Adv Colloid Interface Sci 152:26–38

    CAS  Article  Google Scholar 

  2. 2.

    Prashanth KH, Tharanathan R (2007) Chitin/chitosan: modifications and their unlimited application potential – an overview. Trends Food Sci Technol 18:117–131

    CAS  Article  Google Scholar 

  3. 3.

    Ulery BD, Nair LS, Laurencin CT (2011) biomedical applications of biodegradable polymers. J Polym Sci B Polym Phys 49:832–864

    CAS  Article  Google Scholar 

  4. 4.

    Berger J, Reist M, Mayer J, Felt O, Peppas N, Gurny R (2004) Structure and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications. Eur J Pharm Biopharm 57:19–34

    CAS  Article  Google Scholar 

  5. 5.

    Monteiro OA Jr, Airoldi C (1999) some studies of crosslinking chitosan–glutaraldehyde interaction in a homogeneous system. Int J Biol Macromol 26:119–128

    CAS  Article  Google Scholar 

  6. 6.

    Leung H (2001) Ecotoxicology of glutaraldehyde: Review of environmental fate and effects studies. Ecotoxicol Environ Saf 49:26–39

    CAS  Article  Google Scholar 

  7. 7.

    Takigawa T, Endo Y (2006) Effects of glutaraldehyde exposure on human health. J Occup Health 48:75–87

    CAS  Article  Google Scholar 

  8. 8.

    Sung HW, Huang RN, Huang LLH, Tsai CC (1999) In vitro evaluation of cytotoxicity of a naturally occurring cross-linking reagent for biological tissue fixation. J Biomater Sci Polym Ed 10:63–78

    CAS  Article  Google Scholar 

  9. 9.

    Butler MF, Ng YF, Pudney PDA (2003) Mechanism and kinetics of the crosslinking reaction between biopolymers containing primary amine groups and genipin. J polym sci pol chem 41:3941–3953

    CAS  Article  Google Scholar 

  10. 10.

    Mi F, Shyu S, Peng C (2005) Characterization of ring-opening polymerization of genipin and pH-dependent cross-linking reactions between chitosan and genipin. J polym sci pol chem 43:1985–2000

    CAS  Article  Google Scholar 

  11. 11.

    Mi F, Sung H, Shyu S (2000) Synthesis and characterization of a novel chitosan-based network prepared using naturally occurring crosslinker. J polym sci pol chem 38:2804–2814

    CAS  Article  Google Scholar 

  12. 12.

    Bigi A, Cojazzi G, Panzavolta S, Roveri N, Rubini K (2002) Stabilization of gelatin films by crosslinking with genipin. Biomaterials 23:4827–4832

    CAS  Article  Google Scholar 

  13. 13.

    Mekhail M, Wong KK, Padavan DT, Wu Y, O’Gorman DB, Wan W (2011) Genipin-cross-linked electrospun collagen fibers. J Biomater Sci Polym Ed 22:2241–2259

    CAS  Article  Google Scholar 

  14. 14.

    Moura MJ, Figueiredo MM, Gil MH (2007) Rheological study of genipin cross-linked chitosan hydrogels. Biomacromol 8:3823–3829

    CAS  Article  Google Scholar 

  15. 15.

    Muzzarelli RAA (2009) Genipin-crosslinked chitosan hydrogels as biomedical and pharmaceutical aids. Carbohydr Polym 77:1–9

    CAS  Article  Google Scholar 

  16. 16.

    Song M, Jin J, Hourston DJ (2004) Novel chitosan-based films cross-linked by genipin with improved physical properties. Biomacromol 5:162–168

    Article  Google Scholar 

  17. 17.

    Muzzarelli RA, El Mehtedi M, Bottegoni C, Aquili A, Gigante A (2015) Genipin-crosslinked chitosan gels and scaffolds for tissue engineering and regeneration of cartilage and bone. Mar Drugs 13:7314–7338

    CAS  Article  Google Scholar 

  18. 18.

    Dimida S, Demitri C, De Benedictis VM, Scalera F, Gervaso F, Sannino A (2015) Genipin-cross-linked chitosan-based hydrogels: Reaction kinetics and structure-related characteristics. J Appl Polym Sci 132:42256

    Article  Google Scholar 

  19. 19.

    Winotapun W, Opanasopit P, Ngawhirunpat T, Rojanarata T (2013) One enzyme catalyzed simultaneous plant cell disruption and conversion of released glycoside to aglycone combined with in situ product separation as green one-pot production of genipin from Gardenia fruit. Enzym Microbial Technol 53:92–96

    CAS  Article  Google Scholar 

  20. 20.

    Manickam B, Sreedharan R, Elumalai M (2014) Genipin the natural water soluble crosslinking agent and its importance in the modified drug delivery systems: An overview. Curr Drug Deliv 11:139–145

    CAS  Article  Google Scholar 

  21. 21.

    Zhang Y, Wang QS, Yan K, Qi Y, Wang GF, Cui YL (2016) Preparation, characterization, and evaluation of genipin crosslinked chitosan/gelatin three-dimensional scaffolds for liver tissue engineering applications. J Biomed Mater Res A 104:1863–1870

    CAS  Article  Google Scholar 

  22. 22.

    Sampaio GYH, Fook AC, Fideles TB, Cavalcanti MERRM, Fook MVL (2015) Biodegradable chitosan scaffolds: effect of genipin crosslinking. Materials Science Forum, Trans Tech Publications Ltd 805:116–121

    Article  Google Scholar 

  23. 23.

    Muzzarelli RAA, Tanfani F, Scarpini G, Laterza G (1980) The degree of acetylation of chitins by gas chromatography and infrared spectroscopy. J Biochem Bioph Meth 2:299–306

    CAS  Article  Google Scholar 

  24. 24.

    Prabha G, Narayanan A, Kartik R, Dhamodharan R (2019) Facile preparation of biocompatible macroporous chitosan hydrogel by hydrothermal reaction of a mixture of chitosan-succinic acid-urea. Mater Sci Eng C 104:109845

    Article  Google Scholar 

  25. 25.

    Narayanan A, Dhamodharan R (2015) Super water-absorbing new material from chitosan, EDTA and urea. Carbohydr Polym 134:337–343

    CAS  Article  Google Scholar 

  26. 26.

    Narayanan A, Kartik R, Sangeetha E, Dhamodharan R (2018) Super water absorbing polymeric gel from chitosan, citric acid and urea: synthesis and mechanism of water absorption. Carbohydr Polym 191:152–160

    CAS  Article  Google Scholar 

  27. 27.

    Prabha G, Raj V (2017) Sodium alginate–polyvinyl alcohol–Bovin serum albumin coated Fe3O4 nanoparticles as anticancer drug delivery vehicle: doxorubicin loading and in vitro release study and cytotoxicity to HepG2 and L02 cells. Mater Sci Eng C 79:410–422

    CAS  Article  Google Scholar 

  28. 28.

    Huang Y, He M, Lu A, Zhou W, Stoyanov SD, Pelan EG, Zhan L (2015) Hydrophobic modification of chitin whisker and its potential application in structuring oil. Langmuir 31:1641–1648

    CAS  Article  Google Scholar 

  29. 29.

    King C, Stein RS, Shamshina JL, Rogers RD (2017) Measuring the purity of chitin with a clean, quantitative solid-state NMR method. ACS Sustainable Chem Eng 5:8011–8016

    CAS  Article  Google Scholar 

  30. 30.

    Kumirska J et al (2010) Application of spectroscopic methods for structural analysis of chitin and chitosan. Marine Drugs 8:1567–1636

    CAS  Article  Google Scholar 

  31. 31.

    Lei C, Wang Q, Li L (2009) Effect of interactions between poly(vinyl alcohol) and urea on the water solubility of poly(vinyl alcohol). J Appl Polym Sci 114:517–523

    CAS  Article  Google Scholar 

  32. 32.

    Rodriguez-Lazcano Y, Mate B, Herrero VJ, Escribano R, Galvez O (2014) The formation of carbamate ions in interstellar ice analogues. Phys Chem Chem Phys 16:3371

    CAS  Article  Google Scholar 

  33. 33.

    Brugnerotto J, Lizardi J, Goycoolea FM, Arguelles-Monal W, Desbrieres J, Rinaudo M (2001) An infrared investigation in relation with chitin and chitosan characterization. Polymer 42:3569–3580

    CAS  Article  Google Scholar 

  34. 34.

    Okuyama K, Noguchi K, Miyazawa T, Yui T, Ogawa K (1997) Molecular and crystal structure of hydrated chitosan. Macromolecules 30:5849–5855

    CAS  Article  Google Scholar 

  35. 35.

    Dang QF, Zou SH, Chen XG, Liu CS, Li JJ, Zhou X, Liu Y, Cheng XJ (2012) Characterizations of chitosan-based highly porous hydrogel—The effects of the solvent. J Appl Polym Sci 125:E88–E98

    CAS  Article  Google Scholar 

  36. 36.

    Xu Y, Xia D, Han J, Yuan S, Lin H, Zhao C (2017) Design and fabrication of porous chitosan scaffolds with tunable structures and mechanical properties. Carbohydr Polym 177:210–216

    CAS  Article  Google Scholar 

  37. 37.

    Wang QQ et al (2013) Hydroxybutyl chitosan thermo-sensitive hydrogel: a potential drug delivery system. J Mater Sci 48:5614–5623

    CAS  Article  Google Scholar 

Download references


One of the authors, G. Prabha gratefully acknowledges the Science and Engineering Research Board (SERB) of the Department of Science & Technology (DST), Government of India, for providing funds under National Post-Doctoral Fellowship (PDF/2016/002403) and authors would like to thank University Grant Commission (UGC), Government of India, for providing the funds under the scheme of UGC –Dr. D.S. Kothari Post Doctoral Fellowship (Award No:F.4-2/2006(BSR)/CH/18-19/0110). The authors thank Prof. Ramesh Gardas of the Department of Chemistry for extending the rheology measurement facilities. Special thanks to Dr. Manohar Badiger and Mr. T. Arun of National Chemical Laboratory, Pune, India, for the timely help in x-ray microscopy (micro-CT) imaging and analysis.

Author information



Corresponding author

Correspondence to Dhamodharan Raghavachari.

Ethics declarations

Conflict of interest

The authors declare no competing financial interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 5320 KB)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Govindaraj, P., Raghavachari, D. Fabrication of macroporous soft hydrogels of Chitosan scaffolds by hydrothermal reaction and cytotoxicity to 3T3 L1 cells. J Polym Res 28, 86 (2021). https://doi.org/10.1007/s10965-021-02426-z

Download citation


  • Super water absorption
  • Genipin
  • Macroporous chitosan scaffold
  • Cell compatibility
  • Hydrogel