Effects of genipin cross-linking of chitosan hydrogels on cellular adhesion and viability.

PURPOSE The aim of the present study was to investigate the effects of genipin (Gp) cross-linking of chitosan (CHI) hydrogels on the cell adhesion and viability. METHOD Series of Gp crosslinked CHI hydrogels were prepared by incubation of solutions containing a mixture of Gp and CHI in different ratios. The resulting hydrogels were characterized by scanning electron microscopy (SEM), parallel plate rheometer, contact angle and swelling ratio measurement. The in vitro cytocompatibility of hydrogels was evaluated with L929 fibroblasts by MTT method. The cell adhesion morphology on gel surface was characterized by SEM, and the cell viability was assessed through cell count and flow cytometry analysis. RESULTS It was found that macroporous structure of the CHI hydrogels could be tailored by varying Gp or CHI amount. Gp cross-linking of hydrogels enhanced their storage modulus significantly, and also altered their hydrophilicity and swell properties. The MTT results revealed that the cross-linked hydrogels did not induce cytotoxic effects. Cell count and flow cytometry analysis demonstrated that denser surface milieu of hydrogels could facilitate better cell adhesion and viability. CONCLUSIONS It could be concluded that increased cross-linking density significantly improved the cell adhesion and viability on hydrogel surface. This research provides prospective biocompatible approaches by making gel stiffness modifications to hydrogel scaffolds for the purpose of different tissue engineering.

[1]  J. Iatridis,et al.  Genipin-crosslinked fibrin hydrogels as a potential adhesive to augment intervertebral disc annulus repair. , 2011, European cells & materials.

[2]  C. Albigès-Rizo,et al.  Manipulation of the adhesive behaviour of skeletal muscle cells on soft and stiff polyelectrolyte multilayers. , 2010, Acta biomaterialia.

[3]  F. Grinnell,et al.  The differential regulation of cell motile activity through matrix stiffness and porosity in three dimensional collagen matrices. , 2010, Biomaterials.

[4]  Wenxin Wang,et al.  Thermoresponsive hyperbranched copolymer with multi acrylate functionality for in situ cross-linkable hyaluronic acid composite semi-IPN hydrogel , 2011, Journal of Materials Science: Materials in Medicine.

[5]  Thrimoorthy Potta,et al.  Dual cross-linking systems of functionally photo-cross-linkable and thermoresponsive polyphosphazene hydrogels for biomedical applications. , 2010, Biomacromolecules.

[6]  Ashutosh Kumar Singh,et al.  External stimuli response on a novel chitosan hydrogel crosslinked with formaldehyde , 2006 .

[7]  H. Sung,et al.  In vitro evaluation of the genotoxicity of a naturally occurring crosslinking agent (genipin) for biologic tissue fixation. , 2000, Journal of biomedical materials research.

[8]  Miqin Zhang,et al.  Synthesis and characterization of macroporous chitosan/calcium phosphate composite scaffolds for tissue engineering. , 2001, Journal of biomedical materials research.

[9]  H. Bianco-Peled,et al.  Defining the role of matrix compliance and proteolysis in three-dimensional cell spreading and remodeling. , 2008, Biophysical journal.

[10]  國郎 小笠原,et al.  Chem. Pharm. Bull.(オピニオン) , 2007 .

[11]  K. Ren,et al.  Polyelectrolyte Multilayer Films of Controlled Stiffness Modulate Myoblast Cell Differentiation , 2008, Advanced functional materials.

[12]  Ming-Jium Shieh,et al.  The cardiomyogenic differentiation of rat mesenchymal stem cells on silk fibroin-polysaccharide cardiac patches in vitro. , 2009, Biomaterials.

[13]  P. Gatenholm,et al.  Microporous bacterial cellulose as a potential scaffold for bone regeneration. , 2010, Acta biomaterialia.

[14]  S. Hsu,et al.  Preparation of networks of gelatin and genipin as degradable biomaterials , 2003 .

[15]  Samuel I Stupp,et al.  Self-assembling peptide scaffolds for regenerative medicine. , 2012, Chemical communications.

[16]  Miss A.O. Penney.  (b) , 1974, The New Yale Book of Quotations.

[17]  Y. Takeda,et al.  Studies on the Blue Pigments Produced from Genipin and Methylamine. I. Structures of the Brownish-Red Pigments, Intermediates Leading to the Blue Pigments , 1994 .

[18]  M. Tabrizian,et al.  Effect of genipin cross-linking on the cellular adhesion properties of layer-by-layer assembled polyelectrolyte films. , 2009, Biomaterials.

[19]  S. Waldman,et al.  Genipin Cross-Linked Fibrin Hydrogels for in vitro Human Articular Cartilage Tissue-Engineered Regeneration , 2009, Cells Tissues Organs.

[20]  H. Sung,et al.  In vitro evaluation of cytotoxicity of a naturally occurring cross-linking reagent for biological tissue fixation. , 1999, Journal of biomaterials science. Polymer edition.

[21]  Dong-An Wang,et al.  Therapeutic cell delivery and fate control in hydrogels and hydrogel hybrids. , 2010, Advanced drug delivery reviews.

[22]  N. Vrana,et al.  Cells Tissues Organs , 2015 .

[23]  Eun Hye Kim,et al.  In situ thermal gelling polypeptide for chondrocytes 3D culture. , 2010, Biomaterials.

[24]  C. Brett,et al.  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. , 2009, Analytical chemistry.

[25]  C. Airoldi,et al.  The effectiveness of the protected amino group on crosslinked chitosans for copper removal and the thermodynamics of interaction at the solid/liquid interface , 2009 .

[26]  Riccardo A.A. Muzzarelli,et al.  Genipin-crosslinked chitosan hydrogels as biomedical and pharmaceutical aids , 2009 .

[27]  C. Santana,et al.  Crosslinking of chitosan membranes using glutaraldehyde: Effect on ion permeability and water absorption , 2007 .

[28]  N. Gu,et al.  Poly(ethylene glycol)-cross linked poly(methyl vinyl ether-co-maleic acid)hydrogels for three-dimensional human ovarian cancer cell culture , 2013 .

[29]  S. Hanks,et al.  Anti-apoptotic Role of Focal Adhesion Kinase (FAK) , 2000, The Journal of Biological Chemistry.

[30]  R. Misra,et al.  Biomaterials , 2008 .

[31]  H. Sung,et al.  Feasibility study of a natural crosslinking reagent for biological tissue fixation. , 1998, Journal of biomedical materials research.

[32]  M. Maraschin,et al.  Manipulation of chemical composition and architecture of non-biodegradable poly(ethylene terephthalate)/chitosan fibrous scaffolds and their effects on L929 cell behavior. , 2013, Materials science & engineering. C, Materials for biological applications.

[33]  Allon I Hochbaum,et al.  Rational design of cytophilic and cytophobic polyelectrolyte multilayer thin films. , 2003, Biomacromolecules.

[34]  D. Mooney,et al.  Hydrogels for tissue engineering: scaffold design variables and applications. , 2003, Biomaterials.

[35]  S. Richardson,et al.  Introducing chemical functionality in Fmoc-peptide gels for cell culture. , 2009, Acta biomaterialia.

[36]  S. Boyce,et al.  EDC cross-linking improves skin substitute strength and stability. , 2006, Biomaterials.

[37]  J. Kennedy,et al.  Preparation and characterization of water-soluble chitosan derivative by Michael addition reaction. , 2010, International journal of biological macromolecules.

[38]  John F. Kennedy,et al.  Alginate fibres modified with unhydrolysed and hydrolysed chitosans for wound dressings , 2004 .

[39]  Daniel J. Macaya,et al.  Astrocyte infiltration into injectable collagen-based hydrogels containing FGF-2 to treat spinal cord injury. , 2013, Biomaterials.

[40]  M. Albanna,et al.  Chitosan fibers with improved biological and mechanical properties for tissue engineering applications. , 2013, Journal of the mechanical behavior of biomedical materials.

[41]  H. Bianco-Peled,et al.  Nanostructuring PEG-fibrinogen hydrogels to control cellular morphogenesis. , 2011, Biomaterials.

[42]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[43]  D. Dobrowolski,et al.  Hydrogel membranes based on genipin-cross-linked chitosan blends for corneal epithelium tissue engineering , 2012, Journal of Materials Science: Materials in Medicine.