title: Dependence of corneal keratocyte adhesion, spreading, and integrin β1 expression on deacetylated chitosan coating.
abstract: This study reports, for the first time, the regulation of corneal keratocyte adhesion, spreading, morphology, and integrin gene expression on chitosan coating due to the effects of deacetylation. The degree of deacetylation (DD) in chitosan materials was confirmed by elemental analysis, gel permeation chromatography, and Fourier transform infrared spectroscopy. In this study, chitosan samples with the same molecular weight level but varying DD (74.1 ± 0.5%, 84.4 ± 0.7%, and 94.2 ± 0.5%) were obtained by heat-alkaline treatment under a nitrogen atmosphere. For higher DD groups, the biopolymer carried abundant amino groups since the deacetylation process removed larger amount of acetyl groups from the chitosan molecules. Results showed that the mechanical stability and crystallinity of the chitosan coatings significantly increased with increasing DD value. Fibronectin adsorption, keratocyte adhesion, and cell spreading exhibited a positive correlation with DD due to the chemical functionality of polysaccharides (bearing acetyl and amino groups) and increase of substrate stiffness and crystallinity. In particular, when adhered to chitosan coatings with a DD value of 74.1%, the keratocytes appeared to be fibroblastic, elongated, and spindle shape, indicating a loss of their characteristic dendritic morphology. Furthermore, the gene expression of integrin β1 (i.e., a cell-matrix adhesion molecule) was significantly up-regulated on the chitosan coatings with higher DD, which supports favorable attachment of corneal keratocytes. Our findings suggest that DD-mediated physicochemical properties of chitosan coatings greatly affect cell-substrate crosstalk during corneal keratocyte cultivation.
authors.affiliation: Department of Ophthalmology, Chang Gung Memorial Hospital, Keelung 20401, Taiwan, ROC.
authors.forename: Chi-Chin
authors.initials: CC
authors.lastname: Sun
authors.affiliation: Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan 33302, Taiwan, ROC; Department of Bioengineering, University of Washington, Seattle, WA 98195-5061, USA.
authors.forename: Shih-Feng
authors.initials: SF
authors.lastname: Chou
authors.affiliation: Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan 33302, Taiwan, ROC; Biomedical Engineering Research Center, Chang Gung University, Taoyuan 33302, Taiwan, ROC; Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan, ROC; Center for Tissue Engineering, Chang Gung Memorial Hospital, Taoyuan 33305, Taiwan, ROC. Electronic address: jylai@mail.cgu.edu.tw.
authors.forename: Jui-Yang
authors.initials: JY
authors.lastname: Lai
authors.affiliation: Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan 33302, Taiwan, ROC.
authors.forename: Ching-Hsien
authors.initials: CH
authors.lastname: Cho
authors.affiliation: Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan 33302, Taiwan, ROC.
authors.forename: Chih-Hung
authors.initials: CH
authors.lastname: Lee
mesh_terms: D000327:Adsorption; D002448:Cell Adhesion / Q000187:drug effects; D018929:Cell Culture Techniques / Q000295:instrumentation; D002478:Cells, Cultured; D048271:Chitosan / Q000737:chemistry* / Q000494:pharmacology; D060527:Corneal Keratocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D019012:Integrin beta1 / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D017550:Spectroscopy, Fourier Transform Infrared; D013718:Tensile Strength; D014961:X-Ray Diffraction
chemical_list: D019012:Integrin beta1; D011506:Proteins; D048271:Chitosan
keywords: Cell-substrate crosstalk; Chitosan; Corneal keratocyte; Deacetylation degree