title: Characterization of the modified chitosan membrane cross-linked with genipin for the cultured corneal epithelial cells.
abstract: OBJECTIVE
  To modify a chitosan membrane (CM) by cross-linking the chitosan with genipin, a naturally occurring cross-linker extracted from Gardenia jasminoides fructus, with the aim of developing a new cell culture support and to observe the phenotypes of cultured human corneal epithelial cells (HCECs) on genipin-cross-linked chitosan membrane (GCM).
  
  
  METHODS
  We tested the cross-linking characteristics and mechanical strength of the GCM. CMs modified by cross-linking with different concentrations of genipin were prepared to investigate the rate of membrane degradation. The biocompatibility of the GCMs was investigated by determining the viability of HCECs cultured on them in vitro. The morphology of the HCECs cultured on CM or GCM was analyzed by confocal microscopy and scanning electron microscopy (SEM). Immunocytochemical staining was conducted to determine the phenotypes of the cultured cells.
  
  
  RESULTS
  The fixation index of the GCM was 31 ± 3% after treatment of CM with 0.5mM genipin. A stress-strain test showed that the GCM could tolerate three times the mechanical force of noncross-linked CM. The biodegradation rate of GCM was much slower than for CM. A 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay showed that cell viability was not affected by cross-linking with 5.0mM genipin. SEM showed that the cultured HCECs adhered to and grew well on the surface of the GCM. Immunocytochemical staining showed keratin 3 (K3) and connexin 43 (Cx-43) immunoreactive HCECs on the GCM and their proliferative ability was not significantly affected by strong immunoreactivity of Ki-67 and p63 markers.
  
  
  CONCLUSIONS
  GCM has potential as a scaffold for corneal epithelium in ocular surface surgery and greater mechanical strength and slower degradation than unmodified CM.
authors.affiliation: Department of Ophthalmology, Chang-Gung Memorial Hospital, Linkou, Taiwan.
authors.forename: Ya-Han
authors.initials: YH
authors.lastname: Li
authors.affiliation: Department of Cosmetic Science, Graduate Institute of Health Industry Technology, Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Kwei-Shan, Tao-Yuan, Taiwan.
authors.forename: Ching-Yi
authors.initials: CY
authors.lastname: Cheng
authors.affiliation: Department of Ophthalmology, Chang-Gung Memorial Hospital, Linkou, Taiwan; Chang-Gung University College of Medicine, Taiwan.
authors.forename: Nan-Kai
authors.initials: NK
authors.lastname: Wang
authors.affiliation: Department of Ophthalmology, Chang-Gung Memorial Hospital, Linkou, Taiwan; Chang-Gung University College of Medicine, Taiwan.
authors.forename: Hsin-Yuan
authors.initials: HY
authors.lastname: Tan
authors.affiliation: Department of Ophthalmology, Chang-Gung Memorial Hospital, Linkou, Taiwan; Chang-Gung University College of Medicine, Taiwan.
authors.forename: Yueh-Ju
authors.initials: YJ
authors.lastname: Tsai
authors.affiliation: Department of Ophthalmology, Chang-Gung Memorial Hospital, Linkou, Taiwan; Chang-Gung University College of Medicine, Taiwan.
authors.forename: Ching-Hsi
authors.initials: CH
authors.lastname: Hsiao
authors.affiliation: Department of Ophthalmology, Chang-Gung Memorial Hospital, Linkou, Taiwan; Chang-Gung University College of Medicine, Taiwan.
authors.forename: David Hui-Kang
authors.initials: DH
authors.lastname: Ma
authors.affiliation: Department of Ophthalmology, Chang-Gung Memorial Hospital, Linkou, Taiwan; Chang-Gung University College of Medicine, Taiwan. Electronic address: lkyeh@ms9.hinet.net.
authors.forename: Lung-Kun
authors.initials: LK
authors.lastname: Yeh
mesh_terms: D002478:Cells, Cultured; D048271:Chitosan / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D019573:Epithelium, Corneal / Q000166:cytology*; D006801:Humans; D039823:Iridoids / Q000737:chemistry*
chemical_list: D003432:Cross-Linking Reagents; D039823:Iridoids; D048271:Chitosan; C007834:genipin
keywords: Chitosan membrane; Cross-linking; Genipin; Human corneal epithelial cells