title: Biocompatibility of a fish scale-derived artificial cornea: Cytotoxicity, cellular adhesion and phenotype, and in vivo immunogenicity.
abstract: OBJECTIVE
  To determine whether a fish scale-derived collagen matrix (FSCM) meets the basic criteria to serve as an artificial cornea, as determined with in vitro and in vivo tests.
  
  
  METHODS
  Primary corneal epithelial and stromal cells were obtained from human donor corneas and used to examine the (in)direct cytotoxicity effects of the scaffold. Cytotoxicity was assessed by an MTT assay, while cellular proliferation, corneal cell phenotype and adhesion markers were assessed using an EdU-assay and immunofluorescence. For in vivo-testing, FSCMs were implanted subcutaneously in rats. Ologen(®) Collagen Matrices were used as controls. A second implant was implanted as an immunological challenge. The FSCM was implanted in a corneal pocket of seven New Zealand White rabbits, and compared to sham surgery.
  
  
  RESULTS
  The FSCM was used as a scaffold to grow corneal epithelial and stromal cells, and displayed no cytotoxicity to these cells. Corneal epithelial cells displayed their normal phenotypical markers (CK3/12 and E-cadherin), as well as cell-matrix adhesion molecules: integrin-α6 and β4, laminin 332, and hemi-desmosomes. Corneal stromal cells similarly expressed adhesion molecules (integrin-α6 and β1). A subcutaneous implant of the FSCM in rats did not induce inflammation or sensitization; the response was comparable to the response against the Ologen(®) Collagen Matrix. Implantation of the FSCM in a corneal stromal pocket in rabbits led to a transparent cornea, healthy epithelium, and, on histology, hardly any infiltrating immune cells.
  
  
  CONCLUSIONS
  The FSCM allows excellent cell growth, is not immunogenic and is well-tolerated in the cornea, and thus meets the basic criteria to serve as a scaffold to reconstitute the cornea.
authors.affiliation: Department of Ophthalmology, J3-S, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. Electronic address: T.H.van_Essen@lumc.nl.
authors.forename: T H
authors.initials: TH
authors.lastname: van Essen
authors.affiliation: Department of Research, Aeon Astron Europe B.V., J.H. Oortweg 19, 2333 CH, Leiden, The Netherlands. Electronic address: Lisanne.van.zijl@aeonastron.com.
authors.forename: L
authors.initials: L
authors.lastname: van Zijl
authors.affiliation: Department of Ophthalmology, Antwerp University Hospital, Wilrijkstraat 10, 2650 Edegem, Belgium. Electronic address: Tine.Possemiers@hotmail.be.
authors.forename: T
authors.initials: T
authors.lastname: Possemiers
authors.affiliation: Department of Molecular Cell-biology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. Electronic address: A.A.Mulder@lumc.nl.
authors.forename: A A
authors.initials: AA
authors.lastname: Mulder
authors.affiliation: Department of Research, Aeon Astron Europe B.V., J.H. Oortweg 19, 2333 CH, Leiden, The Netherlands. Electronic address: Sarah.zwart@aeonastron.com.
authors.forename: S J
authors.initials: SJ
authors.lastname: Zwart
authors.affiliation: Department of Research, Body Organ Biomedical Corporation, 5F, No. 153, Section 3, Xinyi Road, Da'an District, Taipei City 106, Taiwan, ROC. Electronic address: Charles.chou@aeonastron.com.
authors.forename: C-H
authors.initials: CH
authors.lastname: Chou
authors.affiliation: Department of Research, Body Organ Biomedical Corporation, 5F, No. 153, Section 3, Xinyi Road, Da'an District, Taipei City 106, Taiwan, ROC. Electronic address: Julio.Lin@aeonastron.com.
authors.forename: C C
authors.initials: CC
authors.lastname: Lin
authors.affiliation: Department of Research, Aeon Astron Europe B.V., J.H. Oortweg 19, 2333 CH, Leiden, The Netherlands. Electronic address: Michael.Lai@aeonastron.com.
authors.forename: H J
authors.initials: HJ
authors.lastname: Lai
authors.affiliation: Department of Ophthalmology, J3-S, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. Electronic address: G.P.M.Luyten@lumc.nl.
authors.forename: G P M
authors.initials: GPM
authors.lastname: Luyten
authors.affiliation: Department of Ophthalmology, Antwerp University Hospital, Wilrijkstraat 10, 2650 Edegem, Belgium. Electronic address: Marie-Jose.Tassignon@uza.be.
authors.forename: M J
authors.initials: MJ
authors.lastname: Tassignon
authors.affiliation: Department of Ophthalmology, Antwerp University Hospital, Wilrijkstraat 10, 2650 Edegem, Belgium; University of Antwerp, Prinsstraat 13, 2000 Antwerpen, Belgium. Electronic address: Nadia.zakaria@gmail.com.
authors.forename: N
authors.initials: N
authors.lastname: Zakaria
authors.affiliation: Department of Molecular Cell-biology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. Electronic address: A.E.L.Ghalbzouri@lumc.nl.
authors.forename: A
authors.initials: A
authors.lastname: El Ghalbzouri
authors.affiliation: Department of Ophthalmology, J3-S, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. Electronic address: M.J.Jager@lumc.nl.
authors.forename: M J
authors.initials: MJ
authors.lastname: Jager
mesh_terms: D000825:Animal Structures / Q000737:chemistry*; D000818:Animals; D001672:Biocompatible Materials / Q000494:pharmacology*; D002448:Cell Adhesion / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003094:Collagen / Q000494:pharmacology; D003315:Cornea / Q000187:drug effects* / Q000276:immunology*; D003319:Corneal Stroma / Q000166:cytology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects; D019573:Epithelium, Corneal / Q000166:cytology; D005260:Female; D005399:Fishes; D005947:Glucose / Q000378:metabolism; D006801:Humans; D010641:Phenotype; D011817:Rabbits; D011916:Rats, Inbred F344; D013718:Tensile Strength / Q000187:drug effects
chemical_list: D001672:Biocompatible Materials; D003094:Collagen; D005947:Glucose
keywords: Artificial cornea; Biocompatibility; Collagen; In vitro; In vivo; Keratoprosthesis