May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Characterization of an in vitro Model of Human Corneal Fibrosis
Author Affiliations & Notes
  • A. E. Hutcheon
    Schepens Eye Research Institute, Boston, Massachusetts
    Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts
  • X. Q. Guo
    Schepens Eye Research Institute, Boston, Massachusetts
    Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts
  • J. W. Ruberti
    Schepens Eye Research Institute, Boston, Massachusetts
    Mechanical and Industrial Engineering, Northeastern University, Boston, Massachusetts
  • J. D. Zieske
    Schepens Eye Research Institute, Boston, Massachusetts
    Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts
  • Footnotes
    Commercial Relationships A.E. Hutcheon, None; X.Q. Guo, None; J.W. Ruberti, None; J.D. Zieske, None.
  • Footnotes
    Support NIH Grant EY05665
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 1490. doi:https://doi.org/
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      A. E. Hutcheon, X. Q. Guo, J. W. Ruberti, J. D. Zieske; Characterization of an in vitro Model of Human Corneal Fibrosis. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1490. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose:: Corneal fibrosis is a vision threatening condition characterized by the presence of corneal myofibroblasts and improper deposition of extracellular matrix components, including type III collagen. In the current investigation, we examined the potential of ascorbic acid stimulated human corneal fibroblasts cultured in the presence of TGF-ß1 as an in vitro model of corneal fibrosis.

Methods:: Human corneal fibroblasts were cultivated in the presence of ascorbic acid-2-phosphate (1mM; p-Asc) to stimulate fibroblast stratification and extracellular matrix secretion. The cultures were allowed to grow for eight weeks in the presence of TGF-ß1 (0, 0.1, 1.0, and 10 ng/ml). The morphology of the cultures was examined using transmission electron microscopy (TEM). In addition, indirect immunofluorescence microscopy was used to localize type III collagen and α-smooth muscle actin (SMA).

Results:: Human corneal fibroblasts stimulated with p-Asc stratified to 10-12 cell layers with a total thickness of approximately 50 microns. Fibroblasts cultured in the presence of 0.1 or 1.0 ng/ml of TGF-ß1 showed a slight increase in overall thickness compared to the no TGF-ß1 control. Fibroblasts cultured in the presence of 10 ng/ml of TGF-ß1 did not show an increase in the number of cell layers; however, the overall thickness of the construct increased by 50-60%. In addition, the fibroblasts did not appear to be as thin and attenuated as in the control. Cultures grown without TGF-ß1 expressed, little, if any, type III collagen or α-SMA. Both type III collagen and α-SMA levels increased with increasing concentration of TGF-ß1.

Conclusions:: Human corneal fibroblasts cultured in the presence of p-Asc stratify and secrete matrix in a manner that mimics the human cornea. TGF-ß1 stimulates cells grown in this model system to differentiate into myofibroblasts and secrete type III collagen. This system may potentially be useful as a model of human corneal fibrosis.

Keywords: wound healing • extracellular matrix • cornea: stroma and keratocytes 
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