March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Important Signals for the Reversion of Myofibroblasts to Quiescent Keratocytes in an In Vitro Corneal Model
Author Affiliations & Notes
  • Elizabeth J. Orwin
    Engineering, Harvey Mudd College, Claremont, California
  • Footnotes
    Commercial Relationships  Elizabeth J. Orwin, None
  • Footnotes
    Support  NIH 1R15EY018248-01A2
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 3554. doi:
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      Elizabeth J. Orwin; Important Signals for the Reversion of Myofibroblasts to Quiescent Keratocytes in an In Vitro Corneal Model. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3554.

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Abstract

Purpose: : The overall goal of this project is to understand and control corneal keratocyte phenotype for application to a tissue-engineered corneal model. An ideal cell population for tissue-engineering is one that can be expanded, thereby inducing repair fibroblasts and myofibroblasts, and then reverted to the quiescent keratocyte phenotype to promote decreased light scatter and overall construct transparency. In this project, we investigated a variety of input signals to the cells in order to assess their effect on cell protein expression and transparency. Signals investigated were extracellular matrix chemistry and microstructure, electromagnetic radiation, and applied strain.

Methods: : Rabbit corneal keratocytes were isolated from rabbit corneas and subcultured in serum-containing media prior to experiments, thereby inducing a mixed population of repair fibroblasts and myofibroblasts. All experiments were conducted for 7 days, and corneal cells subsequently evaluated for protein expression (α-SMA as a marker for the myofibroblast phenotype, and ALDH1A1 as a marker for the quiescent keratocyte phenotype) by Western Blot and confocal microscopy. Cell-matrix constructs were also evaluated for light scatter using optical coherence microscopy (OCM).

Results: : Corneal cell expression of the myofibroblast marker protein α-SMA was reduced on matrices composed of collagen, and further reduced to negligible amounts on aligned 2D fibrous matrices and on 3D matrices. In 2D culture, decreased α-SMA expression was correlated with decreased light scatter in cell-matrix constructs. In addition, culture in 3D collagen matrices yielded partial recovery of the keratocyte phenotype through the expression of the ALDH1A1 protein. Equibiaxial strain in the range of 3-7% also significantly downregulated α-SMA expression in corneal cells. Different wavelengths and intensities of incident light also yielded different levels of expression of α-SMA.

Conclusions: : These studies show that we are able to revert subcultured corneal cell populations to repair fibroblasts and in some cases to the quiescent keratocyte phenotype. These results are important because downregulation of α-SMA will have a significant impact on the overall light scatter from the cells, as induction of α-SMA expression in vitro and in vivo has been correlated to increased light scatter. We are currently working on the design and implementation of a bioreactor system to integrate these input signals into a single experimental test bed. These results have important implications for the understanding of wound healing in the cornea, and ultimately for the understanding of corneal cell response to new surgical techniques and new ophthalmic drugs.

Keywords: cornea: stroma and keratocytes • crystallins • wound healing 
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