December 2002
Volume 43, Issue 13
ARVO Annual Meeting Abstract  |   December 2002
Interactions Between Mechanical Forces and Phenotype for Keratocytes Cultured in a Collagen Matrix
Author Affiliations & Notes
  • M Madgett
    Biomedical Engineering University of Minnesota Minneapolis MN
  • A Hubel
    Biomedical Engineering University of Minnesota Minneapolis MN
  • V Barocas
    Biomedical Engineering University of Minnesota Minneapolis MN
  • E Orwin
    Harvey Mudd College Claremont CA
  • Footnotes
    Commercial Relationships   M. Madgett, None; A. Hubel, None; V. Barocas, None; E. Orwin, None. Grant Identification: Support: Univ. of Minnesota, Academic Health Center Faculty Seed Grant; Minnesota Medical Foundation
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 1705. doi:
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      M Madgett, A Hubel, V Barocas, E Orwin; Interactions Between Mechanical Forces and Phenotype for Keratocytes Cultured in a Collagen Matrix . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1705.

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

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Abstract: : Purpose: The interest in and clinical use of refractive surgery has fueled research in corneal wound healing. While much work has been done to characterize the influence of soluble factors on keratocyte phenotype, little has been done to characterize the interplay between mechanical forces and corneal cell phenotype. The purpose of this investigation is to characterize the cell traction forces exerted by corneal fibroblasts during wound healing and compare them with the cellular phenotype. Methods: Human corneal keratocytes were isolated and cultured in a native fibrillar collagen matrix. The matrix contraction, DNA content, and compressive modulus of the cell-matrix system were determined as a function of time in culture. This data was analyzed using a theoretical model to estimate the traction force exerted by the cells onto the matrix. Sections taken from the cell-matrix system were stained with monoclonal antibody to α-smooth muscle actin (α-SMA), and the percentage of cells expressing α-SMA was determined. The expression of lumican, decorin, collagenase, the alpha 5 integrin, and α-SMA will be quantified using RT-PCR to better assess cellular phenotype. Results: Our results to date indicate that unconstrained collagen matrices contract significantly over the period of migration and repopulation of the matrix. This contraction occurs quickly at first (84% of original area by day 2 and 72% by day 7), and slows over time (66% by day 21). Mechanical testing indicates that our corneal equivalent behaves in a classical viscoelastic behavior in unconfined compression. The cell traction force, defined as the stress on the matrix per cell, was found to be high initially (7.58x10-4 MPa), decrease drastically by day 7 (2.78x10-5 MPa ), increase slightly around day 14 (5.46x10-5 MPa), and then decrease slightly again. Phenotypically, up-regulation of α-SMA in the cells was observed over the initial 7 day period. Later cultures showed an increase in α-SMA expression beginning at day 14. Conclusion: These studies show that the cell traction forces are significantly higher during the first few days of culture in the collagen matrix. This indicates that the cells are exerting more force as they are initially migrating throughout the matrix and remodeling its structure. This period of highest force also corresponds with a period when the cells are increasing their production of α-SMA, indicating a correlation. It was also noted that the timing of the highest cell traction forces in-vitro corresponds to that of corneal reshaping during wound healing in-vivo.

Keywords: 631 wound healing • 374 cornea: stroma and keratocytes • 417 gene/expression 

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