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Roanne R. Jones, Simon G. Alcock, Ian W. Hamley, Che J. Connon, Stem Cells and Nanomaterials Laboratory; Corneal Constructs With Tailored Levels Of Epithelial Differentiation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1846.
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© ARVO (1962-2015); The Authors (2016-present)
It has previously been shown that collagen gels when combined with corneal cells form functional ocular surface constructs1-3. Therefore we seek to develop a series of artificial corneal models with tailored levels of cell differentiation controlled by collagen substrate stiffness.
Compressed collagen gels were prepared as previously described4 and the mechanical properties regulated with compressive load and time. Collagen gels were then characterised using shear rheology, scanning electron microscopy (SEM), atomic force microscopy (AFM) and hydration studies. Limbal epithelial cells (LEC) were isolated from fresh bovine corneas and seeded onto differentially compressed collagen gels (C1, stiff > C5, less stiff). Following 14 days expansion in culture CK3 and NMMII protein expression was determined.
A linear increase in fluid loss is observed in collagen gels in relation to substrate stiffness where stiff collagen gels (C1) with an elastic modulus of 2236 Pa (±767) have a fluid loss of 97% compared to less stiff gels (C5) with a fluid loss of 62%. A high percentage of fluid loss is consistent with densely packed collagen fibres as shown by SEM analysis but similar surface topography shown by AFM. In addition bovine LEC expanded on stiff collagen gels show intense CK3 and NMMII protein expression relating to cell differentiation and interestingly cells expanded on less stiff collagen gels (C5) with an elastic modulus of 50 Pa (±32) gave rise to LEC with minimal CK3 expression.
We conclude that the mechanical properties and hydration of collagen gels can be regulated by compressive load which in turn influences LEC phenotype. These findings form a platform for designing and engineering ocular tissue models with defined levels of cell differentiation.
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