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Britta Dreier, Sara J. Liliensiek, Jennifer Brockman, Paul F. Nealey, Paul Russell, Christopher J. Murphy; The Impact Of Biomimetic Topographic Cues On Human Corneal Epithelial Cell Differentiation. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4391.
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To determine the effect of topographic cues on the differentiation state of human corneal epithelial cells (HCECs).
An immortalized HCEC cell line, hTCEpi, which exhibits normal differentiation characteristics of HCEC cells was utilized for our studies. HTCEpi cells were plated onto NOA81 polyurethane substrates containing areas of planar control or anisotropically ordered ridges and grooves ranging in size from 400 nm to 4000 nm pitch (pitch=ridge+groove width). HTCEpi cells were cultured until confluence or post-confluence was achieved and harvested for total RNA. Quantitative PCR was performed to examine mRNA levels of HCEC differentiation markers including Keratin 14 (K14) and ALDH3A1.
Our results using quantitative PCR indicate that two separate markers of HCEC differentiation including K14 and ALDH3A1 are differentially regulated by topographic cues. K14 is normally expressed in the proliferative or self-renewing layer of HCEC cells. ALDH3A1 is a corneal crystalline and protective factor which is upregulated in suprabasal cells which are exposed to outside damaging environmental factors. HCEC cells exhibit an increase in K14 RNA levels in post-confluent hTCEpi cells on 400 nm pitch. On topographic cues of 1400 nm and 4000 nm, K14 levels decreased. ALDH3A1 is upregulated on HCEC cells with increasing pitch up to 4000 nm.
Results demonstrate that biomimetic topographic cues play a role in influencing known markers of HCEC differentiation. Specifically, levels of K14 indicate a maintenance of a self-renewal state on topographic cues of 400 nm. ALDH3A1 RNA levels were highest on the 4000 nm pitch indicating promotion of a differentiated state. Future experiments will determine the ability of our nanostructured substrates to promote HCEC function essential in the proper recreation of a healthy engineered corneal epithelial tissue, with emphasis on maintenance of both self-renewal and differentiation characteristics, and eventual formation of a three-dimensional multi-layered epithelium with proper biological function.
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