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Che John Connon, James W Foster, Ana M Ionescu, Stem Cells and Nanomaterials Laboratory; Improving Transparency of Tissue Engineered Corneal Constructs Containing Human Keratocytes. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5141.
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Previously we have demonstrated that compressed collagen gels can encapsulate keratocytes in a viable form and that such constructs are well tolerated once implanted into the rabbit cornea (1). We now show that by optimizing the in vitro growth conditions of these tissue engineered constructs we can substantially increase their transparency- a key requirement for their subsequent clinical use.
Compressed collagen gels were constructed as described previously (1) with or without human keratocytes. These were maintained for 9 days in low-glucose (2g/L) media (1%ITS, 1mM ascorbic acid) supplemented with (5%) or without serum. Keratocyte markers keratocan and ALDH1A1 from encapsulated cells were quantified by western blotting. Resulting transparency measurements were made using contrast transfer function (CTF). Contrast plots were obtained from digital images of compressed collagen gel (n=5) backlit by high definition LED screen displaying contrast patterns at regular frequencies (0.43, 0.65, 1.08, 2.16, 3.24 cycles/mm). Maximum and minimum grey values from each contrast plot, obtained by ImageJ, were used to calculate CTF values. These were normalized for construct thickness (calculated from confocal images). The resulting CTF values correspond with the constructs optical quality in terms of transparency.
Serum-free conditions resulted in an increased expression of keratocan and ALDH1A1 from the encapsulated keratocytes when compared to those grown in serum. Within low glucose serum-free conditions collagen constructs containing keratocytes had a significantly greater (p ≤0.001) CTF value (better transparency) than the same constructs without cells (after normalization for tissue thickness). In the presence of serum and cells, the collagen gels contracted, reducing the constructs transparency to a level similar to collagen gels without cells.
The transparency of compressed collagen gels containing human keratocytes can be improved by cultivation in low glucose serum-free media. This increase in transparency (obtained from CTF measurements) was concurrent with measured increases in ALDH1A1 expression; a crystallin proposed to decrease the refractive index of keratocytes in situ. 1) Xiao et al. (2013) J Biomed Mat Res A, DOI:10.1002/jbm.a.34848
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