April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Improving Transparency of Tissue Engineered Corneal Constructs Containing Human Keratocytes
Author Affiliations & Notes
  • Che John Connon
    School of Chemistry, Food and Pharmacy, University of Reading, Reading, United Kingdom
  • James W Foster
    School of Chemistry, Food and Pharmacy, University of Reading, Reading, United Kingdom
  • Ana M Ionescu
    University of Granada, Granada, Spain
  • Footnotes
    Commercial Relationships Che Connon, None; James Foster, None; Ana Ionescu, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 5141. doi:
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    • Get Citation

      Che John Connon, James W Foster, Ana M Ionescu, ; Improving Transparency of Tissue Engineered Corneal Constructs Containing Human Keratocytes. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5141.

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

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Abstract
 
Purpose
 

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.

 
Methods
 

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.

 
Results
 

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.

 
Conclusions
 

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

 
 
Contrast Transfer Function from compressed collagen gels containing human keratocytes in serum-free (left) and 5% serum (right).
 
Contrast Transfer Function from compressed collagen gels containing human keratocytes in serum-free (left) and 5% serum (right).
 
Keywords: 484 cornea: stroma and keratocytes • 480 cornea: basic science • 519 extracellular matrix  
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