J Korean Ophthalmol Soc.  2014 Nov;55(11):1698-1705. 10.3341/jkos.2014.55.11.1698.

Effect of Preservative-Free Healon Eye Drop on Human Corneal Epithelial Cell in Vitro

• Kim HY
• Park YM
• Lee JS

• KMID: 2216621
• DOI: http://doi.org/10.3341/jkos.2014.55.11.1698

Abstract

PURPOSE
To investigate the biologic effects of preservative-free artificial tear drops on cultured human corneal epithelial cells in vitro.
METHODS
The effects of preservative-free artificial tear drops (sodium hyaluronate 0.1% (Kynex(R), Alcon, Seoul, Korea), sodium hyaluronate 0.18% (Kynex2(R), Alcon, Seoul, Korea), and sodium hyaluronate 0.3% (Hyaluni eye drops 0.3%(R), Taejoon, Seoul, Korea)) on human corneal epithelial cells were evaluated. Cellular proliferation was determined using MTT (3- (4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide) and Ki-67 assays. Cellular migration was determined using CD44 and scratch wound assays. Cell damage was determined using the lactate dehydrogenase (LDH) assay and cellular morphologies using electronic microscopy and inverted microscopy.
RESULTS
Proliferation of corneal epithelial cells, as determined by the MTT and Ki-67 assays, was not significantly different between the different eye drops (p > 0.05). The measured value of cellular migration after exposure of the sodium hyaluronate 0.3%, as determined by mean CD44 percentage and scratch wound assay, was higher than that of the sodium hyaluronate 0.1% and sodium hyaluronate 0.18%, but the CD44 value was not significantly different (p > 0.05). The LDH titer tended to increase as the concentration of sodium hyaluronate increased (p > 0.05), but influence on cytoplasm, as determined by electronic microscopy, was not different.
CONCLUSIONS
Among 3 preservative-free artificial tear drops, sodium hyaluronate 0.3% demonstrated increased migration of corneal epithelial cells. As the concentration of sodium hyaluronate in eye drops increased, the corneal cytotoxicity of corneal epithelial cells also increased. However, there was no significant difference among the 3 tear drops.

Keyword

Artificial tear; Cornea; Epithelial cell; Hyaluronic acid

MeSH Terms

Cell Proliferation
Cornea
Cytoplasm
Epithelial Cells*
Humans
Hyaluronic Acid*
L-Lactate Dehydrogenase
Microscopy
Ophthalmic Solutions
Seoul
Tears
Wounds and Injuries
Hyaluronic Acid
L-Lactate Dehydrogenase
Ophthalmic Solutions

Figure

• 

  Figure 1. The mean survival rate of corneal epithelial cell showed 3 preservative free sodium hyalunate eye drops - among kynex®, kynex2®, hyaluni eye drops 0.3%®. The survival rate tended to increase with higher concentrations of sodium hyaluronate and longer epithelial cells exposure to eye drops, but the values were not statistically significant (p > 0.05).

• 

  Figure 2. The graph show the mean percentage of Ki67 positive human corneal epithelial cells in the 3 preservative free sodium hyalunate eye drops - among kynex®, kynex2®, hyaluni eye drops 0.3%®. The percentage of Ki67 positive human corneal epithelial cells tended to increase with higher concentration of sodium hyaluronate, but the values were not statistically significant (p > 0.05).

• 

  Figure 3. This graph show the mean percentage of CD44 positive human corneal epithelial cells in 3 preservative free sodium hyalunate eye drops - among kynex®, kynex2®, hyaluni eye drops 0.3%®. The percentage of CD44 positive human corneal epithelial cells tended to increase with higher concentration of sodium hyaluronate was, but the values were not statistically significant (p > 0.05).

• 

  Figure 4. This show that scratch wound assay of the corneal epithelial cells after 24 hours and 48 hours exposure to control, kynex®, kynex2®, hyaluni eye drops 0.3%®. Corneal epithelial cells were proliferated and moved into collagenous membrane in media. Hyaluni 0.3%® showed most prominent cellular migration activity than other eye drops.

• 

  Figure 5. The lactate dehydrogenase (LDH) leakage was increased as concentration of sodium hyaluronate increased with exposure time, but the values were not statistically significant (p > 0.05). The LDH leakage tended to increase after all sodium hyaluronate eye drops were exposed up to 6 hours and decrease after 24 and 36 hours later.

• 

  Figure 6. Inverted microscopic image of the corneal epithelial cells after 24 hours exposure. A: kynex®, B: kynex2®, C: hyaluni eye drops 0.3%®, D: control. The higher concentration of sodium hyaluronate, the more detached epithelial cell from the media compared with the control.

• 

  Figure 7. Transmission electron micrographs of corneal epithelial cells appeared after 24-hour exposure to A: kynex®, B: kynex2®, C: hyaluni eye drops 0.3%®, D: control. There was no significantly difference among the 3 preservative free hyalunate eye drops - among kynex®, kynex2®, hyaluni eye drops 0.3%®. White arrows indicate the intracellular vacuoles of corneal epithelial cell.

Reference

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