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Purpose: Sodium hyaluronate (hyaluronic acid) is known to promote corneal epithelial wound healing in
vivo and in vitro, in animal experiments. Sodium hyaluronate is the ligand for CD44, a cell surface
adhesion molecule which has been found on normal human corneal epithelial cells. The purpose of this
study was to investigate the effect of sodium hyaluronate on human corneal epithelial cell migration,
proliferation, and CD44 receptor expression.
Methods: Human corneal epithelial cell cultures were established from 32 donor corneoscleral rims and
See end of article for maintained separately in three different culture conditions: (1) culture medium only, (2) sodium
authors’ affiliations
....................... hyaluronate enriched (0.6 mg/ml) medium, and (3) hydroxypropylmethylcellulose enriched (2.5 mg/ml)
medium. The total area of migrating epithelial cell sheets in each case was measured by planimetry on
Correspondence to: days 4, 8, 12, and 16. Cytospin preparations of cells cultured in the different culture conditions were
Professor H S Dua,
Division of Ophthalmology examined immunohistochemically for proliferation and CD44 receptor expression using antibodies
and Visual Sciences, B directed against Ki67 and CD44 respectively.
Floor, Eye Ear Nose Throat Results: Cells cultured in the presence of sodium hyaluronate showed significantly increased migration at
Centre, University days 12 and 16 (Friedmen test: p = 0.0012, day 16; p = ,0.001, day 12) compared with cells cultured in
Hospital, Queens Medical
Centre, Nottingham NG7 the other media. There was no difference in cell proliferation (Ki67) or CD44 expression on cells cultured
2UH, UK; harminder. in the different culture conditions.
[email protected] Conclusions: Sodium hyaluronate promotes migration but not proliferation or CD44 expression on human
Accepted corneal epithelial cells in vitro. The beneficial effect of sodium hyaluronate in corneal wound healing is
22 September 2003 likely to be related to rapid migration of cells leading to rapid wound closure. This may be facilitated by the
....................... adhesion between CD44 on the cells and hyaluronic acid, which coats the surface of the denuded cornea.
T
he corneal epithelium forms an integral part of the ocular in cell to cell and cell to substratum interactions that mediate
surface and is necessary for maintaining a clear and cell migration during re-epithelialisation.21 Studies have also
proper functioning cornea. When compromised, it is shown that CD44 expression is associated with proliferation
therefore important that it is rapidly regenerated. Epithelial of epithelial cells.22–25 The reason for this association is not
healing at the corneal surface involves the centripetal and known.
circumferential (along the limbus) migration of epithelial cell Due to the possible implications of sodium hyaluronate on
sheets from the remaining intact epithelium, proliferation of wound healing in humans, we investigated its effect on
basal epithelial cells surrounding the defect to restore the human corneal epithelial cell migration and proliferation and
normal multilayered architecture of the epithelium, and determined whether its presence caused upregulation of the
anchoring of the newly regenerated epithelium to underlying CD44 receptor.
connective tissue.1–3
Sodium hyaluronate is a naturally occurring glycosamino- MATERIALS AND METHODS
glycan of the extracellular matrix that plays an important role Corneal epithelial cell cultures, obtained from human donor
in development, wound healing, and inflammation.4 Its explants, were maintained in three different culture condi-
viscoelastic properties have rendered it ideally suited for use tions: (1) standard growth medium only (SM), (B) standard
in ophthalmic practice to protect the corneal endothelium growth medium enriched with hydroxypropylmethyl cellu-
and to maintain the anterior chamber depth during lose (HPMC) (Ocucoat, 2.5 mg/ml) (Storz Ophthalmics,
intraocular surgery.4–6 It has also been used in the treatment Clearwater, FL, USA), and (3) standard growth medium
of dry eyes because of its long ocular surface residence enriched with sodium hyaluronate (SH) (Healon, 0.6 mg/ml)
time.7–11 Recent experiments in animals have shown that (Pharmacia & Upjohn, Uppsala, Sweden). The viscosities of
sodium hyaluronate promotes corneal epithelial wound hydroxypropylmethyl cellulose and sodium hyaluronate
healing by stimulating the migration, adhesion, and prolif- enriched media were equal at the concentrations used.
eration of the corneal epithelium.4 12 13 The mechanism of Corneal epithelial cell migration, cell proliferation, and
action of sodium hyaluronate on these cell functions remains number of cells expressing CD44 were compared in the three
controversial.13–16 Human studies have been confined to the in groups.
vivo topical instillation of sodium hyaluronate drops in eyes
with epithelial problems.17 18 Corneal epithelial cell culture
Sodium hyaluronate is a ligand for CD44, a transmem- Primary cultures of human corneal epithelial cells were
brane cell surface adhesion molecule.19 The CD44 receptor has established from 2 mm limbal explants obtained from 32
been characterised on normal human corneal epithelial cells. donor corneoscleral rims following corneal transplantation,
It has enhanced expression in inflammation and allograft using the method described before by Dua et al.26 Donor age
rejection suggesting its importance in corneal epithelial cell
physiology.19 20 Its expression has also been found to correlate Abbreviations: HPMC, hydroxypropylmethyl cellulose; SH, sodium
with corneal re-epithelialisation, suggesting its involvement hyaluronate; SM, standard growth medium; TBS, TRIS buffered saline.
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822 Gomes, Amankwah, Powell-Richards, et al
ranged from 30 to 74 years. Briefly, under aseptic conditions (FITC) conjugated swine antirabbit antibody (Dako) 1:20
and using the dissecting microscope, each donor corneo- dilution for 30 minutes, was used as secondary antibody.
scleral rim was divided into six equal pieces. The endothelial Slides were mounted in glycerol/phosphate buffered saline.
and posterior stromal layer was carefully peeled off and each The proportion of positively labelled cells in each cytospin
explant was placed separately, with the epithelial surface was determined by analysing 200 cells per slide using a
facing upwards, in the centre of a 35 mm Falcon Primaria fluorescent microscope.
tissue culture dish (Becton Dickinson, Oxford, UK). These
were left covered in a laminar flow cabinet at room CD44 (cell adhesion molecule)
temperature for 5 minutes and then covered in growth Expression of CD44 by cells cultured in the different culture
medium (Dulbecco’s MEM/Nut Mix F-12) (DMEM) (Gibco conditions was determined using an antibody directed
BRL, Life Technologies, Paisley, UK) supplemented with fetal against the CD44 receptor. Cytospin preparations were
bovine serum (5%) (Gibco); dimethyl sulfoxide (DMSO) stained using the standard alkaline phosphatase anti-alkaline
(0.5% v/v) (Sigma-Aldrich, Dorset, UK); gentamicin (5 mg/ phosphatase (APAAP) method.27 Following incubation with
ml) (Roussel Laboratories Ltd, Uxbridge, UK); epidermal the primary antibody, mouse antihuman CD44 antibody
growth factor (10 ng/ml) (Gibco); bovine insulin (5 mg/ml) (Dako), 1:40 dilution for 1 hour at room temperature in a
(Gibco) and cholera toxin (0.1 mg/ml) (Gibco). Cultures were moist chamber, slides were stained with rabbit antimouse
incubated at 37˚C, in 5% carbon dioxide in humid air. The immunoglobulin (Dako) and then APAAP complex (Dako) at
epithelial cell morphology of the cultures was evaluated daily 1:50 and 1:100 dilutions respectively for 30 minutes.
by phase contrast microscopy. At day four, cell migration Negative controls with TBS and an irrelevant antibody, were
from the explants was measured and three similar cultures also set up. All antibodies were diluted in TBS and between
were selected and randomly assigned to one of the three each staining, slides were washed three times in TBS. The
culture conditions: standard medium (SM) alone, medium reaction product was developed with Fast Red TR salt
with hydroxypropylmethyl cellulose (HPMC) (Ocucoat, (Sigma) as chromogen for the APAAP. Slides were counter-
2.5 mg/ml) (Storz Ophthalmics), and medium with sodium stained with haematoxylin (Meyer’s). The proportion of
hyaluronate (SH) (Healon, 0.6 mg/ml) (Pharmacia & positive red stained cells in each cytospin preparation was
Upjohn). Thus explants from the same donor rim were determined microscopically by analysing cells in five random
maintained in the three different conditions and compared. high power fields.
Explants were left in the culture dish for the duration of the
incubation. Culture medium was changed every fourth day. Statistical analysis
Epithelial cell migration from the explants was measured on The comparison among migration of groups SH, HPMC, and
days 8, 12, and 16. The advancing edge of the migrating SM was performed separately for each day, through repeated
epithelial cell sheet was outlined on the culture dish and the measures of ANOVA. Repeated measures of ANOVA was also
total area of the sheet was determined by planimetry and used to study intragroup variation for epithelial migration
expressed in square millimeters. separately for SH, HPMC, and SM. The comparisons for
proliferation with Ki-67 and CD44 expression among groups
Intragroup variation of cell migration SH, HPMC, and SM were performed using Friedmans and
Three similar cultures, with respect to area of cell migration Wilcoxon test. The significance level adopted in this study
at day four, from the same donor rim were maintained in was 5% (a = 0.05) and the SPSS system (SPSS Inc, Chicago,
each of the three culture conditions, for 16 days. Thus of nine IL, USA) was used for the statistical analysis.
cultures selected, three were maintained in SM, three with
HPMC, and three with SH. Measurements of area of cell RESULTS
migration were taken on days 8, 12, and 16. Migration
Corneal epithelial cells began to migrate from the limbal
Cytospins explants between the second and third day of culture. The
On the 16th day of culture, cytocentrifuge preparations of cells tended to have a polygonal or cobblestone like
epithelial cells were performed. Adherent cells were first morphology migrating as continuous sheets around the
detached from the culture dish by incubation with Trypsin/ explant and towards the edge of the culture dish. A difference
EDTA (0.05%) (Gibco) at 37 ˚C for 15 minutes. Detached cells in the rate of cell migration with time was detected among
were washed twice in DMEM. The Trypan blue dye exclusion the different corneoscleral rims used. However, no significant
test was carried out to ascertain whether any of the culture intra rim difference was noted amongst the triplicates in the
conditions had an adverse effect on cell membrane integrity. same culture conditions (SH: p = 0.300, HPMC: p = 0.277,
The number of cells in suspension was adjusted to a and SM: p = 0.676). The different culture conditions did not
concentration of 56104 cells/ml. Cytospin preparations of affect cell morphology. Migration rates were however
5000 cells/slide were made by centrifugation in a cytospin for affected by the different culture conditions. On days 4 and
10 minutes at 1000 rpm, 100 ul/cup. Slides were air dried, 8 there was no significant difference in migration of cells
fixed in acetone for 10 minutes and stained. cultured in the different conditions. On day 12, cells cultured
in SH presented a significantly higher migration value than
Immunohistochemistry cells cultured in HPMC (p = 0.007) and SM (p = 0.025).
Ki67 (cell proliferation marker) However there was no difference between cells cultured in
Cell proliferation in the different culture conditions was HPMC and SM (p = 0.630). On day 16, the three groups were
determined by the use of monoclonal antibody, which reacts significantly different among themselves (SH v SM:
with a nuclear antigen Ki67, of proliferating human cells in p = 0.001; SH v HPMC: p = 0.003, and HPMC v SM:
G1-M but not G0 phase. Primary rabbit anti human Ki67 p = 0.010) (fig 1).
antibody (Dako, Carpentiria, CA, USA) was applied to the
cytospin preparations at a 1:40 dilution for 1 hour at room Trypan blue dye exclusion test
temperature in a moist chamber. Excess antibody was The number of cells that excluded tyrpan blue dye was
removed by washing slides in TRIS buffered saline (TBS) consistent in the different culture conditions. The number of
(Sigma). Negative controls with TBS and an irrelevant cells ranged from 97.14 to 99.3% (mean 98.49%) for cells
antibody, were set up alongside test slides. Flourescein cultured in the SM and 97 to 99% (mean 98.40%) and 97 to
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Sodium hyaluronate and corneal epithelium 823
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824 Gomes, Amankwah, Powell-Richards, et al
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Sodium hyaluronate and corneal epithelium 825
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Notes