Sodium hyaluronate (hyaluronic acid) promotes migration of human corneal epithelial cells in vitro

Abstract

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 maintained separately in three different culture conditions: (1) culture medium only, (2) sodium 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 days 4, 8, 12, and 16. Cytospin preparations of cells cultured in the different culture conditions were examined immunohistochemically for proliferation and CD44 receptor expression using antibodies directed against Ki67 and CD44 respectively.

Results: Cells cultured in the presence of sodium hyaluronate showed significantly increased migration at days 12 and 16 (Friedmen test: p = 0.0012, day 16; p = <0.001, day 12) compared with cells cultured in the other media. There was no difference in cell proliferation (Ki67) or CD44 expression on cells cultured in the different culture conditions.

Conclusions: Sodium hyaluronate promotes migration but not proliferation or CD44 expression on human corneal epithelial cells in vitro. The beneficial effect of sodium hyaluronate in corneal wound healing is 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.

Figure 1

Mean area of migrating human corneal epithelial cell sheets in the different culture conditions—standard medium (control), hydroxypropyl methyl cellulose (Occucoat), and sodium hyaluronate (Healon)—over time. Error bars = standard error of mean.

Figure 2

(A) Mean percentage of proliferating (Ki67 positive) human corneal epithelial cells in the different culture conditions—standard medium (control), hydroxypropyl methyl cellulose (Occucoat), and sodium hyaluronate (Healon)—studied on day 16 of culture. Error bars = standard error of mean. (B) Immunofluorescent staining of a cytospin preparation of cells harvested at day 16 of culture in sodium hyaluronate medium, showing Ki67 positive cells. (×400). The staining was identical to that seen with standard medium and hydroxypropyl methyl cellulose (Occucoat) medium.

Figure 3

(A) Mean percentage of CD44 positive human corneal epithelial cells in the different culture conditions—standard medium (control), hydroxypropyl methyl cellulose (Occucoat), and sodium hyaluronate (Healon)—studied on day 16 of culture. Error bars = standard error of mean. (B) Immunostaining (alkaline phosphatase anti alkaline phophatase) of a cytospin preparation of cells harvested at day 16 of culture in sodium hyaluronate medium, showing CD44 positive cells. (×400). Staining was identical to that seen with standard medium and hydroxypropyl methyl cellulose (Occucoat) medium.