IN-VITRO MODEL FOR CORNEAL WOUND-HEALING - ORGAN-CULTURED HUMAN CORNEAS

Healing of linear, non-perforating thermal burns was studied in 56 human corneas in an air/liquid organ culture system in serum free medium or in media supplemented with 10% fetal bovine serum, 10% human serum or 10% human plasma. The extent of epithelial wound healing was determined by measuring epithelial growth into the wound using digitized computer scanning of light micrographs of 1 mu m sections. The cross-sectional area of this epithelial growth entering the wound was significantly greater for corneas incubated with either human serum (16,350 +/- 12,088 mu m(2)/day; p < 0.0001) or human plasma (20,571 +/- 12,276 mu m(2)/day; p = 0.0004) than for those incubated in serum free (1,784 +/- 1,957 mu m(2)/ day) medium. There was no significant difference between epithelial growth in the serum free and fetal bovine serum supplemented (3,779 +/- 2,580 mu m(2)/day) media or between that in human serum and human plasma supplemented media. The thickness of the epithelium adjacent to the wound was greater in corneas cultured in fetal bovine serum supplemented media than in corneas cultured in the presence of human serum. Similarly, the build-up of epithelium at the wound edge for corneas in either serum free or fetal bovine serum supplemented media was significantly greater than for either human serum or human plasma supplemented media. The percentage of basal epithelial nuclei which incorporated bromodeoxyuridine (BrdU) increased during the first three days of culture when it reached a plateau. Comparison of paired wounded and unwounded corneas showed that wounding stimulated an increase in DNA synthesis. The extent of labelling was similar after seven days for both serum free medium and human serum supplemented medium, indicating that a serum-factor was not necessary for this stimulation. Immunohistochemical (IHC) analysis showed that fibronectin was present around the wound edges, in the stroma and along the basement membrane of the epithelium, after seven days of culture in either serum free or human serum containing media. Type IV collagen was found in the basement membrane of both groups but appeared along the wound edge and surrounding the epithelial cell influx into the wound in human serum cultured corneas only. This cultured human cornea system offers a useful model for examining factors which modulate the course of wound healing.