The eye area is talked of a lot in the issued patent:
Migration Assays of Corneal Epithelial Cells
Corneal Epithelial Cell migration assays were carried out in Boyden chamber using 12 .mu.m pore polyester membranes (Poretics, Livermore, Calif.) coated with a 0.1 mg/ml solution of collagen IV in dH.sub.2O (Trevigen, Gaithersburg, Md.). Filters were then dried at least 1 h. Cells were cultured and resuspended in Eagle's Minimal Essential Medium with 0.05 mM Ca.sup.2+. The bottom chamber was loaded with EMEM containing 0.01, 0.1, 10, 100, and 1000 ng/ml of synthetic T.beta.4. Conditioned medium from primary dermal fibroblasts and/or keratinocyte growth factor was added to several wells as a positive control. Cells were added to the upper chamber at a concentration of 50,000 cells per well. Chambers were incubated at 35 C/7% CO.sub.2 for 4-5 hours and the filters were then fixed and stained using Diff-Quik (Baxter Healthcare Corporation, McGraw Park, Ill.). The cells that migrated through the filter were quantitated by counting the center of each well at 10.times. using an Olympus CK2 microscope. Each condition was assayed in triplicate wells and each experiment was repeated four times with different preparations of cells. The results demonstrated that corneal epithelial cell migrated in response to T.beta.4 after 4-5 hours of exposure. Migration was enhanced 2-3 fold over migration in the presence of media alone (FIG. 7) with the highest level of migration seen at 100 ng/ml of T.beta.4.
In vivo Corneal Re-Epithelialization
To determine the effect of T.beta.4 on corneal re-epithelialization in vivo, Rat corneas were de-epithelialized and treated with T.beta.4. Filters were soaked in heptanol, applied to the eye for 30 seconds, and then the epithelium was scraped. Various concentration of T.beta.4 in saline was applied to the eye and at 24 hours the rats were sacrificed. The eyes were fixed, sectioned and the degree of corneal epithelial migration (as measured in pixels) was determined using a microscope with an internal caliper by a masked observer. The results demonstrate that re-epithelialization of the cornea was increased 2-fold over untreated control in the presence of about 1 to 25 .mu.g of T.beta.4 (FIGS. 8 and 9). In addition, it was noted that T.beta.4 treated eyes had reduced inflammation compared to the non-treated corneas.