Cross-linking is the process of chemically joining two or more molecules by a covalent bond. Cross-linking reagents contain reactive ends to specific functional groups (primary amines, sulfhydryls, etc.) on proteins or other molecules. Because of the availability of several chemical groups in proteins and peptides that may be targeted, they are readily conjugated using different cross-linking methods. It is a well established method used in polymer industries for the stabilization of tissues. In medicine some applications of cross linking include chemical cross-linking using glutaraldehyde in the preparation of prosthetic heart valves, papraformaldehyde for tissue hardening in specimens being prepared for examination and UV cross-linking used to harden dentistry fillings.

Not long ago riboflavin/UVA corneal cross linking (CXL) was introduced to ophthalmic practice. Initially, it was aimed for the treatment of ectatic corneal conditions such as keratoconus, pellucid marginal degeneration and post excimer laser keratectesia. This is due to the formation of new covalent bonds between collagen fibrils and hence corneal rigidity is improved. More recently, other applications for its usage are emerging such as bullous keratopathy, infectious keratitis, corneal melting and the stabilization of post RK hyperopic shifts.

The standard protocol for CXL could be considered as moderately invasive as it requires mechanical removal of the epithelium. The drawbacks of this step include:

1. Prolonged surgical time.

2. Increased incidence of herpetic activation.

3. Increased incidence of developing haze.

4. Corneal oedema.

5. Postoperative pain and discomfort for many days.

6. Reduced visual acuity until epithelialization is complete and resolution of corneal oedema.

Figure 1 & 2: Light microscopy at X20 demonstrates the CXL occurance in the treated corneas (Figure 1) in comparison with the control (Figure 2).

This has motivated us to develop a variant of the standard technique in which the epithelium is not removed offering patients a less invasive, safer and faster CXL, while retaining the efficacy of the standard technique. This technique applies a riboflavin solution containing benzalkonium chloride (BAK) directly onto intact epithelium. BAK, the most commonly used preservative in ophthalmic medications, is also a tensioactive substance. A tensioactive substance, surfactant or an active surface agent are chemical substances that change the surface tension value, and hence facilitate the penetration of other substances through biological membranes. Therefore, BAK aids the penetration of riboflavin into the corneal stroma without the need for removing the epithelium.

We demonstrated the results of tensioactive CXL in rabbit corneas at the IV international congress of CXL held in Dresden, Germany last December. In our results the following signs by light microscopy (Figure 1) demonstrated the occurance of CXL in the treated corneas in comparison to control ones:

• The epithelial surface was straighter (retained curvature due to increased stiffness).
• The epithelium was more compact.
• In the stroma, the fibres showed more compact arrangement, and were straight compared to the wavy fibres in the stroma of the control corneas. This effect was noticed in the upper 50% (approximately) of the stroma.
• The deep stroma adjacent to the endothelium looked the same as the control.