Enzymatic Digestion of Porcine Corneas Cross-linked by Hypo- and Hyperosmolar Formulations of Riboflavin/ultraviolet A or WST11/Near-Infrared Light

Abstract

Purpose: To assess enzymatic digestion rate after Riboflavin (RF) and Water-Soluble-Taurine (WST11) based corneal cross-linking (CXL), with or without the addition of high molecular weight dextran (RF-D and WST-D).

Methods: Eighty-eight paired porcine corneas were cross-linked by either RF (n = 11) or RF-D (n = 11) and ultraviolet light (UVA), or WST11 (n = 11) or WST-D (n = 11) and near-infrared (NIR) light, or used as paired control (n = 44). Corneal buttons of treated and paired control eyes were placed in a 0.3% collagenase solution. Time to full digestion and remaining dry sample weight after six hours were compared.

Results: A strong treatment effect was seen with all four formulations, as all controls had been fully digested whilst all treated samples were still visible at the experiment's endpoint. After irradiation, central corneal thickness was significantly higher in samples treated with hypo-osmolar formulations, compared to dextran enriched formulations (P < 0.001). Dry sample weight after digestion was nonsignificantly different between corneas treated by the four different formulations (P = 0.102). Average dry sample weight was 1.68 ± 0.6 (n = 10), 2.19 ± 0.50 (n = 8), 1.48 ± 0.76 (n = 11), and 1.54 ± 0.60 (n = 9) mg, for RF, RF-D, WST11, and WST-D treated samples, respectively. Enzymatic resistance was similar for RF and WST based CXL (P = 0.61) and was not affected by the addition of dextran (P = 0.221).

Conclusions: Both RF and WST11 based CXL significantly increases resistance to enzymatic digestion, with similar effect for hypo-osmolar and hyperosmolar (dextran enriched) formulations.

Translational relevance: Our findings indicate these formulations are interchangeable, paving the way for the development of novel PACK-CXL protocols for thin corneas and deep-seated infections.

Keywords: WST-D; collagenase; corneal collagen cross-linking; keratolysis; riboflavin.

Figure 1.

Study flowchart. Eighty-eight paired porcine eyes were mechanically de-epithelialized. One eye of each pair served as control (n = 44), whereas the contralateral eye was treated by one of the following: WST11 (n = 11) or WST-D (dextran enriched, n = 11) impregnation with consecutive NIR irradiation, or RF (n = 11) or RF-D (n = 11) impregnation with UVA irradiation. Between steps, if applicable, central corneal thickness was measured by ultrasound pachymetry. After treatment, samples were subjected to a collagenase type II digestion assay, during which samples were inspected half-hourly. After six hours dry sample weight of remaining samples (n = 44) was measured to quantify the degree of digestion.

Figure 2.

Mean central corneal thickness (CCT) for control (n = 44) and treated (n = 44, n = 11 per chromophore) corneas. CCT was determined for each sample as the average of five consecutive measurements, and was repeated at different timepoints: (1) epithelium-on, (2) epithelium off, (3) post-chromophore impregnation (treated group only), and (4) postirradiation (treated group only). At baseline, no significant difference in CCT was seen between chromophore groups for both control (P = 0.148) or treated (P = 0.124) corneas. Hypo-osmolar chromophore impregnation induced significant swelling compared to impregnation by hyperosmolar formulations (P < 0.001). CCT in micrometer, error bars indicate 95% confidence interval.