Topical alternatives to therapeutic riboflavin photochemical tissue cross-linking: a comparison of cell toxicity thresholds.

Abstract

 

Purpose

 

As therapeutic corneoscleral cross-linking using riboflavin photochemistry continues to gain widespread clinical application, there exists ongoing interest in developing an alternative approach to such therapy that obviates the need for ultraviolet irradiation exposure and is delivered topically. In addition to nitroalcohols, several agents including glyceraldehyde and genipin have been proposed for such uses. The present study was undertaken in order to compare the cell toxicity of such agents.

 

Methods

 

Toxicity thresholds for nine topical cross-linking agents were tested on four different cell types. The cross-linking agents were: 2-nitroethanol (NE), 2-nitro-1-propanol (NP), 2-methyl-2-nitro-1,3,-propanediol (MNPD), 2-hydroxymethyl-2-nitro-1,3-propanediol (HMPD), 2-bromo-2-nitro-1,3-propanediol (BP=bronopol), genipin (GP), glyceraldehyde (GC), paraformaldehyde (PA-polymeric), and glutaraldehyde (GA-polymeric). The cell types were primary bovine corneal endothelium, human skin fibroblasts (from ATCC), immortalized human corneal epithelium (HCEC - gift from Dr. Peter Reinach), and immortalized human retinal pigment epithelial cells (ARPE-19). The cells were grown in planar culture to confluence and exposed to each agent (0.001mM to 10mM) for 24 hours followed by a 48 hour recovery phase. Toxicity thresholds were then determined using the trypan blue exclusion method.

 

Results

 

The results are summarized in Table 1. Overall, the toxicity levels varied between cross-linking agents by a factor of 100x with the least toxic being NE, glyceraldehyde, and NP. MNPD, HNPD, polymeric PA, and polymeric GA showed intermediate toxicity. The most toxic agents were GP and BP. Toxicity levels were generally in agreement between cell types although in some cases there was as much as a 10x difference for a given compound when comparing between cell types.

 

Conclusions

 

Regarding cell toxicity, there are significant differences between topical cross-linking compounds. In addition, for a given compound, toxicity can vary by cell type. Such differences should be considered when selecting agents to use for topical therapeutic corneoscleral tissue cross-linking.

 

 

NE= 2-nitroethanol GC= glyceraldehyde NP= 2-nitro-1-propanol MNPD= nitrodiol HNPD= nitrotriol PA= paraformaldehyde GA= glutaraldehyde GP= genipin BP= bronopol

 

Keywords: 574 keratoconus • 605 myopia • 519 extracellular matrix