Abstract
Purpose:
As riboflavin photochemical corneal cross-linking continues to gain a foothold in clinical Ophthalmology, development of alternative methods that neither require ultraviolet irradiation nor painful de-epithelialization continue to be an attractive goal. β-nitroalcohols (BNAs) have favorable safety profiles and function as formaldehyde donors via a reverse Henry reaction (rHr) to cross-link collagenous tissues. Because the reaction is reversible, catalytic mechanisms for the forward reaction were tested for tissue cross-linking via the reverse reaction.
Methods:
Using bovine sclera as a collagenous tissue substrate (8x5mm tissue pieces), 60 min incubations (37oC) of the higher order nitroalcohols (1-5mM) 2-methyl-2-nitro-1,3-propanediol (MNPD), 2-hydroxymethyl-2-nitro-1,3-propanediol (HNPD), and 2-bromo-2-nitro-1,3-propanediol (bronopol=BP) [a compound widely used in cosmetics, etc.] were carried out with a variety of potential catalysts. Both NaH2PO4/Na2HPO4 and HCO3 buffers (pH 6.5-9.5) were tested. In addition, three enzymes (D-aminoacylase, transglutaminase, hydroxynitrile lyase from Arabidopsis thaliana) and salmon testes DNA were tested at pH 7.4. Cross-linking effects were evaluated using thermal shrinkage temperature (Ts) analysis as previously described.
Results:
Ts values for uncross-linked control tissues were as previously reported (65.3oC). The BNA cross-linking reaction displays a strong pH dependency, with enhanced tissue cross-linking effects occurring as the pH is raised from 6.5 to 9.5. As compared to control tissue, the Ts using HNPD was shifted 1.5oC at pH 8.3, 3oC at pH 8.4, 3.8oC at pH 8.5, and 5.4oC at pH 9.5, indicating a pH dependent cross-linking effect. Similar shifts were observed using both MNPD and BP. Because Ts shifts of 2-2.5oC are observed using the standard riboflavin photochemical cross-linking technique, this degree of Ts shift represents our target effect. The type of buffer used did not have a bearing on the pH dependent catalytic effect. When reacting at pH 7.4, 37oC, for 1hr, there were no discernible catalytic effects using the three enzymes or DNA.
Conclusions:
Because the cornea can tolerate a pH up to 8.5 without sustaining damage, and the tear film has a fairly limited buffering capacity, modulating the pH of a BNA corneal cross-linking solution could provide a useful way to rapidly strengthen corneal tissue (i.e. within minutes).
Keywords: 574 keratoconus •
605 myopia •
519 extracellular matrix