doi:10.1016/j.exer.2008.06.008 
Copyright © 2008 Elsevier Ltd All rights reserved.
Aliphatic β-nitro alcohols for non-enzymatic collagen cross-linking of scleral tissue
David C. Paik
, a, 
, Quan Wena, Suzanna Airiania, Richard E. Braunsteina and Stephen L. Trokela
aDepartment of Ophthalmology, Edward S. Harkness Eye Institute, Columbia University College of Physicians and Surgeons, New York, NY, USA
Received 16 May 2008;
accepted 12 June 2008.
Available online 19 June 2008.
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Abstract
The success of riboflavin photochemical cross-linking of the cornea in treating keratoconus and post-surgical keratectasia has prompted interest in cross-linking scleral tissue with a potential application to stabilize myopic progression. Applying an UVA light source to the sclera is difficult, particularly in the posterior region. An alternate pharmacologic approach to scleral cross-linking may be possible. The present study was undertaken in order to identify nitrite related compounds capable of inducing scleral tissue cross-linking and to gain information regarding the possible chemical mechanisms involved. 8 × 4 mm strips of porcine and human sclera were incubated in various concentrations of nitrite related agents (1–100 mM) at 37 °C. pH 7.4 was used for all experiments except those involving NaNO2. Following a 24–96 h incubation period, the samples were tested for cross-linking effects using thermal shrinkage temperature (Ts) analysis. Several compounds were studied including NaNO2, 2-nitroethanol, 2-nitro-1-propanol, 3-nitro-2-pentanol, 2-nitrophenol, 2-nitroethane, 2-aminoethanol, isopentyl nitrite, DPTA/NO, DETA/NO, and urea, a nitrous acid trap. The results indicate that short chain aliphatic β-nitro alcohols (2-nitroethanol, 2-nitro-1-propanol, and 3-nitro-2-pentanol) are particularly effective cross-linking agents at pH 7.4, showing both time and concentration dependent effects. Furthermore, nitrosation does not appear to induce tissue cross-linking. In conclusion, aliphatic β-nitro alcohols can cross-link scleral tissue at physiologic pH and temperature. Since β-nitro alcohols are known to have reasonable toxicity profiles, these agents could find utility as pharmacologic cross-linking agents for scleral thinning disease.
Keywords: collagen; sclera; myopia; protein cross-linking; nitrite; β-nitro alcohols; nitrosation; riboflavin photolysis; thermal shrinkage temperature
Fig. 1. (A) Custom box for determination of thermal shrinkage temperature (Ts). (B) Example of delay in shrinkage temperature effect in cross-linked tissues. The top row of each panel shows triplicate samples of control porcine sclera. The middle and lower rows shows 10 and 100 mM 2-nitroethanol (2NE) cross-linked samples, respectively. From left to right are shown serial scans of tissues as the temperature of the water bath is raised progressively. At 62 °C the samples are at baseline size. At 66 °C the control samples have begun to shrink. At 70 °C, the 10 mM samples are shrinking but the 100 mM samples remain at baseline size. At 74 °C, all the samples have shrunken in size.
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Fig. 2. (A) Concentration dependent shift in Ts using 2-nitroethanol (2NE). Porcine scleral strips (8 × 4 mm) were incubated in solutions of 20% dextran containing various concentrations of 2NE at pH 7.4. After 96 h of incubation at 37 °C, thermal shrinkage temperature was determined. Minimal effect was observed at 1 mM 2NE. However, at 10 and 100 mM there was a 4.0 and 7.8 °C shift in T50, respectively. (B) Concentration dependent shift in Ts using 2-nitro-1-propanol (2NProp). The same conditions were employed as in (A) except that 2NProp was used as a cross-linking agent. As with 2NE, there was a concentration dependent shift in Ts noted for 2NProp with 10 and 100 mM producing a 1.8 and 6.7 °C shift in T50, respectively. No effect was seen at 1 mM 2NProp. (C) Concentration dependent shift in Ts using 3-nitro-2-pentanol (3N2Pent). The same conditions were employed as in (A) except that 3N2Pent was used as a cross-linking agent. As with 2NE, there was a concentration dependent shift in Ts noted for 3N2Pent at 10 and 100 mM producing a 2.0 and 5.0 °C shift in T50, respectively. No effect was seen at 1 mM. (D) Ts curves for 2NE which show little to no shift in T50.
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Fig. 3. Time dependent shift in Ts using 2-nitroethanol (2NE) at 100 mM (A) and 1 mM (B). (A) Conditions were as in Fig. 2 except that the time of incubation was varied from 24 to 96 h. A time dependent effect in thermal shrinkage temperature shift was noted for 2NE at 100 mM concentration over the course of 96 h with T50 shifts of 0.6, 5.0, 7.1 and 7.8 °C for 24, 48, 72 and 96 h of incubation, respectively. (B) Time dependent shift in Ts using 2-nitroethanol at 1 mM. Conditions were as in (A) except that the time of incubation was varied from 0 to 14 days using a concentration of 1 mM. In addition, the incubation solution was “exchanged daily” using a 1 mM solution of 2NE. A time dependent shift in Ts was noted for 2NE at 1 mM concentration over the course of 14 days. T50 was shifted 0.9, 2.7 and 5.1 °C for 6, 10 and 14 days of incubation, respectively. The shift in Ts was commensurate to the shift observed using higher concentrations of 2NE (i.e. 10 and 100 mM) for shorter durations (i.e. 24–96 h) (see Fig. 2A).
Fig. 4. Concentration dependent shift in Ts of human sclera using 2-nitroethanol (2NE). Human sclera obtained from the NY Eyebank from a 69-year individual was reacted using conditions as in Fig. 2. Following a 96 h incubation using various concentrations of 2NE (1–100 mM), Ts was determined. T50 was shifted 0.9, 2.0 and 7.7 °C using 1, 10 and 100 mM 2NE, respectively. The concentration dependent effect observed is similar to that observed using porcine sclera as shown in Fig. 2A.
Table 1.
Thermal shrinkage temperature changes induced in porcine (and human) scleral strips by reaction with β-nitro alcohols and related agents
Incubation solution includes 20% Dextran (T500) and 0.2 M NaH2PO4/Na2HPO4 buffer pH 7.4 and 37 °C unless otherwise indicated. Incubation time was 96 h unless otherwise indicated. Temperatures are indicated in °C with SEM (±). Ti for each condition was compared to control values (Student's t-test) in order to determine p-values for statistical significance. Each value is the average of a minimum of three independent determinations. Ti = temperature at 1% absolute shrinkage (i.e. initial), TiΔ = change in Ti as compared to control. T50 = temperature at 50% of maximal shrinkage (or maximal rate of shrinkage change), T50Δ = change in T50 as compared to control. All values were compared to porcine controls to derive p-values except for studies using human sclera which were compared to human controls.
Table 2.
Thermal shrinkage temperature changes induced in porcine sclera by reaction with acidified NaNO2, nitrosating agents, aldehydes, and riboflavin photolysis
Incubation solution includes 20% Dextran (T500) and 0.2 M NaH2PO4/Na2HPO4 buffer pH 7.4 and 37 °C unless otherwise indicated. Incubation time was 96 h unless otherwise indicated. Temperatures are indicated in °C with SEM (±). T50 for each condition was compared to control values (Student's t-test) in order to determine p-values for statistical significance. Each value is the average of a minimum of three independent determinations. Ti = temperature at 1% absolute shrinkage (i.e. initial), TiΔ = change in Ti as compared to control. T50 = temperature at 50% of maximal shrinkage (or maximal rate of shrinkage change), T50Δ = change in T50 as compared to control. All values were compared to porcine controls.
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