Evaluating the Toxicity/Fixation Balance for Corneal Cross-Linking With Sodium Hydroxymethylglycinate (SMG) and Riboflavin-UVA (CXL) in an Ex Vivo Rabbit Model Using Confocal Laser Scanning Fluorescence Microscopy

Abstract

Purpose To develop methods to delineate the relationship between endothelial cell toxicity and tissue fixation (toxicity/fixation) using sodium hydroxymethylglycinate (SMG), a formaldehyde releaser, and riboflavin-UVA (CXL) for therapeutic tissue cross-linking of the cornea. Methods Eleven (11) fresh cadaveric rabbit heads were used for ex vivo corneal cross-linking simulation. Following epithelial debridement, the tissue was exposed to 1/4 Max (9.765mM) or 1/3Max (13.02mM) SMG at pH 8.5 for 30min or riboflavin-UVA (CXL). The contralateral cornea served as a paired control. Post-exposure, cross-linking efficacy was determined by thermal denaturation temperature (Tm) and endothelial damage was assessed using calcein AM and ethidium homodimer staining (Live/Dead Kit). Confocal laser scanning fluorescence microscopy was used to generate live/dead cell counts following a standardized algorithm. Results The ΔTm following CXL, 1/3 SMG, and 1/4 SMG was 2.19±0.91°C, 1.33±0.49 °C, and 1.10 ±0.46 °C, respectively. Endothelial cell damage was expressed as the percent of dead cells/live + dead cells counted per high powered field. The values were 2.95±1.74% (control) and 8.86±11.10% (CXL) [p=0.390]; 0.98±0.20% (control) and 19.53±32.22% (1/3max SMG) [p=0.426]; and 2.70±2.37% (control) and 2.84±2.24% (1/4 max SMG) [p=0.938];. The values for endothelial toxicity were then indexed over the shift in Tm in order to yield a toxicity/fixation index. The values were as follows: 2.70 for CXL, 13.95 for 1/3 max, and 0.13 for 1/4 max. Conclusions Quarter max (1/4 Max = 9.765mM) SMG effectively cross-linked tissue and was non-toxic to endothelial cells. Thus, SMG is potentially a compound that could achieve both desired effects.

Evaluation of Therapeutic Tissue Crosslinking (TXL) for Myopia Using Second Harmonic Generation Signal Microscopy in Rabbit Sclera

“…The previous experiments indicated that a concentration-dependent crosslinking effect could be obtained using SMG, with effects ranging well above those achieved with standard CXL as determined by thermal analysis. 14 …”

Prevention and Management of Myopia and Myopic Pathology

“…119,120 UV-A use with scleral CXL has been associated with decreased dark-adapted ERG amplitudes up to 3 months posttreatment, with apoptotic cells and ultrastructural changes in retina layers also found. 121 111,118,119,[122][123][124][125][126][127][128][129][130] Glyceraldehyde is a non-light-activated chemical shown to increase scleral rigidity, but has ill effects on the neighboring cornea and muscles. 120 Alternatively, topical beta-nitroalcohols (or ''BNAs,'' commonly found in antibiotics and shampoos) and formaldehyde releasers (or ''FARs,'' which are preservatives found in cosmetics, body wash, and ophthalmic solutions; e.g., BLU Gel A multidose artificial tears, SOOFT italia [Montegiorgio FM, Italy]) effectively crosslink corneal and scleral collagen in vitro, and have very good safety profiles (Hoang Q, et al IOVS 2013;54:ARVO E-Abstract 136).…”

“…120 Alternatively, topical beta-nitroalcohols (or ''BNAs,'' commonly found in antibiotics and shampoos) and formaldehyde releasers (or ''FARs,'' which are preservatives found in cosmetics, body wash, and ophthalmic solutions; e.g., BLU Gel A multidose artificial tears, SOOFT italia [Montegiorgio FM, Italy]) effectively crosslink corneal and scleral collagen in vitro, and have very good safety profiles (Hoang Q, et al IOVS 2013;54:ARVO E-Abstract 136). [122][123][124][125][126][127] Specifically, in a comparative toxicity study of nine topical crosslinking agents in four different cell lines with a semiquantitative analysis using five categories of toxicity/fixation (Hoang Q, et al IOVS 2013;54:ARVO E-Abstract 136), the toxicity levels varied by a factor of 10 3 , with the least toxic being mononitroalcohol BNAs, and genipin among the most relatively toxic. 122 Ultimately, regardless of the scleral reinforcement approach used, it is paramount to detect scleral weakness before staphyloma formation, to determine which highly myopic eyes are headed down the path toward staphyloma and MMD formation.…”

Development of a topical tissue cross-linking solution using sodium hydroxymethylglycinate (SMG): viscosity effect

“…The formulation contained various concentrations of 15cP HPMC, while keeping the final SMG concentration at 10 mM and NaHCO 3 at 100 mM. The concentration of 10 mM SMG was chosen based on the results from a previous rabbit cornea study in which this concentration was determined to be effective yet non-toxic [19]. Each formulation was prepared freshly on the day of the experiment.…”

“…Less stable fibrils will result in a lower T m and likewise, more stable fibrils induced by the cross-linking treatment will raise the T m . T m has been used as a surrogate assay for tissue cross-linking efficacy, in lieu of mechanical testing, for many decades in the biomaterials world and we have applied to this simple and efficient technique to our therapeutic tissue cross-linking studies previously [19].…”