In vitro assessment of the biocompatibility of chemically treated silicone materials with human lens epithelial cells.

Cytotoxicity testing is a regulatory requirement for safety testing of new ocular implants. In vitro toxicity tests determine whether toxic chemicals are present on a material surface or leach out of the material matrix. A method of evaluating the cytotoxicity of ocular implants was developed using fluorescent viability dyes. To assess the assay's sensitivity in detecting toxic substances on biomaterials, zinc diethydithiocarbamate (ZDEC) and benzalkonium chloride (BAK) were deposited on silicone surfaces at different concentrations. Human lens epithelial cells (HLEC) were added to the surface of these treated silicone surfaces and were assessed for viability. The viability of both the adherent and non-adherent cells was determined using confocal microscopy with, annexin V, ethidium homodimer, and calcein. Cell metabolism was also evaluated using resazurin and the release of inflammatory cytokines was quantified using a multiplex Mesoscale Discovery platform. Confocal microscopy was shown to be a sensitive assay for evaluating material toxicity, as significant toxicity (p < 0.05) from ZDEC and BAK-treated surfaces compared to the untreated silicone control was detected. Patterns of cytokine release from cells varied depending on the toxin evaluated and the toxin concentration and did not directly correlate with the reduction in cell metabolic activity measured by alamarBlue.

Assessment of biofilm formation of E. meningoseptica, D. acidovorans, and S. maltophilia in lens cases and their growth on recovery media.

PURPOSE: Bacterial biofilm formation in contact lens cases is a risk factor in the development of both microbial and infiltrative keratitis. This investigation evaluated three emerging pathogens: Stenotrophomonas maltophilia, Elizabethkingia meningoseptica, and Delftia acidovorans for biofilm formation and metabolic activity in lens cases. Also, growth of these bacteria on different media was assessed to optimize recovery conditions. METHODS: The three bacteria were incubated in lens cases with different concentrations of tryptic soy broth. Biofilm formation was evaluated by measuring metabolic activity using MTT and enumerating the number of viable bacteria. To determine the optimal recovery media, dilutions of these microorganisms were plated on six different media. The number of colony forming units (CFU) was recorded after 48, 72, and 96 h of incubation at 32°C and 37°C for S. maltophilia, and at 37°C for E. meningoseptica and D. acidovorans. RESULTS: All three microorganisms established biofilms in the lens cases, with significant numbers of CFU recovered. Biofilms of S. maltophilia and E. meningoseptica were metabolically active. Significant reduction in metabolic activity and number of viable S. maltophilia occurred when the incubation temperature was raised from 32°C to 37°C (p<0.05). The metabolic activity of the biofilms increased with greater organic load present. The highest percent recovery for all three organisms was given by Columbia blood agar, followed by chocolate. CONCLUSION: Based on the results, the presence of the three emerging pathogens present in lens cases and from corneal isolates can be accurately determined if proper growth media and incubation temperatures are utilized.

A multi-country assessment of compliance with daily disposable contact lens wear.

PURPOSE: To investigate compliance with daily disposable contact lens (DDCL) wear and investigate re-use of lenses according to country and DDCL material worn. METHODS: Optometrists invited eligible DDCL patients from their practices to participate in a survey on DDCL wear in Australia, Norway, the United Kingdom (UK) and the United States (US). Eligible participants completed an online or paper version of the survey. RESULTS: 805 participants completed the survey (96% online): Australia 13%, Norway 32%, UK 17%, US 38%. The median age was 38 years; 66% were female. Silicone hydrogel (SiHy) DDCLs were worn by 14%. Overall, 9% were non-compliant with DDCL replacement; Australia 18%, US 12%, UK 7% and Norway 4%. There were no differences with respect to sex, years of contact lens wear experience or DDCL material (SiHy versus hydrogels). The primary reason for re-use was "to save money" (60%). Re-use of DDCLs resulted in inferior comfort at insertion and prior to lens removal (p=0.001). 75% reported occasional napping and 28% reported sleeping overnight for at least one night in the preceding month, while wearing their DDCLs. CONCLUSION: Non-compliance with replacement of DDCLs occurred in all countries investigated; the rate was highest in Australia and lowest in Norway. Re-use of DDCLs was associated with reduced comfort. DDCL wearers often reported wearing lenses overnight. It is important for optometrists to counsel their patients on the importance of appropriate lens wear and replacement for DDCLs.

Assessment of variation in bulbar conjunctival redness, temperature, and blood flow.

PURPOSE: To assess the diurnal variation in bulbar conjunctival redness, conjunctival temperature, and conjunctival blood flow. METHODS: Bulbar redness was quantified by CIE u' chromaticity using a SpectraScan PR650 spectrophotometer. Conjunctival temperature was measured using a Tasco-Thi 500 infrared thermometer. Measurements of conjunctival blood flow were obtained using a modified Heidelberg Retinal Flowmeter (HRF). Measurements on 10 subjects were made on a periodic basis over the day and on waking. RESULTS: For each factor measured a cyclical pattern was observed, with highest values on waking, a reduction in values towards mid-day, and then a gradual increase over the remainder of the day. There was a significant effect of time for redness, temperature, and conjunctival blood flow (p < 0.001 for all three variables), with no significant difference in the cyclical pattern between eyes being observed (p = NS). CONCLUSIONS: Diurnal bulbar redness, temperature, and conjunctival blood flow variation may be objectively quantified and all three are lowest during the middle of the day and maximal at the start of the day. This information should be considered when undertaking studies in which redness, temperature, and ocular surface blood flow are important outcome variables and time of day is a potential confounding factor.

In vitro assessment of medical device toxicity: interactions of benzalkonium chloride with silicone-containing and p-hema-containing hydrogel contact lens materials.

PURPOSE: To analyze the interactions of benzalkonium chloride (BAK) with silicone-containing (lotrafilcon A and galyfilcon A) and p-HEMA-containing (etafilcon A and vifilcon A) hydrogel contact lenses and to examine the possibility of using sodium fluorescein permeability assay (SFPA), 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the bovine lens assay in conjunction with confocal laser scanning microscopy (CLSM) as a battery of in vitro alternatives to evaluate the potential toxicity of soft contact lenses. METHODS: Four soft contact lens types (Focus Monthly [vifilcon A], Focus NIGHT & DAY [lotrafilcon A], ACUVUE Advance With Hydraclear [galyfilcon A], and SUREVUE [etafilcon A]) were soaked for 24 hours in various concentrations of BAK (1%, 0.1%, 0.01%, and 0.001%) in 20-mL glass vials. After 24 hours, the lenses were gently washed in Hanks' Balanced Salt Solution (HBSS), placed in 5 mL of HBSS, and incubated for a total of 7 days at 37 degrees C, 5% CO2. BAK released into HBSS (i.e., the extract) was recovered from the vials and used as the test chemical in the SFPA (epithelium integrity), MTT assay (cellular viability), and the bovine lens assay with CLSM (mitochondrial metabolism and optical properties). The amount of BAK extracted from the various contact lenses was measured using an Abbe refractometer. Negative controls consisted of HBSS and contact lenses subjected to the same conditions as the treated contact lenses, but without BAK. RESULTS: Extracts obtained from soaking Focus Monthly lenses in BAK caused the most damage to the epithelium and mitochondrial metabolism. However, at 0.1% BAK extraction, all lens extracts showed increased levels of back vertex distance variability of the cultured bovine lens. CONCLUSIONS: Unexpectedly, lenses extracted with HBSS showed SFPA and MTT assay responses and an observed effect on the bovine lens epithelium visualized by CLSM, indicating that unknown chemical agents may be leached from contact lens polymers.