Abstract: In vitro biocompatibility of 50:50 PDLGA, 85:15 PDLGA, and Inion GTR(TM) membrane was evaluated in cell line cultures from various ocular tissues, in human corneal epithelial cells (HCE), rabbit stromal fibroblasts (SIRC), bovine corneal endothelial cells (BCE), human conjunctival epithelial cells (IOBA-NHC), and human retinal pigment epithelial cells (ARPE-19). To study the toxicity of degradation products, the biomaterials were extracted in phosphate buffered saline at 70 degrees C for 24 h. The cell cultures were exposed to biomaterial extract diluted in medium (1:1-1:8) and the biocompatibility was evaluated by the WST-1 cytotoxicity/cell proliferation test. In all experiments without pH neutralization, cell viability increased with decreasing biomaterial extract volume. The highest extraction ratio 1:1 of PDLGA 50:50 decreased viability from 5-20%, from the control level, depending on the cell type. The corresponding cell viability values for PDLGA 85:15 and Inion GTR membrane ranged from 47-87% and 66-92%, respectively. When the pH of biomaterial extract was neutralized, Inion GTR membrane and PDLGA 85:15 had no effect on viability. BCE, HCE, and IOBA-NHC appeared to the most sensitive cell types, while SIRC and ARPE-19 were more resistant. The results of our in vitro studies suggest that the polymers tested are satisfactorily biocompatible.

Abstract: Synthetic biodegradable polymers have many potential therapeutic applications. In ophthalmology, biodegradable polymers have been used as viscoelastic agents and surgical implants. Other potential applications include controlled release of drugs and growth factors, gene therapy, and tissue engineering. In the present study, in vitro biocompatibility of three biodegradable polymers, 50:50 PDLGA, 85:15 PDLGA, and Inion GTR membrane was evaluated in comparison to tissue culture polystyrene by investigating cell proliferation and potential acute toxicity by the WST-1 cytotoxicity/cell proliferation test, the ATP test, and the lactate dehydrogenase (LDH) test. Evaluations were conducted with cell line cultures from various ocular tissues, human corneal epithelial cells (HCE), rabbit stromal fibroblasts (SIRC), bovine corneal endothelial cells (BCE), human conjunctival epithelial cells (IOBA-NHC), and human retinal pigment epithelial cells (ARPE-19) by direct contact studies by plating the cells on the polymer film specimens in 96-wells. The proliferation results show that cell lines from various ocular tissues attached and grew on PDLGA 50:50, PDLGA 85:15, and Inion GTR membrane. Cytotoxicity experiments with the LDH and ATP tests showed no or extremely slight toxic adverse effects. These polymers have potential to be used as scaffolds in cell transplantation devices or as surgical implants.

Abstract: PURPOSE: To analyse the accuracy of corneal flap thickness created in laser-assisted in situ keratomileusis (LASIK) using the Moria Model 2 (M2) single-use head 90 microkeratome. METHODS: The corneal thickness of 300 (266 myopic and 34 hyperopic) eyes of 150 patients was measured by ultrasonic pachymetry preoperationally and intraoperationally after flap cut. The Moria M2 single-use head 90, intended to create a flap with a thickness of 120 microm, was used in all eyes. The right eye was always operated first and the left eye second, using the same blade. RESULTS: Mean corneal flap thickness was 115.4 microm (standard deviation [SD] 12.5) in the two eyes, 115.7 microm (SD 12.4, range 73-147 microm) in right eyes and 115.1 microm (SD 12.6, range 74-144 microm) in left eyes. Mean horizontal flap diameter was 9.1 mm (SD 0.2) and mean hinge length 4.1 mm (SD 0.1). There were no free flaps, incomplete flaps or flaps with buttonholes in the study. Occasional iron particles were observed in three (1.0%) eyes. CONCLUSIONS: As with most microkeratomes, the single-use head 90 microkeratome cut thinner flaps than were intended. The range of the cuts was relatively wide. However, thin flaps did not increase the rate of flap-related complications. The difference between the first and second eyes was not significant.

Abstract: PURPOSE: To compare the Moria (Antony, France) M2 automated microkeratome with the head 130 to a new disposable single use head to evaluate complications, accuracy, and safety of the procedure. METHODS: Ninety-eight eyes of 49 consecutive patients were operated with the Moria M2 microkeratome. One eye was operated with the metallic head 130 and the other with a plastic single use head, both designed to create a 160-microm flap. Intraoperative flap dimensions were correlated to preoperative parameters and evaluated 1 month postoperatively. RESULTS: With the head 130, mean thickness was 153.3 microm (standard deviation [SD] 13.3, range: 102 to 179 microm). When using a single use head, mean thickness was 148.0 microm (SD 9.8, range: 120 to 170 microm). Occasional iron particles were observed in one eye with both head types. No true epithelial ingrowth was detected in any of the eyes, but epithelial dots at the wound edge occurred in one eye, when using the head 130, but not in the eyes operated with a single use head. CONCLUSIONS: On average, both head types created thinner flaps than attempted. Single use heads produced thinner flaps than the head 130. Accuracy in flap thickness in terms of standard deviation was significantly better in single use heads than in the head 130. Single use heads also had fewer microkeratome-related complications. In clinical practice, the single use head was easier to use because no assembly was required. Plastic single use heads also worked more smoothly than the metallic head 130.

Abstract: Development of age-related macular degeneration (AMD) is associated with functional abnormalities and cell death in retinal pigment epithelial (RPE) cells attributable to oxidative stress. To minimize the adverse effects of oxidative stress, cells activate their defence systems, e.g., via increased expression of heat shock protein (Hsp), activation of stress sensitive AP-1 and NF-kappaB transcription factors. In this study, we examined the accumulation of Hsp70 protein, activation of AP-1 and NF-kappaB transcription factors in human ARPE-19 cells subjected to a 4-hydroxynonenal (HNE)-induced oxidative stress. In addition, the influence of Hsp90 inhibitor geldanamycin (GA) was studied in HNE-treated cells. Mitochondrial metabolic activity and apoptosis were determined to evaluate cell death in the ARPE-19 cells. The ARPE-19 cells showed increased accumulation of Hsp70 protein before of the cytotoxic hallmarks appearing in response to HNE. In contrast, increased DNA-binding activities of AP-1 or NF-kappaB transcription factors were not seen under HNE insults. Interestingly, GA significantly increased cell death in the HNE-treated cells, which was involved in caspase-3 independent apoptosis. This study reveals that the Hsps have an important role in the cytoprotection of RPE cells subjected to HNE-derived oxidative stress.

Abstract: PURPOSE: To evaluate accuracy and predictability and factors that influence the dimensions of the laser in situ keratomileusis (LASIK) corneal flap created with the Moria M2 automated microkeratome (Moria SA, Antony, France). METHODS: The flap thickness of 454 eyes of 243 consecutive patients was measured using subtraction ultrasonic pachymetry during LASIK with the Moria M2 microkeratome head 130 designed to create a 160-microm-thick flap. Flap dimensions were evaluated and measurements were correlated with preoperative parameters. A stepwise regression analysis was used to determine the factors that influenced actual flap thickness. RESULTS: The preoperative spherical equivalent refraction of the 454 eyes ranged from -12.125 diopters (D) to +6.25 D. Patient age ranged from 18 to 57 years (mean age: 31.3 +/- 8.8 years). Mean preoperative keratometric power K1 was 44.31 +/- 1.59 D and K2 was 43.32 +/- 1.54 D. Mean preoperative central comeal thickness was 552.4 +/- 32.5 microm (range: 466 to 665 microm). With an attempted thickness of 160 microm, the Moria M2 flap thickness ranged from 77 to 209 microm (mean: 153.3 +/- 19.0 microm). Mean horizontal flap diameter was 9.2 +/- 0.2 mm and mean hinge length 4.6 +/- 0.3 mm. Increasing flap thickness was found to correlate with increasing preoperative comeal thickness, younger patient age, and flatter preoperative keratometric power K1. CONCLUSIONS: Although the standard deviation of the flap thickness was relatively small, remarkable individual variation was noted. Therefore, the intraoperative calculation of the remaining stromal bed is recommended. Furthermore, the consideration of central corneal thickness, patient age, and preoperative keratometry are helpful parameters to avoid too deep ablation.

Abstract: Protein expression of Spodoptera frugiperda (Sf9) insect cells was characterized upon exposure to environmental stresses typically present in bioreactors including heat shock, oxygen deprivation, shear stress, change of pH, and salinity or ethanol shock. This study fills the void in knowledge as to how bioreactor hydrodynamics, anoxia, small changes in pH as well as salinity alterations due to pH control or exposure to ethanol used in asepsis treatments affect protein expression in Sf9 cells. Heat shock at 43 degrees C induced proteins at 83 kDa, 68-78 kDa and six small heat shock proteins (hsps) at 23-15.5 kDa. Anaerobic conditions in CO2 atmosphere reduced significantly the normal protein synthesis and induced a small subset of heat shock proteins at 70 kDa. Oxygen deprivation in nitrogen atmosphere transiently induces the 70 kDa proteins and had minor effects on the normal protein synthesis. Exposure to increased salinity or ethanol concentration failed to trigger the stress response, but may extensively inhibit the induction of normal proteins even though there was a negligible change in cell viability. Shear stress that had a major reducing effect on cell viability did not change the protein synthesis profile of Sf9 cells. Both long and short term exposures to small pH changes had negligible effects on protein synthesis.

Abstract: PURPOSE: We evaluated 8-year results of excimer laser photorefractive keratectomy (PRK) for myopia in terms of stability and late complications. METHODS: Ninety-two myopic eyes of 55 patients were treated with a single-step method using an Aesculap-Meditec MEL 60 excimer laser with a 5.0-mm ablation zone. Treated eyes were divided into three groups according to preoperative refraction: low myopes (< or = -6.00 D), medium myopes (-6.10 to -10.00 D), and high myopes (>-10.00 D). RESULTS: Change in myopic regression stabilized in all myopia groups within 12 months, although a small myopic shift occurred up to 8 years after PRK. Mean change in refraction between 2 and 8 years was -0.42 +/- 0.48 D for low myopes, -0.37 +/- 0.34 D for medium myopes, and -0.41 +/- 0.50 D for high myopes. The percentage of eyes within +/- 1.00 D of emmetropia 8 years after PRK was 78.3% in the low myopia group, 68.8% in the medium myopia group, and 57.1% in the high myopia group. One eye lost 2 lines of best spectacle-corrected visual acuity due to irregular astigmatism. In 13.0% of eyes, a residual trace corneal haze was observed, which had no effect on visual acuity. Apart from the loss of 2 lines of BSCVA in one eye, there were no other late complications during the study period. CONCLUSIONS: The mean change in refraction between 2 and 8 years was less than -0.50 D, regardless of preoperative refraction, and may be attributed to natural age-related refractive change. The appearance of residual corneal haze after 8 years correlated with the amount of myopic correction. PRK was a safe and stable surgical procedure in this group of patients.

Abstract: This study was undertaken to investigate the use of the in vitro test WST-1, an assay of cell proliferation and viability, for a preliminary safety evaluation of topical ophthalmic preparations. The cytotoxicity of two surfactants, benzalkonium chloride (BAC) and polyoxyethylene-20-stearyl ether (Brij78, PSE) was independently investigated in four laboratories in the EU by using an immortalized human corneal epithelial (HCE) cell line. The HCE cells were exposed to BAC and PSE for 5 min, 15 min, and 1 hour, and the results of the HCE-WST-1 tests were collected and compared. After one-hour exposure, the EC(50) values in BAC-treated cells in the presence of serum ranged between 0.0650 +/- 0.0284 (mean +/- SD) mM, and those in the absence of serum 0.0296 +/- 0.0081 mM. The corresponding values for PSE were 0.0581 +/-.0300 mM and 0.0228 +/-.0063 mM. There were variations in the results between different laboratories, with coefficients of variation ranging from 31 to 121%, mean 58%. The use of one-hour exposure time is to be preferred, and the elimination of serum in the culture medium is recommended to avoid both underestimation of toxic effects and variability of the test results.

Abstract: Alternatives to the Draize rabbit eye irritation test are currently being investigated. Because of morphological and biochemical differences between the rabbit and the human eye, continuous human cell lines have been proposed for use in ocular toxicology studies. Single cell-type monolayer cultures in culture medium have been used extensively in ocular toxicology. In the present study, an SV40-immortalised human corneal epithelial (HCE) cell line was characterised immunohistochemically, by using 13 different monoclonal antibodies to cytokeratins (CKs), ranging from CK3 to CK20. The results from the monolayer HCE cell cultures were compared with those from the corneal epithelium of human corneal cryostat sections. Previous studies have shown that the morphology of the HCE cell is similar to that of primary cultured human corneal epithelial cells, and that the cells express the cornea-specific CK3. In the study reported here, we show that the cell line also expresses CKs 7, 8, 18 and 19. These CKs are typically expressed by simple epithelial cells, and are not found in the human cornea in vivo. Therefore, the monolayer HCE cell line grown in culture medium does not express the CK pattern that is typical of human corneal epithelium. This should be taken into consideration when using HCE cell cultures in similar single cell-type experiments for ocular toxicology.