Methods

MATERIALS PREPARATION

Sample discs (8 mm diameter) were punched from 0.5 mm thick sheet PMMA cast from polymer beads (ICI, London), polypropylene (Goodfellow, Cambridge), PTFE (Goodfellow, Cambridge), and silicone (Goodfellow, Cambridge). Additional PMMA discs were coated with PC1008 (2:1 copolymer of lauryl methacrylate and methacryloyl phosphorylcholine) (Biocompatibles, Farnham). Sample discs were washed sequentially with a 7X-PF detergent (ICN Pharmaceuticals, Hants) and distilled water. The coated discs were then air dried in sterile conditions for 10–15 minutes at room temperature before experimentation. Six discs of each material were used for each of the adhesion assays.

ADSORPTION OF FIBRINOGEN

I¹²⁵ labelled fibrinogen (ICN Pharmaceutical, Hants) was diluted (ratio 1:9) with unlabelled human fibrinogen (Sigma, Dorset) in 12.6 ml of phosphate buffered saline (PBS). A total of 25.2 ng of fibrinogen in 40 μl of solution was pipetted onto each disc in a 24 well plate and incubated at room temperature for 2 hours. The quantity was chosen to enable mid-range readings in the scintillation counter. The discs were then washed (×3) in PBS, transferred to scintillation vials and submerged in scintillation fluid. The scintillation count for each vial was then measured over 1 minute in a liquid scintillation counter (Model 1209 Rack Beta, LKB Ltd, USA).

ADHESION OF FIBRIN

Fresh venous blood (20 ml) was obtained from a healthy adult male volunteer in a citrated vial to prevent clotting. The blood was centrifuged for 10 minutes at 3000 rpm to obtain plasma. Plasma 0.1 ml was diluted with 1 ml of normal saline; 0.1 ml of 0.025M calcium chloride solution was then added to this mixture to initiate clotting; 0.2 ml of this solution, which covered the entire surface of the disc, was immediately pipetted onto centre of each sample disc. These discs were incubated for 2 hours at room temperature to allow the formation and adherence of fibrin, before being washed (×3) in PBS, and transferred to a sterile 24 well plate. Fibrin deposits were dissolved by adding 0.2 ml of 4M guanidine thiocyanate into each well with constant agitation (30 rpm) for 1 hour at room temperature. The tyrosine residue in 0.1 ml of this solution was measured as a quantification marker for dissolved fibrin using a spectrophotofluorometer (Model 7687, American Instrument Company, MD, USA) (excitation wavelength 275 nm; emission wavelength 310 nm).

ADHESION OF SCLERAL FIBROBLASTS

Eye bank scleral rim tissue from adult donors (Moorfields Eye Hospital Eye Bank) was cultured to produce a strain of scleral fibroblasts in Eagle’s minimal essential medium (MEM) (Gibco, Life Technology Ltd, Paisley) supplemented with 10% fetal calf serum, 1% penicillin, and 1% streptomycin. Cells were serially passaged to 6–7 population doubling by standard trypsin-EDTA dispersion. The fibroblasts were then seeded in MEM at a density of 6000 cells/cm² into wells containing the sample discs, and incubated at 37°C under 5% carbon dioxide to allow attachment for 48 hours. This cell density means that the total number of cells delivered was higher than required to cover the entire surface of the disc. The discs were then washed (×3) in PBS and transferred to a new 24 well plate. Intracellular ATP was liberated by incubating each disc with 100 ml of sterile hypotonic lysis buffer (0.01M TRIS-acetate pH 8, 2 mM EDTA). After 30 minutes the ATP solution was diluted 1:1 with an additional buffer (0.2M TRIS-HCl, 0.2 mM EDTA, pH 7.3). ATP levels in this buffered lysate were then measured as a quantification marker for the adherent fibroblasts by ATP dependent luminometry, using a commercial luciferase test kit (BioOrbit-Wallac, Turku, Finland) and a 96 well plate luminometer (Amersham, Bucks).

MACROPHAGE ADHESION

Mononuclear cells were purified using commercial gravity separation media as described below. Fresh venous blood was obtained from a healthy 32 year old male volunteer. The blood was defibrinated by agitation in a vial containing sterile glass beads for 5 minutes. The blood was then diluted 1:1 with macrophage serum free medium (RPMI1640 medium, Gibco, Life Technology Ltd, Paisley) and slowly pipetted into a tube containing a purification medium (LymphoSep, ICN-Pharmaceuticals, Hants). The mixture was centrifuged at 400g for 20 minutes to separate the cells. The central layer containing the mononuclear cells was sampled. The mononuclear cells were then pelleted by a further centrifugation at 400g for 10 minutes. The mononuclear cells were resuspended in RPMI1640 medium and counted using a haemocytometer. Sample discs were placed in a 24 well tissue culture plate and 2×10⁵ cells in 0.5 ml of media are pipetted onto each disc in sequence. The plates were incubated for 24 hours in a 37°C incubator under 5% carbon dioxide. The discs were then washed (×3) with PBS and fixed in 4% paraformaldehyde for 10 minutes at room temperature. The fixed cells were then permeabilised by incubation with a 1% solution of Triton X100 (Sigma, Dorset) for 2 minutes, and macrophages detected using the protocol described below using an avidin-biotin-peroxidase kit (RapidStain, Sigma, Dorset).

Endogenous peroxidase activity in the cells was quenched by 72 hours’ incubation in 9% hydrogen peroxide and repeatedly washed in PBS. Mouse anti-human macrophage (CD68) monoclonal antibody (Dako Ltd, Bucks, diluted 1:100 in PBS + 1% fetal calf serum) was added to each disc and allowed to incubate overnight at 4°C in a humidified chamber. The discs were then washed (×3) in PBS and macrophages observed using a high affinity biotin conjugated anti-mouse antibody and an avidin-peroxidase detection system using AEC as a chromogen. The average number of macrophages (stained red) was determined in 50 randomly selected fields under light microscopy by a second “blind” observer.

DATA ANALYSIS

Results are presented graphically as box plots showing the 10th, 25th, 50th, 75th, and 90th centiles as vertical boxes with error bars. Statistical analysis was performed using spss for Windows software (SPSS Inc, Chicago, IL, USA). The Mann–Whitney non-parametric test was used to detect any statistically significant differences between uncoated PMMA and the other test materials.