IN VITRO EVALUATION OF ADHESIVES 3.1 BOVINE EYE CUP EXPERIMENTS:

The following adhesives remained adherent to the retinal surface of bovine eye cups under the test conditions and when applied to the dried retinal surface of bovine eye cups containing no media:

1. all the cyanoacrylates with or without lophendylate

2. the Tisseel fibrin preparation (both 4 and 500 lU/ml strength thrombin)

3. one of the mussel adhesive proteins (synthetic protein 2, using iron sulphate as the cross linker)

Fibrin preparations 1,2 and 3 formed clots which did not adhere to bovine retina and fibrin made from rabbit plasma gave inconsistent results; 0% adherence using 4 lU/ml thrombin, 25% adherence using 20 lU/ml thrombin and 50% adherence using 500 lU/ml thrombin.

None of the adhesives investigated adhered to the retina of bovine eyes filled with media. This occurred for several reasons:

1. some of the adhesives had a lower density than the media and floated away from the retinal surface (all the cyanoacrylate preparations with or without

lophendylate)

2 . they could be delivered to the retinal surface but would not adhere (medical adhesive silastic type A, medical adhesive 355)

3. aqueous preparations dispersed (fibronectin, laminin, Matrogel)

4. the multicomponent adhesives with an aqueous component also dispersed (fibrin preparations, mussel adhesive protein, polyacrylic acid and poly-F plus)

3.2 TISSUE CULTURE TOXICITY STUDIES:

These investigations were undertaken using Histoacryl, octylcyanoacrylate and Tisseel, as these adhesives adhered to the dried retinal surface of bovine retina.

3.2.1. Cyanoacrylates:

1^ Histoacryl and octylcyanoacrylate added to RPE and glia:

Morphological changes were observed in RPE and glial cells adjacent to the adhesive as early as one hour after application - cells had retracted and rounded up. At three hours these changes were more pronounced (Figure 3.1). At 24 hours cultures containing Histoacryl demonstrated three concentric zones around the adhesive (Figure 3.2, 3.3). Immediately adjacent to the adhesive cells appeared to have been "fixed". Surrounding this zone was a cell free zone, where cells had died and separated from the flask, leaving cytoskeletal remnants (Figure 3.3). The third zone consisted of rounded, damaged cells while cells at a further distance from the adhesive appeared normal. At 24 hours flasks containing octylcyanoacrylate did not show a zone of fixed cells and had a much narrower cell free area (Figure 3.4). Over the next few days the area of cell death increased in flasks to which Histoacryl had been added, RPE cultures reaching a maximum by day 7 and glia by day 2. The area of cell death did not increase in RPE cultures to which octylcyanoacrylate had been added, but did increase slightly by the second day in glial cultures (Table 3.1). The maximum area of cell death surrounding Histoacryl (glia, 390 +/- 136 mm^ and RPE 339 +/- 53 mm^) was significantly different from that surrounding octylcyanoacrylate (glia 20 +/- 14 mm^ and RPE 11 +/- 4 mm2) for both cell types (Anova at p = 0.05)(Figure 3.5). Although the maximum area of cell death surrounding glia with each adhesive was greater than that for RPE, this did not reach significance (Anova at p = 0.05).

Over the next few days the "fixed" cells adjacent to the drops of Histoacryl separated from the flask. Healthy cells from the edge of the cell free zone began to migrate into and repopulate the cell free area (Figure 3.6, 3.7). After 5-7 days cells were also seen to be dividing in this area. This occurred with both cell types and in flasks containing both types of cyanoacrylate. Eventually cells reached the edge of the adhesive. SEM of glial cultures to which octylcyanoacrylate had been added showed cells migrating onto the adhesive (Figure 3.8). A plot of the rate of repopulation of the cell free area for both types of cells and adhesives was undertaken by calculating at each time interval the area of cell death as a

percentage of the area of maximum cell death (Figure 3.9). There was no significant difference between the rate of repopulation by cell type or adhesive type.

2^ Octylcyanoacrylate with and without lophendylate and lophendylate alone added to RPE and glial cultures:

When lophendylate alone was added to glial and RPE cultures no morphological changes were observed in either cell type, with no evidence of cell toxicity or death. Small droplets of lophendylate were visible of the surface of some cells.

The maximum area of cell death was recorded at 24 hours in all cultures except those where octylcyanoacrylate with lophendylate had been added to RPE cells; here the maximum area was recorded on day 4 (Table 3.2 and Figure 3.10). The maximum area of cell death was significantly greater in flasks containing lophendylate mixed with octylcyanoacrylate, and this was true for both cell types (glia, 227 +/- 38 mm^ compared to 125 +/- 60 mm^; RPE 99 +/- 37 mm^ compared to 54 +/-14 mm2)(Anova at p = 0.05). This was particularly true for glia (p less than 0.05, Students t test) compared to RPE (p = 0.05). This experiment confirmed the findings of the previous experiment that glia are more sensitive to the toxic effects of cyanoacrylates than RPE and this time the findings were significant whether lophendylate was added or not (p = 0.001, t test).

3) Histoacryl added to human RPE:

This was a non quantitative experiment in which the same morphological changes and evidence of cell death were observed as for bovine cells.

4) Histoacryl and octylcyanoacrylate with and without lophendylate added to glia grown on glass:

In this experiment only one measurement was made at six hours and the findings of this study were at variance with the previous experiment. The effect of mixing lophendylate with Histoacryl significantly reduced its toxic effect as the area of cell death surrounding Histoacryl was much greater than that surrounding the mixture (346 +/- 51 mm2 compared to 79 +/- 3 mm^) (t test, p = 0.014)(Table 3.3 and Figure 3.11). Reduced toxicity was not seen however, when lophendylate was mixed with octylcyanoacrylate; there was a slight but insignificant increase in toxicity (84 +/- 15 mm2 compared to 115 +/- 30 mm^) (Anova at p = 0.05). Again, Histoacryl alone was more toxic than octylcyanoacrylate alone (t test, p = 0.018).

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Figure 3.1 Light micrograph of bovine retinal glial cells in tissue culture

A Confluent culture of bovine retinal glia, passage 7, before adding 2pL of

Histoacryl adhesive (Magnification x 250). B Same culture as above 3 hours after adding Histoacryl. Area immediately adjacent to adhesive. Cells are rounding up and separating from the flask (Magnification x 250).

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Figure 3.2 Light micrograph of bovine retinal glial cells

A 24 hours after adding 2pL of Histoacryl. A zone of cells immediately adjacent to