Análisis pretest grupo control y grupo experimental

Preliminary migration assays were carried out to ascertain whether MG63 cells and HOBs would migrate under Boyden chamber migration assay conditions and, if so, what the time course of migration would be. Each experiment was carried out 3 times with triplicates and ANOVA was used to test the statistical significance of results with a Dunnet’s post-hoc test. Figure 5(a) shows the migration of MG63 cells over a four hour time course towards plasma fibronectin. Cells were detected on the underside of the membrane at 0.5 hours, suggesting chemotaxis had already started to take place. Movement of MG63 cells continued throughout the time course with significantly more cells migrating at each time point when compared to uncoated controls (p<0.001). It was decided that future migration assays using this method would be carried out for two hours as at this time point many cells had migrated to the underside of the membrane but could still be counted easily under the microscope on all the membranes.

Figure 5(b) shows the migration of HOBs over a four hour time course towards plasma fibronectin. HOBs were not detected on the underside of the membrane until the two hour time point with a mean count of 16 cells.mm^ (SD 10.8) (p<0.001 when compared to uncoated controls). After four hours significantly more cells had migrated when compared to the uncoated controls (p<0.001) with a mean count of 212 cells.mm^ (SD 86).

Figure 5(a) - MG63 migration towards plasma fibronectin over a four hour time course

450 4(X) 350 - g 300 S ^ 250 = 2(X) 2 ^ 150 1(X) 50 0 * 0.5 0.5 uncoaied ! 2 2 uncoaied uncoaied Time (hours) 4 4 uncoaied

The mem brane underside o f B oyden cham ber m igration inserts w ere coated with plasm a

fibronectin. lO^cells/migration insert w ere plated out into the cham bers o f m igration

inserts, including uncoated controls. C ells w ere left to migrate for 0 .5 -4 hours and at each tim e point the ce lls m igrating to the underside o f the m igration assay insert w ere stained w ith Crystal V iolet and counted using a light m icroscope on 2 0 x m agnification. After 0.5 hours ce lls w ere detected on the underside o f the migration insert and migration continued throughout the four hour experim ental period. R esults w ere tested for statistical sig n ifica n ce using A N O V A and the D unnet’s post-hoc test (* p < 0 .0 5 , ***p< 0.001 when com pared to uncoated controls).

Figure 5(b) - HOB migration towards plasma fibronectin over a four hour time course

350 300 250 200 150 100 50 0

I

The mem brane undersides o f B oyden cham ber migration inserts w ere coated w ith type 1 collagen . lO^cells/m igration insert w ere plated out into the cham bers o f migration inserts, including uncoated controls. C ells w ere left to m igrate for 0 .5 -4 hours and at each tim e point the cells m igrating to the underside o f the migration assay m em brane w ere stained with Crystal V iolet and counted using a light m icroscope on 2 0 x m agnification. At tw o hours and four hours a sign ifican t number o f ce lls had m igrated w hen com pared with uncoated controls. R esults w ere tested for statistical sig n ifica n ce using A N O V A and the D unnet’s post-hoc test (* p < 0 .0 5 , ***p < 0.001 when com pared to uncoated controls).

Once the migratory behaviour of both HOBs and MG63 cells had been determined towards plasma fibronectin, further migration assays were carried out towards plasma fibronectin, type I collagen, 120kDa plasma fibronectin fragment (120kDa), 30kDa plasma fibronectin fragment (30kDa) and 45kDa plasma fibronectin fragment (45kDa) and an uncoated control. Each experiment was carried out three times with triplicates and ANOVA was used to test the statistical significance of results with a Durmet’s post-hoc test. Results are shown in figure 5(c). Migration of MG63 cells occurred towards plasma fibronectin, type I collagen and the 120kDa plasma fibronectin fragment with no migration towards the 30kDa and 45kDa plasma fibronectin fragments. Significantly more MG63 cells migrated in response to plasma fibronectin, type I collagen and the 120kDa plasma fibronectin than HOBs (p<0.001)

Figure 5(c) - MG63 and HOB migration towards ECM substrates 500 450 400 350 g 300 % 250 1200 I 150 100 50 0 *** f

_f

The mem brane underside o f B oyden cham ber m igration inserts w ere coated with plasm a fibronectin (pfn) and plasm a fibronectin proteolytic fragm ents (120k D a, 30kD a and 4 5k D a) and type I collagen (co l I). lO^cells/migration insert w ere plated out into the cham bers o f m igration inserts including uncoated controls. C ells w ere left to migrate for 0 .5 -4 hours and at each tim e point the c e lls m igrating to the underside o f the migration assay mem brane w ere stained with Crystal V io let and counted using a light m icroscope on 20x m agnification. Both M G 63 c e lls and H O B s m igrated in response to pfn, col and 120kD a with no migration tow ards 30k D a and 45kD a. M igration o f M G 63 ce lls w ere significantly greater than that o f H O Bs (*** p < 0 .0 0 1 w hen using A N O V A and D unnet’s post-hoc test).

5.4 Results - Migration assays with addition of integrin blocking