Control Contractions

A ceU-fi'ee baseline force was generated as shown in Figure 36a, peak contraction produced by the sponge itself was attained by 8 hours and registered 30 dynes. The rate o f contraction was greatest during the first 4 hours, during which cell-free collagen-GAG sponges contracted at 5.64dynes/hour. Rate o f contraction decreased rapidly over the remaining 16 hours (Figure 36b), resulting in a flattening o f the contractile profile. Such a baseline control was necessary to analyse data from subsequent experiments which employed cell-seeded collagen-GAG sponges. Contractile profiles o f all cell-seeded data was calculated and represented as ‘net force per million cells’, that is the cell-free baseline contraction was subtracted, providing contractile forces generated by the cells alone. Untreated human dermal fibroblasts seeded into the collagen-GAG sponge (Figure 36a) generated a net peak contraction o f 70 dynes (ie: 70 dynes plus 30 dynes due to the cell- free sponge). As with the cell-free baseline control, the initial rate o f contraction (0-4hrs) was rapid reaching 8.14 dynes/hour/million cells, after which it gradually fell to 1.10 dynes/hour/million cells (16-20hrs) (Figure 36b).

a 8 0 6 0 4 0 20 -20 20 time (hours)

-A- sponge aione Q Untreated

b

0-4 8 - C

Time (hours)

12-16 16-20

untreated sponge alone

Figure 36. Figure 36a represents cell-free baseline contraction (sponge alone, cell-free control) and untreated human dermal fibroblast contraction (untreated cell seeded control). This baseline contraction was subtracted from all subsequent cell seeded data in order to calculate and graphically present data as'net force per million cells’. Figure 36b shows the changes in rate of contraction for both cell-free and cell-seeded controls every 4 hours over the 20 hour period monitored.

Several other controls were used regularly throughout the experiments to exclude the effect which certain agents may have had on, in particular the contraction o f the collagen- GAG sponge matrix itself. These included testing the effect o f plasma fibronectin (used to aid cell attachment), vehicle (PBS), blocking antibody, blocking peptide, and depleted sera. Results show that application o f such agents to ceU-fi'ee coUagen-GAG sponges produced no change compared to baseline contraction (Figure 37). Effects of sodium azide (present in several of the antibodies used) on ceU-fi'ee and ceU-seeded coUagen matrices were also examined in order to eUminate the effect such a preservative may have had on contraction. Concentrations o f sodium azide appUed were equivalent to those present in the commercial antisera. Again no changes were observed in either ceU-fi'ee or ceU-seeded set-ups (Figure 38).

0 2 4 6 8 10 12 14 16 18 20

■ cell &ee

T im e (hours) ' cell free phis pFn

Figure 37. Figure 37 shows the effect o f pFn (used to aid ceU attachment) on the contraction o f cell fi-ee sponges. Results show that pFn application to ceU-fi'ee collagen- GAG sponges produced no significant change compared to baseline contraction.

s

I

-20

G 2 4 6 g 10 12 14 16 18 20

T ime (hours)

-Q- ceU free -V- c e n free p k is N a N 3 u n tr e a te d A ceDs tr e a te d with N aN 3

Figure 38. Application o f sodium azide (present in several o f the antibodies used) produced no change in cell free or cell seeded contractile profiles. Concentrations of sodium azide applied were equivalent to those present in the commercial antisera.

3.3.1.2 Contraction generated upon application of anti-aS pi-antibody

To study the role and nature o f integrin-matrix ligand binding during early stage contraction, a series o f inhibitors were added (competing antibodies to integrin and matrix components, and synthetic peptides). Initial studies examined the role the classic fibronectin receptor complex, a 5 p l had on contraction by the addition o f a suitable antibody (refer to Table 3). Several doses o f anti-a5pi-antibody (ranging from 0.2mg/ml to 0.005mg/ml) were added to the experimental system at 0 hours, and inhibited cell- mediated collagen contraction in a dose dependent manner (Figure 39a).

Doses o f 0.2mg/ml and 0.1 mg/ml anti-a5 pi-antibody elicited complete inhibition o f contraction for the full 20 hours. Rates attained using these concentrations remained inhibitory throughout (ie,: negative rates), and failed to register any contraction (Figure 39b). At all doses and in all replicates inhibition was maximal over the first 4 hours, after which there was negligible recovery. However, reduction o f the antibody concentration to 0.005mg/ml brought levels o f contraction back to those seen in untreated controls.

Contraction rates using this particular concentration of antibody was similar to controls, except for the first 4 hours. During this initial period the rate of contraction registered for the antibody treated sample (11.35 dynes/hour/million cells) was greater than that for the untreated control (8.14 dynes/hour/million cells).

a -20 -4 0 0 2 6 8 10 T im e (h o u rs) 12 14 16 18 20 4 * U n treated 0 2mg<ml A 0 Img/ml O 0 005m g/m l b 16-20 T im e (h o u rs) 0 kng/m l 0 0.005m g/m l ESI u n tre a te d 0.2m g/m l

Figure 39. Figure 39a represents contractile curves in response to various doses of anti- aSpl-antibody (against the whole FnR complex). Treatment using this antibody caused a complete inhibition of contraction, with recovery seen only at the lowest dose (0.005mg/ml). The typical contractile curve in the absence of this inhibitory antibody (ie,: untreated cell seeded control) is indicated by the red line. Figure 39b shows the rate of contraction registered over the 20 hours monitored.