Effect of Adaprev and G6P on actin cytoskelaton of rat flexor tendon fibroblasts.

<p>Rat flexor tendons were grown in DMEM containing 10% Fetal Bovine Serum (FBS) and then transferred to media without FBS before either having Adaprev or 600 mM G6P added to the media. Non treatment controls were maintained in DMEM without serum. Cells were maintained under the above conditions for up to 60 minutes and photographed at various times to record the cellular effects. At 15 minutes the cells in both groups show “stress shielding” of the actin cytoskeleton then at 60 minutes similar crenation effects upon both the Adaprev and G6P treated cells were observed.</p

Cell viability following increasing doses of M6P treatment.

<p>A. Live/Dead viability stain used to exhibit cell morphology and cell viability. Living cells uptake calcein AM (green) Ethidium homodimer taken up by dead cells (red). Untreated cells show spindle shaped morphology in confluence whereas treated cells with 50 mM retain their spindle morphology at 45 and 60 minutes of exposure. At 200 mM cells begin to lose their spindle morphology but continue to have cytoplasmic protrusions with evidence of crenation after 120 minutes of exposure. Cells treated with 600 mM showed fewer cytoplasmic protrusions with a considerable shielded appearance after 60 minutes and 2 hours. B. Quantification of the living (green) and dead (red) cells revealed the majority of cells were still viable after all treatments with no significant loss of cellular viability (p>0.05). No significant difference was found between the number of live cells or dead cells found between treatments, dosages or exposure times, except those observed in the negative control (methanol) (p>0.05). Error bars represent standard error of mean.</p

Effect of Adaprev on Rabbit flexor tendon breaking strength <i>in vivo</i>.

<p>No significant difference in breaking strength and tensile strength between treated and non treated flexor tendons (p>0.05). Error bars represent standard error of mean.</p

Phosphorylation of p38 by Adaprev and G6P.

<p>Both Adaprev and G6P significantly increased phosphorylation of p38 when compared with controls with peak activity between 15 and 60 minutes of exposure.</p

Effect of Adaprev on fibroblast migration (Chemokinesis).

<p>A. Control without 10% FBS. B. Control with 10% FBS. C. M6P with 10% FBS. D. G6P with 10% FBS. Concentric rings indicate 50 µm distance from centre with ten cells measured per treatment group. Note 100% of cells within 100 µm distance with no FBS. Note 70% of cells had migrated beyond 100 µm in DMEM and 10% FBS controls. Adaprev and G6P treatment resulted in a reduction of migration with only 20% and 30% of cells migrating beyond 100 µm.</p

Effect of M6P on tendon adhesion area and tendon structural organisation.

<p>A. Effect of M6P at increasing doses on adhesion area at 3 weeks (most synthetic period) and 8 weeks (end of tendon healing). B. Effect of M6P at increasing doses on tendon organisation as measured by quantitative polarisation. Effect of TGFβ-1 at 2.5 ng was used as a positive control but did not significantly increase adhesion formation or improve tendon organisation (p>0.05). All values of treated tendons were compared to contralateral saline controls. Unwounded controls were also measured for polarisation normalisation. Significant reductions in adhesion formation were seen in 50 mM M6P treated group at three weeks and in 600 mM M6P treated groups at eight weeks. Improvement in tendon organisation was significant with 600 mM M6P treatment. Significant differences with p<0.05 on paired t-testing are indicated by an asterisk.</p

Effect of Adaprev exposure time on fibroblast proliferation.

<p>Proliferation activity was normalised to untreated cells and treated cells expressed as a percentage of non-template controls. A. Exposure to Adaprev significantly reduced cell proliferation (p<0.05), which gradually recovered to normal levels in 15 minutes and 30 minutes exposure groups after 120 hours. Longer periods of exposure (45–120 minutes) showed a significant “lag phase” which gradually recovered to near normal levels at 168 hours B. Treatment with G6P showed similar “lag phase” kinetics. Error bars represent standard error of mean.</p

<i>In vivo</i> collagen synthesis and cell proliferation by immunohistochemistry.

<p>Representative samples were taken from 3 week injured flexor tendons. A. Sham treated flexor tendon injury stained with antibodies to Hsp47 (collagen synthesis marker). Arrow indicates adhesion. B Adaprev treated flexor tendon injury. C Quantitative analysis of Hsp47 in tendon and subcutaneous tissues showing no significant difference in cellular Hsp47 expression between the two groups. D. Sham treated flexor tendon injury stained with antibodies to BrdU (cellular proliferation marker). Arrow indicates adhesion. Note good staining of basal layer of skin, hair follicles and on the tendon surface. E. Adaprev treated flexor tendon injury. F. Quantitative analysis of if BrdU in tendon and subcutaneous tissues showing no significant difference in cellular BrdU expression between the two groups (p>0.05). Error bars represent standard error of mean. Scale bar represents 200 µm.</p