Figure 1.
Photographs of a wound on Day 6 in a db/db mouse treated with PDGF-BB.
Area wound closure was calculated as original wound area on Day 0– open wound area on Day ‘X’ (area inside inner circumference indicated by a red line). Area wound contraction was calculated as original wound area on Day 0– area including open wound and new epithelium on Day ‘X’ (area inside outer circumference indicated by a blue line).
Figure 2.
Histopathological evaluations of a wound bed from a db/db mouse treated with PDGF-BB for 10 days and harvested 11 days after wounding.
Scale Bars = 500 µm. (A) Measurement of epithelial gap (dotted black line) and reepithelialization (dotted red lines). The epithelial gap was defined as the distance between the advancing edges of keratinocyte migration measured in millimeters. Length of reepithelialization was defined as the length of the layer of proliferating keratinocytes covering the wound area. This value was obtained by measuring s the distance between the free edge of the keratinocyte layer and the base where the cells were still associated with native, non-affected dermal tissue (*). The black vertical lines denote the area of separation between the pre-existing collagen and wound bed. The final value was the sum of distance in millimeters of both sides. H&E staining. (B) Inflammatory infiltrate in the wound bed. The inflammatory response was assessed using a semi-quantitative scoring system ranging from 0 to 4 where 0 indicates no inflammation, 1 indicates 0–25% of the wound area affected, 2 indicates 25–50% of the wound area affected, 3 indicates 50–75% of the wound area affected, and 4 indicates >75% of the wound area affected. This sample was classified as score 2. H&E staining. (C, D and E) Measurement of the fibrovascular dermal proliferation in the wound bed. Picrosirius red staining. (C) The wound bed area is outlined (green lines), comprising a preset depth of 0.75 mm (this is the average depth of the wounds in the experiment) and the borders between preexisting dermal collagen and newly formed collagen. (D) Under polarized light the bright collagen fibers of the wound bed are highlighted and automatically measured by the software (E), and the final data is expressed as a percentage of outlined wound area.
Figure 3.
Soluble PDGF-BB promotes proliferation of primary foreskin fibroblasts over 5 days, and can be attenuated by blocking PDGF beta receptors.
(A) A trimodal dose dependent response was observed with peak proliferation at 7.5 ng/ml, 7.5 and 50 µg/ml. Results are Mean ± StDev, n = 6. (B) Cell proliferation over 5 days was inhibited for all concentrations of PDGF-BB when PDGF-B receptor in cells were blocked with 10 µg/ml of PDGF beta receptor antibody for 30 min at 37°C prior to cell seeding. Results are Mean ± StDev, n = 6. * P<0.05, *** P<0.001.
Figure 4.
PDGF-BB did not accelerate wound closure, contraction or reepithelialization in the mouse splinted full thickness dermal wound model.
Percentage wound closure (A) and contraction (B) as mean ± SEM in full thickness splinted wounds of db/db mice treated with 0.01% PDGF-BB or vehicle control. Wound area was measured from photographs taken immediately after wounding, and then the area including open wound plus new epithelium was measured from photographs taken day 2, 4, 6, 8, 10, and 11. Percentage wound contraction and closure was calculated as described in methods. Epithelial gap (C) and reepithelialization (D) were measured by histopathological evaluation. All data were expressed as mean ± SEM. No significant differences between groups were found in any comparision.
Figure 5.
PDGF-BB did not modulate collagen deposition and inflammation development at the wound site in the splinted full thickness dermal wound model in mice.
Percentage collagen content (A) and inflammation score (B) as mean ± SEM in full thickness splinted wounds of db/db mice treated with 0.01% PDGF-BB or vehicle control. No significant differences in percentage collagen content or inflammation score were found (P>0.62).