Effects of hydrocellular foam dressing on wound healing in rat skin.

<p>Gross observations (A) and the relative wound areas (B) reveal increased wound contraction induced by hydrocellular foam dressing. Bars are expressed as mean ± SE (n = 4). *<i>p</i> < 0.05 indicates values that are significantly different from the side of film dressing in rat skin.</p

Loading apparatus to apply sustained compressive loading to cells seeded-collagen sponge.

<p>In this representation A indicates an indenter (diameter: 10 mm), B the weights, C a stainless steel plate (thick: 5 mm), D a 12-well plate lid, E culture medium, and F a fibroblast-seeded collagen sponge sample (diameter: 5 mm and thick: 3 mm).</p

Sustained compressive loading did not induce apparent cell proliferation and induced apoptosis through disruption of adhesion.

<p>Fibroblasts were seeded to collagen sponge and incubated for 24: Collagen sponge samples after loading were transferred to new 12-well plates in 1 ml medium containing 100 µl WST-1 reagent per well, and then incubated for 1.5 h. The absorbance of 450 nm was measured. The cell number was shown relative to base line. The results are represented as the mean ± SEM (error bars) of five experiments. D, E, and F: Collagen sponge samples were fixed, dehydrated, cleared, and processed for embedding in paraffin after loading experiments. Sections were prepared at 4-µm thick. Apoptosis assay were performed by TUNEL stain using tissue slides. The number of TUNEL-positive cells was counted in 5 fields in the central area of the collagen sponge (magnification ×10), and the proportion of positive cells to total cells was calculated. The results are represented as the mean ± SEM (error bars) of five experiments. Statistical analysis was performed using the Dunnett’s multiple test: between 2, 4, or 6 h group and 0 h group (A, B, D, and E) or between each of loaded group and nonloaded group (C and F). Statistical significance was taken as <i>p</i><0.05. A value of <i>p</i> was expressed as: *; <i>p</i><0.05, **; <i>p</i><0.01, and ***; <i>p</i><0.001. G, H, and I: H&E staining for confirming cell morphology. Collagen sponge samples were prepared as aforementioned. Sections were prepared at 5-mm thick. The distinctive cell morphology observed in the 6 h–0 mmHg group was spindle-shaped cells (G), whereas that in the 6 h–200 mmHg group was nonspindle-shaped cells (H) along with apoptotic bodies (I). J and K: Immunostaining for the FA structural protein vinculin (green). Nucleus stained by DAPI (blue). Vinculin expression was observed in the 6 h–0 mmHg group (J), whereas it was scarcely observed in the 6 h–200 mmHg group (K). L and M: Immunostaining for actin stress fibers by phaloidin (red). Nucleus stained by DAPI (blue). Actin stress fibers were observed in the 6 h–0 mmHg group (L), but not in the 6 h–200 mmHg group (M). Scale bars = 20 µm for all images.</p

<i>Cd44</i> and <i>Has2</i> were upregulated by 6-h compressive loading, but HA binding proteins and hyaluronidase gene expression were not.

<p>Fibroblasts were seeded to collagen sponge and incubated for 24(□) or 200 mmHg (▪) compression for 6 h. Total mRNA was extracted after WST-1 assay, and mRNA expression was assessed using real-time RT-PCR. The expression of the target genes in the 6 h–200 mmHg group relative to the value in the 6 h–0 mmHg group was calculated by the comparative Ct method using the 18S ribosomal RNA gene as an internal control. The results are represented as the mean ± SEM (error bars) of five experiments. Statistical analysis was performed using the Student’s t test between the 0 mmHg group and the 200 mmHg group, and statistical significance was taken as <i>p</i><0.05. A value of <i>p</i> was expressed as: *; <i>p</i><0.05, **; <i>p</i><0.01, and ***; <i>p</i><0.001. A: <i>Cd44</i>. B: <i>Has2</i>. G: <i>Cox2</i>. J: <i>Vcan</i> and <i>Tnfaip6</i> are HA binding proteins. <i>Hyal1</i>, <i>2</i>, and <i>3</i> are HA degrading enzyme. C and D: Immunostaining for CD44. Representative sections of (C) the 6 h–0 mmHg group and (D) the 6 h–200 mmHg group. E and F: Immunostaining for HAS2. Representative sections of (E) the 6 h–0 mmHg group and (F) the 6 h–200 mmHg group. H and I: Immunostaining for COX2. Representative sections of (H) the 6 h–0 mmHg group and (I) the 6 h–200 mmHg group. Scale bars = 20 µm for all images.</p

Effects of hydrocellular foam dressing on CD44 mRNA levels in periwound epidermis.

<p>CD44 mRNA levels in periwound epidermis were measured by quantitative PCR and expressed as values relative to those of GAPDH. Bars are expressed as mean ± SE (n = 5). *<i>p</i> < 0.05 indicates values that are significantly different from the side of film dressing in rat skin.</p

Effects of hydrocellular foam dressing on PPARα mRNA levels in periwound epidermis.

<p>The PPARα mRNA levels in periwound epidermis were measured by quantitative PCR and expressed as values relative to those of GAPDH. Bars are expressed as mean ± SE (n = 5). *<i>p</i> < 0.05 indicates values that are significantly different from the side of film dressing in rat skin.</p

Effects of hydrocellular foam dressing on level of hyaluronan and Has3 mRNA expression in periwound epidermis.

<p>Hyaluronan levels in periwound epidermis (A) were measured using a QnE hyaluronic acid ELISA assay. Has3 mRNA levels in periwound epidermis (B) were measured by quantitative PCR and expressed as values relative to those of GAPDH. Bars are expressed as mean ± SE (n = 4, 5). *<i>p</i> < 0.05 indicates values that are significantly different from the side of film dressing in rat skin.</p

Full-thickness cutaneous wound model.

<p>Two full-thickness wounds, a diameter of 1.5 cm, were created in the dorsolateral skin of five rats using sterile scissors. Each wound was covered with either a hydrocellular foam dressing (A) or the film dressing (B). A film dressing was used as a secondary dressing in both sides of individual rat and also used to keep the hydrocellular foam dressing attached to the wound.</p

The secretions of HSP90α, HA, and PGE₂ into cell culture medium were increased by compressive loading.

<p>Fibroblasts were seeded to collagen sponge and incubated for 24(A: HSP90α, B: HA, C: PGE₂) was measured by ELISA. A value of concentration was normalized by WST-1 value. The results are represented as the mean ± SEM (error bars) of five experiments. Statistical analysis was performed using the Dunnett’s multiple test between non-loaded group and each of loaded group, and statistical significance was taken as <i>p</i><0.05. A value of <i>p</i> was expressed as: *; <i>p</i><0.05, **; <i>p</i><0.01, and ***; <i>p</i><0.001.</p

Participant characteristics (n = 13).

<p>Participant characteristics (n = 13).</p