The animal protocol used in this study was reviewed and approved by the Pusan National University-Institutional Animal Care and Use Committee (PNU-IACUC; approval no. PNU-2015-0972). Adult male Sprague Dawley rats were purchased from Samtako BioKorea (Osan, Korea), and handled at the Pusan National University Laboratory Animal Resources Center accredited by the Korea Food and Drug Administration (accredited unit no. 00231) and AAALAC International (accredited unit no. 001525). All rats were provided with a standard irradiated chow diet (Purina Mills, Seoungnam, Korea) ad libitum, and were maintained in a specific pathogen-free (SPF) state under a strict light cycle (lights on at 06:00 h and off at 18:00 h) at a temperature of 23±2°C and a relative humidity of 50±10%.

An in vivo wound healing assay was developed, in which seven-week-old Sprague Dawley rats (n=20) were assigned to one of four groups: Vehicle (gauze, GZ)-treated group (n=5); Medifoam liquid bandage (MFLB)-treated group (n=5); LoBSLB-treated group (n=5); and HiBSLB-treated group (n=5). MFLB as a positive control bandage was purchased from Mundipharma (Seoul, Korea). Briefly, animals were anesthetized by intramuscular injection with Zoletile (50 mg/kg body weight) and Rompun (5 mg/kg body weight). Appropriate anesthesia of all rats was monitored by pedal reflex which clamps the tail or the transitional skin of the rat with forceps and corneal reflex that touches the cornea with watery cotton. After, the back skins were shaved with an electrical razor and cleaned with 70% ethanol. Using a biopsy punch (Kasco Co., Sialkot, Pakistan), a round wound of 8 mm diameter and 2–4 mm depth was created by removing the cutaneous tissue in the back shoulder region. The incision wound on each rat was sterilized with 70% ethanol, after which it was treated with GZ, MFLB, LoBSLB or HiBSLB. The pieces (5×4×0.3 mm) of GZ and the three types of LB were replaced every 4 days. During replacement, the condition of the skin wound was observed and photographed with a Canon^® digital camera, while the body weight was measured using an electronic balance (Mettler Toledo, Greifensee, Switzerland). After 12 days, all rats were euthanized using carbon dioxide (CO₂) according to the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition (gradual filling at verified 10–30% displacement rate/min). During this process, CO₂ gas was introduced into the 18.74 l euthanasia chamber at a flow rate of 3.7 l/min of (20% chamber volume/min). Successful euthanization in rats was confirmed by the measurement of several biological signals including heartbeat, movement, breathing and pupillary response to light. The samples of the damaged skin were collected for further histological and western blot analysis. Additionally, blood serum from the abdominal veins, and liver and kidney tissues were collected to analyze the toxicity of the two BSLBs.

Photographic data were used for measuring the wound size (%), which was calculated as follows:

Wound size (%) = WtW0

where Wt is the wound area at time ‘t’ and W₀ is the wound area at the initial time. A multiple comparisons test was performed for clarifying the statistical difference between groups.

At day 12, all animals were fasted for 8 h, after which blood was collected from the abdominal veins of rats and incubated for 30 min at room temperature. Serum was obtained by centrifugation the whole blood, and serum biochemical components, including alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA), were assayed using an automatic serum analyzer (Hitachi 747; Hitachi Ltd., Tokyo, Japan). All assays were measured in duplicate using fresh serum.

The site of application on the cutaneous wound region was collected and fixed in 10% formalin for 48 h, embedded in paraffin wax, and sectioned into 4 µm thick slices. The skin sections were subsequently stained with hematoxylin and eosin (H&E; Sigma-Aldrich; Merck KGaA) and examined by light microscopy for the presence of edema and inflammatory cell accumulation. Additionally, re-epithelialization and thickness of the epidermis were measured using the Leica Application Suite (Leica Microsystems, Wetzlar, Germany). The liver and kidneys collected from all experimental rats were processed using the same protocol applied to treat the skin tissue, and pathological changes were examined after H&E staining, using the Leica Application Suite (Leica Microsystems).

Granulated wound skin tissue were isolated from a subset of groups, homogenized using a PRO-PREP^™ Solution kit (iNtRON Biotechnology, Sungnam, Korea) supplemented with half tablet of a protein inhibitor cocktail (Roche Applied Science, Penzberg, Germany), and the mixture was centrifuged at 13,000 rpm for 5 min. The prepared proteins were then electrophoresed on a 10% SDS-PAGE gel, following which they were transferred onto a nitrocellulose membrane (Amersham Biosciences, Corston, UK) for 2 h at 40 V in transfer buffer (25 mM Trizma-base, 192 mM glycine, and 20% methanol). The efficiency of the transfer and equal protein loading were determined by staining the membrane with Ponceau, while the gel was stained with Coomassie blue (both Sigma-Aldrich; Merck KGaA). Appropriate dilutions of the following primary antibodies were added to the membranes and allowed to hybridize overnight at 4°C: Anti-VEGF (PeproTech, Rocky Hill, New Jersey, USA), anti-EGFR (Abcam, Cambridge, UK), anti-p-EGFR (Cell Signaling Technology, Inc., Danvers, MA, USA), anti-collagen-1 (Abcam), anti-AKT, anti-phospho-AKT (both Cell Signaling Technology, Inc.), and anti-β-actin (Sigma-Aldrich; Merck KGaA). After removing the antibodies, the membrane was washed three times in a solution composed of 10 mM Trizma-base (pH 7.6), 150 mM NaCl (both Sigma-Aldrich; Merck KGaA), and 0.05% Tween-20 (Biosesang, Gyeonggi-do, Korea), for 10 min. The primary antibody conjugated membranes were then incubated with horseradish peroxidase-conjugated anti-secondary antibody for 1 h at room temperature. The membrane was washed again as described above, and developed using an enhanced chemiluminescence detection system (Amersham Biosciences). Finally, the results were quantified using the Image Analyzer System (Eastman Kodak 2000MM; Kodak, Rochester, NY, USA), and the results are expressed as the fold-increase over control values. All results were confirmed by two independent researchers conducting the experiments at least twice.

Collagen distribution was detected by immunohistochemical analysis using light microscopy, as previously described (21). Briefly, the wound skin tissue samples were fixed in 10% formalin for 48 h, embedded in paraffin, and sliced into 4 µm thick sections. These sections were subsequently deparaffinized with xylene, rehydrated, and pretreated for 30 min at room temperature with PBS blocking buffer containing 10% goat serum. The sections were then incubated with primary anti-collagen-1 antibody (Abcam) or anti-p-EGFR (Cell Signaling Technology, Inc.) diluted 1:300 in PBS blocking buffer. The antigen-antibody complexes were subsequently visualized with biotinylated secondary antibody (goat anti rabbit)-conjugated HRP streptavidin (Histostain-Plus kit; Zymed, South San Francisco, CA, USA) at a dilution of 1:1,500 in PBS blocking buffer. Finally, collagen proteins were detected using a stable DAB (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and the Leica Application Suite (Leica Microsystems). Also, the distribution of p-EGFR protein in skin section was analyzed in the same way using anti-p-EGF receptor antibody.

One-way ANOVA (SPSS for Windows, Release 10.10, Standard Version; SPSS Inc., Chicago, IL, USA) followed by Tukey's post hoc test was used to identify significant differences between the Vehicle and the three LB-treated groups. All values are reported as the means ± SD, and P<0.05 was considered to indicate a statistically significant difference.

To determine the stimulatory effects of BSLB on the healing process of wounded skin, a spherical area measuring 8 mm in diameter and 2–4 mm in depth was evaluated for wounded skin covered with LoBSLB and HiBSLB for 12 days. The most significant changes observed in the wound area occurred between days 4–12. In the LoBSLB-treated group, the wound area decreased 79% on day 4, 43% on day 8, and 47% on day 12, when compared to the Vehicle-treated group. Similar results were also observed in the HiBSLB-treated group, with complete disappearance of the wound on day 12 (Fig. 2A and B). Furthermore, the decrease in wound area in the MFLB-treated group was similar to the LoBSLB-treated group (Fig. 2A and B). These results suggest that enhanced healing ability of the cutaneous surgical wound is reasonably attributed to BSLB treatment.

The alterations in tissue regeneration of the wounded skin was determined by the recovery of the epidermis, dermis and hyperdermis of the skin tissue of rats over the designated study duration (12 days). To measure the therapeutic effects of the BSLB on tissue regeneration in the surgical skin wounds, we measured the thickness of the epidermis and the width of re-epithelialization region in the Vehicle, MFLB, LoBSLB and HiBSLB-treated groups. We observed a significant increase in re-epithelialization in the LoBSLB and HiBSLB-treated groups relative to the Vehicle-treated group, with a greater increase rate in the LoBSLB group than the HiBSLB group (Fig. 3A). Additionally, a similar pattern was observed for the thickness of the total epidermis. After day 12 post-surgery, the thickness of the total epidermis was significantly enhanced by about 126 and 139% in the LoBSLB and MFLB-treated groups, respecrively, relative to the Vehicle-treated group; however, constant levels were maintained in the HiBSLB-treated group (Fig. 4A and B). To further determine if the therapeutic effects of BSLB were related with activation of EGFR, we examined for differences in the level of phosphorylation of EGFR in the wound skin of BSLB-treated groups. We observed a significant increase in levels in the HiBSLB-treated group, whereas other groups maintained a constant level (Fig. 3B). Also, a similar pattern was observed in the immunohistochemical analysis for p-EGFR (Fig. 3C). Taken together, these results suggest that BSLB treatment improves tissue regeneration and re-epithelialization in the skin tissue of Sprague Dawley rats through the stimulation of EGFR.

The effect of BSLB treatment on the angiogenesis signaling pathway in the dermis of wounded skin was evaluated by measuring the expression level of VEGF and a member of the downstream signaling pathway, in a subset of groups at day 12. On day 12, the expression of VEGF, an important signaling protein involved in both vasculogenesis and angiogenesis, dramatically decreased in the LoBSLB and HiBSLB-treated groups, as compared with Vehicle-treated group (Fig. 5). A similar decrease was observed in AKT phosphorylation, one of members of the VEGF downstream signaling pathway. The phosphorylation of AKT was lower in the LoBSLB and HiBSLB-treated groups, as compared to the Vehicle-treated group, on day 12. These effects were similarly observed in the MFLB-treated group (Fig. 5). These results therefore indicate that BSLB treatment induces the down-regulation of the angiogenesis signaling pathway during the late stage of the wound skin repair process by regulating the expression of VEGF and AKT phosphorylation.

To evaluate the therapeutic effects of BSLB on the formation of connective tissue in the wounded skin, the expression level of collagen was measured in a subset of groups at different times. We observed a rapid increase in the expression of collagen-1 in the LoBSLB and HiBSLB-treated groups (169 and 137%, respectively) compared with the Vehicle-treated group (Fig. 6A). The wound skin tissue stained for immunohistochemical analysis also showed a similar trend. The collagen protein was densely stained around the dermis of the LoBSLB and HiBSLB-treated groups relative to the Vehicle-treated group (Fig. 6B). These findings indicate that BSLB treatment induces an enhancement in collagen during the late stage of the wounded skin repair process.

The toxicity of BSLB toward the liver and kidneys of Sprague Dawley rats was evaluated by investigating modifications in the metabolic enzymes (in the blood serum) and histopathology (of liver and kidney tissues), using serum biochemical analysis and histological analysis, respectively. Liver toxicity analysis revealed no increase in the levels of the three liver toxicity indicators, specifically ALP, AST and ALT, in both the LoBSLB and HiBSLB-treated groups and Vehicle-treated group. Also, no significant pathological changes such as inflammation, necrosis, apoptosis, or fibrosis were observed in the liver tissue of BSLB-treated groups (Fig. 7). Results for kidney toxicity were similar to the liver toxicity results. The BUN and CREA levels in the serum showed no significant increase in the two BSLB-treated groups, regardless of the dose administered. Furthermore, no specific pathological symptoms, including degeneration and necrosis of the glomerulus and renal tubes, were detected in the kidney tissue of the BSLB-treated groups (Fig. 8). The above results provide strong evidence that treatment with BSLB for a short duration does not induce any significant toxicity in the liver and kidneys of Sprague Dawley rats.