The present study was approved by the ethics committee of the Hellenic State Veterinary Authorities on animal care and use. Twenty-four healthy purpose bred, adult mixed breed dogs 1~4 years old were used (10 castrated males and 14 spayed females). A complete physical examination, complete blood count, serum biochemical analyses, and urinalysis were performed on each dog before the study commenced. The animals were housed in indoor individual runs and had outdoor access twice daily. Commercial dry maintenance diets were given twice daily and water was available ad libitum.

Following the creation of skin defects on both forearms, the animals were randomly divided in three groups of eight dogs each. In group A, skin staples were used for skin stretching. In group B, the needle of an intravenous catheter was inserted along each side of the wound. In group C, a Pavletic skin-stretching device was applied.

The dogs were premedicated with acepromazine (0.05 mg/kg, im, Acepromazine; Alfasan International, The Netherlands) and butorphanol (0.1 mg/kg, iv, Butadors; Richter Pharma, Austria). Anesthesia was induced with 2.5% sodium thiopental (8 mg/kg, iv, Pentothal; Abbott Laboratories, USA) and maintained with isoflurane (1~2%, Isoflo; Abbott Laboratories) in oxygen (1.5 L/min). Preoperatively, cefuroxime (20 mg/kg, iv, Zinacef; GlaxoSmithKline, UK) and carprofen (2 mg/kg, iv, Rimadyl; Pfizer, USA) were administered. Both forelimbs were clipped from the middle of the humerus to the carpus and prepared for aseptic surgery.

On day 1, one skin defect was created by a No. 10 blade on the craniolateral surface of each mid-forearm (Fig. 1). The defect had a length of 5 cm and a width ranging from 3.5~7 cm in order to be equal to 50% of the circumference of the mid-forearm. Undermining of the wound edges at a distance of 2 cm in left extremity was performed. For group A (Fig. 2), skin staples (Proximate plus MD; Ethicon, USA) in groups of two or three were placed circumferentially and perpendicular to the skin edges [9]. The groups of staples were placed 1 cm apart from each other and 0.5 cm away from the wound edge. Simple interrupted polypropylene 0 sutures (Prolene; Ethicon) were passed through the staples on each opposing side of the wound and tied over a cylinder (made from polypropylene thoracic tube cases by cutting them into 10 cm sections and by creating side holes) after passing through the side holes. The cylinder was placed over the wound on top of a sterile non-adherent, semi-occlusive pad (Melolin; Smith & Nephew, UK). For group B (Fig. 3), the needle of an intravenous catheter (catalogue no. 16; Abbott, Ireland) was inserted along each wound side approximately 0.5 cm from the wound edge [1,25]. Simple interrupted polypropylene 0 sutures were passed around the needles on each opposing side of the wound and tied over a cylinder as in group A. For group C (Fig. 4), a Pavletic skin-stretching device (X Banders; M.M. Pavletic, USA) was applied [23,24]. Specifically, three adherent skin pads of the device, measuring 0.5 × 5 cm, were placed next to each other on opposite sides of the defect and attached to the skin by using the cyanoacrylate glue and simple interrupted polyamide 3/0 sutures. The adjustable elastic straps engaged the pads of one side of the defect to the pads of the opposing side.

During suture tightening, tension was measured with a special device that consisted of a dynamometer attached to haemostatic forceps (Fig. 5) provided by the Centre for Research and Technology of Greece. Care was taken to not exceed a limit of 3.5 kg [15]. Tips of the forceps were passed through the suture loops on opposite wound sides and the force required to pull each side of the defect to the midline was measured [19]. Thereafter, the wounds were bandaged with sterile non-adherent, semi-occlusive pads (Melolin; Smith & Nephew) and Elizabethan collars were placed on the dogs. Under general anesthesia, dogs in groups A and B underwent three cycles of intermittent skin stretching (cycle loading) 24 h after creation of the defects (day 2). Forces of 1.5~2 kg were applied for 2~3 min followed by 1-min periods of relaxation [25]. The sutures were tightened once again and tension was measured. On day 3, the skin stretchers were removed with the canines under general anesthesia. For group C, cycle loading was not performed. The elastic strapsattached to the pads of one wound side were stretched prior to engaging the pads of the opposing side. The straps were adjusted every 24 h for a total of 48 h.

On day 3 (48 h after creation of the defects) following removal of the stretchers, primary closure of the skin wounds was performed for groups A and B (Fig. 6) but not group C. Closure was achieved in a two-layer fashion using polyglecaprone 3/0 (Monocryl; Ethicon) in a continuous pattern and polypropylene 2/0 in a simple interrupted pattern. All wounds were dressed using the bandage pads mentioned above. The bandages were changed daily until suture removal on day 15 (groups A and B) or wound healing (group C). Postoperatively, all animals received cefuroxime (20 mg/kg, im, bid, Zinacef; GlaxoSmithKline) for at least 10 days and carprofen (2 mg/kg, per os, bid, Rimadyl; Pfizer) for 5 days.

Following primary wound closure in groups A and B, and wound healing in group C, healing was clinically scored daily until suture removal (day 15). The following modified scale was used: 1 = no visible reaction; 2 = minimal swelling or erythema; 3 = suture line inflammatory reaction at least 1-cm thick with pain or redness; 4 = seroma or abscess formation, and 5 = dehiscence, skin necrosis, or impossible primary closure [10]. Scoring was performed by a clinician (M. Karayannopoulpou) who was not aware of which stretching devices had been applied in each group.

Cutaneous microcirculation was assessed by laser-Doppler flowmetry (LDF). The animals were acclimatized to the study room (20°~22℃) at least 30 min before anesthesia [26]. LDF measurements were performed under general anesthesia using the same protocol as for wound creation, with the dogs' body temperature ranging between 38~39℃. A laser-Doppler velocimeter (Periflux 4001 Master; Perimed, Sweden) with a standard right angle probe was used. Blood perfusion was assessed (Fig. 1) at six predetermined sites (three on the frontal and three on the caudal side of the defect at the upper, middle, and lower part and at a distance of 0.5 cm from the skin stretchers). LDF was performed just before creation of the defect (day 1), 24 h after defect creation, prior to cycle loading and suture tightening (day 2), and 48 h after wound creation (day 3). Each time, five separate readings were recorded at 5-min intervals to obtain a mean value for proper interpretation [26]. Values are expressed in perfusion units (PU).

Samples for histological evaluation were taken on the day of wound creation (from the excised skin; control specimen), on day 3 (following primary wound closure), and at the time of suture removal (day 15). Specimens were obtained using a 6-mm biopsy punch (Biopsy punch; Jørgen Kruuse, Denmark) while the dogs were under general anesthesia as previously described, at a distance of 0.5 cm from the wound margin or suture line (day 3 and 15). The specimens were pinned to a flat cork surface and fixed in 10% neutral-buffered formalin. They were labeled so that the pathologists (D. Psalla, N. Papaioannou) were not aware of the animal number, biopsy day or group corresponding to the specimen. After routine processing (dehydration in a series of alcohols, clearing in xylene and paraffin embedding), the samples were cut into sections 4- to 6-µm thick and stained with Hematoxylin-Eosin (H&E; Merk, Germany) along with Van Gieson stain (Merk) for elastin.

The stained sections were microscopically examined. The degree of cellular infiltration (neutrophils, eosinophils, macrophages, lymphocytes, and plasma and mast cells), edema, collagen orientation, and thickness of the epidermis were evaluated using the following criteria for scoring. For cellular infiltration, sparsely scattered inflammatory cells arranged in a random fashion in the dermis were considered normal (score 0). Detection of three to 10, 11 to 30, or ≥ 31 cells per high power field (400 ×) in the wound tissue was considered mild (score 1), moderate (score 2), or substantial (score 3) infiltration, respectively [13]. Absence of cells separating from collagen was considered a lack of edema (0), slight separation was considered mild edema (1), a separation of 30 to 50 µm was considered moderate edema (2), and a separation of > 50 µm was considered substantial edema (3) [13]. Orientation of collagen bundles (the number of bundles that ran parallel to the skin surface) was graded according to the following semi-quantitative scoring system [28]: orientation exclusively perpendicular to the skin surface (0), orientation predominantly perpendicular to the skin surface (1), random orientation (2), orientation predominantly parallel to the skin surface (3), and orientation parallel to the skin surface (4). Epidermal thickness was evaluated relative to that of normal epidermis of the excised skin on day 0 (control specimen) and assigned a score of 0 (thickness similar to that of normal epidermis), 1 (slightly decreased thickness), 2 (moderately decreased thickness), and 3 (substantially decreased thickness).

Data are expressed as the mean ± standard deviation (SD). Normality of the data distribution was assessed with a Shapiro-Wilk test. To assess any statistically significant differences, variables were compared among all groups as well as between undermined and non-undermined wounds at each time point or between measurement times in each group. A general linear model for repeated measures (for tension and LDF) or the Wilcoxon paired-sample ranks test (for histological parameters) was used to make these comparisons. P values ≤ 0.05 were considered significant. All statistical analyses were performed using SPSS software (ver. 15.0; SPSS, USA).

Mean values for tension in all groups measured on days 1 and 2 are presented in Table 1. Irrespective of measurement time, statistically significant differences in tension values (p < 0.0005) were found among all groups. The lowest and highest mean values were recorded for groups B and C, respectively. Irrespective of group, significantly decreased tension was observed on day 2 compared to day 1 in both undermined (p = 0.001) and non-undermined (p < 0.0005) wounds.

The mean clinical scores for the undermined (left limb) and non-undermined (right limb) wounds during the healing period (from wound closure on day 3 to suture removal on day 15) in groups A and B as well as group C (although primary wound closure was impossible) are presented in Table 2. Wound healing seemed to be better, although not significantly, in group B compared to group A. Overall, no significant differences in clinical scores were observed between the undermined and non-undermined wounds.

Mean blood perfusion values at the six different sites during the examined period in undermined and non-undermined wounds as well as the three groups are presented in Table 3. No statistically significant differences in mean LDF values were found among groups A, B, and C. On day 2, the mean LDF value decreased but not significantly compared to day 1 irrespective of group. On day 3, this value significantly increased compared those recorded on day 1 (p = 0.048) and day 2 (p = 0.012). No significant differences in blood perfusion were found between undermined and non-undermined wounds in any group. Some significant differences in blood perfusion were recorded between the sites of LDF measurements. Irrespective of location in the wound (upper, middle, or lower portion) or undermining of the wound (left or right limb), blood perfusion measurements taken on day 3 differed significantly between the frontal and caudal wound side in groups A (p < 0.0005) and C (p = 0.048).

Mean values of the examined histological variables for the three groups are presented in Table 4. A mild to moderate increase in cellular infiltration was found in all wounds. Additionally, moderate to substantial edema was evident on day 3 in all the groups. On day 15, edema was mild to moderate. Collagen orientation was mainly parallel to the skin surface. Epidermal thickness was decreased on day 3 as well as day 15 to a lesser extent. No statistically significant differences were observed for any of the histological variables.