This study was conducted in accordance with ARRIVE (Animal Research: Reporting of in Vivo Experiments) guidelines. All procedures involving the experimental use of animals were approved by the National Ethics Committee and National Veterinary Administration (licenses No U34401-28/2013/7, approval date 14.10.2013 and No U34401-28/2013/17, approval date 29.7.2014). All procedures complied with the relevant national and European legislation.
Animals and housing
Fourteen female and three castrated male pigs from a Landrace and Large White cross, aged eight to nine weeks and weighing approximately 20 kg (weight estimated with a weight measuring tape for pigs) were used in the study. Following the 3Rs principles (replacement, reduction and refinement), we performed this study in pigs that were primarily included in a neurological study in which the sciatic nerve was surgically exposed. The pigs were reared according to the Council directive for minimum standards for the protection of pigs (2008/120/EC). Three days before they were anaesthetised, they were procured from a certified commercial farm (Globočnik Aleš, Voglje, 4208 Šenčur, Slovenia; KMG-MID SI100205065) free of classical and African swine fever and Aujeszky’s disease. The pigs were vaccinated against Mycoplasma hyopneumoniae and porcine circovirus type 2. The pigs were clinically examined and housed in indoor straw-bedded pens of appropriate size (minimum 2 m2 per animal) in groups of four or five animals originating from the same nest. They were exposed to a natural light/dark cycle and kept at a room temperature of 20 to 23 °C. They were fed commercial pig feed for growers twice a day and had unlimited access to tap water from nipple waterers. Food was withheld from them 12 hours before anaesthesia.
Anaesthesia protocol
Pigs were premedicated with midazolam 0.5 mg/kg, butorphanol 0.5 mg/kg and ketamine 10 mg/kg mixed together and given intramuscularly approximately 5 cm behind and on the level of the base of the ear. An intravenous catheter was placed into an auricular vein, and anaesthesia was induced with propofol, titrated to effect. After endotracheal intubation, anaesthesia was maintained with isoflurane at 1.5 to 2% (vapour setting) in a mixture of oxygen (1 L/min) and air (1 L/min) delivered through a circle breathing system. The pigs breathed spontaneously during anaesthesia.
The pigs were placed in right lateral recumbency on an electrically heated surgery table. Arterial oxygen saturation (SpO2), end-tidal CO2 concentration, oesophageal temperature, ECG and blood pressure (non-invasively) were monitored. Hartmann’s solution was infused intravenously during anaesthesia at a rate of 10 mL/kg/h. The pigs recovered from anaesthesia in a warmed, straw-bedded pen. The endotracheal tube was removed when the palpebral reflex returned.
Postoperative analgesia was provided with butorphanol 0.5 mg/kg administered intramuscularly approximately 5 cm behind and on the level of the base of the ear 4, 8 and 12 hours after the end of anaesthesia. If the pigs showed signs of discomfort or pain later, the administration of butorphanol was prolonged as required. The pigs were offered food 4 to 6 hours after extubation. Pre-surgical antibiotic prophylaxis with amoxicillin-clavulanic acid 9 mg/kg was administrated intramuscularly 30 minutes before the skin incision and then orally once a day for the following 10 days per the requirements of the primary study.
Experimental design
The surgical site was prepared by clipping of the hair immediately preceding the procedure. After removal of the hair, the skin was scrubbed, dried and prepared aseptically using propan-2-ol and benzalkonium chloride (Cutasept F, Bode, Germany), first with the spray technique and two minutes later with the paint technique. Five minutes later, the surgical site was draped, and a 15-cm-long skin incision was performed on the lateral aspect of the femoral region from the trochanter major to the genual region. Superficial gluteal, biceps femoris and vastus lateralis muscles were separated to expose the sciatic nerve. The fascia was approximated with interrupted sutures using 2-0 lactomer 9-1. The subcutaneous tissue was sutured with a continuous suture using 2-0 lactomer 9-1 (dorsal half of the wound) and 2-0 glycomer 631 (ventral half of the wound), and bites were placed 8 to 10 mm apart. The skin was sutured with a continuous subcuticular suture using 3-0 lactomer 9-1 (dorsal half of the wound) and 3-0 glycomer 631 (ventral half of the wound), with six throws placed on each knot to assure the safety and comparison of the knots at the beginning and end of the continuous suture. All surgical procedures were performed by the same surgeon.
Skin incisions were assessed once daily by a blinded researcher unaware of the use and placement of the suture material. They were evaluated for erythema around the wound (absent, mild = less than 5 mm and pink, moderate = between 5-20 mm and red-violet, severe = more than 20 mm and violet), swelling around the wound (absent, mild = less than 5 mm, moderate = between 5-20 mm, severe = more than 20 mm), discharge (absent, mild = small amounts of serous or serosanguineous discharge, moderate = large amounts of serous or serosanguineous discharge or mucous discharge, severe = purulent discharge), and dehiscence (absent, mild = less than 10% of the wound, moderate = 10-30% of the wound, severe = more than 30% of the wound), and the parameters were recorded as absent (0), mild (1), moderate (2) or severe (3). The sum of the parameters, called the clinical reactive score (CRS), was calculated on postoperative days 7 and 14. The highest possible CRS was 12. The pigs were also evaluated for signs of discomfort or pain (agitation, vocalization, changes in facial expression, deviation from normal behaviour and appetite, deviation from normal posture and movement) for 12 hours after extubation and then three times a day.
Fourteen days after surgery, the pigs were sedated and anaesthetised using the same anaesthesia protocol as that used for the initial surgery, after which they were euthanatised with T-61 euthanasia solution 0.3 mL/kg intravenously. The skin around the site of the incision was scrubbed, dried and disinfected twice using propan-2-ol and benzalkonium chloride with the spray technique. The contact time of disinfectant was 2 minutes for the first and 5 minutes for the second skin preparation. Biopsies of the skin and subcutaneous tissue were taken from the dorsal and ventral part of the wound with 2-cm margins of adjacent skin and associated deeper tissue (to the first muscle plane). Samples were halved, and one half was saved in a sterile collecting pot for immediate microbiological evaluation. The other half was fixed in 10% buffered formalin for histopathologic examination. All examiners were blinded.
Histopathologic examination
Tissue sections transverse to the incision were taken and routinely embedded in paraffin, sectioned at 4 μm and stained with hematoxylin and eosin. During histopathologic examination, the tissue response to subcuticularly placed suture material was scored by a system modified from that of Sewel et al. [32] and Fick et al. [13]. For the evaluation of the tissue reaction, seven variables were assessed semi-quantitatively and scores multiplied by their weighting factors: the presence of extravascular neutrophils, macrophages, multinucleated giant cells, lymphocytes, fibroblasts and bacterial colonies, and the overall severity of the inflammatory response to the suture material. The absence of a variable was scored as zero, and the presence of a variable was scored from 1 to 3 (presence of bacterial colonies) or 1 to 5 (all other variables) so that score 1 represented the mildest and scores 3 (for presence of bacterial colonies) and 5 (for all other variables) represented the most severe presentation of the variables. In the next step scores were multiplied by their weighting factors (WF). In the scoring system of Sewel et al. [32] and Fick et al. [13], different WF are applied to different variables so that variations in cell types and cell densities are considered in view of the interpretation of the tissue reaction. The WF were as follows: for the presence of extravascular neutrophils (WF -6), macrophages (WF -1), multinucleated giant cells (WF -2), lymphocytes (WF -2), fibroblasts (WF -1) and bacterial colonies (WF -3), and the overall severity of the inflammatory response to the suture material (total estimated cell number) (WF -3). In the last step, the results of multiplication were summed up to get an aggregate tissue irritation score (ATIS). A more negative ATIS was associated with more severe tissue reaction and poorer wound healing response. The score for the most severe possible ATIS was -84 (Table 3).
Suture extrusion, the presence of hyperplastic epidermis and single small pustules were not included in the scoring system of Sewel et al. [32] and Fick et al. [13].
Table 3. Variables assessed to calculate the aggregate tissue irritation score (ATIS)
Variable
|
Score*
|
Weighting factor
|
Maximum score
|
Extravascular neutrophils
|
0-5
|
-6
|
-30
|
Macrophages
|
0-5
|
-1
|
-5
|
Multinucleated giant cells
|
0-5
|
-2
|
-10
|
Lymphocytes
|
0-5
|
-2
|
-10
|
Fibroblasts
|
0-5
|
-1
|
-5
|
Overall severity of inflammatory response to suture material (total estimated cell number)
|
0-5
|
-3
|
-15
|
Bacterial colonies
|
0-3
|
-3
|
-9
|
ATIS
|
|
|
-84
|
* The absence of the variable was scored as zero, and its presence was semi-quantitatively scored in an order increasing from 1 to 3 (presence of bacterial colonies) or 1 to 5 (all other variables).
Microbiological examination
Skin samples and samples of tissue around the suture material were inoculated onto two different solid media: nutrient agar (Oxoid, Hampshire, UK) supplemented with 5% sheep blood for bacteriological examination and Sabouraud dextrose agar (Oxoid, Basingstoke, UK) with chloramphenicol (100 mg/L) for mycological examination. Blood agar plates were prepared in a duplicate for incubation in aerobic and anaerobic atmosphere. Cultures were incubated at 37 °C for up to four days, checked for microbial growth and subcultures were performed when necessary. Isolated colonies were further examined. The biochemical characteristics were evaluated using commercial kits API Coryne System (bioMerieux, Marcy I’Etoile, France) and the strain was determined to the species level.
Statistical analysis
Normal distribution of data was tested by the Shapiro-Wilcox test. The non-parametric Mann-Whitney test was used to compare the two suture materials with respect to the CRS and ATIS, and the results are presented as median (minimum, maximum). Fisher’s exact test was used to compare suture extrusion, the presence of hyperplastic epidermis and single small pustules. Differences were considered significant at a value of p < 0.05.
The data were analysed with the Statistical Package for the Social Sciences (IBM SPSS Statistics for Windows, Version 22.0, Armonk, NY: IBM Corp.).