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 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, cross from Landrace and Large White, aged eight to nine weeks and weighing approximately 20 kg (weight estimated with weight measuring tape for pigs) were used in the study. Following the principles of 3Rs (replacement, reduction and refinement) we performed this study in pigs that were primarily included in the neurological study in which ischiadic nerve was surgically exposed. Pigs were reared according to the Council directive for minimum standards for the protection of pigs (2008/120/EC). They were procured from a certified commercial farm (Globočnik Aleš, Voglje, 4208 Šenčur, Slovenia; KMG-MID SI100205065) with an average health status three days before they were anaesthetised. 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 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. 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 mixture of oxygen (1 L/min) and air (1 L/min) delivered through a circle breathing system. Pigs were breathing spontaneously during anaesthesia.
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. Pigs recovered from anaesthesia in a warmed, straw bedded pen. Endotracheal tube was removed when the palpebral reflex returned.
Postoperative analgesia was provided with butorphanol 0.5 mg/kg administered intramuscularly 4, 8 and 12 hours after the end of anaesthesia. Pigs were offered food 4 to 6 hours after the 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 due to 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 spray technique and two minutes later with 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 left thigh from trochanter major to the knee and gluteus and biceps femoris muscles were separated to expose ischiadic nerve. The fascia was approximated with interrupted sutures using 2-0 lactomer 9-1, subcutaneous tissue with continuous suture using 2-0 lactomer 9-1 (dorsal half of the wound) and 2-0 glycomer 631 (ventral half of the wound), bites were placed 8 to 10 mm apart. Skin was sutured with 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. All surgical procedures were performed by the same surgeon.
Skin incisions were evaluated once daily by blinded researcher unaware of the use and placement of suture material. Skin incisions were evaluated for erythema, swelling, discharge, and dehiscence, and the parameters 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 worst possible CRS was 12. 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) during 12 hours after the extubation and then three times a day.
Fourteen days after the surgery, the pigs were sedated and induced to anaesthesia using the same anaesthesia protocol as the first time, after which they were euthanatised with T-61 euthanasia solution 0.3 mL/kg. The skin around the site of the incision was scrubbed, dried and disinfected twice using propan-2-ol and benzalkonium chloride with 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.
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 suture material was scored by a system modified from that of Sewel et al. [22] and Fick et al. [13]. In these systems the weighing factors are applied to different variables and thus variations in cell types and cell densities are considered in the interpretation of the tissue reaction. For the evaluation of the tissue reaction, seven variables were assessed semi-quantitatively and scores multiplied by their weighing factors (WF): 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 overall severity of inflammatory response to suture material (total estimated cell number) (WF -3). The absence of the variable was scored as zero, and the presence of variable was scored in increasing order from 1 to 3 (presence of bacterial colonies) or 1 to 5 (all other variables). Scores were multiplied by their weighing factors and summed up to get an aggregate tissue irritation score (ATIS). A more negative ATIS was associated with more severe tissue reaction and the poorer wound healing response. The score for the most severe possible ATIS was -84.
Suture extrusion was not included in a scoring system of Sewel et al. [22] and Fick et al. [13] and was evaluated and analysed separately.
Microbiological examination
Skin samples and samples of tissue around suture material were inoculated onto two different solid media: nutrient agar (Oxoid, Hampshire, UK) supplemented with 5% of 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 strain was determined to species level.
Statistical analysis
Normal distribution of data was tested by Shapiro-Wilcox test. Comparison of the two suture materials regarding CRS and ATIS was made by non-parametric Mann-Whitney test and the results are presented as median (minimum, maximum). Differences were considered significant at a value of p < 0.05. Suture extrusion is presented descriptively.
The data were analysed with the Statistical Package for the Social Sciences (IBM SPSS Statistics for Windows, Version 22.0, Armonk, NY: IBM Corp.).