Clinical and histologic tissue responses of skin incisions closed with glycomer 631 and lactomer 9-1 using subcuticular technique in pigs

doi:10.21203/rs.2.14166/v2

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Clinical and histologic tissue responses of skin incisions closed with glycomer 631 and lactomer 9-1 using subcuticular technique in pigs

https://doi.org/10.21203/rs.2.14166/v2

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published 24 Feb, 2020

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Background: Glycomer 631 and lactomer 9-1 are absorbable suture materials indicated for soft tissue approximation in non-infected wounds. Pigs are often used as surgical models in translational research, however, the reports of tissue reactions to both suture materials in pigs are lacking. The aim of this study was to assess clinical and histologic responses of skin incisions closed with subcuticular technique using glycomer 631 and lactomer 9-1 in pigs. Results: Skin incisions of 17 pigs were closed with glycomer 631 and lactomer 9-1 and clinical reactive score (CRS) including erythema, swelling, discharge, and dehiscence was calculated on postoperative days 7 and 14. Subcuticular tissue reaction was assessed histologically on postoperative day 14 (the presence of extravascular neutrophils, macrophages, multinucleated giant cells, lymphocytes, fibroblasts, bacterial colonies, overall severity of inflammatory response to suture material), and the cumulative score of variables calculated as aggregate tissue irritation score (ATIS). Tissue samples were examined for suture extrusion and evaluated microbiologically. Clinical reactive score did not differ between the suture materials. Only one variable of ATIS, overall severity of inflammatory response, was lower ( p = 0.029) when glycomer 631 was used. Suture extrusion was found in 10/17 of incisions closed by glycomer 631 and in 7/13 of incisions closed by lactomer 9-1. Trueperella pyogenes was isolated from the skin and from the area of tissue reaction in six pigs. Conclusions: No difference in CRS between the suture materials was observed. Glycomer 631 induced less tissue reaction only in terms of overall severity of inflammatory response. Suture extrusion was observed in more than 50% of incisions regardless of the suture material. Trueperella pyogenes was the only pathogen isolated from the tissue surrounding the suture material.

Small Animal Medicine

Large Animal Medicine

Pig

Absorbable suture material

Glycomer 631

Lactomer 9-1

Tissue reaction

Trueperella pyogenes

Primary wound closure is accomplished through subcutaneous tissue apposition followed by either cutaneous or buried, subcuticular suture placement [1].

Cutaneous sutures with the non-absorbable suture materials are easy to perform and provide excellent apposition of wound edges. The disadvantages are the need for removal, bacterial migration along suture tracts, scarring and skin irritation leading to self-mutilation [2].

Buried continuous subcuticular suture pattern with absorbable suture material provides exceptional skin edge apposition which promotes rapid epithelization [3] and does not increase the degree of inflammation in noncontaminated wounds [4]. Swelling and erythema may occur initially because of increased intraoperative tissue handling when compared with cutaneous suture. Further, scar formation is decreased and better cosmetic appearance of the skin closure 10 to 14 days after the surgery may be observed compared to a single interrupted suture pattern [5]. There is no need for suture removal [6] and the risk for self-induced trauma is reduced due to the lack of protruding material for the animal to scratch or lick [3].

Braided suture material causes greater friction than monofilament suture material when pulled through the tissue with a resultant increased tissue trauma around the suture path [1]. It has a greater capillarity, an increased tendency for bacterial colonization and a greater strength as well as pliability when compared with the monofilament suture material of the same size and composition [7].

Both, monofilament glycomer 631 (Biosyn, Covidien, USA) and braided lactomer 9-1 (Polysorb, Covidien, USA) are absorbable suture materials indicated for soft tissue approximation in non-infected wounds. Glycomer 631 is composed of glycolide, dioxanone and trimethylene carbonate. Very low tissue reaction was observed when skin incisions were closed with glycomer 631 with buried vertical intradermal suture pattern in rats [8]. However, when glycomer 631 was compared with polyglycolic acid, polypropylene and tissue adhesive for subcuticular closure technique in rats, fibrotic process and inflammatory cell infiltration were more prominent with glycomer 631 and polypropylene, and foreign body giant cells were present in glycomer 631 group [9]. Lactomer 9-1 is glycolide/lactide copolymer, which is a synthetic polyester composed of glycolide and lactide. It caused marked inflammation around the suture ten days after implantation into the gluteal muscles of rats. Ten days later, the reaction was mild and there was already connective tissue around the suture. Three months after implantation, no sign of tissue response was detected [10]. Subcuticular skin closure with lactomer 9-1 in miniature pigs resulted in 19% cumulative incidence of suture extrusion, with the volume of suture material in the wound being a critical determinant of suture extrusion [11].

Glycomer 631 and lactomer 9-1 are frequently used materials in veterinary medicine, but the reports of tissue reactions to these two materials in pigs are lacking. Because pigs are often used as surgical models in translational research, the purpose of this study was to assess clinical and histologic responses of skin incisions to subcuticular closure with glycomer 631 or lactomer 9-1. Since some other studies have reported fewer clinical and histologically proven reactions to monofilament versus multifilament sutures [3,12], we hypothesised that monofilament glycomer 631 would induce less tissue reaction than braided lactomer 9-1 suture material.

Clinical observations

All pigs recovered uneventfully after the surgery. No signs of discomfort or pain were recognised in the post-operative period and all of them were walking, eating and drinking normally six to eight hours after the surgery. No additional analgesia with butorphanol was required beyond 12 hours after the end of anaesthesia.

During the surgery, both suture materials were handled easily, and a successful wound approximation was achieved. The macroscopic appearance of skin incision on day 7 and 14 postoperatively (Fig. 1) did not differ between the suture materials with respect to CRS (Table 1).

Table 1. Clinical reactive score (CRS) 7 and 14 days after subcuticular skin closure by use of glycomer 631 and lactomer 9-1 in pigs; values are expressed as median (minimum, maximum). Maximum possible CRS is 12.

Postoperative day	Glycomer 631	Lactomer 9-1
7	1 (0, 8)	0 (0, 8)
14	1 (0, 7)	0 (0, 8)

Histopathologic evaluation

The dermal tissue reaction in all samples included a variably wide zone of granulation tissue rich with collagen and was infiltrated predominantly with macrophages and less numerous lymphocytes, which extended from epidermis into subcutis. Suture material was typically found within abscesses, granulomas and pyogranulomas (Fig. 2). Glycomer 631 was found in all samples. Lactomer 9-1 was detected only in 13 out of 17 samples, for which the assessment was carried out and the results of ATIS are presented. In abscesses, the suture material was surrounded with liquefactive necrosis, numerous neutrophils and less numerous macrophages encircled with a rim of fibrous connective tissue capsule. Granulomas were composed of suture material surrounded with macrophages, less numerous Langhans multinucleated giant cells and lymphocytes, and encircled with a rim of fibrous connective tissue capsule. In the surrounding dermis, single multinucleated giant cells or small groups of these cells forming small granulomas were observed. The remains of suture material were noticed even in the cytoplasm of some giant cells. In pyogranulomas, the suture material was encircled by a mixture of neutrophils, macrophages, Langhans multinucleated giant cells and lymphocytes. The difference between glycomer 631 and lactomer 9-1 was observed in only one variable of ATIS. The overall severity of inflammatory response was lower (p = 0.029) with glycomer 631 when compared to lactomer 9-1 (Table 2).

Table 2. Aggregate tissue reactive score (ATIS) 14 days after subcuticular skin closure by use of glycomer 631 and lactomer 9-1 in pigs; values are expressed as median (minimum, maximum).

	Glycomer 631	Lactomer 9-1	Maximum possible score
Extravascular neutrophils	-12 (0, -24)	-12 (0, -30)	-30
Macrophages	-1 (-1, -3)	-2 (-1, -2)	-5
Multinucleated giant cells	0 (0, -2)	-2 (0, -2)	-10
Lymphocytes	-2 (0, -6)	-4 (-2, -8)	-10
Fibroblasts	-2 (-1, -4)	-2 (-1, -5)	-5
Overall severity of inflammatory response to SM^a	-3 (-3, -9)	-6 (-3, -15)	-15
Bacterial colonies	0 (0, -9)	0 (0, -9)	-9
ATIS	-20 (-7, -54)	-25 (-10, -69)	-84
SM = suture material. ^aSignificant difference (p = 0.029) between glycomer 631 and lactomer 9-1. Groups were compared by Mann-Whitney test.

Suture material in the dermis was frequently partially encircled with a rim of stratified squamous epithelium (Fig. 3) or found inside a tract covered with stratified squamous epithelium opened to the skin's surface (Fig. 4). The later was found only once with each suture material while in the remaining samples suture material was not in connection with the skin's surface. The suture extrusion was found in 10/17 samples closed by glycomer 631 and in 7/13 samples closed by lactomer 9-1. Histopathologic evaluation revealed almost complete epithelialization in both groups. The area of the incision was covered with mildly or moderately hyperplastic epidermis in 16/17 samples closed by glycomer 631 and in 11/13 samples closed by lactomer 9-1. Single small pustules were found in the epithelialization area in 3/17 samples sutured with glycomer 631 and in 3/13 samples sutured with lactomer 9-1.

Microbiological examination

Bacterial growth has been observed from the skin and the area of tissue reaction of six pigs and Trueperella pyogenes was the only pathogen isolated. Bacterial cells that morphologically correspond to Trueperella pyogenes, have also been found histologically in the area of tissue reaction of the same pigs (three pigs in which glycomer 631 was used and thee pigs in which lactomer 9-1 was used).

Macroscopic appearance of the skin incisions or CRS on day 7 and 14 postoperatively did not differ between the suture materials used in this study. Similarly, Kirpensteijn et al. [3] did not observe any changes in macroscopic appearance when monofilament poliglecaprone 25 was compared with braided polyglactin 910 for closure of canine skin incisions at 7, 14 and 28 days postoperatively. However, they observed significantly less redness and swelling the day after the surgery when monofilament poliglecaprone 25 suture was used [3]. This difference was attributed to the pronounced tissue reactivity and the increased friction in the tissues related to the multifilament structure of polyglactin 910 suture material [12]. Non-absorbable monofilament polypropylene used for subcuticular pattern in cats resulted in even less reaction when compared to the absorbable monofilament copolymer of glycolide, ε-caprolacton and trimethylene-carbonate material [13].

Another factor which exerts influence upon macroscopic appearance of a skin incision could be the amount of tissue handling necessary for the suture placement. When the subcuticular pattern or simple interrupted pattern using poliglecaprone 25 were investigated and compared with a non-absorbable polypropylene suture used in a simple interrupted pattern, the wounds closed by subcuticular pattern showed significantly more swelling and erythema on the first postoperative day compared to the simple interrupted pattern with either of suture materials [5].

Although histopathologic evaluation of wound healing is more sensitive and precise compared to the macroscopic assessment, none of the methods for histopathologic evaluation is universally accepted [3, 4, 8,14]. They usually compare suture materials by the infiltration of predominant inflammatory cells. Molea et al. [8] reported neutrophil reaction/acute inflammatory reaction to glycomer 631. Predominantly neutrophilic infiltrate was also observed in incisions sutured with polyglactin 910 in continuous subcuticular pattern and with metal staples [14]. Suture material in our study was surrounded with liquefactive necrosis, numerous neutrophils and less numerous macrophages, encircled with a rim of fibrous connective tissue capsule, but there was no difference in the presence of neutrophils in the proximity of both suture materials. The only difference between lactomer 9-1 and glycomer 631 regarding histopathologic evaluation on the day 14 was the lower overall severity of inflammatory response when glycomer 631 was used. Kirpensteijn et al. [3] observed lower mean tissue reaction score with polyglecaprone 25 as compared with polyglactin 910 on the day 7, but this difference was not evident on the day 14 after the skin surgery. Unfortunately, we do not have data for the day 7. The observed differences between suture materials in both studies were probably due to multifilament nature of lactomer 9-1 and polyglactin 910 resulting in more tissue drag by the suture material during insertion. Also, both sutures might incur increased inflammation and possible transient protection of bacteria in their multifilament structure.

The proliferation of the squamous stratified epithelium around suture material, interpreted as suture extrusion, occurs in young, growing pigs, when the suture material with a longer absorption time is used. Polyglycolic acid, polyglactin 910 and polydioxanone may grow outward with the epidermal layer onto the skin surface as the pig grows, which creates skin irritation and potential infection, even after the incision site is healed [15]. Polyglactin 910 had a higher cumulative incidence of suture extrusion than lactomer 9-1 (31% versus 19% at 5 weeks) in juvenile Hanford minipigs, the rate depended on the volume of suture material in the wound. The 5-throw surgeon’s knot had a higher cumulative incidence of suture extrusion than knots with less throws [11]. Suture extrusion occurred in more than 50% of incisions sutured with glycomer 631 or lactomer 9-1 in our study. This finding indicates that suture extrusion in pigs may occur even earlier as stated in the humans, that is at three to four weeks [16], or as late as six months after the surgery [17]. Exact aetiology of suture extrusion is not completely understood; it may occur in tissues with poor blood supply, be related to the degree of suture material’s tissue reactivity, more superficial dermal placement [17] or the volume of suture material in the wound [11]. All these aetiologies could explain the suture extrusion in our study, especially the latter as we used 6-throw knots. Further, tissue reactions to suture materials may be species specific. Extreme reaction of porcine skin tissue against the metal staples that exceeded reactions of metal staples in dogs and cats has been described [14]. Porcine skin is similar to human skin, both are, unlike in rodents and carnivores, tightly attached to the subcutaneous tissues. However, it is also thicker and less vascular than human skin, which makes direct comparison between species difficult [15, 18, 19, 20]. Further, skin on the back is thicker than abdominal skin and perfusion measurements of the gluteal/thigh region [18], which also lack panniculus carnosus [21], showed much lower blood flow when compared to the ventral abdominal region [18]. Jonsson et al. [22] have proven that collagen deposition and wound healing were directly and significantly proportional to wound oxygen tension and perfusion, hence the selection of less vascular region may have had detrimental effect on wound healing and have contributed to unfavourable results when compared with studies where incisions were performed in well vascularised ventral abdominal region [3,5,8,13,14].

Trueperella pyogenes, a Gram-positive, rod-shaped, opportunistic, ubiquitous bacterium, which may be a part of normal skin, oropharyngeal, urogenital and gastrointestinal flora of animals [23], was isolated from the skin and tissue around the suture material of six pigs in our study. In pigs it usually causes abscesses [23, 24], which were noted in the proximity of suture material in this study. The fact that Trueperella pyogenes was isolated from both, skin and tissue surrounding the sutures in the same pigs, suggests that the infection was spread from the skin during or soon after the surgery despite careful aseptic preparation of the skin. The contact time of the antiseptic (propan-2-ol and benzalkonium chloride) was 7 minutes and well exceeded the exposure time recommended by the manufacturer. However, surgical preparation of the skin does not eliminate bacteria from the skin but only reduces the quantity. Epithelial cells bridge the minor gap in sutured wounds in 48 hours [25], until then contamination of suture material is possible. Pigs do not live in sterile environment and the presence of bacteria on the skin and in the incisions would be expected in spite of the efforts to keep the pens as clean as it is possible. Fick et al. [14] observed purulent exudate along the incision closed with polyglactin 910 and with metal staples, and they assigned it to environmental contamination. They detected bacteria via histologic assessment with no overt evidence of clinical infection in 25% of incisions. Even though braided suture materials tend to have greater capillarity effect [7], no difference was observed between glycomer 631 or lactomer 9-1 regarding the bacterial colonization of tissue surrounding the suture material in our study. Possible explanation is coating of the lactomer 9-1 suture with caprolactone/glycolide copolymer and calcium stearoyl lactylate, which is designed to facilitate knot formation [7] and acts as an antimicrobial agent [26]. It is interesting that although pigs were treated with amoxicillin-clavulanic acid in our study, Trueperella pyogenes was isolated from tissue surrounding the suture material in 6 out of 17 pigs. In vitro antimicrobial sensitivity of Trueperella pyogenes isolated from domestic animals proved good sensitivity to amoxicillin (94.6%) [24]. One of the reasons for the antibiotic ineffectiveness can be the fact that Trueperella forms abscesses where the antibiotic is more difficult to penetrate [27].

Our study has several limitations. First, the placement of suture material was not randomised. Lactomer 9-1 was used only in dorsal part of the wound and glycomer 631 only in ventral part of the wound. The blood vessel density in dorsal and ventral parts of the wound may differ and could have an impact on the tissue healing. Also, only the left thigh was used for the incisions and the vascular supply of the skin might be different between the two limbs. Second, only 13 samples of the tissue sutured with lactomer 9-1 were analysed, because in four of them the suture material could not be visualised at histopathological examination. Third, antibiotic prophylaxis with amoxicillin-clavulanic acid was extended to the day 10 after the surgery because of the requirements of the primary study and may have masked the microbiological analysis of tissue samples. Although it is not usual to use antibiotic after clean surgical procedures, the stable environment, inability for postoperative surgical dressing of incisions on the lateral aspect of the thigh and interaction of the pigs outweighed for antibiotic use. In experimental pigs that are not intended for food production, the exceptional use of antimicrobial drugs is allowed according to national and European legislation.

No difference in clinical reactive score between the suture materials was observed. Monofilament glycomer 631 induced less tissue reaction only in terms of overall severity of inflammatory response. Suture extrusion was observed in more than 50% of incisions regardless of the suture material and Trueperella pyogenes was the only pathogen isolated from the tissue surrounding the suture material.

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 from Landrace and Large White cross, 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 sciatic nerve was surgically exposed. 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. 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 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 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 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 would be prolonged as required. 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 femoral region from trochanter major to the genual region. Superficial gluteal, biceps femoris and vastus lateralis muscle were separated to expose the sciatic 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 to assure the safety and comparison of the knots at the beginning and at the end of the continuous suture. All surgical procedures were performed by the same surgeon.

Skin incisions were assessed once daily by blinded researcher unaware of the use and placement of 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 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 anaesthetised using the same anaesthesia protocol as the first time, 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 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. [28] and Fick et al. [14]. 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 -8(Table 3).

Suture extrusion was not included in a scoring system of Sewel et al. [28] and Fick et al. [14] and the results are presented descriptively.

Table 3. Variables assessed to calculate the aggregate tissue irritation score (ATIS)

Variable	Score	Weighing 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

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.).

ATIS – aggregate tissue reactive score

CRS – clinical reactive score

SM – suture material

Ethics approval

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 Slovenian and European legislation.

Consent for publication

Not applicable.

Availability of data and material

The data generated or analysed during the current study are available from

the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interest.

Funding

This study was supported by the Slovenian Research Agency (P3-0043-0381, P4-0053) and the Tertiary funding of the Clinical Department of Anaesthesiology and Intensive Therapy, University Medical Centre Ljubljana, Slovenia. The funders

did not involve in the design of the study and collection, analysis, and interpretation

of data and in writing the manuscript.

Authors’ contributions

TP and AS designed the experiment. TP, MD, TSP and AS performed clinical experiments. TŠ, MG and EC performed histological examination and interpreted the results. IZ performed microbiological examination and interpreted the results. TP performed data analysis. TP, MD, TŠ, MG and IZ drafted the manuscript. TP and TŠ prepared the Figures. AS, TSP and EC reviewed the manuscript. All authors read and approved the final manuscript.

Acknowledgments

For their assistance with the study we want to thank Jerneja Sredenšek from Veterinary Faculty, University of Ljubljana, Slovenia; Milan Števanec, Ivan Blažinovič, Friderik Štendler, Majda Črnak Maasarani, Nataša Pollak Kristl and Marko Slak from the Institute of Anatomy, Faculty of Medicine, University of Ljubljana, Slovenia, and Miha Pintarič from Faculty of Arts, University of Ljubljana.

Authors’ details

¹Small Animal Clinic, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia. ²Institute of Pathology, Wild Animals, Fish and Bees, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia. ³Institute of Microbiology and Parasitology, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia. ⁴Clinical Department of Anaesthesiology and Intensive Therapy, University Medical Centre Ljubljana, Zaloška 7, 1000 Ljubljana, Slovenia. ⁵Institute of Anatomy, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia.

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Clinical and histologic tissue responses of skin incisions closed with glycomer 631 and lactomer 9-1 using subcuticular technique in pigs

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