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

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 deficient. The aim of this study was to assess clinical and histologic responses of skin incisions closed with subcutaneous and subcuticular techniques 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. 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.

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. 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. 3 Background Primary wound closure is accomplished through subcutaneous tissue apposition by either cutaneous or buried, subcuticular suture placement [1]. Cutaneous sutures with the nonabsorbable 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 tissue handling. 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 and 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].
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]. Polysorb is glycolide/lactide copolymer, which is a synthetic polyester composed of glycolide and lactide. It caused marked inflammation around the thread 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 Polysorb in miniature pigs resulted in 19% cumulative incidence of suture extrusion, the volume of suture material in 4 the wound was a critical determinant of suture extrusion [11].
The reports of tissue reactions to glycomer 631 and lactomer 9-1 in pigs are deficient.
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 glycomer 631 or lactomer 9-1 when used for continuous subcutaneous and continuous subcuticular closure in pigs. 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.
During the surgery, both suture materials were handled easily, and 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).

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 5 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).

Microbiological examination
Bacterial growth has been observed from skin and 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).

Discussion
Macroscopic appearance of the skin incisions or clinical reactive scores on day 7 and 14 postoperatively did not differ between the suture materials used in this study. Kirpensteijn et al. [3] observed significantly less redness and swelling the day after the surgery when monofilament poliglecaprone 25 was compared with braided polyglactin 910 for closure of canine skin incisions, however, the difference was not present at later evaluation times.
They attributed it to the pronounced tissue reactivity to the multifilament structure of polyglactin 910 and the increased friction in the tissues caused by it [12]. 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 buried continuous subcuticular pattern and simple interrupted pattern using poliglecaprone 25 were investigated and compared with a nonabsorbable polypropylene suture used in a simple interrupted pattern, the wound 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. However, by day 10 to 14 postoperatively, poliglecaprone 25 used in buried continuous subcuticular pattern was associated with significantly lower wound scores than was the same material used in a simple interrupted pattern [5].
Although histopathologic evaluation of wound healing is more sensitive and precise compared to the macroscopic assessment, none of the methods for histopathologic evaluation of wound healing is universally accepted [3,4,8,13]. 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 [13]. 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 times 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 [14]. When braided suture material was studied in juvenile Hanford minipigs, polyglactin 910 had a higher cumulative incidence of suture extrusion than lactomer 9-1 (31% versus 19% at 5 weeks), 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. In most cases (9 out of 17 in glycomer 631 and 6 out of 13 in lactomer 9-1) the suture material in the dermis was partially encircled with 8 a rim of stratified squamous epithelium and was not in connection with the skin's surface.
In only one animal in each group the tissue tract with suture material that opened to the skin surface was observed. This finding, observed two weeks after suture placement, indicates that suture extrusion in pigs may occur even earlier as stated in the humans, that is at three to four weeks [15], or as late as six months after the surgery [16].
Exact aetiology of suture extrusion is not completely understood; it may occur in tissues with poor blood supply or is related to the degree of suture material's tissue reactivity and more superficial dermal placement [16]. All these aetiologies could explain the suture extrusion in our study. The volume of suture material in the wound could also be the reason for the suture extrusion [11] 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 [13].
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 [17], was isolated from the skin and tissue around the suture material of six pigs in our study. In pigs it usually causes abscesses [17,18], which were noted in the proximity of suture material in this study. The fact that Trueperella pyogenes was isolated Fick et al. [13] observed purulent exudate along the incision closed with braided absorbable suture material (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 9 facilitate knot formation [7] and acts as an antimicrobial agent [20]. 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%) [18]. One of the reasons for the antibiotic ineffectiveness can be the fact that Trueperella forms abscesses where the antibiotic is more difficult to penetrate [21].
Our study has several limitations.

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 m 2 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 (SpO 2 ), end-tidal CO 2 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

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 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. 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.    Abscess with remnants of glycomer 631 (arrowheads). Bar = 100 μm.

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