Eur Surg Res 2015;55:1–11 DOI: 10.1159/000371797 Received: September 24, 2014 Accepted after revision: December 22, 2014 Published online: February 25, 2015

© 2015 S. Karger AG, Basel 0014–312X/15/0552–0001$39.50/0 www.karger.com/esr

Original Paper

Polyvinylidene Fluoride as a Suture Material: Evaluation of Comet Tail-Like Infiltrate and Foreign Body Granuloma Andreas Lambertz a Kai Michael Schröder a Dominik Stefan Schöb a Marcel Binnebösel a Michael Anurov b Uwe Klinge a Ulf Peter Neumann a Christian Daniel Klink a a

Department of General, Visceral and Transplantation Surgery, RWTH Aachen University Hospital, Aachen, Germany; b Department of Physiology, Russian State Medical University, Moscow, Russia

Key Words Polyvinylidene fluoride · Suture material · Biocompatibility · Comet tail-like infiltrate · Foreign body granuloma · Immunohistochemistry · Rat model Abstract Background: Biocompatibility and tissue integration of a surgical suture are decisive factors for wound healing and therefore for the success of sutures. The optimal suture material is still under discussion. Polyvinylidene fluoride (PVDF) is described to have superior properties of biocompatibility and is therefore frequently used as a mesh component. Only little information is available about its use as a suture material. The aim of this study was to evaluate the biocompatibility of PVDF as a suture material in comparison to 5 different established sutures in a rat model. Methods: In 30 male rats, a monofilamental PVDF suture (Resopren®) and 5 established control suture materials [polyester (Miralene®), polytetrafluoroethylene (Gore®), poliglecaprone (Monocryl®), polydioxanone (Monoplus®), polyglactin 910 (Vicryl®), USP size 3–0] were placed in the subcutaneous layer of the abdominal wall without knot or tension. After 3, 7 or 21 days, the abdominal walls were explanted for histopathological and immunohistochemical investigation with special regard to the size and quality of foreign body granuloma and the length of the comet tail-like infiltrate (CTI). Results: The PVDF sutures showed the smallest size of foreign body granuloma (60 ± 14 μm) and the smallest CTI length (343 ± 60 μm) of all polymers after 21 days. Only PVDF (Resopren) and polydioxanone (Monoplus) showed a significant collagen I/III ratio increase between days 3 and 21 (p = 0.009 and p =

Andreas Lambertz, MD Department of General, Visceral and Transplantation Surgery RWTH Aachen University Hospital, Pauwelsstrasse 30 DE–52074 Aachen (Germany) E-Mail alambertz @ ukaachen.de

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A. Lambertz and K.M. Schröder contributed equally to the study.

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Eur Surg Res 2015;55:1–11 DOI: 10.1159/000371797

© 2015 S. Karger AG, Basel www.karger.com/esr

Lambertz et al.: Polyvinylidene Fluoride as a Suture Material: Evaluation of Comet Tail-Like Infiltrate and Foreign Body Granuloma

0.016). The quality of foreign body reaction regarding inflammation, proliferation and fibrotic remodeling was similar between all suture materials. Conclusions: Our data indicate that monofilamental PVDF sutures show a favorable foreign body reaction with small granuloma sizes and CTI length in comparison to established sutures. Its use as a suture material in general surgery could therefore be extended in the future. To reinforce these findings, further clinical studies need to be conducted. © 2015 S. Karger AG, Basel

Introduction

An adequate approximation of tissue using suture materials is a central aspect in general surgery. The success of the suture depends on the favorable combination of several aspects: these are, on the one hand, the patients’ wound healing conditions and, on the other hand, local factors, which are influenced by ischemia due to surgical preparation and tissue compression due to the suture. Furthermore, foreign body reaction induced by the implanted material plays an important role [1]. While the patients’ wound healing conditions cannot be influenced surgically, an atraumatic preparation and adequate knotting tension is generally required. Additionally, various approaches have been made to optimize the surgical technique, for example for midline laparotomy closure [2–4]. Finally, the choice of the optimal suture material determining the foreign body reaction is at least as important but is still under discussion [5]. It should induce a minimum of inflammation and host reaction and should rather support the wound healing process [6]. In the literature, polyvinylidene fluoride (PVDF) is described to have such superior properties of biocompatibility [7, 8]. However, it had not had any impact on general surgery until 2002 when Klinge et al. [9] first used it as a mesh component. Until now, the use of PVDF in meshes has become more and more popular while only little information about its use as a suture material in general surgery is available. Thus, the aim of this study was to evaluate the biocompatibility of PVDF as a suture material in comparison to 5 different established suture materials in a rat model. As a central tool of investigation, we assessed the extent and the cellular quality of foreign body granuloma and the comet tail-like infiltrate (CTI). This way, the reaction of the host tissue to the surface of the foreign body itself has been previously investigated [10]. To have a reliable spectrum of materials in comparison to PVDF, we assembled a set of 5 different suture materials which were monofilamental and polyfilamental as well as absorbable and nonabsorbable sutures to be able to evaluate different aspects influencing the biocompatibility.

Materials and Methods

Animals Thirty male Sprague-Dawley rats with a mean body weight of 450 g were randomly divided into three groups (n = 10 in each group; group 1 was observed for 3 days, group 2 for 7 days and group 3 for 21 days). All animals were kept under standardized conditions: temperature between 22 and 24 ° C; relative humidity 50–60%, and 12 h of light following 12 h of darkness. The animals had free access to food and water.

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The experiments were officially approved by the Animal Care and Use Review Committee of the Russian State Medical University, Moscow, Russia (IACUC, No. 01/02/09/87). All animals received human care in accordance with the requirements of the German Tierschutzgesetz, §8 Abs. 1 and in accordance to the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health (8th edition, NIH publication, USA).

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Eur Surg Res 2015;55:1–11 © 2015 S. Karger AG, Basel www.karger.com/esr

DOI: 10.1159/000371797

Color version available online

Lambertz et al.: Polyvinylidene Fluoride as a Suture Material: Evaluation of Comet Tail-Like Infiltrate and Foreign Body Granuloma

Epidermis

Granuloma

CTI

Suture

CTI

Fig. 1. Schematic diagram of the CTI.

Materials The monofilamental PVDF suture (Resopren®, Resorba, Nürnberg, Germany) was compared to 5 established sutures: polyester (Miralene®, Braun, Melsungen, Germany) and polytetrafluoroethylene (Gore®, Gore, Putzbrunn, Germany) representing nonabsorbable polymers and the following 3 absorbable sutures: poliglecaprone (Monocryl®, Ethicon Inc., Somerville, N.J., USA), polydioxanone (Monoplus®, Braun) and polyglactin 910 (Vicryl®, Ethicon Inc.). All sutures used were of USP size 3–0.

Histological Assessment After formaldehyde fixation, samples were embedded in paraffin and cut in 3-μm sections. All sections were routinely stained with hematoxylin and eosin and processed at the same time to reduce internal staining variations. Histological investigations were performed by two blinded and independent observers. The amount of inflammatory and connective tissue formation was analyzed semiquantitatively by measuring the size of granuloma and the extent of CTI. Granuloma thickness in two different regions and the length of CTI on both sides of the suture parallel to the surface of the abdominal wall were measured (fig. 1–7). The end of the CTI was defined as the point where the two granuloma branches fused. To analyze the host response regarding inflammation, proliferation and fibrotic remodeling, immunohistochemistry was used. After pretreatment of the fixed specimen with microwave and citrate buffer (pH 6) for three times, cells were identified by different monoclonal antibodies. We used the avidin-biotin method (3-step method) to detect the markers. The inflammation was assessed by counting the number of macrophages (CD68 expression) in the foreign body granuloma. Ki67 and COX-2 expression was measured as a marker for cell proliferation. For COX-2, we used a 1:100 diluted polyclonal rabbit antibody of DCS Innovative Diagnostic Systems (Hamburg,

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Surgical Procedure All operations were carried out under general anesthesia, aseptic and sterile surgical conditions and were performed by two different and independent surgeons. The animals received general anesthesia induced by the use of isoflurane, followed by a combination of 0.3 mg/kg medetomidine (Domitor®, Farmos, Turku, Finland; Gräub, Bern, Switzerland) and 100 mg/kg ketamine (Ketamin® 10%, Sanofi-Ceva, Düsseldorf, Germany), which was applied subcutaneously. The rats were fixated in supine position, weighed and their skin was shaved and disinfected with polyvidone iodine solution. Three different suture materials of 2 cm length were randomized and were placed subcutaneously by puncture of the abdominal wall (n = 15 for each suture) like previously described by Klink et al. [10]. After 3, 7 or 21 days (n = 5 for each suture), the animals were euthanized using a lethal dose of pentobarbital sodium (Narcoren®, Rhône Merieux, Laupheim). The entire abdominal wall was resected and immediately fixed in 10% formaldehyde for later histological and immunohistochemical assessment.

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Eur Surg Res 2015;55:1–11 DOI: 10.1159/000371797

© 2015 S. Karger AG, Basel www.karger.com/esr

Color version available online

Lambertz et al.: Polyvinylidene Fluoride as a Suture Material: Evaluation of Comet Tail-Like Infiltrate and Foreign Body Granuloma

Color version available online

Fig. 2. Foreign body granuloma and CTI of PVDF suture after 21 days.

Color version available online

Fig. 3. Foreign body granuloma and CTI of Miralene suture after 21 days.

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Fig. 4. Foreign body granuloma and CTI of Vicryl suture after 21 days.

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Eur Surg Res 2015;55:1–11 DOI: 10.1159/000371797

© 2015 S. Karger AG, Basel www.karger.com/esr

Color version available online

Lambertz et al.: Polyvinylidene Fluoride as a Suture Material: Evaluation of Comet Tail-Like Infiltrate and Foreign Body Granuloma

Color version available online

Fig. 5. Foreign body granuloma and CTI of Gore suture after 21 days.

136,09 μm 18,25 μm

649,55 μm

421,52 μm

14,40 μm

96,07 μm

Fig. 7. Foreign body granuloma and CTI of Monocryl suture after 21 days.

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Color version available online

Fig. 6. Foreign body granuloma and CTI of Monoplus suture after 21 days.

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Eur Surg Res 2015;55:1–11 DOI: 10.1159/000371797

© 2015 S. Karger AG, Basel www.karger.com/esr

Lambertz et al.: Polyvinylidene Fluoride as a Suture Material: Evaluation of Comet Tail-Like Infiltrate and Foreign Body Granuloma

Germany). It was microwave-pretreated with a citrate buffer (pH 6). The secondary antibody was a 1:300 diluted, biotinylated goat-anti-rabbit antibody (Dako, Glostrup, Denmark). The detection of CD68 (macrophages) was performed by using a monoclonal mouse antibody (DPC/Acris, Bad Nauheim, Germany). It was diluted 1: 100 and pretreated with trypsin. The secondary antibody was a biotinylated rabbit-anti-mouse antibody (1: 300) from Dako. Ki67 was detected by using a combination of a primary monoclonal mouse antibody and a secondary biotinylated rabbit-anti-mouse antibody (Dako). The primary antibody was diluted 1:10 and the secondary 1:300. Concerning fibrotic remodeling, c-myc, β-catenin and Notch-3 expression was evaluated. For c-myc, a 1:100 polyclonal rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) and secondarily a biotinylated 1: 500 anti-goat-anti-rabbit antibody (Dako) was used. β-Catenin was identified by a monoclonal mouse antibody (1:500) from Abcam, Cambridge, UK, in combination with a secondary 1:300 biotinylated rabbit-anti-mouse antibody from Dako. Before application, the primary antibody was microwave-pretreated twice with a citrate buffer (pH 6) for 10 min. For Notch-3, we used a 1:100 diluted polyclonal rabbit antibody (Santa Cruz Biotechnology) as primary antibody and a biotinylated goat-anti-rabbit antibody (1: 500) as secondary antibody. This primary antibody also had to be microwave-treated with a citrate buffer (pH 6) before. Finally, measurements of the quality of collagen were performed using Sirius Red staining. These sections were analyzed for the ratio of collagen I/III by cross-polarization microscopy. Therefore, 5-μm sections were stained for 1 h in picrosirius solution (0.1% solution of Sirius Red F3BA in saturated aqueous picric acid, pH 2) [11]. The sections were washed for 2 min in 0.01 N HCl, dehydrated, cleared, and mounted in synthetic resin. In cross-polarization microscopy, thicker collagen type I fibers were stained in red-orange shades, whereas thinner collagen type III appeared as pale-green shades. The expression of all other markers was determined via standard light microscopy (Olympus BX51; Hamburg, Germany). For each sample, 4 regions were examined: 2 regions in the CTI and 2 regions in the granuloma. The percentage of positively stained cells was assessed in an area of 100 × 100 μm with 400-fold magnification and captured by a digital camera (Olympus C-3030, Olympus, Hamburg, Germany). Analysis was performed using a digital image analyzing software (Image-Pro Plus, Media Cybernetics, Silver Spring, Md., USA). Statistical Analysis Statistical analysis was carried out using the Statistical Package for Social Sciences software (SPSS®, Vers. 17.0, Chicago, Ill., USA). Differences between study groups were analyzed by the Kruskal-Wallis test for nonparametric data and in case of significant differences confirmed by the Mann-Whitney test. For numeric data, differences were analyzed by ANOVA and in case of significance confirmed by the t test. p values

Polyvinylidene Fluoride as a Suture Material: Evaluation of Comet Tail-Like Infiltrate and Foreign Body Granuloma.

Biocompatibility and tissue integration of a surgical suture are decisive factors for wound healing and therefore for the success of sutures. The opti...
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