Methods
Animal Studies
The experiments were officially approved by the local Animal Care and Use Review Committee (50.203.2-AC46, 38/02). All animals received humane 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.
A total of 75 male MMP-2/LacZ transgenic CD1-tg mice (mean body weight 28.2 ± 2.0 g) were randomly divided into five groups according to the type of mesh material used. All animals were kept under standardized conditions: temperature between 22°C and 24°C; relative humidity 50–60%; 12 h of light following 12 h of darkness. The animals had free access to food and water. Food was withdrawn 12 h before and after surgery. All operations were carried out under general anesthesia and aseptic and sterile surgical conditions.
MMP-2/LacZ Transgenic Mice Model
The mice strain used as animal model has been described recently. In brief, mouse strain F8 harbour a β-galactosidase reporter gene (LacZ) under control of MMP-2 regulatory sequences -1686/+423 that extend to the middle of the second exon. Thereby cells with MMP-2 promoter activity can be detected by a monoclonal anti-β-galactosidase antibody. A part of this regulatory sequence is the response element-1 (RE-1) that extends from -1282/-1322. In previous data a crucial role for the enhancer element RE-1 was proven in injury-induced MMP-2 transcription of the skin. To determine the significance of the response element-1 (RE-1) in foreign body reaction and tissue integration in response to the surface modified biomaterials, the sequences -1282/-1322 were deleted and F8del mice were created that harbour MMP-2 regulatory sequence −1241/+423, thereby excluding the RE-1. Seventy five of those mice were used for the investigations.
Mesh Material
Overall five different mesh modifications with 0.5 × 0.5 cm in size were implanted: PVDF, a low-weight, large porous and elastic mesh made of polyvinylidenfluoride monofilaments (FEG Textiltechnik mbH, Aachen, Germany) was the basic prosthetic material for the construction of all mesh samples. Plasma-induced graft polymerization was used to modify the surface chemistry and morphology of the PVDF mesh samples. Immediately after treatment, oxygen was introduced into the chamber to generate hydroperoxide as well as other functional groups on the sample surface (FEG Textiltechnik mbH, Aachen, Germany). Thereafter, graft polymerization of polyacrylic acid onto the plasma treated surface of the PVDF mesh samples was performed resulting in a polyacrylic acid monomer layer on the surface (PVDF + PAAc). The antibiotic gentamicin was bound to the active sites of the grafted mesh surface in three different concentrations respectively (PVDF + PAAc +2 μg/mg Gentamicin; PVDF + PAAc + 5 μg/mg Gentamicin, and PVDF + PAAc + 8 μg/mg Gentamicin). The efficacy of the antimicrobial mesh samples investigated by agar diffusion test, the gentamicin release from the mesh surface, and the cytotoxic side effects after direct contact to L929 mouse fibroblasts (BioWhittaker BE71-131F) were tested in a previously published study.
Surgical Procedure and Observation Periods
Operations were carried out under sterile surgical conditions and general anaesthesia by intramuscular administration of ketamine (Ketamin 10%, Sanofi-Ceva, Düsseldorf, Germany) and xylazine (Rompun 2%, Bayer, Leverkusen, Germany). Following the induction of anaesthesia, the skin was shaved and disinfected with polyvidone iodine solution (Braunosan Vet®, B. Braun Vet Care GmbH, Tuttlingen, Germany). Full thickness dermal incisions extending over 1.5 cm were performed 1 cm bilateral of the abdominal midline. Polymers (size: 0.5 × 0.5 cm) were implanted subcutaneously 1 cm distal of the xiphoid. In each animal two of the same polymeric mesh materials were implanted bilateral of the abdominal midline respectively. Following mesh implantation skin closure was obtained with 3/0 polypropylene (Prolene®, Ethicon Inc., Somerville, NJ, USA) single sutures. No additional antibiotic treatment was given before or during the experiments. Throughout the whole observation period all animals were objectively controlled and underwent daily clinical investigation to assess local and systemic complications. 7, 21 and 90 days after mesh implantation n = 5 animals in each group were euthanized by isoflurane (Attane™, MINRAD INC., Buffalo, NY, USA) asphyxation and decapitation. Tissue specimens for histological and immunohistochemical observations were immediately fixed in 10% formaldehyde.
Histological Assessment and Immunohistochemical Analysis
Briefly, all histological and immunohistochemical investigations including the cross polarization microscopy were performed in the same manner as previously described.
Statistical Analysis
Statistical analysis was carried out using the Statistical Package for Social Sciences (SPSS, Version 17.0, Chicago, IL, USA) software. Data were organized according to the types of meshes used (PVDF; PVDF + PAAc; PVDF + PAAc + 2 μg/mg; PVDF + PAAc + 5 μg/mg; PVDF + PAAc + 8 μg/mg), and to the duration of implantation (7, 21, 90 days). Analysis of histological and immunohistochemical parameters were performed using the Mann-Whitney U test. P values of < 0.05 were considered to be significant. All data are presented as mean ± standard deviation if not otherwise mentioned.