Objective
The objective of the study was to compare the influence of collagen-coated vs uncoated polypropylene meshes on the expression of genes critical for wound healing.
Study Design
In 54 rats, abdominal wall defects were created, repaired by polypropylene sutures, and covered by an overlay of coated polypropylene (n = 20), uncoated polypropylene (n = 18), or no mesh (n = 16). Explants were harvested 7 or 90 days after repair and divided for histological, immunohistochemical, and messenger ribonucleic acid (mRNA) analyses. Real-time quantitative polymerase chain reaction arrays were used to profile the expression of 84 genes at the tissue-mesh interface.
Results
One week after implantation, coated mesh elicited a slightly greater inflammatory response and increased mRNA expression of 4 proinflammatory cytokines compared with uncoated mesh. Both materials, however, induced a comparable expression of cytokines and matrix metalloproteinases relative to suture repair 90 days after implantation.
Conclusion
Collagen-coated polypropylene mesh induces elevated inflammatory cytokine expression compared with uncoated mesh early in the healing process, but the response to both meshes is similar 90 days after implantation.
The use of synthetic mesh for the transvaginal repair of pelvic organ prolapse has become increasingly popular as a mechanism to help improve the high failure rates observed with traditional procedures. Polypropylene mesh is currently the most commonly used synthetic graft material in gynecologic surgery. New variants are introduced frequently that are designed to reduce the inflammatory response and improve tissue ingrowth in an attempt to decrease local graft-related complications including erosion, infection, excessive scarring, and resulting dyspareunia. No mesh, however, has demonstrated optimal biocompatibility and incorporation into host tissue, and limited long-term clinical data exist to determine which, if any, of the several variants is the best choice for vaginal surgery.
The host response to implanted mesh follows a cascade of events involved in wound healing including coagulation, inflammation, angiogenesis, epithelialization, fibroplasia, matrix deposition, and contraction. Biocompatibility is determined by the intensity of the foreign body reaction elicited by the mesh material and the host’s ability to resolve the injury to the tissues during implantation. Mesh characteristics such as pore size, chemical composition, filament structure, amount of implanted material, and biodegradability affect the processes of inflammation, angiogenesis, and tissue formation, and consequently may alter the wound healing process.
Inflammatory cells recruited to the site of implantation produce signaling molecules that affect the tissue response to the biomaterial, but the molecular mechanisms directing this foreign body response are poorly understood. Macrophages that adhere to the surface of implanted mesh become activated in an attempt to phagocytose the mesh fibers and fuse to form foreign body giant cells (FBGCs). The subsequent secretion of cytokines, degradative enzymes, and reactive oxygen intermediates by these activated macrophages and FBGCs directs the inflammatory and wound-healing response to the material by influencing the behavior of other cell types including neutrophils (polymorphonuclear leukocytes [PMNs]), monocytes, lymphocytes, and fibroblasts.
Our laboratory recently reported in vivo expression profiles for a wide array of genes involved in angiogenesis, wound healing, and extracellular matrix remodeling after the implantation of a variety of synthetic mesh materials used in inguinal and abdominal hernia repair including polypropylene, polyester, and polytetrafluoroethylene. Evaluation of gene expression profiles and histologic specimens revealed that polypropylene and polyester induced a greater and more persistent inflammatory response than polytetrafluoroethylene, which elicited a response most similar to that induced by suture repair.
This study showed that different meshes induce the differential expression of inflammatory cytokines, matrix metalloproteinases, and growth factors, thereby influencing the extent of the foreign body reaction to these materials. Furthermore, these results suggested that the particular mesh chosen for repair may affect the patient’s wound healing response and clinical outcome. Increased understanding of the host response to implanted materials at the molecular level is needed to develop improved grafts with better wound-healing properties and fewer complications and to develop therapeutic targets intended to optimize the wound-healing response after implantation.
In the current study, we used a rat abdominal wall repair model to determine whether the foreign body response differs at the molecular level for 2 monofilament polypropylene meshes commonly used in vaginal surgery, including an uncoated mesh and a mesh that is coated with a hydrophilic, resorbable film of porcine collagen. The coating is intended to provide a theoretical protective effect by facilitating tissue ingrowth, decreasing the initial inflammatory response during healing, and thus decreasing the risk of mesh erosion, although evidence from clinical and animal studies to support this claim is lacking.
de Tayrac et al found that in the early postoperative period (1 week), coated polypropylene mesh implanted in the sheep vagina was less well integrated into the surrounding tissue and showed increased exudative and acute inflammation than the uncoated mesh. Boulanger et al reported that the complete resorption of the collagen coating occurred at about 15 days, and they found that the coated mesh demonstrated delayed tissue integration compared with uncoated mesh 14 days after implantation in a rat abdominal model. Furthermore, Huffaker et al found a statistically greater percentage of apoptotic cells at the interface of mesh and host tissue in rabbits vaginally implanted with coated mesh.
Materials and Methods
Animals
A total of 54 adult male Sprague Dawley rats weighing approximately 300 g were used. Animals were obtained from Taconic (Germantown, NY) and housed in the Tripler Army Medical Center animal facility. Rats were assigned to abdominal wall repair using coated polypropylene (n = 20), uncoated polypropylene (n = 18), or suture repair with no mesh (n = 16) and were killed at either 7 or 90 days after repair. A power calculation prior to the commencement of the study determined that a sample size of 7 animals per group will allow detection of a 30% difference among means (assuming an SD of 15% of the mean) at an alpha level of 0.05 with 95% statistical power and a calculated effect size of 0.94 using a 1-way analysis of variance (ANOVA) design.
The study protocol was approved by the Institutional Animal Care and Use Committee at Tripler Army Medical Center. Investigators complied with the policies as prescribed in the US Department of Agriculture Animal Welfare Act and the National Research Council’s Guide for the Care and Use of Laboratory Animals. Facilities are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.
Mesh materials and study design
Full-thickness abdominal wall defects were repaired by polypropylene sutures to mimic native tissue repair and, in select cases, were covered by an overlay of monofilament polypropylene mesh coated with porcine collagen (Pelvitex; C. R. Bard, Inc, Covington, GA) or an uncoated monofilament polypropylene mesh (Prolene Soft; Ethicon, Inc, Somerville, NJ). Both macroporous meshes are similar in weight (38 and 41 g/m 2 for coated and uncoated polypropylene, respectively) and pore size (1.5 and 1.7 mm 2 for coated and uncoated polypropylene, respectively). Meshes were cut into uniform strips at the time of surgery by using a precut plastic sterile template. Rats were killed 7 or 90 days after repair, and harvested explants (mesh with underlying abdominal wall) were divided for histological, immunohistochemical, and gene expression analyses.
Surgery and tissue collection
Surgery was performed as described. Briefly, a 3.0 × 0.5 cm longitudinal full-thickness defect was created in the left lateral abdominal wall, which was subsequently closed with a continuous 4.0 polypropylene suture. In animals from the mesh repair groups, a 3.5 × 2.0 cm strip of mesh was fixed to the abdominal wall over the defect using 5.0 polypropylene sutures at its corners with a running 5.0 polypropylene suture (interrun distance 0.5 cm) along all four sides. The subcutaneous tissues and skin were closed with interrupted 3.0 nylon sutures. Rats were fitted with Elizabethan collars to prevent chewing on the incision site.
Animals were evaluated at least twice daily for the initial 48 hours after surgery and at least weekly thereafter for herniation or infection. On days 7 or 90, rats were killed, and the mesh with the underlying full-thickness abdominal wall (or similar region in suture repair animals) was removed. Length and width of the explanted mesh were measured to determine shrinkage (decrease in surface area) of the implant.
Histology
Specimens were fixed in formalin and embedded in paraffin, and serial sections (5 μm) were stained with hematoxylin-eosin, elastic/van Gieson, and Masson trichrome. Inflammation, neovascularization, and fibroblastic proliferation were scored on a scale of 0 to 4 (0, none; 1, minimal; 2, mild; 3, moderate; 4, severe) by a pathologist (J.R.M.) blinded to treatment as described.
In addition, microscopic evaluation was performed to quantify the presence of FBGCs, PMNs, mononuclear cells (MN), newly formed vessels, and collagen deposition (amount and organization) using a semiquantitative scale analogous to that used by others ( Table 1 ). Five nonoverlapping fields per slide were scored at magnification ×400 by 2 blinded investigators (J.R.A. and L.MP.) at the tissue-mesh interface, and average scores were calculated. Digital images were captured using PictureFrame software (Optronics, Goleta, CA) and an Olympus IX71 microscope (Olympus America Inc, Center Valley, PA).
| Category | Score | |||
|---|---|---|---|---|
| 0 | 1 | 2 | 3 | |
| FBGCs a | 0 | 1–5 | 6–10 | >10 |
| PMNs a | 0 | 1–5 | 6–10 | >10 |
| MNs a | 0 | 1–5 | 6–10 | >10 |
| Vascularity a | 0 | 1–3 | 4–10 | >10 |
| Collagen amount | None | Mild | Moderate | Abundant |
| Collagen organization | Totally disorganized | Slightly organized | Moderately organized | Well organized |
a Number per high-power field (original magnification ×400).
Immunohistochemistry
Immunohistochemistry was performed using matrix metalloproteinase (MMP) 9 (Millipore, Temecula, CA) and chemokine (C-X-C motif) ligand 2 (CXCL2) (BioVision Research Products, Mountain View, CA) rabbit polyclonal antibodies as described. These markers were chosen for immunohistochemical analysis after demonstrating marked up-regulation in the expression profiles from mesh groups relative to suture repair. Degree of positive immunostaining was scored on a scale of 0 to 4 (0, none; 1, minimal; 2, mild; 3, moderate; 4, intense) by an investigator blinded to treatment (P.T.N.). Two sections per specimen per antibody were examined to confirm similar staining patterns within each section, and average scores were calculated.
Gene expression profiling
Samples were stored in RNALater (Ambion Inc, Austin, TX) at −20°C until ribonucleic acid (RNA) isolation was performed. Samples were homogenized using an Omni GLH homogenizer (Omni International, Kennesaw, GA), and total RNA was isolated using the Trizol method according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Extracted RNA was further purified using the RT 2 qPCR-grade RNA isolation kit (SABiosciences, Frederick, MD), quantified using a spectrophotometer, and stored at −70°C.
Complementary deoxyribonucleic acid was generated from 1 μg total RNA using the RT 2 first strand kit (SABiosciences) and analyzed using a rat RT 2 profiler PCR angiogenesis array (SABiosciences) by means of a Bio-Rad iCycler real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions. This array is a set of optimized real-time PCR primer assays on 96-well plates for pathway-focused genes as well as appropriate RNA quality controls and internal control housekeeping genes to normalize the data for the amount of RNA added to each reverse transcription reaction.
The rat RT 2 profiler PCR angiogenesis array (SABiosciences) profiles the expression of 84 genes involved in modulating the biological processes of angiogenesis and wound healing. A complete list of genes contained in the array can be viewed on the following link: http://www.sabiosciences.com/rt_pcr_product/HTML/PARN-024A.html . Arrays were repeated with 3-5 different rats in each experimental group using total RNA isolated from a single rat per array.
Statistical analysis
Values were expressed as mean ± SEM. Mesh-induced changes in gene expression analyzed using the RT 2 profiler PCR array (SABiosciences) were calculated using software from SABiosciences. Differentially expressed genes were considered significant using a cutoff value greater than a 4-fold change and P < .01. ANOVA was used to to determine differences in histologic parameters and immunohistochemical staining among experimental groups, and the Tukey’s test for pairwise multiple comparisons was used for post hoc analyses to identify specific differences. Comparisons were also performed using the Student t test. Corresponding nonparametric tests were used when indicated. Statistical analyses were performed using SigmaStat 3.5 software (Systat Software, Inc, Point Richmond, CA), with P < .05 considered significant.
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