Objective
The impact of polypropylene mesh implantation on vaginal collagen and elastin metabolism was analyzed using a nonhuman primate model to further delineate the mechanism of mesh induced complications.
Study Design
Forty-nine middle-aged parous rhesus macaques underwent surgical implantation of 3 synthetic meshes via sacrocolpopexy. Gynemesh PS (n = 12) (Ethicon, Somerville, NJ) and 2 lower-weight, higher-porosity, lower-stiffness meshes (UltraPro [n = 19] [Ethicon] and Restorelle [n = 8] [Coloplast, Minneapolis, MN]) were implanted, in which UltraPro was implanted with its blue orientation lines perpendicular (low stiffness direction, n = 11) and parallel (high stiffness direction, n = 8) to the longitudinal axis of the vagina. Sham-operated animals were used as controls (n = 10). Twelve weeks after surgery, the mesh-tissue complex was excised and analyzed.
Results
Relative to sham, Gynemesh PS had a negative impact on the metabolism of both collagen and elastin—favoring catabolic reactions, whereas UltraPro induced an increase only in elastin degradation. Restorelle had the least impact. As compared with sham, the degradation of collagen and elastin in the vagina implanted with Gynemesh PS was increased with a simultaneous increase in active matrix metalloproteinase (MMP)-1, -8, -13, and total MMP-2 and -9 (all P < .05). The degradation of elastin (tropoelastin and mature elastin) was increased in the UltraPro-implanted vagina with a concomitant increase of MMP-2, and -9 (all P < .05). Collagen subtype ratio III/I was increased in Gynemesh PS and UltraPro perpendicular groups ( P < .05).
Conclusion
Following implantation with the heavier, less porous, and stiffer mesh, Gynemesh PS, the degradation of vaginal collagen and elastin exceeded synthesis, most likely as a result of increased activity of MMPs, resulting in a structurally compromised tissue.
Lightweight polypropylene mesh has been widely used in the surgical repair of pelvic organ prolapse to improve anatomical outcomes. However, mesh-related complications including mesh exposure through the vaginal wall and erosion into adjacent structures, pain, and infection have raised concerns, prompting the Food and Drug Administration to issue 2 public health notifications warning of complications related to prolapse mesh and calling for mechanistic studies.
To date, the impact of mesh on the vagina has not yet been clearly defined, and the mechanism by which mesh complications occur remains unknown. In a well-controlled nonhuman primate sacrocolpopexy model, heavier weight meshes with lower porosity, and higher stiffness were shown to have a profoundly negative impact on the vagina including a decrease in the amount of collagen, elastin, and smooth muscle.
The resulting thinner and biomechanically inferior vagina seemed a perfect scenario for the development of mesh exposure, a process in which mesh becomes visible through the vaginal epithelium. Because collagen and elastin are key structural proteins that maintain the mechanical and structural integrity of the vagina, their content and stability are likely critical factors in the pathogenesis of mesh exposures.
In this study, we aimed to define alterations in collagen and elastin metabolism following the implantation of synthetic meshes varying by weight, porosity, and stiffness. We hypothesized that heavier, less porous, and stiffer meshes would be associated with increased collagen and elastin degradation characterized by increased matrix metalloproteinases (MMPs) and an increased ratio of collagen subtypes III/I.
To test this hypothesis, we compared the impact of 3 distinct polypropylene meshes with varying textile and structural properties: the heavier, less porous and stiffer prolapse mesh (Gynemesh PS; Ethicon, Somerville, NJ) vs 2 lighter, more porous, and less stiff meshes with (Ultrapro; Ethicon) and without (Restorelle; Coloplast, Minneapolis, MN) an absorbable component: poliglecaprone 25. Meshes were implanted via sacrocolpopexy in the rhesus macaque. Because UltraPro is highly anisotropic, it was implanted with its blue orientation lines perpendicular (low stiffness direction) and parallel (high stiffness direction) to the longitudinal axis of the vagina. The production and degradation of collagen and elastin, the collagen subtype III/I ratio, and the levels of MMP-1, MMP-2, MMP-8, MMP-9, and MMP-13 were examined.
Materials and Methods
Mesh
Sterile samples of Gynemesh PS, UltraPro, and Restorelle were obtained. Their structural properties were described previously.
Animals
Animal groups in the current study were the same as those in a previous study except that a new animal was added to the UltraPro Perpendicular group. Parous middle-aged, nonhuman primates (rhesus macaques) were maintained and treated according to protocols approved by the Institutional Animal Care Use Committee of the University of Pittsburgh (no. 1008675) and in adherence to the National Institutes of Health Guidelines (Washington, DC) for the use of laboratory animals.
Surgical procedures
Two animals were excluded from the study at the time of surgery, the first because of a large mass in her right leg and enlarged pelvic lymph nodes and a second with stage IV endometriosis. In the end, a total of 49 animals were used. Thirty-nine animals were implanted with mesh via sacrocolpopexy after hysterectomy : Gynemesh PS (n = 12), UltraPro Perpendicular (n = 11), UltraPro Parallel (n = 8), and Restorelle (n = 8). Ten animals underwent the identical surgery (sham) without insertion of mesh (n = 10). Twelve weeks later, the mesh-tissue complex was harvested en toto, and the epithelium was carefully removed prior to biochemical analyses.
Western blot: precursors of collagen I, and III
Following extraction using a high salt buffer (pH 7.5), the total protein concentration was determined in duplicate (DC protein assay; Bio-Rad Laboratories, Hercules, CA). Proteins at 10 μg/well were separated on 8% polyacrylamide gels and examined by standard procedures of Western blot. Precision plus Protein WesternC standards (Bio-Rad Laboratories) were used to indicate the molecular weight.
Primary antibodies included COL1A1 1:400 (L-19, goat polyclonal; Santa Cruz Biotechnology Inc, Santa Cruz, CA) and COL3A1 1:200 (C-15, goat polyclonal; Santa Cruz Biotechnology). Signal intensity of bands was quantitated via UN-SCAN-IT (version 4.3; Silk Scientific Co, Orem, UT). The blotted membranes were stained with Coomassie Blue, and the protein bands were quantified to represent the loading control for each well.
Protein amounts were expressed as arbitrary units, relative to the loading control and an internal positive control (protein extracts from a human prolapsed vagina) that was loaded in duplicate on each gel.
Western blot: tropoelastin and tropoelastin degradation
Tropoelastin monoclonal antibody at 1:200 (BA-4, mouse; Abcam, Cambridge, MA) and polyclonal antibody at 1:400 (ab21605, rabbit; Abcam) were used to detect tropoelastin at approximately 60 kDa (monoclonal) and tropoelastin degradation products (series of bands <50 kDa), respectively. To reduce the amount of nonspecific binding by the polyclonal antibody, we did the following: (1) established optimal binding conditions utilizing a progressive series of dilutions of the primary antibody and (2) confirmed the absence of nonspecific binding by the secondary antibody by performing parallel blots in which the primary antibody was eliminated.
Western blot: MMP-1, MMP-8, and MMP-13
The primary antibodies including MMP-1 at 1:200 (41-1E5, mouse monoclonal, recognizing both latent and active forms; EMD Millipore, Temecula, CA), MMP-8 at 1:400 (115-13D2, mouse monoclonal, recognizing both latent and active forms, EMD Millipore), and MMP-13 at 1:200 (VIIIA2, mouse monoclonal recognizing both proenzyme and active forms; EMD Millipore) were used. The band detection and quantification were similar to the procedures described above.
Gelatin zymography for MMP-2 and MMP-9
The level of elastin degrading enzymes, MMP-2 and -9, was evaluated via substrate zymography by using 30 μg protein per sample as described.
Interrupted sodium dodecyl sulfate-polyacrylamide gel electrophoresis for collagen subtypes
After protein extraction, the salt-insoluble tissue pellets were used to determine the ratio of collagen subtypes III/I. Following pepsin digestion, samples were isolated on 6% gels by interrupted sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified collagen type I and III (Abcam) and protein standards (prestained SDS-PAGE standards high range; Bio-Rad Laboratories) were also run on the gels to indicate molecular weight. Semiquantification of collagen bands was performed by the densitometric scanning of protein bands corresponding to α1(I) and α1(III) chains on an imaging densitometer (Bio-Rad Laboratories). The relative collagen subtype III/I ratio was determined as α1(III) ×2/α1(I) ×3.
Assay for degradation products of collagen: NTx
Peptides less than 30 kDa were isolated using centrifugal filter units (Amicon Ultra, 30,000 MWCO; Millipore, Billerica, MA). The total amounts of cross-linked N-telopeptides (NTx) including those derived from completely degraded collagen (end degradation products) and those from intermediate collagen degradation fragments (intermediate degradation products) were measured using a standard NTx assay (Osteomark NTx, Princeton, NJ). The values were calculated using a 4-parameter standard curve and expressed as nanomoles per gram protein normalized to collagen content.
The samples were first digested with bacterial collagenase (type I, 2 mg/mL; Worthington Biochemical Corporation, Lakewood, NJ) to degrade the intermediate collagen fragments prior to assaying for the total NTx. The amount of intermediate degradation products was estimated by subtracting the amount of NTx in the end products from total NTx.
Assays for degradation products of mature elastin
The amount of desmosine, a cross-link that is characteristic of mature elastin, was measured in the peptide solution less than 30 kDa via a desmosine cross-link radioimmunoassay (crosslinks per total protein) as previously described.
Statistical analysis
SPSS software (14.0 student version for Windows; SPSS Inc, Chicago, IL) was used for statistical analyses. For normally distributed data, a 1-way analysis of variance was used, followed by the appropriate post-hoc tests including Dunnett for comparison with sham and pair-wise test using the Bonferroni multiple comparison procedure between all groups. For nonparametric data, a Kruskal-Wallis test was used.
Results
Animals had similar age, parity, weight, and Pelvic Organ Prolapse Quantification scores except that the animals in the Restorelle group were heavier ( Table ).
| Groups | Age, y a | Parity b | Weight, kg a | POP-Q stage b |
|---|---|---|---|---|
| Sham | 13.3 ± 2.6 | 3.5 (2, 6) | 7.5 ± 1.3 | 0 (0, 1) |
| Gynemesh | 12.3 ± 2.4 | 4 (2, 5) | 7.9 ± 1.6 | 0 (0, 0) |
| UltraPro Per | 12.0 ± 2.5 | 2 (1.5, 4.5) | 7.4 ± 1.3 | 0 (0, 1) |
| UltraPro Par | 12.9 ± 1.0 | 4 (4, 5.5) | 8.4 ± 1.3 | 0 (0, 0) |
| Restorelle | 13.8 ± 1.7 | 5 (3, 5.3) | 10.0 ± 2.8 | 1 (0, 1) |
| P value c | .43 | .66 | .02 | .30 |
b Median (first quartile, third quartile)
Synthesis of precursors of collagen and elastin
Two bands at approximately 140 kDa and approximately 200 kDa were detected, representing the precursors of collagen Iα1 ( Figure 1 , A). The lower bands (approximately 140 kDa) likely represent soluble collagen I α1 chains prior to cross-linking and incorporation into mature collagen fibrils. Collagen I precursors were significantly increased in all mesh groups relative to sham, with an increase of 66%, 63%, 46%, and 43% in Gynemesh PS ( P = .014), UltraPro Perpendicular ( P = .023), UltraPro Parallel ( P = .026), and Restorelle ( P = .018), respectively. No difference was found between the 2 UltraPro groups ( P = .57).
For precursors of collagen type III, a single band representing collagen III α1 chains was detected at approximately 160 kDa. As shown in Figure 1 , B, collagen III precursors increased 26% with Gynemesh PS ( P = .03) and 29% with UltraPro Perpendicular ( P = .005), whereas no differences were found relative to sham in the other mesh groups (all P > .05). Collagen III precursors were 45% higher in the UltraPro Perpendicular than the UltraPro Parallel group ( P = .004). Bands for tropoelastin were detected at approximately 60 kDa ( Figure 1 , C) with no significant differences found between the mesh groups and sham (all P > .05).
Collagen subtype III/I ratio in salt-insoluble collagen
As shown in Figure 2 , relative to sham (0.20 ± 0.05), the ratio of collagen subtype III/I was 66% higher in Gynemesh PS (0.33 ± 0.04, P < .001) and 55% higher in UltraPro Perpendicular (0.31 ± 0.06, P < .001). No statistical difference was found in UltraPro Parallel (0.24 ± 0.06, P = .08) and Restorelle (0.25 ± 0.09, P = .17) relative to sham. Comparison between the 2 UltraPro groups showed that the ratio was 26% higher in the perpendicular orientation than that in the parallel orientation ( P = .03).
Collagen degradation
NTx, a marker of mature collagen degradation, was assayed for both end products and intermediate products as a measurement of total collagen breakdown. As shown in Figure 3 , consistent with our previous finding demonstrating a decrease in vaginal collagen content following the implantation of Gynemesh PS, the total collagen degradation was increased by 62% in the Gynemesh PS group relative to sham ( P = .007). The other mesh groups were not statistically different from sham (all P > .05). In addition, the intermediate degradation products were increased by 89% in the Gynemesh PS group ( P = .008) relative to sham, whereas no significant increase was found in the other groups. The collagen degradation was not different between the 2 UltraPro groups ( P = .513 and P = .909).
