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Long-term histologic response to synthetic and biologic graft materials implanted in the vagina and abdomen of a rabbit model

  • Lisa M. Pierce
    Affiliations
    Department of Obstetrics and Gynecology, Scott & White Memorial Hospital and Clinic, Texas A&M University Health Science Center College of Medicine, Temple, TX
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  • Arundhati Rao
    Affiliations
    Department of Pathology, Scott & White Memorial Hospital and Clinic, Texas A&M University Health Science Center College of Medicine, Temple, TX
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  • Shannon S. Baumann
    Affiliations
    Department of Obstetrics and Gynecology, Scott & White Memorial Hospital and Clinic, Texas A&M University Health Science Center College of Medicine, Temple, TX
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  • Jocylen E. Glassberg
    Affiliations
    Department of Obstetrics and Gynecology, Scott & White Memorial Hospital and Clinic, Texas A&M University Health Science Center College of Medicine, Temple, TX
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  • Thomas J. Kuehl
    Affiliations
    Department of Obstetrics and Gynecology, Scott & White Memorial Hospital and Clinic, Texas A&M University Health Science Center College of Medicine, Temple, TX

    Department of Pathology, Scott & White Memorial Hospital and Clinic, Texas A&M University Health Science Center College of Medicine, Temple, TX

    Molecular and Cellular Medicine, Scott & White Memorial Hospital and Clinic, Texas A&M University Health Science Center College of Medicine, Temple, TX
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  • Tristi W. Muir
    Affiliations
    Department of Obstetrics and Gynecology, Scott & White Memorial Hospital and Clinic, Texas A&M University Health Science Center College of Medicine, Temple, TX
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Published:March 16, 2009DOI:https://doi.org/10.1016/j.ajog.2008.12.040

      Objective

      We sought to evaluate the effects of graft type, site of implantation, and ovariectomy on the long-term histologic response to graft materials used in pelvic reconstructive surgery.

      Study Design

      In all, 45 rabbits underwent ovariectomy or sham laparotomy and were implanted with polypropylene (PP) mesh (n = 23) or cross-linked porcine dermis (PS) (n = 22) in the vagina and abdomen. Grafts were harvested 9 months later and were processed for histology to evaluate the host inflammatory response and degree of tissue incorporation within the grafts.

      Results

      Polypropylene induced a milder (P < .007), more uniform response than PS, whereas PS elicited a more variable response and degraded by 9 months. Vaginal grafts had higher scores for inflammation (P = .005) and neovascularization (P < .001), and had lower scores for fibroblastic proliferation (P < .001) than abdominal grafts. Ovariectomy had no effect (P > .05).

      Conclusion

      Synthetic and biologic grafts evoke different foreign body responses, which may have implications for surgical outcomes in women.

      Key words

      Because of the high failure rates of traditional procedures for the repair of pelvic organ prolapse using native tissue, current techniques in pelvic reconstructive surgery are being reexamined in an effort to find a mechanism to help improve long-term cure rates.
      • Olsen A.L.
      • Smith V.J.
      • Bergstrom J.O.
      • Colling J.C.
      • Clark A.L.
      Epidemiology of surgically managed pelvic organ prolapse and urinary incontinence.
      • Birch C.
      The use of prosthetics in pelvic reconstructive surgery.
      The use of synthetic and biologic graft materials for the transvaginal repair of pelvic organ prolapse has become prevalent in clinical practice despite a paucity of scientific data evaluating the safety and efficacy of various materials available on the market.
      • Silva W.A.
      • Karram M.M.
      Scientific basis for the use of grafts during vaginal reconstructive procedures.
      Local graft-related complications include erosion, infection, scarring resulting in implant contraction, dyspareunia, and fistula formation.
      • Visco A.G.
      • Weidner A.C.
      • Barber M.D.
      • et al.
      Vaginal mesh erosion after abdominal sacral colpopexy.
      • Kohli N.
      • Miklos J.R.
      Use of synthetic mesh and donor grafts in gynecologic surgery.
      • Baessler K.
      • Hewson A.D.
      • Tunn R.
      • Schuessler B.
      • Maher C.F.
      Severe mesh complications following intravaginal slingplasty.
      For Editors' Commentary, see Table of Contents
      Much of the information about synthetic and biologic grafts comes from research involving the surgical repair of abdominal wall hernias, and thus insufficient knowledge of the vaginal in vivo response to these materials exists.
      • Birch C.
      The use of prosthetics in pelvic reconstructive surgery.
      • Silva W.A.
      • Karram M.M.
      Scientific basis for the use of grafts during vaginal reconstructive procedures.
      The increased vascularity and presence of endogenous microflora in the vagina may have an impact on the host tissue response and biomechanical properties of grafts used in vaginal procedures relative to grafts used in abdominal surgery.
      • Hilger W.S.
      • Walter A.
      • Zobitz M.E.
      • Leslie K.O.
      • Magtibay P.
      • Cornella J.
      Histological and biomechanical evaluation of implanted graft materials in a rabbit vaginal and abdominal model.
      Different characteristics of mesh materials are associated with infection and erosion,
      • Bobyn J.D.
      • Wilson G.J.
      • MacGregor D.C.
      • Pilliar R.M.
      • Weatherly G.C.
      Effect of pore size on the peel strength of attachment of fibrous tissue to porous-surfaced implants.
      • Ziats N.P.
      • Miller K.M.
      • Anderson J.M.
      In vitro and in vivo interactions of cells with biomaterials.
      and, therefore, the same material that has proven to be useful for abdominal surgery may not necessarily be effective for transvaginal surgery.
      Polypropylene (PP) mesh, a permanent material that is neither absorbed nor degraded, is currently the most commonly used synthetic graft material in gynecologic surgery.
      • Birch C.
      The use of prosthetics in pelvic reconstructive surgery.
      The introduction of acellular collagen xenografts, such as porcine dermal (PS) or bovine dermal collagen, porcine small intestinal submucosa, and bovine pericardium, was intended to reduce complications found to occur with the use of synthetic grafts. The host response to xenografts depends mainly on chemical cross-linking and porosity.
      • Trabuco E.C.
      • Klingele C.J.
      • Gebhart J.B.
      Xenograft use in reconstructive pelvic surgery: a review of the literature.
      Noncross-linked xenografts are designed as a scaffold for cellular ingrowth, with eventual replacement of graft material by host connective tissue; however, concern about the use of resorbable xenografts is the loss of strength at the repair site during graft remodeling.
      • Trabuco E.C.
      • Klingele C.J.
      • Gebhart J.B.
      Xenograft use in reconstructive pelvic surgery: a review of the literature.
      Chemical cross-linking is intended to increase resistance to degradation by host collagenases, but the long-term fate of cross-linked xenografts in vivo is presently unknown.
      Prior investigations have used animal models to investigate the properties of natural and synthetic graft materials used in urogynecologic surgery.
      • Dora C.D.
      • DiMarco D.S.
      • Zobitz M.E.
      • Elliott D.S.
      Time dependent variations in biomechanical properties of cadaveric fascia, porcine dermis, porcine small intestine submucosa, polypropylene mesh and autologous fascia in the rabbit model: implications for sling surgery.
      • Walter A.J.
      • Morse A.N.
      • Leslie K.O.
      • Zobitz M.E.
      • Hentz J.G.
      • Cornella J.L.
      Changes in tensile strength of cadaveric human fascia lata after implantation in a rabbit vagina model.
      • Walter A.J.
      • Morse A.N.
      • Leslie K.O.
      • Hentz J.G.
      • Cornella J.L.
      Histologic evaluation of human cadaveric fascia lata in a rabbit vagina model.
      • Yildirim A.
      • Basok E.K.
      • Gulpinar T.
      • Gurbuz C.
      • Zemheri E.
      • Tokuc R.
      Tissue reactions of 5 sling materials and tissue material detachment strength of 4 synthetic mesh materials in a rabbit model.
      • FitzGerald M.P.
      • Mollenhauer J.
      • Brubaker L.
      Donor fascia in urogynecological procedures: a canine model.
      • Badylak S.
      • Kokini K.
      • Tullius B.
      • Simmons-Byrd A.
      • Morff R.
      Morphologic study of small intestinal submucosa as a body wall repair device.
      • Huffaker R.K.
      • Muir T.W.
      • Rao A.
      • Baumann S.S.
      • Kuehl T.J.
      • Pierce L.M.
      Histologic response of porcine collagen-coated and uncoated polypropylene grafts in a rabbit vagina model.
      • de Tayrac R.
      • Alves A.
      • Therin M.
      Collagen-coated vs noncoated low-weight polypropylene meshes in a sheep model for vaginal surgery: a pilot study.
      • Claerhout F.
      • Verbist G.
      • Verbeken E.
      • Konstantinovic M.
      • De Ridder D.
      • Deprest J.
      Fate of collagen-based implants used in pelvic floor surgery: a 2-year follow-up study in a rabbit model.
      Most studies, however, have implanted grafts in the abdomen, whereas very few studies have investigated graft materials, after implantation in the vagina.
      • Hilger W.S.
      • Walter A.
      • Zobitz M.E.
      • Leslie K.O.
      • Magtibay P.
      • Cornella J.
      Histological and biomechanical evaluation of implanted graft materials in a rabbit vaginal and abdominal model.
      • Walter A.J.
      • Morse A.N.
      • Leslie K.O.
      • Zobitz M.E.
      • Hentz J.G.
      • Cornella J.L.
      Changes in tensile strength of cadaveric human fascia lata after implantation in a rabbit vagina model.
      • Walter A.J.
      • Morse A.N.
      • Leslie K.O.
      • Hentz J.G.
      • Cornella J.L.
      Histologic evaluation of human cadaveric fascia lata in a rabbit vagina model.
      • Huffaker R.K.
      • Muir T.W.
      • Rao A.
      • Baumann S.S.
      • Kuehl T.J.
      • Pierce L.M.
      Histologic response of porcine collagen-coated and uncoated polypropylene grafts in a rabbit vagina model.
      • de Tayrac R.
      • Alves A.
      • Therin M.
      Collagen-coated vs noncoated low-weight polypropylene meshes in a sheep model for vaginal surgery: a pilot study.
      The New Zealand White rabbit vagina model has recently been established as a valuable model to investigate host responses to graft implantation.
      • Hilger W.S.
      • Walter A.
      • Zobitz M.E.
      • Leslie K.O.
      • Magtibay P.
      • Cornella J.
      Histological and biomechanical evaluation of implanted graft materials in a rabbit vaginal and abdominal model.
      • Walter A.J.
      • Morse A.N.
      • Leslie K.O.
      • Zobitz M.E.
      • Hentz J.G.
      • Cornella J.L.
      Changes in tensile strength of cadaveric human fascia lata after implantation in a rabbit vagina model.
      • Walter A.J.
      • Morse A.N.
      • Leslie K.O.
      • Hentz J.G.
      • Cornella J.L.
      Histologic evaluation of human cadaveric fascia lata in a rabbit vagina model.
      • Huffaker R.K.
      • Muir T.W.
      • Rao A.
      • Baumann S.S.
      • Kuehl T.J.
      • Pierce L.M.
      Histologic response of porcine collagen-coated and uncoated polypropylene grafts in a rabbit vagina model.
      To date, studies using this vagina model have evaluated very few graft materials with only short-term outcomes (up to 12 weeks). No studies have determined the effects of ovariectomy, and only 1 study has compared vaginal and abdominal graft placement in the same animal.
      • Hilger W.S.
      • Walter A.
      • Zobitz M.E.
      • Leslie K.O.
      • Magtibay P.
      • Cornella J.
      Histological and biomechanical evaluation of implanted graft materials in a rabbit vaginal and abdominal model.
      In the current study we evaluated the effects of graft type (PP vs cross-linked PS), site of implantation (vagina vs abdomen), and ovariectomy on the long-term (9 months) histologic response to synthetic and biologic graft materials commonly used in pelvic reconstructive surgery.

      Materials and Methods

      Animal subjects

      A total of 45 adult New Zealand White female breeder rabbits (∼ 4 kg) were used in this study. Animals were obtained from Myrtle's Rabbitry (Thompson Station, TN) and were housed at our animal facility. Rabbits were assigned randomly to group 1 (PP, intact ovaries, n = 13), group 2 (PP, ovariectomy, n = 10), group 3 (PS, intact ovaries, n = 13), or group 4 (PS, ovariectomy, n = 9). Guidelines for the care and use of these animals, approved by our institutional animal care and use committee, were followed.

      Graft materials and study design

      Rabbits were randomly assigned to undergo ovariectomy or sham laparotomy and were surgically implanted with a macroporous, monofilament PP mesh (Gynemesh; Ethicon Inc, Somerville, NJ) (n = 23) or cross-linked, perforated, acellular PS collagen (PelviSoft Acellular Collagen BioMesh; C.R. Bard Inc, Covington, GA) (n = 22) in the abdomen and vagina (same graft type at both sites). Grafts were harvested 9 months after implantation and underwent histologic evaluation and biomechanical properties testing (tensile testing results are reported in a separate study).
      All rabbits implanted with PP had 2 grafts placed in the abdomen, and the majority of these animals had 2 grafts placed in the posterior vagina. One graft at each site was designated for histology and the other was designated for biomechanical properties testing. Vaginal PP grafts were 0.8 cm wide and ranged in length from 2.9-1 cm, depending on vaginal capacity (1 rabbit had 2 vaginal grafts, both = 2.9 cm long; 9 rabbits had 1 graft = 2.9 cm and the other = 2.0 cm; 6 rabbits had 1 graft = 2.0 cm and the other = 1.0 cm; 2 rabbits had only 1 graft = 2.9 cm; 5 rabbits had only 1 graft = 2.0 cm). Variability in vaginal size and initial higher than expected vaginal erosion rates obtained with the 2.9 cm grafts caused us to modify the size of implanted grafts during the course of this long-term study. Abdominal PP grafts were 0.8 cm wide and either 2.9 cm in length (n = 14 rabbits) or 2.0 cm in length (n = 9 rabbits). Rabbits implanted with PS had 2 grafts (0.8 × 2.0 cm) placed in the abdomen. Preliminary studies determined that the rabbit vagina would not support 2 pieces of PS, so only 1 PS graft (2.0 × 0.8 cm, n = 2 rabbits; 1.2 × 0.8 cm, n = 20 rabbits) was implanted in the vagina that was designated for histologic analysis. Grafts were cut using a sterile template.

      Surgery and tissue collection

      Rabbits were anesthetized as described previously.
      • Huffaker R.K.
      • Muir T.W.
      • Rao A.
      • Baumann S.S.
      • Kuehl T.J.
      • Pierce L.M.
      Histologic response of porcine collagen-coated and uncoated polypropylene grafts in a rabbit vagina model.
      To perform the laparotomy, a ventral midline incision from the umbilicus to the pubis was made, and the peritoneal cavity was entered. The uterus was exteriorized, allowing visualization and access to the ovaries. Ovaries were identified in all animals but were surgically removed in only a subset. The ovarian ligament with ovarian vessels was ligated and transected on each side, and the ovaries were discarded. Abdominal wall fascia and rectus abdominis muscle were closed with silk or Vicryl (Ethicon Inc) sutures. Lateral to the incision, 2 grafts were implanted and secured without tension to the rectus abdominis muscle and fascia with 4 5-0 Prolene (Ethicon Inc) sutures per graft. The abdominal skin was then closed with 3-0 Vicryl (Ethicon Inc) sutures. Grafts were implanted without tension in the posterior vaginal wall between the fibromuscular layer and vaginal epithelium as described.
      • Huffaker R.K.
      • Muir T.W.
      • Rao A.
      • Baumann S.S.
      • Kuehl T.J.
      • Pierce L.M.
      Histologic response of porcine collagen-coated and uncoated polypropylene grafts in a rabbit vagina model.
      Rabbits were evaluated postoperatively by the investigators at 1 week, 2 weeks, 4 weeks, and monthly thereafter for mesh erosion or infection. At 9 months after implantation, rabbits were killed, and graftswere killed designated for histologic analysis were harvested with surrounding host tissues.
      • Huffaker R.K.
      • Muir T.W.
      • Rao A.
      • Baumann S.S.
      • Kuehl T.J.
      • Pierce L.M.
      Histologic response of porcine collagen-coated and uncoated polypropylene grafts in a rabbit vagina model.

      Histology

      Tissues were processed as described.
      • Huffaker R.K.
      • Muir T.W.
      • Rao A.
      • Baumann S.S.
      • Kuehl T.J.
      • Pierce L.M.
      Histologic response of porcine collagen-coated and uncoated polypropylene grafts in a rabbit vagina model.
      Serial full-thickness sagittal sections of posterior vaginal wall/rectum and abdomen were cut at -20°C on a cryostat (8 μm thick) and stained with hematoxylin and eosin, Masson trichrome, and elastin/van Gieson (Sigma, St. Louis, MO), with a minimum of 6 sections per specimen per stain. Inflammation, neovascularization, and fibroblastic proliferation were scored on a scale of 0-4 (0 = none, 1 = minimal, 2 = mild, 3 = moderate, 4 = severe) in hematoxylin and eosin-stained sections by a pathologist (A.R.) blinded to treatment. Digital images were obtained using an epifluorescence microscope with appropriate filters (Olympus Provis AX-70; Olympus America Inc, Melville, NY) and a cooled charge-coupled device camera (Olympus America Inc).

      Statistical analysis

      Values are expressed as means with SE. The paired t test or the corresponding nonparametric Wilcoxon signed rank-sum test was used to compare histologic parameters from abdominal vs vaginal grafts in each animal. The Student t test or the corresponding nonparametric Mann-Whitney rank sum test was used to compare PP vs PS graft materials and grafts from animals with and without ovaries. A P value of < .05 was considered significant.

      Results

      Surgeries were well tolerated in all animals; however, 4 rabbits died before their 9-month planned sacrifice. These included 1 from the PP/intact ovaries group (7 months postimplantation; cause of death: unknown), 1 from the PP/ovariectomy group (8 months postimplantation; cause of death: spinal cord injury), and 2 from the PS/intact ovaries group (2 weeks and 4 weeks postimplantation; cause of death: acute pneumonia and eosinophilia/neutropenia). The rabbit that died at 8 months was included in analysis; the 3 others were excluded.
      By 9 months after implantation, erosion had occurred in the vagina of 6/22 (27%) rabbits implanted with PP (2 with and 4 without ovaries) and 3/20 (15%) rabbits implanted with PS (2 with and 1 without ovaries). Decreasing the length of implanted grafts was associated with a decreased erosion rate. When subdividing rabbits implanted with long (2.9 cm) vs short (2.0 cm) PP grafts, vaginal erosion occurred in 5/12 (42%) rabbits implanted with long pieces and in 1/10 (10%) implanted with short pieces. Vaginal erosion of PS occurred in 2/2 (100%) rabbits implanted with 2.0 cm pieces but in only 1/18 (6%) rabbits implanted with 1.2 cm pieces. Erosion did not occur at abdominal sites. Erosion of PP was initially identified at 1 month (n = 3), 2 months (n = 1), and 5 months (n = 2), and erosion of PS was initially identified at 1 month (n = 3) after graft implantation. None of the rabbits that died before study completion had erosion of PP or PS grafts.
      All PP grafts in the vagina and abdomen were identified at necropsy. In contrast, all PS grafts in the abdomen were identified at necropsy, but 6/20 (30%) vaginal PS grafts were missing although sutures were visible. Histology revealed a replacement of missing PS grafts by host collagen and minimal inflammation, with PS graft remnants observed in some specimens (Figure 1). Missing grafts were obtained from 3 different lots of PS from the manufacturer (lot 06B13-2, n = 3; lot 05B32-9, n = 1; lot 06B25-1, n = 2). Severely eroded grafts that were unable to be processed for histology (n = 1 PP graft) and missing grafts (n = 6 vaginal PS grafts) were not included in analysis. In addition, eroded PP grafts that involved epithelial trapping of graft material (n = 3) were excluded from analysis because the observed acute inflammatory response and debris were thought to be elicited by the trapped epithelium and, therefore, did not reflect the true behavior of the graft material, which was our primary interest. Epithelial trapping occurred in animals in which vaginal grafts were implanted too close to or within the thin rabbit epithelial layer, resulting in capturing of the epithelium within the mesh (ie, the dissection of the vaginal epithelium from the fibromuscular layer in these animals was inadequate for graft placement).
      Figure thumbnail gr1
      FIGURE 1Porcine dermis implanted in vagina
      Replacement of vaginal porcine dermal (PS) xenograft with host collagen and minimal inflammation 9 months after implantation. Graft remnants are visible histologically but graft was not identified at necropsy. Mature elastic fibers (arrows) and smooth muscle (SM) are present in native vaginal tissue but are not incorporated into new host tissue, which is poorly vascularized. (Elastin/van Gieson stain; original magnification: ×200.)
      Pierce. Long-term histologic response to synthetic and biologic graft materials implanted in the vagina and abdomen of a rabbit model. Am J Obstet Gynecol 2009.
      Grafts available for histologic analysis included 37 PP samples and 33 PS samples (Table 1). Overall, PP induced a milder and more uniform chronic inflammatory response than PS, with lower scores for inflammation (P = .007, vaginal and abdominal sites combined) and good host tissue incorporation between PP fibers (Table 2 and FIGURE 2, FIGURE 3, FIGURE 4). Inflammatory cells present in both graft types consisted of multinucleated foreign body giant cells (FBGC), macrophages, lymphocytes, plasma cells, and few neutrophils. Eosinophils were present in 3 PP samples and 7 PS samples. When separating by site, PP had lower scores for inflammation (P = .02) and higher scores for fibroblastic proliferation (P = .04) than PS in the vagina (Table 2). In the abdomen, however, histologic parameters between the graft types were not statistically different (P > .05) (Table 2).
      TABLE 1Samples available for histologic analysis
      Vaginal graftsAbdominal grafts
      Graft typeTotal samples+Ovaries–Ovaries+Ovaries–Ovaries
      PP37961210
      PS3386109
      PP, polypropylene; PS, porcine dermis.
      Pierce. Long-term histologic response to synthetic and biologic graft materials implanted in the vagina and abdomen of a rabbit model. Am J Obstet Gynecol 2009.
      TABLE 2Comparison of histologic parameters between polypropylene and porcine dermis grafts harvested 9 months after implantation
      Graft type and graft siteNo. of samplesInflammationNeovascularizationFibroblastic proliferation
      PP (vagina and abdomen)371.3 (0.1)0.8 (0.1)1.8 (0.1)
      PS (vagina and abdomen)332.0 (0.2)1.0 (0.1)1.4 (0.2)
      PP vs PS (vagina and abdomen)P value
      Mann-Whitney rank sum test.
      .007.24.20
      PP (vagina only)151.5 (0.2)1.1 (0.2)1.5 (0.1)
      PS (vagina only)142.4 (0.3)1.4 (0.2)0.8 (0.2)
      PP vs PS (vagina only)P value
      Mann-Whitney rank sum test.
      .02.23.04
      PP (abdomen only)221.2 (0.1)0.6 (0.1)2.0 (0.2)
      PS (abdomen only)191.7 (0.2)0.7 (0.2)1.9 (0.2)
      PP vs PS (abdomen only)P value
      Mann-Whitney rank sum test.
      .13.61.56
      Scores for inflammation, neovascularization, and fibroblastic response were graded on a scale of 0-4 (none-severe) by a pathologist blinded to treatment. Values are presented as mean (SE).
      PP, polypropylene; PS, porcine dermis.
      Pierce. Long-term histologic response to synthetic and biologic graft materials implanted in the vagina and abdomen of a rabbit model. Am J Obstet Gynecol 2009.
      a Mann-Whitney rank sum test.
      Figure thumbnail gr2
      FIGURE 2PP mesh implanted in vagina and abdomen
      Polypropylene (PP) mesh 9 months after implantation in A, vagina and B, abdomen in same animal. Note increased numbers of inflammatory cells in A, vagina and increased fibrosis (encapsulation of PP fibers [*]) in B, abdomen. (A, Masson trichrome and B, elastin/van Gieson stains; original magnifications: ×200.)
      FBGC, foreign body giant cell; SM, smooth muscle.
      Pierce. Long-term histologic response to synthetic and biologic graft materials implanted in the vagina and abdomen of a rabbit model. Am J Obstet Gynecol 2009.
      Figure thumbnail gr3
      FIGURE 3Variable host response to PS
      Porcine dermis (PS) induces variable inflammatory response and poor host tissue incorporation 9 months after implantation in A and C, vagina and in B and D, abdomen. A, Histiocytes are visibly degrading vaginal PS graft. Graft remnants are visible as deep pink collagen fibers. B, Intact PS graft (deep pink) in abdomen encapsulated by new layer of poorly vascularized collagen (light pink), fat cells, and skeletal muscle fibers. Foreign body giant cells (FBGC) are visible on periphery of graft. C, Intact PS graft in vagina demonstrating minimal inflammation without fibrosis. D, PS graft in abdomen demonstrating strong inflammatory response with infiltration of inflammatory cells into graft. A-D, Different animals. (A and B, Elastin/van Gieson and C and D, Masson trichrome stains; original magnifications: ×200.)
      EPI, vaginal epithelium; SM, smooth muscle.
      Pierce. Long-term histologic response to synthetic and biologic graft materials implanted in the vagina and abdomen of a rabbit model. Am J Obstet Gynecol 2009.
      Figure thumbnail gr4
      FIGURE 4Reduced elastic fibers in vaginal grafts
      New host tissue incorporated into or encapsulating vaginal grafts contains reduced elastin content 9 months after implantation in vagina. A, Polypropylene (PP) mesh. B, Porcine dermis (PS). Note paucity of mature elastic fibers (arrows) in A, vicinity of PP fibers (*) and in B, fibrotic tissue encapsulating and infiltrating PS graft. B, Note increased neovascularization vaginal PS graft relative to abdominal PS graft (, B). (Elastin/van Gieson stain; original magnification: ×200.)
      BV, blood vessel; FBGC, foreign body giant cell; SM, smooth muscle.
      Pierce. Long-term histologic response to synthetic and biologic graft materials implanted in the vagina and abdomen of a rabbit model. Am J Obstet Gynecol 2009.
      The host response to PS was more variable than that to PP. At 9 months after implantation, some PS grafts in the vagina and abdomen were encapsulated by a layer of connective tissue and appeared intact, with minimal inflammation and poor host tissue infiltration, whereas a severe foreign body reaction was observed in other grafts that appeared to be undergoing digestion (Figure 3). Degradation of PS grafts (those possessing a score of ≥ 3 for inflammation, with a strong foreign body reaction infiltrating the graft) was observed histologically in 7/19 (37%) abdominal sites and 8/14 (57%) vaginal sites, not including the 6 absent vaginal PS grafts. If these 6 are included, then 14/20 (70%) vaginal PS grafts were either partially degraded or missing 9 months after implantation. In contrast, 1/22 (5%) abdominal and 1/15 (7%) vaginal PP grafts had a score for inflammation ≥ 3, with FBGC surrounding PP fibers but not digesting them.
      Paired analysis (n = 28) revealed that vaginal grafts had higher scores for inflammation (P = .005) and neovascularization (P < .001) and had lower scores for fibroblastic proliferation (P < .001) than abdominal grafts (Table 3 and Figure 2). These associations were apparent even when separating by graft type (Table 3).
      TABLE 3Comparison of histologic parameters between vaginal and abdominal implantation of polypropylene and porcine dermis grafts
      Site of implantation and graft typeNo. of paired samplesInflammationNeovascularizationFibroblastic proliferation
      Vagina (PP and PS)281.9 (0.2)1.2 (0.1)1.1 (0.1)
      Abdomen (PP and PS)281.3 (0.2)0.6 (0.1)1.9 (0.1)
      Vagina vs abdomen (PP and PS)P value
      Wilcoxon signed rank-sum test for all comparisons unless otherwise indicated;
      .005< .001< .001
      Vagina (PP only)151.5 (0.2)1.1 (0.2)1.5 (0.1)
      Abdomen (PP only)151.0 (0.1)0.5 (0.1)1.9 (0.2)
      Vagina vs abdomen (PP only)P value
      Wilcoxon signed rank-sum test for all comparisons unless otherwise indicated;
      .008.02
      Paired t-test.
      .07
      Vagina (PS only)132.4 (0.3)1.4 (0.2)0.7 (0.2)
      Abdomen (PS only)131.6 (0.3)0.7 (0.2)1.9 (0.2)
      Vagina vs abdomen (PS only)P value
      Wilcoxon signed rank-sum test for all comparisons unless otherwise indicated;
      .11.03< .001
      Paired t-test.
      Scores for inflammation, neovascularization, and fibroblastic response were graded on a scale of 0-4 (none-severe) by a pathologist blinded to treatment. Values are presented as mean (SE).
      PP, polypropylene; PS, porcine dermis.
      Pierce. Long-term histologic response to synthetic and biologic graft materials implanted in the vagina and abdomen of a rabbit model. Am J Obstet Gynecol 2009.
      a Wilcoxon signed rank-sum test for all comparisons unless otherwise indicated;
      b Paired t-test.
      Ovariectomy had no effect on scores for inflammation (P = .32), neovascularization (P = .51), or fibroblastic proliferation (P = .83) for grafts implanted in the vagina (Table 4). For abdominal grafts, scores for inflammation and neovascularization were similar in animals with and without ovaries (P > .05), but animals without ovaries had higher scores for fibroblastic proliferation (P = .02) (Table 4). Further analysis revealed that fibroblastic proliferation was higher in ovariectomized animals compared with those with intact ovaries for PS grafts (2.3 ± 0.2 vs 1.6 ± 0.2, respectively; P = .04) but not for PP (P = .18).
      TABLE 4Comparison of histologic parameters between animals with and without ovaries (polypropylene and porcine dermis grafts were combined in analysis)
      Treatment and graft siteNo. of samplesInflammationNeovascularizationFibroblastic proliferation
      Intact ovaries (vagina and abdomen)391.6 (0.2)0.9 (0.1)1.5 (0.1)
      No ovaries (vagina and abdomen)311.7 (0.2)0.9 (0.1)1.8 (0.2)
      Intact ovaries vs no ovaries (vagina and abdomen)P value
      Mann-Whitney rank-sum test.
      .82.63.10
      Intact ovaries (vagina only)172.1 (0.3)1.3 (0.1)1.2 (0.2)
      No ovaries (vagina only)121.7 (0.3)1.2 (0.2)1.1 (0.2)
      Intact ovaries vs no ovaries (vagina only)P value
      Mann-Whitney rank-sum test.
      .32.51.83
      Intact ovaries (abdomen only)221.3 (0.2)0.7 (0.2)1.7 (0.2)
      No ovaries (abdomen only)191.6 (0.2)0.7 (0.1)2.3 (0.2)
      Intact ovaries vs no ovaries (abdomen only)P value
      Mann-Whitney rank-sum test.
      .24.88.02
      Scores for inflammation, neovascularization, and fibroblastic response were graded on a scale of 0-4 (none-severe) by a pathologist blinded to treatment. Values are presented as mean (SE).
      Pierce. Long-term histologic response to synthetic and biologic graft materials implanted in the vagina and abdomen of a rabbit model. Am J Obstet Gynecol 2009.
      a Mann-Whitney rank-sum test.
      Staining for elastic fibers in vaginal grafts revealed that new host tissue surrounding PP fibers contained fewer mature elastic fibers than was observed in native vaginal tissue (Figure 4, A). A paucity of elastic fibers was also seen in fibrotic tissue encapsulating, infiltrating, or replacing vaginal PS grafts (Figures 1 and 4, B). Elastic fibers were negligible in abdominal grafts (Figures 2, B, and 3, B).

      Comment

      Results from this study revealed that synthetic and biologic grafts evoke different foreign body reactions, which may have implications for surgical outcomes in women. PP induces a milder, more uniform long-term response than cross-linked PS. The increased inflammatory response elicited by PS relative to PP 9 months after implantation is consistent with studies examining the short-term response (6 and 12 weeks) to these materials in the rabbit.
      • Hilger W.S.
      • Walter A.
      • Zobitz M.E.
      • Leslie K.O.
      • Magtibay P.
      • Cornella J.
      Histological and biomechanical evaluation of implanted graft materials in a rabbit vaginal and abdominal model.
      • Krambeck A.E.
      • Dora C.D.
      • Sebo T.J.
      • Rohlinger A.L.
      • DiMarco D.S.
      • Elliott D.S.
      Time-dependent variations in inflammation and scar formation of six different pubovaginal sling materials in the rabbit model.
      We and others have shown that lightweight, macroporous PP meshes induce a mild chronic inflammatory response with good host tissue incorporation within the grafts.
      • Hilger W.S.
      • Walter A.
      • Zobitz M.E.
      • Leslie K.O.
      • Magtibay P.
      • Cornella J.
      Histological and biomechanical evaluation of implanted graft materials in a rabbit vaginal and abdominal model.
      • Huffaker R.K.
      • Muir T.W.
      • Rao A.
      • Baumann S.S.
      • Kuehl T.J.
      • Pierce L.M.
      Histologic response of porcine collagen-coated and uncoated polypropylene grafts in a rabbit vagina model.
      • de Tayrac R.
      • Alves A.
      • Therin M.
      Collagen-coated vs noncoated low-weight polypropylene meshes in a sheep model for vaginal surgery: a pilot study.
      The host response at 9 months after implantation of PP mesh is similar to that observed at 12 weeks.
      • Huffaker R.K.
      • Muir T.W.
      • Rao A.
      • Baumann S.S.
      • Kuehl T.J.
      • Pierce L.M.
      Histologic response of porcine collagen-coated and uncoated polypropylene grafts in a rabbit vagina model.
      Host tissue incorporated into vaginal PP grafts consists of new collagen, fibroblasts, blood vessels, and smooth muscle bundles between mesh fibers but contains a paucity of mature elastic fibers in new connective tissue encapsulating mesh fibers. The reduced elastin content of implanted grafts relative to native vaginal tissue may contribute to graft stiffness and dyspareunia frequently observed in women.
      • Visco A.G.
      • Weidner A.C.
      • Barber M.D.
      • et al.
      Vaginal mesh erosion after abdominal sacral colpopexy.
      • Kohli N.
      • Miklos J.R.
      Use of synthetic mesh and donor grafts in gynecologic surgery.
      • Baessler K.
      • Hewson A.D.
      • Tunn R.
      • Schuessler B.
      • Maher C.F.
      Severe mesh complications following intravaginal slingplasty.
      Findings from this study determined that perforated, cross-linked PS elicits a variable long-term response and may degrade by the process of foreign body reaction. This raises concern clinically, because it is marketed as a “permanent” biologic material that undergoes chemical cross-linking to stabilize the collagen and protect it indeterminately from host degradation. In some individuals, however, cross-linked PS may behave more like a resorbable material that loses strength at the repair site during graft remodeling.
      • Trabuco E.C.
      • Klingele C.J.
      • Gebhart J.B.
      Xenograft use in reconstructive pelvic surgery: a review of the literature.
      Other studies in women and in rabbits have also shown that cross-linked PS elicits a variable long-term host response, which may lead to unpredictable clinical outcomes in patients.
      • Claerhout F.
      • Verbist G.
      • Verbeken E.
      • Konstantinovic M.
      • De Ridder D.
      • Deprest J.
      Fate of collagen-based implants used in pelvic floor surgery: a 2-year follow-up study in a rabbit model.
      • Gandhi S.
      • Kubba L.M.
      • Abramov Y.
      • et al.
      Histopathologic changes of porcine dermis xenografts for transvaginal suburethral slings.
      Histopathologic analyses of cross-linked nonperforated PS (Pelvicol; C.R. Bard, Inc) used for transvaginal suburethral slings in women found that some specimens underwent limited collagen remodeling and had minimal inflammation, whereas others induced a strong foreign body response and contained numerous histiocytes and FBGC that appeared to be engulfing the porcine collagen grafts.
      • Gandhi S.
      • Kubba L.M.
      • Abramov Y.
      • et al.
      Histopathologic changes of porcine dermis xenografts for transvaginal suburethral slings.
      In addition, in cases of recurrent stress incontinence, no graft remnants were observed at 58 and 67 weeks after implantation, and they appeared to be completely replaced by dense connective tissue without evidence of inflammation.
      • Gandhi S.
      • Kubba L.M.
      • Abramov Y.
      • et al.
      Histopathologic changes of porcine dermis xenografts for transvaginal suburethral slings.
      A 2-year study examining nonperforated cross-linked PS grafts implanted in a rabbit abdominal model revealed that these grafts were encapsulated and remained intact for up to 180 days, but after 1 year, half the grafts underwent late-onset degradation by a foreign body reaction.
      • Claerhout F.
      • Verbist G.
      • Verbeken E.
      • Konstantinovic M.
      • De Ridder D.
      • Deprest J.
      Fate of collagen-based implants used in pelvic floor surgery: a 2-year follow-up study in a rabbit model.
      The inconsistency in response may provide an explanation for the unreliable long-term surgical outcomes attained with the use of cross-linked PS grafts in a variety of procedures for pelvic organ prolapse and incontinence.
      • Quiroz L.H.
      • Gutman R.E.
      • Shippey S.
      • et al.
      Abdominal sacrocolpopexy: anatomic outcomes and complications with Pelvicol, autologous and synthetic graft materials.
      • Altman D.
      • Zetterstrom J.
      • Mellgren A.
      • Gustafsson C.
      • Anzen B.
      • Lopez A.
      A three-year prospective assessment of rectocele repair using porcine xenograft.
      • Giri S.K.
      • Hickey J.P.
      • Sil D.
      • et al.
      The long-term results of pubovaginal sling surgery using acellular cross-linked porcine dermis in the treatment of urodynamic stress incontinence.
      In the current study, degradation of PS grafts was observed histologically in 37% of abdominal sites and up to 70% of vaginal sites 9 months after implantation. Macroscopic and/or microscopic degradation of grafts was associated with a more intense foreign body reaction compared with intact PS grafts. Although it is possible that missing vaginal PS grafts eroded out of the animals without the investigators' knowledge in between examinations, circumstances suggest that host tissues replaced these grafts instead. For example: (1) only 1 vaginal graft was implanted; (2) the size of the graft was small; (3) permanent sutures that secured the grafts were present; (4) graft remnants were observed histologically in some missing graft sites; (5) partial degradation also occurred in the abdomen (the abdomen is a less hostile environment in which the degradation process may take longer than in the vagina); and (6) these findings are supported by human
      • Gandhi S.
      • Kubba L.M.
      • Abramov Y.
      • et al.
      Histopathologic changes of porcine dermis xenografts for transvaginal suburethral slings.
      and animal
      • Claerhout F.
      • Verbist G.
      • Verbeken E.
      • Konstantinovic M.
      • De Ridder D.
      • Deprest J.
      Fate of collagen-based implants used in pelvic floor surgery: a 2-year follow-up study in a rabbit model.
      data as mentioned above.
      Erosion of vaginal implants was observed for both graft materials used in this study and did not appear to be affected by ovariectomy, but rather by the size of the implanted material. Excess material may limit the amount of tissue incorporation within the grafts, leading to erosion. Decreasing the length of implanted synthetic and biologic grafts was associated with decreased erosion rates. Although erosion occurred in the vagina overall in 27% of rabbits implanted with PP mesh and 15% of rabbits implanted with cross-linked PS, these rates decreased to 10% and 6%, respectively, in animals implanted with shorter pieces of graft material. This finding suggests that if the amount of material implanted exceeds a certain threshold, it is more likely to erode, even if it elicits a mild inflammatory response. With the increasing popularity of mesh repair kits, clinical studies are needed to better characterize this threshold to minimize erosion rates in women.
      This study importantly provides data to substantiate prior speculation that implantation of graft materials in the vagina elicits a different host tissue response than abdominal implantation. Paired analysis of grafts implanted at both sites in the same animal revealed that vaginal grafts had higher scores for inflammation and neovascularization, and had lower scores for fibroblastic proliferation than abdominal grafts. These associations were apparent for both the PP and PS graft materials. To our knowledge, only 1 other study has examined vaginal and abdominal implantation of graft materials simultaneously in an animal model; this study, however, examined the short-term response (6 and 12 weeks) and was unable to show significant differences in inflammation, neovascularization, or collagen deposition between sites, likely because of a small sample size.
      • Hilger W.S.
      • Walter A.
      • Zobitz M.E.
      • Leslie K.O.
      • Magtibay P.
      • Cornella J.
      Histological and biomechanical evaluation of implanted graft materials in a rabbit vaginal and abdominal model.
      Although ovarian hormones are known to be important in maintaining vaginal tissue structure, contractility, and collagen metabolism,
      • Kim N.N.
      • Min K.
      • Pessina M.A.
      • Munarriz R.
      • Goldstein I.
      • Traish A.M.
      Effects of ovariectomy and steroid hormones on vaginal smooth muscle contractility.
      ovariectomy did not appear to negatively affect host tissue incorporation and healing in vaginal grafts. Ovariectomy did not have a statistically significant effect on scores for inflammation, neovascularization, and fibroblastic proliferation for grafts implanted in the vagina. In the abdomen, however, fibroblastic proliferation was shown to be higher in ovariectomized animals compared with those with intact ovaries for PS xenografts but not for PP mesh; the meaning of this differential effect in the abdomen in response to PS is unclear and warrants further investigation.
      Limitations of this study exist. Data from this study were obtained from a rabbit model using a relatively small sample size. Rabbits possess a high collagenolytic activity and, therefore, may degrade PS grafts more rapidly than do human beings.
      • Claerhout F.
      • Verbist G.
      • Verbeken E.
      • Konstantinovic M.
      • De Ridder D.
      • Deprest J.
      Fate of collagen-based implants used in pelvic floor surgery: a 2-year follow-up study in a rabbit model.
      However, emerging clinical evidence indicates that cross-linked PS also degrades in women by a similar foreign body reaction.
      • Gandhi S.
      • Kubba L.M.
      • Abramov Y.
      • et al.
      Histopathologic changes of porcine dermis xenografts for transvaginal suburethral slings.
      Strengths of the current study include the long-term study design as well as abdominal and vaginal implantation in the same animals.
      In summary, accumulating scientific and clinical data suggest that cross-linked PS grafts are likely inferior to lightweight PP meshes for pelvic organ prolapse and incontinence procedures. PP mesh induces a milder, more uniform response than PS. In contrast, cross-linked PS may undergo long-term degradation by a foreign body response, which may lead to compromised biomechanical properties of grafts and unacceptable surgical failure rates in women. Because vaginal implantation elicits a different host tissue response than abdominal implantation, vaginal models are likely more appropriate than abdominal models for investigations of host responses to graft materials used in vaginal surgery. Additional research is needed in vaginal models to evaluate the long-term safety and efficacy of specific graft materials used in pelvic reconstructive surgery.

      Acknowledgments

      We acknowledge the unconditional donation of Gynemesh Nonabsorbable Prolene Soft Mesh by Ethicon Inc, Somerville, NJ, and PelviSoft Acellular Collagen BioMesh by C.R. Bard Inc, Covington, GA. We also thank the Scott & White Hospital animal care staff for their assistance in management of anesthesia and husbandry of the rabbits used in this study.

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