Hernia DOI 10.1007/s10029-013-1203-7
ORIGINAL ARTICLE
Comparative analysis of histopathologic responses to implanted porcine biologic meshes Y. W. Novitsky • S. B. Orenstein • D. L. Kreutzer
Received: 29 June 2013 / Accepted: 6 December 2013 Ó Springer-Verlag France 2013
Abstract Objectives Biologic mesh (BM) prostheses are increasingly utilized for hernia repairs. Modern BMs are not only derived from different tissue sources, but also undergo various proprietary processing steps—factors that likely impact host tissue responses and mesh performance. We aimed to compare histopathologic responses to various BMs after implantation in a mouse model. Materials and methods Five-mm samples of non-crosslinked [Strattice (ST)], and intentionally crosslinked [CollaMend (CM), Permacol (PC)] porcine-derived biologic meshes were implanted subcutaneously in C57BL/6 mice. 1, 4, 8, and 12 weeks post-implantation, meshes were assessed for inflammation, foreign body reaction (FBR), neocellularization, and collagen deposition using H&E and trichrome stains. Results All meshes induced early polymorphonuclear cell infiltration (highest in CM; lowest in ST) that resolved by 4 weeks. ST was associated with extensive macrophage presence at 12 weeks. Foreign body response was not seen in the ST group, but was present abundantly in the CM and
PC groups, highest at 8 weeks. New peripheral collagen deposition was seen only in the ST group at 12 weeks. Collagen organization was highest in the ST group as well. Both CM and PC groups were associated with fibrous encapsulation and no evidence of integration or remodeling. Conclusions Inflammation appears to be a common component of integration of all biologic meshes studied. Pronounced inflammatory responses as well as profound FBR likely lead to observed encapsulation and poor host integration of the crosslinked BMs. Overall, ST was associated with the lowest foreign body response and the highest degree of new collagen deposition and organization. These features may be key predictors for improved mesh performance during hernia repair. Keywords Biologic mesh Porcine mesh Crosslinked Non-crosslinked Foreign body response Collagen Inflammation Mouse Hernia
Introduction Y. W. Novitsky S. B. Orenstein Department of Surgery, University of Connecticut Health Center, Farmington, CT, USA Y. W. Novitsky (&) S. B. Orenstein Case Comprehensive Hernia Center, Department of Surgery, University Hospitals Case Medical Center, Case Western Reserve University School of Medicine, 11100 Euclid Avenue, Cleveland, OH 44106, USA e-mail: [email protected] D. L. Kreutzer Center for Molecular Tissue Engineering, Department of Surgery, University of Connecticut School of Medicine, Farmington, CT, USA
Throughout the USA and most of the world, herniorrhaphy continues to be the most common surgical procedure performed by general surgeons [1–3]. Successful repair of most hernias requires the use of a prosthetic implant for reinforcement of the defect. Synthetic surgical meshes have been used for over 50 years and continue to be the most prevalent category of meshes utilized today. However, synthetic materials are currently contraindicated in infected or potentially contaminated fields [4–7]. Because of the need for prosthetic implants to support native tissues in contaminated or potentially contaminated fields, biologic meshes (BMs) have been developed to take the place of
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Hernia Table 1 Biologic mesh processing and characteristics Mesh product
Tissue source
Intentional crosslinking
Sterilization method
Decellularization and processing
CollaMendTM
Porcine dermis
Yes (EDC)
Ethylene oxide
NaSulfate, NaHyperchlorite, NaCl, HCl, H2O, EtOH, H2O2, phosphate buffer, detergent, lime
PermacolTM
Porcine dermis
Yes (HMDI)
Gamma radiation
Proprietary enzymatic process
StratticeÒ
Porcine dermis
No
Electron-beam radiation
Non-denaturing detergents, alpha-gal removal
EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, HMDI hexamethylene diisocyanate
non-degradable synthetic meshes where mesh infection is of high concern. Biologic meshes are derived from human dermis, porcine dermis, porcine small intestine submucosa, bovine dermis, or bovine pericardium. Variations between different BM products are brought about not only from tissue source, but also from methods of decellularization, processing, and sterilization. Previous in vitro studies performed in our laboratory have demonstrated significant differences in cytokine induction from BMs derived from similar sources [8–10]. While we believe that such differences are likely attributable to manufacturers’ various proprietary processing methods, other causes are still being elucidated. In fact, chemical collagen crosslinking has been suggested to be one of the most likely deleterious processes in biologic mesh processing. Our study sought to evaluate the histological responses to commonly used BMs following implantation in a mouse model. We hypothesized that crosslinked (XL) porcine meshes will be associated with increased host immune/inflammatory response and adverse histopathological host tissue reaction as compared to non-crosslinked (NXL) matrix.
with protocols approved by the University of Connecticut Health Center Animal Care Committee. The animals were housed within the Center for Laboratory Animal Care at the University of Connecticut Health Center throughout the study. They were maintained on a regular 12/12 light/dark cycle at 74 ± 2 °F with food and water available ad libitum. Surgical implantation
Materials and methods
Prior to midline skin incision, the abdominal hair of each mouse was removed with Nair (Church & Dwight Co. Inc., Princeton, NJ, USA) followed by thorough rinsing with water. Povidone-iodine was used for skin disinfection. A 1-cm skin incision was made in the middle third of the abdomen. Bilateral subcutaneous pockets were developed by blunt dissection and a 5-mm piece of mesh was placed in each of the bilateral subcutaneous spaces. Finally, the skin was closed with a running 5-0 plain gut suture. A sham surgery group was also included. Sham surgeries were performed in a similar fashion; subcutaneous pockets were developed by blunt dissection; however, no mesh was implanted. All mice received subcutaneous injections of buprenorphine (0.1 mg/kg) for post-operative analgesia.
Surgical meshes
Explantation and histological evaluation
Three porcine dermis biologic surgical meshes were utilized in this study: CollaMendTM (CM; CR Bard/Davol Inc, Warwick, RI, USA), PermacolTM (PC; Covidien, Mansfield, MA, USA), and StratticeÒ (ST; LifeCell Corp, Branchburg, NJ, USA). CM and PC are chemically XL porcine dermal matrices, while ST is an NXL porcine dermal matrix (Table 1). Multiple 5-mm circular samples were cut from each mesh using a sterilized metal punch.
All mice were euthanized at 1, 4, 8, and 12 weeks postimplantation. Following euthanasia, the abdomen was resected in toto, with cut specimens including skin, mesh, and abdominal wall. Each specimen was fixed in
Score
Inflammation
Animals
0 1
Thirty-six 6-to 10-week-old C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME, USA) weighing approximately 25–30 g were used. Each animal received two mesh samples. All experiments were performed in accordance
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Table 2 Histological scoring scale Foreign body reaction (FBGCs)
Neocellularization
None
None
None
Mild
Minimal
Minimal (peripheral)
2
Moderate
Moderate
Moderate
3
Severe
Severe
Extensive
FBGCs foreign body giant cells
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formalin, processed, embedded in paraffin and sectioned. The sections were stained with hematoxylin and eosin (H&E) and Gomori’s one-step Trichrome for histological evaluation. The slides were then viewed and assessed by a blinded histopathologist using an established histological scale [7, 11, 12] (Tables 2, 3). Histological parameters included inflammatory response, foreign body reaction, fibrotic response, collagen organization, and neovascularization. After an initial review of all slides to gain a baseline measure of histological parameters, each sample was reevaluated and scored against each other to obtain a semiquantitative measure of tissue responses to the implanted
Table 3 Data expressed as mean (range)
Time point
mesh. For inflammatory response, the degree of infiltration of chronic inflammatory cells, principally lymphocytes and macrophages, at the mesh/host interface was noted. Foreign body reaction was determined by the relative quantity of foreign body giant cells (FBGCs) surrounding the biologic mesh scaffold. Fibrotic change was a function of relative abundance of new collagen deposition at sites of mesh implantation, while collagen organization was determined by factors such as connective tissue density (loose versus dense) and arrangement of collagen bundles (parallel versus haphazard pattern). Statistical analysis was done using Kruskal–Wallis test. A p \ 0.05 was considered as being significant.
Mesh groups ST
p values PM
CM
SHAM
2.25 (2–3)
2.75 (2–3)
0
Inflammatory response 1 week
1 (1–1)
ST vs PM: 0.002 ST vs CM: 0.002 CM vs PM: 0.18
4 weeks
0.75 (0–1)
2.5 (2–3)
2.25 (2–3)
0
ST vs PM: 0.0001 ST vs CM: 0.0017 CM vs PM: 0.6
8 weeks
0.5 (0–1)
2.0 (1–3)
2.5 (2–3)
0
ST vs PM: 0.02 ST vs CM: 0.002 CM vs PM: 0.18
12 weeks
0.5 (0–1)
2.0 (0–3)
2.25 (2–3)
0
ST vs PM: 0.001 ST vs CM: 0.0001 CM vs PM: 0.45
Foreign body response 4 weeks
0.20 (0–1)
1.0 (1–2)
0.75 (0–3)
0
ST vs PM: 0.01 ST vs CM: 0.04 CM vs PM: 0.6
8 weeks
0.30 (0–1)
2.2 (2–3)
2.5 (2–3)
0
ST vs PM: 0.02 ST vs CM: 0.001 CM vs PM: 0.8
12 weeks
0.15 (0–1)
2.0 (1–3)
2.25 (2–3)
0
ST vs PM: 0.0001 ST vs CM: 0.0001 CM vs PM: 0.55
Neocellularization 4 weeks
1 (1–1)
0.15 (0–1)
0.20 (0–1)
0
ST vs PM: 0.04 ST vs CM: 0.04 CM vs PM: 0.9
8 weeks
1.4 (1–2)
0.4 (0–1)
0.5 (0–1)
0
ST vs PM: 0.002 ST vs CM: 0.004 CM vs PM: 0.8
12 weeks Sham group had no associated inflammatory response or foreign body response
1.7 (1–3)
0.5 (0–1)
0.5 (0–1)
0
ST vs PM: 0.0001 ST vs CM: 0.0001 CM vs PM: 1
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Results
Fig. 1 Inflammation at the mesh/implant interface as quantified by macrophage and lymphocyte response. Both XL groups were associated with significantly higher degree of the inflammatory response (p = 0.001). There was no difference between the XL groups
All meshes induced varying degrees of inflammation throughout the study (Fig. 1). Pronounced inflammatory response, demonstrated by extensive proliferation of the mononuclear cells at the mesh/host interface, was associated with both XL meshes (Fig. 2a). The NXL group, on the other hand, was associated with minimal presence of inflammatory cells surrounding the mesh at 4-, 8-, and 12-week time points (Fig. 2b). Importantly, significant inflammatory response was present in both XL groups even 12 weeks after implantation (Fig. 3a, b). With regard to foreign body response, both XL meshes had significantly higher foreign body reaction (Fig. 4). As seen in Fig. 5a, histologic evaluation revealed FBGCs abundantly surrounding the XL mesh borders at the 8-week time point. Alternatively, ST did not display any FBGC accumulation (Fig. 5b).
Fig. 2 Histologic analysis revealed extensive presence of inflammatory cells adjacent to the XL mesh (a) and minimal inflammation at the interface of the NXL grafts (b) at the 4-week time point (H&E, 209)
Fig. 3 Persistent chronic inflammatory response to both XL grafts was seen at the 12-week time point (H&E, 109)
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Hernia Foreign Body Response
Neo -cellularization
3
2
2.5
1.5 2
Collamend 1.5
Collamend 1
Permacol
Permacol 1
Strattice
Strattice 0.5
0.5
0
0
4 wks 4 wks
8 wks
8 wks
12 wks
1 2 wks
Fig. 4 Foreign body response calculated by the presence of the foreign body giant cells was significantly diminished in the NXL group as compared to both XL groups (p = 0.0001). There was no difference between the XL groups
Mesh integration and remodeling was evaluated by macrophage and fibroblast penetration into the mesh scaffold, and new collagen deposition within the mesh. No group demonstrated significant mesh remodeling. However, only the NXL group was associated with any detectable level of mesh integration. Importantly, only ST displayed evidence of macrophage and fibroblast migration into the mesh scaffold at both 8- and 12-week time points. When compared with XL meshes, NXL group had significantly higher neocellularization into the mesh scaffold (Fig. 6). Both CM and PC were associated with moderate to severe fibrosis surrounding the mesh—encapsulation (Fig. 7). Furthermore, new vascularized collagen deposition was seen at the 12-week time point in the NXL group (Fig. 8). Importantly, no penetration of fibroblasts into the mesh was seen in either PC or CM grafts, even at the 12-week time point. (Fig. 9).
Fig. 6 Neocellularization was significantly higher in the NXL group at all three time points. Importantly, it increased at every subsequent time point (p \ 0.001). There was no difference between the XL groups
Sham mice samples demonstrated essentially normal histology. There was no evidence of chronic inflammation, new collagen deposition, or FBGCs’ presence.
Discussion Biologic mesh prosthetics are increasingly being utilized for hernia defect repairs, among other surgical procedures. The purported function of BMs is to serve as a regenerative framework by inducing neovascularization, with subsequent simultaneous BM matrix remodeling and new collagen deposition [5, 13–16]. While an ever increasing list of surgical disciplines is utilizing BMs for various repairs, there continues to be a paucity of data regarding the histopathologic reaction and changes of BMs following implantation [15, 17]. We aimed to characterize and
Fig. 5 Numerous FBGCs (black arrows) were noted to surround the XL graft (a) with no significant presence of those cells seen in the NXL group (H&E, 209) (color figure online)
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Fig. 7 Dense fibrotic encapsulation of PM graft seen at 8 weeks after implantation (Trichrome blue stain, 209) (color figure online)
Fig. 8 XL graft remodeling is manifested by the fibroblast proliferation and new collagen deposition with visible vascular channels (white arrows) (H&E, 209) (color figure online)
compare histopathological responses to commonly used porcine BM scaffolds in a mouse. Inflammation appears to be a common component around implanted biologic prosthetics. The commonly used notion that the biologic meshes are ‘‘inert’’ is clearly without merit. In the present study, all implanted meshes displayed significant inflammatory reactions. Being a necessary component of wound healing, excessive inflammation may often lead to negative recognition. For biologic implants, this would lead to excessive fibrosis with encapsulation or matrix degradation and mesh failure [18]. A profound inflammatory response was present for crosslinked BMs, indicative of inferior biocompatibility of those grafts. Furthermore, crosslinked meshes demonstrated
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heightened foreign body reaction and encapsulation, both signs of deleterious reaction by the host. It was thus not surprising that we detected no evidence of mesh integration nor remodeling of any of the crosslinked grafts as far out as 12 weeks post-implantation. While tissue source may certainly impact how a BM is reacted upon by the recipient, difference in tissue reaction is likely, at least in part, a result of the different methods of processing, decellularization, and sterilization of each product. Manufacturers utilize various proprietary methods and processing solvents that likely influence the innate biochemical and biomolecular structure of the collagen scaffold [17–19]. Subsequently, these matrix alterations may influence foreign recognition and antigen presentation. The resulting processing changes likely influence biocompatibility, foreign body response and immunogenic potential of the implant. In fact, poor outcomes of several biologic meshes in the study by Sandor et al. [20] were linked to the tissue processing technique that may have damaged the biochemical matrix components. Unfortunately, the exact methodology of tissue processing remains proprietary and largely unknown. It, therefore, could not be precisely investigated as an independent risk factor of biocompatibility in this study. Of all the various processing techniques employed by mesh manufacturers, collagen crosslinking appears likely to have the most profound impact on tissue responses to BMs. Intentional crosslinking is utilized to prolong the lifespan of the mesh, using processing techniques that add to the 3-dimensional structure of the collagen to, essentially, mechanically strengthen the matrix and impede degradation of collagen. Of note, the final product of ‘‘terminally’’ crosslinked animal dermis is leather. By using hexamethylene diisocyanate, carbodiimide, glutaraldehyde, or photo-oxidizing agents [21, 22], intentionally crosslinked BMs are believed to remodel over a time course over 12 months or more, versus 4–6 months for the non-crosslinked BMs [23–25]. Even non-intentionally crosslinked products may get crosslinked from gamma irradiation during the sterilization process [26]. In addition, incomplete removal of chemical crosslinking agents could result in cytotoxicity from residues leaching from the BM which may induce prolonged toxic effects and heightened cellular responses [21, 22]. We have previously demonstrated in vitro profound differences in cytokine induction from human mononuclear cells when exposed to porcinebased BMs with crosslinking when compared with NXL meshes [27]. While certain clinical circumstances may utilize XL meshes, such as when ‘‘biologic’’ mesh behavior is not essential, we found that those grafts displayed significant immunologic disadvantage which could diminish their regenerative capacity and overall efficacy of those types of meshes in a clinical setting.
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Fig. 9 Poor neocellularization in the XL groups. Essentially, no penetration of fibroblasts into the graft was seen in either PC (a) or CM (b) grafts, even at the 12-week time point (H&E, 109)
Early cellular and vascular infiltration of a biologic matrix has been seen as critical for mesh integration and regeneration. Monocyte/macrophage penetration of the graft from the surface inward is paramount for fibroblast proliferation and new collagen deposition. In the absence of angiogenesis, remodeling may be impeded and the biologic graft, akin rapidly absorbable meshes, will breakdown, disappear and/or get simply replaced by scar. Butler et al. [28] found that NXL porcine dermis promoted early cellular and vascular infiltration and likely contributed to stronger mesh–musculofascia interface. Xu et al. [29] reported that functional blood vessels paralleled host cell repopulation with clearly delineated channels lined with endothelial cells in human dermis by 1 month after implantation in primates. These findings were confirmed more recently in a sublay biologic mesh study in rats [30]. The authors found that the Alloderm group was associated with 100 % neo-cellularity by 3 months after implantation. Neovascularization was noted to support the cells. Finally, normal, non-denatured collagen pattern was seen, indicative of remodeling and new collagen deposition [30]. We found moderate degree of cellular infiltration in the NXL group. Alternatively, similar to Butler et al. [28], we found essentially no new cellularization and/or vascularization in the XL meshes. In fact, our 4- and 12-week samples were indistinguishable from cellularity standpoint in the latter group. These findings clearly highlight poor biocompatibility of XL grafts and lack of ‘‘biologic’’ behavior of those biologic meshes. It is also important to point out, however, that Deeken et al. [31] have recently suggested, that although crosslinking differentiated biologic meshes with regard to cellular infiltration and neovascularization early on, those histologic features were no
longer impacted by crosslinking at a 1-year time point. While these isolated results are intriguing, it is, however, unclear whether the findings by Deeken et al. are true representations of what happens in humans or is just one of the limitations of long-term ventral hernia/biologic mesh investigation in resilient animal models like minipigs. The final and most important step in biologic mesh placement is graft integration and remodeling with new collagen deposition and tissue regeneration. Melman et al. [32] have suggested that when scaffold degradation is accompanied by cellular infiltration, ECM deposition, and neovascularization, it can be viewed as remodeling. Deeken et al. [31] recently reported that NXL grafts exhibited more favorable remodeling characteristics. Another recent study revealed essential lack of matrix absorption and absence of remodeling of XL grafts after 6-month implantation [30]. Interestingly, most of their CollaMend matrices were completely degraded by bacteria from associated wounds. The mesh samples that did not get infected were found to be essentially intact with no evidence of remodeling and poor integration [30]. Once again, while such mesh performance may at times result in an effective repair in the clinical setting, such behavior of XL biologic meshes can hardly be viewed as regenerative. Instead, given its lack of integration into the host and likely resultant fibrous encapsulation, XL grafts often act as permanent foreign body materials, similar to PTFE-based synthetics. In line with other investigators, we found in our mouse model that crosslinked grafts had no evidence of remodeling nor new collagen deposition (except for a thick layer of fibrous encapsulation) 3 months after implantation.
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We have utilized mice as our model for multiple projects due to the ease of implantation, low cost, and availability of genetic variants of mice for future studies. However, whether the tissue responses in mice are directly comparable to those of humans is debatable. Mice have been used extensively throughout modern scientific research and serve as useful stepping stones towards higher-order animals, and ultimately, human studies. In addition, we view our mouse model as an ideal baseline model for tissue responses to implanted biomaterials. By implanting biomaterials in a relatively benign subcutaneous space, we avoid other variables including direct tissue trauma in the immediate vicinity of the implant. For example, when utilizing a more advanced model with hernia defects, the tissue response may be a result of tissue trauma rather than the biomaterial itself. Finally, because our research is based on an animal model under ideal conditions, it is unclear how much histopathology displayed in our specimens relates to the clinical setting following biologic mesh implantation in humans. Future investigations, including ‘‘humanized’’ mice, mesh implantation during herniorrhaphy, as well as ‘‘stress’’ conditions like contamination and/or systemic immune and metabolic deficiencies are currently under way in our laboratory.
Conclusion All biologic mesh prosthetics induce inflammation at the site of implantation. We found that chemical crosslinking of acellular porcine dermal matrices was associated with a more pronounced host acute and chronic inflammatory response. Moreover, profound foreign body reactions to the XL grafts likely lead to observed encapsulation and poor integration. NXL mesh, on the other hand, was associated with the lowest foreign body response and the highest degree of new collagen deposition and matrix remodeling. Of critical importance, XL grafts had no evidence of new cellularization and no new collagen deposition. It thus appears that benefits of biologic mesh crosslinking may be a theoretical concept that, in fact, is deleterious to BM/host reaction at the site of implantation. Overall, improved biocompatibility of NXL grafts seen in this study may be one of the key predictors of superior performance of this type of biologic mesh during hernia repair. Acknowledgments This study was funded by institutional support from the University of Connecticut Health Center. No direct or indirect industry support was utilized. Conflict of interest Y.W.N. has received honorarium for consulting and/or speaking from LifeCell and CR Bard. SO and DK have nothing to disclose.
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ORIGINAL ARTICLE
Comparative analysis of histopathologic responses to implanted porcine biologic meshes Y. W. Novitsky • S. B. Orenstein • D. L. Kreutzer
Received: 29 June 2013 / Accepted: 6 December 2013 Ó Springer-Verlag France 2013
Abstract Objectives Biologic mesh (BM) prostheses are increasingly utilized for hernia repairs. Modern BMs are not only derived from different tissue sources, but also undergo various proprietary processing steps—factors that likely impact host tissue responses and mesh performance. We aimed to compare histopathologic responses to various BMs after implantation in a mouse model. Materials and methods Five-mm samples of non-crosslinked [Strattice (ST)], and intentionally crosslinked [CollaMend (CM), Permacol (PC)] porcine-derived biologic meshes were implanted subcutaneously in C57BL/6 mice. 1, 4, 8, and 12 weeks post-implantation, meshes were assessed for inflammation, foreign body reaction (FBR), neocellularization, and collagen deposition using H&E and trichrome stains. Results All meshes induced early polymorphonuclear cell infiltration (highest in CM; lowest in ST) that resolved by 4 weeks. ST was associated with extensive macrophage presence at 12 weeks. Foreign body response was not seen in the ST group, but was present abundantly in the CM and
PC groups, highest at 8 weeks. New peripheral collagen deposition was seen only in the ST group at 12 weeks. Collagen organization was highest in the ST group as well. Both CM and PC groups were associated with fibrous encapsulation and no evidence of integration or remodeling. Conclusions Inflammation appears to be a common component of integration of all biologic meshes studied. Pronounced inflammatory responses as well as profound FBR likely lead to observed encapsulation and poor host integration of the crosslinked BMs. Overall, ST was associated with the lowest foreign body response and the highest degree of new collagen deposition and organization. These features may be key predictors for improved mesh performance during hernia repair. Keywords Biologic mesh Porcine mesh Crosslinked Non-crosslinked Foreign body response Collagen Inflammation Mouse Hernia
Introduction Y. W. Novitsky S. B. Orenstein Department of Surgery, University of Connecticut Health Center, Farmington, CT, USA Y. W. Novitsky (&) S. B. Orenstein Case Comprehensive Hernia Center, Department of Surgery, University Hospitals Case Medical Center, Case Western Reserve University School of Medicine, 11100 Euclid Avenue, Cleveland, OH 44106, USA e-mail: [email protected] D. L. Kreutzer Center for Molecular Tissue Engineering, Department of Surgery, University of Connecticut School of Medicine, Farmington, CT, USA
Throughout the USA and most of the world, herniorrhaphy continues to be the most common surgical procedure performed by general surgeons [1–3]. Successful repair of most hernias requires the use of a prosthetic implant for reinforcement of the defect. Synthetic surgical meshes have been used for over 50 years and continue to be the most prevalent category of meshes utilized today. However, synthetic materials are currently contraindicated in infected or potentially contaminated fields [4–7]. Because of the need for prosthetic implants to support native tissues in contaminated or potentially contaminated fields, biologic meshes (BMs) have been developed to take the place of
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Hernia Table 1 Biologic mesh processing and characteristics Mesh product
Tissue source
Intentional crosslinking
Sterilization method
Decellularization and processing
CollaMendTM
Porcine dermis
Yes (EDC)
Ethylene oxide
NaSulfate, NaHyperchlorite, NaCl, HCl, H2O, EtOH, H2O2, phosphate buffer, detergent, lime
PermacolTM
Porcine dermis
Yes (HMDI)
Gamma radiation
Proprietary enzymatic process
StratticeÒ
Porcine dermis
No
Electron-beam radiation
Non-denaturing detergents, alpha-gal removal
EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, HMDI hexamethylene diisocyanate
non-degradable synthetic meshes where mesh infection is of high concern. Biologic meshes are derived from human dermis, porcine dermis, porcine small intestine submucosa, bovine dermis, or bovine pericardium. Variations between different BM products are brought about not only from tissue source, but also from methods of decellularization, processing, and sterilization. Previous in vitro studies performed in our laboratory have demonstrated significant differences in cytokine induction from BMs derived from similar sources [8–10]. While we believe that such differences are likely attributable to manufacturers’ various proprietary processing methods, other causes are still being elucidated. In fact, chemical collagen crosslinking has been suggested to be one of the most likely deleterious processes in biologic mesh processing. Our study sought to evaluate the histological responses to commonly used BMs following implantation in a mouse model. We hypothesized that crosslinked (XL) porcine meshes will be associated with increased host immune/inflammatory response and adverse histopathological host tissue reaction as compared to non-crosslinked (NXL) matrix.
with protocols approved by the University of Connecticut Health Center Animal Care Committee. The animals were housed within the Center for Laboratory Animal Care at the University of Connecticut Health Center throughout the study. They were maintained on a regular 12/12 light/dark cycle at 74 ± 2 °F with food and water available ad libitum. Surgical implantation
Materials and methods
Prior to midline skin incision, the abdominal hair of each mouse was removed with Nair (Church & Dwight Co. Inc., Princeton, NJ, USA) followed by thorough rinsing with water. Povidone-iodine was used for skin disinfection. A 1-cm skin incision was made in the middle third of the abdomen. Bilateral subcutaneous pockets were developed by blunt dissection and a 5-mm piece of mesh was placed in each of the bilateral subcutaneous spaces. Finally, the skin was closed with a running 5-0 plain gut suture. A sham surgery group was also included. Sham surgeries were performed in a similar fashion; subcutaneous pockets were developed by blunt dissection; however, no mesh was implanted. All mice received subcutaneous injections of buprenorphine (0.1 mg/kg) for post-operative analgesia.
Surgical meshes
Explantation and histological evaluation
Three porcine dermis biologic surgical meshes were utilized in this study: CollaMendTM (CM; CR Bard/Davol Inc, Warwick, RI, USA), PermacolTM (PC; Covidien, Mansfield, MA, USA), and StratticeÒ (ST; LifeCell Corp, Branchburg, NJ, USA). CM and PC are chemically XL porcine dermal matrices, while ST is an NXL porcine dermal matrix (Table 1). Multiple 5-mm circular samples were cut from each mesh using a sterilized metal punch.
All mice were euthanized at 1, 4, 8, and 12 weeks postimplantation. Following euthanasia, the abdomen was resected in toto, with cut specimens including skin, mesh, and abdominal wall. Each specimen was fixed in
Score
Inflammation
Animals
0 1
Thirty-six 6-to 10-week-old C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME, USA) weighing approximately 25–30 g were used. Each animal received two mesh samples. All experiments were performed in accordance
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Table 2 Histological scoring scale Foreign body reaction (FBGCs)
Neocellularization
None
None
None
Mild
Minimal
Minimal (peripheral)
2
Moderate
Moderate
Moderate
3
Severe
Severe
Extensive
FBGCs foreign body giant cells
Hernia
formalin, processed, embedded in paraffin and sectioned. The sections were stained with hematoxylin and eosin (H&E) and Gomori’s one-step Trichrome for histological evaluation. The slides were then viewed and assessed by a blinded histopathologist using an established histological scale [7, 11, 12] (Tables 2, 3). Histological parameters included inflammatory response, foreign body reaction, fibrotic response, collagen organization, and neovascularization. After an initial review of all slides to gain a baseline measure of histological parameters, each sample was reevaluated and scored against each other to obtain a semiquantitative measure of tissue responses to the implanted
Table 3 Data expressed as mean (range)
Time point
mesh. For inflammatory response, the degree of infiltration of chronic inflammatory cells, principally lymphocytes and macrophages, at the mesh/host interface was noted. Foreign body reaction was determined by the relative quantity of foreign body giant cells (FBGCs) surrounding the biologic mesh scaffold. Fibrotic change was a function of relative abundance of new collagen deposition at sites of mesh implantation, while collagen organization was determined by factors such as connective tissue density (loose versus dense) and arrangement of collagen bundles (parallel versus haphazard pattern). Statistical analysis was done using Kruskal–Wallis test. A p \ 0.05 was considered as being significant.
Mesh groups ST
p values PM
CM
SHAM
2.25 (2–3)
2.75 (2–3)
0
Inflammatory response 1 week
1 (1–1)
ST vs PM: 0.002 ST vs CM: 0.002 CM vs PM: 0.18
4 weeks
0.75 (0–1)
2.5 (2–3)
2.25 (2–3)
0
ST vs PM: 0.0001 ST vs CM: 0.0017 CM vs PM: 0.6
8 weeks
0.5 (0–1)
2.0 (1–3)
2.5 (2–3)
0
ST vs PM: 0.02 ST vs CM: 0.002 CM vs PM: 0.18
12 weeks
0.5 (0–1)
2.0 (0–3)
2.25 (2–3)
0
ST vs PM: 0.001 ST vs CM: 0.0001 CM vs PM: 0.45
Foreign body response 4 weeks
0.20 (0–1)
1.0 (1–2)
0.75 (0–3)
0
ST vs PM: 0.01 ST vs CM: 0.04 CM vs PM: 0.6
8 weeks
0.30 (0–1)
2.2 (2–3)
2.5 (2–3)
0
ST vs PM: 0.02 ST vs CM: 0.001 CM vs PM: 0.8
12 weeks
0.15 (0–1)
2.0 (1–3)
2.25 (2–3)
0
ST vs PM: 0.0001 ST vs CM: 0.0001 CM vs PM: 0.55
Neocellularization 4 weeks
1 (1–1)
0.15 (0–1)
0.20 (0–1)
0
ST vs PM: 0.04 ST vs CM: 0.04 CM vs PM: 0.9
8 weeks
1.4 (1–2)
0.4 (0–1)
0.5 (0–1)
0
ST vs PM: 0.002 ST vs CM: 0.004 CM vs PM: 0.8
12 weeks Sham group had no associated inflammatory response or foreign body response
1.7 (1–3)
0.5 (0–1)
0.5 (0–1)
0
ST vs PM: 0.0001 ST vs CM: 0.0001 CM vs PM: 1
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Hernia
Results
Fig. 1 Inflammation at the mesh/implant interface as quantified by macrophage and lymphocyte response. Both XL groups were associated with significantly higher degree of the inflammatory response (p = 0.001). There was no difference between the XL groups
All meshes induced varying degrees of inflammation throughout the study (Fig. 1). Pronounced inflammatory response, demonstrated by extensive proliferation of the mononuclear cells at the mesh/host interface, was associated with both XL meshes (Fig. 2a). The NXL group, on the other hand, was associated with minimal presence of inflammatory cells surrounding the mesh at 4-, 8-, and 12-week time points (Fig. 2b). Importantly, significant inflammatory response was present in both XL groups even 12 weeks after implantation (Fig. 3a, b). With regard to foreign body response, both XL meshes had significantly higher foreign body reaction (Fig. 4). As seen in Fig. 5a, histologic evaluation revealed FBGCs abundantly surrounding the XL mesh borders at the 8-week time point. Alternatively, ST did not display any FBGC accumulation (Fig. 5b).
Fig. 2 Histologic analysis revealed extensive presence of inflammatory cells adjacent to the XL mesh (a) and minimal inflammation at the interface of the NXL grafts (b) at the 4-week time point (H&E, 209)
Fig. 3 Persistent chronic inflammatory response to both XL grafts was seen at the 12-week time point (H&E, 109)
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Hernia Foreign Body Response
Neo -cellularization
3
2
2.5
1.5 2
Collamend 1.5
Collamend 1
Permacol
Permacol 1
Strattice
Strattice 0.5
0.5
0
0
4 wks 4 wks
8 wks
8 wks
12 wks
1 2 wks
Fig. 4 Foreign body response calculated by the presence of the foreign body giant cells was significantly diminished in the NXL group as compared to both XL groups (p = 0.0001). There was no difference between the XL groups
Mesh integration and remodeling was evaluated by macrophage and fibroblast penetration into the mesh scaffold, and new collagen deposition within the mesh. No group demonstrated significant mesh remodeling. However, only the NXL group was associated with any detectable level of mesh integration. Importantly, only ST displayed evidence of macrophage and fibroblast migration into the mesh scaffold at both 8- and 12-week time points. When compared with XL meshes, NXL group had significantly higher neocellularization into the mesh scaffold (Fig. 6). Both CM and PC were associated with moderate to severe fibrosis surrounding the mesh—encapsulation (Fig. 7). Furthermore, new vascularized collagen deposition was seen at the 12-week time point in the NXL group (Fig. 8). Importantly, no penetration of fibroblasts into the mesh was seen in either PC or CM grafts, even at the 12-week time point. (Fig. 9).
Fig. 6 Neocellularization was significantly higher in the NXL group at all three time points. Importantly, it increased at every subsequent time point (p \ 0.001). There was no difference between the XL groups
Sham mice samples demonstrated essentially normal histology. There was no evidence of chronic inflammation, new collagen deposition, or FBGCs’ presence.
Discussion Biologic mesh prosthetics are increasingly being utilized for hernia defect repairs, among other surgical procedures. The purported function of BMs is to serve as a regenerative framework by inducing neovascularization, with subsequent simultaneous BM matrix remodeling and new collagen deposition [5, 13–16]. While an ever increasing list of surgical disciplines is utilizing BMs for various repairs, there continues to be a paucity of data regarding the histopathologic reaction and changes of BMs following implantation [15, 17]. We aimed to characterize and
Fig. 5 Numerous FBGCs (black arrows) were noted to surround the XL graft (a) with no significant presence of those cells seen in the NXL group (H&E, 209) (color figure online)
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Hernia
Fig. 7 Dense fibrotic encapsulation of PM graft seen at 8 weeks after implantation (Trichrome blue stain, 209) (color figure online)
Fig. 8 XL graft remodeling is manifested by the fibroblast proliferation and new collagen deposition with visible vascular channels (white arrows) (H&E, 209) (color figure online)
compare histopathological responses to commonly used porcine BM scaffolds in a mouse. Inflammation appears to be a common component around implanted biologic prosthetics. The commonly used notion that the biologic meshes are ‘‘inert’’ is clearly without merit. In the present study, all implanted meshes displayed significant inflammatory reactions. Being a necessary component of wound healing, excessive inflammation may often lead to negative recognition. For biologic implants, this would lead to excessive fibrosis with encapsulation or matrix degradation and mesh failure [18]. A profound inflammatory response was present for crosslinked BMs, indicative of inferior biocompatibility of those grafts. Furthermore, crosslinked meshes demonstrated
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heightened foreign body reaction and encapsulation, both signs of deleterious reaction by the host. It was thus not surprising that we detected no evidence of mesh integration nor remodeling of any of the crosslinked grafts as far out as 12 weeks post-implantation. While tissue source may certainly impact how a BM is reacted upon by the recipient, difference in tissue reaction is likely, at least in part, a result of the different methods of processing, decellularization, and sterilization of each product. Manufacturers utilize various proprietary methods and processing solvents that likely influence the innate biochemical and biomolecular structure of the collagen scaffold [17–19]. Subsequently, these matrix alterations may influence foreign recognition and antigen presentation. The resulting processing changes likely influence biocompatibility, foreign body response and immunogenic potential of the implant. In fact, poor outcomes of several biologic meshes in the study by Sandor et al. [20] were linked to the tissue processing technique that may have damaged the biochemical matrix components. Unfortunately, the exact methodology of tissue processing remains proprietary and largely unknown. It, therefore, could not be precisely investigated as an independent risk factor of biocompatibility in this study. Of all the various processing techniques employed by mesh manufacturers, collagen crosslinking appears likely to have the most profound impact on tissue responses to BMs. Intentional crosslinking is utilized to prolong the lifespan of the mesh, using processing techniques that add to the 3-dimensional structure of the collagen to, essentially, mechanically strengthen the matrix and impede degradation of collagen. Of note, the final product of ‘‘terminally’’ crosslinked animal dermis is leather. By using hexamethylene diisocyanate, carbodiimide, glutaraldehyde, or photo-oxidizing agents [21, 22], intentionally crosslinked BMs are believed to remodel over a time course over 12 months or more, versus 4–6 months for the non-crosslinked BMs [23–25]. Even non-intentionally crosslinked products may get crosslinked from gamma irradiation during the sterilization process [26]. In addition, incomplete removal of chemical crosslinking agents could result in cytotoxicity from residues leaching from the BM which may induce prolonged toxic effects and heightened cellular responses [21, 22]. We have previously demonstrated in vitro profound differences in cytokine induction from human mononuclear cells when exposed to porcinebased BMs with crosslinking when compared with NXL meshes [27]. While certain clinical circumstances may utilize XL meshes, such as when ‘‘biologic’’ mesh behavior is not essential, we found that those grafts displayed significant immunologic disadvantage which could diminish their regenerative capacity and overall efficacy of those types of meshes in a clinical setting.
Hernia
Fig. 9 Poor neocellularization in the XL groups. Essentially, no penetration of fibroblasts into the graft was seen in either PC (a) or CM (b) grafts, even at the 12-week time point (H&E, 109)
Early cellular and vascular infiltration of a biologic matrix has been seen as critical for mesh integration and regeneration. Monocyte/macrophage penetration of the graft from the surface inward is paramount for fibroblast proliferation and new collagen deposition. In the absence of angiogenesis, remodeling may be impeded and the biologic graft, akin rapidly absorbable meshes, will breakdown, disappear and/or get simply replaced by scar. Butler et al. [28] found that NXL porcine dermis promoted early cellular and vascular infiltration and likely contributed to stronger mesh–musculofascia interface. Xu et al. [29] reported that functional blood vessels paralleled host cell repopulation with clearly delineated channels lined with endothelial cells in human dermis by 1 month after implantation in primates. These findings were confirmed more recently in a sublay biologic mesh study in rats [30]. The authors found that the Alloderm group was associated with 100 % neo-cellularity by 3 months after implantation. Neovascularization was noted to support the cells. Finally, normal, non-denatured collagen pattern was seen, indicative of remodeling and new collagen deposition [30]. We found moderate degree of cellular infiltration in the NXL group. Alternatively, similar to Butler et al. [28], we found essentially no new cellularization and/or vascularization in the XL meshes. In fact, our 4- and 12-week samples were indistinguishable from cellularity standpoint in the latter group. These findings clearly highlight poor biocompatibility of XL grafts and lack of ‘‘biologic’’ behavior of those biologic meshes. It is also important to point out, however, that Deeken et al. [31] have recently suggested, that although crosslinking differentiated biologic meshes with regard to cellular infiltration and neovascularization early on, those histologic features were no
longer impacted by crosslinking at a 1-year time point. While these isolated results are intriguing, it is, however, unclear whether the findings by Deeken et al. are true representations of what happens in humans or is just one of the limitations of long-term ventral hernia/biologic mesh investigation in resilient animal models like minipigs. The final and most important step in biologic mesh placement is graft integration and remodeling with new collagen deposition and tissue regeneration. Melman et al. [32] have suggested that when scaffold degradation is accompanied by cellular infiltration, ECM deposition, and neovascularization, it can be viewed as remodeling. Deeken et al. [31] recently reported that NXL grafts exhibited more favorable remodeling characteristics. Another recent study revealed essential lack of matrix absorption and absence of remodeling of XL grafts after 6-month implantation [30]. Interestingly, most of their CollaMend matrices were completely degraded by bacteria from associated wounds. The mesh samples that did not get infected were found to be essentially intact with no evidence of remodeling and poor integration [30]. Once again, while such mesh performance may at times result in an effective repair in the clinical setting, such behavior of XL biologic meshes can hardly be viewed as regenerative. Instead, given its lack of integration into the host and likely resultant fibrous encapsulation, XL grafts often act as permanent foreign body materials, similar to PTFE-based synthetics. In line with other investigators, we found in our mouse model that crosslinked grafts had no evidence of remodeling nor new collagen deposition (except for a thick layer of fibrous encapsulation) 3 months after implantation.
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We have utilized mice as our model for multiple projects due to the ease of implantation, low cost, and availability of genetic variants of mice for future studies. However, whether the tissue responses in mice are directly comparable to those of humans is debatable. Mice have been used extensively throughout modern scientific research and serve as useful stepping stones towards higher-order animals, and ultimately, human studies. In addition, we view our mouse model as an ideal baseline model for tissue responses to implanted biomaterials. By implanting biomaterials in a relatively benign subcutaneous space, we avoid other variables including direct tissue trauma in the immediate vicinity of the implant. For example, when utilizing a more advanced model with hernia defects, the tissue response may be a result of tissue trauma rather than the biomaterial itself. Finally, because our research is based on an animal model under ideal conditions, it is unclear how much histopathology displayed in our specimens relates to the clinical setting following biologic mesh implantation in humans. Future investigations, including ‘‘humanized’’ mice, mesh implantation during herniorrhaphy, as well as ‘‘stress’’ conditions like contamination and/or systemic immune and metabolic deficiencies are currently under way in our laboratory.
Conclusion All biologic mesh prosthetics induce inflammation at the site of implantation. We found that chemical crosslinking of acellular porcine dermal matrices was associated with a more pronounced host acute and chronic inflammatory response. Moreover, profound foreign body reactions to the XL grafts likely lead to observed encapsulation and poor integration. NXL mesh, on the other hand, was associated with the lowest foreign body response and the highest degree of new collagen deposition and matrix remodeling. Of critical importance, XL grafts had no evidence of new cellularization and no new collagen deposition. It thus appears that benefits of biologic mesh crosslinking may be a theoretical concept that, in fact, is deleterious to BM/host reaction at the site of implantation. Overall, improved biocompatibility of NXL grafts seen in this study may be one of the key predictors of superior performance of this type of biologic mesh during hernia repair. Acknowledgments This study was funded by institutional support from the University of Connecticut Health Center. No direct or indirect industry support was utilized. Conflict of interest Y.W.N. has received honorarium for consulting and/or speaking from LifeCell and CR Bard. SO and DK have nothing to disclose.
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