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1.  Strategies to Minimize Adhesions to Intraperitoneally Placed Mesh in Laparoscopic Ventral Hernia Repair 
Fibrin sealant abolished adhesions to DualMesh and prevented adhesions to polypropylene mesh when applied over the entire surface of mesh in this animal study.
Adhesions to mesh/tacks in laparoscopic ventral hernia repair are often cited as reasons not to adopt its evidence-based superiority over conventional open methods. This pilot study assessed the occurrence of adhesions to full-sized Polypropylene and Gore-tex DualMesh Plus meshes and the possibility for adhesion prevention using fibrin sealant.
Two 10-cm to 15-cm pieces of mesh were placed and fixed laparoscopically in pigs (25kg to 55kg). Group I: 2 animals with Polypropylene mesh on one side and DualMesh on other side. Group II: 2 animals with DualMesh on each side with fibrin sealant applied to the periphery of mesh and staples to one side. Group III: 1 animal with 2 pieces of Polypropylene mesh with fibrin sealant applied to the entire mesh. All animals underwent laparoscopy 3 months later to assess the extent of adhesions, and full-thickness specimens were removed for histological evaluation.
More Polypropylene mesh was involved in adhesions than DualMesh. However, with the DualMesh involved in adhesions, more of the surface area was involved in forming adhesions than with Polypropylene mesh. None of the implanted DualMesh had visceral adhesions, while 2 out of 3 Polypropylene meshes had adhesions to both the liver and spleen but none to the bowel. Implanted Polypropylene mesh with fibrin sealant had no adhesions. DualMesh had shrunk more significantly than Polypropylene mesh. Histological evaluation showed absence of acute inflammatory response, significantly more chronic inflammatory response to DualMesh compared to Polypropylene and complete mesothelialization with both meshes. There was extensive collagen deposition between Polypropylene mesh fibers, while fibrosis occurred on both sides of DualMesh with synovial metaplasia over its peritoneal surface akin to encapsulation.
DualMesh caused fewer omental and visceral adhesions than Polypropylene mesh did. Fibrin sealant eliminated adhesions to DualMesh and prevented adhesions to Polypropylene mesh when applied over the entire surface. These results support our current use of DualMesh and fibrin sealant in LVHR.
PMCID: PMC3407463  PMID: 22906336
Laparoscopy; Ventral hernia; Fibrin sealant; Adhesion prevention
2.  Histologic and biomechanical evaluation of a novel macroporous polytetrafluoroethylene knit mesh compared to lightweight and heavyweight polypropylene mesh in a porcine model of ventral incisional hernia repair 
Hernia : the journal of hernias and abdominal wall surgery  2011;15(4):10.1007/s10029-011-0787-z.
To evaluate the biocompatibility of heavyweight polypropylene (HWPP), lightweight polypropylene (LWPP), and monofilament knit polytetrafluoroethylene (mkPTFE) mesh by comparing biomechanics and histologic response at 1, 3, and 5 months in a porcine model of incisional hernia repair.
Bilateral full-thickness abdominal wall defects measuring 4 cm in length were created in 27 Yucatan minipigs. Twenty-one days after hernia creation, animals underwent bilateral preperitoneal ventral hernia repair with 8 × 10 cm pieces of mesh. Repairs were randomized to Bard®Mesh (HWPP, Bard/Davol,, ULTRAPRO® (LWPP, Ethicon,, and GORE®INFINIT Mesh (mkPTFE, Gore & Associates, Nine animals were sacrificed at each timepoint (1, 3, and 5 months). At harvest, a 3 × 4 cm sample of mesh and incorporated tissue was taken from the center of the implant site and subjected to uniaxial tensile testing at a rate of 0.42 mm/s. The maximum force (N) and tensile strength (N/cm) were measured with a tensiometer, and stiffness (N/mm) was calculated from the slope of the force-versus-displacement curve. Adjacent sections of tissue were stained with hematoxylin and eosin (H&E) and analyzed for inflammation, fibrosis, and tissue ingrowth. Data are reported as mean ± SEM. Statistical significance (P < 0.05) was determined using a two-way ANOVA and Bonferroni post-test.
No significant difference in maximum force was detected between meshes at any of the time points (P > 0.05 for all comparisons). However, for each mesh type, the maximum strength at 5 months was significantly lower than that at 1 month (P < 0.05). No significant difference in stiffness was detected between the mesh types or between timepoints (P > 0.05 for all comparisons). No significant differences with regard to inflammation, fibrosis, or tissue ingrowth were detected between mesh types at any time point (P > 0.09 for all comparisons). However, over time, inflammation decreased significantly for all mesh types (P < 0.001) and tissue ingrowth reached a slight peak between 1 and 3 months (P = 0.001) but did not significantly change thereafter (P > 0.09).
The maximum tensile strength of mesh in the abdominal wall decreased over time for HWPP, LWPP, and mkPTFE mesh materials alike. This trend may actually reflect inability to adequately grip specimens at later time points rather than any mesh-specific trend. Histologically, inflammation decreased with time (P = 0.000), and tissue ingrowth increased (P = 0.019) for all meshes. No specific trends were observed between the polypropylene meshes and the monofilament knit PTFE, suggesting that this novel construction may be a suitable alternative to existing polypropylene meshes.
PMCID: PMC3826829  PMID: 21279663
Polypropylene; Knit polytetrafluoroethylene; Ventral hernia repair; Biomechanical evaluation; Histologic response; Tissue remodeling
3.  A Preclinical Evaluation of Alternative Synthetic Biomaterials for Fascial Defect Repair Using a Rat Abdominal Hernia Model 
PLoS ONE  2012;7(11):e50044.
Fascial defects are a common problem in the abdominal wall and in the vagina leading to hernia or pelvic organ prolapse that requires mesh enhancement to reduce operation failure. However, the long-term outcome of synthetic mesh surgery may be unsatisfactory due to post-surgical complications. We hypothesized that mesh fabricated from alternative synthetic polymers may evoke a different tissue response, and provide more appropriate mechanical properties for hernia repair. Our aim was to compare the in vivo biocompatibility of new synthetic meshes with a commercial mesh.
We have fabricated 3 new warp-knitted synthetic meshes from different polymers with different tensile properties polyetheretherketone (PEEK), polyamide (PA) and a composite, gelatin coated PA (PA+G). The rat abdominal hernia model was used to implant the meshes (25×35 mm, n = 24/ group). After 7, 30, 60, 90 days tissues were explanted for immunohistochemical assessment of foreign body reaction and tissue integration, using CD31, CD45, CD68, alpha-SMA antibodies. The images were analysed using an image analysis software program. Biomechanical properties were uniaxially evaluated using an Instron Tensile® Tester.
This study showed that the new meshes induced complex differences in the type of foreign body reaction over the time course of implantation. The PA, and particularly the composite PA+G meshes, evoked a milder early inflammatory response, and macrophages were apparent throughout the time course. Our meshes led to better tissue integration and new collagen deposition, particularly with the PA+G meshes, as well as greater and sustained neovascularisation compared with the PP meshes.
PA, PA+G and PEEK appear to be well tolerated and are biocompatible, evoking an overlapping and different host tissue response with time that might convey mechanical variations in the healing tissue. These new meshes comprising different polymers may provide an alternative option for future treatment of fascial defects.
PMCID: PMC3502256  PMID: 23185528
4.  Laparoscopic Evaluation of Abdominal Adhesions With Different Prosthetic Meshes in Rabbits 
The use of prosthetic materials to reinforce the abdominal wall is associated with a low index of recurrence; however, intraperitoneal placement of a foreign body may lead to adhesions. The present investigation was designed to determine adhesion formation with commercially available meshes implanted laparoscopically in rabbits.
Three different meshes were implanted laparoscopically in 24 rabbits: polypropylene (mesh A), polypropylene and sodium hyaluronate-carboxymethylcellulose (mesh B), and polypropylene and expanded polytetrafluoroethylene (mesh C). Sites of implantation for each mesh (the left lower quadrant, right lower quadrant, and lower midline) were randomly determined so that every rabbit had all 3 meshes implanted. All animals underwent diagnostic laparoscopy after 28 days to grade adhesions and histological analysis of inflammation.
Adhesions were noticed in 46 of the 72 meshes implanted (64%). The number of adhesions was higher for mesh C (87.5%) compared with meshes A (62.5%) and B (41.6%). The severity of adhesions was also higher for mesh C (grade I in 14, II in 6, and III in 1) compared with mesh A (grade I in 10, II in 4, and III in 1 case) and B (all of them grade II). Histological inflammatory reaction was classified as mild in 23 cases of mesh A, 15 of mesh B, and 23 of mesh C. A moderate reaction was found in 1 case of mesh A, 4 cases of mesh B, and 1 case of mesh C. Severe reaction was induced in 5 cases of mesh B. Mesh B induced a higher inflammatory reaction compared with the other meshes.
All meshes induced adhesions of different grades. Mesh B had fewer adhesions and more intense inflammation them did the others.
PMCID: PMC3016035  PMID: 18402740
Adhesions; Hernia repair; Laparoscopy; Mesh; Prosthetic materials; Rabbit
5.  Characterization of the Natural History of Extracellular Matrix Production in Tissue-Engineered Vascular Grafts during Neovessel Formation 
Cells, Tissues, Organs  2011;195(1-2):60-72.
The extracellular matrix (ECM) is a critical determinant of neovessel integrity. Materials and Methods: Thirty-six (polyglycolic acid + polycaprolactone and poly lactic acid) tissue-engineered vascular grafts seeded with syngeneic bone marrow mononuclear cells were implanted as inferior vena cava interposition grafts in C57BL/6 mice. Specimens were characterized using immunohistochemical staining and qPCR for representative ECM components in addition to matrix metalloproteinases (MMPs). Total collagen, elastin, and glycosaminoglycan (GAG) contents were determined. MMP activity was measured using zymography.
Collagen production on histology demonstrated an initial increase in type III at 1 week followed by type I production at 2 weeks and type IV at 4 weeks. Gene expression of both type I and type III peaked at 2 weeks, whereas type IV continued to increase over the 4-week period. Histology demonstrated fibrillin-1 deposition at 1 week followed by elastin production at 4 weeks. Elastin gene expression significantly increased at 4 weeks, whereas fibrillin-1 decreased at 4 weeks. GAG demonstrated abundant production at each time point on histology. Gene expression of decorin significantly increased at 4 weeks, whereas versican decreased over time. Biochemical analysis showed that total collagen production was greatest at 2 weeks, and there was a significant increase in elastin and GAG production at 4 weeks. Histological characterization of MMPs showed abundant production of MMP-2 at each time point, while MMP-9 decreased over the 4-week period. Gene expression of MMP-2 significantly increased at 4 weeks, whereas MMP-9 significantly decreased at 4 weeks.
ECM production during neovessel formation is characterized by early ECM deposition followed by extensive remodeling.
PMCID: PMC3257815  PMID: 21996715
Tissue engineering; Extracellular matrix; Vascular remodeling; Collagen; Elastin
6.  Site controlled transgenic mice validating increased expression from human matrix metalloproteinase (MMP-1) promoter due to a naturally occurring SNP 
Matrix Metalloproteinases (MMPs) comprise a family of more than 20 members, each with the ability to degrade components of the extracellular matrix. The interstitial collagenases have the unique capacity to degrade the stromal collagens, types I, II and III, the body's most abundant proteins. These collagenases include MMP-1, MMP-8, MMP-13 and MMP-14. MMP-1, with a very broad expression pattern, has major roles in mediating matrix destruction in many diseases. We have described a single nucleotide polymorphism (SNP) in the MMP-1 promoter that augments transcription. This SNP is the presence or absence of an extra guanine (G) at -1607 bp, which creates the sequence 5'-GGAA-3'(2G allele), and which is an ETS binding site. Compared to the 1G allele (5'-GAA-3'), the 2G SNP is associated with enhanced transcription of MMP-1 and increased enzymatic activity.
Although murine systems are often used to model human diseases, mice have only distant homologues of human MMP-1. Therefore, we used a technique for the targeted insertion of a single copy of a gene at the HPRT locus to compare expression of the 1G and 2G alleles. We generated transgenic mice with -4372 bp of the human MMP-1 promoter containing either the 1G or 2G SNP in front of the Lac Z (E.coli ß-galactosidase) gene. We measured relative expression of the transgenes in vitro in embryonic stem (ES) cells and in fibroblasts derived from embryonic mice. Our data show modest constitutive expression of ß-galactosidase mRNA and protein from these alleles, with the 2G allele more transcriptionally active than the 1G allele. We conclude that these mice represent a model for integration of a single copy of the human MMP-1 promoter into the murine genome, and could be used to study MMP-1 gene expression in a murine system.
PMCID: PMC2783711  PMID: 19577645
mRNA; ß-galactosidase; gene expression; single nucleotide polymorphism; fibroblasts
7.  Different tissue reaction of oesophagus and diaphragm after mesh hiatoplasty. Results of an animal study 
BMC Surgery  2008;8:7.
Laparoscopic mesh-reinforcement of the hiatal region in the treatment of gastroesophageal reflux disease (GERD) and paraesophageal hernia (PEH) reduces the risk of recurrence. However, there are still controversies about the technique of mesh placement, shape, structure and material. We therefore compared tissue integration and scar formation after implantation of two different polypropylene-meshes in a rabbit model.
A total of 20 female chinchilla rabbits were included in this study. Two different meshes (Polypropylene PP, Polyglecaprone 25 Composite PP-PG) were implanted on the abdominal diaphragm around the oesophagus. After 3 months the implanted meshes were excised en-bloc. Histological and morphological analyses were carried out accordingly proliferation rate, apoptosis and collagen type I/III ratio.
Regarding proliferation rate of oesophagus PP (9.31 ± 3.4%) and PP-PG (13.26 ± 2.54%) differ in a significant (p = 0.0097) way. In the diaphragm we found a significant (p = 0.00066) difference between PP (9.43 ± 1.45%) and PP-PG (18.73 ± 5.92%) respectively. Comparing oesophagus and diaphragm we could prove a significant difference within PP-PG-group (p = 0.0195). Within PP-group the difference reached no statistical significance (p = 0.88). We found analogous results regarding apoptosis.
Furthermore, there is a significant (p = 0.00013) difference of collagen type I/III ratio in PP-PG (12.28 ± 0.8) compared to PP (8.44 ± 1,63) in case of oesophageal tissue. Concerning diaphragm we found a significant difference (p = 0.000099) between PP-PG (8.85 ± 0.81) and PP (6.32 ± 1.07) as well.
The histologic and morphologic characteristics after prosthetic enforcement of the hiatus in this animal model show a more distinct tissue integration using PP-PG compared to PP. Additionally, different wound healing and remodelling capability influence tissue integration of the mesh in diaphragm and oesophagus.
PMCID: PMC2330020  PMID: 18405386
8.  Cellular and Molecular Factors in Flexor Tendon Repair and Adhesions: A Histological and Gene Expression Analysis 
Connective tissue research  2013;54(3):218-226.
Flexor tendon healing is mediated by cell proliferation, migration, and ECM synthesis that contribute to the formation of scar tissue and adhesion. The biological mechanisms of flexor tendon adhesion formation has been linked to TGF-β. To elucidate the cellular and molecular events in this pathology, we implanted live FDL grafts from the reporter mouse Rosa26LacZ/+ in WT recipients, and used histological β-galactosidase (β-gal) staining to evaluate the intrinsic versus extrinsic cellular origins of scar, and RT-PCR to measure gene expression of TGF-β and its receptors, extracellular matrix (ECM) proteins, and MMPs and their regulators. Over the course of healing, graft cellularity and β-gal activity progressively increased, and β-gal-positive cells migrated out of the Rosa26LacZ/+ graft. In addition, there was evidence of influx of host cells (β-gal-negative) into the gliding space and the graft, suggesting that both graft and host cells contribute to adhesions. Interestingly, we observed a biphasic pattern in which Tgfb1 expression was highest in the early phases of healing and gradually decreased thereafter, whereas Tgfb3 increased and remained upregulated later. The expression of TGF-β receptors was also upregulated throughout the healing phases. In addition, type III collagen and fibronectin were upregulated during the proliferative phase of healing, confirming that murine flexor tendon heals by scar tissue. Furthermore, gene expression of MMPs showed a differential pattern in which inflammatory MMPs were highest early and matrix MMPs increased over time. These findings offer important insights into the complex cellular and molecular factors during flexor tendon healing.
PMCID: PMC3697755  PMID: 23586515
Flexor tendon; Tendoplasty; Autograft; Allograft; Adhesions; Tenocytes; Transforming Growth Factor; Extracellular Matrix; Matrix Metalloproteinase
9.  Three-year results from a preclinical implantation study of a long-term resorbable surgical mesh with time-dependent mechanical characteristics 
Hernia  2011;16(2):191-197.
The purpose of this study was to evaluate the biocompatibility, local tissue effects and performance of a synthetic long-term resorbable test mesh (TIGR® Matrix Surgical Mesh) compared to a non-resorbable polypropylene control mesh following implantation in a sheep model.
Full-thickness abdominal wall defects were created in 14 sheep and subsequently repaired using test or control meshes. Sacrifices were made at 4, 9, 15, 24 and 36 months and results in terms of macroscopic observations, histology and collagen analysis are described for 4, 9, 15, 24 and 36 months.
The overall biocompatibility was good, and equivalent in the test and control meshes while the resorbable mesh was characterized by a collagen deposition more similar to native connective tissue and an increased thickness of the integrating tissue. The control polypropylene mesh provoked a typical chronic inflammation persistent over the 36-month study period. As the resorbable test mesh gradually degraded it was replaced by a newly formed collagen matrix with an increasing ratio of collagen type I/III, indicating a continuous remodeling of the collagen towards a strong connective tissue. After 36 months, the test mesh was fully resorbed and only microscopic implant residues could be found in the tissue.
This study suggests that the concept of a long-term resorbable mesh with time-dependent mechanical characteristics offers new possibilities for soft tissue repair and reinforcement.
PMCID: PMC3895198  PMID: 21972049
Preclinical; Hernia; Mesh; Implant; Resorbable; Absorbable; Degradable; Soft tissue; Collagen
10.  Behaviour of a New Composite Mesh for the Repair of Full-Thickness Abdominal Wall Defects in a Rabbit Model 
PLoS ONE  2013;8(11):e80647.
Composite biomaterials designed for the repair of abdominal wall defects are composed of a mesh component and a laminar barrier in contact with the visceral peritoneum. This study assesses the behaviour of a new composite mesh by comparing it with two latest-generation composites currently used in clinical practice.
Defects (7x5cm) created in the anterior abdominal wall of New Zealand White rabbits were repaired using a polypropylene mesh and the composites: PhysiomeshTM; VentralightTM and a new composite mesh with a three-dimensional macroporous polyester structure and an oxidized collagen/chitosan barrier. Animals were sacrificed on days 14 and 90 postimplant. Specimens were processed to determine host tissue incorporation, gene/protein expression of neo-collagens (RT-PCR/immunofluorescence), macrophage response (RAM-11-immunolabelling) and biomechanical resistance. On postoperative days 7/14, each animal was examined laparoscopically to quantify adhesions between the visceral peritoneum and implant.
The new composite mesh showed the lowest incidence of seroma in the short term. At each time point, the mesh surface covered with adhesions was greater in controls than composites. By day 14, the implants were fully infiltrated by a loose connective tissue that became denser over time. At 90 days, the peritoneal mesh surface was lined with a stable mesothelium. The new composite mesh induced more rapid tissue maturation than PhysiomeshTM, giving rise to a neoformed tissue containing more type I collagen. In VentralightTM the macrophage reaction was intense and significantly greater than the other composites at both follow-up times. Tensile strengths were similar for each biomaterial.
All composites showed optimal peritoneal behaviour, inducing good peritoneal regeneration and scarce postoperative adhesion formation. A greater foreign body reaction was observed for VentralightTM. All composites induced good collagen deposition accompanied by optimal tensile strength. The three-dimensional macroporous structure of the new composite mesh may promote rapid tissue regeneration within the mesh.
PMCID: PMC3827430  PMID: 24236192
11.  What do we know about titanized polypropylene meshes? An evidence-based review of the literature 
Hernia  2013;18(4):445-457.
Despite the vast selection of brands available, nearly all synthetic meshes for hernia surgery continue to use one or other of three basic materials: polypropylene, polyester and ePTFE. These are used in combination with each other or with a range of additional materials such as titanium, omega 3, monocryl, PVDF and hyaluronate. This systematic review of all experimental and clinical studies is aimed at investigating whether titanized meshes confer advantages over other synthetic meshes in hernia surgery.
Materials and methods
A search of the medical literature from 2002 to 2012, as indexed by Medline, was performed, using the PubMed search engine ( The search terms were: hernia mesh, titanium coating, lightweight mesh, TiMesh, mesh complications. All papers were graded according to the Oxford hierarchy of evidence.
Patients operated on with the Lichtenstein technique performed using the lightweight titanium-coated mesh have a shorter convalescence than those with the heavy-weight mesh Prolene. For inguinal hernias operated on with the TAPP technique and using a lightweight titanium-coated mesh in comparison to a heavy-weight Prolene mesh, the early postoperative convalescence seems to improve. Titanized meshes do exhibit a negative effect on sperm motility 1 year after a TEP operation, but not after 3 years. The laparoscopic IPOM technique with a titanium-coated polypropylene mesh was associated with less postoperative pain in the short term, lower analgesic consumption and a quicker return to everyday activities compared with the Parietex composite mesh.
In clinical studies, the titanium-coated polypropylene mesh shows in inguinal hernia repair certain benefits compared with the use of older heavy-weight meshes.
PMCID: PMC4113678  PMID: 24253381
Titanized polypropylene meshes; TiMesh; Mesh biocompatibility; Mesh fixation
12.  Ventralight ST and SorbaFix Versus Physiomesh and Securestrap in a Porcine Model 
Ventralight ST with SorbaFix fixation exhibited more favorable strength of tissue ingrowth and histologic response with similar mesh contracture and adhesion characteristics compared with Physiomesh fixed with Securestrap.
Background and Objectives:
The objective of this study was to compare mesh contracture, adhesion characteristics, tissue ingrowth, and histologic response of Ventralight ST/SorbaFix (C.R. Bard/Davol, Warwick, RI, USA) with Physiomesh/Securestrap (Ethicon, Somerville, NJ, USA) in a porcine model of laparoscopic ventral hernia repair.
Standard laparoscopic technique was used to bilaterally implant meshes in 10 female Yorkshire swine. Each animal received either two Ventralight ST meshes (oval shaped, 10.2 × 15.2 cm) or two Physiomesh meshes (oval shaped 10 × 15 cm), one on either side of the midline. The meshes were fixated to the intact peritoneum with either SorbaFix (for animals receiving Ventralight ST) or Securestrap (for animals receiving Physiomesh). There were 5 animals in each group, yielding 10 of each mesh-fixation combination. Mesh contracture, adhesion characteristics, tissue ingrowth, and histologic response were evaluated after 14 days by image analysis, mechanical testing, and histologic staining (hematoxylin-eosin, Masson trichrome, picrosirius red, and von Willebrand factor).
Ventralight ST/SorbaFix and Physiomesh/Securestrap exhibited a similar percentage of mesh contracture, percentage of adhesion coverage, adhesion tenacity, collagen deposition, and levels of necrosis (P > .05 in all cases). However, Ventralight ST/SorbaFix exhibited significantly less inflammation (P = .0001), fibrosis (P = .0017), hemorrhage (P = .0001), and angiogenesis (P = .0032) and significantly greater strength of tissue ingrowth (P = .0003) than Physiomesh/Securestrap after the 14-day implantation period.
Ventralight ST/SorbaFix exhibited more favorable strength of tissue ingrowth and histologic response and similar mesh contracture and adhesion characteristics compared with Physiomesh/Securestrap over a short-term 14-day implantation period in a preclinical porcine model.
PMCID: PMC3866058  PMID: 24398196
Absorbable fixation; Tensile strength; Adhesions; Ventral hernia; Tissue ingrowth; Absorbable barrier mesh
13.  Tumor necrosis factor-α-accelerated degradation of type I collagen in human skin is associated with elevated matrix metalloproteinase (MMP)-1 and MMP-3 ex vivo 
Tumor necrosis factor (TNF)-α induces matrix metalloproteinases (MMPs) that may disrupt skin integrity. We have investigated the effects and mechanisms of exogenous TNF-α on collagen degradation by incubating human skin explants in defined serum-free media with or without TNF-α (10 ng/ml) in the absence or presence of the nonselective MMP inhibitor GM6001 for 8 days. The basal culture conditions promoted type I collagen catabolism that was accelerated by TNF-α (p < 0.005) and accomplished by MMPs (p < 0.005). Levels of the collagenases MMP-8 and MMP-13 were insignificant and neither MMP-2 nor MMP-14 were associated with increased collagen degradation. TNF-α increased secretion of MMP-1 (p < 0.01) but had no impact on MMP-1 quantities in the tissue. Immunohistochemical analysis confirmed similar tissue MMP-1 expression with or without TNF-α with epidermis being the major source of MMP-1. Increased tissue-derived collagenolytic activity with TNF-α exposure was blocked by neutralizing MMP-1 monoclonal antibody and was not due to down-regulation of tissue inhibitor of metalloproteinase-1. TNF-α increased production (p < 0.01), tissue levels (p < 0.005) and catalytic activity of the endogenous MMP-1 activator MMP-3. Type I collagen degradation correlated with MMP-3 tissue levels (rs = 0.68, p < 0.05) and was attenuated with selective MMP-3 inhibitor. Type I collagen formation was down-regulated in cultured compared with native skin explants but was not reduced further by TNF-α. TNF-α had no significant effect on epidermal apoptosis. Our data indicate that TNF-α augments collagenolytic activity of MMP-1, possibly through up-regulation of MMP-3 leading to gradual loss of type I collagen in human skin.
PMCID: PMC4300401  PMID: 25457675
Aging; Cytokine; Extracellular matrix proteins; Protease inhibitors; UK370106; C-terminal telopeptide of type I collagen; Type I C-terminal collagen propeptide
14.  Assessment of adhesion formation after laparoscopic intraperitoneal implantation of Dynamesh IPOM mesh 
Formation of adhesions after laparoscopic hernia repair using the intra-peritoneal onlay mesh (IPOM) procedure can lead to intestinal obstruction or mesh erosion into intestinal lumen. The aims of this study included: measurement of adhesion formation with Dynamesh IPOM after laparoscopic intraperitoneal implantation, and assessment of the occurrence of isolated adhesions at the fastening sites of slowly absorbable sutures.
Material and methods
Twelve healthy pigs underwent laparoscopic implantation of 2 Dynamesh IPOM mesh fragments each, one was fastened with PDSII, and the other with Maxon sutures. An assessment of adhesion formation was carried out after 6 weeks and included an evaluation of surface area, hardness according to the Zhulke scale, and index values. The occurrence of isolated adhesions at slowly absorbable suture fixation points was also analyzed.
Adhesions were noted in 83.3% of Dynamesh IPOM meshes. Adhesions covered on average 37.7% of the mesh surface with mean hardness 1.46 and index value 78.8. In groups fixed with PDS in comparison to Maxon sutures adhesions covered mean 31.6% vs. 42.5% (p = 0.62) of the mesh surface, mean hardness was 1.67 vs.1.25 (p = 0.34) and index 85.42 vs. 72.02 (p = 0.95).
The Dynamesh IPOM mesh, in spite of its anti-adhesive layer of PVDF, does not prevent the formation of adhesions. Adhesion hardness, surface area, and index values of the Dynamesh IPOM mesh are close to the mean values of these parameters for other commercially available 2-layer meshes. Slowly absorbable sutures used for fastening did not increase the risk of adhesion formation.
PMCID: PMC3701981  PMID: 23847671
adhesions; surgical meshes; laparoscopy
15.  Protective constriction of coronary vein grafts with knitted nitinol 
Different flow patterns and shear forces were shown to cause significantly more luminal narrowing and neointimal tissue proliferation in coronary than in infrainguinal vein grafts. As constrictive external mesh support of vein grafts led to the complete suppression of intimal hyperplasia (IH) in infrainguinal grafts, we investigated whether mesh constriction is equally effective in the coronary position.
Eighteen senescent Chacma baboons (28.8 ± 3.6 kg) received aorto-coronary bypass grafts to the left anterior descending artery (LAD). Three groups of saphenous vein grafts were compared: untreated controls (CO); fibrin sealant-sprayed controls (CO + FS) and nitinol mesh-constricted grafts (ME + FS). Meshes consisted of pulse-compliant, knitted nitinol (eight needles; 50 μm wire thickness; 3.4 mm resting inner diameter, ID) spray attached to the vein grafts with FS. After 180 days of implantation, luminal dimensions and IH were analysed using post-explant angiography and macroscopic and histological image analysis.
At implantation, the calibre mismatch between control grafts and the LAD expressed as cross-sectional quotient (Qc) was pronounced [Qc = 0.21 ± 0.07 (CO) and 0.18 ± 0.05 (CO + FS)]. Mesh constriction resulted in a 29 ± 7% reduction of the outer diameter of the vein grafts from 5.23 ± 0.51 to 3.68 ± 0 mm, significantly reducing the calibre discrepancy to a Qc of 0.41 ± 0.17 (P < 0.02). After 6 months of implantation, explant angiography showed distinct luminal irregularities in control grafts (ID difference between widest and narrowest segment 74 ± 45%), while diameter variations were mild in mesh-constricted grafts. In all control grafts, thick neointimal tissue was present [600 ± 63 μm (CO); 627 ± 204 μm (CO + FS)] as opposed to thin, eccentric layers of 249 ± 83 μm in mesh-constricted grafts (ME + FS; P < 0.002). The total wall thickness had increased by 363 ± 39% (P < 0.00001) in CO and 312 ± 61% (P < 0.00001) in CO + FS vs 82 ± 61% in ME + FS (P < 0.007).
In a senescent non-human primate model for coronary artery bypass grafts, constrictive, external mesh support of saphenous veins with knitted nitinol prevented focal, irregular graft narrowing and suppressed neointimal tissue proliferation by a factor of 2.5. The lower degree of suppression of IH compared with previous infrainguinal grafts coincided with a lesser reduction of calibre mismatch in the coronary grafts.
PMCID: PMC3708718  PMID: 23295444
Vein grafts; Coronary bypass; Knitted nitinol mesh; Intimal suppressive
16.  Effects of Molecular Weight and Loading on Matrix Metalloproteinase-2 Mediated Release from Poly(Ethylene Glycol) Diacrylate Hydrogels 
The AAPS Journal  2012;14(3):482-490.
Herein, we report on continued efforts to understand an implantable poly(ethylene glycol) diacrylate (PEGDA) hydrogel drug delivery system that responds to extracellular enzymes, in particular matrix metalloproteinase-2 (MMP-2) to provide controlled drug delivery. By attaching peptide as pendant groups on the hydrogel backbone, drug release occurs at an accelerated rate in the presence of active protease. We investigated MMP-2 entry and optimized parameters of the drug delivery system. Mesh size for different PEGDA molecular weight macromers was measured with PEGDA 3,400 hydrogels having a mesh size smaller than the dimensions of MMP-2 and PEGDA 10,000 and PEGDA 20,000 hydrogels having mesh sizes larger than MMP-2. Purified MMP-2 increased release of peptide fragment compared to buffer at several loading concentrations. Cell-stimulated release was demonstrated using U-87 MG cells embedded in collagen. GM6001, an MMP inhibitor, diminished release and altered the identity of the released peptide fragment. The increase in ratio of release from PEGDA 10,000 and PEGDA 20,000 hydrogels compared to PEGDA 3,400 hydrogels suggests MMP-2 enters the hydrogel. PEGDA molecular weight of 10,000 and 15 % (w/V) were the optimal conditions for release and handling. The use of protease-triggered drug delivery has great advantage particularly with the control of protease penetration as a parameter for controlling rate of release.
PMCID: PMC3385811  PMID: 22535508
cancer; chemotherapy; controlled drug delivery; enzyme-triggered drug delivery; matrix metalloproteinase-2; poly(ethylene glycol) diacrylate, hydrogel
17.  Inhibition of matrix metalloproteinases improves left ventricular function in mice lacking osteopontin after myocardial infarction 
Molecular and cellular biochemistry  2008;322(1-2):53-62.
Osteopontin (OPN) plays an important role in left ventricular (LV) remodeling after myocardial infarction (MI) by promoting collagen synthesis and accumulation. This study tested the hypothesis that MMP inhibition modulates post-MI LV remodeling in mice lacking OPN. Wild-type (WT) and OPN knockout (KO) mice were treated daily with MMP inhibitor (PD166793, 30 mg/kg/day) starting 3 days post-MI. LV functional and structural remodeling was measured 14 days post-MI. Infarct size was similar in WT and KO groups with or without MMP inhibition. M-mode echocardiography showed greater increase in LV end-diastolic (LVEDD) and end-systolic diameters (LVESD) and decrease in percent fractional shortening (%FS) and ejection fraction in KO-MI versus WT-MI. MMP inhibition decreased LVEDD and LVESD, and increased %FS in both groups. Interestingly, the effect was more pronounced in KO-MI group versus WT-MI (P<0.01). MMP inhibition significantly decreased post-MI LV dilation in KO-MI group as measured by Langendorff-perfusion analysis. MMP inhibition improved LV developed pressures in both MI groups. However, the improvement was significantly higher in KO-MI group versus WT-MI (P<0.05). MMP inhibition increased heart weight to body weight ratio, myocyte cross sectional area, fibrosis and septal wall thickness only in KO-MI. Percent apoptotic myocytes in the non-infarct area was not different between the treatment groups. Expression and activity of MMP-2 and MMP-9 in the non-infarct area was higher in KO-MI group 3 days post-MI. MMP inhibition reduced MMP-2 activity in KO-MI with no effect on the expression of TIMP-2 and TIMP-4 14 days post-MI. Thus, activation of MMPs contributes to reduced fibrosis and LV dysfunction in mice lacking OPN.
PMCID: PMC2711544  PMID: 18979185
Osteopontin; MMPs; Extracellular matrix; apoptosis; heart failure
18.  Interstitial remodeling in β1-adrenergic receptor transgenic mice 
Basic Research in Cardiology  2006;102(2):183-193.
Inhibition of proteolytic MMP activity could be a therapeutic approach to prevent ventricular dilatation by diminishing collagen matrix turnover and interstitial fibrosis. We investigated the time-course of MMP/TIMP activity during transition from hypertrophy to ventricular dilatation in transgenic mice with myocyte overexpression of the human β1-adrenergic receptor (β1TG). These β1TG mice were studied at 3 (normal function), 5 (hypertrophy) and 12 (ventricular dilatation) months of age compared to age-matched controls (WT).
Picro Sirius red staining and real-time PCR were performed for total collagen and for collagen type I and III quantification, respectively. MMP-activity assays (zymography), immunoblotting and real-time PCR experiments were done for gelatinase- (MMP-2, -9), collagenase- (MMP-1, -13), membrane-type MMP- (MT1- MMP; MMP-14) and TIMP expression measurements. To investigate β1-integrin activity, integrin-linked kinase (ILK) expression was measured by immunoblotting.
Compared to WT with normal cardiac function, interstitial collagen type I and III mRNA and protein expression increased 3.6-fold in β1TG at 5 months of age with moderate fibrosis and cardiomyocyte hypertrophy and 17-fold in β1TG at 12 months of age with severe fibrosis and ventricular dilatation. Protein expression of the collagenases MMP-1 and -13 as well as the gelatinase proMMP-2 increased in the β1TG group with cardiac hypertrophy. Maximal activity of the gelatinase MMP-2 (3.5-fold vs.WT) was measured in β1TG at 12 months of age with severe fibrosis and ventricular dilatation, accompanied by coexpression of MT1- MMP (3.8-fold vs.WT) colocalized to the cell membranes.
These data provide evidence that sympathetic overactivation can trigger interstitial matrix remodeling and fibrosis by induction of MMP/TIMP activity. In particular gelatinolytic MMP-2 activity accompanies ventricular dilatation and the development of heart failure.
PMCID: PMC2779411  PMID: 17122889
heart failure; myocardial fibrosis; cardiac remodeling; β1-adrenergic receptors; matrix metalloproteinases
19.  Changes in the expression of MMP2, MMP9, and ColIV in stromal cells in oral squamous tongue cell carcinoma: relationships and prognostic implications 
Type IV collagen (ColIV) is the most important scaffold for the basement membrane (BM) proteins, and plays an important role in regulating and limiting tumour invasion and metastasis.
Here, we observed the changes in morphology and distribution of type IV collagen (ColIV) in the basement membrane (BM) surrounding nests of carcinoma in 48 patients with oral tongue squamous cell (OTSCC). We examined the correlation between the expressions of ColIV, MMP-2 and MMP-9 and the prognosis of OTSCC patients. The intensity and patterns of expression were assessed immunohistochemically using anti-human mouse monoclonal MMP-2, MMP-9 and Col IV antibodies. Statistical analyses were performed to determine the prognostic correlations of ColIV, MMP-2, and MMP-9 levels.
MMP-2 and MMP-9 expressions in OTSCC were higher than those in normal oral mucosa and dysplastic oral mucosa group(MMP-2 iOD: 66.40 ± 24.20, 134.69 ± 37.08, and 357.79 ± 116.78; MMP-9 iOD: 88.05 ± 23.85, 307.13 ± 93.22, and 791.31 ± 260.52; in normal, dysplastic oral mucosa, and tumour tissues, respectively, P < 0.01); however, ColIV immunoreactivity was lower (ColIV iOD: 406.87 ± 62.95, 247.83 ± 42.30, and 151.92 ± 38.17 in normal, dysplastic oral mucosa, and tumour tissues, respectively, P < 0.01). High tumour and stromal MMP-2 and MMP-9 expression was significantly associated with positive lymph node status. Col IV expression was associated with positive lymph node status (P < 0.05), and have negatively correlated with the expression of MMP-2 and MMP-9. Overall survival was significantly shorter in patients with high tumour and stromal MMP-2 and MMP-9 expression, and tended to be shorter in patients with low ColIV expression.
Degradation of ColIV was closely related to increased MMP-2 and MMP-9 expression; MMP-9 have more important function than MMP-2 during the cancer development. Monitoring changes in the expression of ColIV, MMP-2, and MMP-9 may be a useful technique for assessing prognoses in OTSCC patients.
PMCID: PMC3490717  PMID: 23107277
Oral tongue squamous carcinoma; MMPs; ColIV; Immunohistochemistry; Prognosis
20.  Does PRP enhance bone integration with grafts, graft substitutes, or implants? A systematic review 
Several bone implants are applied in clinical practice, but none meets the requirements of an ideal implant. Platelet-rich plasma (PRP) is an easy and inexpensive way to obtain growth factors in physiologic proportions that might favour the regenerative process. The aim of this review is to analyse clinical studies in order to investigate the role of PRP in favouring bone integration of graft, graft substitutes, or implants, and to identify the materials for which the additional use of PRP might be associated with superior osseo- and soft tissues integration.
A search on PubMed database was performed considering the literature from 2000 to 2012, using the following string: ("Bone Substitutes"[Mesh] OR "Bone Transplantation"[Mesh] OR "Bone Regeneration"[Mesh] OR "Osseointegration"[Mesh]) AND ("Blood Platelets"[Mesh] OR "Platelet-Rich Plasma"[Mesh]). After abstracts screening, the full-texts of selected papers were analyzed and the papers found from the reference lists were also considered. The search focused on clinical applications documented in studies in the English language: levels of evidence included in the literature analysis were I, II and III.
Literature analysis showed 83 papers that fulfilled the inclusion criteria: 26 randomized controlled trials (RCT), 14 comparative studies, 29 case series, and 14 case reports. Several implant materials were identified: 24 papers on autologous bone, 6 on freeze-dried bone allograft (FDBA), 16 on bovine porous bone mineral (BPBM), 9 on β-tricalcium phosphate (β-TCP), 4 on hydroxyapatite (HA), 2 on titanium (Ti), 1 on natural coral, 1 on collagen sponge, 1 on medical-grade calcium sulphate hemihydrate (MGCSH), 1 on bioactive glass (BG) and 18 on a combination of biomaterials. Only 4 papers were related to the orthopaedic field, whereas the majority belonged to clinical applications in oral/maxillofacial surgery.
The systematic research showed a growing interest in this approach for bone implant integration, with an increasing number of studies published over time. However, knowledge on this topic is still preliminary, with the presence mainly of low quality studies. Many aspects still have to be understood, such as the biomaterials that can benefit most from PRP and the best protocol for PRP both for production and application.
PMCID: PMC3870962  PMID: 24261343
PRP; Implant integration; Platelets; Bone; Regenerative medicine
21.  Collagen Fibril Growth During Chicken Tendon Development: Matrix Metalloproteinase-2 and Its Activation 
Cell and tissue research  2009;336(1):79-89.
The role of matrix metalloproteinases (MMPs) in collagen fibrillogenesis during development was studied in the well-characterized chicken metatarsal tendon. Collagen fibrils are initially assembled as intermediates and the mature fibrils assemble by linear and lateral growth from intermediates. We hypothesize that this involves the turnover of fibril-associated molecules mediated by expression and activation of matrix metalloproteinase-2 (MMP-2). We demonstrate changes in the ratio of full-length to truncated MMP-2 during tendon development, consistent with enzyme activation. The level of full length proMMP-2 remains relatively unchanged, however, the truncated form of MMP-2 is highest prior to and during fibril growth. Membrane type matrix metalloproteinase-3 (MT3-MMP, MMP-16) is fibroblast-associated and involved in the regulation of MMP-2 as well as in direct matrix turnover. The ratio of full-length proMT3-MMP/truncated (active) MT3-MMP has a pattern similar to that of full-length proMMP-2/truncated (active) MMP-2 during tendon development. Regulation of proMMP-2 activation involves complex formation with active MT3-MMP and TIMP-2. The constant low TIMP-2 expression seen in tendon development is consistent with this role. Isolation of collagen fibrils from pre-fibril growth tendons (14 day) in the presence of activated MMP-2 is associated with premature fibril growth seen as increased fibril diameters compared to controls. These data implicate MMP-2/MT3-MMP in the initiation of and progression of fibril growth, matrix assembly and tendon development. This may involve turnover of fibril-associated molecules involved in regulating linear and lateral growth, such as small leucine-rich proteoglycans and fibril-associated collagens. Activation of proMMP-2 dependent on MT3-MMP would allow a focal control of turnover.
PMCID: PMC2746393  PMID: 19221802
Matrix Metalloproteinases; MMP-2; Collagen Fibril Growth; Tendon; Development
22.  Repair of Abdominal Wall Defects with Biodegradable Laminar Prostheses: Polymeric or Biological? 
PLoS ONE  2012;7(12):e52628.
Biological and synthetic laminar absorbable prostheses are available for the repair of hernia defects in the abdominal wall. They share the important feature of being gradually degraded in the host, resulting in place the formation of a neotissue. This study was designed to assess the host tissue’s incorporation of collagen bioprostheses and a synthetic absorbable prosthesis.
Partial defects were created in the abdominal walls of 72 New Zealand rabbits and repaired using collagen bioprostheses Tutomesh® and Strattice® or a synthetic prosthesis Bio-A®. Specimens were collected for light microscopy, collagens gene and protein expression, macrophage response and biomechanical resistance at 14, 30, 90 and 180 days post-implantation.
Tutomesh® and Bio-A® were gradually infiltrated by the host tissue and almost completely degraded by 180 days post-implantation. In contrast, Strattice® exhibited material encapsulation, no prosthetic degradation and low cell infiltration at earlier timepoints, whereas at later study time, collagen deposition could be observed within the mesh. In the short term, Bio-A® exhibited higher level of collagen 1 and 3 mRNA expression compared with the two other biological prostheses, which exhibited two peaks of higher expression at 14 and 90 days. The expression of collagen III was homogeneous throughout the study and collagen I deposition was more evident in Strattice®. Macrophage response decreased over time in biomeshes. However, in the synthetic mesh remained high and homogeneous until 90 days. The biomechanical analysis demonstrated the progressively increasing tensile strength of all biomaterials.
The tissue infiltration of laminar absorbable prostheses is affected by the structure and composition of the mesh. The synthetic prosthesis exhibited a distinct pattern of tissue incorporation and a greater macrophage response than did the biological prostheses. Of all of the laminar, absorbable biomaterials that were tested in this study, Strattice® demonstrated the optimal levels of integration and degradation.
PMCID: PMC3528658  PMID: 23285119
Journal of vascular surgery  2009;49(3):10.1016/j.jvs.2008.11.001.
Saphenous vein grafts suffer from neointima formation following bypass surgery. Matrix metalloproteinases (MMPs) play important roles in this process. We examined MMP-3 for its therapeutic potential to prevent smooth muscle cell migration and neointima formation in venous bypass grafts using adenovirus-mediated gene transfer.
Human aortic smooth muscle cells (hAoSMC) were transduced with adenoviral vectors encoding β-galactosidase (Ad.βgal) or human MMP-3 (Ad.hMMP-3), and characterized for migration in the amniotic membrane stroma as an in vitro model of the vascular wall. Cholesterol-fed New Zealand White rabbits underwent jugular vein bypass grafting into carotid arteries. Before insertion, grafts were incubated ex vivo with either Ad.βgal or Ad.hMMP-3. Transgene expression was characterized by immunohistochemistry and in situ zymography. Grafts (n=6) were explanted after 28 days and intimal hyperplasia was quantified.
Migration of hAoSMC was significantly reduced when transduced with Ad.hMMP-3 compared to controls (p<0.001). Immunocytochemistry of Ad.hMMP-3 transduced venous grafts localized this protein to the intima. In situ-zymography showed increased MMP activity in the intima of Ad.hMMP-3 transfected grafts. Stenosis degree (p=0,001), intima/media-ratio (p=0,023) and lesion thickness (p=0,003) were significantly reduced in grafts transduced with Ad.MMP-3 in comparison to controls. There was no difference inside control groups.
MMP-3 overexpression inhibits formation of intimal hyperplasia in arterialized vein grafts. Adenovirus mediated gene transfer of MMP-3 may be of clinical use to prevent vein graft stenosis following bypass surgery.
PMCID: PMC3816542  PMID: 19268777
matrix-metalloproteinases; gene therapy; bypass; vascular diseases; surgery
24.  Matrix metalloproteinase-9 in the initial injury after hepatectomy in mice 
AIM: To investigate the role of matrix metalloproteinase (MMP)-9 in the pathogenesis of postoperative liver failure (PLF) after extended hepatectomy (EH).
METHODS: An insufficient volume of the remnant liver (RL) results in higher morbidity and mortality, and a murine model with 80%-hepatectomy was used. All investigations were performed 6 h after EH. Mice were first divided into two groups based on the postoperative course (i.e., the PLF caused or did not), and MMP-9 expression was measured by Western blotting. The source of MMP-9 was then determined by immunohistological stainings. Tissue inhibitor of metalloproteinase (TIMP)-1 is the endogenous inhibitor of MMP-9, and MMP-9 behavior was assessed by the experiments in wild-type, MMP-9(-/-) and TIMP-1(-/-) mice by Western blotting and gelatin zymography. The behavior of neutrophils was also assessed by immunohistological stainings. An anti-MMP-9 monoclonal antibody and a broad-spectrum MMP inhibitor were used to examine the role of MMP-9.
RESULTS: Symptomatic mice showed more severe PLF (histopathological assessments: 2.97 ± 0.92 vs 0.11 ± 0.08, P < 0.05) and a higher expression of MMP-9 (71085 ± 18274 vs 192856 ± 22263, P < 0.01). Nonnative leukocytes appeared to be the main source of MMP-9, because MMP-9 expression corresponding with CD11b positive-cell was observed in the findings of immunohistological stainings. In the histopathological findings, the PLF was improved in MMP-9(-/-) mice (1.65% ± 0.23% vs 0.65% ± 0.19%, P < 0.01) and it was worse in TIMP-1(-/-) mice (1.65% ± 0.23% vs 1.78% ± 0.31%, P < 0.01). Moreover, neutrophil migration was disturbed in MMP-9(-/-) mice in the immunohistological stainings. Two methods of MMP-9 inhibition revealed reduced PLF, and neutrophil migration was strongly disturbed in MMP-9-blocked mice in the histopathological assessments (9.6 ± 1.9 vs 4.2 ± 1.2, P < 0.05, and 9.9 ± 1.5 vs 5.7 ± 1.1, P < 0.05).
CONCLUSION: MMP-9 is important for the process of PLF. The initial injury is associated with MMP-9 derived from neutrophils, and MMP-9 blockade reduces PLF. MMP-9 may be a potential target to prevent PLF after EH and to overcome an insufficient RL.
PMCID: PMC3662942  PMID: 23716982
Matrix metalloproteinase; Shear stress; Sinusoidal injury; Hepatectomy; Portal hypertension
25.  Active synovial matrix metalloproteinase-2 is associated with radiographic erosions in patients with early synovitis 
Arthritis Research  2000;2(2):145-153.
Serum and synovial tissue expression of the matrix metalloproteinase (MMP)-2 and -9 and their molecular regulators, MMP-14 and TIMP-2 was examined in 28 patients with inflammatory early synovitis and 4 healthy volunteers and correlated with the presence of erosions in the patients. Immunohistological staining of MMP-2, MMP-14 and TIMP-2 localized to corresponding areas in the synovial lining layer and was almost absent in normal synovium. Patients with radiographic erosions had significantly higher levels of active MMP-2 than patients with no erosions, suggesting that activated MMP-2 levels in synovial tissue may be a marker for a more aggressive synovial lesion.
In cancer the gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] have been shown to be associated with tissue invasion and metastatic disease. In patients with inflammatory arthritis the gelatinases are expressed in the synovial membrane, and have been implicated in synovial tissue invasion into adjacent cartilage and bone. It is hypothesized that an imbalance between the activators and inhibitors of the gelatinases results in higher levels of activity, enhanced local proteolysis, and bone erosion.
To determine whether the expression and activity levels of MMP-2 and MMP-9, and their regulators MMP-14 and tissue inhibitor of metalloproteinase (TIMP), are associated with early erosion formation in patients with synovitis of recent onset.
Patients and method:
A subset of 66 patients was selected from a larger early synovitis cohort on the basis of tissue availability for the study of synovial tissue and serum gelatinase expression. Patients with peripheral joint synovitis of less than 1 years' duration were evaluated clinically and serologically on four visits over a period of 12 months. At the initial visit, patients underwent a synovial tissue biopsy of one swollen joint, and patients had radiographic evaluation of hands and feet initially and at 1year. Serum MMP-1, MMP-2, MMP-9, MMP-14, and TIMP-1 and TIMP-2 levels were determined, and synovial tissue was examined by immunohistology for the expression of MMP-2 and MMP-9, and their molecular regulators. Gelatinolytic activity for MMP-2 and MMP-9 was quantified using a sensitive, tissue-based gel zymography technique. Four healthy individuals underwent closed synovial biopsy and their synovial tissues were similarly analyzed.
Of the 66 patients studied, 45 fulfilled American College of Rheumatology criteria for rheumatoid arthritis (RA), with 32 (71%) being rheumatoid factor positive. Of the 21 non-RA patients, seven had a spondylarthropathy and 14 had undifferentiated arthritis. Radiographically, 12 of the RA patients had erosions at multiple sites by 1 year, whereas none of the non-RA patients had developed erosive disease of this extent. In the tissue, latent MMP-2 was widely expressed in the synovial lining layer and in areas of stromal proliferation in the sublining layer and stroma, whereas MMP-9 was expressed more sparsely and focally. MMP-14, TIMP-2, and MMP-2 were all detected in similar areas of the lining layer on consecutive histologic sections. Tissue expression of MMP-14, the activator for pro-MMP-2, was significantly higher in RA than in non-RA patients (8.4 ± 5 versus 3.7 ± 4 cells/high-power field; P = 0.009). In contrast, the expression of TIMP-2, an inhibitor of MMP-2, was lower in the RA than in the non-RA samples (25 ± 12 versus 39 ± 9 cells/high-power field; P = 0.01). Synovial tissue expressions of MMP-2, MMP-14, and TIMP-2 were virtually undetectable in normal synovial tissue samples. The synovial tissue samples of patients with erosive disease had significantly higher levels of active MMP-2 than did those of patients without erosions (Fig. 1). Tissue expression of MMP-2 and MMP-9, however, did not correlate with the serum levels of these enzymes.
With the exception of serum MMP-2, which was not elevated over normal, serum levels of all of the other MMPs and TIMPs were elevated to varying degrees, and were not predictive of erosive disease. Interestingly, MMP-1 and C-reactive protein, both of which were associated with the presence of erosions, were positively correlated with each other (r = 0.42; P < 0.001).
MMP-2 and MMP-9 are thought to play an important role in the evolution of joint erosions in patients with an inflammatory arthritis. Most studies have concentrated on the contribution of MMP-9 to the synovitis, because synovial fluid and serum MMP-9 levels are markedly increased in inflammatory arthropathies. Previously reported serum levels of MMP-9 have varied widely. In the present sample of patients with synovitis of recent onset, serum MMP-9 levels were elevated in only 21%. Moreover, these elevations were not specific for RA, the tissue expression of MMP-9 was focal, and the levels of MMP-9 activity were not well correlated with early erosions. Although serum MMP-2 levels were not of prognostic value, high synovial tissue levels of MMP-2 activity were significantly correlated with the presence of early erosions. This may reflect augmented activation of MMP-2 by the relatively high levels of MMP-14 and low levels of TIMP-2 seen in these tissues. We were able to localize the components of this trimolecular complex to the synovial lining layer in consecutive tissue sections, a finding that is consistent with their colocalization.
In conclusion, we have provided evidence that active MMP-2 complexes are detectable in the inflamed RA synovium and may be involved in the development of early bony erosions. These results suggest that strategies to inhibit the activation of MMP-2 may have the potential for retarding or preventing early erosions in patients with inflammatory arthritis.
PMCID: PMC17808  PMID: 11062605
early synovitis; erosion; metalloproteinase; matrix metalloproteinase-2; rheumatoid arthritis

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