Inhibition of the activity of matrix metalloproteinase 2 by triethylene glycol dimethacrylate

The aim of this study was to evaluate the effect of different concentrations of triethylene glycol dimethacrylate (TEGDMA) on the inhibition of matrix metalloproteinase 2 (MMP-2). Mouse gingival explants were cultured overnight in DMEM and the expression of secreted enzymes was analyzed by gelatin zymography in buffers containing 5 mM CaCl₂ (Tris-CaCl₂) in 50 mM Tris-HCl buffer with the addition of TEGDMA at different concentrations (0.62%, 1.25%, 2.5%, or 5.0% (v/v)). The gelatinolytic proteinase present in the conditioned media was characterized as matrix metalloproteinase by means of specific chemical inhibition. The matrix metalloproteinases present in the conditioned media were characterized as MMP-2 by immunoprecipitation. The eletrophoretic bands were scanned and the transmittance values were analyzed. Data was plotted and submitted to linear regression to investigate MMP-2 inhibition as a function of TEGDMA concentration. Three major bands were detected in the zymographic assays. These bands were characterized as MMP-2. Zymogene (72 kDa), intermediate (66 kDa) and active forms of MMP-2 (62 kDa) were inhibited by TEGDMA in a dose-dependent way. These findings suggest that TEGDMA could inhibit MMP-2 expression even at small concentrations.     

Inhibition of human pulpal gelatinases (MMP-2 and MMP-9) by zinc oxide cements

The interaction between metal ions and the oral environment is a major subject matter in dental research. Matrix metalloproteinases (MMPs) have been implicated in several pathological and physiological processes such as, periodontal tissue destruction, root caries, dentin calcification and pulpal inflammation. The aim of this work was to test the effect of zinc released from zinc oxide-eugenol (ZOE) cements, on the activity of the major pulpal gelatinolytic MMPs. Pulpal explants were cultured overnight in Dulbecco's Modified Eagle Medium and the activity of secreted enzymes was analysed by gelatin zymography in buffer conditioned with diverse ZOE cements. Phenanthroline, a zinc chelator, was used to revert the inhibition of MMPs caused by zinc. The major gelatinolytic proteinases present in the conditioned media were characterized as MMP-2 and MMP-9 by immunoprecipitation. All ZOE cements inhibited MMPs activity, whereas phenanthroline could partially revert the inhibition caused by plain ZOE and Intermediate Restorative Material (IRM).     

Regulation of matrix metalloproteinase-2 production by cytokines and pharmacological agents in human pulp cell cultures.

Type IV matrix metalloproteinases (MMPs) are members of the family of MMPs and are thought to play an important role in degradation of extracellular components. Human pulp cells can secrete and produce these enzymes. Recent evidence shows that MMPs may play a role in pulpal inflammation. To date little is known regarding the regulation of MMPs in human pulp cell cultures. The purpose of this study was to determine the effects of cytokines (interleukin-1 and transforming growth factor-beta (TGF-beta), protein synthesis inhibitor cycloheximide (CD), and protein kinase C inhibitors (H7 and Go6976) on the secretion and production of MMPs by human pulp cell cultures using gelatin zymography. The main gelatinase secreted by human pulp cells migrated at 72 kDa and represented MMP-2. Minor gelatinolytic bands were also observed at 92 kDa regions that correspond to MMP-9. After an 8-day culture period TGF-beta, CD, H7, and Go6976 were found to depress MMP-2 production. The inhibition decreased in an order of CD > H7 > TGF-beta > Go6976. IL-1 was found to elevate MMP-2 production. Human pulp cells, however treated with either cytokines or pharmacological agents had no effect on the pattern of MMP-9 produced or secreted in either cell extracts or conditioned medium fractions. These observations suggest that the cytokines and pharmacological agents can regulate MMP-2 produced by human pulp cells. Inflammatory cytokines stimulate the production of elevated levels of MMP-2 and MMP-2 might play a role in pulpal inflammation. In addition agents that target protein synthesis or the protein kinase C pathway in human pulp cells inhibit MMP-2 production, and such inhibition may contribute to the pathogenesis of pulpal inflammation. Such inhibition might contribute to therapeutic efficacy.     

Inhibition of human gingival gelatinases (MMP-2 and MMP-9) by metal salts.

OBJECTIVES: The interaction between metal ions and the oral environment is a major subject matter in dental research. Matrix metalloproteinases (MMPs) have been implicated in several pathologic oral processes such as periodontal tissue destruction, root caries, tumour invasion and temporomandibular joint disorders. The aim of this work was to test the effect of Zn, Cu, Sn and Hg ions on the activity of the major gingival gelatinolytic MMPs. METHODS: Gingival explants were cultured overnight in DMEM and the activity of secreted enzymes was analyzed by gelatin zymography in buffers containing different metal ion concentrations. The major gelatinolytic proteinases present in the conditioned media were characterized as MMP-2 and MMP-9 by immunoprecipitation with specific antibodies. The eletrophoretic bands were scanned and the transmittance values were analyzed with the Sigmagel software (Sigma). RESULTS: ZnSO4 was a strong inhibitor of MMP-2 (I50 = 15 microM) and MMP-9 (I50 = 40 microM), whereas CuSO4, HgSO4 and SnCl2 showed less efficient inhibition potential. SIGNIFICANCE: Our findings show that the activity of oral tissue MMPs may be modulated by metal ions present in the oral environment. Therefore, the accumulation of metals in connective tissue may interfere with the formation and resorption of the extracellular matrix components.     

Levels and molecular forms of MMP-7 (matrilysin-1) and MMP-8 (collagenase-2) in diseased human peri-implant sulcular fluid

OBJECTIVES: Matrix metalloproteinases (MMPs) play crucial role in various tissue destructive inflammatory processes by degrading almost all peri-cellular and basement membrane components. MMP-8 (collagenase-2) is the major MMP in periodontitis. MMP-7 (matrilysin-1), in addition to its ability to degrade matrix and basement membrane components, activates other latent pro-MMPs and defensins, host cell-derived antimicrobial cryptidins. The aim of the present study was to characterize the relationship, levels and molecular forms of MMP-8 and MMP-7 in diseased peri-implant sulcular fluid (PISF). MATERIALS AND METHODS: Seventy-two human dental implant fluid samples were collected with filter paper strips from peri-implant sulci from healthy and untreated diseased implant sites. Gingival index (GI) and/or bone resorption (BR) were also recorded. Western immunoblot method with polyclonal anti-human-MMP-8 and monoclonal anti-human-MMP-7 antibodies was used, and immunoreactivities were quantified with computer scanning program. The effects of MMP inhibitors (doxycycline, chemically modified tetracycline-3, clodronate, CTT-peptide and marimastat) were studied on the activity of recombinant human matrilysin-1 (MMP-7) using beta-casein degradation assay. RESULTS: The levels of active forms of MMP-8 and MMP-7 were significantly elevated in diseased PISF in relation to healthy PISF. Furthermore, MMP-8 and MMP-7 levels correlated significantly to each other and GI. MMP-8 was present not only as bands corresponding to 75-kDa polymorphonuclear leukocyte (PMN) -type pro- and 65-kDa active forms, but also as 55-kDa non-PMN-type pro- and 45-kDa active forms. Immunoreactivities > 80 kDa most likely represented dimeric and/or inhibitor-bound MMP-8 complexes and the low molecular weight (< 30 kDa) species were apparently degraded fragments. In diseased PISF, 19-21-kDa active MMP-7 and 28-30-kDa pro-MMP-7 species were detected, and the active 19-21-kDa forms of MMP-7 predominated in diseased PISF. Doxycycline (50 micro m and 250 micro m), chemically modified non-antimicrobial tetracycline (CMT-3) (50 micro m and 100 micro m), clodronate (a bisphosphonate, 20 micro m and 500 micro m) and the cyclic CTT (CTTHWGFTLC)-peptide (125 micro m and 250 micro m), all known broad-spectrum or selective MMP-inhibitors, did not inhibit the activity of human recombinant MMP-7; only marimastat (1 micro m and 5 micro m) inhibited MMP-7. DISCUSSION: Increased immunoreactivities of the active MMP-8 and MMP-7 species in PISF from diseased peri-implantitis lesions eventually reflect the stage and course of peri-implantitis; MMP-7 may potentially act as MMP-8 and defensin activator in diseased PISF. CONCLUSION: The elevated levels of MMP-8 and matrilysin-1/MMP-7 were identified in active forms in diseased PISF, but MMP-7 was less prominent. MMP inhibitors, potential future tissue protective drugs, seemingly do not interfere with the defensive antibacterial action of MMP-7.     

Effects of alendronate on human osteoblast-like MG63 cells and matrix metalloproteinases

ObjectiveThe objective of this study was to examine the effects of alendronate on the expression and activity of matrix metalloproteinases (MMPs) and the expression of the tissue inhibitors of MMPs (TIMPs) from human osteoblast-like MG63 cells.Materials and methodsMG63 cells were exposed to various concentrations of alendronate. Cell proliferation and cytotoxicity were evaluated by water-soluble tetrazolium-1 and lactate dehydrogenase, respectively. MG63-mediated collagen degradation was assessed utilising Type I collagen assays. Conditioned media and membrane extracts were collected for Western blot analyses of select MMPs and TIMPs. Gelatin zymography gels were incubated with alendronate to assess its effects on MMP-2 activity.ResultsAlendronate affected MG63 proliferation and cytotoxicity at concentrations equal to/or greater than 10^?5 M (all p < 0.05). There were no significant differences in the collagen degrading ability of treated cells at non-toxic levels vs. untreated cells. Alendronate had no effects on the expression of MMP-2 or MT1-MMP (membrane type-1 MMP) in the conditioned media or membrane extracts, and of MMP-1 or TIMP-2 in the conditioned media. TIMP-2 in the membrane extracts was not detectable. MMP-2 activity in the zymograms was inhibited by 10^?3 and 10^?2 M alendronate.ConclusionAlendronate at 10^?5 M or higher was toxic to the cells. Alendronate at 10^?8 to 10^?6 M did not alter the expression of MMP-1, MMP-2, MT1-MMP or TIMP-2, as well as did not alter collagen degradation. Alendronate inhibited MMP-2 activity at 10^?3 and 10^?2 M in the zymograms. In conclusion, non-toxic levels of alendronate (10^?8 to 10^?6 M) did not alter MMP expression in MG63 cells or inhibit MMP-2 activity.     

Inhibitory effect of procyanidin oligomer from elm cortex on the matrix metalloproteinases and proteases of periodontopathogens

OBJECTIVES: The purpose of this study was to evaluate a partially purified extract (elm extract) from the Ulmi cortex (Ulmi macrocarpa Hance) and its active ingredient, a mix of procyanidin oligomers (3 to 12 flavan-3-ol monomers, an average molecular weight of 1,518 with an average polymerization degree of 5.3) for a possible inhibitory effect against proteases. BACKGROUND: Host-derived matrix metalloproteinases (MMPs) and bacterial proteases play important roles in the gingival tissue destruction that is a characteristic of periodontitis. The inhibitors of these proteases may be developed into therapeutic agents against periodontitis. METHODS: The inhibitory effects were assessed by gelatin zymography. The MMPs tested were originated from the gingival crevicular fluid (GCF) of adult periodontitis patients and from the conditioned media of cultured periodontal ligament (PDL) cells, which provided the proMMP-2 and activated MMP-2 when treated with a periodontopathogen, Treponema lecithinolyticum. Bacterial enzymes tested were secreted forms from two major periodontopathogens, Porphyromonas gingivalis and Treponema denticola. In addition, the inhibitory effects on trypsin-like enzymes from these two periodontopathogens were assayed by the n-benzoyl-DL-arginine-naphthylamide (BANA) test. RESULTS: The elm extract and the procyanidin oligomer (100-1,000 microg/ml) exhibited potent inhibitory effects on the MMPs in GCF (chiefly MMP-8 and MMP-9), the pro and active forms of MMP-2, and secreted and trypsin-like enzymes from T. denticola and P. gingivalis. CONCLUSIONS: These results suggest that elm cortex should be considered as a potential agent against periodontal diseases, due to its inhibitory action on MMPs and the proteases of periodontopathogens.     

Inhibition of ameloblastoma invasion in vitro and in vivo by inhibitor of metalloproteinase-2 activity

Background:  Ameloblastoma is an odontogenic benign tumor characterized by local invasiveness and most of its local recurrences clinically result from local invasion. This study used matrix metalloproteinase-2 (MMP-2) inhibitor I (MMP-2I) to investigate the role played by MMP-2 activity in the local invasiveness of ameloblastoma.
Methods:  The cells and xenografts of ameloblastoma were treated with MMP-2I and treatment group were compared with the control group. In vitro , the invasive activity of tumor cells was assayed in transwell cell culture chamber. Gelatinolytic activity of gelatinases and MMP-2/tissue inhibitor of matrix metalloproteinase (TIMP-2) protein expression was detected using gelatin zymography and flow cytometry. The cell viability and adhesion were evaluated using methyl thiazol tetrazolium. In vivo , bilateral subrenal capsule xenograft transplantation of ameloblastoma was performed in 10 nude mice and the invasion of ameloblastoma into the renal parenchyma was observed.
Results:  Active-MMP-2 of conditioned media was significantly lower in treatment group than in the control group. Accordingly, potential of in vitro cell invasion, adhesion and in vivo tumor invasion were also significantly lower in the treatment group than in the control group.
Conclusions:  Inhibitor of MMP-2 activity suppressed the local invasive capability of ameloblastoma by decreasing MMP-2 activity. MMP-2 activity is in relation with invasive capacity of ameloblastoma.     

Stimulation of epithelial cell matrix metalloproteinase (MMP-2, -9, -13) and interleukin-8 secretion by fusobacteria

Background/aims:  Bacterial pathogens involved in periodontal diseases exert their destructive effects primarily by stimulating the host cells to increase their secretion of proinflammatory cytokines and matrix metalloproteinases (MMPs). This study aimed to determine the epithelial cell matrix metalloproteinase and interleukin-8 (IL-8) secretion upon exposure to fusobacteria.
Methods:  Eight different oral and non-oral Fusobacterium strains were incubated with HaCaT epithelial cells. Gelatin zymography and Western blot analysis were performed to detect collagenase 3 (MMP-13), gelatinase A (MMP-2), gelatinase B (MMP-9), and IL-8 secretion by epithelial cells.
Results:  All Fusobacterium strains, especially Fusobacterium necrophorum ATCC 25286, Fusobacterium nucleatum ATCC 25586, and Fusobacterium varium ATCC 51644, increased MMP-9 and MMP-13 secretion. Fusobacterium simiae ATCC 33568, and to a lesser extent F. nucleatum and F. necrophorum , increased epithelial MMP-2 secretion. F. nucleatum and F. necrophorum also increased IL-8 secretion. F. varium ATCC 27725, a strain that only weakly stimulated MMP production, strongly increased the IL-8 production, suggesting that their expression is differently regulated.
Conclusion:  We conclude that the pathogenic potential of fusobacteria may partly result from their ability to stimulate secretion of MMP-9, MMP-13, and IL-8 from epithelial cells.     

Identification and characterization of gelatinases/type IV collagenases in jaw cysts

The molecular mechanisms of jaw cyst expansion probably involve interactions of matrix metalloproteinases (MMPs) and the tissue inhibitors of MMPs (TIMPs). In this study, molecular species of gelatinases present in neutral salt extracts of cyst walls and cyst fluids were characterized by functional activity measurements (type I gelatin and α-casein zymography) and immunologically (Western-blotting). The effects of various protein thiol-group or cysteine-switch reactants involved in the activation of collagenases were studied on cyst gelatinases and a gelatinases purified from human gingival fibroblasts (72 kD MMP-2), gingival keratinocytes (92 kD MMP-9) and polymorphonuclear neutrophilic leukocytes (92 kD MMP-9). Western-blottings revealed the presence of both 92 kD (MMP-9) and 72 kD (MMP-2) gelatinases in cyst wall extracts and cyst fluids. Western-blot studies further suggested that jaw cyst gelatinases were only in part complexed with and thus inhibited by TIMP-1 or TIMP-2, suggesting that both MMP-9 and MMP-2 may participate in cyst expansion. MMP-2 was also partially fragmented to a 68 kD form and additional lower molecular weight proteinases (<60 kD) were detected by α-casein zymography and by Western-blotting, suggesting proteolytic fragmentation. MMP-9 was at least partially activated by all protein-thiol group reactants and rather resistant to oxidative inhibition by hypochlorite (NaOCl); in contrast, MMP-2 was activated by APMA but not at all by gold thioglucose (GTG) and was clearly inactivated by hypochlorite (NaOCl). This indicates MMP-specific sensitivity to oxidative agents, but more specifically to preferential oxidative activation of PMN 92 kD MMP-9 and oxidative inactivation of the fibroblast-type 72 kD MMP-2.     

Regulation of matrix metalloproteinase production by cytokines,pharmacological agents and periodontal pathogens in human periodontal ligament fibroblast cultures

Matrix metalloproteinases (MMPs). produced by both infiltrating and resident cells of the periodontium, play a role in physiologic and pathologic events. It is recognized that an imbalance between activated MMPs and their endogenous inhibitors leads to pathologic breakdown of the extracellular matrix during periodontitis. To date, little is known about the regulation of MMP synthesis and secretion in human periodontal ligament fibroblasts (PDLFs). The purpose of this study was to examine the effects of cytokines, pharmacological agents (protein synthesis inhibitor and protein kinase C inhibitors) and predominant periodontal pathogens (Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis) on MMP production in human PDLFs using gelatin zymography. The gelatin zymograms revealed that the main gelatinase secreted by human PDLFs migrated at 72 kDa and represents MMP-2. Minor gelatinolytic bands were also observed at 92 kDa regions that correspond to MMP-9. We found that A. actinomycetemcomitans, P. gingivalis and IL-1alpha can elevate MMP-2 secretion in human PDLFs. These results indicate that periodontal pathogens and inflammatory cytokines play an important role in tissue destruction and disintegration of extracellular matrix in periodontal diseases. Thus, activation of MMPs may be one of the distinct host degradative pathways in the pathogenesis of periodontitis. In addition, H7, staurosporine, cycloheximide and TGF-beta could suppress MMP-2 production. Agents that target protein synthesis or the protein kinase C pathway in human PDLFs inhibit MMP-2 production, and such inhibition may contribute to the pathogenesis of periodontal inflammation. Taken together, these findings suggest a possible new therapeutic approach, involving the use of drugs that modify host-response mechanisms to suppress or inhibit MMP-mediated tissue destruction.     

Matrix metalloproteinase inhibitors reduce collagen gel contraction and alpha-smooth muscle actin expression by periodontal ligament cells

Background and Objective:  Orthodontic tooth movement requires remodeling of the periodontal tissues. The matrix metalloproteinases (MMPs) degrade the extracellular matrix components of the periodontal ligament, while the tissue inhibitors of metalloproteinases (TIMPs) control their activity. Synthetic MMP inhibitors have been developed to inhibit MMP activity. In this study, periodontal ligament cells in contracting collagen gels served as a model for enhanced periodontal remodeling. The effect of MMP inhibitors on gel contraction and on MMP and TIMP expression was analyzed.
Material and Methods:  Human periodontal ligament cells were cultured in three-dimensional collagen gels and incubated with the MMP inhibitors BB94, CMT-3, doxycycline and Ilomastat. Gel contraction was determined using consecutive photographs. The relative amounts of MMPs and TIMPs were analyzed using substrate zymography and mRNA expression using quantitative polyermase chain reaction.
Results:  All MMP inhibitors reduced MMP activity to about 20% of the control activity. They all reduced contraction, but CMT-3 and doxycycline had the strongest effect. These inhibitors also reduced MMP-2, MMP-3 and α-smooth muscle actin mRNA expression. The expression of MMP-1 mRNA seemed to be increased by CMT-3. No effects were found on the amounts of MMPs and TIMPs.
Conclusion:  Synthetic MMP inhibitors strongly reduced gel contraction by periodontal ligament cells. This was primarily caused by an inhibitory effect on MMP activity, which reduces matrix remodeling. In addition, α-smooth muscle actin expression was reduced by CMT-3 and doxycycline, which limits the contractile activity of the fibroblasts.     

Zymographic analysis and characterization of MMP-2 and -9 forms in human sound dentin

The role and function of dentin matrix metalloproteinases (MMPs) are not well-understood, but they may play a key role in dentinal caries and the degradation of resin-bonded dentin matrices. To test the null hypothesis that MMP-9 is not found in dentin matrix, we used gelatin zymography to extract and isolate all molecular forms of gelatinolytic MMPs in demineralized mature sound dentin powder obtained from extracted human molars, characterizing and identifying the enzymes by Western blotting. Gelatinolytic MMPs were detected in extracts of demineralized dentin matrix and identified as MMP-2 and MMP-9. Acidic extracts (pH 2.3) yielded 3-8 times more MMP activity than did EDTA (pH 7.4). Their activation may contribute to dentin matrix degradation, which occurs during caries progression and following resin bonding. Inhibition of MMP-2 and -9 proteolytic activity may slow caries progression and increase the durability of resin-dentin bonds.     

Tissue inhibitor of metalloproteinase-2 inhibits ameloblastoma growth in a new mouse xenograft disease model

J Oral Pathol Med (2010) 39 : 94–102
Background:  Ameloblastomas are odontogenic neoplasms characterized by local invasiveness. This study was conducted to develop a new animal model of ameloblastoma and to address the role of tissue inhibitor of metalloproteinase-2 (TIMP-2) and matrix metalloproteinase-2 (MMP-2) in the growth and invasiveness of ameloblastomas.
Method:  Donated fresh human ameloblastoma tissue was finely minced, screened, and subcutaneously implanted in three locations on each of 10 BALB/c-nu/nu nude mice. Newly established tumors on each mouse were injected with: (i) transfection reagent; (ii) liposome and transfection reagent; or (iii) liposome, transfection reagent, and the expression plasmid pcDNA3.1(+)/green fluorescent protein (GFP)-TIMP-2. Tumors were monitored for 5 weeks and excised for histopathology, RNA, and protein analyses.
Results:  The ameloblastoma xenografts were established with high frequency and contained a variety of typical features, validating this new model system. Xenografts injected with the TIMP-2 expression plasmid showed reduced growth, increased TIMP-2 mRNA and protein, and decreased MMP-2 protein compared with the control groups.
Conclusions:  We successfully established a new experimental model of ameloblastoma consisting of subcutaneous human xenografts in nude mice. In addition, we demonstrated the successful introduction of the TIMP-2 gene in tumor xenograft cells in vivo , resulting in xenograft growth inhibition. This growth inhibition may have resulted from TIMP-2 overexpression specifically inhibiting MMP-2 protein expression and activity.     

Expression of matrix metalloproteinases 2 and 9 in odontogenic myxoma in vivo and in vitro

Odontogenic myxoma is a benign neoplasm, which presents local invasiveness and tendency for recurrence. Matrix metalloproteinases (MMPs) 2 and 9 are involved in tumor invasion. Thus, the aim of this study was to analyze the expression and activity of these MMPs in odontogenic myxoma in vivo and in vitro. Three cases of odontogenic myxoma and cultured cells derived from this tumor (Mix1 cell line) were used. The detection and activity of two MMPs (2 and 9) were performed by immunohistochemistry in formalin-fixed, paraffin-embedded sections of odontogenic myxoma and immunofluorescence of the cultured cells and, by gelatin zymographic analysis of Mix1 conditioned media, respectively. MMPs 2 and 9 were detected in vivo and in vitro. The zymographic assay detected latent MMP-2 as well as latent and active MMP-9. Based on our findings, we suggest that MMPs may be involved in local invasiveness of the odontogenic myxoma. MMP-9 is not only secreted by odontogenic myxoma but also has enzyme activity with no further stimulation. Other MMPs were not analyzed; however, our results suggest that the invasive behaviour of odontogenic myxoma could be related at least to MMP-9.