1) Activated matrix metalloproteinase-8 in saliva as diagnostic test for periodontal

disease? A casecontrol study

Abstract Untreated periodontal disease may influence general health. However, how may a physician,
who is not trained in periodontal probing, detect untreated peri- odontitis? Activated matrix
metalloproteinase-8 (aMMP- 8) in saliva correlates with periodontal probing param- eters. Thus, sensitivity
and specificity of a chair-side test for aMMP-8 to detect periodontitis were evaluated. Thirty cases
[untreated chronic periodontitis (ChP); 15 general- ized moderate and 15 generalized severe] and 30
controls[probing depths (PD) 3 mm, vertical probing attachment level (PAL-V) 2 mm at <30 % of
sites) were examined periodontally (PD, PAL-V, bleeding on probing). Sub-sequently, the aMMP-8 test
was performed. The test kitbecomes positive with 25 ng/ml aMMP-8 in the sam- ple. The aMMP-8 test
was positive in 87 % of ChP and in 40 % of controls. That corresponds to a sensitivity of 87 % and a
specificity of 60 %. The sensitivity to detect gener- alized severe ChP was 93 % (60 % specificity).
Backward stepwise logistic regression analysis to explain positive aMMP-8 tests identified exclusively ChP
with an odds ratioClinicalTrials.gov Identifier: NCT02280122.* Peter Eickholz eickholz@med.unifrankfurt.deSusan Izadi Borujeni susan_izadi@hotmail.comMatthias Mayer dr.matthiasmayer@t-online.de1
Department of Periodontology, Centre for Dentistry and Oral Medicine (Carolinum), Johann Wolfgang
Goethe- University Frankfurt/Main, Theodor-Stern-Kai 7 (Haus 29), 60596 Frankfurt am Main,
Germany2Private Practice, Arndtstrae 14, 60325 Frankfurt am Main, Germanyof 9.8 (p < 0.001).
Positive results of the aMMP-8 test sig- nificantly correlate with generalized ChP. The aMMP-8 test may
be used by physicians to detect periodontitis in their patients.
2) Matrix metalloproteinases and myeloperoxidase in gingival crevicular fluid provide
site-specific diagnostic value for chronic periodontitis
AIM: To identify the diagnostic accuracy of gingival crevicular fluid (GCF) candidate biomarkers to
discriminate periodontitis from the inflamed and healthy sites, and to compare the performance of two
independent matrix metalloproteinase (MMP)-8 immunoassays.\n\nMATERIALS AND METHODS: Cross
sectional study. GCF (N = 58 sites) was collected from healthy, gingivitis and chronic periodontitis
volunteers and analysed for levels of azurocidin, chemokine ligand 5, MPO, TIMP-1 MMP-13 and MMP-14
by ELISA or activity assays. MMP-8 was assayed by immunofluorometric assay (IFMA) and ELISA.
Statistical analysis was performed using linear mixed-effects models and Bayesian statistics in R and
Stata V11.\n\nRESULTS: MMP-8, MPO, azurocidin and total MMP-13 and MMP-14 were higher in
periodontitis compared to gingivitis and healthy sites (p < 0.05). A very high correlation between MPO
and MMP-8 was evident in the periodontitis group (r = 0.95, p < 0.0001). MPO, azurocidin and total
levels of MMP-8, MMP-13 and MMP-14 showed high diagnostic accuracy (0.90), but only MMP-8 and
MPO were significantly higher in the periodontitis versus gingivitis sites. MMP-8 determined by IFMA
correlated more strongly with periodontal status and showed higher diagnostic accuracy than
ELISA.\n\nCONCLUSIONS: MPO and collagenolytic MMPs are highly discriminatory biomarkers for sitespecific diagnosis of periodontitis. The comparison of two quantitative MMP-8 methods demonstrated
IFMA to be more accurate than ELISA.
3) Temporal expression of metalloproteinase-8 and -13 and their relationships with
extracellular matrix metalloproteinase inducer in the development of ligature-induced
periodontitis in rats
BACKGROUND AND OBJECTIVE: Matrix metalloproteinases (MMPs) play important roles in extracellular
matrix degradation and may be regulated by extracellular matrix metalloproteinase inducer (EMMPRIN).
The aim of this study was to investigate the temporal expression and localization of MMP-8 and MMP-13
during the development of ligature-induced periodontitis in rats, and to analyze the correlations of
EMMPRIN with MMP-8 and MMP-13 in periodontitis.\n\nMATERIAL AND METHODS: Periodontitis was
simulated in rats by ligaturing the cervix of the lower first molars, as described in our previous method.
The rats were killed 0, 3, 5, 7, 11, 15 and 21 d after ligation. Micro-computed tomography examinations
were performed to detect alveolar bone loss. Semiquantitative western blotting was used to assess the
temporal changes in the levels of MMP-8, MMP-13 and EMMPRIN proteins in gingival tissue.
Immunohistochemistry was applied to detect the expression and locations of MMP-8 and MMP-13 in
gingival tissue and alveolar bone.\n\nRESULTS: Alveolar bone loss showed an exponential increase from
days 3 to 11, followed by a slower rate of loss at subsequent study time points. MMP-8 showed a rapid
increase of expression from baseline to a peak on day 3, a gradual decrease from days 5 to 7 and then
stabilized thereafter. MMP-8 was predominantly located in neutrophil-like cells. Statistically, the expression
of MMP-8 was not correlated with the expression of EMMPRIN. The expression of MMP-13 and of
EMMRPIN increased from days 3 to 7, and showed a moderate decrease thereafter. The immunoreactivity
of MMP-13 was mainly detected in monocytes/macrophages, on the alveolar bone surface, in osteoclasts

and in gingival epithelial cells. Statistically, MMP-13 had a strong, positive correlation with EMMPRIN (r =
0.855, p < 0.01).\n\nCONCLUSION: The levels of expression of MMP-8 and MMP-13 are temporally varied
at different periods during the development of experimental periodontitis. The level of expression of
EMMPRIN is closely associated with the expression of MMP-13, but not with the expression of MMP-8. In
addition, MMP-13 might be involved in alveolar bone destruction, as well as in physiological bone
remodeling.
4) Matrix metalloproteinase-8 expression in periodontal tissues surgically removed
from diabetic and non-diabetic patients with periodontal disease
BACKGROUND: Although it is known that periodontal matrix metalloproteinase-8 (MMP-8) expression is
associated with periodontal disease, the information concerning the periodontal MMP-8 expression in
diabetic patients with periodontal disease is insufficient.\n\nMATERIALS AND METHODS: Periodontal tissue
specimens were collected from seven patients without periodontal disease and diabetes (Group 1), 15
patients with periodontal disease alone (Group 2) and 10 patients with both periodontal disease and
diabetes (Group 3). The frozen sections were prepared and MMP-8 protein expression was detected using
immunohistochemistry and quantified. For in vitro study, human U937 mononuclear cells were preexposed to normal or high glucose and then treated with lipopolysaccharide (LPS).\n\nRESULTS: The
nonparametric Kruskal-Wallis test showed that the difference in MMP-8 protein levels among the three
groups were statistically significant (p = 0.003). Nonparametric analysis using Jonckheere-Terpstra test
showed a tendency of increase in periodontal MMP-8 levels across Group 1 to Group 2 to Group 3 (p =
0.0002). In vitro studies showed that high glucose and LPS had a synergistic effect on MMP-8
expression.\n\nCONCLUSION: Our current study showed an increasing trend in MMP-8 protein expression
levels across patients without both periodontal disease and diabetes, patients with periodontal disease
alone and patients with both diseases.
5) Salivary levels of matrix metalloproteinase (MMP)-9 and tissue inhibitor of matrix
metalloproteinase (TIMP)-1: A pilot study about the relationship with periodontal
status and MMP-9-1562C/T gene promoter polymorphism
Objective: Chronic periodontitis (CP) has been linked with an imbalance in the MMP-9/TIMP-1 ratio. A
reasonable biologic explanation for this link is that the MMP-9 transcriptional activity can be modulated
by MMP-9-1562C/T gene promoter polymorphism contributing to periodontal breakdown. This study
aimed to assess the relationship between salivary MMP-9/TIMP-1 balance, MMP-9-1562C/T genotype and
periodontal clinical status. Design: Sixty-nine CP subjects and 54 healthy controls (HC) were selected.
Periodontal status was assessed by criteria based on probing depth, clinical attachment level, extent, and
severity of periodontal breakdown. Salivary levels of MMP-9 and TIMP-1 were analysed using ELISA and
MMP-9-1562C/T genotype using the polymerase chain reaction-restriction fragment length polymorphism
(PCR-RFLP) method. The association between salivary levels of MMP-9, TIMP-1 and MMP-9/TIMP-1 ratio
with CP was assessed individually and adjusted for confounding using a binary logistic regression model.
Results: Significantly higher levels of both markers and their ratios were detected in the CP group in
comparison to healthy controls. Synchronously, weak-to-moderate positive significant correlations
between salivary biomarkers and clinical parameters were observed. After binary logistic regression
analysis, salivary levels of MMP-9 > 20 ng mL-1, TIMP-1 > 64 ng mL-1 as well as MMP-9/TIMP-1 ratio >1
were independently associated with CP. Nevertheless, the MMP-9-1562C/T gene promoter polymorphism
was not associated with the different degrees of chronic periodontitis and did not have influence on the
salivary levels of biomarkers. Conclusion: The findings when considered within the limitations of this study
may indicate that although a dominant expression of MMP-9 over TIMP-1 in saliva might reflect the
periodontal clinical status, the functional polymorphisms in the promoter of the MMP-9-1562C/T gene
from the Colombian population are not linked neither with significant salivary MMP-9 variations in these
individuals nor periodontal clinical status. 2010 Elsevier Ltd. All rights reserved.
6) Detection of gingival crevicular fluid MMP-8 levels with different laboratory and
chair-side methods
OBJECTIVE: The aim of the study was to compare four methods for gingival crevicular fluid (GCF) matrix
metalloproteinase (MMP)-8 detection. METHODS: Matrix metalloproteinase-8 levels from 20 GCF samples
from two periodontally healthy subjects, 18 samples from two patients with gingivitis and 45 samples
from six patients with moderate to severe periodontitis, altogether 83 samples, were analysed using (1)
a time-resolved immunofluorometric assay (IFMA), (2) an MMP-8 specific chair-side dip-stick test, (3) a
dentoAnalyzer device and (4) the Amersham ELISA kit. Western immunoblot using same monoclonal antiMMP-8 as in IFMA and dentoAnalyzer was used to identify molecular forms of MMP-8 in GCFs. RESULTS:
Correlation between IFMA and dentoAnalyzer results calculated with Spearman's correlation coefficient

was 0.95 (P = 0.01). The chair-side dip-stick test results were well in line with these assays.
Periodontitis sites with unstable characteristics were differentiated with these methods. The Amersham
ELISA results were not in line with the findings by other methods. CONCLUSIONS: Immunofluorometric
assay and dentoAnalyzer can detect MMP-8 from GCF samples and these methods are comparable. Using
Western immunoblot, it was confirmed that IFMA and dentoAnalyzer can detect activated 55 kDa MMP-8
species especially in periodontitis-affected GCF. dentoAnalyzer is among the first quantitative MMP-8
chair-side testing devices in periodontal and peri-implant diagnostics and research.
7) Matrix metalloproteinase-8 concentration in shallow crevices associated with the
extent of periodontal disease
OBJECTIVE: The aim of this study was to analyse the association between matrix metalloproteinase-8
(MMP-8) concentration in shallow, mostly non-bleeding gingival crevices, and the extent of periodontal
disease. MATERIAL AND METHODS: Plaque, bleeding on probing (BOP), probing pocket depth (PPD) and
attachment level (AL) were assessed clinically in 48 patients with chronic periodontitis. MMP-8
concentrations in gingival crevicular fluid (GCF) from four shallow (PPD<or=3 mm), and four diseased
sites and in serum, were measured by enzyme-linked immunosorbent assay. RESULTS: The mean
concentration of MMP-8 in GCF from shallow crevices was 11.8 +/- 12.8 ng/ml and from diseased sites
was 150.1 +/- 91.8 ng/ml. In subjects with moderate to high plaque scores, a statistically significant
association was found between MMP-8 concentration from shallow crevices and the extent of AL>or=4
mm (p=0.028) and AL>or=6 mm (p<0.001). CONCLUSION: The above association between MMP-8
concentration in shallow crevices and attachment loss provides a new aspect to future studies of MMP-8
as a prognostic marker for periodontal disease.
8) Characterization of progressive periodontal lesions in chronic periodontitis
patients: Levels of chemokines, cytokines, matrix metalloproteinase-13, periodontal
pathogens and inflammatory cells
BACKGROUND AND AIMS: Periodontitis is an infection with an episodic nature of tissue support
destruction. The aim of this work was to determine the levels of chemokines, cytokines, matrix
metalloproteinase-13, periodontal pathogens and inflammatory cells in periodontal sites characterized by
active periodontal connective tissue destruction. MATERIAL AND METHOD: Fifty-six patients with
moderate or advanced severity of chronic periodontitis were selected. Periodontitis was characterized by
at least six sites with probing depth > or =5 mm, clinical attachment level > or =3 mm and radiographic
bone loss. Periodontitis progression was determined by the tolerance method. Receptor activator for
nuclear factor kappa B-ligand (RANK-L), monocyte chemoattractant protein-1 (MCP-1), tumour necrosis
factor-alpha (TNF-alpha), IL-1beta, MMP-13, Actinobacillus actinomycetemcomitans, Porphyromonas
gingivalis, Tannerella forsithia and inflammatory cells levels were determined. Statistical analysis was
performed using the Stata 7.0 software. Data were expressed as mean+/-SD and paired samples t-test
and chi(2) tests were used. RESULTS: Higher RANK-L, IL-1beta and MMP-13 activity levels were observed
in active sites (p<0.05). The proportion of P. gingivalis, A. actinomycetemcomitans, T. forsythia and the
number of CD4(+) T were higher in active than in inactive sites (p>0.05). CONCLUSION: The detection of
periodontopathic bacteria, host matrix metalloproteinases and cytokines in periodontitis patients with
lesions undergoing episodic attachment loss could partially explain the mechanisms associated with the
destruction of the supporting tissues of the tooth.

9) MMP-13 and TIMP-1 determinations in progressive chronic periodontitis

Matrix metalloproteinase (MMP)-13 is a collagenase involved in extracellular matrix degradation either by
its direct degradative effects or by processing bioactive substrates. The aim of this study was to
determine the levels of MMP-13 and tissue inhibitor of metalloproteinase (TIMP)-1 in gingival crevicular
fluid (GCF) and gingival biopsies obtained from active and inactive sites during chronic periodontitis
progression. MATERIALS AND METHODS: This was a longitudinal study in which chronic periodontitis
patients with moderate to severe disease were included and followed until they developed progression
determined by the tolerance method. GCF samples were obtained from periodontitis, active, inactive and
healthy sites and additional gingival biopsies were taken from active and inactive sites. MMP-13 and
TIMP-1 determinations were carried out by immunodot blots and immunowestern blots. RESULTS: In
progressive periodontitis, MMP-13 and TIMP-1 remained unchanged between active and inactive sites, but
as the TIMP-1 relative levels increased together with MMP-13 elevation in inactive samples, an inverse
correlation was observed in active sites. Besides, MMP-13 was undetectable in healthy controls.
CONCLUSION: Chronic periodontitis is characterized by increased MMP-13 expression. During disease
progression, active sites tended to decrease TIMP-1 levels in association with MMP-13 elevation.
10) Functional polymorphisms in the matrix metalloproteinase-9 gene in relation to
severity of chronic periodontitis.
BACKGROUND: Members of the matrix metalloproteinase (MMP) family are implicated in the chronic
remodeling in periodontal diseases. Therefore, we performed a case-control study to investigate a
plausible association between susceptibility to chronic periodontitis (CP) and the polymorphisms in the
MMP-9 (gelatinase B) gene. METHODS: Using polymerase chain reaction with subsequent restriction
analyses, MMP-9 -1562C/T and R+279Q variants were determined in 304 subjects (169 patients with
CP, 76 with a mild to moderate form and 93 with severe generalized CP, and 135 age- and gendermatched unrelated control subjects). RESULTS: The distribution of the MMP-9 -1562C/T and R+279Q
genotypes and alleles did not significantly differ between cases and controls. However, the frequency of
the T variant at position -1562 was marginally higher in patients with severe disease compared to those
with mild to moderate forms (P <0.05; P(corr) >0.05). In further analysis, the -1562T allele was
associated with a severe form of CP in men (odds ratio: 3.87; 95% confidence interval: 1.40 to 10.65;
P <0.01 and P(corr) <0.05 for allele; P <0.05 and P(corr) >0.05 for genotype) but not in women.
CONCLUSION: These findings suggest that genetic polymorphisms of the MMP-9 gene are not associated
with the susceptibility to CP in the Czech population; however, the promoter variant may influence the
severity of the disease in men.
11) Expression of Matrix Metalloproteinases (MMP-8 and -9) in Chronic Periodontitis
Patients With and Without Diabetes Mellitus
Background: Matrix metalloproteinases (MMPs) are in-volved in a number of physiological events, and they
are the major players in collagen breakdownduring periodontal tissue destruction. Diabetes mellitus (DM)
has been associated with altered collagen metabolism and increases the response of the periodontal
tissue to pathogenic microorganisms, thereby in- creasing the severity of periodontal disease. The aim of
this studywas to assess the expression ofMMP-8 and -9 in gingival tissues of diabetic chronic
periodontitis (CP), non-diabeticCP, and healthy patients. Methods: The plaque index (PI), gingival index
(GI), prob-ing depth (PD), and clinical attachment level (CAL) were assessed. The levels of MMP
expression in gingival tissue ex- tracts were measured by gelatin zymography and Western blotting. The
structural changes in tissues were analyzed by histological examination and collagen estimation. Results:
Higher levels of PI, GI, CAL, and deeper PD wereobserved in CP patients with DM than CP patients
without DM. The average concentration of MMP-9 was increased three-fold, and the MMP-8 was increased
two-fold in CP pa- tients with DM compared to CP patients without DM. A high infiltration of
inflammatory cells and less collagen were ob- served in the histologic analysis of gingival biopsies from
diabetic patients. Conclusions: An increased concentration of MMP-8 and -9in the gingival tissue of
diabetic CP patients suggests that the expression of theseMMPscontributes to the failure of the heal- ing
process in the diabetic condition. Treatment strategies directed toward the inhibition of these MMPs
could lead to an improved healing rate in CP patients. J Periodontol 2006; 77:1803-1808.

12) Expression of matrix metalloproteinases (MMPs) and tissue

metalloproteinases (TIMPs) in healthy and diseased human gingiva

inhibitors

of

BACKGROUND: The purpose of this study was to quantify the amount of matrix metalloproteinases such
as MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13 and tissue inhibitors of metalloproteinases TIMP-1
and TIMP-2 expressed by human gingival explants in culture media and the area fraction (AA%) of
gingival collagen fibers according to the degree of inflammation, to investigate a possible correlation
between these enzymes and collagen loss. METHODS: Gingival tissue specimens from 6 healthy controls
(group 1), 17 patients with mild gingival inflammation (group 2), 10 patients with moderate gingival
inflammation (group 3), and 9 patients with severe gingival inflammation (group 4) were placed in organ
culture for 3 days. The MMPs and TIMPs in the culture media were quantified using zymography, dot
blotting, and Western blotting. Paraffin gingival sections were stained with sirius red F3Ba for
visualization of collagen fibers, then the area fraction (AA%) occupied by the gingival fibers was
determined by automated image analysis. RESULTS: The AA% occupied by collagen fibers significantly
decreased from group 1 (53%) to group 4 (35%). The decrease in collagen fibers was inversely
correlated with the significant increase in MMP-1, MMP-9, and MMP-13 (dot blotting analysis), with the
increase of the active form of MMP-2, and with the active form and proform of MMP-9 (zymography
analysis). CONCLUSION: The present study showed that metalloproteinases, particularly MMP-2, MMP-9,
MMP-1, and MMP-13, are involved in the gingival extracellular matrix degradation during periodontitis.
13) Inflammatory mediator response as a potential risk marker for periodontal
diseases in insulin-dependent diabetes mellitus patients.
The gingival crevicular fluid (GCF) and monocytic secretion of prostaglandin E2 (PGE2) and interleukin 1
beta (IL-1 beta) were measured in a group of 39 insulin-dependent diabetes mellitus (IDDM) patients and
64 systemically healthy individuals. Diabetics were divided into Group A (gingivitis or mild periodontal
disease) and Group B (moderate or severe periodontal disease). Diabetics had significantly higher GCF
levels of both PGE2 and IL-1 beta as compared to non-diabetic controls who were matched with regard
to periodontal disease severity (P < 0.00001 and P = 0.0005, respectively). Within the diabetic
population, the GCF levels of these inflammatory mediators were almost 2-fold higher in Group B as
compared to Group A (P = 0.01, P = 0.006, respectively for GCF-PGE2 and IL-1 beta). Furthermore,
diabetics as a group had a significantly higher monocytic PGE2 and IL-1 beta production in response to
various concentrations of both Escherichia coli and Prophyromonas gingivalis lipopolysaccharide (LPS) as
compared to non-diabetic patients with adult periodontitis (P = 0.0001). LPS dose-response curves
demonstrated that monocytes from Group B diabetics produced approximately 3 times more PGE2 than
Group A monocytes; however, there was no significant difference in monocytic IL-1 beta secretion within
the IDDM patients. The levels of GCF or monocytic mediators did not correlate with age, race, or
glycosylated hemoglobin (HbA1C) levels. Our data suggest that the high GCF and monocytic secretion of
PGE2 and IL-1 beta in IDDM patients may be a consequence of a systemic response trait and that the
presence of Gram-negative infections such as periodontal diseases may interact synergistically to yield
high local levels of these mediators and a more severe periodontal condition.
14) Role of matrix metalloproteinases in human periodontal diseases.
Matrix metalloproteinases (MMP) are a family of proteolytic enzymes that mediate the degradation of
extracellular matrix macromolecules, including interstitial and basement membrane collagens, fibronectin,
laminin, and proteoglycan core protein. The enzymes are secreted or released in latent form and become
activated in the pericellular environment by disruption of a Zn(++)-cysteine bond which blocks the
reactivity of the active site. The major cell types in inflamed and healthy periodontal tissues (fibroblasts,
keratinocytes, endothelial cells, and macrophages) are capable of responding to growth factors and
cytokines, as well as to products released from the microbial flora by induction of transcription of 1 or
more MMP genes. Cytokines that are likely to regulate expression of MMP genes in periodontal tissues
include IL-1, TNF-alpha, and TGF-alpha. In addition, triggered PMN leukocytes which express only 2 MMP
(PMN-CL and Mr 92K GL) release these enzymes from specific granule storage sites in response to a
number of stimuli. The evidence that MMP are involved in tissue destruction in human periodontal
diseases is still indirect and circumstantial. Cells isolated from normal and inflamed gingiva are capable of
expressing a wide complement of MMP in culture and several MMP can be detected in cells of human
gingiva in vivo. In addition, PMN-CL and Mr 92K GL are readily detected in gingival crevicular fluid from
gingivitis and periodontitis patients. Osteoclastic bone resorption does not appear to directly involve
MMP, but a body of evidence suggests that bone resorption is initiated by removal of the osteoid layer
by osteoblasts by means of a collagenase-dependent process.