Biomarkers in periodontal

disease

Presented by:Shilpa ShivanandII MDS

Contents IntroductionDefinition of BiomarkerCandidates for biomarkers in periodontal diseaseSources of biomarkersMicrobiological markersImmune and inflammatory markersCytosolic enzymes as markers Enzymes released from host cells as markersConnective tissue degradation products as markersBone resorption products as markersConclusion References

Introduction Periodontal disease is a bacteria-induced chronic inflammatory disease affecting the soft and hard supporting structures encompassing the teeth.

When left untreated, the ultimate outcome is alveolar bone loss and exfoliation of the involved teeth.

Traditional periodontal diagnostic methods include assessment of clinical parameters and radiographs.

Though efficient, these conventional techniques are inherently limited in that only a historical perspective, not current appraisal, of disease status can be determined.

For these reasons a large proportion of recent periodontal research has been concerned with finding and testing potential markers of periodontal disease.

Advances in the use of oral fluids as possible biological samples for objective measures of current disease state, treatment monitoring, and prognostic indicators have boosted saliva and other oral-based fluids to the forefront of technology.

Definition A biomarker is an objective measure that has been evaluated and confirmed either as an indicator of physiologic health, a pathogenic process, or a pharmacologic response to a therapeutic intervention.

(Biomarker definition workshop, Clin. Pharmacol. Ther 2001)

Advantages of diagnostic tests using biomarkers

Some appear to be predictive of disease activity in longitudinal studies, e.g GCF bacterial proteases.

Commercial tests are simple to use.

Results of chairside test kits available in short time.

Chairside kits produce visual result which can be shown to patients.

Candidated for Biomarkers Microorganisms

And their products

Inflammatory and

Immune products

Enzymes releasedfrom host cells

Connective tissuedegradation

products

Bone resorptionproducts

Biomarkers ofPeriodontal

disease

Sources

Sources

Blood or

serumSaliva

Subgingivalplaquesample

Tissues GCF Exhaled air

Potential problems with GCF collection

Contamination

Sampling time

Volume determination

Recovery from strips

Data reporting

Blood, saliva, plaque

Changes its nature..Initial sample- interstitial

Prolonged- serum

Significant problem- evaporationEspecially vol collected- <1μl

Vol-small, error- major significance

conflicting results

Data presented as conc and total enzyme activity-used to

identify differences between disease activity

Saliva as sourceFactors derived from GCF and subgingival plaque are found in whole saliva holds greater promise as diagnostic test.

Sampling is less complicated and less invasive.

Salivary diagnostic tests can aid in screening large populations.

Salivary diagnostic test would be patient specific and could be useful in evaluation of patients in the maintenance phase of care.

Microorganisms and their productsNo: of periodontal pathogens have been implicated..

Red complex- Tanerella forsythensis, Porphyromonas gingivalis, and Treponema denticola progression CP

Actinobacillus actinomycetemcomitans AgP

The rationale for the use of microbial analysis for periodontitis monitoring is to target pathogens implicated in disease to

identify specific periodontal diseases, identify antibiotic susceptibility of infecting organisms

colonizing diseased sites, and predict disease activity.

Microbial markers

Microorgannisms Microbial products

Toxins Enzymes VSCs

Determinationof bacterial

species

Microscopy Culture Immuno-logical

Direct &Indirect

IFAFlow

cytometry ELISA Membraneassay

Latexagglutination

PCRCheckerboard

DNA-DNAhybridisation

DNA probe

Evaluasite Chairside detection of 3 periodontal pathogens using immunoassay- A.a, P.g, P.i

This assay is a sandwich configuration...detects antigens..

Color/ intensity of the reaction- no: of bacteria.

The limit of detection for the 3 species ranges upwards from approximately 5x104 to 105 bacterial cells.

Toxin detectionToxins are biochemically active substances, released by microorganisms and have a particular effect on host cells.

TypesExotoxins are proteins, often enzymes produced inside of the cell & cause damage only upon release from the cell. Gram positive bacteria produce exotoxins. Their effects are limited but specific.Endotoxins are non diffusible, lipid polysaccharide complex, produced by gram negative bacteria. They can cause endotoxic shock

Tests for identification of endotoxins :

LAL (Limulus Ameobocyte lysate): using LAL reagent prepared from horseshoe crab blood

was shown to be the most sensitive and specific means of measuring bacterial endotoxins. The chromogenic test, introduced in 1977, is a modification that enables endotoxin concentration to be measured as a function of color intensity rather than by turbidity or gelation in the reaction mixture.

ELISA – not very sensitive

BANA assayBacteria-- trypsin-like enzymes such T.forsythensis, T.denticola and P.gingivalis.

Advantages- Since some of these species grow poorly in cultures and account for a significant proportion of the protease activity of the subgingival flora, these enzyme assays provide a rapid and inexpensive method of screening samples of these bacteria.

Plaque sample

Paper strip with

BANA

Chair side test kit- Perioscan

Limitation-

Can not distinguish between relative proportions of these 3 bacteria…Aa, T forsythia, P gingivalis.

Its utility as a diagnostic method is uncertain due to its low reliability to predict clinical assessment of disease progression.

It may have more value when performed in combination with other chair side microbiological tests such as microscopy.

Volatile sulphur compounds

by products of gram –ve anaerobic bacterial metabolism of sulphur containing amino acids.

Bacteria which produce them – P.gingivalis, P.intermedia, P.melaninogenica, B.forsythus, T.denticola, F.nucleatum.

Volatile sulphur compounds include- hydrogen sulphide, methyl mercaptan, dimethyl sulphide, dimethyl disulphide.

Studies have shown that levels of these compounds are higher in chronic periodontitis patients

than healthy controls. (Yaegaki and Sanda, 1992)

Diamond probe/ Perio 2000 system

Features of periodontal probe with detection of volatile sulphur compounds in periodontal pocket.

The sulphide readings of the probe have been shown to relate to clinical parameters of disease severity

Polychronopoulou, 1998

Disadvantages-

Clinical relevance of the results obtained was hampered by the poor sensitivity of the probe at the low and high ranges of its scale. This resulted in the majority of the readings at both apparently healthy and diseased sites being zero.

Also there are no longitudinal studies of the relationship of volatile sulphur compounds to periodontal disease progression and therefore its diagnostic potential is unknown.

Disadvantages of microbial analysis

Polymicrobial nature of the disease: therefore difficult to choose the particular bacterial species to assay as a marker in any particular case.

Most are not predictive of disease activity: The only bacterial factors that have so far been shown to have good predictive ability are GCF arg-gingipain/arg-gingivain and bacterial dipeptidylpeptidases (DPPs). We need to know which site to sample: some sites progression is more compared to others…They only detect the bacteria that are looked forSpecial lab required..Cost…

Inflammatory and immune products

Bacteria in dental plaque trigger inflammatory and immune host responses which, along with the direct effects of the bacteria, cause most of the tissue destruction.

A number of substances are released from inflammatory and immune cells into the tissues and many of these pass into GCF and are thus easily available for analysis.

Immune products

Inflammatorymarkers

Immunoglobulins

Complement

Arachidonic acid derivatives

Cytokines

Immunoglobulins Patients with various forms of periodontal disease produce antibodies to antigens from periodontopathic bacteria which can be detected in serum, saliva, gingival tissue, GCF.

Secretory IgA is actively secreted into saliva, IgG and IgM pass into saliva mainly from GCF.

Levels of IgG and IgA are very low in healthy patients.

Salivary IgG is increased to 34% in moderate and 57% in advanced periodontitis patients

Sandholm and Gronblad, 1984

Serum and saliva concentrations of IgG and IgA reduce following periodontal treatment of chronic periodontitis probably because of reduction in antigenic stimulus.

Specific IgA antibody to A.a is present in saliva of refractory periodontitis patients and this specific antibody is found to be raised in only 19% of chronic periodontitis patients.

The total IgG in GCF does not correlate with the disease severity or progression and indeed may be lower at progressive sites.

Lamster 1992

A study compared IgG subclasses in GCF at progressive and stable sites. It found that the concentration of IgG1 and IgG4 subclasses was significantly higher at progressive sites.

Reinhardt et al 1989

Elevations in P.gingivalis specific IgG1,2,4 in rapidly progressive and adult periodontitis have been reported

Kinane et al, 1999

Sakai et al 2001 investigated P.gingivalis-specific IgG subclasses in adult periodontitis patients and controls.

Results showed-IgG1 levels-significantly higher in patient groups compared to controls. IgG2-significantly higher in untreated group compared to other groups. IgG4 levels-significantly higher in the maintained patients compared to the untreated group. Patients from maintained group with high IgG1 levels and low IgG4 levels showed greater bone loss. This work suggests that a persistently high P.gingivalis IgG2 level after periodontal treatment may be indicative of recurrent or persistent periodontal destruction at patient level.

Complement Complement proteins are present in GCF from sites with inflammation.

Total activation (classic and alternate) may be assessed by C3 conversion and has been studied in GCF by means of an experimental gingivitis model.

Split fragments C3 and Factor B have been detected during experimental gingivitis

Patters et al, 1989

Cytokines Cytokines are involved in cell to cell communications of most of the cells in the body and are present in all tissues and body fluids including serum, saliva and GCF.

IL-1 and TNFα have proinflammatory effects of relevance to periodontal pathology which include stimulation of PGE2 and collagenase production.

IL-1α and β are present in GCF from patients with periodontitis, but in extremely low concentrations at healthy sites. Their levels are reduced following SRP.

TNF-α is also present in GCF but does not correlate with probing depths or gingival inflammation and its total amount is inversely related to tissue inflammation.

Rossomando et al 1990

The levels of IL-1 and IL-6 have also been studied in refractory periodontitis. There were no significant differences in the mean level of IL-1 in refractory or stable sites but refractory sites produced significantly more IL-6. Reinhardt et al, 1993

Levels of IL-8 in GCF have been shown to significantly reduce following periodontal treatment along with the corresponding and related reductions in PMN elastase and putative pathogens.

Prostaglandins PGE2 has proinflammatory and immunoregulatory effects and its concentration in gingival tissue is sufficient to elicit significant effects on cell responses and functions. In bone organ culture it stimulates osteoclastic bone resorption. It may thus play a significant role in periodontal pathology.

PGE2 levels are low in health and non detectable at many sites.

Offenbacher et al 1993

In naturally occurring gingivitis there is a modest rise in GCF PGE2 levels to about 32ng/ml and higher in experimental gingivitis (53ng/ml). Untreated periodontitis patients have significantly higher levels than gingivitis patients.

In one study, following SRP, the periodontitis patients were divided into two groups, those that experienced no further attachment loss and those which experienced one or more sites >3mm attachment loss. The mean GCF PGE2 levels were higher in latter group than that of former group. Also the group which experienced significant attachment loss at one or more sites in the following 6months had significantly higher mean GCF PGE2 levels of 113ng/ml. This observation lead to the basis that GCF PGE2 is predictive for periodontal disease activity.

Offenbacher et al 1986

Levels >66ng/ml were found to be predictive of further possible loss of attachment and this level is used as a cut off value in positive and negative screening test.

Leukotrienes Evidence indicates that LTB4 is elevated in inflamed deeper periodontal tissues.

Offenbacher et al 1991 reported higher levels of LTB4 in GCF obtained from clinical sites associated with adult periodontitis, alveolar bone loss, juvenile periodontitis, and severe periodontitis.

These workers concluded that LTB4 may be a good marker for inflammation but would not be considered to be a marker of specific tissue destruction.

Enzymes released by dead cells- Cytosolic enzymes

Since dead cells are integral and essential component of periodontal destruction they are released and passed with the inflammatory exudates into GCF.

2 enzymes—

AST (Aspartate Aminotransferase Enzyme)LDH (Lactate Dehydrogenase)

ASTIn longitudinal studies, GCF AST levels have been related to confirmed attachment loss

Persson et al 1990, Chambers et al 1991

Elevated GCF AST levels were strongly associated with disease active sites in contrast to disease inactive sites.

There is as yet no evidence to indicate that GCF AST levels are predictive of disease activity, because the positive correlations were made at the time of attachment loss rather than before it.

Commercial Test Kit: Periogard (colgate) AST in GCF test kit

The only commercial test kit based on factors released from tissue degradation is that based on GCF AST, which is released from dead cells.

GCF in buffer(trimethamine Hydrochloride)

mixture ofL-aspartic and

L-ketoglutaric acid

AST

oxalacetate and glutamate

Color change

Test is positive at >800 mIU

AST activity

Chair side kit for AST- Pocket watch:Principle:

Other kit for AST: Perimonitor

cysteine sulfinic acid β-sulfinyl pyruvate

AST

Decomposes, Releases inorganic

sulphite Malachite green

pink colored rhodamine B dye show through.

Colorless

LDHLDH has been correlated with probing depth and other clinical indices of disease severity in cross-sectional studies.

In both cases the level of correlation was less than for β-glucuronidase, which was included in the same studies Lamster et al 1988

Disadvantages of immune & inflammatory markersThe choice of the most appropriate biomarker may still be difficult at the present state of knowledge. IL-2 and IL-6 and possibly IL-lβ have shown promising results in this regard. However, further research is needed to confirm this.

If a moiety is associated with inflammation this may mask its association with destructive disease: All the inflammatory mediators are associated with gingival inflammation and this association could produce false association with disease activity.

Cost

Bacterial enzymesEnzyme unique to one or more of the relevant bacterial species.

Role of bacterial enzymes in periodontal pathology by: Reducing the effectiveness of host defenses or By degrading host tissues

Priodontopathic bacteria

Proteolytic enzymes

Hydrolytic enzymes

Proteolytic enzymes Pdl c.t proteins peptides a.a

Dipeptidyl-peptidases

Aminopep-tidases

Chemotrypsin like

Trypsin like

Elastase like enzyme

Collagen degrading

Proteolytic enzymes

Spirochetes, Capnocytophaga

Pg, T denticola, B forsythus, other spirochetes

Pg, Aa, Spirochetes

Pg, P intermedia, Capnocytophaga

Capnocytophaga, T denticola

T denticola, Capnocytophaga

Hydrolytic enzymes Nonproteinaceous components of ct…

Hydrolyticenzymes

Hyaluronidase&

chondroitinasePhospho-lipases

Acid & alkalinephosphatases

Neuramina-dases

Hyaluronidase and chondroitinase activities are produced by C. ochracea, F. nucleatum, P. gingivalis and T. denticola. These could hydrolyze the glycosaminoglycan components of proteoglycans in the extracellular matrix. Neuraminidase (sialidase) activity, which is found in B. forsythus, Prevotella melaninogenica and P. gingivalis, might attack sialoproteins in the epithelium, thereby increasing its permeability to bacterial products. Phospholipases from Porphyromonas, Prevotella and Bacteroides species which damage the surface of epithelial and other cells. Strong acid and alkaline phosphatase activities are present in Porphyromonas, Prevotella, Bacteroides and Capnocytophaga species

Various studies…Eley and Cox, 1992-

determined the correlation of bacterial protease activity and their conc

(Cathepsin-like, elastase-like, tryptase-like, trypsin-like and dipeptidyl peptidase IV-like)

probing attachment loss and bone loss at both patient level and site level.

Eley and Cox, 1995-following treatment there were marked reductions in

clinical parameters & enzyme activities (DPP, trypsin like) and concentrations.

Significant reduction- pt & site level

Eley and Cox, 1996- 2-year longitudinal study showed

significant correlation between gingipain

and bacterial DPPs in GCF and periodontal

attachment loss in CP pts

Enzymes of tissue originInflammation PMNs, macrophages, lymphocytes and mast cells enzymes in their lysosomes...

released during function die

Tryptase

DPPs

Cathepsin D Cathepsin G

Cathepsin B

Elastase

Collagenase

Proteolyticenzymes

Acid phosphatase

Alkaline phosphatase

β-glucuronidase

Aryl sulphatase

MPO

Hydrolyticenzymes

Collagenases Part of family MMPs…

Synthesized by Neutrophils- MMP-8,9…. TIMP Macrophages- MMP-1,9,13…. TIMP Fibroblasts- MMP-1,2,3…. TIMP Keratinocytes…

MMP-1,2,8,9 in both GCF and saliva

Predominantly- MMP-8,9 as they are produced by PMNs.

MMP-8,9 main collagen-degrading enzymes in inflamed tissue during gingivitis and adult periodontitis.

Therefore these enzymes as good indicators for periodontal inflammation.

MMPs in saliva…Higher levels of MMP-8, 9 CP

MMP-8 levels correlate with indices of disease severity

Lower TIMP-1 untreated CP pts Hayakawa et al 1994

By contrast, LJP salivary MMP-1 predominates; but the levels < untreated or treated CP or healthy pts

Ingman et al 1993

Also increased levels of TIMP-1 LJP cases compared with healthy or CP pts

MMPs in GCF…GCF collagenase ≈ severity of inflammation in naturally occurring and experimental gingivitis Kowaski et al 1979

Collagenase levels ≈ amount of attachment loss in ligature induced periodontitis in dogs and

Latent enzyme predominated at healthy and gingivitis sites Kryshalskyi et al, 1986

Similarly in human periodontitis, GCF collagenase ≈ gingival inflammation, PD, bone loss Golub et al 1976

Total enzyme and active enzyme levels higher in diseased sites; enzyme inhibitor levels lower

Larivee et 1986

GCF levels of MMP-8,9 higher in CP pts Ingman et al 1996

MMP-8 concentration reduced following pdl treatment.

Also this enzyme gave more significant correlations with clinical parameters and fell more after successful periodontal treatment than either elastase or cathepsin B Chen et al 2000

Relationship between MMPs and LJP same as in saliva Ingman et al 1993

Detection of MMPsEarlier studies used biochemical assays with collagen substrates to detect collagenase and relate to disease severity…

Previous studies- assumption – it measured neutrophil collagenases

But it reflected the combined action of several collagenolytic enzymes…

Kiili et al 2002 developed immunological assay that can detect different MMPs-- possible to assay them individually.

Using these techniques, it has been shown that MMP-8 relates positively to indices of periodontal severity and significantly reduces following treatment.

Therefore, MMP-8 might give better results in a longitudinal study to those given by a mixture of enzymes in previous using biochemical assays.

Immunological assays: high-affinity antibodies that can recognize a given MMP.

Eg. monoclonal antibodies to human MMP-8 have been developed…. Similarly antibodies to MMP-1,13…

ELISA Immunofluoroscence Immunoblot- used to assess the presence of active

MMPs Immunodot Gel capture methods Neo-epitome antibody approach- An indirect method in which neo-epitope antibodies that

specifically recognize degradation fragments of either matrix proteins or the MMPs themselves are examined.

Substrate degradation products: measure primarily the ability of the MMPs to degrade the substrate.

The enzyme activity is estimated on the relative abundance of substrate degradation products measured

Advantage of these assays: can measure MMP inhibitors..

Dot assay Sod. dodecyl sulfate polyacrylamide gel electrophoresis Radioactivity Zymography Fluorescence

ELISAs may be more adaptable to use in office settings than other assays

improved sensitivity, ease of use, moderately rapid throughput, the ability to quantify enzyme levels and the flexibility to test for multiple MMPs in single samples

PeriocheckFor neutral proteinases- collagenases

GCF sampleIn paper strip

Collagen gel withblue dye

43 c

Cystein proteinasesIncludes- family of Cathepsins B, L, H

Act at- acid pH and are primarily involved in intracellular degradation

Role- degrade extracellular components including collagen.

Produced primarily by fibroblast, macrophages. Its inhibitors:

α2-macroglobulin Cystatins

Cystein proteinases in GCFGCF levels of cathepsin B, L ≈ gingival inflammation, PD, attachment level and bone loss.

Eley and Cox 1992Levels of cathepsin B, L reduce following pdl treatment. Zero or very low levels healthy sites; high levels at periodontitis sites

Eley and Cox 1993

There has been only one longitudinal study of GCF cathepsin B activity and periodontal attachment loss

Eley and Cox 1996

The GCF levels were shown to be predictive of attachment loss in diagnostic testing.

Serine proteinase- elastaseCatalytic site of this enzyme- contain a serine amino acid-

essential for the enzyme activity.

Act at- neutral or slightly alkaline pH.

They are secreted upon release of azurophilic granules during neutrophil phagocytosis, stimulation, and cell lysis.

Inhibitors in tissues- α1-proteinase inhibitor (α1-PI) (fibroblasts, macrophages), α2-macroglobulin (α2-M) (fibroblasts, macrophages), secretory leucocyte protease inhibitor (SLPI) (mast cells) skin anti-leucoproteinase (SKALP) (epithelial cells).

Elastase in saliva…Salivary elastase levels low in periodontally healthy patients

Mean elastase level rose gps gingivitis early periodontitis moderate periodontitis advanced periodontitis.

Levels ≈ indices of disease severity and with the no: of deep pockets

Uitto et al, 1996Levels decreased following pdl treatment.In LJP lower salivary levels than in untreated CP and in fact similar to healthy controls

Ingram et al, 1993

Elastase in GCF…GCF elastase levels ≈ gingival inflammation, PD, CAL and bone loss

Its level reduces following treatment Eley and Cox 1992

Zero or very low levels healthy sites, low to moderate gingivitis; very high periodontitis

Eley and Cox 1993Similar results in 2 longitudinal studies:

Palcanis et al 1992 a 6-month longitudinal study using a test kit system

Eley and Cox 1996 2 years

Tryptase Tryptase is found in mast cells. It is released on degranulation.

Role: Cleaves the 3rd component of complement and can

activate latent collagenase.

Stimulate the release of collagenase form gingival fibroblasts and in inflamed gingival tissues mast cell degranulation occurs.

GCF tryptase levels ≈ gingival inflammation, PD, CAL and bone loss and its level also significantly reduces following treatment

Eley and Cox 1992

Zero or very low levels at healthy sites, low to moderate gingivitis sites; very high levels periodontitis Eley and Cox 1993

A commercial test system suitable for chairside (PrognostiK) has been developed.

Cox et al 1990

PrognostikFor serine proteinase, in GCF samples.

The biochemical technology for this test system was developed by Enzyme System Products/Prototek, Dublin. California. USA for Dentsply.

Dipeptidylpeptidase (dpp) 4 types of DPPs are present- DPP II is active at acid pH and DPP IV is active at alkaline pH, both are present in gingival tissue and GCF.

DPP II lysosomal enzyme in fibroblasts, macrophages.

Function: cleave glycylprolyl residues and may play a role in collagen degradation after the action of other enzymes.

DPP II and IV ≈ clinical parameters of disease severity and significantly reduce following periodontal treatment. Eley and Cox 1992

Eley and Cox, 1995 2-year longitudinal study, by of both II and IV ≈ attachment loss and 3 months previously.

Thus both GCF DPP II and IV appear to be good predictors of future progressive attachment loss.

A test system suitable for chairside use has been developed and has been shown to produce similar results to the laboratory system.

Protease inhibitors2 main endogenous protease inhibitors: (serum, saliva and GCF)

α1-proteinase inhibitor (α1 - PI) and α2–macroglobulin (α2 – M)

Levels of α1- PI in saliva and GCF do not significantly vary…

Levels of α2 – M higher in CP pts > gingivitis pts >healthy pts

Pederson et al 1995

Other inhibitors- Cystatins types-- A, C, S, SA, SN and D.

Cystatin C general distribution, produced by many cells

Cystatins S, SA, SN and D produced by glandular acinar cells and are mainly found in glandular secretions, including saliva.

Cystatin A by inflammatory and is the main Cystatin in GCF.

Total salivary Cystatins higher in CP > gingivitis> healthy pts.

Heskens et al 1993

Levels of Cystatin C higher CP

Saliva from healthy patients contains mainly Cystatin S whilst that from CP patients contains both S and C.

Heskens et al 1994, 1996

β-glucuronidase and arylsulphatase

Source: lysosomal , in PMNs.

β -glucuronidase acid hydrolase ; considered to be a marker for primary granule release by these cells.

Role: Degrades proteoglycans and glycoproteins.

Both these enzymes in GCF ≈ with gingival inflammation, PD and alveolar bone loss.

Levels higher in diseased sites ; drops following pdl treatment.

Lamster 1992

β -glucuronidase ≈ spirochetes, P. g, P.i in the subgingival flora but negatively associated with cocci.

Lamster 1992

Lamster et al 1988 : β -glucuronidase activity- association with loss of attachment- at 3month interval

The association with disease activity has been confirmed in a multicentre trial and showed a total predictive value of 90%.

Lamster 1992

Alkaline phosphatase Source: found in PMNs.

distributed - bone, intestine, kidney, liver, placenta, PMN.

Function: It probably plays role in calcification and its determination in serum is of practical importance in bone disease.

Alkaline phosphatase in serum is only 50% that of GCF. In serum, the enzyme is associated with bone disease, and its elevation in GCF could reflect changes of alveolar bone in localized areas.

Ishikawa and Cimasoni 1970 in periodontitis patients this enzyme in GCF levels ≈ PD but not with bone loss.

Chapple et al 1994 higher in diseased periodontal sites

However, its predictive value is low

Acid phosphataseIt is present in inflammatory cells and has been detected in GCF.

Binder et al 1987

However, levels DO NOT correlate- disease severity or activity ...

MPOSource: PMNs, antibacterial enzyme…

Salivary MPO levels higher in CP than in healthy; also reduce following treatment

Guven et al 1996

Similarly with GCF MPO levels

However, MPO activity was not found to correlate with clinical indices of disease severity.

Cao and Smith, 1989

Lysozyme Antibacterial enzyme

Source: basic and azurophil granules of PMNs.

Levels lower in CP and type I DM than healthy control Markannen et al 1986

Salivary lysozyme levels not to vary between LJP and healthy

Levels in GCF higher in LJP compared to healthy; reduce to normal following treatment.

Suomalainen et al 1996

Lactoferrin It is antibacterial agent produced by inflammatory cells.

Role: due to its high affinity for iron locks the available sources required for bacterial growth.

Levels of salivary and GCF lactoferrin higher in LJP; and decreased to normal levels following periodontal treatment

Suomalainen et al 1996

Connective tissue degradation markers

Consequence of the disease process breakdown of the extracellular matrix (results from the catabolic response of bacterial and host challenge)

Break down products GCF could pick up during its passage through inflamed tissue….

Component Breakdown product

Collagen

Hydroxyproline

Collagen cross links

N-propeptide

Proteoglycan Glycosaminoglycans (GAGs)

GAGs Heparin sulphate

Chondroitin-4-sulphate

Chondroitin-6-sulphate

Fibronectin More intact molecules are present in samples from healthy and treated sites than from diseased sites.

Lopatin et al 1989

There have been no longitudinal studies of this molecule.

GAGsMost common GAG nonsulfated hyaluronic acid, sulfated heparan sulfate, chondroitin-4 sulfate and chondroitin-6 sulfate.

chondroitin-4-sulfate most common GAG in periodontium but distributions differ.

dermatan sulfate rare in bone, cementum; common in pdl and gingiva. probably reflecting a functional involvement of the molecule in the mineralization process.

Function: Proteoglycans bind most collagens as well as fibronectin. On degradation of pdl tissues, GAGs are released GCF.

Embery et al 1982 The non-sulphated GAG, hyaluronic acid was present in all samples, and was the only major GAG found in chronic gingivitis patients.

Sulphated GAG, c-4-sulphate, in GCF from sites with untreated advanced periodontitis, JP around teeth undergoing orthodontic movement, teeth subject to occlusal trauma

The presence of c-4-sulphate in GCF may be a sensitive method of indicating active phases of destructive periodontal disease.

Embery G et al 1982 In contrast to periodontitis, GCF collected from sites of gingivitis usually contain only the nonsulfated hyaluronic acid.

Last & Embery 1987 suggested -- hyaluronic acid may be a marker of nonactive sites; found that sites of ANUG recover their hyaluronic acid levels after antibacterial treatments.

Last et al 1991; Beck et al 1991 Studies on the levels of C-4-S in GCF from sites with endosseous dental implants, where forces on the supporting bone induced changes in C4S quantity, lend support to the fact that C4S being a bone marker

The GAGs in GCF from individual sites of defined clinical conditions have been investigated with cellulose acetate electrophoresis.

Unfortunately -- difficult to design a diagnostic test based on electrophoretic profile techniques.

Large sample of GCF is required.... micropipette for 15 min..

Bone resorption products- markers

Osteonectin and bone phosphoprotein (N-propeptide)Role: imp in initial phase of mineralization.

Bone phosphoprotein an amino propeptide extension of alpha 1 chains of type I collagen, appears to be involved in the attachment of connective tissue cells to the substratum.

Bowers et al 1989Both detected in GCF from CP pts.

Total amount increases with PD Bowers et al 1989

They therefore may be associated with periodontal disease severity.

Osteocalcin Most abundant non-collagenous protein of mineralized tissues.

Source: osteoblasts. It is a small calcium-binding protein of bone.

Function: promotes hydroxyapatite binding and accumulation of

bone. chemotactically attracts osteoclast progenitor cells and

blood monocytes.

Elevated levels in blood during periods of rapid bone turnover osteoporosis and fracture repair.

Thus has been suggested as a possible marker for bone resorption and hence periodontal disease progression.

Serum osteocalcin presently a valid marker of bone turnover when resorption and formation are coupled and

specific marker of bone formation when formation and resorption are uncoupled.

Kunimatsu et al 1993 GCF osteocalcin ≈ clinical parameters in a cross-

sectional study of patients with periodontitis. also reported that osteocalcin could not be detected in

patients with gingivitis

In contrast, Nakashima et al 1994 GCF osteocalcin levels higher in both periodontitis and gingivitis patients.

Contradicting results osteocalcin has a potential role as a bone specific marker of bone turnover but not as a predictive indicator for periodontal disease.

Can be assayed using polyclonal or monoclonal antibodies by an ELISA or radioimmune assay.

An ELISA technique could be simplified and developed for use in a diagnostic test.

Cross-linked carboxyterminal telopeptide of type I collagen

During synthesis of bone collagen, pyridinoline cross links

telopeptide region of α1 collagen molecule ------ helical region of another such molecule.

Crosslink- mechanical stability...

digestion with bacterial collagenase fragments are formed pyridinoline cross linked carboxyterminal telopeptide of type I collagen (CTP)

CTP ≈ bone turnover in myxoedema, thryrotoxicosis, primary hyperparathyroidism, postmenopausal osteoporosis.

Elevated CTP has also been shown to coincide with the bone resorptive rate

Eriksen et all 1993

GCF CTP levels ≈ PD, radiological bone loss, papillary bleeding index, plaque index.

GCF levels were 100 times higher than serum reference levels.

Talonpoika and Hamalained 1994

Shibutani et al 1997 Another pyridinoline cross-link, deoxypyridinoline

Minor cross-link abundantly in bone and dentin; absent in cartilage.

Shibutani et al study in an experimentally induced periodontitis model in beagle dogsGCF deoxypyridinoline levels ≈ bone resorption

Contrast- Griffiths et al 1998 could not detect pyridinoline nor deoxypyridinoline cross-links in GCF during orthodontic tooth movement in adolescents, suggesting that the sensitivity of specific assay protocols or of bone resorptive situations may affect cross-link measurements.

CTP ≈ clinical parameters and putative periodontal pathogensPalys et al 1998

Golub et al 1997 reductions after periodontal therapy

Therefore- GCF CTP levels as a diagnostic marker of periodontal disease activity have produced promising results to date..

Controlled human longitudinal trials are needed fully establish the role of CTP as a predictor of periodontal tissue destruction, disease activity, and response to therapy in periodontal patients.

Osteopontin OPN -- single-chain polypeptide-- kidney, blood, mammary gland, salivary glands, and bone.

OPN -- in bone matrix, is highly concentrated at sites where osteoclasts are attached to the underlying mineral surface….

OPN-- produced by both osteoblasts and osteoclasts,

Function--it holds a dual function in bone maturation….

GCF OPN ≈ PD measures of periodontally healthy and diseased patients.

Kido et al 2001

GCF OPN concentrations ≈ progression of disease; on nonsurgical pdl treatment levels were significantly reduced.

Sharma et al 2006

Rankl and OPGReceptor activator of nuclear factor κB ligand (RANK-L) cytokine involved in the regulation of osteoclastogenesis in bone remodeling and inflammatory osteolysis.

Lacey et al 1998 In vivo treatment of mice with RANKL activates osteoclasts bone loss…

Osteoprotegerin (OPG) a secreted glycoprotein, is a decoy receptor for RANKL

OPG binds to RANKL the cell-to-cell signaling between marrow stromal cells and osteoclast precursors is inhibited osteoclasts are not formed

Simonet et al 1997; Yasuda et al 1998

Thus, RANKL and decoy receptors OPG expressed by bone-associated cells play important roles during osteoclast formation by balancing induction and inhibition

GCF RANK-L increased in CP patients, supporting its role in the alveolar bone loss developed in the disease.

Rolando Verna et al 2004

RANKL levels low in health and gingivitis groups; increased in CP.

OPG higher in health compared to gingivitis and periodontitis.

There were no differences in RANKL and OPG levels between CP and GAP groups

Bostanci N et al 2007

GCF RANKL and OPG levels were oppositely regulated in periodontitis, but not gingivitis, resulting in an enhanced RANKL/OPG ratio.

This ratio was similar in all three periodontitis groups and may therefore predict disease occurrence.

The ratio of RANKL to OPG in GCF was higher for pdl patients

Growth factorsSeveral growth factors are concentrated in the organic matrix of bone and released during bone resorption play a role in bone remodeling through regulation of the coupling process

Mogi et al 1999 GCF concentrations of EGF and TGF-a. No significant differences in EGF conc. Lower TGF-a-- severe pdl disease.

Mechanism- could be- TGF-a is involved in the wound healing process, the lack of this factor may result in a delayed periodontal regenerative response during disease progression.

Lower EGF -- deep periodontal sites compared to shallow sitesTaken together, the results appear equivocal regarding the use of EGF and the related growth factor TGF-a as markers of alveolar bone loss.

Chang et al 1996

Patients with failing implants were found to exhibit higher GCF PDGF levels at both failing and stable implant sites than control patients presenting with stable implants. TGF-b was not clinically detectable in any sites in this investigation.

In contrast, higher GCF TGF-b levels were reported in sites with deeper periodontal pockets than less involved sites in periodontal patients.

Skaleric et al 1997

Booth V et al, 1998- cross sectional studyGCF levels of VEGF – higher diseased sites.

Summary of commercially available chair side test

kits available in India

Conclusion Through the biomarker discovery process, new therapeutics have been designed linking therapeutic and diagnostic approaches together, especially in the area of host modulatory drugs for periodontal disease treatment.

New diagnostic technologies, such as microarray and microfluidics, are now currently available for risk assessment and comprehensive screening of biomarkers.

The future is bright for the use of rapid, easy-to-use diagnostics that will provide an enhanced patient assessment that can guide and transform customized therapies for dental patients, leading to more individualized, targeted treatments for oral health.

References Periodontics- 5th edition, BM Eley and JD MansonClinical periodontology- Carranza, 10th edition Analysis of saliva for periodontal diagnosis- review. JCP 2000:27:453GCF: Biomarkers of periodontal tissue activity- review. Adv dent res 8 (2):329,1994Diagnostic biomarkers for oral and periodontal diseases. DCNA 2005;49(3):551Oral fluid based biomarkers of alveolar bone loss in periodontitis. Ann NY Acad Sci 2007;1098:230Matrix molecules and growth factors as indicators of periodontal disease activity. Giannobile et al, Periodontology 2000, Vol. 31, 2003, 125–134

Potential for gingival crevice fluid measures as predictors of risk for periodontal diseases. Champane et al, Periodontology 2000, Vol. 31, 2003, 167–180

Advances in periodontal diagnosis 8. Commercial diagnostic kits based on GCF proteolytic and hydrolytic enzyme levels. B.M. Eley and S.W. Cox, British Dental Journal 1998; 184: 373-376

Laboratory diagnosis of periodontal infections. Periodontology 2000, Vol. 7, 1995, 69-82

Microbiological diagnostic testing in the treatment of periodontal diseases. Periodontology 2000, Vol. 34, 2004, 49-56.

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