Presented by

Bibina George

BIOMARKERS

• Periodontal Diagnosis

• Clinical diagnostic parameters

• probing depths, bleeding on probing, clinical attachment levels, plaque index, and radiographs assessing

alveolar bone level

• Require a 2- to 3-mm threshold change before a site can be identified as having experienced a significant

anatomic event. Goodson JM.

• Advances in oral and periodontal disease diagnostic research are moving toward methods whereby periodontal risk can be identified and quantified by objective measures such as biomarkers

Chapple I. Periodontal diagnosis and treatment-where does the future lie? Periodontology 2000; 51:9-24

INTRODUCTION:

• Early recognition of the microbial challenge to the host

• To determine the presence of current disease activity, predict sites vulnerable for

future breakdown

• To assess the response to periodontal intervention

• Haffajee et al 1983

• Ultimately improve the clinical management of periodontal patients.

NEED FOR ADVANCED DIAGNOSTIC INDICATORS

• Biomarkers -“cellular, biochemical, molecular, or genetic alterations by which a normal, abnormal, or simply biologic process can be recognized or monitored”

NIH BIOMARKER DEFINITIONS WORKING GROUP, 1998

• Also described as measures of health & disease that define the underlying biological basis to a conditionpresenting as a characteristic clinical phenotype & are potentially more objective than clinical indices.

Beck et al. 2000

• A substance that is measured objectively and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention

Biomarkers Definitions Working Group.2001

DEFINITION:

CLASSIFICATION

Diagnostic

Prognostic

• Predicts if

subject will be

susceptible to a

disorder

Predisposition

• Incidence and

progression of

disease

Sensitivity of subject to disease

EARLY

SURROGATE

LATE

SOURCES

Khiste SV, Ranganath V, Nichani AS, Rajani V. Critical analysis of biomarkers in the current periodontal practice. J Indian Soc Periodontol 2011;15:104-10

ORAL FLUIDS

Mucosal

Tramsudate

GCF Saliva

Gary C. Armitage 2004

GCF SALIVA SERUM

- More than 65 GCF constituents have been evaluated as potential diagnostic markers of periodontal disease progression(Armitage et al 2004 )

- Non-invasive collection method- Detection of RNA in the salivary fluid

Zimmermann BG - eg: Salih E et al.

- Inflammatory mediators are released into circulation- Not the preferred source today due to lack of definitive evidence- Mainly used for genetic risk marker identification - More invasive than GCF or saliva

BIOMARKERS FOR PERIODONTAL DISEASES

Biomarkers Examples

EnzymesAlkaline phosphatise, Amino peptidase, Trypsin, â galactosidase, ââ glucoronidase, Gelatinase, Esterase, Collagenase Kininase

Immunoglobulins Ig A, Ig G, Ig M,

ProteinCystatin, Fibronectin, Lactoferrin, Vascular endothelial growth factors, Plateletactivating factors, Epidermal growth factors

Phenotypic marker Epithelial keratin

Host cell Leukocytes (PMN’S)

Ion Calcium

Hormones Cortisol

Bacteria Aa, Pg, Pi, C. rectus, T. denticola, B. forsythus , mycoplasma

Volatile CompoundsHydrogen sulphide, Methyl mercaptan, Picolines, Pyridines.

Possible Salivary Biomarkers

• LPS-HOST DEFENSE

•MMP-1 & MMP-8,IL 1 &TNF-α

PROTEOMIC APPROACH

• IL-1 and TNFα, the anti-inflammatory cytokine IL-10

and the Fc gamma receptors

• Yoshie et al

• Reactive O2 species; 8-OHdG

GENOMIC APPROACH

• Bacteria present due to plaque, tongue, GCF, Pdl

pocket..

MICROBIAL APPROACH

DISCOVERY & AN OVERVIEW

Alkaline Phosphatase (ALP):

• Mixed saliva of adult periodontitis

patients revealed the highest enzyme

activities with ALP

• Associated with alveolar bone loss, a

key feature of periodontal disease.

Interleukin (IL) 1β:

• Proinflammatory cytokine

• Functions:

- Osteoclastic activity in periodontitis

• Influences:

- Immune cell recruitment

- Cell proliferation

- Tissue destruction

- Vascular smooth muscle cell contraction

INDIVIDUAL SALIVARY BIOMARKER

Acta Odontol Scand.2015 Jul;73(5):343-7

• Synthesised in Liver

• Systemic marker and indicator of acute phase of an inflammatory response.

• Circulating CRP reaches saliva via GCF or salivary glands.

• Christodoulides N et al reported that high levels CRP are associated with chronic

and aggressive periodontal diseases.

C-reactive protein (CRP) :

Journal of Indian Society of Periodontology. Jan-Feb 2013:17(1):36-41

8-OHdG

• Most stable product of ROS

• Marker of mitochondrial DNA

damage caused by premature

oxidation in the gingival tissues of

periodontitis patients

{Canakci CF et al}

IL-17

• Proinflammatory cytokine produced by

T-helper 17 cells

• Stimulate various cell types to produce

other inflammatory cytokines and

chemokines

• Preserves immune homeostasis

• Supports immune responses (Th1) and

combines with receptor activator of

RANK and RANKL, resulting in

osteoclastic bone resorptionYang et al

J Periodontol.2015

• Key enzyme in extracellular collagen matrix degradation,

• Origin: PMNs during acute stages of periodontal disease

• Significantly increased the risk of periodontal disease

• MMP-8 is not only an indicator of disease severity, but also disease activity.

• MMP-1 (interstitial collagenase) also appeared to be activated in periodontitis.

MATRIX METALLOPROTEINASES

• Possible markers for the progression of periodontitis fall into three general

categories:

1. Host-derived enzymes and their inhibitors

2. Tissue breakdown products

3. Inflammatory mediators and host response modifiers

GCF BIOMARKERS

INFLAMMATORY MEDIATORS AND HOST-RESPONSE MODIFIER IN GINGIVAL

CREVICULAR FLUID

Biomarker Type Function

Interferon alpha Cytokine AntiviralIncreased MHC Cl-I expression

Interferon gamma Cytokine Macrophage Activation,Th2 suppression

IgA Antibody Antigen Neutralization

IgG, IgG1,IgG2, G3,G4, IgM Antibody Antigen Neutralization

IL-1 ra (Receptor Antagonist) Cytokine Antagonist of IL-1

IL-1 Cytokine Regulates immune and inflammatory reactions, stimulates bone resorption

IL-4 Cytokine Antiinflammatory, macropgeinhibitor,Th2 differentiation

Pro inflammatory Cytokines

• Monocytes, macrophages, fibroblasts and endothelial cells respond to plaque

microorganisms by secreting chemokines and inflammatory cytokines ( TNF-α,

PGE2, IL-1β and IL-6)

(Beck et al 1998)

• Can be obtained from GCF and saliva (IL1, TNF-α)

TNF -α

• Origin: Mononuclear phagocyte.

• The main stimulus for release is the Lipopolysaccharide of bacterial cell walls.

• Functions:

Bone resorption

Inhibit bone collagen synthesis.

Induce collagenases

Stimulate osteoclast differentiation in the presence of M-CSF

• Act synergistically with cytokines and induces release of IL-1.

TNF – β (Lymphotoxin)

• 25 KD glycoprotein

• Activated T cells

• 28% homology to TNF-

• Functions:

CTL stimulation

Osteoclast activation of PMNLs and

Antiviral activity.

• 17 KD

• Origin: Stimulated macrophages

• (+) cytoxic T lymphocytes (CTL).

• ↑ in periodontitis

Gorska et al

• ↑ bone resorption and CT

degradation by (+) PGE2 and

Collagenase

Morimoto Y et al,2008

• Shapira et al 2001, Ejeil AL et al 2003

TNF - à

INTERLEUKINS

IL-1

Stimulate adhesion

molecule and

chemokine

expression

Enhance

Osteoclast

formation and

activity

Induce matrix

metalloproteinase

expression

Stimulate

production of

inflammatory

mediators(PGE

Stimulate apoptosis

of matrix producing

cells

Inflammation Bone loss

Connective Tissue

Breakdown

Limit repair of periodontium

Mechanism by which IL-1 could contribute to the net loss of periodontal tissues

IL-6

• Produced by various cells such as activated monocytes or macrophages, endothelial

cells, activated T-cells, and fibroblasts.

• Earlier Names: B- cells stimulatory factor II, interferon B2 and plasmacytoma growth

factor.

• Functions :

o B cells : Promote growth and facilitate maturation of the B cells causing immunoglobulin

secretion.

o Osteoclast formation and activity

o ↑ in sites of gingival inflammation and plays a role in bone resorption.

• On measuring the level of IL-1β, IL-2, IL-4, IL-6 and TNF- α in GCF by ELISA

assays :

• Results showed high individual variability of cytokine profiles, and no association

between cytokine concentrations and clinical parameters of periodontitis

Gorska et al

• Disease activity thought to be related to Th1-Th2 cytokine profile

• Th2 cytokines like IL4, IL5, IL6 associated with progressive disease

Seymour 2002

• Sakai et al 2006, Yucel et al 2008, Liu et al 1996, Homlund et al 2004,

Engebretson et al.2002, Ozmeric 1998, Toker et al 2008, Tsai et al 2007 reported

increased GCF and salivary levels of proinflammatory cytokines like IL 1, IL6, IL8

in periodontal disease.

• Th1 cytokines IFN γ, IL2 associated with progressive lesion

Berglundh 2003

• In the absence of evidence, there is no single inflammatory cytokine that can be

used as a marker as yet.

Biomarker Type Function

IL-6 Cytokine Regulator of t and B cell Acute phase protein

IL-8 Cytokine Recruitment and activation of neutrophils

IL-15 Cytokine Anti-apoptotic effect on cells

Lactoferrin Acute Phase Protein Antibacterial, creates Fe limiting environment

TNF - alpha Cytokine Delays neutrophil apoptosis

Leukotriene B4 Proinflammatory mediator Stimultes chemotaxis, adhesion, oxidative burst, degranulation

Prostaglandin E2 Immune Mediator Multiple pro inflammatory and immunomodulatory effects

Pradeep AR, Manjunath SG et al. J Periodontol. 2007;78:2325-30.

Kim DM et al. J Periodontol. 2007 ;78:1620-6.

Goodson et al. in 1974

PGE2

• In untreated cases ,gcf levels not able to distinguish between progressive and non

progressive sites Offenbacher-94

• Reduction in GCF levels after nonsurgical therapy Sengupta 1990

• Attachment loss at one or more sites after 6 months had significantly higher mean GCF

PGE2 levels of 113 ng/ml at the baseline.

• Levels greater than 66 ng/ml were found to be predictive of further possible loss of

attachment and this level was used as a cut off value in a positive and negative

screening test.

• Membrane-derived lipid mediator formed from arachidonic acid.

• Source : PMNLs

• Tsai et al 1998, Back et al.2006, Pradeep et al 2007,Emingil 2001 reported difference

in levels in health,disease and post treatment.

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

• Offenbacher et al. (1991)

• AR Pradeep et al

LtB4

CONNECTIVE TISSUE BREAKDOWN

Beta Glucuronidase

• Degradation of the connective tissue ground substance – glycoproteins, proteoglycans

• Marker for primary granule release from PMNL

• May be obtained from both saliva and GCF

• Also positively associated with spirochetes, P. gingivalis, P.intermedia and negatively

associated with cocci.

• Lamster et al 1988 showed that sites that showed the higher β -glucuronidase activity

at baseline and again at 3 months had the highest association with loss of attachment.

• Lamster et al 1988, Harper et al 1989, Wolff et al, 1997, Lamster and Novak 2002

Neutrophil elastase

• Serine endopeptidase found in primary granules

• Marker of intracrevicular PMN activity

• Degrades collagenous & non collagenous substrates, activates collagenases.

• Seen either adjacent to junctional epithelium or in granulation tissue at the

advancing front of the lesion.

• Anti bacterial activity

• Palcanis et al 1992 did a 6-month longitudinal study using a test kit system, and showed

significant differences of total elastase activity at baseline in progressive and non-

progressive sites assessed 2-6 months later.

• Eley and Cox 2006 did a longitudinal study for 2 years and found levels above critical

values for total elastase activity and enzyme concentration present at all Rapid

Attachment Loss sites, both at the time of attachment loss and 3 months previously

(predictive time).

Cathepsin B

• Intracellular cysteine proteinases

• Intracellular collagenolytic breakdown

• Activates MMPs

• Source: Macrophages, fibroblasts, osteoblasts

• Eley & Cox (1996) compared the GCF contents of cathepsin B to probing

depths to determine the success of cathepsin B as a marker for active disease.

• 75 patients with moderate chronic periodontitis were tested; with a high degree of specificity and

sensitivity (99.8% and 100%, respectively)

• Cathepsin B was able to better identify active pocket destruction than other markers in GCF..

• Kunimatsu et al 1990, Eley & Cox (1996), Chen et al 1998 reported its ability to

distinguish Progressive & Non progressive disease and aid in Prognosis/Therapy

• Loos et al 2005

Matrix Metallo Proteinases

• Family of homologous zinc endopeptidases, that collectively cleave most if not all

constituents of ECM

• Key mediators of tissue destruction (Kinane et al 2000)

• Source: Macrophages, neutrophils, fibroblasts and keratinocyte

Gingival fibroblast-MMP8

Monocytes and macrophges:MMP9

Gingival fibroblast , epithelial cells:MMP2

MMP-9

• Degrades collagen extracellular

ground substance

• Teng et al : 2x ↑ in MMP-9 with

recurrent attachment loss

• Metronidazole : ↓ MMP-9

• Most prevalent MMP found in diseased

periodontal tissue and GCF

• Mancini and co-workers: 18x ↑ MMP-8 in

patients experiencing active periodontal

tissue breakdown

• ↑ in disease and ↓ by 60% during the 2-

month protocol of LDD

Golub et al

• ↑ in PISF from periimplantitis lesion

• Collagen –I & II breakdown

MMP-8 (Collagenase 2)

Matrix Metallo Proteinases

Aspartate Amino transferase (AST)

Source :Epithelial cells, gingival and PDL fibroblasts

Evidence of cell death within the periodontal tissues and, hence, possibly disease

activity.

• Chambers et al.1988, Chambers et al. (1991), Smith et al 1998, Persson et al 1992,

Oringer 2001 reported greater AST activity in periodontal disease

• Chair side Test

Pocketwatch

Perioguard

MARKERS OF BONE LOSS

Pyridinoline cross links (ICTP) levels

• Class of collagen degradation molecules that include pyridinoline, deoxy pyridinoline,

N telopeptide and C telopeptide.

• 12 to 20 kd fragment of bone type I collagen released by digestion with trypsin or

bacterial collagenase

• Specific biomarkers for bone resorption

Eriksen et al

• Palys et al. related ICTP levels to the subgingival microflora of various disease states

on GCF.

• Levels differed significantly between health, gingivitis, and periodontitis subjects

• Related modestly to several clinical disease parameters.

• Strongly correlated with whole subject levels of several periodontal pathogens including T.

forsythensis, P. gingivalis, P. intermedia, and T. denticola.

• Golub et al.

• Elevated GCF ICTP levels at baseline, especially at shallow sites, were found to be

predictive for future attachment loss as early as 1 month after sampling.

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.

• Kunimatsu et al 1993

GCF osteocalcin has positive correlation with 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.

OSTEONECTIN AND BONE PHOSPHOPROTEIN

(N-PROPEPTIDE)

• 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 1989

• Both detected in GCF from CP pts.

• Total amount increases with PD

Bowers et al 1989

• They therefore may be associated with periodontal disease severity.

Osteopontin

• Found in bone matrix

• Highly concentrated at sites where osteoclasts are attached to the underlying

mineral surface

• Source: 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

GAGs

• Most 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

Rankl and OPG

• 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

• The development of rapid point-of-care (POC) chairside diagnostics has the

potential for the early detection of periodontal infection and progression to

identify incipient disease and reduce health care costs. However, validation of

effective diagnostics requires the identification and verification of biomarkers

correlated with disease progression (Ramseier CA et al., 2009).

Chairside kit

COMMERCIAL DIAGNOSTIC KITS

IJCDS • FEBRUARY, 2011 • 2(1) © 2011 Int. Journal of Clinical Dental Science

lab-on-a-chip (LOC) assay system (micro-total-analysis

systems(lTAS); assess the levels of interleukin-1 (IL-1), C-

reactive protein (CRP), and matrix metalloproteinase-8

(MMP-8) in whole saliva. biomarker-based identification of

oral cancer

Grover V et al.Journal of Oral Diseases.Vol 2014

AVAILABLE TEST

KITS AND THEIR

EVALUATION

IJCDS • FEBRUARY, 2011 • 2(1) © 2011 Int. Journal of Clinical Dental Science

• OBESITY RELATED

• Chemerin - an adipokine ; CP & DM Pradeep et al,2015

• Leptin - The decreasing leptin level in GCF and gingival tissue was associated with a deteriorated

periodontal status, and smokers also showed reduced GCF leptin levels in recent studies

• SYSTEMIC DISEASE RELATED

• Progranulin – CP & type 2 DM. Pradeep et al 2013

Capsase 3 - GCF and the serum concentration of caspase-3 proportionally increases with the progression of

periodontal disease Pradeep et al 2014

• IL-29 - antiviral IL-29 level was highest in GCF of aggressive periodontitis patients while that of chronic

periodontitis lying in between. After non-surgical periodontal therapy, IL-29 levels increased both in chronic andaggressive periodontitis patients a potential therapeutic agent in treating periodontitis.

Shivaprasad BM ,Pradeep AR 2013

RECENT BIOMARKERS

• Though several products show potential benefit; which gives a clue as to which tissue

components are at risk, most of the test kit, or biomarkers yield little or no additional

information, at high costing.

• It is also clear that no single marker has been able to fulfil all the criteria necessary for

assessment of the clinical state of the periodontium.

• Future research should be directed possibly at the production of "marker packages"

• As of now various efforts are on to develop an ideal test, but actual use as a chairside

diagnostic is still illusive. Therefore the development of a wide spectrum of markers is the

primary goal of periodontal research

CONCLUSION:

1.Periodontology 2000 vol.39,50,51,70

2.Kolokythas A et al. Salivary biomarkers associated with bone deterioration in patients with medication-related osteonecrosis of the jaws. J Oral Maxillofac Surg : 2015.

3.Christodoulides N et-al. Lab-on-a-chip methods for point-of-care measurements of salivary biomarkers of periodontitis. Ann. N.Y. Acad. Sci.2007: 1098: 411–428 .

4.Carranza’s CLINICAL PERIODONTOLOGY .11th Edition

5.Pradeep A.R et-al. Correlation of human S100A12 (EN-RAGE) and high-sensitivity C-reactive protein as gingival crevicular fluid and serum markers of inflammation in chronic periodontitis and type 2 diabetes. Inflamm. Res. (2014) 63:317–323.

6.Gingival crevicular fluid and plasma levels of neuropeptide Substance-P in periodontal health, disease and after nonsurgical therapy. Pradeep AR, Raj S, Aruna G, Chowdhry S. J Periodontal Res. 2009;44:232-7.

REFERENCES: