MINTU M KUMAR 2 ND YEAR PG DEPARTMENT OF PERIODONTICS

 Introduction Gingival sulcus What is GCF? History Functions

Its formation
Methods of collection GCF flow Composition Conclusion References
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 Gingival crevice fluid (GCF) is a complex mixture of

substances derived from serum, leukocytes, structural cells of the periodontium and oral bacteria

These substances possess a great potential for serving as

indicators of periodontal disease and healing after therapy

 A shallow crevice or space around the tooth bounded by

the surface of the tooth on one side and epithelium lining the free margin of the gingiva on the other

V shaped

Ideal conditions..0

 Tissue fluid that seeps through the crevicular and

junctional epithelium. It is increased in the presence of inflammation.

(Glossary of periodontal terms, 2001,4th edition)

 A fluid occuring in minute amounts in the gingival

crevice, believed by some authorities to be an inflammatory exudate and by others to cleanse material from the crevice, containing sticky plasma proteins which improve adhesions of the epithelial attachment, have antimicrobial properties, and exert antibody activity.

 Pioneering work was done by Waerhaug ,Brill and Krasse

The pioneer research of Waerhaug in the early 1950s was

focused on the anatomy of the sulcus and its transformation into a gingival pocket during the course of periodontitis (Waerhaug 1966)
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 In the late 1950s and early 1960s a series of groundbreaking

studies by Brill et al. laid the foundation for understanding the physiology of GCF formation and its composition (Brill 1962)

Brill(1950) confirmed the presence of GCF in humans and

considered it a transudate. He injected fluorescin dyes i.m in dogs and demonstrated the dye in the sulcular fluid.

 Loe,Holm-Pederson considered GCF as a inflammatory

exudate.
The studies of Le et al. contributed to this understanding

and started to explore the use of GCF as an indicator of periodontal diseases

Egelberg continued to analyze GCF and focused his studies

on the dentogingival blood vessels and their permeability as they relate to GCF flow

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 The GCF studies boomed in the 1970s. Dentogingival structure and physiology was created by the

outstanding electron microscopic studies of Schroeder and Listgarten

Presence and functions of proteins, especially enzymes in

GCF were first explored by Sueda, Bang and Cimasoni

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 In a healthy gingiva ,little or no GCF is collected. Cimasoni (1983) identified and classified 40 products in the

GCF.
Lamster et al(1985) focused on biochemical mediators of

inflammation ,tissue degrading enzymes in GCF as markers of inflammatory response.

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 Ohlsson, Golub and Uitto discovered that collagenase and

elastase in GCF are derived primarily from human cells, most notably neutrophils, and that their activity is correlated with gingival inflammation and gingival pocket depth

Migration of neutrophils and their function in gingival tissue

and GCF were clarified by the excellent investigations of Attstrm et al.

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 In 1974 the first edition of the monograph The Crevicular

Fluid by Cimasoni was published

The review by Griffiths examines the hypothesis of Alfano

which states that in health GCF represents the transudate of gingival tissue interstitial fluid but in the course of gingivitis and periodontitis GCF is transformed into true inflammatory exudate.

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 An increase in GCF flow may have physical protective effects

through flushing the pocket, as well as facilitating the passage of Immunoglobulins

Such treatment approaches would aim to enhance the hosts

natural specific and non-specific responses

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 Is GCF an inflammatory exudate?

The initial investigations of GCF attempted to relate

its formation to the inflammatory changes in the connective tissues underlying the sulcular and junctional epithelia

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 Changes were primarily an increased permeability of the

blood vessels, which was induced by chemical or mechanical means

These early studies monitored the appearance in GCF of

substances introduced into the parenteral circulation

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 Early experiments showed that systemically administered

fluorescein appeared in the GCF collected from healthy gingival crevices in dogs [Brill & Krasse 1958]
The samples were collected using filter paper strips held in

place for three minutes and these experiments were extended to human volunteers

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 Since the other oral epithelia had not allowed the passage of

the fluorochrome it was concluded that differences in permeability must exist between these oral epithelia and the epithelium lining the gingival pocket
Subsequent experiments in dogs showed that the flow of

gingival fluid increased markedly following stimulation of the gingivae by tooth brushing or by chewing, or after intravenous injection of histamine or the development of inflammation

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 This led to the conclusion that some irritation, whether

chemical or mechanical, was necessary to induce the production of GCF and that it should therefore be considered as a pathological phenomenon

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Alfano (1974) and from the hypothesis postulated by Pashley (J Periodontal Res 1976) The initial, pre-inflammatory fluid was considered to be a transudate as a result of an osmotic gradient and, on stimulation, this changed to become an inflammatory exudate.

Bacterial plaque would result in the accumulation of high molecular weight molecules. supported experimentally (Alfano et al, J Dent Res 1976), by the application of phosphate-buffered saline containing 10mg/ml of homologous serum albumin resulted in a 100% increase in the volume of GCF produced.
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 Most difficult hurdle scarcity of GCF.

Methods include :

1.Absorbing paper strips. -Intra sulcular method(BRILL) -Extra sulcular method (LOE and HOLM PEDERSON) 2.Crevicular washings.(CIMASONI) 3.Micropipettes.(BJORN and BRILL) 4.Twisted threads.(WEINSTEIN et al)
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 Used to study GCF from clinically normal gingiva One method uses an appliance consisting of a hard

acrylic plate covering maxilla with the soft borders and a groove following the gingival margins and it is connected to four collection tubes
Second method uses two injection needles fitted on

within the other such that during sampling needle is at the bottom of the pocket and collecting one is at the gingival margin
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 It permits the collection of fluid by micropipettes

Capillary tubes of standardized length and diameter

are placed in the pocket and their content is later centrifuged and analyzed

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 Used by Weinstein

Threads were placed in the gingival crevice around the

tooth and the amount of fluid collected was estimated by weighing the sample thread

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 the distance the fluid had migrated up the strip

area of filter paper wetted by the GCF

staining the strips with ninhydrin to produce a

purple color (Cimasoni ,1983).

2g fluorescein given systemically (Weinstein et al,

Periodontics 1967).

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 weighing of strips before and after sample

collection,(Valazza et al,1972)
requires a very sensitive balance to estimate the

very small amounts of fluid which may be collected from a healthy crevice.

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1.GCF collected on a strip is measured by staining with Ninhydrin dyes, then measured on an enlarged photograph with magnifying glass or microscope.

2.The fluid can also be collected on a blotter (PerioPaper)and analysed with an electronic transducer(Periotron). The wetness of the strip affects the flow of electric current & gives a digital readout
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 Each machine needs its own calibration, as

machines differ markedly in their range.

Serum is a suitable material to generate a calibration

curve.

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 An accurate syringe and some form of

standardization of dispensing duplicate volumes are essential.

Duplicate volumes are required, but it is not

necessary to add additional replicates.

A full range of volumes from 0.1 to 1.2l is

necessary to generate an accurate curve.

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1.

CONTAMINATION

2. SAMPLING TIME 3. VOLUME DETERMINATION

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 An important determinant in the ecology of the

periodontal pocket or sulcus.

Creates a flushing action and an isolation effect. Determines the growth level of subgingival

microorganisms
Potential marker for periodontal disease activity.

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 GCF flow (or flow rate) is the process of fluid moving

into and out of the gingival crevice or pocket

Small stream, usually only a few microliters per hour Fluid flow is a rate measure Mathematically symbolized as dV/dt, the first derivative

of volume with respect to time

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 The three components of gingival crevice fluid

(GCF) flow are the resting volume, the influx and the efflux.
Since the resting volume is constant over the

measurement period and losses from evaporation or absorption are minimal, GCF flow can be measured by considering either the influx or efflux.
Measurement of GCF influx is the most easily

accomplished.
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 In the gingival environment at equilibrium, the influx

(fi=dV1/dt) should equal the efflux (fo=dVo/dt) so that measurement of either could be considered the GCF flow.
Washout ratio (fi/Vr) is the number of times the

pocket volume is replaced in unit time.

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 Measurement (Vs), the sample volume, is neither

the influx (fi=dV1/dt) nor the efflux (fo=dVo/dt) but the sum of the resting volume and the influx increment, Vr+fit over the collection period Dt (i.e. Vs=Vr+fit).
The resting volume (Vr) is the result of forming a

pool of fluid in the crevice or pocket

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 Method 1: Remove resting volume Method 2: Measure combined resting volume and

influx for varying time Periods

Method 3: Measure the equilibrium concentration of a

marker substance pumped into a pocket at a constant rate

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Goodson JM. J Dent Res 1989

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Curtis et al. J Clin Periodontol 1988

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Chapple et al. J Clin Periodontol 1996

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Persson GR, Page RC. J Periodontal Res 1990

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(Lamster, J Periodontol 1985 )

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(Holborow et al, J Dent Res 1990)

Comparison of gingival crevice fluid (GCF) flow changes with probable pocket depth (PD), clinical attachment level (AL) and bleeding on probing (BOP) following periodontal therapy by scaling and root planing with tetracycline fiber placement 54

Darany et al, J Periodontol,1992

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CELLS ELECTROLYTES

BACTERIA EPITHELIAL CELLS LEUKOCYTES CALCIUM SODIUM FLUORINE MAGNESIUM BACTERIAL ENZYMES CYTOTOXIC SUBSTANCES METABOLIC END PRODUCTS LIPOPOLYSACCHARIDS ACUTE PHASE PROTEINS CYTOKINES IMMUNOGLOBULINS LYSOSOMAL ENZYMES PROSTAGLANDINS COMPLEMENTS FIBRIIN FIBRINONECTIN COLLAGENS OSTEOCALCIN OSTEONECTIN PROTEOGLYCANS

MICROBIAL PLAQUE PRODUCTS

INFLAMMATORY CELL PRODUCTS

HOST TISSUSES

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 collagen-degrading enzymes from P. gingivalis,

A.actinomycetemcomitans and spirochaetes (Robertson et al,J Periodontal Res 1982)

elastase-like enzyme from spirochaetes (Uitto et al, J

Periodontal Res 1986) and Capnocytophaga species (Gazi et al, 1997)

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 trypsin-like proteases from P. gingivalis, B. forsythus,

T. denticola and other spirochaetes (Gazi et al,1997)

chymotrypsin- like enzymes from T. denticola and

Capnocytophaga species (Gazi et al, 1997)

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 aminopeptidases from Capnocytophaga species (Gazi

et al, 1997) and T. denticola (Gazi et al, 1997)

dipeptidylpeptidases from P. gingivalis, P. intermedia

and Capnocytophaga species (Gazi et al, 1997)

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 Hyaluronidase and chondroitinase activities -C.

ochracea, F. nucleatum, P. gingivalis and T. denticola (Seddon ,1989).

Neuraminidase (sialidase) activity-B. forsythus,

Prevotella melaninogenica and P. gingivalis (Moncla et al. J Clin Microbiol 1990),

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 phospholipases -Porphyromonas, Prevotella and

Bacteroides species (Bulkacz et al, J Periodontal Res 1985).

strong acid and alkaline phosphatase activities -

Porphyromonas, Prevotella, Bacteroides and Capnocytophaga species (Laughton et al,. J Clin Microbiol 1982).

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 The number of neutrophils increases from 7X104 to

20X104 per ml during the conversion of a healthy sulcus into a diseased gingival pocket.
the GCF flow rate adjacent to the tooth surface

increases from 1 to 5ml/min (Cimasoni, 1974).

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 Azurophilic (primary) -elastase, cathepsin G, urokinase,

myeloperoxidase, lysozyme and mannosidase, as well as hydrolases active at acidic pH, including cathepsin B, cathepsin D and -glucuronidase. four types of defensins (HNP1 to HNP4)
Specific (secondary)-lactoferrin, neutrophil collagenase

[matrix metalloproteinase-8 (MMP-8)], and lysozyme,

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 gelatinase (tertiary) -gelatinolytic MMP-9.

neutral proteinases- to facilitate neutrophil migration

and to create an environment for effective defense against microbes

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 MMP-8 (collagenase-2)- degrade interstitial collagens

MMP-9 (gelatinase B).- degrading several extracellular

matrix proteins, including basement membrane (type IV) collagen

can inactivate, 1-proteinase inhibitor thereby increasing

the tissue serine proteinase activity

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 MMP-8 and -9 are the main collagen-degrading

enzymes in GCF and saliva (Mkel et al,1994),

Mainly responsible for collagen degradation in inflamed

tissue during gingivitis and adult periodontitis (Lee et al,. J Periodontal Res 1995)
These enzymes are good indicators for periodontal

inflammation

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 Urokinase-type plasminogen activator (uPA) and its

receptor in gelatinase granules and primary granules (Borregaard , 1995).

Plasminogen activators are important regulators of

various physiological events, such as cell migration, wound healing, tissue remodeling, fibrinolysis, and inflammation (Vassalli , 1991).

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 MMP-9
Membrane-type matrix metalloproteinases Matrilysin (MMP-7)

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 MMP-1 (collagenase-1), MMP-13 (collagenase-13),

MMP-2 (gelatinase A), MMP-3 (stromelysin-1), and MT1-MMP (MMP-14).

Lysosomal cysteine proteinases, especially cathepsins

B and L, are involved in the intracellular collagen degradation of fibroblasts during normal connective tissue turnover (Everts et al, 1996).

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 GCF from gingivitis sites of adult periodontitis

patients showed multiple inflammatory mediators, with elevated IgG levels and lower IgA levels (Ebersole et al, 1991).

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 Grbic et al. (1995) demonstrated that IgA levels were

significantly increased in GCF from gingivitis sites when compared to that from periodontitis sites
Inflammatory/immune mediators and cytokines,

including prostaglandins, leukotrienes, interleukin-1 (IL-1), IL-6, tumor necrosis factor-, and IL-8 and other inflammatory/ immune mediators and cytokines, are measurable in GCF (Ebersole ,. J Periodontal Res 1993).

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 IgG being the primary immunoglobulin in GCF (Smith

et al, J Periodontal Res 1985).

The presence of all subclasses of IgG have been

identified in GCF, with IgG1 and/or IgG4 levels in GCF elevated relative to serum concentrations (Mackler et al,1979).

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 Powell et al. (1994) showed the presence of all

subclasses in GCF, although, the IgG4 subclass was elevated relative to serum concentrations in adult periodontitis.

GCF IL-6 levels correlated with bleeding, pocket

depth, and disease active sites (Geivelis, J Periodontol 1993);

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 Elastase and -glucuronidase activity in crevicular

fluid is related to the severity of periodontal disease (Lamster et al,J Clin Periodontol 1995).

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 The risk ratio of clinical attachment loss in patients

with high -glucuronidase activity in crevicular fluid is about 10-fold (Lamster et al,. J Clin Periodontol 1994).
Significantly higher elastase values were measured in

sites with progressing periodontal disease than in nonprogressing sites (Armitage, J Periodontol 1994).

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 Most of these substances that are released in the

tissues pass into the GCF and fall into 3 general categories :
1.

Inflammatory mediators and host-response modifiers Host-derived enzymes and their inhibitors Tissue breakdown products
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2. 3.

 Immune response

Antibody: Total immunoglobulin and IgG subgroups complement Inflammatory response Arachidonic acid derivatives, e.g., PGE2

Cytokines, e.g., IL-1, IL-2, IL-4, IL-6, TNF-

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Bone-specific proteins Osteonectin Bone phosphoprotein (N-propeptide) Osteocalcin Telopeptides of type 1 collagen

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 GCF prostaglandin E2 levels (ng/ ml) were

20.37.9 (controls), 49.44.8 (gingivitis), 42.312.3 (adult periodontitis), 81.620.3 (juvenile periodontitis), 90.015.9 (refractory periodontitis),

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 GCF interleukin-1 - elevated in all forms of periodontitis

compared to health or gingivitis. GCF interleukin- 1 levels were 16.85.3 for healthy controls , 115.852.6 for gingivitis patients, 263.273.7 for adult periodontitis patients, 836.8284.2 for juvenile periodontitis patients 457.9157.9 for refractory periodontitis patients expressed in ng/ml. (Salvi et al,1992)

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 Amount of GCF is greater during inflammation,

mastication of coarse food, tooth brushing, gingival massage, ovulation, hormonal contraceptives and smoking.

Circadian Periodicity-increases from 6AM TO

10PM,decreases thereafter.

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 Sex Hormones-increases GCF flow due to increase

vascular permeability.
Periodontal Therapy-increase in GCF during healing Smoking- immediate transient increase in GCF flow. Drugs are excreted in GCF(Tetracycline and

Metronidazole).

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 Fluorometry of MMP

ELISA for enzymes and IL-1beta,

RIA for COX derivatives Procollagen III

High pressure liquid chromatography for timidazole

Direct and Indirect Immunodot tests for acute phase

proteins.

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 IL-1 alpha and beta Binds PMN and Macrophages to endothelial cells Stimulates production of PGE2 , Releases lysosomal

enzymes
Stimulates bone resorption.

Antibodies are difficult to detect in GCF, but

consensus is that a reduction is detrimental and its presence is protective.

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 GCF -is an ideal medium for monitoring changes during

development of periodontal diseases.

It is collected non-invasively and is site specific.

It contains products of host, plaque and their

interactions.
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 It is dynamic and its composition are closely related

to the environment of periodontal tissues, hence an early marker for periodontitis.

Transudate or inflammatory exudate.

Normal gingiva no GCF.

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 GCF -Does not throw light on attachment and

bone loss.
Lacks the gold standard against which the test can

be evaluated.

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 GCF provides a unique window for analysis of

periodontal condition.
Several tests have been developed that are aimed at

specifically and sensitively revealing the metabolic status of periodontal tissues

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 The origin, composition and the clinical significance

of GCF are now well known with more precision and have significantly helped our understanding of the pathogenesis of periodontal disease.
Thus, the collection and analysis of GCF samples

provides a non- invasive means to assess the pathophysiological status of the periodontium in a sitespecific manner.

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 Clinical periodontology, Carranza. 10th edition Periodontology 2000, Vol. 30, 2002. Gingival crevice

fluid
Biology of Periodontal connective tissue, P. Mark

Bartold & A. Sampath Narayanan

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Thank you

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