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Journal of Investigative Periodontology 3 (6) Special Issue: 45-63, 2010

Gingival Crevicular Fluid (GCF): An oral

biomarker in the diagnosis and

quantification of periodontal diseases

Punit Vaibhav PatelFaculty of Dental Sciences,Institute of Medical Sciences,

Banaras Hindu University (B.H.U.),

Varanasi,-05, U.P., India

Abstract

Periodontitis is a disease characterized by loss of connective tissue attachment and bone aroundthe teeth in conjunction with the formation of periodontal pockets due to the apical migration of

the junctional epithelium. Early diagnosis and treatment of progressive periodontitis is important

because of the irreversible nature of this disease The long-term aim is that treatment andprevention of periodontal disease will be founded on diagnostic tests based on aetiopathogenic

factors rather than just clinical experience. Clinical measurements used in diagnosis ofperiodontal diseases are often of limited usefulness in that they are indications of previous

periodontal disease rather than the present disease activity. Biochemical mediators in oral fluidslike saliva and gingival crevicular fluid (GCF) are highly beneficial in the determination of

current periodontal status. These substances known as biomarkers help in determination of

inflammatory mediator levels, as they are good indicators of inflammatory activity. This reviewhighlights recent advances in the use of salivary and gingival crevicular fluid (GCF) biomarker-

based disease diagnostics that focus on the identification of active periodontal disease.

IntroductionThe presence of sulcular or gingival crevicular fluid has been known since the 19 thcentury but

its composition and possible role in oral defense mechanisms were elucidated by the pioneering

work of Waerhuag , Brill and Krasse in the 1950s.1The 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 .In the late 1950s and early 1960s a series of groundbreaking studies by Brill et al. laid the foundation for understanding the physiology of GCFformation and its composition.The studies of Le et al. contributed to this understanding and

started to explore them 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 permeabilityas they relate to GCF flow The GCF studies boomed in the 1970s. The rationale for

Review Article Journal of Investigative Periodontology


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understanding dentogingival structure and physiology was created by the outstanding electron

microscopic studies of Schroeder and Listgarten .2 Presence and functions of proteins,

especially enzymes in GCF were first explored by Sueda, Bang and Cimasoni . Ohlsson, Goluband 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 . In 1974 the first edition of the monograph The Crevicular Fluid byCimasoni was published. This comprehensive revie gave a big boost to GCF studies and towards

the end of the first millenium the research on GCF increased dramatically.3-9

How is GCF formed?Brill et al 1959systemically administered fluorescein in dogs GCF collected using filter paper

strips Fluorescein appeared in the GCF collected Sample collected from other oral epithelia had

not allowed the passage of the fluorochrome it was concluded that differencesin permeability must exist between these oral epithelia and the epithelium lining the gingival

pocket.

Egelberg in 1966, in a first experiment obtained an increased permeability of the blood vessels of

healthy gingivae by the use of three different methods: -

topical application of histamine, gentle massage of the gingiva by means of a ball-ended amalgam plugger and scraping of the gingiva crevice by means of a blunted dental explorer.Immediately after the application of histamine large amounts of gingival fluid were obtained andmarked vascular labeling could be observed at the same time. Massage and scraping were also

immediately followed by appearance of large amounts of gingival fluid.

Formation of GCF is Physiological or Pathological process?

Subsequent experiments showed that the flow of gingival fluid increased markedly followingstimulation of the gingivae by

Tooth Brushing By Chewing After Intravenous Injection Of Histamine Or The Development Of Inflammation .

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 apathological phenomenon.

Is GCF a transudate of interstitial fluid?

An alternative theory arose from the work of Alfano and Pashley (1974-76) which suggestedthat the initial fluid produced could simply represent interstitial fluid which appears in the

crevice as a result of an osmotic gradient. This initial, pre-inflammatory fluid was considered to

be a transudate, and, on stimulation, this changed to become an inflammatory exudate. Amechanistic analysis of gingival fluid production which analyzed the physiology of the gingival

crevice area with respect to the factors described Originally by Starling was

made by Pashley (1976). The model proposed by Pashley , predicted that GCF Production isgoverned by the passage of fluid from capillaries into the tissues (capillary filtrate) and theremoval of this fluid by the lymphatic system (lymphatic uptake). When the rate of capillary

filtrate exceeds that of lymphatic uptake, fluid will accumulate as edema and/or leave the area as

GCF

The mathematical model proposed by Pashley


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through an intact epithelium were reviewed by Squier and

Johnson.

Three routes have been described:1. Passage Form CT Into The Sulcus:2. Passage From The Sulcus Into The CT3. Passage Of Substances Through Pathological or

Experimentally Modified Gingival Sulcus

Methods of collection

Several techniques have been employed for the Collection ofGCF and the technique chosen will Depend upon the

objectives of the study as each Technique has advantages and

disadvantages.

Gingival washing methodsIn this technique the gingival crevice is perfused with an

isotonic solution, such as Hanks balanced salt solution, usually of fixed volume. The fluid

collected then represents a dilution of crevicular Fluid and contains both cells and solubleconstituents such as plasma proteins. Two different techniques have been used. The simplest

involved the instillation and re-aspiration of 10ml of Hanks balanced salt solution at the

interdental papilla . This process was repeated 12 times to allow thorough mixing of the

transport solution and GCF. This technique could therefore be applied either to individualinterdental units or to multiple units

which were then pooled. A more complicated method involved the construction of a customizedacrylic stent which isolated the gingival tissues from the rest of the mouth. The tissues were then

irrigated for 15min, with A saline solution, using a peristaltic pump, and the diluted GCF was

removed.

AdvantagesThe washing technique is particularly valuable for harvesting cells from the gingival crevice

region.

Disadvantages

1. The production of customized acrylic stents is complicated and technically demanding.2. It is therefore a technique of limited application and has been restricted to the study of GCFobtained from only a few individuals.

3. It has also usually only been applied to the maxillary arch, presumably because of thedifficulties of producing a technically satisfactory appliance for the mandibular arch.

4. It is also a disadvantage that GCF from individual sites cannot be analyzed.5. In contrast, the simpler technique may be applied to individual sites or groups of sites

which may be categorized as healthy or inflamed.

6. The major disadvantage of this technique is that all fluid may not be recovered during theaspiration and re-aspiration procedure.

7. Thus accurate quantification of GCF volume or Composition is not possible as the precisedilution factor cannot be determined.

Capillary tubing or micropipettes

Following the isolation and drying of a site, capillary tubes of known internal diameter arinserted into the entrance of the gingival Crevice. GCF from the crevice migrates into the tube

by capillary action and because the internal diameter is known the volume of fluid collected can


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produced at a given site. The amount of GCF collected on a strip was assessed by the distance

the fluid had migrated up the strip. This was often taken as a simple linear measurement, but a

more accurate value was achieved by assessing the area of filter paper wetted by the GCFsample.Further accuracy was achieved by staining the strips with ninhydrin to produce a purple

color in the area where GCF had accumulated. A similar result was shown with 2g fluorescein

given systemically to each patient 2 hours prior to the collection of GCF, following which thestrips were examined under ultraviolet light (Weinstein 1961). They found that fluorescein

labeling was 100 times more sensitive than ninhydrin for staining protein. The introduction of an

electronic measuring device, The Periotron , has allowed accurate determination of The GCFvolume and subsequent laboratory Investigation of the sample composition.

The instrument measures the effect on the electrical current flow of the wetted paper strips. It

has two metal jaws which act as the plates of an electrical condenser. If a Dry strip is placedbetween the jaws, the capacitance is translated via the electrical circuitry and registers zero on

the digital readout. A wet strip will increase the capacitance in proportion to the volume of

fluid and this can be measured as an increased value in the readout. The technique is rapid and

has no discernible effect upon the GCF sample. Three models of PeriotronA have been produced(the 600, 6000 and now the 8000) and each one has been shown to be an efficient means of

measuring the volume of fluid collected on filter paper strips. An alternative approach, involving

the weighing of strips before and after sample collection, has been adopted by some workers .

This has been successful but requires a very sensitive balance to estimate the very smallamounts of fluid which may be collected from a healthy crevice.

Problems with GCF collectionThe major sources of contamination of GCF samples would be blood, saliva, or plaque. Frank

blood contamination is usually dealt with by discarding the sample and removing the data from

analysis. The presence of dental plaque on filter paperstrips used for collecting GCF has been shown to have a marked effect on the volume recorded.

Experiments where dental plaque was applied directly to filter paper strips showed that a large

plaque mass contained a considerable amount of fluid which would influence volumedeterminations, which would be expressed as volume of GCF. Careful isolation should be

performed in an effort to minimize the potential for saliva contamination. Alpha-amylase wasused in an assay to confirm, or refute, the presence of the contamination of GCF samples withsaliva (Griffiths 1992). Application of the Assay to saliva samples collected using a good

isolation and drying technique confirmed that the likelihood of a significant contribution from

saliva was small

Sampling timeThe early literature from the PeriotronA suggested that filter paper strips should be left in place

for 5 seconds. Alternative approaches to sampling techniques have been developed which

included either leaving the strip in place for a longer period or the use of a sequence of repeatedstrips, with Possible recovery periods in between. Other approaches have also included

collecting until a Minimum volume has been recovered. This latter approach

has sometimes resulted in collection times of 2030min.The problem with prolonged collectiontimes isthat the nature of the GCF sample collected is likelyto change with the protein

concentration of the initialGCF collected comparable to interstitial fluid,whereas prolonged

sampling at the site resulted inprotein concentrations approaching those of serum.

Volume determination


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Evaporation is considered to be a significant problem in accurate volume determination of GCF

samples. This is particularly the case as the total volumes collected are usually Less than 1ml and

more often than not are less than 0.5 ml. In practical terms it may be necessary to determine aParticular volume (for example 0.2ml) below which it is not possible to analyze the sample.

Recovery from strips. Having collected the GCF sample and determined its volume the samples

are usually then required for some investigation of the composition of GCF. To achieve this it isnecessary to recover the GCF from the filter paper strips and initial work indicated that protein

recovery was close to 100% using a centrifugal elution technique (Cimasoni 1988). A variety of

other methods of elution have been employed, but it is essential in all instances to determine thepercentage recovery from the original samples.

Data reporting

Constituents found within GCF samples have eitherbeen reported as absolute amount (mg),concentrations(mg/ml) or either of these two measurements with

reference to pocket depth or duration ofsample collection.

Gingival crevice fluid flow

Gingival crevice fluid (GCF) flow is an important Determinant in the ecology of theperiodontal pocketor sulcus.

It creates a flushing action and an isolationeffect. In addition, it probably determines the growth

level of subgingival microorganisms and is a potentialmarker for periodontal disease activity.

The first important characteristic associated With GCF flow is its flushing action. Substances

put into the periodontal pocket are rapidly washed out. The second important characteristicassociatedwith GCF flow is the isolation effect. Substancesfrom the outside do not easily

penetrate the Periodontal pocket. The concentration of immunoglobulin G (IgG) found in GCF

from periodontal pockets is approximately 100 times that found in

saliva . Clearly, this could not be the case if saliva gained ready access to the periodontal pocket.One may reasonably conclude That the net outward GCF flow inhibits retrograde salivaryflow

and that salivary contents do not generally enter the periodontal pocket.

This creates a relative isolation of the periodontal pocket from the rest of the oral cavity.The nutrient effect of GCF flow has been suggestedby associations between bacterial numbers or

composition and GCF flow in several studies .Although it is widely suggested that accumulationof bacteria leads to increased GCF flow, it has not beenso widely appreciated that increased GCFflow maysupport larger bacterial plaque masses.GCF flow (or flow rate) is the process of fluid


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moving into and out of the gingival crevice or pocket .It is a small stream, usually only a few

microliters per hour. GCF in a gingival sulcus or periodontal pocket is like a spring-fed pond.Fluid enters the pond from an unseen source and leaves by flowingout at the margin. Fluid flow

is a rate measure. It is the volume that crosses a defined boundary over a given time,

mathematically symbolized as dV/dt, the first derivative of volume with respect to time.In thegingival environment at equilibrium, the influx (fidVi/dt) should equal the efflux (fodVo/dt)so that measurement of either could be considered the GCF flow. As a practical matter, however,

only the influx is measured. The gingival sulcus or pocket also has a resting volume (Vr) through

Which the GCF flows. The washout ratio (fi/Vr) is the number of times the pocket volume isreplaced in unit time.The resting volume (Vr) is the result of forming apool of fluid in the crevice

or pocket. The incrementin volume as a result of GCF flow (fiDt) results becausethe GCF flow

cannot be turned off while takingthe measurement.There are at least two approaches tomeasuring GCF

flow using conventional methods for measurementof GCF volume.The concept is that by

pumping a marker substance into a Periodontal pocket at a constant rate, an equilibrium

concentrationwill be established which is the result of the fluid flow rate and thepump delivery rate.

An intrapocket drug delivery system is a small pump that can deliver a marker substance

into the periodontal pocket. Although many devices of this type have been developed , onlyone, the Tetracycline fiber, establishes and maintains the constantconcentration required for

this method.Application of this method requires that a measuredlength of tetracycline fiber

be placed into a periodontalpocket and the equilibrium concentration be measured at a latertime.

To date, no published study has been conducted to evaluate GCF flow in this manner. The

measurement of tetracycline concentration requires two measurements, the GCF volume ofthe sample and the amount of Tetracycline in the sample.

COMPOSITION OF GCF:

The GCF consists of:

1. Cellular elements2. Electrolytes


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3. Organic compounds4. Metabolic and bacterial products5. Enzyme and enzyme inhibitors

Cellular Elements:1. The cellular elements found in the gingival fluid include bacteria, desquamated epithelial

cells, and leukocytes (PMNs, lymphocytes and monocytes, erythrocytes) which migrate

through the sulcular epithelium.

The major cellular components of the gingival crevicular fluid

The anatomical origin of the cells of the gingival crevice fluid (GCF)

With the accumulation of bacterialplaque in the vicinity of the gingival sulcus, the flow of GCF

increases. Cells that originate from plaque and Their metabolic by-products can induce theegress of inflammatorycells from the gingival plexus of blood vessels, located in the

connective tissue, just below the Junctional epithelium.

Electrolytes: Potassium, sodium calcium magnesium and fluoride have been studied in gingival fluid. Most studies have shown a positive correlation of calcium and sodium concentrations and

the sodium to potassium ratio with inflammation.

According to Krasse and Egelberg (1962) the sodium and potassium ratio of fluid frominflamed pockets was more than two times that of fluid from healthy sulci.

According to Kaslick et al (1970) a significantly higher sodium: potassium ratio was alsofound in the moderately inflamed as compared to the nearly normal gingivae.

It has also been reported that in case of deep pockets sodium values tend to be lower,while the values for potassium tended to increase. In more severe cases of periodontitis,

the higher number of degenerating epithelial, CT and blood cells contribute to increase

the potassium concentration of exudate by liberating their intracellular content.

(iii) Organic Compounds:

Carbohydrates, proteins and lipids have been investigated. Glucose hexosamine andhexuronic acidare two of the compounds found in gingival fluid. Glucose concentration

in gingival fluid is 3-4 times greater than that in serum.

This is interpreted not only as a result of metabolic activity of adjacent tissues, but also asa function of the local microbial flora.

The total protein content of gingival fluid is much less than that of serum. Nosignificant correlations have been found between the concentration of proteins in the


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gingival fluid and the severity of gingivitis, pocket depth and extent of bone loss.

Proteins namely , , 2 and 1 globulins, transferrin, albumin, immunoglobulins suchas IgG, IgM and IgA, complement components such as C1, C4, C3, C5, have been reportedto be present in GCF.

Proteins Include: - fibrinogen, ceruloplasmin, - lipoprotein, transferrin, 1 antitrypsin and 2 macroglobulin.

(iv) Metabolic And Bacterial Products:Metabolic and bacterial products identified in gingival fluid include

lactic acid, urea, hydroxy proline, endotoxins, prostaglandins, cytotoxic substances,

hydrogen sulphide and antibacterial factors.

(v) Enzyme And Enzyme Inhibitors:Various enzymes known to be present in gingival fluid include: -

Acid phosphatase, alkaline phosphatase, pyrophosphatase, - Glucoronidase, lysozyme, Hyaluronidase, Proteolytic enzymes such as Mammalian proteinases which includes Cathepsin

D,Elastase, Cathepsin G, Plasminogen activators, Collagenase and bacterial proteinases

(i.e. endo and exopeptidases), and lastly lactic dehydrogenase serum proteinase inhibitors

such as 2 macroglobulin, 1 antitrypsin, 1 antichymotrypsin have also beenknown to be present in GCF.

The primary reason for this intense interest is the realization that the most widely used method

for assessing disease progression is not precise enough to detect small amounts of periodontaldamage. The general approach used in most investigations has been to first determine in cross sectional studies if a specified component of GCF is either, present in Periodontitis and absent in

health or gingivitis.

Strongly related to the severity of periodontitis, the potential markers have been grouped intothree general categories:


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Inflammatory mediators and products Host derived enzymes Tissue breakdown products

(i) Inflammatory Mediators And Products:

(a) Prostaglandin E2 (PGE2 ):PGE2 is a product of the cyclooxygenase pathway. Elevated levels of PGE2in GCF were foundin patients with periodontitis compared to patients with gingivitis. PGE2 levels were three times

higher in patients with juvenile periodontitis compared to adult periodontitis.

(b) Cytokines:Cytokines are potent local mediators of inflammation that are produced by variety of cells.Cytokines that are present in GCF and have been investigated as potential diagnostic makers for

periodontal disease include: - interleukin - 1, 1, interleukin 6, interleukin 8 and tumor

necrosis factor (TNF -).Both IL - 1 and IL - 1 have pro-inflammatory effects anddepending on a variety of factors can stimulate either bone resorption or formation.It has alsobeen reported that in adult periodontitis patients, a higher percentage of sites are positive for IL -

1(87%) and IL - 1(56%) IL-6 has also been associated with bone resorption. GCF from siteswith progressing periodontitis contains elevated amounts of IL-6.IL-8 was formerly called

monocyte-derived neutrophil chemotactic factor. GCF from sites with periodontitis containssignificantly more total IL-8 than GCF from healthy sites.

(c) Antibacterial Antibodies:Several reports indicate that certain antibacterial antibodies and antibody subclasses are

significantly higher in GCF than in serum.This suggests that local production of specific

antibody might occur in response to infecting bacteria in periodontal pockets. In one of thestudies, GCF from unstable or progressing sites had significantly elevated levels of Ig G1 and Ig

G4. One of the other study demonstrated that GCF from progressing sites had depressed levels of

IgA thus suggesting the protective role of IgA.

(d) Auto Antibodies:Some reports have demonstrated that sera from patients with periodontitis have elevated levels of

antibody to type I collagen compared to control sera. Presence of anti-desmosomal antibodies insera and GCF from patients with periodontitis has also been demonstrated. To date, the possible

use of these autoantibodies as diagnostic markers has not been investigated.

(e) Total Protein:Several reports suggest that, compared to periodontally healthy controls, GCF from sites

withperiodontitis has significantly elevated levels of total protein.Some study has reported thatGCF from inflamed sites in patients with periodontitis havesignificantly lower protein

concentrations than GCF from inflamed sites in patients with gingivitis alone.

(2) Host Derived Enzymes:

(a) Aspartate Aminotransferase:

It has been demonstrated that on a total amount per-site basis, the GCF levels of aspartate

aminotransferase increase during the development of experimental gingivitis.In ligature inducedexperimental periodontitis studies in dogs, the GCF levels of AST increase during the


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development of attachment loss and bone resorption.The association between GCF AST levels

and the risk of progression of periodontitis has led to the commercial development of a chairside

test. Some uncertainity remains regarding the ability of the test to reliably distinguish betweenprogressing sites and those that are inflamed but not progressing.

(b) Alkaline Phosphatase:Its been confirmed that the levels of ALP in GCF are significantly higher than those in serum

thus suggesting the local production of enzyme.Cross-sectional data indicate that GCF from sites

with gingivitis or periodontitis have significantly higher concentrations of ALP than healthysites.

(c) -Glucuronidase:-Glucuronidase is a lysosomal enzyme but its presence in GCF is probably due to contribution

from numerous host cells. GCF levels of -Glucuronidase were positively corelated to depth of

periodontal pockets and bone loss. In experimental gingivitis, -glucuronidase andarylasulphatase activity in GCF was significantly higher in gingivitis and periodontitis than in

healthy patients.

Elastase:It has been demonstrated that the GCF levels of functional and antigenic elastase increase during

the development of experimental gingivitis. It was also noted that as the experimental gingivitisdeveloped, the GCF content of the two major inhibitors of neutrophil elastase (i.e. 2-

macroglobulin and 1 proteinase inhibitor) also increased.The value of GCF Neutrophil elastase has also been evaluated as a possible screening device to

identify periodontitis sites as increased risk of developing additional attachment loss.

(e) Cathepsins:

Cathepsins B, H and L are cysteine proteinases that play an important role in intracellular proteindegradation. Significant decreases in the GCF levels of Cathepsin B/L activity have been noted

after scaling and root planing.

(f) Trypsin like and Other Enzymes:Besides the trypsin-like enzymes other host derived GCF enzymes with potential diagnostic

importance include: immunoglobulin degrading enzymes, glycosidases, dipeptidyl peptidases

and myeloperoxidase. On a concentration and total activity per site basis, the GCF levelsdipeptidyl peptidase II and IV were significantly elevated at the progressing sites

(g)Collagenases / Gelatinases / Neutral proteinases / Stromelysins:GCF from sites with adult or juvenile forms of periodontitis exhibit significantly elevated

collagenolytic activities compared to GCF from healthy or gingivitis sites.In ligature-induced

periodontitis in the beagle dog, GCF collagenase activity increased to maximum values within

weeks after ligature placement, active collagenase was elevated during active periodontitis andactive collagenase was strongly corelated with attachment loss. Latent collagenase and

collagenase inhibitors were prominent during gingivitis.

(3) Tissue Breakdown Products:


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(a) Glycosaminoglycans (GAGs):The GAGs in GCF that have been most examined as possible diagnostic markers for periodontal

diseases are: Chondroitin 4 sulfate, chondroitin 6 sulfate and hyaluronic acid.The appearance of C-4-S in GCF has been suggested as a marker for bone resorption associated

with periodontal disease or orthodontic tooth movement. But no studies have been conducted to

determine its role in the progression of periodontitis.

(b) Hydroxyproline:

It is a prominent aminoacid of collagen and its appearance in GCF has been preliminaryinvestigated as a marker for the destruction of periodontal connective tissue. Data from one

cross-sectional study in humans indicate that GCF hydroxyproline levels cannot distinguish

between sites with gingivitis or periodontitis. Because of this it is not an attractive candidate as apotential marker for the progression of periodontitis.

(c) Fibronectins:These are a large group of heterogenous glycoproteins present in blood and connective tissues.

Data from most studies indicate that GCF fibronectin is not a promising diagnostic marker.

(d) Connective Tissue Proteins:Increased GCF levels of the aminoterminal propeptide of type I collagen have been reported at

periodontitis sites. On a concentration basis, the amount of osteocalcin in GCF does not appearto be different at sites with gingivitis or periodontitis.Osteonectin another non-collagenous

protein of bone and a variety of other tissues, has been reported to be elevated in GCF at siteswith severe periodontitis. Neither Osteocalcin nor Osteonectin levels in GCF have been

systematically evaluated as diagnostic markers for periodontitis.Biochemical markers of the

progression of periodontitis

The composition of the gingival crevice fluid (GCF) is the result of the interplay between the

bacterial biofilm adherent to the tooth surfaces and the cells of the periodontal tissues.

The collection of GCF Is a minimally invasive procedure and the analysis of specific constituents

in the GCF provides a quantitative biochemicalindicator for the evaluation of the localcellularmetabolism that reflects a persons periodontal health status.

Many studies have looked at the association between total amount or Concentration levels of

different constituents of GCF and periodontal health status. Since host response is a criticaldeterminant in periodontal disease pathogenesis, the measure of inflammatory

mediator levels in the GCF has been used to evaluate risk: Risk for a tooth, or more precisely a

site, to lose clinical attachment and alveolar bone, or risk for an individual to develop

periodontal disease.

Pathogenesis of periodontitis; Periodontitis is a chronic inflammatory response to the

subgingival bacteria, producing irreversible periodontal Tissue destruction and tooth loss. Theprogression of periodontitis is chronic, with cyclic periods of exacerbation and remission, and

may remain unnoticed with Minimal symptoms in the early stages. Periodontitis is Diagnosed

clinically by loss of attachment between the tooth and the supporting tissues (clinical attachmentloss), by deepening of the pocket between The root of the tooth and the supporting tissues

(pocket depth), and/or by radiographic evidence of bone loss.


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Lipopolysaccharide is a key microbial stimulus that will trigger the host response at

periodontal disease Sites. Locally, Lipopolysaccharide triggers monocytes to releaseinflammatory mediators (prostaglandin E2, thromboxane B, interleukins -1, -6 and -8, tumor

necrosis factor, And collagenase) that increase local destruction of the connective tissues

structural elements. Therefore, levels of monocytic inflammatory mediators (includingprostaglandin E2, interleukin-1, and tumor necrosis factor) in GCF may well represent the ideal

markers of disease activity at a site level.

Elevated GCF levels of neutrophil Markers (including neutrophil elastase, b-glucuronidase, andleukotriene B4) may reflect acute episodes of localized tissue destruction.Taken together, these

monocytic and neutrophilimediator levels in the GCF may also give an indication of the quality

of the host response, andof the level of risk for the individual to develop periodontaldisease.Many studies have reported that GCF Prostaglandin E2 levels are significantly elevated

in patients suffering from severe forms of diseases (juvenile periodontitis and refractory

periodontitis) compared to healthy controls or patients suffering from a mild form of the disease

(gingivitis or chronic adult periodontitis).

It seems that GCF prostaglandin E2 levels are significantly elevated in gingivitis patients

compared to controls (seymour 2001). This would indicate that the determination of GCF

prostaglandin E2 levels is a good indicator of inflammatoryactivity.Patients with mild forms of adult periodontitis did not Have much higher levels than

gingivitis patients, althoughGCF prostaglandin E2 levels seemed to increase with increasingdisease severity. Studies by others haveconfirmed that GCF prostaglandin E2 levels are

elevatedin periodontitis patients compared to controls and gingivitis

patients (preshaw 1999,Tsai 1998). In addition, various periodontal treatmenttherapiesinduced a

decrease in GCF prostaglandin E2 levels GCF interleukin-1 levels are also significantly Elevatedin all forms of periodontitis compared to Health or gingivitis. In the Salvi et al. study, 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, and 457.9157.9 for refractory periodontitis patients , expressed in mg/ml.

Overall individual response to bacterial challenge

One way to investigate the idea that GCF Inflammatory mediator levels reflect the overall

ability of an individual to produce inflammatory mediators is to evaluate the individualsability to produce inflammatory mediators. This can be accomplished by measuring the

levels of inflammatory mediators released from isolated peripheral blood monocytes.

Clearly, monocytes isolated from periodontitispatients produce higher levels of prostaglandinE2 than healthy controls, and monocytes isolated from patients suffering from severe forms

ofperiodontal diseases produced even higher levels ofprostaglandin E2 than monocytes

isolated from Patients suffering from milder forms of the disease.Interestingly, levels ofprostaglandin E2 released By isolated peripheral blood monocytes were highlycorrelated to

the levels of prostaglandin E2 Measured in the GCF .


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CLINICAL SIGNIFICANCE:

(a) General Health And Gingival Fluid:

(i) Circadian Periodicity:

There is a gradual increase in gingival fluid amount from 6:00AM to 10:00PM andadecrease afterwardOn the other hand in the studies conducted by a group of

investigators, there are no systematic differences between the flow of fluid measured at

9:00a.m and that of the fluid collected at 3p.m

(ii) Gingival Fluid Flow And Sex Hormones:Clinical investigations have shown an exacerbation of gingivitis during pregnancy (loe1965) during the menstrual cycle (Lemann 1948) and at puberty (Sutcliffe 1972). Female

sex hormones increase the gingival fluid flow, probably because they enhance

vascularpermeability.Pregnancy, ovulation and hormonal contraceptives all increase

gingival fluid production.

(iii)Gingival Fluid in Diabetic Patients:Ringelberg et al in 1977 described a higher flow rate of gingival fluid in a group of

diabetic children, when compared to the flow rate measured in a group of childrenwithout diabetes.

In healthy individuals Hara and Le found exudate glucose values upto 6 times those ofserum. Kjellman (1970) reported glucose values much lower in gingival fluid when

compared to serum, this being true for both healthy and diabetic patients.

(b) Measurement of Gingival Inflammation:Brill was the first to suggest that measurements of the fluid recovered from gingival

pockets by means of filter paper strips might be used for determining the degree of

inflammation of the gingival.

Presence or Absence of Fluid In healthy sulci:

According to Egelberg when a deep intracrevicular technique of collection is used, a lowbut definite amount of gingival fluid can be collected from clinically healthy sulci. Withthis technique, the mechanical irritation supplied by the filter paper will elicit an increase

of vascular permeability and a subsequent production of fluid.According to Loe and

Holm Pedersen when the filter paper strip is not inserted deeply into the sulcus butplacedat its very entrance, no or very little fluid can be collected from healthy sulci.

(ii) Gingival Fluid As a Sign of Subclinical Inflammation:Le and Holm-Pedersen (1965) in a study found a gradual increase of the amount of fluid

collected with their own technique during the period of no cleansing, and reported thatflow from the gingival crevice was regularly demonstrated before clinical gingivitis was

observable. The increase in amount of fluid preceding the earliest clinical evidence of

inflammation during the development of gingivitis has also been confirmed by severalother studies.

(iii)Gingival Fluid flow as Related to Histological Inflammatory Changes:It is generally agreed that the histological signs of inflammation as recorded on biopsies

of marginal gingiva, are not well co-related with the intensity of gingival fluid


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flow.Muhlemann and Mazor found a significant and positive co-relation between the

exudate scores and both the clinical recordings of gingival inflammation and the

roundcell infiltration.Sulcus fluid and the number of connective tissue cells or bloodvessels were, however not co-related.

(c) Drugs In Gingival Fluid:Drugs that are excreted through the gingival fluid may be used advantageously in

periodontal therapy. Bader and Goldhaber were able to show that

intravenouslyadministered tetracycline in dogs rapidly emerges within the sulcus.Ciancio et al in 1976 measured the concentration of tetracycline in blood and gingival

fluid of 5 adult patients with advanced periodontitis, who were given 1g of tetracycline

HCL daily for 2 weeks and 0.5g for 10 weeks. The concentration of the drug ingingivalfluid was only 1/10 of that found in serum.In a second study from the same laboratory the concentrations of the drug were found to

be 5 times higher in samples of gingival fluid as compared to the concentrations in blood

(d) Influence Of Mechanical Stimuli:Chewing and vigorous gingival brushing stimulate the oozing of gingival fluid. Even the

minor stimuli represented by intrasulcular placement of paper strips increase the

production of fluid. Smoking produces as immediate transient but marked increase in thegingival fluid flow.

(e) Periodontal Therapy And Crevicular Fluid:There is an increase in gingival fluid production during the healing period after

periodontal surgery. According to Arnold et al 1966 this increase was probably the result

of the inflammatory reaction from gingival trauma and the loss of an intact epithelial

barrier, especially considering the fact that fluid had been collected by deepintracrevicular technique.

Suppipat et al in 1978 sampled gingival fluid 14, 21, 28 and 35 days after gingivectomy

and found an increase in gingival fluid flow during the first 2 weeks after surgeryfollowed by a gradual decrease. This decrease was same when using mechanical or

chemical plaque control.

RECENT CONCEPTS ON THE MECHANISM OF GINGIVAL FLUID

PRODUCTION:

(i) Effect of Histamine:Brill in 1959 and Egelberg in 1966 showed an increase in gingival fluid production after

intravenous or topical administration of histamine respectively. It is known, however, that

histamine can modify capillary filtration co-efficients, transmural capillary pressure and

transmural osmotic pressure but, for the moment, the relative contribution of each of thethree changes to the overall effect of increased rate of gingival fluid production by

histamine is unknown.

(ii) Preinflammatory Gingival Fluid:Most of the investigators agree that the gingival fluid flow increases several days prior to

detectable clinical inflammation.

(iii) Concentration Of Proteins In Gingival Fluid:Hattingh and Ho in 1980 confirmed that fluid collected from cases of inflamed gingivae


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contains the same concentration of total proteins as serum but indicates also that the

concentration of proteins in the minute amounts of fluid collected in the absence of

clinical inflammation is lower and similar to that of extracellular fluids.

(iv) Passage of fluid and PMNL in the sulcus:The passage of fluid is governed, at least at the initiation of the exudation process by

osmotic gradients, whereas that of cells is governed by chemotactic factors thussuggesting their independent phenomena.

v) Inflammatory Changes Of The Basal Membrane:

During inflammation the basal membrane becomes thinner and even to partiallydisappear. Such ultra structural lesions may decrease the coefficient of filtration of the

junctional epithelium, thus allowing more fluid to enter the sulcus.

(vi) Morphology Of The Junctional Epithelium:According to Pashley the loose organization of the junctional epithelium will also have

an influence on its coefficient of filtration and may explain the relative ease with which

large molecules and even cells can permeate this epithelial covering.

(viii) Mechanical Stimuli And Gingival Fluid:

Pressure sources such as mastication and tooth brushing may cause increase of gingival

fluid production

CONCLUSION:

It is obvious that GCF provides a unique window for analysis of periodontal condition.Collection of GCF is a noninvasive and relatively simple Procedure.The potential of GCF in

early detection Of periodontitis and healing of periodontal tissues Following therapy was already

understood in the 1950s.However, we still do not have a practical and accurate periodontal

indicator based on GCF. Several tests have been developed that areaimed at specifically andsensitively revealing the metabolic status of periodontal tissues. Unfortunately only a handful of

GCF tests have made their way into clinical practice. Many longitudinal studies are required

which will eventually pave the way for thedevelopment of practical GCF indicators that will aidin the accurate diagnosis and appropriate treatment ofperiodontal diseases

References1. Patel PV, Kumar S, Kumar V, Vidya GD. Quantitative cytomorphometric analysis of

exfoliated normal gingival cells. J Cytol 2011;28:66-72.2. Gujjari GK, Gujjari AK, Patel PV, Shubhashini PV. Comparative evaluation of ultraviolet

and microwave techniques for toothbrush decontamination. J Int Soc Prevent Communit

Dent 2011;1:20-6

3. Patel PV, Kumar S, Vidya GD, Patel A, Holmes JC, Kumar V. Cytological Assessment ofHealing Palatal Donor Site wound and Grafted Gingival Wound after Application of

Ozonated Oil: An Eighteen Months Randomized Controlled Clinical Trial. Acta Cytol.

2012;56(3):277-84.

4. Patel PV, Kumar V, Kumar S,GD V, Patel A. Therapeutic effect of topical ozonated oil onthe epithelial healing of palatal wound sites: a planimetrical and cytological study. Journal

of Investigative and Clinical Dentistry 2011;2: 248258.


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5. Patel PV, Kumar GS, Patel A. Periodontal abscess: a review. Journal of Clinical andDiagnostic Research. 2011;5:404-409

6. Patel PV, Shruthi S, Kumar S. Clinical effect of miswak as an adjunct to tooth brushing ongingivitis. J Indian Soc Periodontol 2012;16:84-8.

7. SB Vishwanath, Kumar V, Gujjari S, Sashikumar P, Sashikumar Y, Patel PV, Correlationof periodontal status and bone mineral density in postmenopausal women-a digital

radiographic and quantitative ultrasound study, Indian J Dent Res 2011; 22 (2), 271-276

8. Patel PV, Patel A, Kumar S, Holmes JC. Effect of subgingival application of topicalozonated olive oil in the treatment of chronic periodontitis: a randomized, controlled,

double blind, clinical and microbiological study. Minerva Stomatol 2012;61:381-98.

9. S S, Patel PV, Kumar S. Management of a Persistent Periapical Lesion due toApicomarginal Defect Associated with Root End Fracture in an Endodontically Treated

Tooth: A Clinical Report.J Clin Diagn Res 2012;6:1593-6.

10.Patel PV, Gujjari S, Patel A, Acquired Immune Deficiency Syndrome (AIDS)in PeriodontalPractice: A pactitioners handbook 1

sted. LAP LAMBERT Academic Publishing; Heinrich-

Bocking-Str. 6-8, 661 21 Saarbrucken,Germany: 2012

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