GCF

Contents
Introduction Leukocyte in dento-

Sulcular fluid
Composition Formation Collection Gingival crevicular

gingival area Periodontal therapy and crevicular fluid Conclusion References

flow Clinical Significance of gingival crevice fluid Cellular and humoral activity
2

Introduction
Oral tissue is constantly subjected to mechanical

and bacterial aggressions

Saliva, epithelial surface, & initial stages of the

inflammatory response and gingival crevicular fluid resistance

Sulcular fluid
Composition and possible role in oral defence

mechanism-waerhaug, brill and krasse in 1950

passage of fluid from the bloodstream through the

tissues and exiting via the gingival sulcus

Inflammatory exudate, not a continuous

transudate
Strictly normal gingiva -little or no fluid
4

Composition
use - to detect or diagnose active disease or to

predict patients at risk for periodontal disease

Enzymes and other compound

Acid phosphatase- lysosomal marker- attack nucleic acid & techoic aci(component of bacterial cell wall)10- 2o times than serum2 sources- mammalian (PMN & desquamating epithelial cells) & bacteria 2. Alkaline phosphatase- osteoblasts, fibroblasts, and neutrophils, plaque bacteria- twice as that of serumpositivecorrelation
1. 3.
5

l Antitrypsin- Acute phase protein-proteinase inhibitors

Composition contd
5.

Aspartate aminotransferase (glutamateoxaloacetate transaminase)-present in virtually all cells during the development of attachment loss and bone resorption

Chondroitin sulfatase 6. Citric acid 7. Cystatins

5.

Composition contd
9.

Cytokines (interleukins)
1. IL-l2. IL-1

B cells, neutrophils, fibroblasts, and epithelial cells-can stimulate either bone resorption or formation-Chronic periodontitis- IL-1(87%) , IL-1(56%)

3. IL-6 -macrophages, fibroblasts, and endothelial cells-

proinflammatory

Cytokines
4. IL-8 -macrophages and a wide variety of other

cells-neutrophil chemotaxis 5. Tumor necrosis factor -macrophages and Thelper cells- GCF from sites with a gingival index = 0 had higher levels of TNF a than sites with gingivitis 6. Prostaglandin E2 (PGE2)- increase vascular permeability and induce bone resorptionjuvenile periodontitis contains more PGE2 than GCF from individuals with adult periodontitis
8

Composition contd
10. Endopeptidases:
1. Cathepsin D- cysteine proteinases that play an 2. 3.

4.

5.

important role in intracellular protein degradation Cathepsin B/L Cathepsin G-serine proteinase Elastase-Neutrophil -serine proteinase stored in the azurophil granules -higher total NE activity than GCF from healthy or gingivitis sites Plasminogen activators

Composition contd
6. Collagenase- neutrophils, macrophages,

fibroblasts, keratinocytes, and osteoclasts 7. Tryptasel ike enzyme 8. Trypsin like enzyme- P. gingivalis 9. Elastase l - proteinase inhibitor
10. Exopeptidases
11. Fibrin 12. Fibronectin-important cell-binding functions during wound

healing, phagocytosis, and cell migration

10

Composition contd
11. -Glucuronidase- primary granules of neutrophils,-

elevated at sites with more severe periodontal disease

12. Myeloperoxidase

13. Dipeptydil peptidase IVlike enzyme

- host-derived GCF enzymes - predictors of the progression of periodontitis

11

11. Glycosidases-host-derived
1.

1-Fucosidase

2. 3. 4. 5.

Sialidase -N-acetylglucosaminidase -Galactosidase Mannosidase

12. Hyaluronidase

12

Composition contd
17. Immunoglobulins (IgG, IgA, IgG4, IgM) 17. Lactate dehydrogenase

18. Lactoferrin-secondary granules of

neutrophils
19. Transferrin- serum
20. Lactic acid
13

21. Lysozyme 22. 2-Macroglobulins- proteinase inhibitors 23. Medullasin(osteocalcin) 24. Thromboxane 25. -defensins - protect the host against bacterial

infiltration at critical sites of potential infection

14

Composition contd
Cellular elements
Bacteria Desquamated

epithelial cells Leukocytes

15

Composition contd
Electrolytes
Potassium-crevicular exudate greatly exceeds that of

serum . Kaslick et al(1970)- no significant difference between potassium concentration in normal and moderately inflamed samples Mean conc.- 9.54 Sodium Moderately inflamed gingiva- 137-150 mEq/l Slightly negative corelation of crevicular fluid with inflammation
Calcium
16

10 mEq/L- normal gingiva 15.9mEq/L- moderately inflamed gingiva

Composition contd
Organic Compound
carbohydrates Glucose hexosamine hexuronic acid compounds

glucose concentration in GCF is 3-4 times greater than that in serum

Proteins total protein content of GCF is much less than that of serum Average conc.- 6.9g/100ml No correlation - Bang and Cimasoni(1971)
17

Composition contd
Metabolic product Lactic acid Hydroxyproline Urea Endotoxin Cytotoxic substance- H2S( bacterial origin) 50 % destruction in 6H and 100 % in 48 hrs

18

Formation of GCF

Differences between the oral sulcular epithelium and the junctional epithelium
1. size of the cells in the junctional epithelium is, relative to the tissue volume, larger than in the oral epithelium

2. intercellular space -wider than in the oral epithelium

3. number of desmosomes smaller than in the oral 19 epithelium

Formation of GCF
existence of gingival crevice fluid (GCF),

for over 100years(GB black-1899)

Principal questions - whether the initial fluid

produced represents a transudate or is an exudate

Increase in GCF flow -physical protective effects - through flushing the pocket, - facilitating the passage of immunoglobulins
20

Formation- inflammatory exudate ?

initial investigations- inflammatory changes in the

connective tissues underlying the sulcular and junctional epithelia

Increased permeability of the blood vessels,

which was induced by chemical or mechanical means

Brill (1958)-Systemically administered fluorescein

appeared in the GCF collected from healthy gingival crevices in dogs

21

Formation- inflammatory exudate ?

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 Conclusion some irritation, whether chemical or mechanical, was necessary to induce the production of GCF

22

 2 function

-flushing effect of GCF, which was shown to be capable of removing carbon particles and bacteria which had been introduced into the gingival crevice
-transporting antibacterial substances, either of host origin or those introduced into the circulation such as antibiotics

23

1966-Egelberg Experiments -a suspension of carbon particles-

(intravenous)into dogs killed and their gingival tissue was examined histologically
healthy (control) specimens, the carbon particles

remained within the capillaries and small venules

acute inflammation -particles could be seen in the

intercellular spaces
24

 Changes in the capillary permeability of healthy

gingivae -either mechanically by massage of the gingivae with a ball-ended burnisher - chemically by the topical action of histamine

chronically inflamed gingivae - increase in GCF

production in response to -air drying - systemic histamine, whereas - healthy gingivae only occasionally responded to these stimuli
25

GCF as transudate of interstitial fluid

Alfano-1974
clinically healthy gingival crevice

- bacterial plaque would result in the accumulation of high molecular weight molecules
- permeate the intercellular regions of the

epithelium - but would then be limited by the basement membrane

26

2. produce an osmotic gradient which would induce the flow of interstitial fluid from the connective tissue to the gingival sulcus
Phosphate - buffered saline containing 10mg/ml

of homologous serum albumin which resulted in a 100% increase in the volume of GCF produced

27

GCF as transudate of interstitial fluid

Pashley-1976 - jpr

Factors Filtration coefficients of the lymphatic and capillary endothelium Osmotic pressure within the different compartment
28

capillaries

Lymph vessel

 GCF protein concentration -

inflamed gingivae -similar to that of serum Most proteins were significantly lower in GCF, but with a strong covariation between the proteins Suggesting that GCF represents an inflammatory exudate of serum
consistent with the

hypothesis of Alfano- Initial transudte 29 Later- true exudate -

Curtis-jpr 1990-25,6-16

Methods of collection

Gingival washing methods 2. Capillary tubing or micropipettes 3. Absorbent filter paper strips
1.

30

Gingival washing methods

gingival crevice is perfused with an isotonic

solution- Hanks balanced salt solution fluid collected - a dilution of crevicular fluid and contains both cells and soluble constituents such as plasma proteins
instillation and re-aspiration,of 10ml of Hanks balanced salt solution -repeated 12 times (thorough mixing)

31

 valuable for harvesting cells from the gingival

Gingival washing methods

crevice region Disadvantage production of customized acrylic stents is complicated and technically demanding Only been applied to the maxillary arch
All fluid may not be recovered during the aspiration and re-aspiration procedure

32

Capillary tubing or micropipettes

-isolation and drying of a site -capillary tubes of known internal diameter are inserted into the entrance of the gingival crevice

provides an undiluted sample of native GCF - volume can be accurately assessed

33

Capillary tubing or micropipettes

difficult to collect an adequate volume of GCF

in a short period, unless the sites are inflamed and contain large volumes of GCF
difficulty of removing the complete sample

from the tubing

34

Absorbent filter paper strips Advantages

quick and easy to use can be applied to individual sites least traumatic when correctly used (a) Extracrevicular method (b) intracrevicular method superficial [Le & Holm Pederson] (c) intracrevicular method deep [Brill ]
35

Methods of estimating the volume collected

In many early studiesamount of GCF collected on a strip was assessed by the distance the fluid had migrated up the strip

Further accuracy staining the strips with ninhydrin to produce a purple color in the area where GCF had accumulated

A similar result- 2g fluorescein given systemically to each patient 2hours prior to the collection of GCF - following which the strips were examined under ultraviolet light

fluorescein labeling- 100 x more sensitive than ninhydrin for staining protein
36

Inevitable delay in measuring the strip variation in the reported volume evaporation 2. Staining of the strips for protein labeling prevents further laboratory investigations of the components of GCF (limiting to volume determination only) electronic measuring device ( Periotron)
1.

accurate determination of the GCF volume n sample composition

37

Periotron
2 metal jaws - act as the plates of an electrical

condenser
If a dry strip is placed between the jaws

- capacitance is translated via the electrical circuitry and registers zero on the digital readout
wet strip -increased value in the readout Three models of PeriotronA -(the 600, 6000 and now
38

the 8000)

Periotron
limitations inability to measure volumes of GCF greater than 1.0ml calibration of the Periotron error between repeat samplings attributed to - Periotron itself - fluid evaporation - syringe used to dispense replicate volumes - dispensing method
39

Recommendations for calibration of Periotron

each machine needs its own calibration serum - suitable material to generate a calibration

curve
an accurate syringe and some form of

standardization of dispensing duplicate volumes essential

a full range of volumes from 0.1 to 1.2ml is

necessary to generate an accurate curve

40

Association of GCF with health or disease

Initial experiments in healthy crevices intracrevicular sampling technique (inserting strips until resistance is felt) and yielded -detectable levels of GCF
If filter paper strips - placed just at the entrance to

the gingival crevice GCF - seldom detected

The association of an increased volume of GCF

with an increase in the severity of inflammation

41

Different finding of GCF

in the flow of GCF- together with an tendency to bleed -earliest signs of inflammation of the gingivae GCF volumes -sign of subclinical inflammation

histological observations gingival connective tissue, totally free of inflammatory cells, probably does not exist (nor can it be achieved)
42

Problems with GCF collection and data interpretation

Contamination- blood, saliva, or plaque-Alphaamylase Sampling time-5 seconds-

risk of going off-scale, protein concentrations approaching those of serum Gel electrophoresis -electrophoretic bands in the early samples of GCF
Volume determination less than 1l and more often than not are less than 0.5 l 43

Recovery from strips- entrapment within, or binding of GCF proteins to the filter papers 100% -centrifugal elution technique

Data reporting Inherent problems of accurate determination of GCF volume- concentration -not an appropriate method of data presentation Total amount of enzyme activity - preferred

44

Gingival crevicular flow

flushing action and an isolation effect- determines

the growth level of subgingival microorganisms - potential marker for periodontal disease activity
Charcoal particles and even bacteria rapidly

removed from periodontal pocket

GCF - blood ultrafiltrate but accumulates

elements of the metabolism from both bacterial and host cells

45

Methods of measurement
Most commonly measured by placing a calibrated

filter paper strip at the opening of the gingival crevice or periodontal pocket
sample volume= sum of the resting volume and the influx increment (Vr+fi dt) 3 methods

Three component
46

Method 1: Remove resting volume

Filter paper strips - placed for 20s and

25s was allowed before the next 20-s sample was

taken. Entire sequence - repeated five times for each subject visit

47

48

Method 2: Measure combined resting volume and influx for varying time periods

resting volume -constant

and that a sufficiently long time between measurements has been allowed to elapse so that the pocket or sulcus volume will have returned to a steady state10 min
49

Method 3: Measure the equilibrium concentration of a marker substance pumped into a pocket at a constant rate
pumping a marker substance into a periodontal

pocket at a constant rate, an equilibrium concentration will be established-result of the fluid flow rate and the pump delivery rate
tetracycline fiber
establishes and maintains the constant concentration

50

 equilibrium concentration (Ce) of approximately

1590mg/ml=1.6mg/ml for 10 days

23cm-long fiber is sufficient to treat

approximately two teeth

Rate of tetracycline delivery: dq/dt = 2g/cm/h * 11.5cm = 23 g/h

GCF flow rate is: Fi=[(dq/dt)/Ce]=[(23 g/h)/(1.6 g/ml)] =14.4ml/h

51

Clinical significance
Following periodontal therapy by scaling and root planing with tetracycline fiber placement

52

 healthy subjects -GCF flow rates of 38 l/h

Pockets with intermediate periodontal disease

have GCF flow - 20 l/h

GCF flow at sites (advanced periodontal

disease) - 137 l/h

healthy subjects have resting volumes - 0.06 l
Pockets with periodontal disease have resting

volumes from 0.4 to 1.5 l

53

Clinical significance
Circadian periodocity
Deep intracrevicular technique- average flow was

greater in the evening and minimal early in the morning

Orifice technique no systematic differences

between the flow of fluid measured at 9 a.m. and 3 p.m.

54

Clinical significance

Flow & sex hormone

Formicola(1970)-

estrdiol injected subcutaneously will accumulate in gingival tissue at levels higher than those found in the uterus

El Attar (1971)- progesterone and estrogen are

metabolized in vitro in normal and inflamed human gingiva

Hormone - decrease the stability of lysosomal
55

membranes

Clinical significance
In pregnancy- excessive amount of lysosomal

enzymes could be released in gingival tissue and could make it more vulnerable to bacterial aggression
Lindhe et al(1971)- female sex hormone cause

increase in gingival vascular permeability

Lindhe & Bjorn(1967)- gradual & statistical

56

signifcant increase in amount of exudation was recorded in women receiving pills compared to control

 Hugoson (1970)- pregnancy did not influence

gingiva which were originally healthy Parallel to GI score, Gingival exudate reached maximum value during last trimester and decreased to minimum 20 wks after delivary
Holm-pederson and Loe-

previously instructed in oral hygiene and given initial prophylaxis -no differences in the mean flow of exudate during pregnancy as compared to mean found post partum after cessation of the lactation period
57

Clinical significance- diabetics

In presence of a similar index, no difference in the

scores of gingival inflammation and pocket dethdiabetic and control

Increase in width of basement membrane of

capillaries, small arteries and venules, splitting of this basement membrane with an increased intendity in its staining

58

Clinical significance- diabetics

Hara and Loe (1969) exudate- 6 times

than serum
Kjellman(1970)- lower in gingival fluid

compared to serum( healthy gingiva)healthy and diabetic patients

Ficara et. Al (1975) similar
59

concentration of glucose in gingival fluid and serum

Clinical significance drugs

Di iodofluorescein- more rapid entry than

electrolyte and albumin- fat soluble

Tetracyclin iv- rapidly emerges within sulcus of

dog Cancio et al(1976)- 2 wks after drug administration- 1/10 of that found in serum Minocyclin- 5 times higher than serum
Stephen(1985)- ampcillin, cephlexin, tetracyclin,

erythromycin, clindamycin, rifampicin lower than serum and greater than saliva
60

Permeability of epithelium
intercellular spaces -18 % volume of JE and 12 %

of sulcular epithelium Degree of permeability of oral mucosa doesnot seem to depend on degree of keratinization
Passage from connective tissue into the sulcus Brill and Krasse with Na fluoresceinplasma Plasma protein- present in GCF Fat soluble- more rapid rate of entry into the gingival sulcus of rabbits---Brown-Grant (1962,1966)
61

Permeability of epithelium
Passage from sulcus into connective tissue Substance with molecular weight 111(histamin)- 200000 (dextran)
relative barrier to the penetration of

foreign materials from sulcus into the CT

62

Passage of substance through pathological or experimentally modified gingival sulcus

Thilander(1964) -permeability of skin

and mucous membrane altered by chemical stimulation

Inflammation- enlargement of

intecellular space of junctional epithelium and thinning and partial destruction of basal lamina
63

Passage of substance through pathological or experimentally modified gingival sulcus

Fine & Stuchell (1977) passage of fluorescent

latex particles of 0.75mm from gingival sulcus into CT is greater in highly inflamed areas
Stallard and Awwa(1969) application of

hyalurinidase & collagenase on the marginal region of monkeys- allows penetration of foreign material( tryptan blue) into the CT

64

Passage of substance through pathological or experimentally modified gingival sulcus

Bacterial collagenase cannot penetrate thru JE

unless pretreated with hyaluronidase ( streptococcal hyaluronidase)

Nutritional deficiency ( ascorbic acid or iron) might

alter sulcular permeability- Alfano(1980)

65

Increased
by mastication of coarse foods, toothbrushing gingival massage ovulation hormonal contraceptives smoking

66

Cellular and Humoral Activity in Gingival Crevicular Fluid

cellular immune response includes the appearance of cytokines in GCF but there is no clear evidence of a relationship between cytokines and disease Interleukin-1 alpha (IL-l) and IL-1
Increase the binding of PMNs and monocytes/macrophages to endothelial cells, Stimulate the production of prostaglandin E2 (PGE2) and release of lysosomal enzymes, and stimulate bone resorption
67

 significant local elevations in immunoglobulins

occur in periodontitis resulting from extensive local production -low levels in GCF from healthy sites
plasma cells - dominated by IgG cells,

- followed by IgA cells; no IgM cells are found in tissues from patients with aggressive periodontitis

68

 Interferon- - inhibit bone resorption Antibody presence in GCF significant role in

periodontal disease
Antibody response - protective role

69

Leukocyte in dento-gingival area

predominantly PMNs

travel across the epithelium'"' to the gingival

sulcus, where they are expelled

91.2% to 91.5% -PMNs 8.5% to 8.8'% - mononuclear cells

Ratio of T lymphocytes to B lymphocytes -normal - 3:1 found in peripheral blood -1:3 in GCF
70

 Leukocytes - detected in both clinically healthy

and diseased tissues of experimental animals and humans

Under inflamed conditions, 60% or more of the

junctional epithelium space can be occupied by neutrophils

major route of entry into the oral cavity for these

cells was via the gingival sulcus

71

 PMNs appeared in the gingival sulcus 2030min

after being present in the blood and Scully noted a peak neutrophil concentration within the sulcus after 1h
approximately 80% of crevicular PMNs, obtained

from the first two sulcular washings, remain functional, while 99% of the cells from the final two washings were still viable
72

 Aggressive periodontitis-

dysfunction might be a localized phenomenon because in those patients with aggressive periodontitis, the decreased phagocytosis was isolated to the diseased site, while healthy sites in the same individuals showed normal neutrophil function Interestingly, no differences were noted in the number of neutrophils recovered from all sitesMurray & Patters-J Periodontal Res 1980:
73

Leukocyte in dento-gingival area

Attracted by different plaque bacteria but can also be found in the dentogingival region

of germfree adult animals.

migration may be independent of an increase i n

vascular permeability viable and have phagocytic and killing capacity protective mechanism against the extension of plaque into the gingival sulcus main port of entry of leukocytes in to the oral cavity is the gingival sulcus
74

Periodontal therapy and crevicular fluid

Scaling and root planing- minimum at 14 days after

treatment -( by 10 factor)
Gingivectomy- strike increase after 1 wk Minimum after 5 wk

75

If oral prophylaxis given prior to surgery-gradual decrease after 4 wk Suppipat et al(1978)- Loe and Holme techniqueincrease in crevicular fluid during first 2 weeks followed by gradual decrease

Summary
Experimentally in dog healthy- few PMN cells

migrating
At the beginning fluid contain low concentration

76

of protein representing interstitial fluid Latter stage- inflammatory exudate containing higher amount of total protein Permeability of junction epithelium and sulcular epithelium depend degree of inflammation More than 90% of leukocyte- PMN ( some of them r viable and posses capcity to phagocytose) O. 5 to 2.4 ml / day

References
1.

2. 3.

4.

5. 6.
77

Jeffrey L. Ebersole. Humoral immune responses in gingival crevice fluid: local and systemic implications Periodontology 2000, Vol. 31, 2003, 135166 Carranza's Clinical Periodontology. Tenth Edition Lindhes Clinical Periodontology and oral Implantology. 5th edijtion A. Refaie, O. Anuksaksathiem, G. Singh, J. Moran, A.E. Dolby.Antibody to Collagen Type I in Gingival Crevicular Fluid. J Periodontol 1990;60:289-292. Polson AM, Goodson JM. Periodontal diagnosis, current status and future needs. J Periodontol 1985: 56: 2534 Gary C. Armitage: periodontal disease:diagnosis Annals : 37-215 :section 1B

Gareth S. Griffith. Formation, collection and significance of gingival crevice fluid. Periodontology 2000, Vol. 31, 2003, 3242 7. J. Max Goodson. Gingival crevice fluid flow. Periodontology 2000, Vol. 31, 2003, 4354 8. Andrew J. delima & thomas E. Van Dyke. Origin and function of the cellular components in gingival crevice fluid. Periodontology 2000, Vol. 31, 2003, 5576 9. G. cimasoni. Crevicular fluid updated. 1983 10. Barry M. Eley and Stephen W. Cox.Cathepsin B/L-, Elastase-, Tryptase-, Tlypsin- and Dipeptidyl Peptidase IV-Like Activities in Gingival Crevicular Fluid: A Comparison of Levels Before and After Periodontal Surgery in Chronic Periodontitis Patients. Journal of Periodontology 1992 May (412 - 417)
6.
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