Gingival Crevicular Fluid

1

GCF

Contents

2

IntroductionSulcular fluid

CompositionFormationCollection Gingival crevicular

flowClinical Significance

of gingival crevice fluid

Cellular and humoral activity

Leukocyte in dento-gingival area

Periodontal therapy and crevicular fluid

ConclusionReferences

Introduction

3

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

4

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

Composition

5

use - to detect or diagnose active disease or to predict patients at risk for periodontal disease

1. Acid phosphatase- lysosomal marker- attack nucleic acid & techoic aci(component of bacterial cell wall)- 10- 2o times than serum-

2 sources- mammalian (PMN & desquamating epithelial cells) & bacteria

2. Alkaline phosphatase- osteoblasts, fibroblasts, and neutrophils, plaque bacteria- twice as that of serum- positivecorrelation

3. αl –Antitrypsin- Acute phase protein-proteinase inhibitors

4. Arylsulfatase

Enzymes and other compound

Composition contd…

6

5. Aspartate aminotransferase (glutamate-oxaloacetate transaminase)-present in virtually all cells –

during the development of attachment loss and bone resorption

6. Chondroitin sulfatase7. Citric acid8. Cystatins


7

9. Cytokines (interleukins)

1. IL-lα-2. IL-1 β

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

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

Cytokines

8

4. IL-8 -macrophages and a wide variety of other cells-neutrophil chemotaxis

5. Tumor necrosis factor α-macrophages and T-helper 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 resorption-

juvenile periodontitis contains more PGE2 than GCF from individuals with adult periodontitis


9

10. Endopeptidases:1. Cathepsin D- cysteine proteinases that play

an important role in intracellular protein degradation

2. Cathepsin B/L3. Cathepsin G-serine proteinase 4. Elastase-Neutrophil -serine proteinase

stored in the azurophil granules -higher total NE activity than GCF from healthy or gingivitis sites

5. Plasminogen activators


10

6. Collagenase- neutrophils, macrophages, fibroblasts, keratinocytes, and osteoclasts

7. Tryptasel ike enzyme8. Trypsin like enzyme- P. gingivalis 9. Elastase–α l - proteinase inhibitor10. Exopeptidases11. Fibrin12. Fibronectin-important cell-binding functions during

wound healing, phagocytosis, and cell migration

11

11. β-Glucuronidase- primary granules of neutrophils,- elevated at sites with more severe periodontal disease

12. Myeloperoxidase

13.Dipeptydil peptidase IV–like enzyme- host-derived GCF enzymes - predictors of

the progression of periodontitis


12

11. Glycosidases-host-derived 1. α 1-Fucosidase2. Sialidase3. β -N-acetylglucosaminidase4. β -Galactosidase5. β –Mannosidase

12. Hyaluronidase


13

17.Immunoglobulins (IgG, IgA, IgG4, IgM)

18.Lactate dehydrogenase

19.Lactoferrin-secondary granules of neutrophils

20.Transferrin- serum

21.Lactic acid

14

21. Lysozyme22. α2-Macroglobulins- proteinase inhibitors 23. Medullasin(osteocalcin)24. Thromboxane25. β-defensins - protect the host against

bacterial infiltration at critical sites of potential infection


15

Cellular elements Bacteria Desquamated

epithelial cellsLeukocytes


16

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.- 9.54Sodium – Moderately inflamed gingiva- 137-150 mEq/lSlightly negative corelation of crevicular fluid with

inflammation

Calcium10 mEq/L- normal gingiva15.9mEq/L- moderately inflamed gingiva


17

Organic Compoundcarbohydrates

Glucose hexosamine hexuronic acid compounds

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

total protein content of GCF is much less than that of serum

Average conc.- 6.9g/100mlNo correlation - Bang and Cimasoni(1971)


18

Metabolic productLactic acidHydroxyprolineUreaEndotoxinCytotoxic substance- H2S( bacterial origin)50 % destruction in 6H and 100 % in 48 hrs

Formation of GCF

19

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 epithelium

Formation of GCF

20

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

Formation- inflammatory exudate ?

21

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

Formation- inflammatory exudate ?

22

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

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

Conclusion

23

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

24

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

25

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

GCF as transudate of interstitial fluid

26

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

27

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

GCF as transudate of interstitial fluid

28

Pashley-1976 - jpr

Factors-Filtration

coefficients of the lymphatic and capillary endothelium

Osmotic pressure within the different compartment

Lymph vessel

capillaries

29

GCF protein concentration -inflamed gingivae -similar to that of serum

Most proteins were significantly lower in GCF, but with a strong co-variation between the proteins

Suggesting that GCF represents an inflammatory exudate of serum

consistent with the hypothesis of Alfano-

- Initial –transudte - Later- true exudate

Curtis-jpr 1990-25,6-16

Methods of collection

30

1. Gingival washing methods2. Capillary tubing or micropipettes3. Absorbent filter paper strips

Gingival washing methods

31

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)

32

valuable for harvesting cells from the gingival crevice region

Disadvantage production of customized acrylic stents is

complicated and technically demandingOnly been applied to the maxillary arch•All fluid may not be recovered during the aspiration and re-aspiration procedure

Gingival washing methods

Capillary tubing or micropipettes

33

-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

Capillary tubing or micropipettes

34

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

Absorbent filter paper strips

35

Advantages•quick and easy to use• can be applied to individual sites • least traumatic when correctly used(a)Extracrevicular method

(b) intracrevicular method ‘superficial’ [Löe & Holm Pederson]

(c) intracrevicular method ‘deep’[Brill ]

Methods of estimating the volume collected

36

In many early studies-amount 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

37

1. 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)

accurate determination of the GCF volume n sample composition

Periotron

38

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 the 8000)

Periotron

39

limitations – inability to measure volumes of GCF greater

than 1.0ml

calibration of the Periotron – error between repeat samplingsattributed to - Periotron itself- fluid evaporation- syringe used to dispense replicate volumes- dispensing method

Recommendations for calibration of Periotron

40

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

Association of GCF with health or disease

41

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

Different finding of GCF

42

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)

Problems with GCF collection and data interpretation

43

Contamination- blood, saliva, or plaque-Alpha-amylase

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 determinationless than 1μl and more often than not are less than 0.5

μl

44

Recovery from strips- entrapment within, or binding of GCF proteins to the filter papers

100% -centrifugal elution technique

Data reportingInherent problems of accurate determination

of GCF volume- concentration -not an appropriate method of data presentation

Total amount of enzyme activity - preferred

Gingival crevicular flow

45

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

Methods of measurement

46

Most commonly measured by placing a calibrated filter paper strip at the opening of the gingival crevice or periodontal pocket

Three components

sample volume= sum of the resting volume and theinflux increment (Vr+fi dt)

3 methods

Method 1: Remove resting volume

47

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

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

49

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 state- 10 min

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

50

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

51

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 = 2μg/cm/h * 11.5cm = 23 μ g/h

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

Clinical significance

52

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

53

healthy subjects -GCF flow rates of 3–8 μl/h

Pockets with intermediate periodontal disease have GCF flow - 20 μ l/h

GCF flow at sites (advanced periodontal disease) - 137 μl/h

healthy subjects have resting volumes - 0.06 μl

Pockets with periodontal disease have resting volumes from 0.4 to 1.5 μl


54

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.


55

Flow & sex hormoneFormicola(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 membranes


56

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

57

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

Clinical significance- diabetics

58

In presence of a similar index, no difference in the scores of gingival inflammation and pocket deth- diabetic 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

Clinical significance- diabetics

59

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 concentration of glucose in gingival fluid and serum

Clinical significance – drugs

60

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

Permeability of epithelium

61

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 GCFFat soluble- more rapid rate of entry into

the gingival sulcus of rabbits---Brown-Grant (1962,1966)

Permeability of epithelium

62

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

Passage of substance through pathological or experimentally modified gingival sulcus

63

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


64

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


65

Bacterial collagenase –cannot penetrate thru JE unless pretreated with hyaluronidase ( streptococcal hyaluronidase)

Nutritional deficiency ( ascorbic acid or iron) might alter sulcular permeability- Alfano(1980)

66

Increased

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

Cellular and Humoral Activity in Gingival Crevicular Fluid

67

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-lα) and IL-1β

•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

68

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

69

Interferon-γ - inhibit bone resorption

Antibody presence in GCF – significant role in periodontal disease

Antibody response - protective role


70

predominantly PMNstravel 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

71

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

72

PMNs appeared in the gingival sulcus 20–30min 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

73

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 sites-Murray & Patters-J Periodontal Res 1980:


74

Attracted by different plaque bacteriabut 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

Periodontal therapy and crevicular fluid

75

Scaling and root planing- minimum at 14 days after treatment

-( by 10 factor)

Gingivectomy-- strike increase after 1 wk

Minimum after 5 wkIf oral prophylaxis given prior to surgery-gradual

decrease after 4 wkSuppipat et al(1978)- Loe and Holme technique-

increase in crevicular fluid during first 2 weeks followed by gradual decrease

Summary

76

Experimentally in dog –healthy- few PMN cells migrating

At the beginning – fluid contain low concentration 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

77

1. Jeffrey L. Ebersole. Humoral immune responses in gingival crevice fluid: local and systemic implications Periodontology 2000, Vol. 31, 2003, 135–166

2. Carranza's Clinical Periodontology. Tenth Edition

3. Lindhe’s Clinical Periodontology and oral Implantology. 5th edijtion

4. 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.

5. Polson AM, Goodson JM. Periodontal diagnosis, current status and future needs. J Periodontol 1985: 56: 25–34

6. Gary C. Armitage: periodontal disease:diagnosis Annals : 37-215 :section 1B

78

6. Gareth S. Griffith. Formation, collection and significance of gingival crevice fluid. Periodontology 2000, Vol. 31, 2003, 32–42

7. J. Max Goodson. Gingival crevice fluid flow. Periodontology 2000, Vol. 31, 2003, 43–54

8. Andrew J. delima & thomas E. Van Dyke. Origin and function of the cellular components in gingival crevice fluid. Periodontology 2000, Vol. 31, 2003, 55–76

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)

Gingival Crevicular Fluid

Documents

Transcript of Gingival Crevicular Fluid