GINGIVAL CREVICULAR

FLUID

CONTENTS Introduction History Gingival vasculature and permeability Mechanisms of GCF production Assessment of GCF Composition GCF as a diagnostic marker Analysis of components Commercial diagnostic kits Clinical significance Conclusion References

INTRODUCTIONComplex mixture of substances

Serum

WBCs

Cells

Bacteria

HISTORYSince 50 years….

RESEARCHERS STUDIES

Waerhaug (1952) Sulcus pocket

Brill et al (1962) Physiology and composition

Löe and Holm-Pederson (1965)

Indicator of periodontal diseases

Egelberg (1966) Gingival vasculature and permeability

Schroeder (1969), Listgarten (1966)

Dentogingival structure

Sueda, Bang and Cimasoni (1969)

Presence and functions of proteins in GCF

Ohlsson (1973), Golub(1976) & Uitto(1978)

Collagenase & Elastase in GCF & its co-relation with inflammation.

Gingival vasculature & permeability

Gingival vasculatureCapillary units

Inflammation - looping

Network below epithelium

Egelberg 1966 Arranged in a flat layer Superficial position Diameter > 7 m

Egelberg 1966 – slight irritation of the sulcular area

Increase in vascular permeability

“Vascular labeling”1. Carbon particles2. Histamine3. Ball-ended plugger4. Blunted explorer

Injected carbon particles into

dogs

Healthy samples - particles remained

within the capillaries

Acute inflammation - particles seen in the intercellular spaces

Brill and Krasse 1958 Sodium fluorescein Evans blue, India ink and saccharated

iron oxide

Substances penetrating the sulcular epithelium M. Wt. <1000 kDAlbumin, Thymidine,

Histamine, Phenytoin, Endotoxin

Mechanisms of GCF production

Existence of GCF - over 100 years (Black GV 1899)

Subject of controversy

Transudate Inflammatory

exudate

CONCEPTS OF GCF PRODUCTION

Brill & Krasse 1958, Brill & Bjorn 1959

and Egelberg 1966 Production of fluid

is related to an inflammatory permeability

of vessels underlying the sulcular & junctional

epithelium.

Alfano’s hypothesis (1974)

GCF is a pre-inflammatory fluid, which is

osmotically mediated.

In healthy gingiva….

Increase accumulation of macromolecules , they

will diffuse intercellularly to basement membrane,

an osmotic gradient is created and flow of gingival

fluid is generated

This is not an inflammatory exudate, but may

progress to a secondary inflammatory exudate.

Pashley’s hypothesis (1976) Mathematical model based on Starling factors

Gingival fluid production is modulated by…capillary filtration & lymphatic uptake.

Capillary fluid > Lymphatic uptake oedema/GCF

More fluid gingival tissue compliance low.

In health, Oncotic pressure sulcular compartment >

interstitial fluid, net produc of ging fluid will increase

In inflammation,

Oncotic pressure in sulcular = tissue

compartment Protein content in GCF =

serum

This cancels their role in fluid production

(Bang & Cimasoni 1971)

In inflammation, capillary pressure more than

osmotic gradient determines fluid production,

thereby supporting Alfano’s hypothesis.

Assessment of GCF

METHODS USED FOR COLLECTION

ESTIMATION OF THE SAMPLE

VOLUME OF GCF

PROBLEMS ASSOCIATED WITH COLLECTION

METHODS USED FOR COLLECTION

1. Absorbing paper strips

2. Pre-weighed twisted threads

3. Capillary tubes / Micropipettes

4. Gingival washings

5. Other methods

Absorbing paper strips

Whatman No. 1 and Munktell No. 3

1.5 mm wide

1. Intrasulcular method

Brill’s technique, 1962

Löe and Holm-Pederson technique, 1965

2. Extrasulcular method

3. Modified method

Rudin et al 1970

Valazza et al 1972

Advantages

Pre-weighed twisted threads(Weinstein et al 1967)

Capillary tubes / Micropipettes (Krause and Egelberg, 1962) Advantages - provides undiluted sample- ‘native’ GCF

Disadvantages Collection of fluid Obtaining sample

Gingival washings

Method given by Skapski and Lehner, 1976

Hamilton’s microsyringe

10 l of Hanks balanced solution

Takamori & Oppenheim method, 1970

Acrylic plate to cover maxilla

Groove for plastic tubes

4-6 ml solution – 15 min – peristaltic pump

Disadvantages

Modified method Ejection needle Collection needle

Other methods

Platinum loops

Microspatules

Transparent / Plastic strips

ESTIMATION OF THE SAMPLE

1. Appreciation by direct viewing & staining

2. Weighing the strip

3. Periotron

Appreciation by direct viewing & staining

1. Staining with 0.2 – 2% Ninhydrin. • Transparent ruler (Egelberg 1964)

• Sliding caliper (Bjorn et al 1965)

• Calibrated magnifying glasses (Oliver et al

1969)

• Microscope with an eye piece graticule

(Wilson et al 1971)

• Photometric planimetric technique (Farsam

et al 1977)

• Specially designed inexpensive paper strip

viewer (Wilson et al 1978)

2. 2gm of sodium fluorescein ( Weinstein et al, 1967)

Disadvantages

Weighing of the strip

Weinstein et al 1967…pre-weighed twisted

threads

Cimasoni et al…pre-weighed paper strips in

sealed micro centrifugation plastic tubes.

Periotron Developed by Harco electronics: “HAR 600

Gingival Crevice Fluid Meter”

One jaw has +ve charge & another –ve

charge……kept apart by dry insulating paper

strip

Digital read out

Advantages

Disadvantages

1. Vol > 1 µl unable to measure.

2. Position of the strip influences readings.

VOLUME OF GCF COLLECTED

CHALLACOMBE(1980)- Isotope dilution

method-In anterior( 0.24- 0.43 μl)-

posterior( 0.43- 1.56 μl)

CHALLACOMBE(1980)-suggested the total

volume GCF secreted in the mouth per day-

0.5 -2.4 ml fluid per day.

PROBLEMS ASSOCIATED WITH COLLECTION

Contamination Sampling time

Volume determination

Recovery from strips

Data reporting

Contamination Blood Saliva Plaque

Sampling time

Early literature suggests 5 seconds

Adequate volume : 20–30 min

Longer periods – increase in volume

Longer periods – change in nature of fluid

Volume determination

Scarcity of material : 0.5 – 1 l

Evaporation

Percentage of error

Use of vasoconstrictors (Hakkarainen and

Ainamo 1981)

Recovery from strips

Data reporting

Earlier work – concentration & total enzyme activity

Lately - total amount of enzyme activity

Absolute amount (mg)

Concentration (mg/ml)

Composition CELLULAR ELEMENTS

ELECTROLYTES

ORGANIC COMPOUNDS

METABOLIC AND BACTERIAL PRODUCTS

ENZYMES AND ENZYME INHIBITORS

CELLULAR ELEMENTSEpithelial cells, Leukocytes and bacteria

Epithelial cells - Lange and Schroeder 1971 Cells of the sulcular epithelium

Flattened Cytoplasmic filaments

Cells originating from the junctional epithelium Found at the bottom of the sulcus Coronal to the sulcus bottom

Role of inflammation

Rate of renewal

Structural characteristics of desquamating

cells

reduction in acid phosphatase activity

( Cornaz et al 1974)Increased

permeability of lysosomal

membranes

Progressive degeneration of

the cellular components

Leukocytes Differential leukocyte count in the sulcus

GCFPeripheral

blood

Neutrophil 95 – 97 % 60%

Monocyte 2-3% 5-10%

Lymphocyte 1 – 2 % 20-30%

T cells 24% 50-75%

B cells 58% 15-30%

Mononuclear phagocyte

18%

T : B 1 : 2.7 3 : 1

Role of inflammation - in number( Egelberg 1963)

Phagocytic function of PMNsAgP < CP

Bacteria

Poor correlation to severity of gingival

inflammation and depth of pocket (Krekeler

and Ferck, 1977)

ELECTROLYTES

Sodium Normal GCF - 158 mEq/l

Inflammation - 207 to 222 mEq/l

Follows circadian periodicity( Kaslick et al 1970)

pocket depth Na

Potassium Mean concentration in GCF - 9.54 mEq/l

GCF > serum

Increases towards the middle of the day

severity of periodontitis pocket depth

2x

Sodium : Potassium Ratio Diseased tissues ratio

Accumulation of intracellular potassium

GCF < ECF ( Krasse & Egelberg, 1962)3.9 28:1

Other ions Fluoride : GCF = Plasma( Whitford et al,1981)

Calcium : Normal gingiva – 10mEq/l Inflammed gingiva – 15.9 mEq/l with inflammation GCF (30-50 x) > Serum (Biswas et

al,1977)

iPO4 : 4.2 mg / 100ml of GCF

Mg : 0.8mEq/L

I : 40% of the concentration in saliva

ORGANIC COMPOUNDS

Carbohydrates Glucose, hexosamine and hexuronic acid (Hara

and Loe 1969)

Glucose : GCF > serum

Hexosamine & Hexuronic acid – no correlation with variation in gingival inflammation

Increased in: Inflammation Diabetes

3-6x

Proteins GCF < serum

IgG, IgA - plasma cells

Complements tissue damage(Schenkein & Genco,1977) Chemotactic attraction of PMNs Release of lysosomal enzymes Degranulation of mast cells

Albumin , fibrinogen, ceruloplasmin, ß-lipoproteins &

transferrin ( Mann & Stoffer, 1964)

Bradykinin (Rodin et al 1973)

Lipids

Serum, saliva, bacteria and host tissue

Phospholipids and neutral lipids

METABOLIC AND BACTERIAL PRODUCTS Lactic acid – inflammation, flow

Hydroxyproline

Prostaglandins

Urea and pH Inversely related to severity of

inflammation GCF > saliva, serum pH: 7.54 - 7.89 (Bang and Cimasoni)

Endotoxins

Lipopolysaccharides(LPS) of cell wall of gm-ve

bacteria released from autolysing bacteria cells

Highly toxic to gingival tissues & possible

pathogenic factor in periodontal disease.

Shapiro 1972…+ve correlation b/w LPS conc. &

ging inflam

Cytotoxic substances - H2S

Antibacterial factors

Crevicular fluid was found to be as potent as leukocyte

extract in lysing Staph aureus, Strep faecalis & A.

viscosus. Strep. Mutans seemed more resistant.

Sela et al 1980….lytic agents are the lysosomal

enzymes present in GCF

A peroxidase mediated antimicrobial system has also

been shown in human crevicular fluid

Growth stimulating factors – Lactobacilli

( Takamori,1963)

ENZYMES AND ENZYME INHIBITORS

Acid phosphatase Lysosomal enzyme Present in azurophil granules Sources : PMNs, desquamating epithelial

cells Associated with connective tissue

catabolism Can also attack teichoic acid Acts at a pH of 4 to 5 Poor correlation with periodontal disease

(Cimasoni, 1983)

Alkaline phosphatase Sources : PMNs, bacteria Plays a role in calcification

Pyrophosphatase Role in calculus formation Conc. Is positively correlated – amount of

calculus

– glucoronidase

Lysozomal enzyme

Primary granules of PMNs

Sources : macrophages, fibroblasts, endothelial cells, bacteria

Plays a role in the catabolism of mucopolysaccharides

Lysozyme Major source : PMNs

Bactericidal - hydrolyzes β-1,4–glycosidic bonds of peptidoglycans of bacterial cell wall

Activity : GCF, saliva > serum( Brandtzaeg & Mann,1964)

May contribute to the formation of pocket

Accelerates release of bacterial enzymes(Sela ,1976)

Hyaluronidase

Lysosomal enzyme

Splits β-1,4–N– acetylglucosaminide links in hyaluronic acid and chondroitin sulphate

pH : 3.5 – 4.1

Increases – Gram positive bacteria, inflammation (Tynelius- Brathall & Attstrom,1972)

Lactate dehydrogenase

Pyruvate Lactate

GCF > blood

LDH

10-20x

Proteolytic enzymes

Mammalian proteinases

Bacterial proteinases

Proteinase inhibitors

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GINGIVAL CREVICULAR

FLUID

CONTENTS Introduction History Gingival vasculature and permeability Mechanisms of GCF production Assessment of GCF Composition GCF as a diagnostic marker Analysis of components Commercial diagnostic kits Clinical significance Conclusion References

GCF as a diagnostic marker

BACTERIA AND THEIR PRODUCTS

INFLAMMATORY AND IMMUNE PRODUCTS

ENZYMES RELEASED FROM DEAD CELLS

CONNECTIVE TISSUE DEGRADATION PRODUCTS

PRODUCTS OF BONE RESORPTION

BACTERIA AND THEIR PRODUCTS

Bacterial proteases Trypsin-like protease

Arg-gingipain/Arg-gingivain Excellent predictor (Eley & Cox,1996)

Dipeptidylpeptidase (DPP) Good predictor of future progressive

attachment loss

INFLAMMATORY AND IMMUNE PRODUCTS

Immunoglobulin Total Ig correlates with adjacent gingival tissue Does not correlate with disease severity

(Lamster 1992, Page 1992) Reduction in specific antibody risk for disease

(AgP, ANUG) (Lamster et al,1992)

Correlation may exist between IgG levels to P. gingivalis & severity perio disease(Gmur et al 1986)

IgG2 levels recurrent or persistent destruction

Cytokines Interleukins - relevance to periodontal

pathology

bacteria inflammation IL-8 PMN elastase secretion

IL-1α and β Do not correlate with probing pocket depths

IL-1 Associated with progressive attachment loss

IL-2, IL-6 Might predict and associate with progressive attachment loss

IL-6 Produced more at refractory sites

IL-8 Reduce significantly after treatment

Prostaglandins (PGE2)

Health - low, undetectable Naturally occurring gingivitis - ~ 32 ng/ml Experimental gingivitis - ~ 53 ng/ml Periodontal disease activity - > 66 ng/ml

(Offenbacher et al 1986)

HYDROLYTIC ENZYMES RELEASED FROM DEAD CELLS

Enzymes Degrade phagocytosed

material

Degrade gingival tissue

Proteolytic enzymes

•Collagenase•Elastase•Cathepsin G •Cathepsin B•Cathepsin D•Tryptase•DPP II & IV

Hydrolytic enzymes

•Aryl sulphatase• – glucuronidase•Alkaline phosphatase•Acid phosphatase•Myloperoxidase•Lysozyme•Lactoferrin

Collagenase

Gingivitis - correlate with severity of inflammation

Human periodontitis - increase with increasing clinical features (Golub et al,1976)

Untreated chronic periodontitis : MMP-8, -9 (Ingman et al, 1996; Makela et al, 1994)

Chen et al 2000 - MMP-8 levels reduce following therapy, also decrease in inhibitor levels

-glucuronidase

Positively associated with Spirochaetes P. gingivalis P. intermedia Lactose-negative black pigmenting

bacteria

Negative with cocci (Lamster 1992)

Alkaline phosphatase

Cross sectional study – correlation with

pocket depth but not with bone loss

( Ishikawa & Cimasoni,1970)

Active sites > serum (Binder et al 1987) ,

associated with periodontal disease activity

20x

Lysozyme in chronic periodontitis ( Markannen et

al,1986) in AgP patients

Pseudocholinesterase Serum > GCF > saliva Significantly higher in AgP

Levels significantly correlate with disease severity and reduce following treatment

Cysteine proteinases - Cathepsins B and L

Aspartate proteinases - Cathepsin D Serine proteinases - Elastase, tryptase DPP II and IV -glucuronidase Aryl sulphatase Myeloperoxidase Lactoferrin Pseudocholinesterase

CYTOSOLIC ENZYMES RELEASED FROM DEAD CELLS

Aspartate transaminase Marker of tissue necrosis and cell death

GCF AST - correlate with clinical indices of disease severity (Imrey et al 1991)

Longitudinal studies confirmed attachment loss (Persson et al 1990, Chambers et al 1991)

No evidence that it can be a predictor for disease severity / activity

Lactate dehydrogenase

Cross-sectional studies - Correlated with PPD and other clinical indices

Longitudinal study - disease activity

CONNECTIVE TISSUE DEGRADATION PRODUCTS

GAGs Hyaluronic acid – chronic gingivitis

Chondroitin-4-sulphate Untreated advanced periodontitis AgP

Sulphated GAGs Teeth undergoing orthodontic movement Teeth subject to occlusal trauma Healing extraction wounds

PRODUCTS OF BONE RESORPTION

Osteonectin & Bone phosphoproteins

Increase with site probing depth (Bowers et al 1989)

May be associated with disease severity

No longitudinal studies done

Osteocalcin

Possible marker for bone resorption and disease progression

Kunimatsu et al 1993 – first to study

Moderate predictive value for future bone loss as measured by radiography

Cross-linked carboxyterminal telopeptides of type 1 collagen

Elevated CTP coincides with bone resorptive rate (Eriksen et al, 1993)

Has been detected in GCF in periodontitis patients as well as experimental periodontitis in dogs

Analysis of componentsTESTS

COMPONENTS ANALYSED

Fluorometry MMPs

ELISA Enzyme levels and IL-1

RIA COX derivatives and procollagen III

HPLC Timidazole

Direct/indirect immunodot tests Acute phase proteins

Commercial diagnostic kitsPERIOCHECK

Detects collagenase

Paper strip + (collagen gel blue colour on + blue dye) strip

Intensity proportional to amount of enzyme present

43C

PROGNOSTIK

Detects elastase

Paper strip + 7 Aminotrifluoro methylcoumarin( AFC)

Substrate (MeOSuc-Ala-Ala-Pro-Val-AFC) Detects elastase Linked to AFC

If elastase is present 4 – 8 mins → releases AFC → green fluorescence

Intensity proportional to amount of enzyme present

PERIOGARD

To detect AST (Persson et al 1995)

Uses paper point GCF samples

Strip placed in suitable wells 2 drops of reagent + 2 drops of a solution

After 9 mins, substrate / detection solution mixed

After 10 mins, results - colorimetric detection

Potential diagnostic tests

For PGE2

Nakashima et al, 1994 - ELISA assay utilizing a monoclonal rabbit anti-PGE2 antibody to assay GCF PGE2

For osteocalcinCan be assayed using polyclonal or

monoclonal antibodies by an ELISA or RIA.

For β-glucoronidase A diagnostic kit based on GCF -

glucuronidase is being commercially developed by Abbott Laboratories, North Chicago, USA.

Based on colour detection systems

For cysteine and serine proteinase Based on colour detection systems

Clinical significance

Circadian Periodicity

Mechanical Stimulation

Inflammation

Smoking

Sex Hormones vascular permeability flow

Diabetic patient High flow rate (Ringelberg et al 1977) More glucose

Periodontal Therapy GCF during healing period after surgery

After gingivectomy: 1st wk - At 5 wks – preoperative

levels

After first flap procedure : 4 weeks later, levels lesser than preoperative

following SRP and curettage1 week after SRP: After second SRP: lower values are sustained (Gwinnett 1978)

Drugs in GCF Advantageous in therapy

Tetracyclines (Bader and Goldhaber, 1966) 1/10th conc. in GCF compared to serum

Minocycline (Ciancio et al 1980) GCF > blood

Metronidazole (Eisenberg 1991)

5x

Conclusion

Monitoring periodontal disease – complicated

task.

Analysis GCF constituents- extremely useful-

simplicity & non invasive.

Thorough knowledge- Better aid for

diagnosis.

Newman, Takei, Klokkevold, Carranza. 10th edition. Carranza’s Clinical Periodontology. W. B. Saunders Company.

Velli-Jukka Uitto. Gingival crevicular fluid. Periodontology 2000 2003, Vol. 31.

G. Cimasoni. Volume 12. Monographs in Oral Science - Crevicular Fluid Updated. S. Karger.

BM Eley, JD Manson. 5th edition 2004. Periodontics. Wright Publishers.

Bartold PM, Narayanan AS. Periodontal connective tissues. Quintessence books.

References

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