This document discusses gingival crevicular fluid (GCF), including its history, mechanisms of production, assessment, and composition. GCF forms as a transudate or inflammatory exudate from gingival vasculature. It contains cellular elements, electrolytes, proteins, enzymes, and metabolic/bacterial products. Enzymes in GCF like acid phosphatase are associated with connective tissue breakdown. The composition of GCF allows it to serve as a diagnostic marker for periodontal disease.
4. HISTORY
Since 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.
5. Gingival vasculature & permeability
Gingival vasculature
Capillary units
Inflammation - looping
Network below epithelium
Egelberg 1966
Arranged in a flat layer
Superficial position
Diameter > 7 m
6. Egelberg 1966 – slight irritation of the sulcular area
Increase in vascular permeability
“Vascular labeling”
1. Carbon particles
2. Histamine
3. Ball-ended plugger
4. Blunted explorer
Injected carbon
particles into dogs
Healthy samples - particles
remained within the
capillaries
Acute inflammation -
particles seen in the
intercellular spaces
7. Brill and Krasse 1958
Sodium fluorescein
Evans blue, India ink and saccharated iron oxide
Substances penetrating the sulcular epithelium
M. Wt. <1000 kD
Albumin, Thymidine,
Histamine, Phenytoin,
Endotoxin
8. Mechanisms of GCF production
Existence of GCF - over 100 years (Black GV 1899)
Subject of controversy
Transudate Inflammatory
exudate
9. 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.
10. 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.
11. 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
12. 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.
13. Assessment of GCF
METHODS USED FOR COLLECTION
ESTIMATION OF THE SAMPLE
VOLUME OF GCF
PROBLEMS ASSOCIATED WITH COLLECTION
14. METHODS USED FOR COLLECTION
1. Absorbing paper strips
2. Pre-weighed twisted threads
3. Capillary tubes / Micropipettes
4. Gingival washings
5. Other methods
15. 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
22. ESTIMATION OF THE SAMPLE
1. Appreciation by direct viewing & staining
2. Weighing the strip
3. Periotron
23. 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)
24. 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.
25. 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
27. 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.
28. PROBLEMS ASSOCIATED WITH COLLECTION
Contamination Sampling time
Volume
determination
Recovery from
strips
Data reporting
29. 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
30. Volume determination
Scarcity of material : 0.5 – 1 l
Evaporation
Percentage of error
Use of vasoconstrictors (Hakkarainen and Ainamo 1981)
Recovery from strips
31. Data reporting
Earlier work – concentration & total enzyme activity
Lately - total amount of enzyme activity
Absolute amount
(mg)
Concentration
(mg/ml)
32. Composition
CELLULAR ELEMENTS
ELECTROLYTES
ORGANIC COMPOUNDS
METABOLIC AND BACTERIAL PRODUCTS
ENZYMES AND ENZYME INHIBITORS
33. CELLULAR ELEMENTS
Epithelial 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
34. 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
35. Leukocytes
Differential leukocyte count in the sulcus
GCF Peripheral 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
36. Role of inflammation - in number( Egelberg 1963)
Phagocytic function of PMNs
AgP < CP
37. Bacteria
Poor correlation to severity of gingival inflammation
and depth of pocket (Krekeler and Ferck, 1977)
38. ELECTROLYTES
Sodium
Normal GCF - 158 mEq/l
Inflammation - 207 to 222 mEq/l
Follows circadian periodicity( Kaslick et al 1970)
pocket depth Na
39. Potassium
Mean concentration in GCF - 9.54 mEq/l
GCF > serum
Increases towards the middle of the day
severity of periodontitis
pocket depth
2x
40. Sodium : Potassium Ratio
Diseased tissues ratio
Accumulation of intracellular potassium
GCF < ECF ( Krasse & Egelberg, 1962)
3.9 28:1
41. 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
42. 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
43. 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)
45. 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)
46. 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
47. 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)
48. 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)
49. Alkaline phosphatase
Sources : PMNs, bacteria
Plays a role in calcification
Pyrophosphatase
Role in calculus formation
Conc. Is positively correlated – amount of calculus
50. – glucoronidase
Lysozomal enzyme
Primary granules of PMNs
Sources : macrophages, fibroblasts, endothelial cells,
bacteria
Plays a role in the catabolism of mucopolysaccharides
51. 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)
57. 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
58. 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
59. 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
60. 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
61. 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
63. 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
64. 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
65. -glucuronidase
Positively associated with
Spirochaetes
P. gingivalis
P. intermedia
Lactose-negative black pigmenting bacteria
Negative with cocci (Lamster 1992)
66. 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
67. Lysozyme
in chronic periodontitis ( Markannen et al,1986)
in AgP patients
Pseudocholinesterase
Serum > GCF > saliva
Significantly higher in AgP
68. 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
69. 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
72. 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
73. 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
74. 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
75. Analysis of components
TESTS 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
76. Commercial diagnostic kits
PERIOCHECK
Detects collagenase
Paper strip + (collagen gel blue colour on
+ blue dye) strip
Intensity proportional to amount of enzyme present
43C
77. 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
78. 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
79. Potential diagnostic tests
For PGE2
Nakashima et al, 1994 - ELISA assay utilizing a
monoclonal rabbit anti-PGE2 antibody to assay GCF
PGE2
For osteocalcin
Can be assayed using polyclonal or monoclonal
antibodies by an ELISA or RIA.
80. 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
82. Sex Hormones
vascular permeability flow
Diabetic patient
High flow rate (Ringelberg et al 1977)
More glucose
83. 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 curettage
1 week after SRP:
After second SRP: lower values are sustained
(Gwinnett 1978)
84. 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
85. Conclusion
Monitoring periodontal disease – complicated task.
Analysis GCF constituents- extremely useful- simplicity
& non invasive.
Thorough knowledge- Better aid for diagnosis.
Starlings hypothesis states that the fluid movement due to filtration across the wall of a capillary is dependent on the balance between the hydrostatic pressure gradient and the oncotic pressure gradient across the capillary
α-globulin, ceruloplasmin, -lipoprotein and transferrin
Similarity in composition of GCF and plasma support the concept that it is an inflammatory exudate
Since the same enzyme is present in both desquamated epithelial cells and oral bacteria.
Chondroitin 4 sulphate - Metabolic activity of the deeper-seated periodontal tissues, notably alveolar bone.