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.

2. 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

3. INTRODUCTION
 Complex mixture of substances
Serum
WBCs
Cells
Bacteria

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

16. 3. Modified method
Rudin et al 1970
Valazza et al 1972
 Advantages

17. 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

18. Gingival washings
 Method given by Skapski and Lehner, 1976
 Hamilton’s microsyringe
 10 l of Hanks balanced solution

19.  Takamori & Oppenheim method, 1970
 Acrylic plate to cover maxilla
 Groove for plastic tubes
 4-6 ml solution – 15 min – peristaltic pump

20.  Disadvantages
 Modified method
 Ejection needle
 Collection needle

21. Other methods
 Platinum loops
 Microspatules
 Transparent / Plastic strips

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

26.  Advantages
 Disadvantages
1. Vol > 1 µl unable to measure.
2. Position of the strip influences readings.

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)

44. Lipids
 Serum, saliva, bacteria and host tissue
Phospholipids and neutral lipids

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)

52. 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)

53. Lactate dehydrogenase
 Pyruvate Lactate
 GCF > blood
LDH
10-20x

54. Proteolytic enzymes
 Mammalian proteinases
 Bacterial proteinases
 Proteinase inhibitors

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

62. 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)

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

70. Lactate dehydrogenase
 Cross-sectional studies - Correlated with PPD and
other clinical indices
 Longitudinal study - disease activity

71. 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

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
43C

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

81. Clinical significance
 Circadian Periodicity
 Mechanical Stimulation
 Inflammation
 Smoking

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.

86.  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