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  • 2. CONTENTS      Introduction Definition Function History Formation  Permeability of junctional and oral sulcular epithelium  Methods of collection  Problems during collection
  • 3.  Composition of GCF  Brief review of pathogenesis of periodontitis  Use of GCF inflammatory mediators as      indicators of risk for Periodontal diseases Commercially available diagnostic kits Clinical significance Conclusion Recent findings in GCF References
  • 4. Defence mechanism of oral cavity  Defence mechanism Specific 1. Humoral immunity 2. cell mediated immunity Non-specific 1.saliva 2.sulcular fluids 3.Higher tissue turnover 4.Intact epithelial barrier 5.Presence of normal flora 6.Local antibody production 7.Migrating leukocytes
  • 5. Anatomy of the gingival crevice The gingival sulcus is the shallow crevice or space around the tooth , bounded by the surface of the tooth on one side and the epithelial lining the free margin of the gingiva on the other. gingival crevicular fluid Monday, December 02, 2013 5
  • 6. Definition A fluid occurring in minute amounts in gingival crevice, belived by some authorities to be an inflammatory exudate and by others to cleanse material from the crevice, containing sticky plasma proteins which improve adhesions of the epithelial attachment, have antimicrobial properties and exert antibody activity. (from Jablonski, illustrated Dictionary of Dentistry, 1982)
  • 7. Functions : 1) Cleanse material from the sulcus 2) Contain plasma proteins that may improve adhesion of the epithelium to the tooth. 3) Possess antimicrobial properties. 4) Exert antibody activity in defense of the gingiva. gingival crevicular fluid Monday, December 02, 2013 7
  • 8. Studies on gingival crevice fluid (GCF) extend over a period of about 50 years  The pioneer research of Waerhaug (1952) was focused on ----- the anatomy of the sulcus and its transformation into a gingival pocket during the course of periodontitis.  Studies by Brill et al.(1962) laid the foundation for understanding the physiology of GCF formation and its composition.  The studies of Löe et al.(1965) ----- use of GCF as an indicator of periodontal diseases. gingival crevicular fluid Monday, December 02, 2013 8
  • 9.  Egelberg continued to analyze GCF and focused his studies on the dentogingival blood vessels and their permeability as they relate to GCF flow.  Attstrom R, Egelberg J. presence of leukocytes in gingival crevice during developing gingivitis in dogs. JPR 1971 : 6; 110 -114.  The GCF studies boomed in the 1970s. The rationale for understanding dentogingival structure and physiology was created by the outstanding electron microscopic studies of Schroeder and Listgarten. gingival crevicular fluid Monday, December 02, 2013 9
  • 10.  It was soon understood that enzymes released from      damaged periodontal tissue possessed an enormous potential for periodontal diagnosis. Presence and functions of proteins – Sueda, Bang and Cimasoni. Collagenases and Elastase in GCF are derived from human cells - Ohlsson, Golub, Uitto. Goodson thoroughly studied major issues in GCF flow rate and its method of collection. Flow rate of GCF may increase about 30 times in periodontitis patients than compared to healthy sites. Resting volume also increases with the formation of pockets. gingival crevicular fluid Monday, December 02, 2013 10
  • 11. In 1974 the first edition of the monograph  The Crevicular Fluid by Cimasoni was published. This comprehensive review gave a big boost to GCF studies and towards the end of the first millennium the research on GCF increased dramatically. gingival crevicular fluid Monday, December 02, 2013 11
  • 12. Timeline gingival crevicular fluid Monday, December 02, 2013 12
  • 13. Formation of GCF GCF is formed at the rate of 0.5- 2.4 ml/day. There are 2 theories that suggest the formation of GCF. Theory 1 (Brill and Egelberg) Increase in the permeability of vessels seepage of fluids in sulcus Formation of GCF
  • 14.  Theory 2  From the work of Alfano (1974) and from the hypothesis postulated by Pashley (1976) which suggested that the initial fluid produced could simply represent interstitial fluid which appears in the crevice as a result of an osmotic gradient. This initial, pre-inflammatory fluid was considered to be a transduate and on stimulation, this changed to become an inflammatory exudate.
  • 15.  The model proposed by Pashley predicted that GCF production is governed by passage of interstial fluid from capillaries tissues lymphatic system). When the rate of when capillary filtrate exceeds that of lymphatic uptake, fluid will accumulate as edema and/or leave the area as GCF gingival crevicular fluid Monday, December 02, 2013 15
  • 16.  Factors modulating:  Filtration coefficient of the lymphatic and capillary endothelium  Osmotic pressure within the different compartments.  Therefore, even in health also, if the osmotic pressure of the sulcular fluid exceeds that of the tissue fluid, (possibly because of accumulation of plaque derived molecules) there will be net increase in the flow of GCF. gingival crevicular fluid Monday, December 02, 2013 16
  • 17. PERMEABILITY OF JUNCTIONAL AND ORAL SULCULAR EPITHELIA  Substances that have been shown to penetrate the sulcular epithelium include albumin, Endotoxins, thymidine, histamine, phenytoin, per oxidase.  The main pathway for the transport of substances across the junctional and sulcular epithelia seems to be the intercellular spaces which according to Schroeder and Munzel – Pedrazzoli (1970) form 18% of the total volume of the junctional epithelium and 12% that of the oral sulcular epithelium. gingival crevicular fluid Monday, December 02, 2013 17
  • 18.  According to Squier (1973) the degree of permeability of the oral mucosa does not seem to depend upon its degree of Keratinization. The mechanisms of penetration through an intact epithelium were reviewed by Squier and Johnson.  Three routes have been described:  Passage Form CT Into The Sulcus:  Passage From The Sulcus Into The CT:  Passage Of Substances through pathological or experimentally modified gingival sulcus. gingival crevicular fluid Monday, December 02, 2013 18
  • 19.  Brill was also first to show the presence of plasma proteins in the gingival fluid.  The fundamental observations of Brill have been confirmed in other experiments, where it was shown that extraneous materials such as India Ink, labeled albumin or labeled fluorescein, tetracycline and saccharated iron oxide could be seen to pass from the gingival vessels into the gingival sulcus or pocket gingival crevicular fluid Monday, December 02, 2013 19
  • 20. Methods of collection
  • 21. Absorbent filter paper strips These strips are placed for 3 mins and GCF sample is collected by 2 methods. a) Intrasulcular or Brill technique – within the sulcus b) Extracellular or Loe & Holm pederson technique- at its entrance.
  • 22. Evaluation of amount of fluid collected by paper strips 1. 2. Direct viewing and staining Weighing of the strip 1.  Direct viewing and staining: Alcoholic solution of ninhydrin (0.2%) -blue purple pink Measured with – transparent scales, calipers, caliberated magnifying glass  gingival crevicular fluid Monday, December 02, 2013 23
  • 23.  Disadvantages of staining method:  Cannot be used chair side.  Inevitable delay in measurement may result in increase variation due to evaporation of the fluid.  Staining of the strips for protein labeling prevents further lab investigations. 2. Weighing of strips  Sealed micro centrifugation plastic tube.
  • 24. PERIOTRON An electronic method has been devised for measuring gingival fluid absorbed on paper strips by Harco electronics called Periotron (Dental product division Winnipeg, Manitoba, Canada). • latest and standard method for measuring gingival fluid absorbed on paper strips. gingival crevicular fluid Monday, December 02, 2013 25
  • 25.  Developed by Harco electronics  Principle:- The wetness of the paper strip affects the flow     of an electronic current It has 2 metal jaws which acts as the plates of an electrical condenser. When a dry strip is placed  zero reading is obtained A wet paper strip will increase the capacitance in proportion to the volume of fluid and this can be measured as an increased value in the readout. Three models  600, 6000 and 8000. gingival crevicular fluid Monday, December 02, 2013 26
  • 26. Advantages  Simple procedures, can be viewed directly.  Quantitative assessment of fluid can be obtained.  Evaporation is kept minimum.     Disadvantages Contamination can occur. In case of evaporation, has to be repeated many times. Care should be taken to insert paper strip in standardized position. inability to measure the volume of GCF greater than 1.0µl.
  • 27. Pre-weighed twisted threads Thread is placed in the gingival crevice around the tooth and the amount of fluid collected is estimated by weighing the sample thread. Used by WEINSTEIN et al. gingival crevicular fluid Monday, December 02, 2013 28
  • 28. Micropipettes/ capillary tubing  Krasse and Egelberg  Principle- collection of fluid by capillary action. After isolation and drying of collection site, capillary tubes of known diameter are inserted into the entrance of gingival crevice, GCF migrates into the tube by capillary action. As diameter is known, the amount of GCF can be calculated by measuring the distance which the GCF has migrated. And finally, their content is then centrifuged and analyzed.
  • 29. Disadvantages  Collection of fluid is difficult due to viscosity of the fluid.  Recovery of sample is demanding.  Long collection period.  May cause trauma as prolonged holding of pipette is required.
  • 30. Crevicular washings  The Method Of Oppenheim: This method uses an appliance consisting of a hard acrylic plate covering the maxilla with soft borders and a groove following the gingival margins, connected to four collection tubes.  The washings are obtained by rinsing the crevicular areas from one side to the other, using a peristaltic pump. gingival crevicular fluid Monday, December 02, 2013 31
  • 31.  ADVANTAGES:  Useful for longitudinal studies.  Permits collection without disturbing the integrity of the marginal tissues.  Contamination is least.  DISADVANTAGES:  Complex procedure.  Represents a dilution of crevicular fluid. gingival crevicular fluid Monday, December 02, 2013 32
  • 32.  The method of Skapski And Lehner:  This method uses two injection needles fitted one within the other such that during sampling the inside, or ejection, needle is at the bottom of the pocket and the outside, or collecting, one is at the gingival margin. The collection needle is drained into a sample tube by continuous suction. gingival crevicular fluid Monday, December 02, 2013 33
  • 33.  ADVANTAGES  Useful for cases of clinically normal gingiva.  Useful for studying the number and state of cells and bacteria form the crevicular area.  DISADVANTAGES  Does not permit absolute quantitative assessment as the dilution factor cannot be determined. gingival crevicular fluid Monday, December 02, 2013 34
  • 34. Problems during GCF collection  Scarcity of material that can be obtained from sulcus.  Contamination The major sources of contamination of GCF sample would be blood, saliva, or plaque.  Sampling time The problem with prolonged collection time is that the nature of the GCF sample collected is likely to change with the protein concentration of the initial GCF collected. gingival crevicular fluid Monday, December 02, 2013 35
  • 35.  Volume determination  Recovery from strips  Data reporting Constituents found within GCF samples have either been reported as absolute amount (mg) or in concentrations (mg/ml).
  • 36. Composition of GCF enzymatic Host derived Nonenzymatic Cellular component electrolytes Bacteria derived Organic organic component
  • 37. Host derived enzymes Acid phosphates Alkaline phosphatase Alpha 1 antitrypsin Arylsulphatse Aspartate aminotransfarase Chondroition sulphate Citric acid Cystatins B-glucuronidase Cathepsin Matrix metalloproteins Elastase gingival crevicular fluid Monday, December 02, 2013 38
  • 38. Bacteria derived enzymes  Acid phosphatase  Alkaline phosphatase  Collagenase  Hyaluronidase  Phospholipse-A  Phospholipase-C gingival crevicular fluid Monday, December 02, 2013 39
  • 39. Cellular elements Bacteria Desquamated epithelial cells Leucocytes ( PMN’S, monocytes/macrophages) Electrolytes Potassium Calcium Sodium Organic compounds Carbohydrates-Glucosehexosamine -Hexuronic acid Proteins gingival crevicular fluid Monday, December 02, 2013 40
  • 40. Metabolic end products  Lactic acid  Urea  Hydroxyproline  Endotoxins  Cytotoxic substances  Hydrogen sulphide gingival crevicular fluid Monday, December 02, 2013 41
  • 41. gingival crevicular fluid Monday, December 02, 2013 42
  • 42. Electrolytes  Potassium, sodium, calcium, magnesium and fluoride have been studied in gingival fluid. Most studies have shown a positive correlation of calcium and sodium concentrations as well as sodium to potassium ratio with inflammation. gingival crevicular fluid Monday, December 02, 2013 43
  • 43. Organic Compounds  Carbohydrates, proteins and lipids have been investigated. Glucose hexosamine and hexuronic acid are two of the compounds found in gingival fluid. Glucose concentration in gingival fluid is 3-4 times greater than that in serum.  This is interpreted not only as a result of metabolic activity of adjacent tissues, but also as a function of the local microbial flora. gingival crevicular fluid Monday, December 02, 2013 44
  • 44. The total protein content of gingival fluid is much less than that of serum. No significant correlations have been found between the concentration of proteins in the gingival fluid and the severity of gingivitis, pocket depth and extent of bone loss.  Proteins namely , , and 2 1 globulins, transferrin, albumin, immunoglobulins such as IgG, IgM and IgA, complement components such as C1, C4, C3, C5 have been reported to be present in GCF.  Proteins Include: - fibrinogen, ceruloplasmin, lipoprotein, transferrin, 1 – antitrypsin and 2 – macroglobulin. gingival crevicular fluid Monday, December 02, 2013 45
  • 45.  According to Armitage (2004), more than 65 GCF constituents have been evaluated as potential diagnostic markers of periodontal disease progression.
  • 46. Pathogenesis of periodontal diseases  Periodontal diseases are caused by a localized inflammatory reaction in response to a bacterial infection of the teeth, and are manifested by an alteration of the integrity of the tissues supporting the teeth.
  • 47.  studies have brought a paradigm change in periodontal disease pathogenesis.  These findings indicate that the microorganisms associated with disease are also found at healthy or nonprogressing sites (albeit at lower levels).  The level of plaque control (oral hygiene) is not associated with an individual’s disease severity or extent.  The presence of a certain level of specific pathogens (such as Porphyromonas gingivalis) makes a statistically significant but small contribution in multivariate models of disease.
  • 48.  The host immune and inflammatory response to the microbial challenge is a critical determinant of susceptibility to develop the destructive disease, under the influence of multiple behavioral, environmental, and genetic factors.  Hence, although disease progression is episodic in nature on a tooth site level, the risk of developing periodontal disease is principally patient-based rather than site-based.
  • 49.  As plaque matures, becoming more pathogenic, in parallel, host inflammatory response evolves from an acute to a chronic one.  Gram negative periodontal pathogens can evade the host clearance mechanisms (complement, antibodies and neutrophils), while shedding vesicles containing microbial toxins, proteases and endotoxins( Lipopolysachcharides).
  • 50.  LPS(Lipopolysachcharides) penetrates tissues stimulates monocytes secretes mediators of inflammation( PGE2, thromboxane B2, IL-1, 6 & 8, TNF and collagenases)
  • 51. Mediators of inflammation activate vascular smooth muscles, fibroblasts, more monocytes, and osteoclasts to produce MMPS Stimulates bone resorption
  • 52.  This inflammatory cascade produces clinical inflammation, attachment loss, pocket formation, bone loss.  Along side monocytic synthesis of inflammatory mediators antigen presentation also occurs.  This arm of host response triggers adaptive immune response with an initial (T helper type1 ) response.  T – helper type 1 response consists of proinflammatory IL-2, TNF α, Interferon γ.
  • 53.  T – helper type 1 response later shifts to T – helper type 2 dominant response consisting of antiinflammatory, ILs – 4,5,6,10,13 and production of immunoglobulins.  This is consistent with a shift from T- helper type 1 to T- helper type 2 between transitions from gingivitis to periodontitis as described by Seymour and Gemmal (1994).  However other studies indicate that in active periodontal disease progression, there is dominant T- helper type 1 response versus T – helper type 2 which being consistent with periodontal disease stability (non progression).
  • 54.  So, inflammatory cytokines can be detected within GCF and serve as an indicator of local immunoregulatory and inflammatory status.  In addition, collagen breakdown products such as Hydroxyproline and Pyridinoline cross- linked carboxyterminal telopeptide fragments of type 1 collagen are found in GCF that serves as direct measure indicator of connective tissue catabolism for both soft and hard tissues.
  • 55.  Elevated GCF levels of neutrophil markers i.e. neutrophil elastase, β-glucuronidase and leukotriene B4 reflects Acute episodes of localized tissue destruction  Taken together, these monocytic and neutrophilic mediator levels in the GCF may also give an indication of quality of the host response, and of the level of risk for the individual to develop periodontal disease.
  • 56. Prostaglandin E2 (PGE2 ):  PGE2 was first identified in GCF by Goodson et al. in 1974.  PGE2 is a product of the cyclooxygenase pathway. Elevated levels of PGE2 in GCF were found in patients with periodontitis compared to patients with gingivitis. PGE2 levels were three times higher in patients with juvenile periodontitis compared to adult periodontitis.
  • 57.  Offenbacher et al (1986) showed that there were differences in the GCF concentration of PGE2 in patients with gingivitis compared with periodontitis. Subsequently, it was found that there was a correlation between increased PGE2 concentration and clinical attachment loss in patients who were diagnosed with moderate to severe periodontitis. gingival crevicular fluid Monday, December 02, 2013 58
  • 58. Cytokines  Cytokines are potent local mediators of inflammation that are produced by variety of cells. Cytokines that are present in GCF and have been investigated as potential diagnostic makers for periodontal disease include:  interleukin - 1 , 1 ,  interleukin – 6,  interleukin – 8 and  tumor necrosis factor (TNF - ).  Both IL - 1 and IL - 1 have pro-inflammatory effects and depending on a variety of factors can stimulate either bone resorption or formation.
  • 59.  It has also been reported that in adult periodontitis patients, a higher percentage of sites are positive for IL - 1 (87%) and IL - 1 (56%) IL-6 has also been associated with bone resorption. GCF from sites with progressing periodontitis contains elevated amounts of IL-6.  IL-8 was formerly called monocyte-derived neutrophil chemotactic factor. GCF from sites with periodontitis contains significantly more total IL-8 than GCF from healthy sites
  • 60.  Proinflammatory cytokines in particular IL-1, may play an integral role in the aetiology of periodontal disease.  Lieu et al (1996) demonstrated that with an increase in gingival index and probing, there was a corresponding increase in IL-1 in both the gingival tissue and GCF.  Engebretson et al through a longitudinal study suggested that GCF IL-1 expression is genetically influenced and not solely a result of local clinical parameters. Also, a GCF level of IL8 was found to be higher in periodontal diseases and was influenced by local IL-1 activities. gingival crevicular fluid Monday, December 02, 2013 61
  • 61. Total Protein  Several reports suggest that, compared to periodontal healthy controls, GCF from sites with periodontitis has significantly elevated levels of total protein.  Some study has reported that GCF from inflamed sites in patients with periodontitis have significantly lower protein concentrations than GCF from inflamed sites in patients with gingivitis alone
  • 62. Host – Derived Enzymes (a) Aspartate Aminotransferase:  Aspartate aminotransferase enzyme (AST) is one of the components of GCF that is released and can be detected as a result of cell death.  Significant associations between GCF levels of AST and clinical measurements have been determined, and a test system, the PeriogardTM periodontal tissue monitors (PTM), has been developed (Persson et al. 1990.
  • 63.  The commercial chair side test do not have the ability to reliably distinguish between progressing sites and those that are inflamed but not progressing.
  • 64. Alkaline phosphatase  In the periodontium, alkaline phosphatase is a very important enzyme as it is part of the normal turnover of periodontal ligament, root cement formation and maintenance, and bone homeostasis.  It is produced by many cells, including fibroblasts, osteoblasts and osteoclasts, but the main source of alkaline phosphatase in gingival crevice fluid is neutrophils. gingival crevicular fluid Monday, December 02, 2013 65
  • 65.  Similar levels of alkaline phosphatase in GCF have been found in gingival health and experimental gingivitis, but a longitudinal study demonstrated that elevated alkaline phosphatase levels preceded clinical attachment loss and that the total amount of alkaline phosphatase in GCF was significantly higher in active sites (Nakashima 1996).
  • 66. Beta – glucuronidase  Beta-glucuronidase is a lysosomal enzyme that is active in the hydrolysis of glycosyl bonds of intercellular ground substances.  It is highly conceivable and periodontal disease activity is associated with increased levels of beta-glucuronidase in gingival crevice fluid gingival crevicular fluid Monday, December 02, 2013 67
  • 67.  ß glucuronidase is a glycoprotein of about 3,32,000 dalton. It is a homo tetramer comprised of four identical subunits. It has high sensitivity and specificity when related to occurrence of clinical attachment loss. This enzyme also proved to be a good predictor of the response to treatment and the risk for future periodontal breakdown (Lamster et al 1998).  Subjects without active disease did not have elevated betaglucuronidase in gingival crevice fluid. In relation to attachment loss, they observed beta-glucuronidase to have a sensitivity and specificity of 89% and 89%, respectively.
  • 68. Elastase  Neutrophil elastase, sometimes referred to as granulocyte elastase, is an abundant proteinase released from the azurophilic granules of neutrophils and as such is an indicator of neutrophil activity.  Neutrophil elastase is a serine proteinase, active in the degradation of microbiological components in conjunction with, or without, phagocytosis. At the same time, when released extracellularly, this enzyme can degrade host intercellular matrix components, including elastin, fibronectin, and collagen. gingival crevicular fluid Monday, December 02, 2013 69
  • 69.  Elastase levels in GCF increase with induction of experimental gingivitis, and decrease when plaque removal is reinstituted.  In a longitudinal study, Eley and Cox (1996) demonstrated that increased elastase in GCF was predictive of periodontal attachment loss. Long-term observation of adult patients with periodontitis undergoing supportive periodontal therapy showed a positive correlation of elastase in GCF with clinical attachment loss.  Smokers display higher levels of elastase than nonsmokers.Soder B(2002)
  • 70. Cathepsin B  Cathepsin B is an enzyme active in proteolysis; it belongs to the class of cysteine proteinases. The cellular source of cathepsin B in gingival crevice fluid seems to be mainly macrophages.  Cathepsin B activity has been found in gingival crevice fluid in adult periodontitis. It seems to be increased in periodontitis but is not increased in gingivitis, even though the flow of gingival crevice fluid is more or less equal in these two periodontal conditions. gingival crevicular fluid Monday, December 02, 2013 71
  • 71.  Kunimatsu et al (1990) observed that levels of cathepsin B were increased in periodontitis when compared to gingivitis, despite similar GCF flow.  Furthermore, GCF levels of cathepsin correlate significantly with clinical parameter before and after periodontal treatment suggesting a use for this enzyme in assessment of treatment outcomes. Cathepsin G may contribute to periodontal tissue destruction directly and indirectly, via proteolytic activation of latent neutrophil procollagenase (promatrix metalloproteinase-8).  Eley & Cox have further investigated cathepsin B and evaluated its use as a predictor of attachment loss.
  • 72. (g) Collagenases/ Gelatinases/ Neutral proteinases/ stromelysins:  GCF from sites with adult or juvenile forms of periodontitis exhibit significantly elevated collagenolytic activities compared to GCF from healthy or gingivitis sites.  In ligature-induced periodontitis in the beagle dog, GCF collagenase activity increased to maximum values within weeks after ligature placement, active collagenase was elevated during active periodontitis and active collagenase was strongly correlated with attachment loss. Latent collagenase and collagenase inhibitors were prominent during gingivitis.
  • 73. (3) Tissue Breakdown Products: (a) Glycosaminoglycans (GAG’s):  The GAG’s in GCF that have been most examined as possible diagnostic markers for periodontal diseases are:  Chondroitin – 4 – sulfate,  chondroitin – 6 – sulfate and  hyaluronic acid.  The appearance of C-4-S in GCF has been suggested as a marker for bone resorption associated with periodontal disease or orthodontic tooth movement. But no studies have been conducted to determine its role in the progression of periodontitis.
  • 74. (b) Hydroxyproline:  It is a prominent aminoacid of collagen and its appearance in GCF has been preliminary investigated as a marker for the destruction of periodontal connective tissue. Data from one cross-sectional study in humans indicate that GCF hydroxy proline levels cannot distinguish between sites with gingivitis or periodontitis. Because of this it is not an attractive candidate as a potential marker for the progression of periodontitis.
  • 75. (c) Fibronectin: These are a large group of heterogeneous glycoprotein present in blood and connective tissues. Data from most studies indicate that GCF fibronectin is not a promising diagnostic marker.
  • 76. (d) Connective Tissue Proteins:  Increased GCF levels of the amino terminal propertied of type I collagen have been reported at periodontitis sites. On a concentration basis, the amount of osteocalcin in GCF does not appear to be different at sites with gingivitis or periodontitis.  Osteonectin another non-collagenous protein of bone and a variety of other tissues, has been reported to be elevated in GCF at sites with severe periodontitis. Neither Osteocalcin nor Osteonectin levels in GCF have been systematically evaluated as diagnostic markers for periodontitis.
  • 77. MARKERS FROM MICROBIAL PLAQUE It is evident that since periodontal disease is associated mainly with the presence of certain bacteria which are recognized as the principal etiological agents, then factors derived from such bacteria may be useful indicators of their presence and metabolic activity.
  • 78. Lipopolysaccharides (Endotoxins)  These molecules are found in the outer membrane of the cell wall of Gram-negative bacteria. The presence of endotoxins has been positively correlated with gingival inflammation (Simonet al, 1971)  when measured in GCF and in combination with clinical and histological studies. The level of endotoxin is related to the number of Gram-negative bacteria.  Lipopolysaccharides (LPS) vary in their structure depending on bacterial source.
  • 79. Bacterial Enzymes  Perhaps bulk of the work on bacterial enzymes has been carried out on proteolytic enzymes or proteinases.  The most studied would be the trypsin-like proteinase of P.gingivalis.
  • 80.  Similar trypsin-like enzymes are also associated with Treponema denticola (Makinin et al, 1987).  Bacterial collagenases are also identified with Clostridium histolyticum and Streptococcus mutans.  The presence of such enzymes in GCF correlates with the levels of these bacteria in the periodontal pocket and also with the severity of the attachment loss.
  • 81. Enzymes Associated with Tissue Destruction Lactoferrin  This is an antimicrobial agent with distribution in PMNs and secretory fluids similar to that of Lysozyme. The antibacterial properties of Lactoferrin are due to its high affinity for iron, thus locking available sources required for bacterial growth.  Lactoferrin showed better correlation with clinical indices than PMNs (Adonogianaki et al, 1993).
  • 82. Friedman et al (1983) found that Lactoferrin increased two fold in GCF in sites showing gingivitis periodontitis, and localized juvenile periodontitis. It has also been reported that the ratio of Lactoferrin to Lysozyme may be more representative and a useful diagnostic assay of periodontal inflammation.
  • 83. Myeloperoxidase This enzyme has also been shown to give good correlation with an inflammatory response where it is found in the primary granules of PMN (Smith et al, 1986).
  • 84.  Several products show potential benefit, particularly those directly from specific regions of the periodontium which give a clue as to which tissue components are at risk. It is clear that no single marker will fulfill all the criteria necessary for assessment of the clinical state of the periodontium, and future research should be directed at the production of "marker packages". The development of a wide spectrum of marker factors will be a primary goal of periodontal research.
  • 85.  COMMERCIALY AVAILABLE DIAGNOSTIC KIT  Periocheck - Neutral Proteinases - Approved by FDA  Periogard - AST  Prognostik- Elastase - Not Approved by FDA and ADA  Biolise - Elastase  Pocket watch - AST  TOPAS – Toxicity Pre-screening assay (bacterial toxins and proteases  MMP dipstick method - MMPs  Under development, for B - glucornidase and proteinases gingival crevicular fluid Monday, December 02, 2013 86
  • 86.  The components of gingival crevice fluid are analyzed     with regard to their potential utility in fulfilling the following aims: (BRUNO G. LOOS & STANLEY T JOA) AIM 1: To detect a case of periodontitis i.e., to distinguish periodontitis from health and gingivitis AIM 2: To classify a case of periodontitis, i.e., chronic periodontitis or aggressive periodontitis AIM 3: To plan treatment for the patient on the basis of the level of disease activity AIM 4: To monitor the treated patient based on the level of disease activity gingival crevicular fluid Monday, December 02, 2013 88
  • 87. gingival crevicular fluid Monday, December 02, 2013 89
  • 88. Clinical significance  Circadian Periodicity: There is a gradual increase in gingival fluid amount from 6:00AM to 10:00PM and a decrease afterward. gingival crevicular fluid Monday, December 02, 2013 90
  • 89.  GCF and sex hormones Clinical investigations have shown an exacerbation of gingivitis during pregnancy (loe 1965), during menstrual cycle (------Lemann 1948) and at puberty (Sutcliffe 1972). Female sex hormones increase the gingival fluid flow, probably because they enhance vascular permeability. Pregnancy, ovulation and hormonal contraceptives all increase gingival fluid production. gingival crevicular fluid Monday, December 02, 2013 91
  • 90.  GCF and drugs Drugs that are excreted through the gingival fluid may be used advantageously in periodontal therapy. Bader and Goldhaber demonstrated that intravenously administered tetracycline in dogs rapidly emerges within the sulcus. gingival crevicular fluid Monday, December 02, 2013 92
  • 91.  Ciancio et al (1976) measured the concentration of tetracycline in blood and gingival fluid of 5 adult patients with advanced periodontitis, who were given 1g of tetracycline HCL daily for 2 weeks and 0.5g for 10 weeks. The concentration of the drug in gingival fluid was 1/10 of that found in serum.  In a second study from the same laboratory the concentrations of the drug were found to be 5 times higher in samples of gingival fluid as compared to the concentrations in blood. gingival crevicular fluid Monday, December 02, 2013 93
  • 92.  Stephen et al (1980) measured the conc. of ampicillin, cephalexin, tetracycline, erythromycin, clindamycin and rifampicin in serum, saliva and GCF after a single dose administration. Except on one occasion, individual GCF antibiotic conc. were equal to or considerably greater than those found in saliva. But they were, however, always much lower than the concentration found in serum.  Metronidazole is another antibiotic that has been detected in human GCF. (Eiserbeng et-al 1991). gingival crevicular fluid Monday, December 02, 2013 94
  • 93.  GCF in diabetic patients Ringelberg et al in 1977 described a higher flow rate of gingival fluid in a group of diabetic children, when compared to the flow rate measured in a group of children without diabetes. In healthy individuals Hara and Löe found exudate glucose values up to 6 times those of serum. Kjellman (1970) reported glucose values much lower in gingival fluid when compared to serum, this being true for both healthy and diabetic patients. gingival crevicular fluid Monday, December 02, 2013 95
  • 94.  Periodontal therapy and GCF There is an increase in gingival fluid production during the healing period after periodontal surgery. According to Arnold et al 1966 this increase was probably the result of the inflammatory reaction from gingival trauma and the loss of an intact epithelial barrier, especially considering the fact that fluid had been collected by deep intracrevicular technique. Suppipat et al in 1978 sampled gingival fluid 14, 21, 28 and 35 days after gingivectomy and found an increase in gingival fluid flow during the first 2 weeks after surgery followed by a gradual decrease. This decrease was same when using mechanical or chemical plaque control. gingival crevicular fluid Monday, December 02, 2013 96
  • 95.  Influence of mechanical stimulation Chewing and vigorous gingival brushing stimulate the oozing of gingival fluid. Even the minor stimuli represented by Intrasulcular placement of paper strips increase the production of fluid. gingival crevicular fluid Monday, December 02, 2013 97
  • 96.  Smoking and GCF Smoking produces as immediate transient but marked increase in the gingival fluid flow. Mcluaghlin WS et al 1993 gingival crevicular fluid Monday, December 02, 2013 98
  • 97. CONCLUSION In conclusion one can say that the origin, the composition and the clinical significance of gingival fluid are now known with more precision and have significantly helped our understanding of the pathogenesis of periodontal disease. Up to now, for instance, none of the multiple components analyzed in the fluid has improved clinical judgment of the rate of progress of gingivitis and periodontitis or of the rate of repair of these conditions.
  • 98. Recent findings in GCF  GCF resistin level as a potential inflammatory marker for periodontitis with type2 diabetes mellitus.(Gokhale NH et al.2013).  OPG concentrations in GCF decreases proportionally with the progression of periodontal disease, that is gingival inflammation and clinical attatchment loss (CAL) (Bandari P et al. 2012).
  • 99.  IL-23 level in GCF is directly proportional to the severity of periodontal affliction suggesting its possible role in periodontal inflammation. (Himani GS 2013).  Periodontal treatment downregulates protease-activated receptor2. (VTE Alves 2013)
  • 100. References  CARRANZA,s Clinical Periodontology. 10th edition.  Griffiths. Formation, collection and significance of GCF. Periodontal 2000 2003; 31:32 – 42.  J. Max Goodson. Gingival crevicular fluid. Periodontal 2000 2003;31:43 – 54.  Catherine M.E. et al. Potential for gingival crevice fluid measures as predictors of risk for Periodontal disease. Periodontology 2000 2003;31:167-80.  BRUNO G. LOOS & STANLEY T JOA. Host-derived diagnostic markers for periodontitis: do they exist in gingival crevice fluid? Periodontology 2000 2005;39: 53– 72.