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Gingival Crevicular Fluid: Composition, Collection, and Clinical Significance, Lecture notes of Biochemistry

University of JamestownBiochemistry

An in-depth analysis of Gingival Crevicular Fluid (GCF), also known as sulcular fluid. the definition, formation, function, composition, and methods of collection of GCF. It also discusses the clinical significance of GCF and its use as a diagnostic tool for periodontal disease. various methods for estimating GCF amount and the problems associated with its collection.

What you will learn

  • What are the main components of GCF and how is it collected?
  • What is the clinical significance of GCF and how is it used as a diagnostic tool?
  • How is GCF formed and what is its function?
  • What are the methods for estimating GCF amount and what are their challenges?
  • What is Gingival Crevicular Fluid (GCF) and where is it located?

Typology: Lecture notes

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Gingival Crevicular Fluid
Lecture: 12 Dr. Ali A Abdulkareem
Introduction
Gingival crevicular fluid (GCF), also known as sulcular fluid, is defined as physiologic fluid secreted
in the gingival crevice that is classified as inflammatory exudate during disease or serum
transudate during health. GCF secreted in minute amount in healthy state, which is increased in
response to inflammation.
Gingival crevice is a V-shaped space bounded by tooth from one side and epithelium lining the
free margin of gingiva on the other side. Waerhaug (1950) studied pathologic transformation of
gingival sulcus into periodontal pocket. In the same period, work of Brill et al. established the
understanding the physiology of gingival crevicular fluid formation and its composition. Later,
Loe et al. (1965) used gingival crevicular fluid as an indicator of periodontal disease. Brill and
Krasse (1958) had first demonstrated the seepage of GCF. They introduced filter paper into the
gingival sulci of dogs previously injected intramuscularly with fluorescein; within 3 minutes, the
fluorescent material was recovered on the paper strips. This indicated the passage of fluid from
the bloodstream through the tissues and exiting via the gingival sulcus. Later, other studies
showed little or no fluid can be collected from the gingival crevice in health condition. Recently,
it is agreed that GCF is secreted as transudate due to osmotic gradient in health and it changes
into exudate upon in response to inflammation during periodontal disease.
Formation of GCF
Epithelial lining of the sulcus characterizes by permeability that allows traversing of the molecules
through intercellular spaces. Previous studies showed that wide range of molecules could
penetrate the epithelial barrier including albumin and endotoxin.
Pashley et al. (1976) suggested a model that explains the flow of fluid into gingival crevice area.
This model suggested that GCF production is controlled by the passage of fluid from capillaries
into the tissues (capillary filtrate) which is removed by lymphatic system. However, when the role
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Gingival Crevicular Fluid

Lecture: 12 Dr. Ali A Abdulkareem

Introduction Gingival crevicular fluid (GCF), also known as sulcular fluid, is defined as physiologic fluid secreted in the gingival crevice that is classified as inflammatory exudate during disease or serum transudate during health. GCF secreted in minute amount in healthy state, which is increased in response to inflammation. Gingival crevice is a V-shaped space bounded by tooth from one side and epithelium lining the free margin of gingiva on the other side. Waerhaug (1950) studied pathologic transformation of gingival sulcus into periodontal pocket. In the same period, work of Brill et al. established the understanding the physiology of gingival crevicular fluid formation and its composition. Later, Loe et al. (1965) used gingival crevicular fluid as an indicator of periodontal disease. Brill and Krasse (1958) had first demonstrated the seepage of GCF. They introduced filter paper into the gingival sulci of dogs previously injected intramuscularly with fluorescein; within 3 minutes, the fluorescent material was recovered on the paper strips. This indicated the passage of fluid from the bloodstream through the tissues and exiting via the gingival sulcus. Later, other studies showed little or no fluid can be collected from the gingival crevice in health condition. Recently, it is agreed that GCF is secreted as transudate due to osmotic gradient in health and it changes into exudate upon in response to inflammation during periodontal disease. Formation of GCF Epithelial lining of the sulcus characterizes by permeability that allows traversing of the molecules through intercellular spaces. Previous studies showed that wide range of molecules could penetrate the epithelial barrier including albumin and endotoxin. Pashley et al. (1976) suggested a model that explains the flow of fluid into gingival crevice area. This model suggested that GCF production is controlled by the passage of fluid from capillaries into the tissues (capillary filtrate) which is removed by lymphatic system. However, when the role

of capillary fluid exceeds that of lymphatic uptake, the fluid accumulate as edema and leave the area as GCF (Fig 1). According to this model, the flow of GCF can be explained as following: A- In the absence of inflammation: In health state, there is a low vascular pressure and low permeability of basement membrane. Subsequently, this will reduce the flow of GCF associated with increased fluid uptake by lymphatics. B- During inflammation: Presence of dental biofilm leads to an increase in osmotic gradient, which is followed by increased leakage of proteins. This will cause increase in hydrostatic pressure and vascular permeability thereby exceeding the capacity of lymphatics to drain fluids leading to upregulation of GCF flow. i ii Figure 1. i) In healthy condition, the majority of interstitial fluid is drained by lymphatic system and only small amount leak into gingival crevice forming transudate. ii) During periodontal disease, the amount of leaked fluid from blood vessels is beyond the drainage capacity of lymphatics, leading to formation of inflammatory exudate.

1 - Absorbing paper strip Paper strips are available in standard sizes that can absorb volume of GCF ranging between 1.2-2 μl. Absorbing papers can be used in two ways a. Intra-crevicular method: the paper strip is placed superficially at the entrance of gingival crevice (Loe and Holm-Pederson technique) to minimize irritation to the sulcular epithelium. The other approach is to insert the paper strip gently within the gingival crevice until minimal resistance is felt (Brill’s technique). Brill’s method cause more irritation to the tissue and may stimulate oozing of the fluid (Fig 2). Typically, absorbing papers are inserted no more than 1– 2 mm into the sulcus/ pocket with gentle placement until mild resistance is felt, and held in place for 30 seconds. i ii Figure 2. i) Loe and Holm-Pederson technique, positioning of paper strip at gingival margin without penetrating gingival sulcus; (ii) Brill’s technique, positioning of paper strip into gingival sulcus-P3.

b. Extra-crevicular method: The paper strip is adapted on the gingiva & the hard tissue of the tooth in this region so the paper strip will absorb the seepage of GCF (Fig 3). Figure 3. Positioning of paper strip overlying the tooth, gingiva, and alveolar mucosa for obtaining GCF 2 - Crevicular washing The Gingival sulcus is perfused with an isotonic solution of fixed volume. Three methods of gingival washing are available: a- Instillation and re-aspiration of 10 μl of Hank’s balanced salt solution at the interdental papilla. It is repeated several times to allow thorough mixing of transport solution and GCF. The fluid collected represents a dilution of GCF that contains both cells and soluble constituents such as plasma proteins. b- Second method is more complicated which involves the construction of a customized acrylic stent that isolates the gingival tissue from the rest of the mouth. The tissues are irrigated with a saline solution, using a peristaltic pump and the diluted GCF is removed. The stent is made of hard acrylic plate covering the maxilla with soft borders and a groove following the gingival margin, connected to four collection tubes. This method lacks accuracy, technically demanding and limited to use for maxillary arch only. c- Third method is a modification that uses two injection needles fitted one within the other such that during sampling, the inner needle (or ejection) is at the bottom of the

  • The collection of fluid is difficult because the viscosity of fluid makes the aspiration difficult.
  • Difficult to hold capillary tube at the entrance of gingival crevice for such lengthy periods
  • It is also difficult to remove the complete sample from the tubing Figure 5. Gingival crevicular fluid collection by micro-capillary pipettes Methods for estimating GCF amount 1 - The wetted area of paper strip can be made visible by staining with alcoholic solution of ninhydrin (concentration between 0.2 & 2%); this stain has specificity to for amino acids & gives a blue or purple color. The stained part is then measured on an enlarged photograph or with a magnifying glass or a microscope using ruler or Vernier (Fig 6). The shortcomings of this method include evaporation may affect estimation of GCF volume beside it is not easily applicable chairside. Figure 6. Linear measurement of paper strip area stained by ninhydrin solution.

2 - An electronic method has been devised for measuring the fluid collected on a “blotter” (Periopaper), employing an electronic transducer (Periotron). Periotron is an electronic instrument that measures the effect of wetness of filter paper strips on the capacitance between the ‘jaws’ of the device, between which the filter paper is placed after the sample has been collected. The wetness of the paper strip affects the flow of an electronic current and gives a digital readout (Fig 7). The readings obtained by Periotron can be converted into corresponding clinical conditions and scores recorded by gingival index (Table 2) Figure 7. Electronic device (Periotron) for measuring the amount of fluid collected on filter paper. Periotron reading Level of gingival inflammation Gingival index 0 - 20 Healthy 0 21 - 40 Mild 1 41 - 80 Moderate 2 81 - 200 Severe 3 Table 2. Translation of Periotron value into associated clinical conditions and gingival index 3 - Weighting the strip: pre-weighted strip is inserted into the gingival crevice & then determined the amount of fluid collected by weighting the sample. 4 - Isotope dilution method to measure extremely small amount of GCF present in a particular space at any given time.

  • Lactoferrin (LF): Level of LF significantly decrease following periodontal surgery. Hence, LF levels in GCF could serve as a useful marker for monitoring of periodontal treatment results
  • C-reactive protein (CRP): the level of CRP increases as the disease progress from gingivitis to periodontitis.
  • Substance-P: level of this biomarker significantly increase at sites with periodontal destruction and reduce following periodontal treatment. Clinical significance of GCF The amount of GCF is greater when inflammation is present and is sometimes proportional to the severity of inflammation. However, level of GCF may increase due to other reasons. These factors could be summarize as follow: 1 - Circadian periodicity: there is gradual increase in GCF amount from 6 AM to 10 PM & decrease afterward. 2 - Sex hormones: gingival inflammation increases during pregnancy, menstrual cycle, hormonal contraceptive and puberty. Female sex hormones increase the GCF flow because they enhance the vascular permeability. 3 - Mechanical stimulation: GCF production is not increased by trauma from occlusion but is increased by mastication of coarse foods, toothbrushing and gingival massage. 4 - Smoking: It produces an immediate transient but marked increase of GCF. 5 - Periodontal therapy: there is an increase in GCF production during the healing period after periodontal surgery. This increase was probably the result of inflammatory reaction from gingival trauma and the loss of an intact epithelial barrier. GCF Composition in Relation with Systemic Diseases Systemic conditions are associated with changes in the levels of certain biomarkers that could be detected in GCF.
  • Increase glucose in diabetic patient.
  • Increase urea, alteration of protein in kidney disease.
  • Increase lactic acid in liver disease.
  • Increase calcium in hyperparathyroidism.
  • Increase alkaline phosphatase in bone disease (Ricket's, Paget's disease) Drugs and GCF Medications that are excreted through the GCF may be used advantageously in periodontal therapy. Tetracyclines are one of these medications & are effective in treating refractory periodontitis because of their concentration in the gingival crevice is 2-10 times than that in serum. This allows a high drug concentration to be delivered into periodontal pockets & inhibit the growth of Aggregatibacter actinomycetem comitans and other anaerobic bacteria. Metronidazole is another antibiotic that has been detected in human GCF. Ampicilin, cephalexin, rifampicin was also detected. References
  1. Fiorini T, Susin C, Da Rocha J, Weidlich P, Vianna P, Moreira CHC, et al. Effect of nonsurgical periodontal therapy on serum and gingival crevicular fluid cytokine levels during pregnancy and postpartum. Journal of periodontal research. 2013;48(1):126-33.
  2. Kinney JS, Morelli T, Oh M, Braun TM, Ramseier CA, Sugai JV, et al. Crevicular fluid biomarkers and periodontal disease progression. Journal of clinical periodontology. 2014;41(2):113-20.
  3. Kudva P, Saini N, Kudva H, Saini V. To estimate salivary aspartate aminotransferase levels in chronic gingivitis and chronic periodontitis patients prior to and following non-surgical periodontal therapy: A clinico-biochemical study. Journal of Indian Society of Periodontology. 2014;18(1):53.
  4. Lagos MLP, Sant'Ana ACP, Greghi SLA, Passanezi E. Keratinized gingiva determines a homeostatic behavior of gingival sulcus through transudation of gingival crevice fluid. International journal of dentistry. 2011;2011.
  5. Newman MG, Takei H, Klokkevold PR, Carranza FA. Carranza's clinical periodontology-e-book: Expert consult: Online: Elsevier health sciences; 2014.
  6. SALONEN JI, PAUNIO KU. An intracrevicular washing method for collection of crevicular contents. European Journal of Oral Sciences. 1991;99(5):406-12.
  7. Wassall RR, Preshaw PM. Clinical and technical considerations in the analysis of gingival crevicular fluid. Periodontology 2000. 2016;70(1):65-79.