What the components of gingival crevicular fluid tell us
BY Diana J. Lamoreux, RDH, BS, MEd
Gingival tissue is constantly subjected to mechanical and bacterial assault. Resistance to these forces is provided by the saliva, gingival crevicular fluid (GCF), epithelial surface keratinization, and initial stages of inflammation. Analysis of minute amounts of GCF reflects clinical changes occurring within the gingiva. The potential for using GCF as a diagnostic and prognostic tool has become apparent over the past few decades. Recent studies have demonstrated that qualitative and quantitative measurements of GCF reflect the extent of gingival and periodontal inflammation and deterioration.
The origin, composition, and clinical significance of gingival crevicular fluid have been studied over the past 60 years. (See History) The constituents of GCF originate from serum, gingival tissues, and from both host response and bacterial cells present in the sulcus or pocket and surrounding tissues. GCF is collected for analysis via absorbent paper filter strips, reweighted twisted threads, and micropipettes.
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In 2004, Armitage et al. identified more than 65 GCF constituents as potential diagnostic "markers." Biomarkers are measurable indicators of some biological state or condition. Due to impressive advancements, biomarkers can now be measured and evaluated to examine pathogenic processes.
Analysis of gingival crevicular fluid focuses on inflammatory markers such as prostaglandin E2 (PGE2), tumor necrosis factor (TNF), alkaline phosphatase, ß-glucuronidase, elastase, and aspartate aminotransferase (AST). Inflammatory indicators differentiate between states of health, gingivitis, and periodontal disease. For example, the presence of the enzyme elastase is highly correlated with collagen and clinical attachment loss. The hope is to utilize the information gleaned to direct future diagnosis and treatment planning.
Research findings have significantly facilitated the understanding of the pathogenesis and progression of periodontal disease. Recent longitudinal trials have focused on:
• The relationship of inflammatory markers found in GCF to the risk for developing periodontal disease.
• Identifying the severity of periodontal disease in patients with the condition (distinguishing destructive from quiescent lesions, chronic versus aggressive).
• Comparing health, gingivitis, and periodontitis gingival crevicular fluid components.
• Detection of early changes that could indicate the onset of disease. Other trials have investigated GCF bacterial types.
Further, the investigation of inflammatory markers in the GCF may assist in defining how certain systemic diseases (e.g., diabetes mellitus) can modify periodontal disease, and how periodontal disease can influence certain systemic disorders (e.g., atherosclerosis, preterm delivery, diabetes mellitus, and some chronic respiratory diseases).
Clinical significance of gingival crevicular fluid
Gingival crevicular fluid is an extravascular, serumlike fluid secreted from the underlying connective tissue into sulcular spaces. GCF, part of the body's defense mechanism, transports inflammatory mediators, antibodies, and certain systemically administered drugs (e.g., doxycycline, metronidazole). GCF can be found in the healthy gingival sulcus, but it is commonly sited in the gingival or periodontal pocket.
Gingival crevicular fluid begins to form and flow after 24 hours without biofilm removal and significantly increases in the presence of inflammation. GCF is called a serum transudate or suppuration (depending on the author) when it is a clear, serous liquid without larger molecules, such as proteins and cell debris. GCF is known as a purulent exudate (pus) when it is cloudy and contains larger molecules, such as living and dead polymorphonuclear leukocytes, bacteria, necrotic tissue, proteins, and enzymes. Gingival crevicular fluid reflects the components of the serum, the cellular response of the periodontium, and contributions from the gingival crevice. The components can vary greatly within and among individuals.
Although purulent exudate is a striking sign of inflammation, it does not indicate the severity or rate of progression of inflammation, probing depth, or clinical attachment loss (CAL). Some pockets form pus, and some do not, regardless of depth. Suppuration correlates with attachment loss only 2% to 30% of the time. Thus, it is not a reliable indicator of active periodontal damage. The last 60 years of GCF research has made progress identifying the level of damage to the periodontium in people with periodontal disease. Given laboratory assessment results of the GCF components, treatment can then be focused and monitored based on the disease activity level.
Assessing the data obtained via GCF collection allows a number of conclusions to be drawn concerning the potential diagnostic value of GCF constituents:
• A dramatic host inflammatory response is associated with progressive disease in periodontal patients.
• Collection of GCF using absorbent strips is a reproducible and reliable collection technique.
• The total amount of mediators can be reported when timed samples are collected.
• Current technology offers a practical diagnostic test that is feasible for private dental offices.
Gingival crevicular fluid components and analyses
The composition and flow rate of GCF serve as a barometer of the intensity of inflammation. GCF is composed of enzymatic and nonenzymatic components, prostaglandin E2, and cytokines, such as interleukins and tumor necrosis factor. It is evident that since periodontal disease is associated with the presence of certain bacteria, recognized as principal etiological agents, then factors derived from such bacteria may be useful indicators of metabolic activity.
Enzymes are large biological molecules responsible for thousands of metabolic processes. They are highly selective catalysts that accelerate metabolic reactions. Most are proteins. Enzymatic components found in GCF are either host-derived or bacteria-derived, and the nonenzymatic components include cellular elements, electrolytes, and organic material, such as albumin (see "Enzymatic Components of GCF" sidebar).
Prostaglandins stimulate bone resorption and were first identified in 1974; elevated levels are found in the GCF of patients with periodontitis, but not in the sulci of individuals with gingivitis. PGE2 levels are three times higher in younger patients with periodontitis than adults with the disease. A correlation has been found between increased levels of PGE2 concentration and clinical attachment loss in patients who were diagnosed with moderate-to-severe periodontitis. Biomarkers of bone resorption can also be evident in GCF during orthodontic treatment.
Cytokines are a broad category of small proteins responsible for cell signaling and are produced and released by a variety of cells including macrophages, lymphocytes, fibroblasts, and endothelial cells. They are potent local mediators of inflammation. Cytokines in GCF that indicate the presence of periodontal disease include interleukins and TNF. Cytokines play an important role in the host response to infection and inflammation. Cytokines that have been investigated as potential diagnostic markers for periodontal disease are: Interleukins-1a and -b, -6, and -8, and TNF. Interleukin-6 has been associated with progressive periodontitis and bone resorption; interleukin-8 is significantly higher in individuals with periodontal disease. Anti-inflammatory interleukins such as I-4, I-10 and I-15 are found to be deficient during active disease. In health, interleukins are more in balance.
Enzymatic components of GCF
• Alkaline phosphatase is an important enzyme produced by fibroblasts and neutrophils. It plays a role in the turnover of the periodontal ligament, root cementum formation, and bone homeostasis. Elevated levels found in GCF precede CAL; levels are higher at sites undergoing progressive change. The presence of alkaline phosphatase is considered a diagnostic marker for active periodontitis.
• ß-glucuronidase is an enzyme that is active in the hydrolysis of intercellular ground substance. Therefore, destructive periodontal activity would be associated with increased GCF levels. Healthy patients had no ß-glucuronidase in their GCF. Higher levels have been found in patients with CAL.
• Elastase is an abundant enzyme released from neutrophils and is an indicator of neutrophil activity. Elastase works apart and along with phagocytosis - it can degrade elastin and collagen. Levels increase in gingivitis. Its presence is predictive of periodontal attachment loss. Smokers have higher levels than nonsmokers.
• Aspartate aminotransferase (AST) presence in the GCF is indicative of cell death. PerioGard offers a monitor that measures AST.
• Hyaluronidase aids in the destruction of connective tissue and has properties similar to collagenase. It is also produced by some bacteria/periodontal pathogens. See below.
Bacteria-derived enzymes: The presence of these enzymes directly corresponds to the type and quantity of the bacteria in the sample.
• Acid and alkaline phosphatase break up the molecular bonds of phosphate groups in an acidic or alkaline oral environment, respectively.
• Collagenase is the most plentiful protein in the oral cavity. Collagenase deteriorates portions of the periodontium that contain collagen.
• Hyaluronidase. See above.
Nonenzymatic components of GCF:
• Cellular elements such as desquamated epithelial cells (indicative of a high turnover rate of the epithelium), PMNs, monocytes, and macrophages are all present in GCF during an inflammatory episode.
• Electrolyte levels vary depending on the degree of inflammation. Increased concentrations of potassium, calcium, sodium, and magnesium are seen in the GCF of patients with severe periodontitis.
• Organic components found in GCF are carbohydrates such as hexuronic acid and proteins such as fibrinogen, albumin, globulins, and lipids. Carbohydrates are three to four times more abundant in the GCF of inflamed sites.
Collection of gingival crevicular fluid
The collection and analysis of GCF are relatively noninvasive methods to assess the host response in health and the pathophysiology of gingivitis and periodontitis. It is a viable and reliable diagnostic tool. The fluid can be collected using absorbent filter paper strips, reweighted twisted threads (Weinstein), micropipettes (Krasse and Egelberg, collected via capillary action), or crevicular washings (Oppenheim). Volume and constituents of the fluid are calculated during assay.
Fluid initially accumulates at the gingival margin. Extracrevicular samples are taken at the gingival margin; intracrevicular samples are taken in the sulcus or pocket until resistance is felt (Brill method). Some research projects utilize samples taken at a constant depth, such as 4 mm deep at each site.
The disadvantage of the intracrevicular method, especially with micropipettes, is that it introduces a degree of irritation, which can trigger fluid flow. The crevicular washing technique employs a peristaltic pump that permits collection without disturbing sulcular tissues. This method is time consuming, difficult when fluid is viscous, and dilutes the GCF. All collection methods are challenging due to the trauma inflicted and the possible contamination of the samples with blood, saliva, or biofilm. Methods of estimating the volume collected in samples include:
• Absorbent strips - A Periotron machine, an electronic method, is used to measure the distance the fluid has migrated up the strip and indicates volume collected. Strips can also be stained and viewed with UV light to calculate the volume of GCF.
• Reweighted twisted threads - The fluid collected is weighed and analyzed.
• Micropipettes - The fluid is collected, centrifuged, weighed, and analyzed.
It is important to note that because of a lack of uniformity in the methods used for collection and quantitation, comparisons between different studies in the literature often have not been feasible. Several companies manufacture and distribute supplies for collecting GCF, and some conduct analysis of and tabulate the data of the GCF components in samples. Kits available for collection are:
• Periocheck (approved by the FDA)
• PerioGard (AST)
• Biolise (Elastase)
• Prognostik (Elastase)
• PocketWatch (AST)
• TOPAS (bacterial toxins and proteases)
• MMP dipstick (MMPs)
Gingival crevicular fluid flow
Substances, depending on their molecular size, are transported across the junctional and sulcular epithelium due to the permeability of these structures. The details of this process were discovered during animal research in 1970; prior to that, fluid appearing in the sulcus or pocket was thought to be because of change in osmotic pressure. The flow of the gingival crevicular fluid is from the surrounding tissues into the crevice and vice versa, as needed, depending on oral health status. In the mid '70s, research defined preinflammatory fluid as a transudate and an inflammatory exudate upon stimulation.
By the early '80s, scientists confirmed that a fluid was often present in minute amounts in the gingival crevice. Some believed it to be an inflammatory exudate; others thought the fluid served other purposes - to clean material from the crevice, to improve adhesion of the epithelial attachment with sticky plasma proteins, and to offer antimicrobial properties in defense of the gingiva.
What is now common knowledge is that the gingival crevicular fluid forms when bacterial biofilm constituents stimulate the site and inflammatory biomarkers infiltrate. The biomarkers found in GCF are byproducts of the breakdown of collagen and biochemical material released by macrophages and neutrophils, etc. The fluid passes through permeable epithelium and flows into the crevice via capillaries. When biofilm is not removed, the body sends mechanisms of defense via the GCF into the sulcus to begin fighting off offending agents. GCF components are removed from these areas filled with undisturbed biofilm and inflammatory mediators through the lymphatic system. When the rate of capillary infiltrate exceeds that of lymphatic uptake, fluid accumulates in the sulcus or pocket. Even in health, if the osmotic pressure of the sulcular fluid exceeds that of the tissue fluid, there will be a net increase in the flow of GCF (see "Conditions that affect flow rate"). Biofilm-derived molecules initiate this process.
Gingival crevicular fluid is now regarded as a promising medium for the detection of biomarkers and the corresponding levels of periodontal disease activity. Monitoring GCF flow, components, and stage of disease activity can be useful diagnostically - early diagnosis and disease prevention, assessing the severity of gingival and periodontal inflammation, the effectiveness of oral hygiene regimens, the response of tissues to periodontal therapy, and the efficacy of antibiotics as adjuncts to periodontal therapy. Introduction of GCF-based diagnostic tests can provide clinicians with an improved quantitative and qualitative means of evaluating patients' dental health status.
Conditions that affect flow rate are:
• Chewing and vigorous brushing temporarily increases the rate of flow.
• Postperiodontal surgery healing increases flow for approximately five weeks.
• Placing a test strip or micropipette in the sulcus or pocket temporarily increases the production of GCF.
• Gradual increases in flow rate have been noted between 6 a.m. and 10 p.m. Flow decreases for the following eight hours.
• Pregnancy, ovulation, and hormonal contraceptives all increase gingival fluid production (which explains why gingival health status sometimes changes during these periods).
• When a patient irrigates, a dramatic decrease of inflammatory mediators and increase in anti-inflammatory agents in the GCF occurs.
• The flow rate of GCF increases up to 30 times in periodontally involved versus healthy sites.
• Smoking produces an immediate and marked increase in gingival fluid flow.
• Research has demonstrated that GCF flow rate is about equal in periodontitis versus gingivitis.
GCF research has advanced far in a short period of time. Regardless, many questions remain.
Still to be researched and developed are:
• The relationship between the likelihood of developing periodontitis in patients with gingivitis markers.
• The level of test accuracy needed to make using GCF sample testing reasonable, realistic, and justifiable.
• The need for such tests as perceived by clinicians.
• How clinicians can utilize and implement test results into private-practice treatment plans.
• The development of a wide spectrum of marker factors, or "marker packages," since no single marker fully assesses the clinical state of the periodontium. RDH
DIANA J. LAMOREUX, RDH, BS, MEd, graduated from Ohio State University in 1972, practiced dental hygiene for over 30 years, was a part-time clinical instructor in the Cleveland area since 1981, and retired in December 2011.
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