Relevance of gingival crevice fluid components in assessment of periodontal disease - A critical analysis.

Monitoring infections which are episodic, site specific, localized or generalized, initiated by a number of microorganisms and assessed with measurement devices that have inherent shortcomings presents a difficult task. The lack of precise clinical criteria for assessment of periodontal disease has led to a search for alternative means of determining active disease sites, predicting future sites of breakdown and evaluating response to therapy. This paper highlights the potential array of biomarkers present in gingival crevice fluid (GCF) and provides an insight of which of these factors has the greatest diagnostic potential. The rationale behind using GCF as sampling source is also described. Finally it discusses the possible use of predictive chair side diagnostic tests in periodontitis.

INTRODUCTION

Periodontal disease is a chronic bacterial infection characterized by persistent inflammation, connective tissue breakdown and alveolar bone destruction. Contributing inflammatory mediators and tissue destructive molecules have been detected in the gingival tissues, gingival crevice fluid (GCF) and saliva of patients affected by periodontitis, and qualitative changes in the composition of these biomarkers could have diagnostic and therapeutic significance.[1]

WHY GCF MARKERS — RATIONALE OF GCF SAMPLING

GCF contains a rich array of cellular and biochemical molecules associated with disease activity. In terms of application to diagnostic tests, sampling of crevice fluid provides advantages that are analogous to drawing of blood.[2] It is noninvasive, site specific about teeth, comparatively easy to obtain and offers one of the most accessible entrees to any tissue in the body as means of assessing the disease state.[3] Furthermore, it is an inflammatory exudate that reflects ongoing events in the periodontal tissues that produce it. This has led to extensive search of GCF components that might serve as potential diagnostic and prognostic markers for determining progression of periodontitis.

Various substances are measured in GCF, and reports have suggested that some of these could be used to detect or predict periodontal disease activity[4] ; however, differences in experimental design among various investigations have made it difficult to identify which parameter has the most promising potential as a diagnostic marker.

In this article, we review the biological basis of these various GCF components that could be used as potential diagnostic markers and evaluate their efficiency in predicting future disease. Finally to evaluate if these patients are better off as a result of such testing.

However, the crevicular fluid sampling has some limitations [Figure 1]. They are as follows:

Figure 1

GCF as a source of biomarkers

Nonaccurate and nonreproducible

No uniform consensus on choice of collection device, its placement and collection time

Potential depletion of sample by prolonged collection

Potential contamination by serum components and loss of sample from the collection device

Variability in calculation of data as absolute measures or as flow rates

The fluid is collected by various means, depending upon the nature of the analyte under investigation, e.g., filter paper for enzyme assays and micro-capillary tubes for connective tissue metabolites.

Thus there is no consensus obtained on which protocol has the lowest bias, the highest repeatability and the strongest validity. Although some studies have shown that sampling for 30 seconds or less by placement of paper strips at the site seems to be the most ideal, provided a sample of sufficient size to perform the analysis can be obtained.[5]

PATHOGENESIS OF PERIODONTITIS

Bacterial pathogens are primary etiologic factors in initiation of periodontitis.[5] They trigger the local inflammatory response, which leads to accumulation of Polymorphoneutrophils, macrophages, lymphocytes and mast cells, which are very important in protecting the body against infection. These cells contain lysosomal enzymes, which are normally used to degrade phagocytozed material but are capable of degrading gingival tissue components if released. Such enzymes may be released by inflammatory cells during their function or when they degenerate or die. Cells and tissues in the vicinity of these enzymes may be damaged, and this process is known as bystander damage.

The main tissues damaged in this process are the connective tissue components like collagens and proteoglycans, and the breakdown of these tissues around the inflammatory cells helps the spread of these cells through the tissues.

Most of these substances that are released in the tissues pass into the GCF and fall into 3 general categories[5-7]:

Inflammatory mediators and host-response modifiers

Host-derived enzymes and their inhibitors

Tissue breakdown products

Potential immune and inflammatory mediators

Immune response

Antibody: Total immunoglobulin and IgG subgroups complement

Inflammatory response

Arachidonic acid derivatives, e.g., PGE2

Cytokines, e.g., IL-1, IL-2, IL-4, IL-6, TNF-A

Bone-specific proteins

Osteonectin

Bone phosphoprotein (N-propeptide)

Osteocalcin

Telopeptides of type 1 collagen

THE SEARCH FOR CLINICALLY USEFUL GCF MARKER IN PROGRESSION OF PERIODONTITIS — CHALLENGES AND OPPORTUNITIES

When assessing the clinical value of a new assay, one must initially determine, in cross- sectional studies, if the selected marker is present in periodontitis and absent in health or if it is directly related to the severity of periodontitis.[8]

Most proposed GCF markers can easily distinguish between healthy and inflamed sites but cannot distinguish between gingivitis and periodontitis as most of these components are some sort of inflammatory markers and are significantly elevated in both conditions. Finally only longitudinal studies can distinguish progressive sites from nonprogressive ones.

The next step in the evaluation of a new assay involves a demonstration that the suspected marker decreases in response to therapeutic intervention such as scaling and root planing, wherein the clinician must be able to decide whether or not the treatment is completed.

Various studies state that neutral proteases, glycosaminoglycans and PGE2 reduce post treatment.[9] However, the difficulty is to identify post-treatment sites that might still be at high risk for progression, and the clinician has to rely on clinical judgement for this purpose.

Once the assay has been shown to have a significant relationship to periodontitis, longitudinal studies are conducted to determine if there is an association with disease progression. Neutral proteinases, cathepsin-B and B-glucornidase[9] show positive relation.

However, the biggest roadblock to the longitudinal evaluation of a new periodontal test is the lack of clear “gold standard” for disease activity. In longitudinal studies, a measurable loss of attachment over the course of the study is generally recognized as the criterion for progression.

If a fully validated site-specific GCF test to determine the progression of periodontitis were to be discovered, in what clinical situations would it be most useful? High on the list would be, in patients who have been treated and are in maintenance phase of therapy.

Preliminary reports suggest that GCF level of cathepsin-B, dipeptidyl peptidases, matrix-metalloproteinases and collagenase shows some promise as potentially useful markers for the risk of disease progression in maintenance patients.[9] However, uncertainties regarding which sites to be tested still exist.

If one actually has a validated GCF marker of progression, the final evaluation step would be to conduct randomized controlled clinical trials designed to determine if use of the test resulted in better treatment outcomes.

However, these types of studies are nonexistent, and thus a validated marker has not yet emerged.

The components of gingival crevice fluid are analyzed with regard to their potential utility in fulfilling the following aims [Table 1]:[10]

Table 1

Potential diagnostic markers in GCF

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, e.g., to determine when renewed maintenance care is needed based on the level of disease activity

Its potential diagnostic utility is listed as follows:

WHICH OF THE GCF MARKERS HAVE THE MOST PROMISE ?

Are they good indicators of risk of periodontal disease at individual level?

Traditional diagnostic tests are subjective, retrospective and not enough to detect small degrees of periodontal damage. Furthermore, they cannot identify susceptible individuals nor can they differentiate between disease-active and disease-non-active sites. GCF based tests provide some hope in this regard, but which of these components have the greatest promise to act as diagnostic markers is not clearly known.

Among the inflammatory mediators

Studies claim that GCF PGE2 is predictive for periodontal disease activity. Levels greater than 66 ng/mL were found to be predictive of further possible loss of attachment with a sensitivity of 0.76 and specificity of 0.96 with an overall predictive value of 0.92 to 0.95,[1112] But it cannot clearly differentiate between gingivitis and periodontitis nor between active and inactive sites

Specific antibody or total immunoglobulin in GCF appears to be of no use in distinguishing between stable and progressive sites because —

Thus specific antibodies in gingival tissues and serum are important in modulating the pathology of periodontal diseases; but with the present level of knowledge, they do not offer a means of either identifying patients at risk for active disease or of predicting active sites within particular patients.[5]

The total immunoglobulin in GCF does not correlate with disease severity or progression and indeed may be lower at progressive sites than nonprogressive sites.[13]

Reduction in specific antibody in serum and consequently GCF in patients with existing disease can place them at risk for further disease progression.[13]

Among the cytokines, studies have shown association of elevated levels of IL-1B with gingival inflammation and severities of periodontitis. However, within a group of patients with similar levels of disease, differences were detected in GCF interleukin levels between groups of patients with different IL-1 gene polymorphisms.

Thus the most likely diagnostic marker of the inflammatory and immune factors is GCF PGE2.

Among the host-derived enzymes

Alkaline phosphatase, beta-glucuronidase, cathepsin-B, collagenase-2 (MMP-8), gelatinase (MMP-9), elastase and dipeptidyl peptidases II and IV may have potential diagnostic utility for treatment planning and for monitoring treated patients.[10]

In addition, cathepsin-B, collagenase-2 (MMP-8), dipeptidyl peptidases II and IV, and elastase seem promising for distinguishing periodontitis from gingivitis.[10]

No factor in GCF has been identified which can distinguish between aggressive and chronic periodontitis, although a multitude of these markers have been studied in both types of disease.

Among tissue breakdown product

Chondriotin-4-sulfate and bone-specific GAGs are the most promising potential markers as they reflect the degradation of bone. However, currently there are no longitudinal studies to evaluate the diagnostic and prognostic value of these markers.

CLINICAL USEFULNESS OF PREDICTIVE DIAGNOSTIC TEST

The potential markers are usually detected by laboratory assays in cross-sectional and longitudinal studies. They need to be simplified and modified to make an assay system suitable for use in a dental surgery.

COMMERCIALLY AVAILABLE DIAGNOSTIC TEST KITS

Periocheck — Neutral Proteinases — Approved by FDA[14]

Periogard — AST[15]

Prognostik- Elastase — Not Approved by FDA and ADA[14]

Biolise — Elastase[16]

Pocket watch — AST

TOPAS — Bacterial toxins and proteases

MMP dipstick method — MMPs[17]

Under development, for B — glucornidase and proteinases[14]

CONCLUSION

Today, no single GCF marker or combination of GCF markers is available to determine whether periodontal treatment is sufficient and⁄ or necessary to prevent further periodontal breakdown.

Some of the host tissue enzymes and tissue breakdown products are the most promising potential GCF markers of disease progression, especially of those diseases that degrade the bone. Future research on these bone markers will provide promising results.

Optimally, tests should be available in the form of chair-side or home-use dipstick tests, which can be self performed and which may indicate that a person is at risk for periodontal tissue loss and thus needs professional attention.

As was so aptly stated by Dr. Irwin Mandel at a clinical trial conference in 1986, “Instead of a magic bullet for treatment of periodontitis, we now seek the magic marker. There is reason to remain confident that markers of disease progression and enhanced subject susceptibility do exist and will be found. Nevertheless, no marker or any test based on a marker alone will provide us with the magic answer. At best, such tests will be useful as objective adjuncts to our traditional diagnostic procedures.”