Gingival Crevicular Fluid as a Biomarker for Periodontal Disease

Gingival crevicular fluid (GCF) is found within the physiologic gingival sulcus and pathologic periodontal pocket.¹ Considered an inflammatory exudate,¹ GCF seeps through the connective tissue within the gingiva and lining of the sulcus. Research has shown that GCF directly relates to periodontal disease. In health, the flow rate of GCF is close to nothing;¹ however, in a periodontal pocket, its composition changes with the presence of inflammation. Thus, increased GCF flow may be a sign of periodontal disease.¹ While many factors contribute to periodontal conditions, this article will discuss how testing GCF can aid in the assessment and diagnosis of the stages of periodontal disease — and its correlation to systemic disease. 

This inflammatory exudate includes serum, inflammatory mediators, antibodies, and materials formed from the breakdown of tissue.² These components can be divided into several sections, such as cellular elements, electrolytes and organic compounds. The cellular elements include epithelial cells, leukocytes and bacteria from dental plaque.² The electrolytes include sodium and potassium in a 3:9 ratio, fluoride, calcium, iodine and phosphorus.² The organic compounds are comprised of carbohydrates, proteins, immunoglobulins, cytokines and metabolic bacterial products.² Produced at a rate of a few microliters per hour,² the fluid acts as a host defense mechanism by flushing bacteria away from the sulcus through the route of diffusion from the basement membrane to the junctional epithelium and into the sulcus.² Several factors can stimulate the flow of GCF, including pocket depth, gingival inflammation, mobility, periodontal surgery, enzymes, sex hormones, contraceptives and smoking.² Because the amount of GCF changes in the periodontium, this fluid can be used as a biomarker to identify the various stages of periodontal disease. 

Research shows there are many biomarkers in GCF that are currently used to diagnose periodontal disease progression.^3,4 Numerous studies suggest that interleukin-1 beta (IL-1β), IL-2, IL-6, IL-8, IL-17 and tumor necrosis factor-alpha (TNF-α) are reliable inflammatory biomarkers in patients with periodontal disease.¹ The most studied biomarker is the bone-resorbing cytokine IL-1β.¹ Previous studies demonstrated the volume of IL-1β in GCF was elevated in active sites of periodontal disease and declined after periodontal treatment.¹ Thus, GCF provides a laboratory tool for assessing periodontal disease activity. 

Longitudinal studies have found high levels of biomarkers, such as matrix metalloproteinase-8 (MMP-8), MMP-9 and IL-1β in patients with high sensitivity and periodontal disease progression.^3,4 As a sensitive and unbiased biomarker, MMP-8 helps with the early diagnosis of periodontitis. Furthermore, in a cross-sectional study, mild gingivitis and mild periodontitis were used to compare MMP-8 levels acting as an inflammatory marker in periodontal disease.³ The results demonstrated that MMP-8 concentrations are higher in individuals with mild periodontitis than in subjects with gingivitis or gingival health.³ These studies show how GCF can act as a biomarker regarding the progression of periodontal disease and loss of alveolar bone. 

TESTING 

Multiple options exist for the noninvasive collection of GCF, which include the use of pre-weighed twisted threads, the absorption method, microcapillary pipetting, and the washing method.^2,4 The use of pre-weighed twisted threads involves placing threads into the gingival crevice around the tooth, where the fluid is collected. The threads are weighed to determine the amount of fluid collected.² The absorption method uses paper strips that are dipped into the sulcus.^2,4 This is the least traumatic, most efficient and easiest way to obtain a GCF sample.⁴ With the microcapillary pipetting method, the pipette remains in the fluid for approximately 10 minutes, and the resulting levels of each biomarker indicate the severity of periodontitis.⁴ The washing method, which is not common due to its high rate of contamination, extracts GCF through the injection of two needles, which can lacerate the tissue and contaminate the GCF sample with blood.⁴ 

It is important to note the use of more than one biomarker to determine inflammation activity in periodontal disease is highly recommended.^2,4 The two preferred cytokines for determining inflammation in the periodontium are IL-1β and MMP-8.⁴ Conversely, more research is needed to determine the most reliable and accurate technique that can be used as the universal standard.⁴ Additionally, it is unclear how stress and genetic differences among different ethnic groups affect the biomarkers in GCF. 

DIAGNOSTIC USE IN ORTHODONTICS 

The diagnostic use of GCF testing also aids orthodontic therapy. During orthodontic treatment, teeth are moved through a controlled mechanical force, which creates a biological reaction in the periodontium.⁵ This biological reaction is reflected in the patient’s GCF levels.⁵ The specific biomarkers of bone alkaline phosphatase and osteoprotegerin create a new understanding of bone growth and remodeling.⁵ Bone deposition-related biomarkers, including MMP-8, can be used as a guide in identifying the results of orthodontic forces in the periodontal ligament and alveolar bone.⁵ 

A study with prepubertal participants used alkaline phosphatase as a biomarker in GCF to determine the activity of active alveolar bone formation during a retention phase of rapid maxillary expansion.⁵ The research revealed an increase of alkaline phosphatase activity in the GCF during months three and six.⁵ This confirmed that alkaline phosphatase can be used as a diagnostic method for assessing orthodontic movement, as it was found to be more active in sites of movement.⁵

An advantage of using GCF as a biomarker in orthodontics is ease of testing; additionally, quick sample testing avoids the use of radiographic exposure.⁶ However, further research regarding GCF biomarkers’ clinical applicability is needed to confirm its diagnostic accuracy.⁶ 

APPLICATIONS IN IMPLANTOLOGY

In patients with dental implants, GCF, called peri-implant fluid, can be collected from implant sites to determine its profile and note specific markers. When compared to mildly inflamed sites, GCF shows higher levels of neutral proteases at moderately to severely inflamed implant sites.² Moreover, IL-1β levels in these sites have been shown to be approximately three times higher than levels found in healthy sites.² Future studies that focus on the equilibrium of chemistry changes in GCF could aid in early detection of peri-implant disease.

EVIDENCE OF SYSTEMIC DISEASE

Systemic conditions and modifiers represent an increased risk factor for periodontal disease. These conditions include, but are not limited to, diabetes, human immunodeficiency virus (HIV), obesity, atherosclerosis and tobacco use. Diabetes mellitus is categorized as a multifactorial disease due to its disruption of the regulation of the endocrine and metabolic pathways.⁷ Both periodontal disease and diabetes are inflammatory ­diseases that involve a variety of cells, including endothelial cells, adipocytes and cytokines.⁷ It has been shown that periodontal disease can lead to poor glycemic control, and play a role in severe alveolar bone loss.⁷ Studies show the levels of IL-1β, TNF-α and prostaglandin in GCF are significantly higher compared to non-diabetic controls with a similar periodontal status.⁷ Moreover, studies that investigated glycemic control and IL-1β in GCF levels in patients with type 2 diabetes and periodontitis found a significant correlation in both periodontal measures and glycemic control measures, with increased IL-1β in GCF.⁷ Additionally, GCF levels of IL-1β and prostaglandin increased in individuals with diabetes as their severity of periodontal disease increased.⁷

Specific biomarkers in GCF taken from patients with HIV present a profile conducive to determining periodontal disease progression. Studies show an increase of the IgG antibody in the GCF, along with IL-1β in deep periodontal pockets.⁸ High levels of IL-1β and IL-6 are associated with periodontal disease in patients with HIV, as opposed to uninfected patients, which suggests these high cytokine levels are responsible for the periodontal lesions observed in those infected with HIV.⁹ The presence of HIV in leukocytes found in GCF suggests these cells could be the intraoral source of the HIV virus.¹⁰ However, more studies are needed to verify this.

Nevertheless, biomarkers in GCF from patients with HIV affirm the role of cytokines in the advancement of periodontitis in seropositive patients.¹¹

There is evidence in GCF of the relationship between obesity and periodontal disease. Cetiner et al¹² reported a positive correlation between periodontal disease and visfatin levels in GCF. Visfatin is an adipocytokine that plays an important role in immune functions as a growth factor, enzyme and pro-inflammatory mediator.¹² Research also shows visfatin increases the number of sites with periodontal disease.¹² Additionally, the levels of visfatin and IL-6 in GCF are higher in obese patients as compared to nonobese individuals and in persons with periodontitis, as compared to healthy periodontal sites.¹² However, investigators also found that levels of visfatin can decrease in some obese groups, suggesting that obesity and periodontitis can independently, or together, change pro-inflammatory adipocytokines levels in GCF.¹² Thus, levels of visfatin and IL-6 in GCF in relation to the pathogenesis of obesity and periodontal disease serve as two markers that may be used to monitor the progression of both diseases.

Smoking is known to increase disease risk, including periodontal disease risk. In gingival tissue, microcirculation of gingival blood flow and GCF volume are greatly reduced in smokers, but have been found to recover after smoking cessation.¹³ The cytokine profiles in GCF suggest smoking has an effect on the cytokine network.¹⁴ Compared to nonsmokers, research shows smokers display higher levels of IL-8, but lower levels of IL-4.¹⁴ Levels of IL-6 were found to be elevated in patients with periodontitis who also smoked, and IL-1β concentration was also lower in smokers than nonsmokers.¹⁴ 

Although there are limited data, GCF levels of inflammatory mediators have been studied in relation to cerebrovascular diseases. Research suggest higher levels of leukotriene and cysteinyl-leukotrienes exist in GCF in patients with atherosclerosis.¹⁵ Though more study is needed, these biomarkers can indicate inflammation that increases the risk for atherosclerosis associated with periodontal disease.¹⁵

SALIVARY MEDIATORS 

Saliva is a combination of oral fluids, including secretions from salivary glands, bronchial and nasal secretions, serum and blood derivatives from oral wounds, bacteria and bacterial byproducts, viruses, fungi, desquamated epithelial cells, food, cellular components and GCF.¹⁶ The analysis of GCF components in saliva may aid the development of a simple diagnostic test for periodontal disease.¹⁶ Analysis of saliva offers some advantages when used for diagnostic purposes. One advantage is that saliva can be easily collected in a noninvasive manner and without the need for special equipment.¹⁶ In addition, salivary analysis may provide a feasible, cost-effective approach for large-scale screening of patients.¹⁶

The inflammatory marker β-glucuronidase (βG) in GCF has been known to identify patients at risk for periodontal disease.¹⁷ Studies show a strong positive correlation between salivary levels of βG and an increase in attachment loss or probing depth at multiple sites (e.g., ≥ four sites with ≥ 5 mm probing depth).¹⁷ The available data provide evidence that increased salivary levels of βG, a GCF marker that has been linked to an increased risk of periodontitis, could be used for diagnostic screening for periodontal conditions.¹⁷

CHAIRSIDE TESTING

The typical clinical methods for assessing a patient’s periodontal condition tend to uncover the damage the periodontium has already endured. They reveal disease history, as opposed to disease activity. It is important to find a diagnostic tool that can predict and assess active periodontal disease.⁴ Such methods have already been developed, such as MMP-8 chairside testing that is beneficial during the maintenance phase of periodontal therapy.⁴ The analysis of GCF could be used in collaboration with clinical assessments to better examine periodontal disease activity. Select GCF components are proven to act as biomarkers for periodontal disease, which include host-derived enzymes, host-response modifiers, and tissue breakdown products.⁴ Collecting GCF and analyzing its composition can help determine periodontal disease activity and predict future disease progression.

Discussing the importance of oral health and educating patients about effective self-care are important tools in preventing periodontal disease. Additionally, equipping patients with the knowledge to recognize the initial visible changes in the gingiva when periodontal disease first progresses can motivate patients to seek treatment early. Implementing diagnostic techniques — such as chairside GCF testing — during routine checkups can help promote early detection, treatment, and the arrest of periodontal disease. Increased flow of GCF can be used as a biomarker in conjunction with traditional diagnostic methods to detect periodontal disease.¹⁸ In addition, clinicians can utilize biomarker analysis to help customize periodontal treatment and provide individualized recommendations. 

FUTURE DIRECTION OF PERIODONTAL DIAGNOSIS

The complex nature of periodontal disease makes identifying a single diagnostic marker for disease detection and prediction challenging. Today’s medical models use genetic, genomic, environmental and clinical diagnostic testing to individualize patient care.¹ Utilization of this approach in oral healthcare has the potential to support highly individualized diagnosis, prognosis and personalized periodontal therapy.¹ Personalized treatment based on analyzing a combination of markers may provide a more accurate assessment of the periodontal condition and could be useful in identifying disease progression.¹ Using oral fluids, such as saliva and GCF, as periodontal diagnostic tools to aid decisions regarding disease susceptibility, site-specific risk of disease progression, and treatment modalities holds promise in advancing periodontal assessment and therapy.¹

CONCLUSION

The analysis of GCF through its microbial and host interactions associated with the onset and progression of periodontal disease has the potential to expand our understanding and facilitate the discovery of diagnostic, prognostic and therapeutic markers.¹⁸ The two main biomarkers, IL-1β and MMP-8, are present in healthy and diseased gingiva; thus, GCF testing can support periodontal risk assessment, diagnosis and treatment planning. There are various options to obtain GCF through atraumatic techniques, which include pre-weighed twisted threads,  the absorption method and microcapillary pipetting.

While there are other ways to diagnose the progression of periodontitis, GCF testing is an alternative that is proven to show periodontal disease activity. In short, when used as a screening and diagnostic tool, it can help support improved outcomes. 

REFERENCES

1. Ghallab NA. Diagnostic potential and future directions of biomarkers in gingival crevicular fluid and saliva of periodontal diseases: review of the current evidence. Arch Oral Biol. 2018;87:115–124.
2. Subbarao KC, Nattuthurai GS, Sundararajan SK, Sujith I, Joseph J, Syedshah YP. Gingival crevicular fluid: an overview. J Pharm Bioallied Sci. 2019;11:135. 
3. Kasuma N, Oenzil F, Darwin E, Sofyan Y. The analysis of matrix metalloproteinase-8 in gingival crevicular fluid and periodontal diseases. Indian J Dent Res. 2018;29:450–454.
4. Majeed ZN, Philip K, Alabsi AM, Pushparajan S, Swaminathan D. Identification of gingival crevicular fluid sampling, analytical methods, and oral biomarkers for the diagnosis and monitoring of periodontal diseases: a systematic review. Dis Markers. 2016;2016:1804727. 
5. Perinetti G, Franchi L, Castaldo A, Contardo L.  Gingival crevicular fluid protein content and alkaline phosphatase activity in relation to pubertal growth phase. Angle Orthod. 2012;82:1047–1052. 
6. de Aguiar MC, Perinetti G, Capelli J. The gingival crevicular fluid as a source of  biomarkers to enhance efficiency of orthodontic and functional treatment of growing patients. Biomed Res Int. 2017;3:1–7. 
7. Pooja S, Varghese S. Gingival crevicular fluid level of interleukin 1 in chronic periodontitis with diabetes mellitus. Drug Invention Today. 2019;12:1–4.
8. Grbic JT, Lamster IB, Mitchell-Lewis D. Inflammatory and immune mediators in crevicular fluid from HIV-infected injecting drug users. J Periodontol. 1997;68:249–255. 
9. Baqui AA, Meiller TF, Jabra‐Rizk MA, Zhang M, Kelley JI, Falkler WA Jr. Enhanced interleukin 1β, interleukin 6 and tumor necrosis factor α in gingival crevicular fluid from periodontal pockets of patients infected with human immunodeficiency virus 1. Oral Microbiol Immunol. 2000;15:67–73.
10. Suzuk T, Tai H, Yoshie H, et al. Characterization of HIV‐related periodontitis in AIDS patients: HIV‐infected macrophage exudate in gingival crevicular fluid as a hallmark of distinctive etiology. Clin Exp Immunol. 1997;108:254–259.
11. Alpagot T, Font K, Lee A. Longitudinal evaluation of GCF IFN‐γ levels and periodontal status in HIV+ patients. J Clin Periodontol. 2003;30:944–948.
12. Çetiner D, Uraz A, Öztoprak S, Akça G. The role of visfatin levels in gingival crevicular fluid as a potential biomarker in the relationship between obesity and periodontal disease. J Appl Oral Sci. 2019;27:e20180365.
13. Morozumi T, Kubota T, Sato T, Okuda K, Yoshie H. Smoking cessation increases gingival blood flow and gingival crevicular fluid. J Clin Periodontol. 2004;31:267–272.
14. Kamma JJ, Giannopoulou C, Vasdekis VG, Mombelli A. Cytokine profile in gingival crevicular fluid of aggressive periodontitis: influence of smoking and stress. J Clin Periodontol. 2004;31:894–902.
15. Bäck M, Airila-Månsson S, Jogestrand T, Söder B, Söder PO. Increased leukotriene concentrations in gingival crevicular fluid from subjects with periodontal disease and atherosclerosis. Atherosclerosis. 2007;193:389–394. 
16. Lamster IB, Ahlo JK. Analysis of gingival crevicular fluid as applied to the diagnosis of oral and systemic diseases. Ann NY Acad Sci. 2007;1098: 216­–229. 
17. Lamster IB, Oshrain RL, Harper DS, Celenti RS, Hovliaras CA, Gordon JM. Enzyme activity in crevicular fluid for detection and prediction of clinical attachment loss in patients with chronic adult periodontitis: six month results. J Periodontol. 1988;59:516–523.
18. Tsuchida S, Satoh M, Takiwaki M, Nomura F. Current status of proteomic technologies for discovering and identifying gingival crevicular fluid biomarkers for periodontal disease. Int J Mol Sci. 2018;20:86.

From Decisions in Dentistry. April 2021;7(4):40–43.