|Year : 2014 | Volume
| Issue : 2 | Page : 89-92
A new dawn in modern perio-diagnostics: Saliva and gingival crevicular fluid
Harpreet Singh Grover, Rohit Saini, Pearl Bhardwaj, Amit Bhardwaj
Department of Periodontology, Faculty of Dental Sciences, SGT University, Gurgaon, Haryana, India
|Date of Web Publication||18-Jun-2015|
Dr. Amit Bhardwaj
Department of Periodontology, Faculty of Dental Sciences, SGT University, Gurgaon, Haryana
Source of Support: None, Conflict of Interest: None
Periodontal disease is a chronic microbial infection. There is a need for the evolvement of contemporary diagnostic tests to improve the clinical management. Molecular arrays for the detection of biomarkers on the different stages of the disease help in understanding various mediators of the disease process. This evolutionary process has promoted the discovery of new biomarkers. A periodontal diagnostic tool provides pertinent information for differential diagnosis and localization of disease. Human saliva is an easily accessible biochemical fluid, which is similar to blood in various biological aspects. Gingival crevicular fluid is an inflammatory exudate that represents serum components overlaid with products from local physiologic phenomena, and has diagnostic value.
Keywords: Biomarkers, diagnostic indicators, gingival crevicular fluid, saliva
|How to cite this article:|
Grover HS, Saini R, Bhardwaj P, Bhardwaj A. A new dawn in modern perio-diagnostics: Saliva and gingival crevicular fluid. J Dent Allied Sci 2014;3:89-92
|How to cite this URL:|
Grover HS, Saini R, Bhardwaj P, Bhardwaj A. A new dawn in modern perio-diagnostics: Saliva and gingival crevicular fluid. J Dent Allied Sci [serial online] 2014 [cited 2021 Sep 27];3:89-92. Available from: https://www.jdas.in/text.asp?2014/3/2/89/159091
| Introduction|| |
Periodontal disease is a chronic microbial infection that elicits inflammation-mediated loss of the periodontal ligament (PDL) and alveolar bone that supports the teeth. Because of its increasing prevalence and associated co-morbidities, there is a need for the evolvement of contemporary diagnostic tests that can ascertain the presence of active periodontal disease, predict its future progression, and evaluate the response to periodontal therapy, thereby improving the clinical management of periodontal patients. 
| Periodontal Diagnostic Indicator|| |
A periodontal diagnostic tool provides pertinent information for differential diagnosis, localization of disease, and severity of infection. These diagnostics, in turn, serve as a basis for treatment planning and provide a means for assessing the effectiveness of periodontal therapy. 
| Saliva as Diagnostic Indicator|| |
Human saliva is an easily accessible biochemical fluid, which is similar to blood in various biological aspects. Besides, it possesses a simple and noninvasive collection with low-cost storage and easily storage nature. The main constituent of this hypotonic bio-fluid is water comprising 99.5% of the total volume, with the rest of 0.5% being amino acids, histatins, cystatins, defensins, statherins, lysozyme, proline-rich proteins, carbonic anhydrases, peroxidases, lactoferrin, mucins, secretory immunoglobulins (Igs), lipids, together with various ions such as potassium, calcium (Ca), chloride, sodium, and phosphates.  Saliva, an important physiologic fluid, containing a highly complex mixture of substances, is rapidly gaining popularity as a diagnostic tool. Saliva, as a mirror of oral and systemic health, is a valuable source for clinically relevant information because it contains biomarkers specific for the unique physiologic aspects of periodontal diseases.  Saliva is also particularly useful for the detection of viral infection, especially HIV, due to the noninvasive method of collection, monitoring of hormone levels, especially of steroids and for the detection of banned abusive substances.
Immunoglobulins are important specific defense factors of saliva. The predominant Ig in saliva is secretory IgA, which is derived from plasma cells in the salivary glands and IgG and IgM which are in lesser amount found in saliva. IgA, IgG, and IgM influence the oral microbiota by interfering with the bacterial adherence or by inhibiting bacterial metabolism.  Saliva from treated periodontitis patients had higher IgA and IgG levels to periodontal pathogens Porphyromonas gingivalis and Treponema denticola than as compared to saliva from control subjects. 
It is an antimicrobial enzyme with the ability to cleave chemical bonds in the bacterial cell wall, and can lyse some bacterial species by hydrolyzing glycosidic linkages in the cell wall peptidoglycan. Patients with low levels of lysozyme in saliva are more susceptible to plaque accumulation, which is considered a risk factor for periodontal disease. 
This enzyme removes toxic hydrogen peroxide produced by oral microorganisms and reduces acid production in the dental biofilm, thereby decreasing plaque accumulation and the establishment of gingivitis and caries. Patients with periodontal disease have demonstrated high levels of this enzyme in saliva. 
Calcium is the ion that has been most intensely studied as a potential marker for periodontal disease in saliva. An elevated Ca concentration in saliva was characteristic of patients with periodontitis. 
- Lactoferrin is an iron-binding glycoprotein produced by salivary glands, which inhibits microbial growth by sequestering iron from the environment, thus depriving bacteria of this essential element. Lactoferrin is strongly upregulated in mucosal secretions during gingival inflammation and is detected at a high concentration in saliva of patients with periodontal disease compared with healthy patients. 
- Histatin is a salivary protein with antimicrobial properties and is secreted from parotid and submandibular glands. It neutralizes the endotoxic lipopolysaccharides located in the membrane of Gram-negative bacteria. It is an inhibitor of host and bacterial enzymes involved in the destruction of the periodontium. 
- Fibronectin is a glycoprotein that mediates adhesion between cells and is involved in chemotaxis, migration, inflammation, and wound healing and tissue repair. 
- Cystatins are physiological inhibitors of cysteine proteinases, and may function by modulating enzyme activity in the periodontium. 
- Other factors like platelet activating factor and amino acids like proline have positive correlation with periodontal inflammation. 
Growth factors such as epidermal growth factor (EGF) and vascular endothelial growth factor are also proven to stimulate epithelial cells and helps in wound healing respectively.  Growth factors are natural cell products that are released or activated when cell division is needed. This action typically occurs during such events as wound healing or tissue regeneration.
Activated platelets at the wound margins release several growth factors such as platelet-derived growth factor (PDGF), transforming growth factor (TGF)-alpha, EGF etc. Cells adjacent to the injured site also are induced to release growth factors such as insulin-like growth factor-I, PDGF, and TGF-alpha within a few hours after injury. In periodontal regeneration, the coronal re-establishment of the PDL is required together with corresponding cementum and supporting alveolar bone. Thus, agents which promote periodontal ligament fibroblast proliferation and migration as well as collagen biosynthesis would appear to be mediators for enhancing new PDL formation. 
Volatile sulfur compounds primarily hydrogen sulfide and methyl mercaptan, associated with oral malodor act as possible diagnostic markers and contributory factors in periodontal disease. 
Markers of periodontal soft tissue inflammation
Pro-inflammatory cytokines, such as prostaglandin E2, interleukin (IL)-1 beta, IL-6, and tumor necrosis factor-alpha are released from cells of the junctional epithelium, connective tissue fibroblasts, macrophages, and polymorphonuclear leukocytes. 
Markers of alveolar bone loss
These are proteases that participate in the degradation of many components of the extracellular matrix (ECM), like collagen. They also catalyze the activation reaction of osteoclasts. Matrix metalloproteinase 9 (MMP-9), is expressed by keratinocytes, fibroblasts, osteoclasts, and macrophages and is responsible for degrading a variety of the ECM during physiological and pathological conditions. The severity of periodontitis may be associated with increased metalloproteinase activity. Therefore, the cooperative action of some MMPs, such as MMP-9 and MMP-8, can induce the destruction of periodontal ECM molecules.  MMP-13 may be useful for diagnosing and monitoring the course of periodontal disease as well as for tracking the efficacy of therapy. 
Given the specificity and sensitivity for bone resorption, pyridinoline cross-links, like pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen represent a potentially valuable diagnostic aid for periodontal disease. 
Alkaline phosphatase (ALP) is a membrane-bound glycoprotein produced by a relatively wide variety of cells including osteoblasts, leukocytes, and fibroblasts in the periodontium and gingival crevice. The enzyme has an important responsibility in the bone metabolism. It is one of the important enzymes of PDL, root cementum and maintenance, and bone homeostasis. Increased ALP activity has been reported in the PDL due to renewal of the tissue as well as in pathological cases.  In special circumstances, like alveolar bone loss due to periodontitis, salivary ALP activity may show a significant rise, which is an important biomarker for periodontal problems. 
Systemic markers like C-reactive protein have been associated with various types of periodontal diseases and with other inflammatory biomarkers. 
| Gingival Crevicular Fluid as a Diagnostic Indicator|| |
Gingival crevicular fluid (GCF) is an inflammatory exudate that seeps into gingival crevices or periodontal pockets around teeth with inflamed gingiva. It represents serum components overlaid with products from local physiologic phenomena, such as connective tissue destruction and bone loss, and has diagnostic value. It is a convenient, noninvasive, and efficient means to sample biomarkers of inflammation and bone resorption in the oral cavity. 
| Inflammatory Mediators as Markers in Gingival Crevicular Fluid|| |
Host cell-derived enzymes like MMPs are an important group of neutral proteinases implicated in the destructive process of periodontal disease that can be measured in GCF. The neutrophils are the major cells responsible for the MMP release at the infected site, specifically MMP-8 (collagenase-2) and MMP-9 (gelatinase-B). Although MMP-8 is able to potently degrade interstitial collagens, MMP-9 degrades several ECM proteins. Rapid chair-side test based on the immunologic detection of elevated MMP-8 in GCF is used to diagnose and monitor the course and treatment of periodontitis. 
The amounts of IL-1α, IL-1β, and IL-1 receptor antagonist from GCF are quantified by ELISA. It is observed that bone resorption activity and levels of IL-1α, IL-1β, and IL-1 receptor antagonists are significantly higher in GCF from diseased sites compared with healthy sites but did not relate to defect morphology. 
| Proteolytic and Hydrolytic Enzymes as Markers in Gingival Crevicular Fluid|| |
A tissue destruction biomarker released from necrotic cells in GCF, is associated with periodontitis severity. 
Alkaline phosphatase is considered to be an important indicator of bone formation and is a phenotypic marker for osteoblast cells. The presence of ALP in the saliva and GCF is usually indicative of inflammation and/or destruction of the periodontal tissues. The level of ALP is positively correlated with the severity of the periodontal disease. 
Lactate dehydrogenase activity is higher in patients with increased probing depth than individuals with healthy periodontium. 
It is evaluated as a predictor of attachment loss. It may have a potential use in distinguishing periodontitis from gingivitis and in planning treatment and monitoring treatment outcomes. 
| Bone Related Biomarkers in Gingival Crevicular Fluid|| |
Increased osteocalcin levels in oral fluids are seen during an increased periodontal disease activity. 
It plays a role in immune regulation through its ability to inhibit Ig production and, of particular interest, its role as a pro-inflammatory protein for neutrophil recruitment and activation. Calprotectin GCF levels in patients with periodontal disease were higher than those in healthy subjects. 
Osteonectin is a single-chain polypeptide that binds strongly to hydroxyapatite and other ECM proteins including collagens. Osteonectin appeared to be the more sensitive marker for detection of periodontal disease status. 
Osteopontin-gingival crevicular fluid
Osteopontin (OPN) concentrations increased proportionally with the progression of disease. When nonsurgical periodontal treatment is provided, GCF OPN levels are significantly reduced. 
| Cell Death and Connective Tissue Degradation Products in Gingival Crevicular Fluid|| |
It plays a role in a variety of cellular activities, including cell-cell and cell-ECM adhesion. Fibrinogen (FN) and its fragments have also been thought to have a role in inflammation.  Specific FN fragments are markers for the periodontal disease status and support the role of FN fragments as potential components in the pathogenesis of periodontal disease. 
The sulcular fluid appears to be rich in metabolic and degradative products of the proteoglycans found in various periodontal tissues. The presence of raised levels of sulfated glycoaminoglycans in GCF reflects the active destruction of periodontal tissue, most likely, the alveolar bone. 
Prostaglandin E 2
Its concentration in GCF becomes significantly high during the active phase of periodontitis. 
It is involved in the degradation of the connective tissue ground substance and levels increases by six-fold in periodontal diseases. 
It is a marker of the intracrevicular PMN activity. Its levels increase in GCF in periodontitis. 
Collagen in gingival crevicular fluid
Variations in collagen product levels resulting from disease states are reflected in the GCF and it shows the active status of the periodontal disease process. 
Increased levels of proteoglycans in GCF are seen in the active phase of periodontitis. Increased amounts chondroitin 4 and 6 sulfate isomers and hyaluronan in GCF indicate the increased periodontal disease activity. 
Glycoprotein concentration increases in GCF along with the increased disease process and decreases in concentration when the healing process takes place in periodontium. 
Glycosylated IgG will be measured by measuring levels of gal-deficient IgG in GCF and serum from patients with periodontitis and nonperiodontitis controls using lectin enzyme-linked immunosorbent assay. 
| Conclusion|| |
There are ample possibilities for the future use of oral fluids such as saliva and GCF in biotechnology and health care applications, especially in the field of perio-diagnostics. Both saliva and GCF offer a noninvasive method for periodontal diagnosis which until recently was a domain of blood and its products. From physical measurements by periodontal probing to molecular arrays for the detection of biomarkers on the different stages of the disease, substantial improvements have been made in the understanding of the mediators implicated on the initiation, pathogenesis, and progression of periodontitis. In the field of periodontal diagnosis, there has been a steady progress during the last few decades to develop tools to monitor periodontitis, from physical measurements such as periodontal probing to molecular assays for the detection of biomarkers in salivary and crevicular fluid. At the same time, this evolutionary process has promoted the discovery of new biomarkers and the development of new therapeutic approaches mainly using host modulation.
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Conflict of interest
There are no conflicts of interest.
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