| Abstract|| |
Background: There are no studies comparing the gingival crevicular fluid (GCF) cytokines expression with its corresponding values from the same tissue's sites. Such comparison might be of great value since most of the cytokine function is related to cell and/or tissue receptors.
Aims: Our aim was to use minimally invasive biopsies to evaluate the expression of interferon-gamma, interleukin 1 (IL-1) β, IL-6, IL-17A, IL-17F, and their correlation with the expression in gingival fluid in patients with chronic periodontitis.
Materials and Methods: The collection of gingival fluid comprised 22 samples from 11 patients (mean age 46.73 ± 10.16 standard deviation years) with chronic periodontitis. The collection of biopsies comprised 22 samples from the same patients. Gingival fluid and biopsy were taken from the same site in one shallow and one deep site per patient. Gingival fluid samples were collected with periopaper ® and analyzed using Luminex ® . Biopsies were taken with a 2 mm diameter punch and analyzed for the same mediators using immunohistochemistry.
Results: The gingival fluid showed higher amounts for IL-1-β in deep sites. Immunohistochemical markers were observed in the analyzed cells groups, both in deep and shallow sites, without significant differences between them. In the comparative analysis between immunohistochemical markers and GCF, IL-1-β showed high concordance in shallow and deep sites.
Conclusions: The use of a standardized punch of 2 mm diameter for periodontal tissue biopsies seems to be suitable for immunohistochemistry analysis and showed that the GCF may not express all the markers in the same proportion at the corresponding tissue.
Keywords: Biopsy, gingival crevicular fluid, inflammation mediators, periodontitis
|How to cite this article:|
Lomba KB, de Souza Breves Beiler TC, Sete MC, Pires FR, da Silva Figueredo CM. Use of minimally invasive gingival biopsies in the study of inflammatory mediators expression and their correlation with gingival fluid in patients with chronic periodontitis. Indian J Dent Res 2015;26:126-30
Periodontitis has been proposed as having an etiological or modulating role in cardiovascular, cerebrovascular disease, diabetes, respiratory disease, and adverse pregnancy outcome; several mechanisms have been proposed to explain or support such theories. One of these is based on the potential for the inflammatory phenomenon of periodontitis to have effects by the systemic dissemination of locally produced inflammatory mediators. 
|How to cite this URL:|
Lomba KB, de Souza Breves Beiler TC, Sete MC, Pires FR, da Silva Figueredo CM. Use of minimally invasive gingival biopsies in the study of inflammatory mediators expression and their correlation with gingival fluid in patients with chronic periodontitis. Indian J Dent Res [serial online] 2015 [cited 2019 Sep 22];26:126-30. Available from: http://www.ijdr.in/text.asp?2015/26/2/126/159134
Cellular and humoral immune responses, especially T-cells responses, have been considered essential in the pathogenesis of several diseases such as rheumatoid arthritis, chronic intestinal diseases, and also in periodontitis. , Adaptive immune responses against specific pathogens are enhanced by the differentiation of CD4 + lymphocytes in groups of effector T-cells, for instance, Th1, Th2, and Th17. These subsets eliminate infected cells and modulate inflammatory response through the release of cytokines and other proteins, which may lead to host's damage. 
The analysis of gingival crevicular fluid (GCF) has been the main source of information to study cytokine expression in periodontal tissues. GCF has different components which are incorporated from its flow through the periodontal tissue to the gingival sulcus.  Higher GCF volume showed a positive correlation with the presence of supragingival plaque, inflammation and a higher degree of tissue destruction, ,,, however there are no studies comparing the GCF cytokines expression with its corresponding gingival tissues. Such comparison might be of great value since most of the cytokine function, mainly the chemokines, are related to cell and/or tissue receptors. Although soluble, it is reasonable that most of its expression might occur in the tissues and the GCF amounts represent only a small part of it.
Gingival biopsies generally come from various sources, such as areas adjacent to teeth undergoing extractions, , interdental papillae , regions undergoing gingivectomy for different reasons. , All of them are very invasive leaving scars and/or defects, making it almost impossible to be used in a large scale for diagnosis. Herein we suggest a very small punch to collect gingival tissue. We hypothetized that minimally invasive biopsies might be an alternative to study cytokine expression in periodontal diseased sites. Therefore, our aim was to compare the expression of interleukin 1 (IL-1)-β, IL-6, IL-17, and interferon-gamma (IFN)-γ in minimally invasive biopsies and GCF from patients with chronic periodontitis.
| Methods|| |
Gingival fluid and biopsies collection
The collection of gingival fluid comprised 22 samples from 11 patients (mean age 46.73 ± 10.16 standard deviation years) with chronic periodontitis, classified according to the American Academy of Periodontology.  The collection of biopsies comprised 22 samples from the same patients. Gingival fluid and biopsy were taken from the same site in one shallow site and one deep site per patient. The characteristics of these patients are shown in [Table 1]. All subjects were seeking for dental treatment at the Rio de Janeiro State University (UERJ), Rio de Janeiro, Brazil. The study protocol was approved by the Ethics Committee of the University Hospital Pedro Ernesto (UERJ, Rio de Janeiro, Brazil- CEP/HUPE 2081/2008). Eleven samples were collected from deep sites (PPD ≥5 mm CAL ≥3 mm) and 11 from shallow sites (PPD ≤3 mm CAL ≤1 mm).
Before GCF collection, the supragingival plaque was gently removed, teeth were dry with air jets and isolated with cotton rolls. PerioPaper® (ProFlow, Inc., Amityville, NY, USA) was inserted into the periodontal pocket or sulcus until slight resistance was felt and left in place for 30 s. For each individual, the paper strip of each site was stored in Eppendorf containing 150 μL of phosphate buffered saline. After elution for 40 min at room temperature, the paper strips were removed, the samples centrifuged at 8000 rpm for 5 min, the supernatant collected and frozen immediately at −70°C until analysis.
Gingival tissue samples were obtained after the collection of gingival fluid. The selected site for gingival biopsy was anesthetized and the pocket depth measured with the periodontal probe, being externally established its corresponding point. Samples were collected from the bottom of the gingival sulcus or periodontal pocket. The tissue was removed with a 2 mm diameter punch before subgingival scaling. Immediately after collection, all samples were stored in eppendorfs with 1 ml of 10% formaldehyde for fixation and sent to the laboratory of Oral Pathology, Faculty of Dentistry UERJ, Brazil.
Quantification of interferon-γ interleukin-1-β, interleukin-6, interleukin-17A, interleukin-17F in gingival crevicular fluid by Luminex ®
Levels of cytokines were determined using an immunoassay with multiplex bead. Fifty microliter samples of gingival fluid were analyzed using a commercially available kit (Lincoplex ® , Millipore, Missouri, USA) on a Luminex analyzer (Luminex, MiraiBio, Alameda, CA) according to the manufacturer's instructions. Levels of cytokines analyzed were expressed as the total amount per site (pg/site).
For the morphological study, histological slides containing 5 μm thick cuts (microtome Leica ® ) of the material included in paraffin blocks and examined under an optical microscope (Nikon ® Eclipse E200). The histological sections were stained with routine hematoxylin and eosin. The characteristics of the epithelium and connective tissue in the aspects of standard and location of inflammatory infiltrate were evaluated in increments of ×10 and ×40.
Immunohistochemistry was performed in sections of 3 μm thick using biotin-streptavidin method the following antibodies were used: Polyclonal rabbit anti-human IFN-γ (H-145, SC-8308, Santa Cruz Biotechnology Inc., California, USA, dilution 1:200); Monoclonal antibody anti-human IL-6 (SC-130326, Santa Cruz Biotechnology, Inc., California, USA, dilution 1:500) mouse; polyclonal rabbit anti-human IL-1-β (SC-7884, Santa Cruz Biotechnology, Inc., California, USA, dilution 1:100); Rabbit polyclonal anti-human IL-17 (SC-7927, Santa Cruz Biotechnology, Inc., California, USA, dilution 1:200). Positive and negative (omission of primary antibody) controls (oral and nasal polyp epithelium) were used in all reactions. The slides were observed under an optical microscope (Nikon ® Eclipse E200) with a ×10 and ×40 objective lens. The evaluation of the staining pattern of cytokines was performed by analyzing the distribution of the antibody in the tissue, epithelial and inflammatory cells (plasma cells and macrophages), fibroblasts and endothelial cells were evaluated. This analysis was performed by two examiners. Intra and inter calibration was performed analyzing 10 slides, twice at different times, with 90% of agreement for the staining scores. Immunoreactive cells showed the presence of brownish staining in the nucleus and/or cytoplasm, regardless the intensity of the marking.
Staining was evaluated semiquantitatively, considering the absence of staining (0), focal/weak (1) marking, moderate/severe (2) marking.
Wilcoxon Statistic test was used to compare GCF total amounts of the analytes between shallow and deep sites. Kruskal-Wallis test was used for comparisons between the scores of the slides of shallow and deep for each cell group. These tests were applied to the groups: Deep sites (n = 11), shallow sites (n = 11). The percentage of agreement between immunohistochemistry and GCF was presented by comparative analysis between immunohistochemical markers and gingival fluid.
| Results|| |
Gingival crevicular fluid's analysis
Deep sites presented significantly higher levels of IL-1-β. For the others cytokines, no significant difference was observed [Table 2].
|Table 2: Medians and interquartile range of GCF in periodontitis patients, for IL, IFN-γ, IL-1β, IL-6, IL-17A, and IL-17F |
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Gingival samples from shallow sites had a stratified squamous keratinized epithelium surface, with moderate lymphocytes and light plasma inflammatory infiltrates. Deep sites exhibited a diffuse distribution of inflammation with moderate to intense lymphocytic infiltrate.
[Figure 1] and [Figure 2] illustrate the degrees of immunohistochemical staining for IFN-γ, IL-6, IL-17, and IL-1-β in epithelium (yellow arrows), and connective tissue (red arrows) of a shallow and deep sites in a periodontitis patient. There was a predominance of weak staining (score 1) in the epithelium, fibroblasts, and macrophages for IFN-γ, IL-6, IL-17 in shallow, and deep sites. There was 100% staining for IL-1-β (scores 1 and 2) in the epithelium in shallow and deep sites. For plasma and endothelial cells, all deep sites were positive for IL-17.
|Figure 1: Histological H and E, section (a) and immunohistochemical expression for interferon-γ (b); IL-1-β (c); IL-6 (d); and IL-17 (e), in gingival tissue from periodontitis patient shallow site, stained in the epithelium (↑) and connective tissue (↑) (immunoperoxidase, ×40)|
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|Figure 2: Histological H and E section (a) and immunohistochemical expression for interferon-γ (b); IL-1-β (c); IL-6 (d); and IL-17 (e), in gingival tissue from periodontitis patient deep site, stained in the epithelium (↑) and connective tissue (↑) (immunoperoxidase, ×40)|
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Concordance analysis between immunohistochemical markers and gingival fluid
[Table 3] presents the percentage of agreement between immunohistochemistry and GCF for shallow sites (n = 11), deep sites (n = 11) and for the analysis of the whole group (n = 22). This analysis was based on the presence or absence of staining for each site (median of cell groups in the immunohistochemical analyzes), and the presence or absence of each marker in GCF, considering the concordance between them.
| Discussion|| |
The screening tool of greatest benefit is cautious oral examination and sound clinical judgment. The clinician's knowledge and training may eliminate the need for biopsy in cases where lesions are clinically definable. 
Immunohistochemistry did not show significant differences between shallow and deep sites for IL-1-β (evaluated on epithelial cells, fibroblast, and macrophage, as it is not expressed in plasma cells and endothelium), as well as for IFN-γ, IL-6 and IL-17. Shallow and deep sites presented 100% marking in epithelium for IL-1-β. Furthermore, macrophages presented 100% marking for IL-1-β in deep sites. 100% marking of IL-17 in plasma and endothelium were also observed in deep sites. IFN-γ was observed in all cells groups for the two groups analyzed. Thus, according to the methodology adopted, it was not possible to determine significant differences in immunohistochemical markers between shallow and deep sites.
Regarding IL-17, an amplifier of the inflammatory response, similarly to the study of Adibrad et al.,  it was found a positive staining for shallow and deep sites in immunohistochemistry. Lester et al.  evaluated several cytokines in biopsies of interdental papillae of sites with mild, moderate, and severe attachment loss; the results showed differences between groups, but they were not able to establish a logical proportion among cytokines in the tissue and the degree of periodontal involvement.
When assessing the results of the gingival fluid, there was a significant difference between shallow and deep periodontal sites for IL-1-β. This result pointing significantly higher amounts of IL-1-β in deep sites, reinforces the methodology used in the collection and cytokines measurement. Several studies found higher IL-1-β levels in GCF of inflamed sites, and its reduction after periodontal therapy, suggesting IL-1-β as a biomarker of periodontitis. ,,
In the study of Sjögren and Anderson,  insertion of a cutaneous microdialysis catheter into normal dermis has been shown to induce the production of IL-1-β, IL-6 and IL-8 in an innate response to minimal trauma. The analysis of biopsies, collected with a 4 mm diameter punch, was performed semiquantitatively, similarly to the immunohistochemical analysis performed in this study. The microdialisate assessed by Luminex showed, in comparison to biopsies, more than 50% concordance for IL-1-β and IL-6 with higher markers in microdialisato and 60% for IFN-γ, with zero (0) values for all samples in the microdialisate and therefore more positive marks on biopsy. IFN-γ, tumor necrosis factor-α and IL-10 showed similar results and therefore the authors were not able to fully correlate the marks in epithelial tissue with the contents of microdialisate. In our study, despite the methodological and statistics differences in relation to the study of Sjögren and Anderson, we can point out some similarities. In relation to IFN-γ, we observed similar behavior, since it showed positive staining in gingival biopsies but showed zero values for most assessments in gingival fluid, with 59% agreement in the group. IL-6 showed 68% concordance in the group and IL-17 showed 56%. However, high concordance between gingival fluid and biopsies for IL-1-β was found. It was positive in both biopsies and gingival fluid in 93% of the group.
When this agreement percentage was evaluated separately between shallow and deep sites, we observed a higher percentage of agreement in the group of deep sites for all four markers analyzed, reaching 100% for IL-1-β, 81% for IL-6, 75% for IL-17, and 63% for IFN-γ. This result could be related to increased epithelial permeability in deep sites, as that increases with the progression of inflammation. 
The differences between cytokines in biopsies and GCF may be due its undetection in GCF, given the high specificity of the applied techniques. Similar point was suggested in studies of Sjögren and Anderson  and Adibrad et al.,  where the absence of detection in external environment could be related to some hypotheses, such as the cytokines secreted were bound to their membrane receptors and thus remain in the tissue; cytokines that would be released to the external environment but would be consumed by regulatory processes; or would not be absorbed by the absorbent paper (PerioPaper ® ). Thus, it seems plausible that the gingival fluid not exactly express the markers present in the tissue, with variations depending on the marker, and even local inflammatory conditions. More specific studies on the epithelium and connective tissue, addressing the cell junctions, as well as functional analyzes of the periodontal pocket epithelium may be important in periodontics.
| Conclusion|| |
The use of a standardized 2 mm diameter punch for periodontal tissue biopsies seems to be suitable for immunohistochemistry analysis and showed that the GCF may not express all the markers in the same proportion at the corresponding tissue.
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Dr. Carlos Marcelo da Silva Figueredo
Department of Integrated Clinical Procedures, Rio de Janeiro State University, Rio de Janeiro
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]