Correlation of mast cells in periodontal diseases.

BACKGROUND: Among the cells involved in immune and inflammatory responses in periodontal disease, mast cells have been shown to be capable of generating a large number of biologically active substances. Mast cells are mobile, bone-marrow-derived, granule-containing immune cells that are found in all connective tissue and mucosal environments and in the peripheral and central nervous systems. Mast cells are able to phagocytose, process and present antigens as effectively as macrophages. The present study was undertaken to quantify the mast cells in health and disease, whether they correlate degree of inflammation and clinical features of periodontium.
MATERIALS AND METHODS: Ten cases of localized chronic periodontitis (CP), 10 cases of dental plaque-induced gingivitis (DPIG) and 10 cases of clinically healthy gingival tissues were selected. Samples were obtained from patients undergoing for periodontal surgery in CP and DPIG. In health group third molar impacted and covered with operculum without any inflammation were selected. Sample fixed in 10% buffered formalin and stained with Toludine blue stain and observed under binocular microscope.
CONCLUSION: In human periodontal disease there is an increase in the number of mast cells that may be participating either in the destructive events or in the defense mechanism of periodontal disease via secretion of cytokines.

INTRODUCTION

Bacterial plaque has been implicated as the primary etiological factor in inflammatory periodontal disease, but recently several studies have focused on the role of the immune system in the evolution of periodontal disease, indicating that bacterial antigens trigger an immunopathological reaction and that the susceptibility of the patient determines the ultimate outcome of the disease process.[1] Among the cells found in the periodontal tissues, mast cells have been detected in both healthy and inflamed gingiva, in different numbers at various sites.[2] Mediators derived from mast cells are stored in secretory granules and released by degranulation or can be newly generated when mast cells are properly activated.

Mast cells are involved in numerous activities ranging from control of vasculature to tissue injury and repair, allergic inflammation and host defense. The significant contribution of mast cell mediators to tissue damage and propagation of inflammatory responses make the control of mast cell activity vital to the management of many inflammatory diseases.[3]

The aim of this study was to quantify the mast cells in health and disease, whether they correlate degree of inflammation and clinical features of periodontium.

MATERIALS AND METHODS

A total 10 cases of chronic periodontitis (CP) [Figure 1] characterized by advanced loss of periodontal support, 10 cases of dental plaque-induced gingivitis (DPIG) [Figure 2] and 10 cases of clinically healthy gingival tissues [Figure 3] were selected who reported to the Department of Periodontics, Rural Dental College, Loni, Maharashtra. The American Academy of Periodontology guidelines were followed for classification of the periodontal disease and conditions.[4] The patients included in the present study had no history of systemic disease.

Figure 1

Chronic periodontitis

Figure 2

Dental plaque-induced gingivitis

Figure 3

Periodontally healthy group

Ten samples with periodontally healthy tissues (PD<3 mm with no bleeding on probing)

Ten samples with plaque-induced gingivitis (PD < 3 mm and CAL < 1 mm with bleeding on probing)

Ten samples with moderate to advanced CP (PD and CAL > 4 mm with bleeding on probing).

After obtaining the informed consents from all human adult participants included, the Ethical committee of Pravara Institute of Medical Sciences Research cell approved the project and verified the protocol. In health group patients undergoing third molar extraction were included. Thorough irrigation was done in the operculum area with the help of hydrogen peroxide and normal saline. Also, the patients were instructed to use the 0.2% chlorhexidine (hexidine) mouthwash twice daily.

Tissue sample was obtained from patients undergoining for periodontal surgery in CP and DPIG. Each patient underwent periodontal surgery, independently of this study, as a part of their routine periodontal treatment (crown lengthening and full-thickness mucoperiosteal flap/debridement) by one surgeon in an identical manner and technique. Informed consent was obtained from the patients to collect, preserve and analyze the gingival tissues for this study. Biopsies were obtained with a scalpel-blade (no.15) as the incisional biopsy method from suitable sites immediately after diagnosis, from the deepest sites of interproximal pocket at the time of surgeries. The specimens were immediately fixed in 10% formalin for further processing and then dehydrated, cleared and embedded in paraffin.

Histochemistry and staining method

Sections measuring 5 μm in thickness by microtome were deparaffinized and hydrated with water. Toludine blue staining was performed with a 1% Toludine Blue (TB) (A1111.01. AD, Synth, Sao Paulo, Brazil) for 30 min. diluted in phosphate buffer (pH = 4-6) for 45 s. Then the tissue samples were placed at 37°C in incubator. After rinsing in phosphate buffer for 1-min. sections were blotted carefully, quickly dehydrated through 70, 96% ethanol and absolute alcohol to xylene and mounted in synthetic resin and observed under binocular microscope.[5]

Quantitative and statistical analysis

Number of positively stained mast cells in the periodontal tissues was determined in 25 consecutive microscopic high-power (×800; objective ×64; eyepiece ×12.5; tube factor ×1) fields (each field has an area of 0.017 gg 25 mm2, obtained from the mathematical expression A = (πd2)/4, where π =3.14, d=0.15) in a representative section of each specimen. Mast cell counts in inflammatory cell infiltrate of diseased tissue and periodontally healthy tissue areas have been performed [Figures 4–6].[5]

Figure 4

Histological section of mast cells in periodontally healthy group

Figure 6

Histological section of mast cells in chronic periodontitis

Histological section of mast cells in dental plaque-induced gingivitis

The results of TB-stained mast cells were expressed as mean ± s. d. of n observations per mm2. Also the comparative analysis of the number of mast cells/mm2 between periodontally healthy and diseased group was performed. The statistical study was performed using ANOVA followed by Student's t-test. A P value of less than 0.05 was considered statistically significant.

RESULTS

Table 1 shows comparison of mean values of quantification of mast cells in periodontally health and gingivitis group. Mean value of periodontally healthy group is in between 5.8 and 9.5 ± standard deviation (SD) 1.67-5.45 mast cells/mm2. Whereas, mean value of gingivitis group is in between 11.2 and 19.2 ± SD 4.45 and 5.16 mast cells/mm2. As P<0.01, there is highly significant difference in between periodontally healthy and DPIG group.

Table 1

Comparison of mean values of quantification of mast cells in periodontally healthy and gingivitis group

Table 2 shows comparison of mean values of quantification of mast cells in Periodontally Healthy and CP group. Mean value of periodontally healthy group is in between 5.8 and 9.5 ± SD 1.67 and 5.45 mast cells/mm2, whereas mean value of CP group is in between 19.2 and 26.8 ± SD 5.14 and 6.14 mast cells/mm2. As P<0.01, there is highly significant difference in between periodontally healthy and CP group.

Table 2

Comparison of mean values of quantification of mast cells in periodontally healthy and chronic periodontitis group

Table 3 shows comparison of mean values of quantification of mast cells in DPIG and CP group. Mean value of DPIG group is in between 11.2 and 19.2 ± SD 4.45 and 5.16 mast cells/mm2, whereas, mean value of CP group is in between 19.2 and 26.8 ± SD 5.14 and 6.14 mast cells/mm2. As P<0.01, there is highly significant difference in between DPIG and CP group.

Table 3

Comparison of mean values of quantification of mast cells in gingivitis and periodontitis group

Thus, quantitative analysis of mast cells with Toludine blue staining revealed statistically significant difference among the three groups examined.

DISCUSSION

Mast cells originate from pluripotential hematopoietic cells in the bone marrow, undergo part of differentiation in this site, then enter the circulation and complete their differentiation in peripheral mucosal or connective tissue microenvironments [Figure 7].[6] Mast cells play important role in mucosal inflammation, host defense and tissue repair. When triggered by locally produced cytokines or bacterial products for e.g., lipopolysaccharides, the cells can release large number of prestored mediators.[7] Limited attention has been given to the role of mast cells may play in periodontal diseases. Mast cells are scattered throughout gingival CT, often in close association with endothelial cells, but are found sub and intraepithelially. In inflamed and in healing gingiva, number of mast cells are found to be increased. Mast cells are characterized by oval to round nuclei and cytoplasm densely packed with bright red granules. They can be stained with Giemsa stain or Toludine blue stain. Mast cells may be round, oval or spindle shape with abundant cytoplasm or thin and elongated resembling fibroblast. Each mast cell typically contains between 80 and 300 granules. When activated, mast cells may either undergo explosive degranulation and then resynthesize their granules or they may release solitary granules into their environment on an ongoing basis, a process termed ‘piecemeal degranulation’ that has been observed in both the oral mucosa and skin.

Figure 7

Origin of mast cells

Mast cell mediators

Following degranulation, mast cell mediators are deposited in large quantities in the extracellular environment, where they exert effects on endothelial cells and other cell types. Mast cells may subsequently synthesize and secrete additional mediators that are not preformed in their granules. As described by Walsh, et al., 1991b key mediators that are preformed in mast cells are the serine proteases tryptase, chymase and cathepsin G, histamine, heparin, serotonin, acid hydrolases, and the cytokines tumor necrosis factor-α (TNF) and interleukin-16.[8] Following activation, mast cells can synthesize a range of mediators, including the interleukins IL-1, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, and IL-16, together with granulocyte-macrophage colony-stimulating factor (GM-CSF), platelet-activating factor (PAF), RANTES, macrophage inflammatory protein (MIP-1a), and the arachidonic acid metabolites prostaglandin 2 and leukotriene C4.[9]

As discussed by Gunhan, et al.[10] in inflamed and in healing gingiva, numbers of mast cells increased. Walsh, et al., have showed that mast cell numbers were dramatically increased in inflamed sites of periapical granulomas and lichen planus when compared with nonlesional sites indicating higher activity of that cells in that area.

The results of this study suggest that mast cell counts may be associated with periodontitis. This finding indicates the role of mast cells in chronic periodontal tissue breakdown. One of the biological and biochemical factors is histamine, which breaks down the tissue barrier, causes edema and helps cellular infiltration. In addition, mast cells are believed to contain most of the body's histamine.[11] Another reason is that the expression of matrix metalloproteinases (MMPs) 1, 2, and 8 are strongest in mast cells. MMPs are crucial in the degradation of the main components in extracellular matrices.[12] Furthermore, tryptase can cleave the third component of collagen and activate latent collagenase that can participate in tissue destruction in periodontitis. Furthermore, it has been indicated that tryptase activity is confined to mast cell granules.

A change from gingivitis to periodontitis involves a shift from predominantly T-cell lesion to a B-cell/plasma cell lesion. Mast cells seem to be able to present antigens to T cells. The resultant T-cell activation would activate mast cells, leading to both degranulation and cytokine release.

In our study, we found that increase in number of mast cells in inflamed site as compared to periodontally healthy site, suggesting important dynamic alterations in the migration and localization of mast cells in the evolution of periodontal disease, which need to be more precisely speculated [Figure 8].[6] Based on our results, the increase of mast cells has called attention with respect to the possible participation of mast cells in the defense mechanism and destructive events both as effector and responsive cells in chronic inflammation, as well as the possible functional relationship between mast cells and immunocompetent cell populations in periodontal lesions.

Figure 8

Comparison of mean values of quantification of mast cells

In summary, periodontitis is not unidirectional, but rather it is interactive; the same cells that produce the destructive proinflammatory cytokines can also produce mediators that activate the healing process.

CONCLUSIONS

In the present study CP cases had higher mast cell counts compared to gingivitis sites or healthy tissues. Increased mast cell counts in the progressing sites of periodontal diseases may indicate the importance of these cells in the progression of CP.

Future prospects

Because of the importance of periodontal diseases, inadequate studies and possible relationships between mast cells and pathogenesis of periodontal diseases, further research is needed to elucidate the cellular interactions and immunological and dynamic aspects of the disease so that the pathogenesis of periodontitis might be elucidated more clearly and effective treatment approaches can be suggested.

The therapeutic implications of the findings and suggestions herein presented include strategies directed toward the possible use of drugs to influence mast cell secretion and thereby prevent inflammation and maintenance of chronicity, or even with the aim of improving periodontal regeneration.