Quantitative analysis of mast cell count and density in chronic periodontal disease.

BACKGROUND: Mast cells play a crucial role in activation of acquired immune response to inflammatory conditions of periodontal diseases. They promote inflammation by releasing pro-inflammatory mediators and bring about angiogenesis, degeneration of the extracellular matrix, and tissue remodeling. Since there is little literature regarding the role of mast cells in periodontitis, the present study was aimed to evaluate mast cell count (MCC) and density in periodontitis.
MATERIALS AND METHODS: A total of eighty participants, Group I (n = 40) healthy participants and Group II (n = 40) participants with moderate chronic periodontitis, were included in the study. Tissue samples of 5 micron were obtained from each participant and were fixed in 10% formalin. Inflammation assessment was carried out after staining the sections with hematoxylin/eosin (H and E) followed by toluidine blue and mast cells were counted.
RESULTS: MCC in healthy group (1.32 ± 0.43) was significantly smaller than periodontitis group (10.28 ± 1.15) and also mast cell density in healthy group (98.08 ± 37.40) was smaller than periodontitis group (803.43 ± 89.94) with P < 0.0001.
CONCLUSIONS: It could be concluded that participants with chronic periodontitis have a higher MCC and density when compared with healthy participants.

INTRODUCTION

Periodontitis is a chronic inflammatory disease with pathogenic microorganisms implicated as the primary etiologic factor leading to loss of cementum, periodontal ligament, and alveolar bone.[1] Constant bacterial deposits on teeth cause chronic inflammatory response which ranges from reversible gingivitis to irreversible and destructive periodontitis ultimately leading to tooth loss if left untreated. Immune system plays a remarkable role in the identification of periodontitis. Immunopathologic reaction triggered by bacterial antigens mediates the host response to microorganisms which, in turn, determines the extent of severity of the disease.[2]

Mast cells are large connective tissue cells with cytoplasm densely laden with granules which can stain metachromatically. Along the walls of blood vessels, they are precursors of heparin and histamine.[3] Moreover, mast cells also express leukotrienes, prostanoids, proteases, cytokines, and chemokines that attract neutrophils to the site of infection and thus stimulate host defence mechanism.[45] Myint et al., 2002, found a high mast cell count (MCC) equal to and often surmounting the macrophages in the inflamed periodontal lesions.[6]

Thus, tremendous activities of mast cells range from host defense to tissue damage through its mediators. These cells are responsible for gingival homeostasis and matrix metalloproteinases (MMPs), and therefore, contribute to periodontitis.[7]

Role of mast cell in systemic disease and oral disease such as oral squamous cell carcinoma, lichen planus, oral submucous fibrosis, leukoplakia, and many others is a well-known fact, but there is a paucity of literature on the role of mast cell in chronic periodontitis. Thus, the present study was aimed to evaluate the mast cells' count and density in periodontal health and disease and examine the relationship between the MCCs and periodontal diseases.

MATERIALS AND METHODS

Study group

A total of eighty participants with age range between 20 and 60 years including both males and females visiting the Department of Periodontics and Implantology were recruited. The study was approved by the Institutional Ethics Committee and a written informed consent was obtained from all participants willing to participate. Participants were divided into two groups with forty participants each. Group 1 involved all periodontally healthy participants (periodontal probing depth [PPD] ≤3 mm and with no bleeding on probing) undergoing premolar extraction for orthodontic treatment or crown lengthening procedure. Group 2 involved participants with moderate chronic periodontitis (PPD ≥4 mm and bleeding on probing) undergoing periodontal treatment (full-thickness mucoperiosteal flap/debridement). Participants having drug-induced gingival enlargement and subjects having pregnancy tumor or any other types of systemic disease were excluded from the study.

Biopsy

An informed consent was obtained from all the participants to collect, preserve, and analyze the gingival tissues for this study. From each patient who was to undergo independent periodontal surgery, small tissue samples were obtained from free marginal gingiva and fixed in 10% formalin.

Histological technique

After extraction of the specimens, they were immediately fixed in 10% formalin for further processing, dehydrated, cleared, and then embedded in paraffin. Five-micron sections were obtained from each sample. The sections were mounted on slides, dried, and subsequently deparaffinized in three changes of xylol and rehydrated in three changes of 95% ethyl alcohol and distilled water. Inflammation assessment was carried out by staining the sections with hematoxylin/eosin (H and E) and toluidine blue (TB) for mast cell counting. TB staining with 1% TB was performed for 30 min diluted in phosphate buffer (pH = 4–6) for 45 s. The samples were then incubated at 37°C. Sections were rinsed in phosphate buffer for 1 min and blotted carefully, dehydrated through 96% ethanol, absolute alcohol and xylene. The prepared sections were then examined under the Olympus Light Microscope [Figures 1 and 2].

Figure 1

Histological section of mast cell in periodontitis group, arrow (→) shows mast cells; toluidine blue stain was used for staining

Figure 2

Histological section of mast cell in healthy group, arrow (→) shows mast cells. Toluidine blue stain was used for staining

Quantitative analysis of mast cells

The positively stained mast cells in the sample tissues were detected by selecting five random fields in high-power microscope (×800; objective ×64; eyepiece × 12.5; tube factor ×1). The area of each field was 0.0176625mm2 as derived from the mathematical expression (A = (πd2)/4, where π =3.14, d = 0.15). Using the inflammatory cell infiltrate of the tissue samples, the MCCs were obtained. Based on this, mast cell density (MCD) was carried out using formula.[8] Figure 1 shows histological section of mast cell in periodontitis).

Statistical methods

The data on demographic and periodontal parameters were obtained on forty participants each from healthy and periodontitis groups. The demographic parameter such as age and periodontal parameters were expressed in terms of mean and standard deviation. The comparison of each periodontal parameter between two groups was performed using t-test for independent samples. A Pearson's correlation coefficient was obtained between PPD and MCC as well as PPD and MCD. The statistical significance was evaluated at 5% level and the analysis was performed using R-3.0.0 programming language.

RESULTS

The data on participants from healthy and periodontitis groups were analyzed as stated in methods. The mean age of patients in healthy group was 21.18 ± 5.25 years, which was significantly smaller than that of periodontitis group with a mean age of 46.73 ± 11.05 years as indicated by P < 0.0001. The gender distribution was statistically insignificant (P = 0.990) with 23 and 24 males in healthy and periodontitis groups, respectively. The periodontal parameters were analyzed between two groups [Table 1]. It shows that the mean plaque index for healthy group (0.52 ± 0.32) was significantly smaller than that of periodontitis group (1.60 ± 0.30) with P < 0.0001. Further, PPD was also significantly smaller in healthy group (1.51 ± 0.30) as compared to periodontitis group (4.09 ± 0.45) with P < 0.0001. MCC in healthy group (1.32 ± 0.43) was significantly smaller than periodontitis group (10.28 ± 1.15), and also MCD in healthy group (98.08 ± 37.40) was smaller than periodontitis group (803.43 ± 89.94) with P < 0.0001.

Table 1

Descriptive statistics for different parameters according to healthy and periodontitis group

A strong positive correlation (0.9275) was observed between PPD and MCC, which was highly significant (P< 0.0001). Furthermore, PPD showed strong positive correlation (0.9276) with MCD with P < 0.0001 [Graphs 1 and 2].

Graph 1

Pearson's correlation was obtained between probing pocket depth and mast cell count in Group I and Group II. The correlation coefficient obtained was 0.9275 with P < 0.0001 indicating highly significant positive relationship between the two parameters. In other words, density as one parameter increases, other also increases, as evident from the scatter plot. Similar was the finding between probing pocket depth and mast cell count. The coefficient obtained was 0.9276 (P < 0.0001). MCC – Mast cell count; PPD – Probing pocket depth; MCD – Mast cell density

Graph 2

Bar chart with error bars showing the mean mast cell count and mast cell density according to Group I and Group II

DISCUSSION

Human mast cells are developed from the reservoir hematopoietic stem cells in the bone marrow.[9] They are also found in the connective tissues and mucosal tissues of the oral cavity. Mast cells are involved in many pathological conditions such as asthma, gingivitis, periodontitis, and oral lichen planus with their numbers and MMPs expression significantly increased in chronic periodontitis.[10] Thus, mast cell analysis can serve as a useful histological marker for periodontitis.

Zhao et al., in 2002, reviewed the interaction between mast cells and T-cells and stated that they are related to the disease process, initiation, vaso-induction, and effector phase of oral lichen planus.[11] Furthermore, Madhuri et al., in 2007, observed an increase in the MCC in OSMF.[12] Apart from that Kinra et al., 2012, observed a tremendous increase in MCC and density in precancerous conditions and lesions, potentially malignant as well as malignant oral lesions.[13] Besides evaluating the role of mast cell in periodontal health and disease, the present study aimed at deriving MCD by mast cell counting for a more precise result.

Results of the current study indicate an increase in MCC and density in periodontitis condition when compared with healthy condition, which is consistent with the result of studies conducted by Lagdive et al., Kenett, Myint et al., and Naesse et al.[61415] These findings stipulate the role of mast cells in periodontal tissue breakdown by the release of numerous mast cell mediators e.g. histamine that breaks tissue barriers and helps in cellular infiltration.[16] According to Gruber et al., in 1989, mast cell serine proteinases such as tryptase and chymase cleave fibrinogen and activate latent collagenase and pro-MMP-3. Accumulation of mast cell tryptase also causes activation of MMP-2, whereas chymase may degrade basal membrane and neuropeptidase and activate latent interleukin (IL)-1 beta and MMP-9.[1718] Mizutani et al., in 1991, observed that mast cells lie close to blood vessels in dermis; therefore, chymase-mediated conversion of 31 kD IL-1 beta.[19]

In the present study, peridontitis had higher MCCs compared to healthy tissue. In a study carried out by Myint et al., 2002,[6] there was an increase in a number of mast cell in chronic periodontitis having HIV infection. Similar results were found in a study conducted by Naesse et al., in 2003, in which MCCs were significantly higher in chronic periodontitis group compared to healthy group in both HIV-positive and HIV-negative patients.[15]

Mean MCC and MCD found in the present study were 10.28 and 803.43 in periodontitis which was significantly higher in comparison to MCC and MCD of healthy group.

Conversely, Gemmell et al., 2004, observed a decrease in the MCC in chronic periodontitis cases compared to healthy/gingivitis samples. This discrepancy could be due to the grouping of samples of clinically healthy and marginal gingivitis patients and the use of probing depth for defining the type of the disease.[20]

Toluidine blue was simple to use although the technique had several limitations. Nevertheless, several immunohistochemical and immunofluorescence assays have yielded similar results since almost 75% of mast cells are formalin sensitive.[21]

Although the correlation between the mast cells and severity of inflammation has been evaluated by numerous investigations, the conflicting results obtained are due to the differences in the histological techniques and a general lack of adequate controls. More consistent results have been observed over the course of time, with the advanced and improved techniques and precise controls.

Limitation

A small sample population of this study

The results of the study were not correlated with either gingival crevicular fluid, salivary, or serum biomarkers.

CONCLUSIONS

In the present study, periodontitis group has higher MCC and MCD when compared with healthy participants. The increase in the MCCs in the sites with periodontal diseases thus accentuates these cells in the disease progression.

Thus, this mast cell analysis can serve as an important chair-side diagnostic marker for periodontitis evaluation in a very cost-effective manner. Further studies, with larger sample size, should aim to establish a correlation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.