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ORIGINAL ARTICLE
Year : 2015  |  Volume : 6  |  Issue : 3  |  Page : 161-165

Histomorphometric analysis of gingival tissue in smokers and nonsmokers


1 Department of Periodontics, KVG Dental College and Hospital, Sullia, Karnataka, India
2 Department of Oral Pathology and Microbiology, MCODS, Mangalore, Karnataka, India

Date of Web Publication4-Aug-2015

Correspondence Address:
Jitendra Kumar
Department of Periodontics, KVG Dental College and Hospital, Sullia, Karnataka
India
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DOI: 10.4103/0976-433X.156214

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  Abstract 

Background: Smoking has been recognized as a major risk factor for periodontal diseases. Periodontal diseases are known to cause inflammatory vascular and epithelial changes in the gingiva on its own even in the absence of smoking. Hence, the present study is conducted to compare the epithelial and vascular changes in the gingiva of smokers and nonsmokers suffering from chronic periodontitis. Aim of Study: The purpose of this study was to evaluate and compare the epithelial and microvasculature changes in the gingiva of smokers and nonsmokers with chronic periodontitis. Materials and Methods: The study population consisted of 15 smokers and 15 nonsmokers. Gingival tissue biopsies with diameter of 5 mm including both epithelium and connective tissue were harvested from periodontal surgical sites and sent for histopathological examination. The images of the histological section were interpreted using ImageJ analyzer system. Statistical Analysis: Student's t-test was used to compare the results. Results: Mean area of lumen and blood vessel density among smokers was lesser than nonsmokers, and thickness was found to be more in nonsmokers. Conclusion: The present study concludes that mean thickness of gingival epithelium, mean blood vessel density and lumen area were higher in nonsmokers compared to smokers. Percentage of small-sized and medium-sized blood vessels was higher in smokers.

Keywords: Chronic periodontitis, gingival epithelium, gingival microvasculature, smoking


How to cite this article:
Dayakar MM, Kumar J, Pai GP, Natarajan S. Histomorphometric analysis of gingival tissue in smokers and nonsmokers. SRM J Res Dent Sci 2015;6:161-5

How to cite this URL:
Dayakar MM, Kumar J, Pai GP, Natarajan S. Histomorphometric analysis of gingival tissue in smokers and nonsmokers. SRM J Res Dent Sci [serial online] 2015 [cited 2019 Dec 9];6:161-5. Available from: http://www.srmjrds.in/text.asp?2015/6/3/161/156214


  Introduction Top


Smoking has been recognized as a major risk factor for periodontal diseases. Various studies have shown that smoking has a profound effect on the prevalence, extent, and severity of periodontitis. [1] It has been reported that altered microvasculature of the gingival connective tissue and increased thickness of the epithelium among smokers masks the signs of inflammation. [2] Various investigators have suggested that nicotine increases rate of proliferation of gingival epithelium, thus increasing epithelial thickness among smokers. [3] There are controversies about the effect of smoking on the gingival tissue. Some reports indicated that smokers had more gingival inflammation; [4] some found no difference [5] and others observed less gingival bleeding and inflammation in smokers than nonsmokers. [6]

It is known fact that inflammation resulting from periodontitis brings about vascular changes as well as epithelial proliferation independent of smoking status. [6] Hence, the present investigation is designed to study the epithelial thickness, vascular density, and vascular lumen area in gingiva of smokers suffering from chronic periodontitis and comparing it with the gingiva of nonsmokers with chronic periodontitis.

Aim of study

The purpose of this study was to evaluate and compare the epithelial and microvasculature changes in the gingiva of smokers and nonsmokers with chronic periodontitis.


  Materials and Methods Top


The present study was conducted at K.V.G. Dental College and Hospital, Sullia, D.K. Thirty patients who visited our out-patient department and who were diagnosed as cases of chronic periodontitis with attachment loss of ≥3 mm or probing depth ≥5 mm and with good systemic health were selected for the study. Individuals who had smoked average 5-10 cigarettes per day for more than 2 years were considered in the smoker group. The study population consisted of 15 smokers and 15 nonsmokers. Gingival tissue biopsies with diameter of 5 mm including both epithelium and connective tissue were harvested from periodontal surgical sites and sent for histopathological examination. The images of the histological section were interpreted using ImageJ analyzer processing system.

Inclusion criteria

Smoker's group

  1. Systemically healthy individuals.
  2. Probing pocket depth of ≥5 mm or clinical attachment loss of ≥3 mm.
  3. Smokers who had smoked average 5-10 cigarettes per day for more than 2 years.


Nonsmokers group

  1. Systemically healthy individuals.
  2. No history of smoking.
  3. Probing pocket depth of ≥5 mm or clinical attachment loss of ≥ 3 mm.


Exclusion criteria

  1. Patients under immunosuppressive therapy.
  2. Patients with a history of systemic diseases.


Tissue preparation

The biopsy specimens were collected and fixed in N/10 formalin solution. After which, tissues were dehydrated in graded alcohol of 70%, 80%, 95%, and followed by absolute alcohol than three washes with xylene, each of 30 min. Then tissue is kept for three changes of molten paraffin wax of 1 h each. After that tissue is embedded in paraffin wax and tissue is serially sectioned. And the section is stained with hematoxylin and eosin. The sections were mounted onto the slide with Distrene di butyl phthalate xylene.

Assessment

All slides were coded, and microvessel density, lumen area of blood vessels was assessed under ×40 magnification using ImageJ analysis software [Figure 1] and [Figure 2].
Figure 1: Scale used for converting dimension of the pixel into millimeter scale (155.61 pixels = 0.1 mm)

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Figure 2: Measurement of blood vessels number, area and diameter of microvasculature using ImageJ analyzer software

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The epithelial thickness was measured in ten different areas from the surface of the epithelium to the basement membrane in a chosen field, and the average obtained was considered as the epithelial thickness per slide [Figure 3].
Figure 3: Measurement of gingival epithelium thickness using Image J analyzer software, 10 measurements were taken for epithelium thickness and average of all ten measurements was taken as the thickness of the epithelium

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Ten areas of equal dimensions were randomly selected beneath the epithelium for assessing the vascular density and lumen area. Based on lumen area, vessels were classified into three categories as small (≤380 μm 2 ), medium (381-760 μm 2 ), and large (≥761 μm 2).

Statistical analysis

Student's t-test was used to compare the epithelial thickness among smokers and nonsmokers. Independent Student's t-test was used to compare the number and the area of the blood vessels. Area was significantly higher in the nonsmokers. Large vessels were significantly lower in the smoker group. Small and medium vessels were higher in the smoker group.


  Results Top


In the present study, a total of 30 patients were recruited. They were divided into two groups. Group I smokers and Group II nonsmokers consisted of 15 patients in each group. Mean area of lumen and blood vessel density among smokers was 664.597 ± 387.600 μm 2 and 7.27 ± 3.283 μm 2 , respectively, 892.522 ± 118.789 μm 2 and 8.6 ± 2.354 μm 2 among nonsmokers, respectively [Table 1] and [Figure 4]. Increased vascular density and lumen size was found in nonsmokers group than in smokers group [Figure 5]. Difference in area of the lumen was statistically significant (P = 0.044) while vascular density was statistically insignificant (P = 0.212). Mean epithelial thickness was 0.344 ± 0.007 and 0.398 ± 0.033 μ among smokers and nonsmokers, respectively. Thickness was found to be more in nonsmokers, but no statistically significant difference was found between the two groups [Figure 6].
Figure 4: Comparison of blood vessels lumen area in nonsmoker and smoker in µ m2 (P = 0.044)

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Figure 5: Comparison of number of blood vessels in nonsmoker and smoker (P = 0.212)

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Figure 6: Comparison of epithelium thickness in smoker and nonsmoker in µ m (P = 0.265)

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Table 1: Comparison of blood vessel number, lumen area and number of small-, medium- and large-sized blood vessels in smokers and nonsmokers

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Lumen area and number of the vessels were measured using ImageJ analyzer, and blood vessels were classified as small, medium, and large [Table 1]. The results showed that average number of small-sized blood vessels and medium-sized blood vessels were 4.0 ± 1.512 and 3.0 ± 1.927 among smokers and 2.4 ± 1.844 and 2.4 ± 1.298 among nonsmokers, respectively. Both small-sized and medium-sized blood vessels were higher in smokers, but results were statistically significant for small-sized blood vessels only. Number of large-sized blood vessels was higher in nonsmokers (4.0 ± 2.035) compared to smokers (1.47 ± 1.552) but it was statistically insignificant [Table 1] and [Figure 7].
Figure 7: Comparison of number of small, medium, and large blood vessels in nonsmoker and smoker

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  Discussion Top


Periodontitis is a complex interrelationships between infectious agents and host factors. Environmental and genetic risk factors may modify the expression of disease. Among the environmental risk factors, tobacco smoking has been found to be associated with an increased prevalence and severity of periodontal disease. [7] Smokers have demonstrated a decreased inflammatory response and reduced gingival bleeding to plaque accumulation. As inflammation may cause changes in the vascular density and epithelial thickness independent of smoking status, [8] the present investigation attempted to study the vascular density and gingival epithelium thickness of smokers suffering from chronic periodontitis in comparison with nonsmokers who were also suffering from chronic periodontitis. There are controversies regarding the effect of smoking on gingival tissue. Some reports have indicated that smokers had more gingival inflammation; [4] some found no difference [5] and others observed less gingival bleeding and inflammation in smokers than nonsmokers. [1]

Recent studies have reported that there is an increase in the gingival thickness of smokers. [9] But our results showed that there is a decrease in the thickness of gingival epithelium of smokers suffering from chronic periodontitis when compared to nonsmokers with chronic periodontitis, but the results were not statistically significant. According to Gültekin et al. increase in thickness of the epithelium was not associated only with smoking; periodontal status and inflammation seemed to be more important factors. [3]

The result of our study reveals that the mean blood vessel density was 7.27 ± 3.283 and 8.6 ± 2.354 μm 2 among smokers and nonsmokers, respectively. Though the difference among the mean values was not statistically significant but present results are in accordance with the results of Mirbood et al., who studied the vessel density of gingiva of smokers and nonsmokers using immunohistochemistry and failed to notice any significant difference in the vascular density. [10] Though contradictory findings were reported by Bergström et al. They noticed a significant decrease in the vascular density among smokers in comparison with nonsmokers, but they have compared the vascular density among diseased gingiva of smokers and healthy gingiva of nonsmokers. [2] Lumen area of the vessels was measured using ImageJ analyzer, and blood vessels were classified as small, medium, and large [Table 1]. The results showed that average number of both small-sized and medium-sized blood vessels was higher in smokers, but results were statistically significant for small-sized blood vessels only. Number of large-sized blood vessels was higher in nonsmokers compared to smokers, but it was not statistically significant [Table 1] and [Figure 7]. Similar results for higher number of small blood vessels have been reported by Mirbood et al., who have measured the lumen area using immunohistochemistry method and they found cigarette smokers show a higher percentage of smaller blood vessels and a lower percentage of larger vessels, but similar vascular density, to nonsmokers. [10] According to Elaziz et al. study number of microvessels/mm 2 was not statistically significant among the three groups although smokers had slightly increased number of microvessels. [11] Hence, the results of our study indicate decrease in epithelial thickness and increased vascular density and lumen size in nonsmokers group than in smokers group both having chronic periodontitis [Figure 5]. Difference in area of the lumen was statistically significant (P = 0.044) while difference in vascular density was statistically insignificant (P = 0.212). Although the increase in number of small (P = 0.015) and medium size vessels (P = 0.326) among smokers in comparison with nonsmokers was observed. However, these differences were not statistically significant. This may be due to the cross-sectional study design and smaller sample size.

An investigation with interventional study designed to know the effect of periodontal therapy and cessation of smoking and large sample size is required to clearly evaluate the effects of smoking on the vasculature as well as thickness of gingival epithelium.


  Conclusion Top


The present study concludes that mean thickness of gingival epithelium, mean blood vessel density, and blood vessel lumen area were higher in nonsmokers compared to smokers. Percentage of small-sized and medium-sized blood vessels were higher in smokers and large-sized blood vessels were higher in nonsmokers compared to smokers but no statistically significant difference was found between thickness of gingival epithelium, vascular density among smokers and nonsmokers. So further, longitudinal studies are required with a larger sample size and advanced histopothological methods to clearly understand the association of smoking with blood vessels and epithelial thickness.

 
  References Top

1.
Johnson GK, Hill M. Cigarette smoking and the periodontal patient. J Periodontol 2004;75:196-209.  Back to cited text no. 1
    
2.
Bergström J, Persson L, Preber H. Influence of cigarette smoking on vascular reaction during experimental gingivitis. Scand J Dent Res 1988;96:34-9.  Back to cited text no. 2
    
3.
Gültekin SE, Sengüven B, Karaduman B. The effect of smoking on epithelial proliferation in healthy and periodontally diseased marginal gingival epithelium. J Periodontol 2008; 79:1444-50.  Back to cited text no. 3
    
4.
Arowojolu MO, Fawole OI, Dosumu EB, Opeodu OI. A comparative study of the oral hygiene status of smokers and non-smokers in Ibadan, Oyo state. Niger Med J 2013;54:240-3.  Back to cited text no. 4
[PUBMED]  Medknow Journal  
5.
Preber H, Kant T, Bergström J. Cigarette smoking, oral hygiene and periodontal health in Swedish army conscripts. J Clin Periodontol 1980;7:106-13.  Back to cited text no. 5
    
6.
Rivera-Hidalgo F. Smoking and periodontal disease. Periodontol 2000 2003;32:50-8.  Back to cited text no. 6
    
7.
Page RC, Kornman KS. The pathogenesis of human periodontitis: An introduction. Periodontol 2000 1997;14:9-11.  Back to cited text no. 7
    
8.
Haber J, Wattles J, Crowley M, Mandell R, Joshipura K, Kent RL. Evidence for cigarette smoking as a major risk factor for periodontitis. J Periodontol 1993;64:16-23.  Back to cited text no. 8
    
9.
Hock J, Niki K. A vital microscopy study of the morphology of normal and inflamed gingiva. J Periodontal Res 1971;6:81-8.  Back to cited text no. 9
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10.
Mirbood SM, Ahing SI, Pruthi VK. Immunohistochemical study of vestibular blood vessel density and internal circumference in smokers and non smokers. J Pak Med Assoc 1994;44:210-2.  Back to cited text no. 10
    
11.
Elaziz AA, Elrahman RA, Amer AE. Effect of smoking on periodontal vasculature in smokers and nonsmokers chronic periodontitis patients. Egypt Dent Assoc 2007;53:2-4.  Back to cited text no. 11
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

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