Effect of smoking on gingival microvasculature: A histological study.

BACKGROUND: Smoking has been reported as a major risk factor for periodontal disease. Studies have demonstrated decreased bleeding on probing and reduced inflammatory response among smokers, which has been attributed to the alterations in gingival microvasculature, gingival epithelium. In previous investigations, vascular and epithelial changes have been studied in the gingival tissues of smokers suffering from periodontitis and compared with healthy periodontium of non smokers. Inflammation is known to cause vascular and epithelial changes in the gingiva on its own in the absence of smoking. Hence, in the present investigation, an attempt is made to study and to compare the vascular and epithelial changes in the gingiva of smokers and non smokers suffering from chronic periodontitis.
AIM OF STUDY: The purpose of this study was to evaluate the vascular and epithelial changes in gingiva of smokers and non smokers with chronic periodontitis. STUDY
DESIGN: Cross sectional study
MATERIALS AND METHODS: Gingival biopsies were obtained from 33 male patients (18 smokers and 15 non smokers) who were undergoing periodontal therapy or extraction. The sections were stained with eosin and hematoxylin. Vascular density, area of the lumen, and epithelial thickness were assessed using histomorphometric image analysis. STATISTICAL
METHOD: Data was analyzed using student unpaired 't' test, Karl Pearsons correlation, and Chi-square test.
RESULTS: The mean blood vessel density for smokers was 12.388±6.472 and for non smokers was 14.800±4.91. The mean lumen area of the vessels among smokers and non smokers was 19.290±8.775 μm(2) and 20.044±7.896 μm(2), respectively. The mean epithelial thickness among smokers was 150.551±32.994 μ and 134.941±30.63 μ for non smokers.
CONCLUSION: Based on the present histomorphometric study, it could be concluded that smokers have less vascular density and reduced lumen area and increased epithelial thickness than non smokers. However, these changes were not statistically significant.

INTRODUCTION

Smoking has been reported as a major risk factor of periodontal disease. Epidemiological studies have shown that smoking has a profound effect on prevalence, extent, and severity of periodontitis.[1] Clinical parameters including probing pocket depth, clinical attachment loss were found to be increased in smokers compared to non smokers. However, it is interesting to note the decrease in bleeding on probing and reduced inflammatory response for plaque accumulation among smokers as compared to non smokers.[23] These changes have been implicated, by various investigators, to altered microvasculature of the gingival connective tissue and increased thickness of epithelium among smokers, which mask the signs of inflammation.[4-6] It is reported that smokers exhibit reduced vascular density and reduction in the lumen area of the gingival vessels.[7] Studies have suggested that nicotine increases rate of proliferation of gingival epithelium, thus increasing epithelial thickness among smokers.[8] However, these investigations have compared gingival vascular and epithelial changes of smokers suffering from periodontitis with those of healthy gingiva of non smokers. It is known that inflammation brings about vascular changes like increased density and dilatation as well as epithelial proliferation independent of smoking status.[9] Hence, the present investigation is designed to study the vascular density, lumen area, and epithelial thickness in the gingiva of smokers suffering from chronic periodontitis and compare it with the gingiva of non smokers with chronic periodontitis.

MATERIALS AND METHODS

The present study was conducted at M. R. Ambedkar Dental College, Bangalore. The study protocol was approved by institutional ethical committee and an informed consent was obtained from all the patients recruited for the study after explaining the purpose of the study. Thirty three male patients who visited out- patient department and who were diagnosed as cases of chronic periodontitis with attachment loss of ≥3 mm and probing depth ≥5 mm and with good systemic health were selected for the study. Individuals who had smoked average ≥10 cigarettes per day for more than three years were considered in the smoker group and number of pack years for each patient was calculated. The study population consisted of 18 smokers and 15 non-smokers. Gingival tissue biopsies with diameter of 5 mm including both epithelium and connective tissue were harvested from periodontal surgical sites and from extraction sites with probing pocket depth ≥5 mm and attachment loss of ≥3 mm.

Inclusion criteria

Smokers group

Systemically healthy individuals with chronic periodontitis

Probing pocket depth of ≥5 mm

Clinical attachment loss of ≥3 mm

In smokers group patients who smoked >10 cigarettes for more than three years

Non-Smokers group

Systemically healthy individuals with chronic periodontitis

No history of smoking

Probing pocket depth of ≥5 mm

Clinical attachment loss of ≥3 mm

Exclusion criteria

With history of systemic diseases

Patients under medications

Patients under immunosuppressive therapy

Tissue preparation

The biopsy specimens collected were fixed in N/10 formalin solution. Tissues were processed and were embedded in fresh paraffin wax. Specimens were serially sectioned at four micron thickness and were cut at right angle to the oral vestibular epithelium. The sections were washed in 100% and then 70% isopropyl alcohol for five minutes each. After which, two washes with xylene, each five minutes were given. It was then rinsed with distilled water for five minutes, immersed in hematoxylin for five minutes, rinsed again with distilled water and alcohol, and finally immersed in eosin one dip. The sections were mounted on to the slide with Distrene di butyle phalate xylene (DPX).

Assessment of microvessel density and epithelial thickness

Under the blinded protocol for smoking history, all slides were coded and microvessel density, lumen area of blood vessels were assessed under ×20 magnification using Magnus Pro 3.0 image analysis software. After applying micro grid, two areas of equal dimensions of 200 μm2 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 (≤33 μm2), medium (34-64 μm2), and large (≥67 μm2) [Figures 1 and 2].

Figure 1

After application of grid on selected field

Figure 2

Counted blood vessels in selected square lattice

The epithelial thickness was measured in three different areas from the surface of 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

Selected section for measuring epithelial thickness

Statistical analysis

Student's ‘t’ test was used to compare the vascular density, lumen area, and epithelial thickness among smokers and non smokers. Chi-square test was used to compare the percentage of small, medium, and large blood vessels. Correlation was assessed between pack years and number of blood vessels and epithelium thickness using Karl Pearson correlation. P<0.05 was considered to be statistically significant. Data was analyzed using Statistical Package for Social Sciences (SPSS) Version 15.

RESULTS

In the present study, a total of 33 patients were recruited. They were divided into two groups. Group I smokers consisted of 18 patients with mean age of 46±10.44 years; group II non smokers consisted of 15 patients with mean age of 47±7.82 years. [Table 1] shows the mean age distribution.

Table 1

Mean age distribution among two groups

Mean blood vessel density and area of lumen among smokers was 12.388±6.472 μm2 and 19.290±8.775 μm2, respectively; 14.800±4.901 μm2 and 20.044±7.896 μm2 among non smokers, respectively. Increased vascular density and lumen size was found in non smokers group than in smokers group. No statistical significant difference was found among the parameters [Table 2, Figures 4 and 5].

Table 2

Mean comparison of epithelial thickness, number of blood vessels and area of lumen

Figure 4

Number of blood vessels in smokers and non smokers

Figure 5

Area of lumen in smokers and non smokers

Mean epithelial thickness was 150.551±32.994 μ and 134.941±30.631 μ among smokers and non smokers, respectively, and thickness was found to be more in smokers. No statistical significant difference was found between the two groups [Table 2, Figure 6].

Figure 6

Epithelial thickness in smokers and non smokers

The percentage of small sized blood vessels was 83.9% among smokers and 82.9% among non smokers. Percentage of medium and large sized blood vessels among smokers was 14.8% and 1.3% which was less than the percentage found in non smokers, 15.3% and 1.8%. The difference was statistically not significant [Table 3, Figure 7].

Table 3

Comparison between percentage of small, medium, and large blood vessels

Figure 7

Blood vessel size in smokers and non smokers

There was a moderate negative correlation found between the number of pack years and number of blood vessels counted among smokers. There was a weak positive correlation between epithelium thickness and pack years among smokers. But the difference was not statistically significant. [Table 4, Figures 8 and 9].

Table 4

Correlation between pack years and epithelium thickness and number of blood vessels

Figure 8

Correlation between no. of blood vessels and pack years

Figure 9

Correlation between epithelial thickness and pack years

DISCUSSION

Smoking today is considered to be associated with periodontal disease. A position paper by American Academy of Periodontology has implicated smoking to be a risk factor, affecting the extent and severity of periodontitis.[10]

Smokers have demonstrated a decreased inflammatory response to plaque accumulation and reduced gingival bleeding.[13] This altered inflammatory response among smokers has been attributed to alteration in the gingival vasculature which includes decreased vascular density, lumen area of gingival vessels, and epithelial thickness.[56] Previous studies, which have implicated alteration in the vascular density and lumen area of gingival vessels and thickening of epithelium, have compared the inflamed gingival tissues of smokers with healthy gingival tissues of non smokers.[78]

As inflammation is known to bring about changes in the vascular density and lumen area as well as epithelial thickness independent of smoking status,[11] the present investigation attempted to study the vascular density as well as lumen area in the gingival tissues of smokers suffering from chronic periodontitis in comparison with non smokers who were also suffering from chronic periodontitis.

Age also affects the gingival vasculature and epithelial thickness.[12-14] To overcome this, age matched patients have been selected for this study. It is presumed that the results of this investigation will go a long way in understanding the role of vascular and epithelial changes in the pathogenesis of periodontal disease among smokers.

The result of our study reveals that the mean blood vessel density was 12.388±6.472 and 14.800±4.901 among smokers and non smokers, respectively. The difference among the mean value was not statistically significant.

Present results are in accordance with the results of Mirbood et al., who studied the vessel density in vestibular gingiva of smokers and non smokers using immunohistochemistry and failed to notice any significant difference in the vascular density in the gingiva of smokers and non smokers.[7] Contrary to our results, Persson et al., and Scardina et al., have reported a significant decrease in the vascular density among smokers in comparison with non smokers, but they have compared the vascular density of diseased gingiva of smokers to the healthy gingiva of non smokers.[415]

In our investigation, the lumen area of the vessels was measured using image analysis software and blood vessels were classified depending upon the lumen area as small, medium, and large. The results indicated that the percentage of small sized blood vessels were higher in smokers than in non-smokers and the percentage of medium and large sized blood vessels were higher in non smokers compared to smokers; however, these differences were not statistically significant. Similar results have been reported by Mirbood et al., who have measured the lumen area using immunohistochemistry method.[7]

In recent years, an interest is being evinced in the thickness of gingival epithelium of smokers and studies have reported that there is increase in the gingival thickness of smokers and this is being implicated as one of the factors responsible for masking the clinical signs and symptoms of inflammation among smokers.[616] However, the increased thickness in these studies was compared with healthy gingival specimens of non smokers.

Our results showed that there is an increase in the thickness of gingival epithelium of smokers suffering from chronic periodontitis when compared to non smokers with chronic periodontitis. These results are in accordance with studies of Cunha et al.[6] In the present investigation, an attempt was made to find out the correlation, if any, between pack years and vessel density and epithelial thickness. We found moderate negative correlation between pack years and the number of blood vessels and a positive correlation between pack years and epithelial thickness. The results of our study did indicate a reduction in vascular density and an increase in epithelial thickness among smokers in comparison with non smokers. 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 design to know the effect of periodontal therapy and cessation of smoking and large sample size is warranted to clearly evaluate the effects of smoking on the vasculature as well as gingival epithelium.

CONCLUSION

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