The Effect of Smoking on Clinical Presentation and Expression of TLR-2 and CD34 in Oral Lichen Planus Patients

Clinical and Immunohistochemical Study

Nermine Raouf Amin; Nermin Yussif; Enji Ahmed


BMC Oral Health. 2020;20(129) 

In This Article


Patients' Selection

OLP patients were recruited from the out-patient clinic of Oral Medicine and Periodontology Department, Faculty of Dentistry, Cairo University. For control patients, tissue samples were taken from those undergoing operculectomy.

The aim of the present study is to compare the immunohistochemical expression of TLR-2 and CD34 in OLP between smokers and non-smokers. According to Klosek et al.[16] and Using G-power program, the effect size between both groups was found to be 2.16 using power of 80 and 5% significance level giving a total sample size of 15 patients (5 patients per group). This number was to be increased to a total sample size of 21 (7 patient per group) to compensate for possible losses during the follow up. The previous sample size was exceeded in this work to be 20 patients for each group.

All patients fulfilled the WHO's clinical diagnostic criteria for OLP[17] which is the presence of bilateral, more or less symmetrical lesions and a lacelike network of slightly raised gray-white lines (reticular pattern). Erosive, atrophic, bullous and plaque-type lesions were accepted only as a subtype in the presence of reticular lesions elsewhere in the oral mucosa.

The patients were examined clinically using spot light and magnifying glass for oral lesions, and natural light for skin lesions.

All patients fulfilled the WHO's histopathologic diagnostic criteria for OLP[18] which is the presence of a well-defined band-like zone of cellular infiltration (mainly lymphocytes) that is confined to the superficial part of the connective tissue, liquefaction degeneration in the basal cell layer and absence of epithelial dysplasia.

Inclusion Criteria. Systemically free OLP patients as evaluated by the aid of the Dental Modification of the Cornell Medical Index to standardize their systemic condition[18] were included in this study. Both sexes, smokers and non-smokers were also included.

Exclusion Criteria. Patients showing signs of malignancy as induration of the lesion, loss of flexibility or rolled edges and patients who had any other visible lesion than OLP were excluded from this study. Patients who had received any medication for at least 3 months before the biopsy taking except that for OLP, patients who had any systemic disease, pregnant and lactating women were also excluded from this study.

Ethical Procedures. Each subject signed an informed written consent form. The Research Ethics Committee of Faculty of Dentistry, Cairo University revised and approved this research on 25/6/2019 with the number (19-6-30).

Grouping of Patients

Patients were divided into three groups; 20 individuals in each group as follows:

Group I: Control group.

Group II: Non-smoker patients with OLP.

Group III: Smoker patients with OLP.

III-Clinical assessment:

The following clinical criteria were evaluated for groups II and III.

Pain Score. The symptomatology score was assessed using visual analogue scale (VAS), which consists of 10 scores in which the patient marked the point along the line that represented his pain. The scale was measured from no pain to the end of scale (0 = no pain, 10 = extremely painful).[19]

Clinical Score. A clinical score was given to all OLP lesions during exacerbation according to the clinical severity on a scale that ranged from 0 to 5 according to the criteria set by Thongprasom et al.,[20] as follows:

0: No lesion, normal mucosa.

1: White straie, no erythematous area.

2: White straie with atrophic area less than 1cm2.

3: White straie with atrophic area more than 1cm2.

4: White straie with erosive area less than 1 cm2.

5: White straie with erosive area more than 1 cm2.

Score for each patient was calculated by recording a score for each lesion in the oral cavity separately, then calculating the average of these scores.

Biopsy Taking

Biopsy was taken from OLP lesions. For small lesions (< 4–5 mm in diameter), an excisional biopsy was taken. For large lesions (> 4–5 mm in diameter), an incisional biopsy was performed taking part of the lesion with part of the adjacent normal mucosa.[21] Tissue samples were obtained under ring block anesthesia from all groups and were placed immediately in 10% neutral buffered formalin fixative.

Histopathological Preparation

A Hematoxylin and Eosin (H&E) stained slide was prepared to confirm the diagnosis. Two sections were mounted on positively charged slides. One for the application of the primary antibody and the other one served as a negative control.

Immunohistochemical Staining

Antibodies (TLR-2 and CD34). TLR-2 antibody (mouse monoclonal primary antibody, sc-21,759, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted at 1:100 and a ready to use CD 34 (mouse monoclonal primary antibody, AM353–5 M, Biogenex, USA) were used in this work.

Steps of Basic Immunostaining Procedures.[22] Immunostaining for TLR-2 and CD34 was performed using Ventana Bench mark autostainer (Ventana Medical Systems, Tucson, AZ, USA) at Pathology Department, National Cancer Institute; Cairo University, as follows:

Deparaffinization and hydration of the tissue sections were done in descending grades of alcohol each for 10 min. Tissue sections were boiled in 10 mM citrate buffer, pH 6.0 for 10–20 min followed by cooling at room temperature for 20 min (antigen retrieval step). The sections were then incubated in 0.3% hydrogen peroxide for 30 min to block the endogenous peroxidase activity. The sections were washed before the application of 100 ml of TLR-2 antibody at dilution of (1:100) under incubation temperature of 30 °C for 80 min and CD34 antibody under incubation temperature of 30 °C for 20 min, followed by application of the secondary antibody for 30 min. Diaminobenzidine tetrahydrochloride (DAB) was applied to sections for 15 min at room temperature. Sections were counterstained with Mayer's Hematoxylin which was applied for 8 min and then a bluing reagent was applied for 4 min. Slides were extracted and arranged in racks. They were washed in tap water for 5 min and then dehydrated in ascending grades of alcohol each for 5 min. Slides were cleared in xylene and then cover slips were applied and mounted using Distyrene Plasticizer Xylene (DPX) mounting agent.

N.B. Immunohistochemical staining was carried out in one batch for standardization.

Immunohistochemical Assessment

Transmission Light Microscope. The immunostained sections were examined using low and high power fields by the light microscope.

Image Analysis Computer System. The image analyzer computer system applying the software Leica Quin 500* {Leica Microsystems LTD. CH9435 Meerbrugg Type: DFC295 (12730469), Input:12v/170 MA, Serial number: 0557060916, Switzerland} was used for measuring the area percent of positive TLR-2 and CD34 immunoexpression in high power fields (× 400 magnification). Area percent was calculated from three fields per patient. Fields were randomly chosen from well stained sections that properly represented the histopathology of OLP. Mean area percent values for both markers were calculated for the studied groups.

Statistical Analysis

The obtained data was presented as mean ± standard deviation (SD) values. P values< 0.05 were considered significant. Statistical analysis was performed by using a computer program IBM SPSS. Student t-test was used to compare between two groups regarding the clinical and pain scores. One Way Analysis of Variance (ANOVA) test was used to compare between three groups followed by Tukey's post hock test for pairwise comparison between each two groups regarding TLR-2 and CD34 immunoexpression.