|Year : 2022 | Volume
| Issue : 1 | Page : 19-23
Cytokeratins: A potential biomarker among smokers – An observational study in Indian population
Parvez Hasib Shaikh, Manish Mukherjee, Partha Roy, Sunny Bhatia, Priyanka Prakash
Department of Periodontology, Army Dental Centre (Research and Referral), New Delhi, India
|Date of Submission||24-Jan-2021|
|Date of Acceptance||26-Apr-2021|
|Date of Web Publication||05-Apr-2022|
Parvez Hasib Shaikh
Department of Periodontology, Army Dental Centre (Research and Referral), New Delhi
Source of Support: None, Conflict of Interest: None
Introduction: Gingival epithelium is made up of stratified squamous epithelium consisting of specific cytokeratins (CKs), as intermediate filament proteins of the cytoskeleton. These CKs are involved in transduction signals and transport of nutrients from inside to outside the cell and vice versa. Their expression varies in diseased and inflammatory conditions and can serve as prognostic markers in various diseases.
Objectives: The objective of the study is to evaluate the alteration of CK pattern and topographical distribution of individual CK among smokers and nonsmokers of Indian population.
Subjects and Methods: Gingival tissue samples from smokers and nonsmokers were obtained after taking their consent and segregated as healthy and inflamed into four groups depending on probing depth, clinical attachment loss, and bleeding on probing based on well-defined inclusion criteria such as young, systemically healthy participants and exclusion criteria such as old edentulous patients suffering from systemic disease. Each tissue sample was blinded and subjected for histopathological and immunohistochemical analysis.
Results: Immunohistochemical analysis for CKs CK19, CK8, and CK18 for their pattern and topographical distribution in gingival tissues were obtained using specific antibodies. Histopathological examination was done to study aberration in tissue morphology.
Conclusions: Data were collated from all the groups and CK was analyzed for decreased or increased expression, topographical distribution, in various cell layers, and association with other diseases among Indian population was evaluated.
Keywords: Cytokeratins, gingival epithelium, keratins, smokers
|How to cite this article:|
Shaikh PH, Mukherjee M, Roy P, Bhatia S, Prakash P. Cytokeratins: A potential biomarker among smokers – An observational study in Indian population. J Dent Def Sect. 2022;16:19-23
|How to cite this URL:|
Shaikh PH, Mukherjee M, Roy P, Bhatia S, Prakash P. Cytokeratins: A potential biomarker among smokers – An observational study in Indian population. J Dent Def Sect. [serial online] 2022 [cited 2022 May 18];16:19-23. Available from: http://www.journaldds.org/text.asp?2022/16/1/19/342638
| Introduction|| |
Gingival diseases have burdened the human race since early civilization and this is evidence enough to fathom the importance of diagnosing and treating them. Gingiva is frequently subjected to inflammation leading to gingivitis, periodontitis, or tooth loss in the absence of timely intervention. Changes induced by inflammation in the epithelium and the connective tissue have been demonstrated by quantitative and qualitative light and electron microscopy. There are adequate studies implicating the role of cytokines, matrix metalloproteinases, and white blood cells in gingival inflammation. However, there are very few studies exploring the role of keratins. Keratins are intermediate filament-forming proteins with specific physicochemical properties produced in vertebrate epithelia. Intermediate filaments are a part of the cytoskeleton that constitutes the cytoplasm. Although constituents of the cytoskeleton exist such as microtubules and microfilaments, the intermediate filaments are much more stable and retain their characteristic form even when cell undergoes apoptosis. Role of cytokeratins (CKs) has been reported in dysplastic lesions such as leukoplakia, oral submucous fibrosis, and squamous cell carcinoma. Studies have also shown differences in CK expression in healthy gingiva as compared with inflamed gingiva. However, there are no studies showing the influence of CKs on the gingival epithelium among smokers in Indian population. Among the various risk factors, responsible for progression of periodontal disease smoking is one of the environmental risk factors that are harmful to almost every organ in the body. A departmental study was undertaken with an aim to evaluate the expression of tissue-specific keratins in gingival epithelium with an objective to study the alteration of pattern and topographical distribution of CK8/18 and CK19 in gingival epithelium among smokers and nonsmokers.
| Subjects and Methods|| |
The study was conducted in a tertiary care dental center on patients reporting at the central outpatient department complex from November 2018 to March 2019. The study was conducted according to Helsinki Declaration and was approved by ethical committee of the institute. Before participation in the study, all eligible participants were informed about the study and possible risk and benefits of their participation. Informed written consent from the participants in this study was obtained before inclusion in this study.
To be included in the study, participants had to fulfill following criteria: systemically healthy individuals between age range of 15 years and 65 years were selected for the study. Current smokers and nonsmokers were selected as per the centre for disease control (CDC) guidelines. Patients giving history of smoking ≥100 cigarettes in their lifetime were defined as smokers and those who had history of smoking <100 cigarettes in their lifetime were defined as nonsmokers.
Completely edentulous patients, pregnant or lactating women, and patients on medication with possible influence on periodontium were excluded from the study.
Clinical parameters that were assessed included bleeding on probing (BOP), clinical attachment levels (CALs), probing depth (PD), and gingival index. Oral hygiene status and gingival inflammation were assessed by full mouth plaque score (FMPS) and full-mouth bleeding scores at four sites per tooth as percentage values. PD and CAL were recorded at six sites per tooth using a periodontal probe (UNC 15). Healthy sites were defined as those sites having PD of <3 mm, no CAL and no BOP; whereas inflamed sites included PD of more than 3 mm, with CAL and BOP.
Based on the above inclusion and exclusion criteria, the study included 40 gingival tissue samples that were segregated into four groups as follows:
- Group A: nonsmokers and periodontally healthy (n = 10 sites)
- Group B: nonsmokers and inflamed gingiva (n = 10 sites)
- Group C: smokers and inflamed gingiva (n = 10 sites)
- Group D: smokers and periodontally healthy (n = 10 sites).
All the parameters were evaluated by single examiner (PS). Clinically healthy tissue samples were obtained from patients reporting for procedures such as crown lengthening, extraction of teeth for orthodontic purpose, and impacted third molar extractions having soft tissue coverage [Figure 1]; whereas, tissue samples of clinically inflamed gingiva were obtained during gingival/periodontal surgical procedures [Figure 2]. The gingival samples collected were formalin-fixed and paraffin-embedded [Figure 3]. They were coded (blinded) and subjected for histopathological and immunohistochemical analysis by independent examiner. From each sample, cryostat sections were made that allowed for histological staining with hematoxylin and eosin stain and immunohistochemical analysis of CK8/18 and 19 using monoclonal antibodies.
The slides were incubated with primary monoclonal antibodies of CK8/18 and CK19. The slides were further incubated with horseradish peroxidase polymer secondary antibody for 30 min. To visualize the color reaction, slides were subsequently incubated with a chromogen for 10 min and later were dehydrated, in xylene and then mounted.
| Results|| |
Hematoxylin and eosin stains did not reveal any aberrations in the soft tissue morphology. In all the tissue samples, continuity of the basement membrane was noticed without any dysplastic changes; however, hypertrophy of the epithelium was noticed in some sections of superficial layers.
In some of the samples, inflammatory infiltration was recognized in subepithelial lamina propria that sometimes extended into the deeper connective tissue of the gingiva [Figure 4].
Evaluation of immunostaining
The expression of CK8/18 [Figure 5] and CK19 [Figure 6] was determined independently by independent examiner who was blinded about the samples and the groups.
The expression of CKs in the epithelium was assessed as weakly positive when expressed only in basal layer and strongly positive when expressed in suprabasal layers.
Weak immunoreactions of CK8/18 and CK19 were noted in the oral mucosa of tissue samples of Group A and Group B. A strong reaction of suprabasal layers was found in the tissue samples of Groups C and D [Table 1] and [Table 2].
Data were analyzed by SYSTAT 13 software, (Chicago, Illinois, US). Friedman's two-way analysis of variance was done for CK8/18 and CK19. An intergroup analysis was done that showed statistically significant difference between Groups A and C and Groups A and D for CK8/18 and CK19 [Table 3], [Table 4] and [Graph 1], [Graph 2].
Strong expression of CK8/18 and CK19 was noticed in Groups C and D in comparison with Group A, and weak expression of CK8/18 and CK19 was seen in Group A as compared to other groups.
| Discussion|| |
Transition from health to disease results from complex interplay among the host, pathogenic bacteria, and commensal bacteria. Small amounts of bacterial plaque can be controlled by the body's defense mechanisms without destruction; however, when dysbiosis happens, as a result of increased susceptibility, high bacterial load, or pathogenic infections, periodontal destruction occurs. The susceptibility of the host is determined by genetic factors, environmental and behavioral factors, such as smoking. Smoking dramatically increases host susceptibility to periodontal breakdown.
Studies show that in smokers, the ability to rebound from a dysbiotic state and re-establish a health-compatible community is impaired. Cigarette smoke has shown to modify the expression of Porphyromonas gingivalis genes directly, including several virulence-associated genes. In smoking, due to release of various end products such as nicotine and aromatic hydrocarbons, there is stimulation of sympathetic ganglion leading to release of catecholamines that causes vasoconstriction. As a sequela to this, there is a reduction in blood flow to gingiva resulting in reduced gingival bleeding. In a study conducted by Prakash et al., vascular caliber was found to be reduced among smokers, and there was an increase in the epithelial thickness. Thus, BOP which is one of the most striking features of periodontal disease is masked among smokers. In the absence of any significant clinical changes, coupled with continuous exposure to smoking, there is rapid periodontal destruction in a chronic smoker. In such situations, one has to rely on patients' history which may be inaccurate. Herein comes the role of keratins as they are stable and retain their characteristic form even in adverse conditions. They can serve as potential biomarkers that may have prognostic and therapeutic.
The present study has revealed marked differences in expression of CK8/18 and 19 between smokers and nonsmokers and this difference was found to be statistically significant. These CKs are normally present in basal layers, and their expression in suprabasal layers is generally associated with some aberration in the tissues. The results of the present study are comparable to a study conducted by Lima et al. wherein the authors evaluated the CK profile by exfoliative cytology. In their study, they found a strong expression of CK19 among smokers in comparison to nonsmokers similar to the present study. In another study conducted by Chen et al., CK19 was markedly increased in the basement membranes and suprabasal layers of the oral epithelium of periodontitis tissues. The authors in their study found that the intensity of CK19 staining was significantly increased in the epithelium of patients with periodontitis but not in those with gingivitis. They observed that enhancement of CK19 expression and altered topographic distribution of CK19-positive cells in the periodontitis group were highly associated with the breakdown of gingival tissues and periodontal pocket formation. Thus, CKs can serve as potential biomarkers to intercept destructive changes before significant clinical signs and symptoms appear in the periodontium.
| Conclusions|| |
In the present study, there is a strong expression of CK8/18 and CK19 among smokers in inflamed and healthy groups as compared to nonsmokers. However, it was a pilot study small sample size. Multicentric studies with larger sample size are required to further evaluate the impact of CKs on periodontium. CK8/18 and CK19 have also been expressed in oral malignancies. Studies have shown them to be potential predictive biomarkers for oral malignancies as well.
The expression of tissue-specific CK8/18 and CK19 in smokers of Indian population with periodontitis therefore may be indicative of an ominous outcome. In the era of evidence-based medicine as we navigate our way to dissect, decipher, and infer clinical conditions, such stable markers may offer greater probability of early diagnosis of periodontal diseases and oral malignancies.
We wish to acknowledge Dr. Partha Roy, Col Anupam Chattoraj, Dr Rajni Parmar, and Wg Cdr Sudip Indu for guiding and facilitating the conduct of study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4]