|Year : 2014 | Volume
| Issue : 6 | Page : 734-738
Comparative evaluation of salivary soluble CD44 levels in periodontal health and disease
Sumeet Kaur, Savitha Narayanswamy, Alampalli Viswanathamurthy Ramesh
Department of Periodontics, The Oxford Dental College, Hospital and Research Centre, Bengaluru, Karnataka, India
|Date of Submission||02-Oct-2010|
|Date of Acceptance||14-Apr-2014|
|Date of Web Publication||19-Dec-2014|
Department of Periodontics, The Oxford Dental College, Hospital and Research Centre, Bengaluru 560 068, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context: Inflammation, immunoactivation, and malignant diseases are associated with increased plasma levels of soluble CD44 (sCD44). Serum sCD44 has been recognized as a diagnostic marker in smoking-induced diseases. Aim: (1) To assess the levels of salivary sCD44 in periodontal health and disease. (2) To compare the levels of salivary sCD44 in smokers and nonsmokers. (3) To assess if salivary sCD44 levels could be used as a diagnostic marker for periodontitis. Setting and Design: A total of 60 patients were divided into three groups viz. Group A - healthy, Group B - aggressive periodontitis and Group C - chronic periodontitis (Subdivided into C1 - chronic periodontitis smokers and C2 - chronic periodontitis nonsmokers). Materials and Methods: The plaque index, gingival index (GI), probing depth and clinical attachment level; along with the radiographs were recorded. The saliva sample collected at baseline was stored at −80°C. The sCD44 levels were analyzed using ELISA. Statistical Analysis Used: ANOVA test and Mann-Whitney's test was used to compare readings between all the groups and Pearson correlation was calculated for CD44 and all the clinical parameters in each group. Results: Highest mean sCD44 was recorded in Group C2 followed by Group C1. The GI was positively correlated with CD44 levels in chronic periodontitis group. Contrary to previous reports nonsmokers subjects had higher CD44 levels as compared to smoker. Conclusion: Soluble CD44 levels were positively correlated with periodontal disease. Thus, salivary sCD44 could be considered as a one of the biomarker for periodontitis that is, aggressive and chronic periodontitis.
Keywords: Aggressive periodontitis, diagnostic marker, nonsmokers, soluble CD44
|How to cite this article:|
Kaur S, Narayanswamy S, Ramesh AV. Comparative evaluation of salivary soluble CD44 levels in periodontal health and disease. J Indian Soc Periodontol 2014;18:734-8
|How to cite this URL:|
Kaur S, Narayanswamy S, Ramesh AV. Comparative evaluation of salivary soluble CD44 levels in periodontal health and disease. J Indian Soc Periodontol [serial online] 2014 [cited 2019 Jun 19];18:734-8. Available from: http://www.jisponline.com/text.asp?2014/18/6/734/147409
| Introduction|| |
Periodontitis is an immune modulatory disease and triggers inflammation mediated loss of the periodontal ligament and alveolar bone that supports the teeth. The goal of periodontal diagnostic procedure is to provide useful information to the clinician regarding present periodontal disease, type, location, and severity, which serves as a basis for treatment planning, and provide essential data during periodontal maintenance and disease monitoring phases of treatment.
Patients with periodontitis have elevated circulating inflammatory markers that can be correlated to the severity of disease. During inflammation, human soluble CD44 (sCD44), a cell surface adhesion molecule is elevated.  CD44 is a family of glycoproteins encoded by a single gene and is expressed on the surface of a wide range of cell types, such as leucocytes, fibroblasts, erythrocytes, myocytes, and epithelial cells. It plays a major role in multiple physiologic functions such as cell to cell adhesion, cell matrix interaction, lymphocyte recruitment to inflammatory sites, and tumor metastasis through adhesion to hyaluronon; the principal ligand. Its analogy with soluble ICAM-1, defines it as an immunomodulator  with the role in the immune system and connective tissue maintenance. Recently, sCD44, an adhesion molecule has been considered for its role as a potential biomarker for periodontal diseases.
Studies carried out by Haberhauer et al.,  Kittl et al. and Kato et al. have shown that CD44 levels are raised in subjects with systemic inflammatory conditions, such as rheumatoid arthritis and bronchitis. Serum sCD44 concentration is significantly elevated in smokers compared with nonsmokers.  Various studies have identified CD44 in saliva as well. ,, In the oral cavity, the most common form of periodontal disease that is, chronic periodontitis is also associated with raised levels of salivary sCD44.  Many studies have shown that the smoking status of an individual influences the levels of CD44 in saliva as well. , Also smoking is a well established risk factor for periodontitis. 
Thus, this study aims to assess the salivary sCD44 levels in periodontal health and disease thereby considering its role as a diagnostic marker for periodontitis and to compare the levels of salivary sCD44 in smokers and nonsmokers.
| Materials and Methods|| |
A total of 60 subjects were included in the study and were categorized into three groups viz. healthy controls (Group A), aggressive periodontitis (mild to severe cases; Group B) and chronic periodontitis (Group C), with 20 subjects in each group. The chronic periodontitis group was further divided into chronic periodontitis smokers (C1 Group) and chronic periodontitis nonsmokers (Group C2) consisting of 10 patients each. The inclusion criteria: Patients aged between 18 and 55 years, systemically healthy with no history of periodontal treatment in last 6 months or antibiotic usage in previous 3 months. Pregnant and lactating women, patients on medication (e.g. corticosteroids, anti-inflammatory drugs, anticancer drugs, and immune modulators) or suffering from infectious conditions other than periodontitis were not included in the study.
The clinical parameters assessed were plaque index (PI) (Silness and Loe, 1964),  gingival index (GI) (Loe H and Silness J, 1963),  probing pocket depth and clinical attachment levels; using Williams periodontal probe. Orthopantomogram along with standardized intraoral periapical radiographs were taken for Group B and C patients using long cone paralleling technique.
Recording of all clinical parameters was completed 1 day before the collection of samples. At the second visit, saliva samples were collected during morning hours  by Navazesh's cotton roll method. ,
The samples were centrifuged at 2000 × g and the supernatants was collected and stored immediately at −80°C until the biochemical assay was carried out. Saliva samples were analyzed for the levels of all sCD44; normal and variant isoforms (total sCD44) by using sandwich-type ELISA. CD44 ELISA kit, manufactured and distributed by Gen-Probe Diaclone SAS, France was used in this study.
Descriptive statistical analysis was carried out using Bonferroni, Kruskal-Wallis test, Mann-Whitney test and the Correlation test to evaluate the results.
| Results|| |
The overall mean [PI, [Table 1] at baseline in Group A was 0.75 ± 0.36, in Group B was 1.56 ± 0.61, in Group C1 was 1.57 ± 0.27 and in Group C2 it was highest with a mean of 1.80 ± 0.40. Higher mean [GI, [Table 2] was recorded in nonsmokers-chronic periodontitis group followed by aggressive periodontitis group, smokers-chronic periodontitis group and healthy group, respectively. Highest mean probing depth [PD, [Table 3] was recorded in aggressive periodontitis group (4.02 ± 1.18 mm) followed by nonsmokers-chronic periodontitis group (3.74 ± 0.67 mm), smokers-chronic periodontitis group (3.73 ± 0.77 mm) and healthy group (1.96 ± 0.39 mm), respectively.
Higher mean clinical attachment loss [CAL, [Table 4] was recorded in smokers-chronic periodontitis (3.87 ± 2.11) followed by aggressive periodontitis group (3.79 ± 1.95) and nonsmoker chronic periodontitis group (3.08 ± 0.88), but the difference between them was not statistically significant (P > 0.05).
Highest mean CD44 [Table 5] was recorded in Group C2 (14.25 ± 8.77 ng/ml) followed by Group C1 (10.12 ± 5.37 ng/ml), Group B (8.94 ± 6.47 ng/ml), and Group A (1.66 ± 0.61 ng/ml), respectively. The difference in mean CD44 between the groups was found to be statistically significant (P < 0.01).
Correlation between CD44 and other parameters in aggressive periodontitis group [Table 6] showed a moderate and positive correlation between CD44 and PI and a weak correlation between CD44 and GI, but both these relationship were not statistically significant. Moderate positive correlation was found between CD44 versus CAL (P < 0.01) and CD44 versus PD (P < 0.05) and the correlation was not statistically significant.
|Table 6: Correlation between CD44 and other parameters in aggressive periodontitis group|
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Strong positive correlation [Table 7] was found between CD44 and GI in Group C (r/ρ = 0.685) and it was statistically significant (P < 0.01).
|Table 7: Correlation between CD44 and other parameters in chronic periodontitis group|
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| Discussion|| |
The objective was to evaluate the salivary sCD44 values in two major forms of periodontitis which are inflammatory in nature viz. aggressive and chronic periodontitis and to compare them with healthy subjects (control group).
The study included 40 males and 20 females. The overall mean CD44 levels in females were higher (8.732 ng/ml) than to males (6.79 ng/ml). This finding was in accordance with the study by Sjφberg et al.  who found higher serum CD44 levels in females as compared to that of males. The female hormones that is, estrogen and progesterone result in depression of cell mediated immunity, increase specific receptors in gingival tissues and alters the rate and pattern of collagen production. Furthermore, the destruction of gingival mast cells by the increased sex hormones and the resultant release of histamine and proteolytic enzymes contribute to exaggerated inflammatory response to local factors in females.  CD44 is present on the surface of these mast cells and are released in circulation on their destruction thereby explaining higher CD44 levels in females as compared with males. 
Gingival index comparison within the groups showed that the Group C had a higher GI as compared with Group B. These findings were consistent with the higher PI score in chronic patients as compared with aggressive patients. In the chronic periodontitis group, the smokers (Group C1) had lower baseline gingival scores than that of nonsmokers (Group C2). This could be due to the fact that the, smokers have a reduced capacity to mount and maintain an effective defense mechanism producing lower inflammatory response to a given plaque challenge.  Furthermore, Mavropoulos et al. attributed the reduced GI to the vasoconstrictive properties of nicotine, which contribute to gingival vascular dysfunction.
In this study, CD44 levels increased with increasing probing depth and clinical attachment level, thereby pointing to the already established finding that the destruction of the collagen matrix in periodontal diseases is due to increased inflammatory response and immunomodulation of the host.
This study noted highest levels of salivary sCD44 (ng/ml) in chronic periodontitis ranging from 2.50 (ng/ml) to 26.04 (ng/ml) with a mean value of 12.19 ± 7.29 in comparison to aggressive periodontitis; 8.94 ± 6.47 ng/ml. CD44 is implicated in cellular matrix changes by the cell to cell and cell matrix interaction. It induces adhesive interaction between lymphocytes and gingival fibroblasts that is, mediated by the CD44 - hyaluronic acid (HA) complex, which regulates cell - cell adhesion and cell migration and proliferation, required for tissue morphogenesis and repair. Thus, salivary sCD44 - HA complex plays an important role in chronic inflammation. Increased salivary sCD44 can be attributed to increased inflammatory response and the chronicity of the disease. The results obtained in this study are in accordance with studies done by Ghallab and Shaker,  Leonardi et al. (2006), Murakami et al. (1997). This further explains higher levels of CD44 in chronic periodontitis cases.
In order to further throw light on the role of smoking on levels of salivary sCD44, the comparison between smokers (10.12 ± 5.37 ng/ml) and nonsmokers (14.25 ± 8.77 ng/ml) was done, but the finding in our study did not match with the previous reports ,, where CD44 levels was higher in smokers as compared to nonsmokers. Elevated concentrations of soluble adhesion molecules reflect ongoing inflammatory processes. However, increasing evidence suggests that specific soluble adhesion molecules are also immunomodulatory.  Moreover, in this study self-reported smoking habits were taken into consideration, which may be unreliable and analysis of dose-dependent relationships may be further complicated because of variable factors in the smoking habits of the individual, such as frequency and depth of inhalation. This is supported by the finding in a study done by Scott et al.  where he found that both the subjects with highest and the lowest level of cotinine smoked equal number of cigarettes daily. Cotinine, a major catabolite of nicotine is shown to be a specific and accurate biomarker of current smoking status. The increase in the level of CD44 in nonsmokers as compared to smokers can also be due to gender distribution that is, the chronic periodontitis smoker group consisted of 100% males, whereas the nonsmoker group consisted of 40% females. This can be explained by our previous finding that females have a higher concentration of salivary sCD44 as compared to their male counterparts.
So far no evidence has been provided for salivary sCD44 levels in aggressive periodontitis patients and this study is first of its kind to assess its level in such subjects. In our study, we found that the CD44 levels in aggressive periodontitis group were lower than Group C (chronic periodontitis), but was higher than the healthy group (Group A). This can be explained on the basis of genetic susceptibility and hence altered immunologic responses to specific bacterial pathogens in aggressive periodontitis subjects. Thus, emphasizing that these individuals have pathogenic mechanisms other than pathways mediated by salivary sCD44.
This study has not considered the effect of periodontal therapy on salivary sCD44 levels. Furthermore, smoking status of an individual should have been assessed by cotinine levels, which would be more reflective and precise. CD44 adhesion molecule is known to play a role in acute conditions such as acute pulmonary inflammation, acute ischemic injuries, and acute asthmatic attack thus its role in acute oral conditions need to be studied. Longitudinal as well as interventional studies with adequate sample size and age and gender distribution are necessary to validate substantial changes in salivary sCD44 levels. These studies will help in assessing periodontal disease severity and monitor its progression and also determine the therapeutic significance of sCD44 levels as a diagnostic marker for periodontitis.
| Conclusion|| |
Salivary sCD44 might be considered as a diagnostic biomarker of periodontal destruction in both chronic and aggressive periodontitis patients and may offer an important diagnostic aid along with clinical parameter assessment.
| ACKNOWLEDGeMENT|| |
Sincere thanks to Mr. S. Syed Fazil Ahamed, Research Associate, Department of Infectious Disease Unit, St. John Research Institute, Bangalore for his co-operation and support in performing the required laboratory procedures during the period of the study.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]