Assessment of membrane-organizing extension spike protein as a biomarker for periodontal disease by comparing its level in gingival crevicular fluid in individuals with and without chronic severe periodontitis - A pilot study.

BACKGROUND: Membrane-organizing extension spike protein (Moesin) is a cytoskeletal protein expressed in the gingival crevicular fluid (GCF) which may play a role in the immune response in periodontal disease. The objective of this study was to evaluate whether Moesin can be used as a biomarker for periodontal disease.
MATERIALS AND METHODS: Thirty patients satisfying the required inclusion criteria were selected from those reporting to the out patient (OP) of the department of periodontics and divided into two groups: Group A - systemically healthy controls with no periodontitis and Group B - systemically healthy controls with chronic severe periodontitis. Periodontal parameters were recorded. GCF was collected, and Moesin levels in the two groups were assessed using enzyme-linked immunosorbent assay. Scaling and root planing (SRP) was done in Group B patients who were reviewed, and samples were collected again after 4 weeks and analyzed.
RESULTS: At baseline, the mean GCF Moesin level in Group A was 666.95 ± 471.872 pg/ml, while in Group B, it was found to be 27435.35 ± 14179.77 pg/ml, which showed a high statistically significant difference on comparison. The mean GCF Moesin level in patients with chronic severe periodontitis was 27435.35 ± 14179.77 pg/ml at baseline, and on review 1 month after SRP, it was found to have undergone a statistically significant reduction to 27161.23 ± 14161.57 pg/ml (P = 0.001).
CONCLUSION: Within the limits of this study, it can be concluded that Moesin can serve as a potential biomarker for periodontal disease.

INTRODUCTION

Periodontitis is defined as an inflammatory disease of the supporting tissues of the teeth caused by specific microorganisms or groups of specific microorganisms, resulting in progressive destruction of the periodontal ligament and alveolar bone with periodontal pocket formation, gingival recession, or both.[1] A biological marker (biomarker) is an objective characteristic that can be applied as a diagnostic, staging, or prognostic tool for a disease and also to assist in monitoring the clinical response to treatment. Many such biomarkers of periodontal disease have been identified in the gingival crevicular fluid (GCF) present within the gingival crevice.

Among the recently identified proteins in the GCF, membrane-organizing extension spike protein (Moesin) was of particular interest because it was shown to be involved in lipopolysaccharide (LPS)-induced intracellular signaling through toll-like receptor (TLR) pathways that induce inflammatory responses, which in turn can exacerbate periodontal disease progression. Tsuchida et al. in 2014 have presented a hypothesis that the protein–protein interaction among Moesin, CD44, and several other key coregulated factors, such as members of the CD14 and TLR pathways, contributes to periodontal disease.[2]

According to the study done by Tsuchida et al., Western blotting revealed that Moesin expression in GCF was higher in patients with severe periodontal disease than healthy controls.[2] To the best of our knowledge, the current literature does not have any studies in which enzyme-linked immunosorbent assay (ELISA) was used to estimate the Moesin levels in GCF. Furthermore, they have not assessed the effect of nonsurgical periodontal therapy (scaling and root planing [SRP]) on the GCF Moesin level.

Thus, this study was conducted to evaluate whether Moesin can be used as a biomarker for periodontal disease by comparing its level in GCF in healthy controls and patients with chronic severe periodontitis as well as in the chronic severe periodontitis patients before and after SRP.

MATERIALS AND METHODS

Source of data

Since the only publication assessing the potential use of Moesin as a biomarker for periodontitis does not have the necessary data required to calculate the sample size for this study, this was conducted as a pilot study with a sample size of 15 individuals in each group. Thirty individuals (16 males and 14 females) aged 30–50 years (mean age: 40.8 years) were recruited voluntarily from the outpatient section of the department of periodontology. A written informed consent was obtained from the individuals after explaining the procedure. The study was conducted in the department of periodontology of our institution from March 2018 to May 2018 after approval by the Institutional Ethical Committee.

Selection criteria

Inclusion criteria

The inclusion criteria of this study were as follows:

Presence of at least 16 natural teeth

Systemically healthy controls with chronic periodontitis with >30% sites having clinical attachment loss >5 mm for the test group

Systemically healthy controls with no periodontitis, no clinical attachment loss, no probing depth >3 mm for the control group

Age group of 30–50 years.

Exclusion criteria

The exclusion criteria of this study were as follows:

Individuals having any systemic diseases

Individuals with a history of any drug allergies

History of antimicrobial or anti-inflammatory therapy within the past 6 months

History of periodontal therapy within the past 6 months

Individuals who smoke or chew any form of tobacco or alcoholics

Pregnant or lactating women

Postmenopausal women

Immunocompromised individuals.

Subject grouping

A total of thirty individuals were selected according to the selection criteria and divided into two groups:

Group A – systemically healthy controls with no periodontitis, no clinical attachment loss, and no probing depth >3 mm

Group B – systemically healthy controls with chronic severe periodontitis as defined by clinical attachment loss >5 mm (based on the classification given by the 1999 International Workshop for the Classification of Periodontal Diseases organized by the American Academy of Periodontology).

Clinical evaluation

The severity and amount of gingival inflammation were assessed using the Gingival Index (Loe and Silness) and Simplified Oral Hygiene Index (OHI-S) while the clinical attachment level (CAL) and pocket probing depth (PPD) were used to evaluate the periodontal tissue destruction. These parameters were assessed at baseline in Group A and Group B and 1 month after nonsurgical periodontal therapy in Group B alone.

In this study, Michigan “O” probe with Williams marking was used to measure the pocket depth and CAL at six sites per tooth (mesiobuccal/labial, mid-buccal/labial, distobuccal/labial, mesiolingual/palatal, mid-lingual/palatal, and distolingual/palatal) in all the teeth, excluding third molars. To ensure reproducibility during examinations, a customized acrylic stent was used as a reference to determine the site and angle of insertion of periodontal probe.

Site selection and collection of gingival crevicular fluid

The site showing the greatest clinical attachment loss was selected for GCF sample collection in Group B. In Group A, GCF sample was collected from any one of the maxillary posterior teeth. Prior to GCF collection, the site was air-dried and isolated with cotton rolls. Supragingival plaque was removed gently without manipulating the gingival margin, to avoid contamination and obstruction of the micropipette.

GCF samples were collected from the gingival sulcus with the help of Thermo Scientific micropipette F2 (Thermo Scientific Finnpipette™ F2 Variable Volume Single-Channel Pipettes) using extracrevicular method. A standardized volume of 1-μL GCF was collected from each site and those sites which did not express sufficient volume of GCF or micropipettes contaminated with blood/saliva/plaque were disposed. GCF was collected at baseline from both Group A and Group B. Nonsurgical periodontal therapy comprising full-mouth SRP was performed for Group B using ultrasonic scalers and area-specific Gracey curettes. GCF was collected from the same site in Group B individuals 1 month after completion of SRP. To estimate the Moesin levels in the GCF samples, a commercially available Human Moesin ELISA Kit (Cloud - Clone Corp., Houston, USA) was used. Collected samples were immediately transferred to Eppendorf tubes and stored at − 20°C until the time of the assay.

Statistical analysis

Statistical tests were performed using statistical software (IBM SPSS 20 [SPSS Inc., Chicago, USA]). For all continuous variables, the results are either given in mean standard deviation and for categorical variables as percentage. To test the statistical significance of the difference in the GCF Moesin levels in Group A and Group B at baseline, the Mann–Whitney U-test was applied. To assess the significance of the Moesin levels as well as the periodontal parameters in the intragroup comparison (in Group B before and 1 month after SRP), the Wilcoxon signed-rank test was applied.

RESULTS

At baseline, the mean GCF Moesin level in systemically healthy controls with no periodontitis was 666.95 ± 471.872 pg/ml, while in patients with chronic severe periodontitis, it was found to be 27435.35 ± 14179.77 pg/ml. Intergroup comparison using the Mann–Whitney U-test showed a highly significant statistical difference (P < 0.001) [Table 1 and Figure 1].

Table 1

Intergroup comparison of mean Moesin levels in gingival crevicular fluid of Group A (healthy controls) and Group B (patients with chronic severe periodontitis)

Moesin level in GCF (pg/ml)	Mean±SD
	Group A	Group B
Baseline	666.95±471.872	27,435.35±14,179.77
P	<0.001*	<0.001*

*Statistical significance is taken as P<0.05. GCF – Gingival crevicular fluid; SD – Standard deviation; P – Probability value

Figure 1

The intergroup comparison of mean membrane-organizing extension spike protein levels in gingival crevicular fluid of Group A (healthy controls) and Group B (patients with chronic severe periodontitis)

After SRP, clinical parameters, such as CAL and PPD, usually will show improvement within 3–4 weeks.[3] All the clinical parameters which were evaluated showed a statistically significant reduction 1 month after SRP as anticipated. The mean values for OHI-S, gingival index, probing depth, and CAL were reduced from 2.16 ± 0.60, 1.61 ± 0.23, 6.93 ± 0.96, and 7.67 ± 1.29 mm at baseline to 1.62 ± 0.61, 1.29 ± 0.22, 5.27 ± 0.96, and 5.87 ± 1.35 mm, respectively, when assessed 1 month postoperatively [Table 2 and Figure 2].

Table 2

Comparison of periodontal parameters and gingival crevicular fluid Moesin level in Group B before and after nonsurgical periodontal therapy

Group B	Mean±SD
	Probing depth (mm)	OHI-S	Clinical attachment level (mm)	Gingival index	Moesin level (pg/ml)
Baseline	6.93 (0.96)	2.16 (0.60)	7.67 (1.29)	1.61 (0.23)	27,435.35 (14,179.77)
1 month	5.27 (0.96)	1.62 (0.61)	5.87 (1.35)	1.29 (0.22)	27,161.23 (14,161.57)
P	<0.001*	0.001*	<0.001*	0.001*	0.001*

*Statistical significance is taken as P<0.05. GCF – Gingival crevicular fluid; SD – Standard deviation; OHI-S – Simplified Oral Hygiene Index; P – Probability value

Figure 2

Comparison of mean probing depth, oral hygiene index, clinical attachment level, and gingival index in Group B before and after nonsurgical periodontal therapy

The mean GCF Moesin level in patients with chronic severe periodontitis was 27435.35 ± 14179.77 pg/ml at baseline. One month following SRP, it was found to have undergone a statistically significant reduction to 27161.23 ± 14161.57 pg/ml (P = 0.001) [Table 2 and Figure 3].

Figure 3

Intragroup comparison of mean gingival crevicular fluid membrane-organizing extension spike protein levels in Group B before and after nonsurgical periodontal therapy

DISCUSSION

Periodontitis is a highly prevalent chronic inflammatory disease affecting the gingiva, periodontal ligament, cementum, and alveolar bone.[4] It has become a major concern for the health-care system as it has surpassed dental caries as the most common cause for tooth loss worldwide.[5] Periodontitis has an estimated prevalence of approximately 50% among adults, with 10%–30% displaying severe forms of disease referred to as generalized periodontitis characterized by an involvement of 30% of the teeth that is present in the oral cavity.[6] The traditional clinical indices often fall short as they are not useful in identifying the disease activity and predicting the prognosis or the underlying causes/events involved in periodontal tissue destruction. Thus, periodontal researchers are constantly in the search for more precise biomarkers of periodontal disease to replace or supplement the routinely assessed clinical parameters.[7]

GCF contains a rich array of cellular and biochemical factors which have been shown to have potential value as prognostic or diagnostic factors of periodontal health and disease. GCF is preferred over saliva for assessment of biomarkers as it is more site specific and hence more reliable.[7] However, the possibility of analyzing a single GCF sample for multiple biomarkers was hindered by technological limitations until quite recently. Over the course of time, technological advancements have made possible the identification and/or quantification of multiple such substances in individual GCF samples and therefore exhibited the significance of investigating combinations or panels of potential biomarkers.[8]

Tsuchida et al. developed a novel protocol for effective protein extraction from GCF and then conducted gel-based and gel-free proteome analyses of GCF from patients with chronic severe periodontitis in which several proteins were discovered. Amid these proteins, we focused on Moesin, a disease-associated protein belonging to the Ezrin, Radixin, and Moesin family of proteins that have seldom been studied in relation to periodontal disease.[9]

Moesin, a cytoskeletal protein, may play a role in LPS signaling through CD14 and TLR pathways.[10] In a study conducted by Tsuchida et al. in 2014, Moesin expression was found to be significantly higher in chronic severe periodontitis patients as compared to healthy controls. Furthermore, Moesin levels were found to increase in a dose-dependent manner when human gingival fibroblasts were exposed to increasing concentrations of LPSs.[2] Zawawi et al.[11] reported that Moesin appears to be increasingly expressed in LPS-stimulated macrophages. Thus, Moesin may either be a by-product or a mediator of the pathophysiology of periodontal disease.

In this study, at baseline, the mean GCF Moesin levels in systemically healthy controls with no periodontitis (Group A) and patients with chronic severe periodontitis (Group B) showed a highly significant statistical difference between the two groups (P < 0.001). This is in accordance with the results from the study conducted by Tsuchida et al. in 2014.[2]

The first phase of periodontal treatment comprising SRP primarily aims at the elimination or reduction of bacterial infection and the control of periodontal plaque-associated inflammation.[12] All the clinical parameters which were evaluated showed a statistically significant reduction 1 month after SRP as expected. This is in accordance with the results of a case report published by Carnio et al. in 2015, which showed a significant reduction in clinical parameters and improvement in the overall as well as individual prognosis of teeth when a patient with chronic severe periodontitis was treated with nonsurgical periodontal therapy and periodic maintenance visits alone.[13]

The mean GCF Moesin level in patients with chronic severe periodontitis was found to have undergone a statistically significant reduction 1 month after SRP (P = 0.001).

The difference between the GCF Moesin levels of healthy controls and patients with chronic severe periodontitis as well as the reduction in the Moesin levels after nonsurgical periodontal therapy indicates that Moesin could be potentially used as a biomarker for periodontal disease. Hence, from the results of the present study, it is observed that the level of Moesin expression in GCF is significantly higher in chronic inflammatory conditions (chronic severe periodontitis) and reduces with decrease in the severity of inflammation (after SRP).

Even though there are multiple biomarkers already established in periodontal disease, none of these have been identified as an ideal biomarker for periodontal disease. Moesin is a newly discovered disease-associated protein that has been studied in relation to periodontal disease only in one study (using Western blot technique).

In this study, we have used ELISA for estimation of Moesin levels which are much more affordable and cost-effective than Western blot. Furthermore, the ELISA kit for Moesin is economically more feasible than those for many other already discovered biomarkers for periodontal disease. Thus, this study was conducted as a step toward the potential development of Moesin as an ideal biomarker for periodontal disease.

CONCLUSION

Within the limits of this study, it can be concluded that Moesin can serve as a potential biomarker for periodontal disease. Further studies using a larger sample size are required to verify the findings of this study as well as to understand the role of Moesin in periodontal disease.

Limitations

This study was conducted as a pilot study with a small sample size of only 15 individuals in each group. Hence, the results from this study cannot be generalized to the whole population. Hence, further studies on a larger scale should be conducted to evaluate the role of Moesin as a biomarker for periodontal disease

Systemically healthy controls were included only on the basis of the medical history given by the individual. Thus, it has not been verified whether the individual has any systemic condition which has not been diagnosed or intentionally or unintentionally not been included when giving the medical history.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.