Estimation of variation in serum and gingival crevicular fluid levels of mannose-binding lectin after periodontal therapy in chronic periodontitis patients

Abstract

Background:

Mannose-binding lectin (MBL), a serum protein, has a role in the activation of the complement cascade and plays an important role in innate immune defense. MBL levels is said to be increased in various immunoinflammatory conditions systemically. The present study analyzes the serum and gingival crevicular fluid (GCF) levels of MBL in patients suffering from chronic periodontitis and also to find out the alteration in its levels after nonsurgical periodontal therapy in serum and GCF.

Materials and Methods:

A total of 20 subjects suffering from chronic periodontitis with an age range of 20–60 years participated in the study. GCF as well as serum samples was collected from each patient at two different time intervals: before nonsurgical periodontal therapy and 1 week after scaling and root planing (SRP). MBL levels were detected using the enzyme-linked immunosorbent assay test. Gingival index (GI), plaque index (PI), oral hygiene index (OHI-S), probing pocket depth, and clinical attachment level were assessed at baseline and 4 weeks after the treatment.

Results:

The present study showed that both serum and GCF MBL levels decreased after periodontal treatment, with a statistically significant decrease in the values for GCF. Furthermore, PI, GI, Oral Hygiene Index–Simplified, and periodontal probing depth showed a statistically significant decrease in the values after periodontal therapy (4 weeks) as compared to baseline.

Conclusion:

This cross-sectional study demonstrated statistically significant reductions in GCF MBL levels, along with a decrease in mean serum MBL levels, following SRP in patients with chronic periodontitis. Assessment of MBL levels can serve as a significant biomarker in the diagnosis of periodontal disease and its treatment outcome.

INTRODUCTION

Periodontal disease is a multifactorial chronic inflammation affecting the tissues around the teeth, with dental plaque being the primary etiological factor.[1] Various microorganisms with special emphasis on Gram-negative bacteria (Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythia) have been incriminated as the most potent periodontal disease-causing agents.[2] They are present at the gingival margin and also subgingivally, which can cause imbalance in the homeostasis of the host and its microbiota, thereby increasing the vulnerability to periodontal diseases.[3]

The virulence factors of these bacteria release toxins in the body, which further causes the release of various pro-inflammatory cytokines in the body such as interleukin-1β (IL-1β), IL-8, and tumor necrosis factor-α (TNF-α). Despite being the host factors, these play the most important role in the destruction of periodontal tissues and alveolar bone resorption.[4] For maintaining healthy periodontal tissues, a proper balance is needed between the host defense factors and oral microbiota. This is managed by the release of various salivary immunoglobulins, antimicrobial peptides of the epithelium, and killing systems of neutrophils (oxidative and nonoxidative), as well as with the activation of the complement system with its lectin and/or classical pathways. These form the defense system against the various periodontopathic bacteria.[5,6,7,8,9,10,11]

Mannose-binding lectin (MBL) is produced in the liver and is present in the circulation as a soluble glycoprotein. After forming a complex with serine proteases associated with it, it can activate the lectin pathway of the complement system.[10] This activation of the complement system leads binding of the MBL complex to various Gram-positive and Gram-negative bacteria.[12] Another important property of MBL is opsonization of pathogenic microorganisms directly and thus acting as one of the acute-phase proteins in chronic infections.[13]

The micro-organisms such as Neisseria meningitidis, Candida Species, Aspergillus fumigatus, and Staphylococcus aureus have mannose groups attached on their surface, which allows the host MBL to recognize the carbohydrate structure that is present on the cell surface of these micro-organisms.[14,15,16] Interestingly, it has been found that periodontal pathogens such as A. actinomycetemcomitans and P. Gingivalis also have polysaccharides-containing mannan on their cell surfaces. Thus, MBL can bind to these periodontal pathogens and help in the process of phagocytizing them.[17,18,19,20,21]

An average plasma level value of MBL was found to be 1.2–1.6 mg/ml, and the subjects with levels below 0.5–1.00 mg/ml are considered to be MBL deficient. The plasma levels of MBL are reported to increase during the immunoinflammatory processes.[22,23,24] When compared to C-reactive protein, MBL is considered a weak acute-phase protein due to modest increase in its levels.[25] Recognition of pathogens by MBL is only the first step in the process of host defense against infection. After binding to target pathogenic bacteria, MBL serves as a dual role of recruiting the complement system as well as phagocytes.[26] Therefore, MBL can be considered as one of the biomarkers in causing periodontal destruction.

Gingival crevicular fluid (GCF) is a potential source for assessing periodontal disease biomarkers, which plays an important role in the diagnosis as well as in periodontal treatment outcomes.[27,28] Previous research has shown the variations of serum MBL levels in the patients suffering from periodontal disease.[29,30] So far, no substantial evidence is available on MBL levels in GCF and its effect on periodontal disease and treatment outcomes. Therefore, the current study was undertaken to assess and evaluate the serum and GCF levels of MBL in patients suffering from chronic periodontitis and also to find out alteration in its levels (if any) after nonsurgical periodontal therapy in both serum and GCF.

MATERIALS AND METHODS

Source of data

The present study was conducted on the patients visiting the outpatient department of periodontology and implantology. The selected subjects were categorized based on the clinical condition of periodontium, according to the classification system given by the American Academy of Periodontology for periodontal disease and conditions, in 2017.[31] The research was started after obtaining the ethical clearance from the institute ethical committee review board (IEC/99/2021).

The sample size of 20 was calculated using G*Power software version 3.1, with an effect size of 0.5, α = 0.05, and 95% power. This provided a 95% confidence interval for detecting clinically meaningful differences in MBL levels. Furthermore, subjects should be having at least two teeth in each quadrant with a pocket depth of ≥5 mm. Patients suffering from systemic illnesses, pregnant women and lactating mothers, alcoholics, and current smokers, who were on analgesic, antibiotic therapy, or periodontal therapy in the last 6 months, were excluded from the study.

Intervention/procedure

The participants were explained about the procedure and the possible outcome in their vernacular language as well as written informed consent was obtained. The patient’s statistics were recorded on the customized case history pro forma.

GCF as well as serum samples were collected from each patient at two different time intervals: before nonsurgical periodontal therapy and 1 week after scaling and root planing (SRP). For all the patients, SRP was done within 48 h. The collected samples were stored at −20°C till the enzyme-linked immunosorbent assay (ELISA) test was performed.

Gingival crevicular fluid sample collection

Subjects selected for GCF sample collection were made to sit on a dental chair in proper position and illumination. The site was dried and separated to prevent saliva contamination. The GCF sample collection was procured from sites having the highest amount of inflammation and the sites having the deepest probing pocket depths (PPDs).

A 50 μl micropipette, which was obtained from Sigma Aldrich Company (catalog number-Z717290), was inserted into gingival crevice for without causing trauma to the tissue (passively). The placement of the pipette was for 3–5 min into the sulcus, and thus, GCF sample was collected into the pipette. The collection of the GCF occurred due to the capillary action and negative pressure that was created by the micropipette. After the collection of the GCF sample, it was carefully placed into the microcentrifuge vials of 1.5 ml and sealed. The patient code was mentioned onto the vials via numbering method, and the samples were stored for further analysis.

Precautions were taken for GCF collection, such as micropipettes contaminated with blood or saliva were excluded from the study, and repeated collection of GCF from the same site was avoided to prevent trauma and bias that would have been caused due to the sample collected from traumatized sites.

Serum sample collection

For analysis, 3 ml of the patients’ blood was collected from the anticubital fossa by venipuncture using a 5 ml syringe. Collected blood was transferred into test tubes and then subjected to low-speed centrifugation for 10 min to obtain serum. Using 1000 μl micropipette, 1 ml of serum collection was done, which was transferred into microcentrifuge vials. The samples were stored appropriately for ELISA testing.

Gingival index (GI), plaque index (PI), oral hygiene index (OHI-S), PPD, and Clinical Attachment level (CAL) were assessed at baseline and 4 weeks after the treatment.

ELISA method for quantifying MBL levels: a commercially available soluble MBL ELISA (KRISHGEN BIO SYSTEMS catalog number was KBH0335) kit was used for quantitative determination of human MBL protein in serum and GCF. The required quantity of GCF and serum samples were coated on the wells of the ELISA kit based on the dilution factor of the reagents used. Upon the completion of the ELISA protocol, the wells were subjected to colorimetric analysis. Using the standard curve, the MBL levels in GCF and serum were tabulated.

For the data analysis, statistical software SPSS version 19.0 (IBM Corporation, New York, USA) was used. Mean and standard deviation were used to give the results on continuous variables. Paired ‘t’- test was applied for statistical evaluation of the results. P < 0.05 was considered to be statistically significant.

RESULTS

A total of 20 subjects suffering from chronic periodontitis group were included in the study. The mean age group of subjects included in this study was 40.1 ± 10.9 years. The values of mean GI reduced from 2.65 ± 0.48 at baseline to 0.40 ± 0.50 30 days after SRP. This decrease in the values of GI after periodontal therapy was statistically significant. Similar was the reduction in the values of PI and Oral Hygiene Index–Simplified. Mean PI values before the therapy were found to be 2.35 ± 0.67, which were significantly reduced to 0.15 ± 0.36 after 30 days. OHIs values also observed a statistically significant decrease in the mean values from baseline, i.e. 2.68 ± 0.78–1.05 ± 0.75 postoperatively [Table 1]. The mean PPD levels showed a statistically significant reduction from pretreatment values (4.91 ± 2.69) to 30 days posttherapy (1.05 ± 0.75). There was a decrease in CAL levels from baseline, i.e. 2.42 ± 0.78 to posttreatment values, i.e. 2.32 ± 1.38; however, the results of CAL could not achieve statistical significance [Table 1].

Table 1.

Comparison of clinical parameters at baseline and 4 weeks after periodontal therapy

Clinical parameters	Mean	n	SD	t	P
GI (baseline)	2.65	20	0.48	18.2	0.0001*
GI (posttreatment)	0.40	20	0.50
PI (baseline)	2.35	20	0.67	15.9	0.0001*
PI (posttreatment)	0.15	20	0.36
OHI-S (baseline)	2.68	20	0.78	6.6	0.0001*
OHI-S (posttreatment)	1.05	20	0.75
PPD (baseline)	4.91	20	2.69	8.9	0.0001*
PPD (posttreatment)	1.55	20	1.49
CAL (baseline)	2.42	20	1.34	1.8	0.07
CAL (posttreatment)	2.32	20	1.38

*P<0.05 – statistically significant. t – Paired t-test; n – Sample size; SD – Standard deviation; GI – Gingival index; PI – Plaque index; OHI-S – Oral Hygiene Index–Simplified; PPD – Pocket probing depth; CAL – Clinical attachment level

After SRP, MBL levels showed a statistically significant reduction (P < 0.02) in the GCF (0.39 ± 0.35) as compared to the pretreatment values (0.82 ± 0.71). Similarly, MBL values in serum showed a mean decline of 0.55 ± 0.41 from their baseline values of 0.83 ± 0.41, but the results did not achieve statistical significance (P > 0.08) [Table 2].

Table 2.

Comparison of mean mannose-binding lectin levels in gingival crevicular fluid and serum at baseline and 7 days after periodontal therapy

MBL levels	Mean	n	SD	t	P
GCF (baseline)	0.82	20	0.71	2.4	0.02*
GCF (posttreatment)	0.39	20	0.35
Serum (baseline)	0.83	20	0.41	1.8	0.08
Serum (posttreatment)	0.55	20	0.41

*P<0.05 – Statistically significant. t – Paired t-test; n – Sample size; SD – Standard deviation; MBL – Mannose-binding lectin; GCF – Gingival crevicular fluid; P – Probability value

DISCUSSION

The present study showed that both serum and GCF MBL levels decreased after periodontal treatment, with a statistically significant decrease in the values for GCF. Furthermore, PI, GI, Oral Hygiene Index–Simplified, and periodontal probing depth showed a statistically significant decrease in the values after periodontal therapy (4 weeks) as compared to baseline.

Till date, various studies have shown controversial results for serum MBL values in chronic periodontitis patients. Caribé et al. assessed the serum MBL levels before and after nonsurgical periodontal therapy in chronic periodontitis patients. A significantly reduced serum MBL levels were seen after periodontal treatment.[32] Similarly, Louropoulou et al. found elevated serum MBL levels in chronic periodontitis patients as compared to healthy individuals and even in MBL-deficient individuals.[30] However, in a study done by Maffei et al., MBL levels were analyzed in varying degrees of chronic periodontitis patients, and also, the severity of periodontitis was assessed in MBL-deficient patients (MBL values <0.8 microg/ml). The levels of MBL were not found to be elevated in patients suffering from periodontitis, and decrease in MBL levels was not related to periodontitis severity.[29]

A. Actinomycetemcomitans and P. gingivalis are the most common bacteria implicated in severe periodontal destruction. The cell surfaces of these microorganisms contain polysaccharides which contain mannan. MBL gets attached to the surface of these bacterial cells. In this process, MBL also interacts with various serine proteinases derived from the serum known as MASPs (MBL-associated serine proteinases), which leads to the formation of the MBL complex.[14] This entire process parallels the early steps for activating the classical complement pathway, i.e. the interaction of the complement factor C1q with complement factors C1s and C1r. The MBL complex and its attachment to mannan-containing polysaccharide patterns on bacterial cell surface, interact with C4 in the complement system. This leads to the activation of the classical complement system through MBL pathway in an antibody-dependent manner, and the recruitment of various phases of phagocytosis takes place.[33]

To the best of the author’s knowledge, there is very limited literature available evaluating MBL levels in GCF as well as serum. The periodontal treatment leads to reduced inflammation and also systemic improvement of various inflammatory biomarkers. An increase in serum concentrations of MBL levels is associated with a higher risk of cardiovascular diseases.[34] MBL levels were decreased in both serum and GCF after periodontal therapy, which leads to a reduced immunoinflammatory burden systemically. The results of the current study could be validated with long-term trials with multiple interval assessments of MBL levels in periodontitis patients.

CONCLUSION

This cross-sectional study demonstrated statistically significant reductions in GCF MBL levels, along with a decrease in mean serum MBL levels, following SRP in patients with chronic periodontitis. This also substantiates that GCF is a more sensitive and site-specific biomarker than serum, making it a better tool for the early detection and monitoring of periodontal disease. Although the pathogenesis is still a continuous enigma, biomarkers will essentially give us the idea about the extent and direction of inflammation. So far, various biomarkers have been isolated and marketed as a diagnostic tool in the field of periodontics; the literature on MBL levels in GCF and serum is scarce. The results of the present study on MBL levels can be significant valuable biomarker in the diagnosis of periodontal disease and its treatment outcome.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.