Effect of scaling and root planing on gingival crevicular fluid levels of adrenomedullin in chronic periodontitis patients with and without diabetes mellitus type 2: A clinico-biochemical study.

Background: Increased plasma adrenomedullin levels are usually observed in certain conditions such as diabetes mellitus (DM). Aims and
Objectives: To gauge adrenomedullin values in the gingival crevicular fluid (GCF) of healthy individuals, chronic periodontitis (CP) patients with and without DM, and diabetics without any periodontal involvement, prior and after periodontal therapy. Materials and
Methods: A total of 120 subjects were segregated into four categories of 30 each: Group 1 (healthy subjects), Group 2 (CP without DM), Group 3 (patients with CP and DM), and Group 4 (diabetics without any periodontal involvement). GCF was collected from all the patients. Nonsurgical periodontal therapy was done in Group 2 and Group 3 patients, followed up for 6 weeks, and GCF was obtained again from the same site. Enzyme-linked immunosorbent assay was used to evaluate the samples.
Results: Adrenomedullin level in the GCF was high in diabetics without any periodontal involvement than in healthy individuals and significantly elevated in those with CP and DM. Reduced adrenomedullin was noted 6 weeks postperiodontal therapy.
Conclusion: Adrenomedullin levels being higher in diabetics without any periodontal involvement than healthy controls suggest a link between DM and adrenomedullin. Furthermore, infection plays a noteworthy role in the elevation of adrenomedullin, indicating its role in periodontal disease progression.

INTRODUCTION

Periodontitis is inflammation of the gingiva and periodontal apparatus, which could be caused by microorganisms causing damage of periodontal ligament and alveolar bone.[12] Literature reveals that Porphyromonas gingivalis, Treponema denticola, and Treponema forsythia are the classic organisms related with periodontal pathogenesis.[345] Disease progression is further dependent on other aspects such as smoking, genetics, and hormonal imbalances.[6] The imbalance of one such hormone that has great implications on the integrity of periodontal tissue is insulin.

Compilation of literature from many studies revealed periodontitis as a possible complication (sixth) of diabetes mellitus (DM).[78] Periodontal destruction depends on the microorganisms efficiency to breach the host–epithelial barrier, which is protected by gingival crevicular fluid (GCF) flow and various antimicrobial peptides. Adrenomedullin is one such potent antimicrobial peptide originally identified by Kitamura et al. (1993) from the extracts of human pheochromocytoma.[9] A broad variety of cells and tissues produce adrenomedullin such as adrenal medulla, kidney, lungs, and endothelial cells. Its plasma level is usually elevated in certain conditions such as DM, periodontal disease (PD), and renal diseases.[101112131415]

However, whether treatment of PD may cause any alterations in the adrenomedullin value is not known. Therefore, with a hypothesis of altered adrenomedullin levels in diabetes patients, we carried this study to determine adrenomedullin levels in healthy controls, patients with chronic periodontitis (CP) with and without DM, and diabetic subjects without any periodontal involvement and also to tabulate the variation in the adrenomedullin values prior to and after therapy (scaling and root planing).

MATERIALS AND METHODS

Sample selection

Our prospective study included 120 subjects. The study was carried out in the Department of Periodontics, Bapuji Dental College and Hospital, Davangere (Karnataka state), from January 2015 to December 2015. The sample size was obtained using Cohen effect size “r” table developed in 1988 (alpha = 0.05, power of the study = 0.8, effect size “r” = 0.6, number of groups = 4, estimated sample size per group will be 28 rounded off to 30).

We obtained ethical committee clearance and written informed consent from all the individuals according to the Declaration of Helsinki. We followed the criteria of 1999 International World Workshop for Classification of Periodontal Diseases and Condition. Subjects without gingivitis and probing depth <3 mm were listed as controls. HbA1c levels of diabetic subjects were assessed chairside using Chek diagnostics A1CNow® test kit (FDA cleared: Globalmart). Type 2 DM diagnosis was according to the HbA1c level (7%–8%).[1617] Individuals with other systemic diseases such as hypertension, subjects on antibiotic therapy in the past 3 months, those who had periodontal treatment in the preceding 6 months, subjects on immunosuppressive therapy, pregnant and lactating mothers, and smokers were excluded.

The selected 120 individuals were made into four groups randomly.

Group 1: (Healthy subjects) Subjects without clinical gingivitis and probing depth of <3 mm (n = 30)

Group 2: (CP without DM) Subjects with probing depth >5 mm in 30% of the sites and presence of bone loss radiographically (n = 30)

Group 3: (CP with DM) Subjects with probing depth >5 mm in 30% of the sites, bone loss evident radiographically, and well-controlled type 2 DM (HbA1c level 7%–8%) (n = 30)

Group 4: (DM without any periodontal involvement) Subjects with <3 mm probing depth, without clinical inflammation or loss of attachment and well-controlled type 2 DM (HbA1c level 7%–8%).

After taking history, clinical and radiographic evaluation was done. Sterile mouth mirror and UNC-15 graduated periodontal probes were used to clinically examine the periodontal status.

Clinical parameters

Patient selection, examination, and periodontal treatment were done by single examiner to obtain reproducibility and prevent the examiners bias. Clinical parameters such as gingival index (GI), plaque index (PI), and probing depth (gingival margin to base of sulcus (healthy) or pockets (CP) were noted for all teeth whereas clinical attachment level (CAL) was also measured. UNC-15 probe was placed along the tooth. To attain a more precise measurement of the preexisting attachment level, a customized acrylic stent was prepared for each subject using self-cure acrylic resin and placed on the study casts to reduce distortion during follow-up measurements.[18]

Gingival crevicular fluid collection

With cotton rolls, isolation of the selected sites was done, and drying with air, supragingival plaque was removed gently not contacting marginal gingiva to prevent contamination or blockage of the microcapillary pipette. Samples contaminated with blood or saliva were discarded. GCF was collected by placing white color-coded 1–5 μL calibrated microcapillary pipettes (Sigma-Aldrich Chemical Company, St. Louis, USA). Using extracrevicular (unstimulated) method, 1 μL of GCF was collected, which was considered as standard volume [Figure 1].

Figure 1

Gingival crevicular fluid collection in patients with chronic periodontitis with diabetes mellitus (Group 3)

Periodontal treatment

Nonsurgical periodontal therapy was done for Group 2 and Group 3 subjects after collection of GCF. With ultrasonic scaling machine-driven ultrasonic scalers (Cavitron Bobcat Pro, Dentsply), calculus was thoroughly removed. After a week, hand instruments (Gracey curettes, Hu-Friedy, Chicago, IL, USA) were used to accomplish root planing under local anesthesia to remove subgingival calculus, thereby providing smooth root surface. Root surface smoothness was evaluated using a shepherd hook explorer (Ecolite Manufacturing Company, Spoken Valley, USA).

Postoperatively, all the subjects were instructed to brush daily with fluoridated toothpaste using bass technique. Antibiotics or mouth rinsing products were not prescribed. Subjects with large embrasure spaces were instructed for using dental floss and interdental brushes, to prevent retention of food particles. After 6 weeks, clinical parameters were recorded [Figure 2] and GCF collected from the same site in Group 2 and Group 3 subjects and transferred to plastic tube without delay and stored at −80°C till the time of the assay.

Figure 2

Clinical attachment level measurement in Group 2 after 6 weeks of nonsurgical periodontal treatment

Enzyme-linked immunosorbent assay kit and principle of assay

The Biolinkk Biosystems Adrenomedullin enzyme-linked immunosorbent assay (ELISA) kit (Biolinkk Company, New Delhi, India) for the quantitative measurement of adrenomedullin was used. This uses a double-antibody sandwich ELISA. The procedure followed was based on the manufacturer's instructions [Figure 3]. The sensitivity was 5.118 ng/L. The amount of adrenomedullin was calculated by measuring the optical density values under 450 nm wavelength by the sample curve.

Figure 3

Color change in wells after incubation

Statistical analysis

Statistical Package for the Social Services version 20 (IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: USA, IBM Corp) for Windows software was used for analysis.[19] The mean and standard deviation were calculated for all parameters and GCF adrenomedullin levels. Since GI, PI, probing depth and CAL were not normally distributed, pair-wise comparison of groups was done by Mann–Whitney U test. Wilcoxon signed-rank test was used in Group 2 and Group 3 for comparison of values before and following intervention. Clinical correlation among the parameters and GCF adrenomedullin levels was compared by Spearman correlation test. A P < 0.05 was considered statistically significant.

RESULTS

Our study subjects were aged between 30 and 60 years (mean age of 40.5 ± 2.9). There were 65 males (54%) and 55 females (46%). Age and gender in all the groups did not show any significant difference (P = 0.362).

Baseline intergroup comparison of clinical parameters

Intergroup comparison of clinical parameters was highly significant (P < 0.001). Pair-wise comparison showed that except for Group 1 versus Group 4 and Group 2 versus Group 3, all the other pair-wise comparisons demonstrated significant difference (P < 0.001). All the parameters, i.e., GI, PI, probing depth, and CAL were improved significantly after the nonsurgical intervention in Group 2 and Group 3.(P < 0.001) [Tables 1 and 2].

Table 1

Comparison of clinical parameters that is plaque index gingival index, probing depth, clinical attachment level and adrenomedullin levels compared at baseline and 6 weeks postperiodontal therapy in Group 2

Groups	Mean±SD
	PI	GI	PD (mm)	CAL (mm)	GCF AM level (pg/ml)
Group 2 at baseline	2.19±0.50	2.11±0.47	6.83±1.70	9.67±1.51	522.27±199.5
Group 2 6 weeks posttherapy	1.02±0.018	1.02±0.23	5.06±1.36	7.10±1.21	249.97±68.28
Difference	1.17**	1.09**	1.77**	2.57**	272.3**
Wilcoxon signed-rank test	−4.708	−4.788	−4.350b	−4.794b	−4.782b

Values are presented as mean±SD. Wilcoxon signed ranks test: *P-value: *P<0.05, (significant), **P<0.001, (highly significant). SD=Standard deviation, GI=Gingival index, PI=Plaque index, PD=Probing depth, CAL=Clinical attachment level, GCF=Gingival crevicular fluid, AM=Adrenomedullin, bP<0.001; Significant

Table 2

Comparison of clinical parameters that is plaque index, probing depth, clinical attachment level and adrenomedullin levels compared at baseline and 6 weeks postperiodontal therapy in Group 3

Groups	Mean±SD
	PI	PD (mm)	CAL (mm)	GCF AM level (pg/ml)	GI
Group 3 at baseline	2.43±0.46	7.3±1.69	10.07±1.63	596.80±112.95	2.40±0.33
Group 3 6 weeks posttherapy	1.31±0.48	5.26±1.18	7.53±1.04	279.13±142.15	1.49±1.8
Difference	1.12**	2.04**	2.54**	317.67**	0.91**
Wilcoxon signed-ranks	−4.788	−4.417	−4.65	−4.782	−4.711

Values are presented as mean±SD. Wilcoxon signed ranks test. *P-value: *P<0.05, (significant), **P<0.001 (highly significant). SD=Standard deviation, GI=Gingival index, PI=Plaque index, PD=Probing depth, CAL=Clinical attachment level, GCF=Gingival crevicular fluid, AM=Adrenomedullin

Clinical correlation

Clinical correlation was done between adrenomedullin levels and the GI, PI, probing depth, and CAL in Group 2 and Group 3, and a negative association was seen before the periodontal treatment. Overall, there was weak positive association found between probing depth, PI, GI, and adrenomedullin levels, 6 weeks after therapy with the exception of CAL and PI in Group 2; CAL and probing depth in Group 3 had negative correlation after periodontal therapy.

DISCUSSION

Several investigators[2021] have proven that nonsurgical periodontal treatment leads to substantial decrease in inflammation and pocket depth as was seen in our study. On comparing the CAL in these subjects initially and 6 weeks posttherapy, a highly significant gain in the attachment level was seen.

Adrenomedullin is a peptide with multifunctions such as strong antimicrobial effect. In the past, several studies reported its influence on periodontal pathogenesis and glucose metabolism.[2021]

We also observed low adrenomedullin values in those without CP (Group 1 and Group 4) when compared to CP subjects. However, the adrenomedullin levels were very high in DM subjects than controls (P = 0.001). Wong et al.[21] stated that hyperglycemia augments adrenomedullin release and expression. Hayashi et al.[22] also found increased plasma concentration of adrenomedullin in diabetics, which they stated was due to increase in endothelial damage.[21] This elevation in diabetics might be due to the combination of both the factors that is hyperglycemia along with endothelial damage.

GCF concentration of adrenomedullin was elevated in CP individuals. Hayashi M et al.[22] had studied the adrenomedullin expression against periodontal bacteria and concluded that the byproducts of periodontal bacteria such as lipopolysaccharides upregulated levels of adrenomedullin. Lundy et al.[23] compared the adrenomedullin levels at diseased and normal sites and found elevated levels at the diseased site. They attributed this to the pro-inflammatory nature of the adrenomedullin. Similarly, when we compared adrenomedullin in CP patients with or without diabetes, we noticed significant rise in adrenomedullin levels. Our hypothesis was further strengthened Hussain et al. and Zang et al. findings,[2425] who established a possible association between elevated adrenomedullin values with different forms and periodontal disease severity. The above findings were also consistent to the findings reported by Suchetha et al. and Ertugrul et al.[1415]

Indeed, dental plaque acts as potent stimulators for adrenomedullin. Plaque control and maintenance aid in substantial decrease in the inflammation and pro-inflammatory cytokines.[20] Our observations substantiated these facts that bacteria and their toxic products cause inflammation and act as potent stimulators for adrenomedullin and we noticed reduced adrenomedullin 6 weeks after periodontal management in CP subjects. The variation in the mean GCF adrenomedullin concentration in CP after treatment was statistically highly significant. Our observations were in accordance to the observations made by Pawar et al.,[26] who reported decline in MMP-3 and 13 levels after periodontal therapy.

Since this study is first of its nature, to the best of our knowledge, reduced adrenomedullin levels establishes its role as an antimicrobial peptide, but further studies are required to establish its exact role.

A negative correlation was seen among the parameters before the periodontal therapy. Overall, there was weak positive correlation found between probing depth, GI, PI adrenomedullin values, 6 weeks after treatment with the exclusion of negative association between CAL and PI in Group 2 and between CAL and probing depth in Group 3. These different observations might be due subject variation and difference in the stage of disease activity in each individual and also the response of adrenomedullin to increase in disease severity. This further helps us substantiate the point that adrenomedullin might not be the only modifying factor in the PD activity.

Limitations

Limited sample size and hence results may perhaps not be accorded to the entire population. In addition, differing levels of disease activity in each individual can modify the final result. Further, subjects with different blood glucose levels (HbA1c 7%–8%) were chosen, and hence, the pin-point role of adrenomedullin in glycemic control cannot be established.

CONCLUSION

We found that adrenomedullin is universally present both in healthy and diseased sites, but its presence is higher at the diseased site. We also found increased adrenomedullin levels in diabetic subjects without any periodontal involvement than healthy controls and decline in adrenomedullin levels after treatment in CP subjects. Further, we noticed a decline in the values for all the parameters when evaluated with baseline values 6 weeks after the periodontal treatment, once again substantiating the significance of phase-I therapy.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.