Salivary tumor necrosis factor-alpha levels in periodontitis associated with diabetes mellitus after low level laser therapy as an adjunct to scaling and root planning: A randomized clinical trial

Abstract

Context:

Tumor necrosis factor-alpha (TNF-α) has been shown to play a critical role in the pathogenesis of periodontitis and diabetes. Nonsurgical periodontal therapy was shown to reduce inflammation and improves glycemic status. Recently, adjunctive low level laser therapy (LLLT) has been shown to alter the inflammatory process.

Aim:

To evaluate and compare the alteration in TNF-α levels before and after treatment in patients with periodontitis with and without type 2 diabetes mellitus (T2DM).

Settings and Design:

Randomised clinico-biochemical study was carried out for 8 weeks from September 2019 to December 2020.

Materials and Methods:

Sixty-four participants were divided into Groups A (periodontitis) and B (periodontitis associated with T2DM), based on probing depth ≥5 mm, clinical attachment level ≥2 mm, and history of T2DM. Later were subdivided into A1, A2, B1, B2, based on assigned treatments. Clinical periodontal parameters and salivary TNF-α levels were evaluated and compared at baseline to 8 weeks.

Statistical Analysis:

Multiple group comparisons were done using analysis of variance, intra group comparisons were made using t-tests.

Results:

Comparison of periodontal parameters and salivary TNF-α levels from baseline to 8 weeks showed statistically significant difference (P < 0.05) in all groups, indicating a positive effect of scaling and root planing (SRP) and adjunctive LLLT.

Conclusion:

Both SRP and SRP with adjunctive LLLT effectively altered TNF-α levels, correlating reduced periodontal inflammation.

INTRODUCTION

Periodontitis (P) is an inflammatory response caused by pathogenic microorganisms of longstanding dental biofilm resulting in progressive damage to tooth-supporting structures.[1] The occurrence of rapid periods of destruction is observed in periodontitis, mainly influenced by systemic or environmental factors due to host and bacterial interactions, compromising host defence system.[2]

A complex bidirectional relationship between type 2 diabetes mellitus (T2DM) and periodontitis creates a vicious circle exacerbating both diseases when present simultaneously in the same individual.[3,4]

Global burden of disease study (2017) postulated that severe periodontal disease was the 11^th most common condition in the world, ranging from 20% to 50%.[5] Oral health survey by dental council of India reported 17.5% and 21.4% of periodontitis between the ages 35–44 and 65–74 years respectively.[6] In India, out of 77 million people with T2DM, 75% of individuals suffer from periodontitis.[7,8]

Periodontitis and T2DM, according to Southerland et al. (2006) share common pathogenesis involving an increased inflammatory reaction on the local and systemic level,[9] with elevated production of pro-inflammatory cytokines.

Tumor necrosis factor-alpha (TNF-α), a macrophage-released pro-inflammatory cytokine and a prototypical ligand of the TNF superfamily, is one of the pro-inflammatory factors. It's a pleiotropic molecule that plays a key role in inflammation, the development of the immune system, apoptosis, and lipid metabolism.[10]

Various therapeutic modalities of periodontitis include scaling and root planing (SRP), disinfectants,[11] antibiotics,[12] and surgical techniques.[13] Recently, few studies reported a reduction in TNF-α levels in periodontitis after SRP.[14,15,16] Many had addressed periodontal treatment on metabolic control and TNF-α level of individuals with T2DM in serum and gingival crevicular fluid (GCF).[17,18,19,20,21,22]

However, SRP has certain physical limitations, such as the inability to access interproximal and furcation areas and deep periodontal pockets.[23] To surmount the limitations of SRP and reduce the bacterial load, laser therapy (LT) has been proposed as a treatment strategy for periodontitis. All available laser wavelengths are being used in dentistry as an adjunct to SRP.[24]

Low-level laser therapy (LLLT) has a bio-stimulatory, anti-infective, and anti-ablation effect.[25] Studies on LLLT adjunct to SRP have shown decreased TNF-α levels in GCF and gingival tissue level of periodontitis subjects by suppressing the bacterial presence and bio stimulating the healing tissue through an anti-inflammatory effect.[26,27]

Salivary cytokines have also been increasingly related to periodontal status and oral inflammatory burden in recent times. Since systemic inflammation influences salivary inflammatory burden, it is easy, rapid, and noninvasive as a salivary biomarker.

In the literature, very few studies have attempted to correlate the levels of TNF-α in periodontitis and T2DM adjunct to LLLT.[27,28,29]

Hence in the present study, an attempt was made to evaluate and compare salivary TNF-α levels in periodontitis associated with and without T2DM before and after SRP alone and SRP with adjunctive LLLT.

MATERIALS AND METHODS

The study was designed and conducted between September 2019 and December 2020, before commencement of the study institutional ethical clearance was obtained.

Inclusion criteria consisted of; body mass index (BMI) normal (18.5–24.9) based on WHO criteria.[30] Periodontitis with a probing depth (PD) of ≥5 mm, clinical attachment level (CAL) of ≥2 mm.[31] Periodontitis and well-controlled T2DM.

Participants who underwent LT in the past 3 months. Surgical or nonsurgical therapy within 6 months before the study. Pregnancy, lactating women. Autoimmune disorders (rheumatoid arthritis,[32] ankylosing spondylitis,[33] inflammatory bowel disease, psoriasis,[34] refractory asthma.[35] With Oro mucosal abnormalities. Tobacco habit in any form, and the presence of prosthetic teeth were excluded to avoid confounding effect.

For allocation of the participants, determined a Power analysis with 95% confidence intervals and a sample size of n = 64.

Participants were grouped based on PD, CAL and history of T2DM under medication as periodontitis and as T2DM associated with periodontitis. Based on computer-generated randomisation tables, these groups were subdivided into A1, A2, B1, B2 as follows:

1. Group A: Participants with periodontitis alone

2. Group B: Participants with periodontitis and T2DM

3. Group A1: Participants of Group A receiving SRP alone

4. Group A2: Participants of Group A receiving SRP + LLLT

5. Group B1: Participants of Group B receiving SRP alone

6. Group B2: Participants of Group B receiving SRP + LLLT.

All the eligible participants were thoroughly informed about the nature, potential risks, and benefits of participation obtained informed consent and were subjected to full mouth SRP (FM-SRP) in single sitting within 24 h under local anaesthesia. Participants in Groups A2 and B2 were recalled within 1 week and were subjected for LLLT. Oral hygiene instructions were given to all the participants to follow strict maintenance protocol during the study period.

Random blood sugar (RBS) level was evaluated at baseline and 8 weeks after treatment, using glucometer, by capillary finger-prick method.[36,37]

Demographic information was collected, including the (1) Age; (2) Gender; (3) The BMI was calculated by dividing the body weight (in kilograms) by the square of the height (in meters).

Periodontal parameters were measured and recorded at baseline and 8 weeks after treatment. The following clinical periodontal parameters were included in the study: Plaque index (PI),[38] Bleeding index (BI),[39] PD, and CAL using a sterile mouth mirror, explorer, and UNC-15 probe (Hu-friedy's).[31]

PD and CAL were measured to the nearest millimeters at four sites per tooth i.e., around mesiobuccal, mid buccal, distobuccal, and lingual surfaces, using UNC-15 probe (Hu–friedy).[31] Obtained PD by measuring the distance from the gingival margin to the base of the periodontal pocket.[31] Obtained CAL by measuring the distance from the Cemento enamel junction to the base of the periodontal pocket.

LLLT was done with the wavelength of 630–670 nm and 0.8 W of power output, equipped with a probe tip, was applied externally with light contact to the gingival tissues corresponding to pockets apical-coronal direction for 15s per site including both lingual, buccal and interproximal sites. The Laser tip was discarded after each session.

Three milliliters of unstimulated saliva were collected at baseline and 8 weeks after treatment from all the participants into sterile tubes using the method that described by Navazesh.[40] The samples were centrifuged for 20 min at 2000–3000 rpm and kept at-80°C until the experiment. Salivary TNF-α levels were measured by the enzyme-linked immunosorbent assay technique.

Participants in Groups A2 and B2 were recalled within 1 week and were subjected for LLLT. All the clinical parameters, and salivary TNF-α levels were evaluated between 7 and 8 weeks after SRP and LLLT.

Four individuals lost their follow-up after treatment; finally included 60 participants for analysis. Representation of study methodology is shown in [study Flow Chart 1].

Flow Chart 1.

Study flow chart. TNF-α – Tumor necrosis factor-alpha; RBS – Random Blood Sugar

All the clinical and biochemical parameters were subjected to statistical analysis using the Jamovi version (1.2.27). Basic descriptions were presented in the form of mean and standard deviation. Multiple group comparisons were done using analysis of variance. For intragroup comparisons, t-tests were used. The level of significance was set at P < 0.05 for all tests.

RESULTS

Demographic characteristics such as gender, the mean and standard deviation of age, BMI in all the groups. represented in [Table 1].

Table 1.

Demographic data representing mean and standard deviation of gender, age, body mass index in all the groups

Demographic variables	Groups
	A1	A2	B1	B2
Gender
Males	7	5	9	7
Females	9	9	5	9
Age, mean±SD	47.56±6.17	43.86±7.63	46.43±7.93	45±9.15
BMI, mean±SD	23.3±1.46	22.5±1.90	22.2±1.86	22.5±1.72

T2DM – Type 2 diabetes mellitus; A1 – Periodontitis participants receiving scaling and root planing alone; A2 – Periodontitis participants receiving scaling and root planing with adjunctive low-level laser therapy; B1 – Periodontitis participants with T2DM receiving scaling and root planing alone; B2 – Periodontitis participants with T2DM receiving scaling and root planing with adjunctive low level laser therapy; BMI – Body mass index; SD – Standard deviation

Intragroup comparison of clinical (PI, BI, PD CAL) parameters, Salivary TNF-α and RBS levels from baseline and 8 weeks after treatment. All parameters showed a significant (P < 0.05) difference represented in [Tables 2, 2a, 2b].

Table 2.

Intragroup comparison of clinical parameters from baseline and 8 weeks after treatment

Groups	Clinical parameters	Time interval	n	Mean±SD	Mean difference	95% CI		t	df	P
						Lower	Upper
A1	PI	Baseline	16	1.73±0.45	1.04	0.75	1.33	7.73	15.0	<0.001*
		8 weeks	16	0.69±0.21
	BI	Baseline	16	1.90±0.63	1.46	1.142	1.77	9.86	15.0	<0.001*
		8 weeks	16	0.44±0.17
	PD	Baseline	16	6.05±0.63	3.19	2.79	3.54	18.1	15.0	<0.001*
		8 weeks	16	2.88±0.41
	CAL	Baseline	16	6.06±0.66	2.18	2.79	3.56	17.7	15.0	<0.001*
		8 weeks	16	2.88±0.41
A2	PI	Baseline	14	1.71±0.51	1.11	0.815	1.41	8.04	13.0	<0.001*
		8 weeks	14	0.69±0.21
	BI	Baseline	14	2.29±0.42	1.17	0.908	1.44	9.59	13.0	<0.001*
		8 weeks	14	1.12±0.48
	PD	Baseline	14	5.98±0.59	2.99	2.57	3.54	15.3	13.0	<0.001*
		8 weeks	14	2.99±0.43
	CAL	Baseline	14	2.99±0.43	3.03	2.58	3.49	14.4	13.0	<0.001*
		8 weeks	14	2.99±0.43
B1	PI	Baseline	14	2.00±0.66	1.62	1.26	1.97	9.92	13.0	<0.001*
		8 weeks	14	0.38±0.14
	BI	Baseline	14	1.96±0.62	1.54	1.53	1.92	8.66	13.0	<0.001*
		8 weeks	14	0.43±0.17
	PD	Baseline	14	5.93±0.97	2.93	2.50	3.52	14.9	13.0	<0.001*
		8 weeks	14	3.00±0.48
	CAL	Baseline	14	6.15±0.94	3.03	2.68	3.38	18.9	13.0	<0.001*
		8 weeks	14	3.12±0.59
B2	PI	Baseline	16	2.14±0.57	1.83	1.55	2.10	14.30	15.0	<0.001*
		8 weeks	16	0.31±0.14
	BI	Baseline	16	1.84±0.55	1.39	1.086	1.69	9.84	15.0	<0.001*
		8 weeks	16	0.45±0.12
	PD	Baseline	16	5.87±0.94	3.01	2.50	3.52	12.6	15.0	<0.001*
		8 weeks	16	2.86±0.44
	CAL	Baseline	16	5.97±1.00	3.04	2.55	3.53	13.3	15.0	<0.001*
		8 weeks	16	2.93±0.49

*Paired t-test, value of P is considered statistically significant P<0.05. n – Number of participants; SD – Standard deviation; t – t statistic value; df – Degrees of freedom; P – Probability; T2DM – Type 2 diabetes mellitus; A1 – Periodontitis participants receiving scaling and root planing alone; A2 – Periodontitis participants receiving scaling and root planing with adjunctive to low-level laser therapy; B1 – Periodontitis participants with T2DM receiving scaling and root planing alone; B2 – Periodontitis participants with T2DM receiving scaling and root planing with adjunctive low level laser therapy; PI – Plaque index; BI – Bleeding index; PD – Probing depth; CAL – Clinical attachment level; CI – Confidence interval

Table 2a.

Intragroup comparison of salivary tumor necrosis factor-alpha levels from baseline and 8 weeks after treatment

Groups	Time interval	n	Mean±SD	Mean difference	95% CI		t	df	P
					Lower	Upper
A1	Baseline	16	14.42±2.53	5.14	0.59	1.74	4.33	15.0	<0.01*
	8 weeks	16	9.13±1.72
A2	Baseline	14	14.43±1.85	4.38	1.04	2.20	6.02	13.0	<0.001*
	8 weeks	14	10.04±1.86
B1	Baseline	14	21.05±2.60	6.87	4.87	8.22	8.45	13.0	<0.001*
	8 weeks	14	14.18±2.43
B2	Baseline	16	21.59±2.25	7.99	4.45	11.52	4.82	15.0	<0.001*
	8 weeks	16	12.36±2.29

*Paired t-test, value of P is considered statistically significant P<0.05. n – Number of participants; SD – Standard deviation; t – t statistic value; df – Degrees of freedom; P – Probability; T2DM – Type 2 diabetes mellitus; A1 – Periodontitis participants receiving scaling and root planing alone; A2 – Periodontitis participants receiving scaling and root planing with adjunctive low-level laser therapy; B1 – Periodontitis participants with T2DM receiving scaling and root planing alone; B2 – Periodontitis participants with T2DM receiving scaling and root planing with adjunctive low level-laser therapy; CI – Confidence interval

Table 2b.

Intragroup Comparison of Random blood sugar Levels from baseline and 8 weeks after treatment

Groups	Time interval	n	Mean±SD	Mean difference	95% CI of the difference		t	df	P
					Lower	Upper
A1	Baseline	16	95.37±3.48	1.75	0.46	3.03	2.91	15.0	0.01*
	8 weeks	16	93.62±4.33
A2	Baseline	14	94.92±3.66	1.29	0.66	1.90	4.50	13.0	<0.001*
	8 weeks	14	93.64±3.69
B1	Baseline	14	167.07±13.28	2.79	1.74	3.83	5.77	13.0	<0.001*
	8 weeks	14	164.28±11.97
B2	Baseline	16	165.25±11.97	2.56	1.31	3.81	4.39	15.0	<0.001*
	8 weeks	16	162.68±11.17

*Paired t test, value of P is considered statistically significant P<0.05. n – Number of participants; SD – Standard deviation; t – t statistic value; df – Degrees of freedom; P – Probability; T2DM – Type 2 diabetes mellitus; A1 – Periodontitis participants receiving scaling and root planing alone; A2 – Periodontitis participants receiving scaling and root planing with adjunctive low-level laser therapy; B1 – Periodontitis participants with T2DM receiving scaling and root planing alone; B2 – Periodontitis participants with T2DM receiving scaling and root planing with adjunctive low level-laser therapy; CI – Confidence interval

Intergroup comparison of clinical parameters at baseline, showed significant difference (P < 0.05), in salivary TNF-α levels and RBS. whereas PI, BI, PD, CAL did not show any difference represented in [Tables 3, 3a, 3b].

Table 3.

Intergroup comparison of clinical parameters at baseline

Parameter	Study groups	n	Mean±SD	ANOVA
				F	P
PI	A1	16	1.73±0.45	2.24	0.09 (NS)
	A2	14	1.71±0.51
	B1	14	2.00±0.66
	B2	16	2.14±0.57
BI	A1	16	1.90±0.63	1.88	0.14 (NS)
	A2	14	2.29±0.42
	B1	14	1.96±0.62
	B2	16	1.84±0.55
PD	A1	16	6.05±0.63	0.14	0.94 (NS)
	A2	14	5.98±0.59
	B1	14	5.93±0.97
	B2	16	5.87±0.94
CAL	A1	16	6.06±±0.66	0.13	0.94 (NS)
	A2	14	6.02±0.62
	B1	14	6.15±0.94
	B2	16	5.97±1.00

P>0.05 statistically NS. n – Number of participants; SD – Standard deviation; ANOVA – Analysis of variance; F – Fisher in ANOVA test; NS – Nonsignificant; P – Probability; PI – Plaque index; BI – Bleeding index; PD – Probing depth; CAL – Clinical attachment level; T2DM – Type 2 diabetes mellitus; A1 – Periodontitis participants receiving scaling and root planing alone; A2 – Periodontitis participants receiving scaling and root planing with adjunctive low-level laser therapy; B1 – Periodontitis participants with T2DM receiving scaling and root planing alone; B2 – Periodontitis participants with T2DM receiving scaling and root planing with adjunctive low level-laser therapy

Table 3a.

Intergroup comparison of salivary tumor necrosis factor-alpha levels at baseline

Parameter	Study groups	n	Mean±SD	ANOVA
				F	P
TNF-α	A1	16	14.42±2.53	48.4	<0.001*
	A2	14	14.43±1.60
	B1	14	20.92±2.55
	B2	16	21.59±2.25

*Value of P is considered statistically significant P<0.05. TNF-α – Tumor necrosis factor-alpha; n – Number of participants; SD – Standard deviation; ANOVA – Analysis of variance; F – Fisher in ANOVA test; P – Probability; T2DM – Type 2 diabetes mellitus; A1 – Periodontitis participants receiving scaling and root planing alone; A2 – Periodontitis participants receiving scaling and root planing with adjunctive low-level laser therapy; B1 – Periodontitis participants with T2DM receiving scaling and root planing alone; B2 – Periodontitis participants with T2DM receiving scaling and root planing with adjunctive low level-laser therapy

Table 3b.

Intergroup comparison of random blood sugar levels at baseline

Parameter	Study groups	n	Mean±SD	ANOVA
				F	P
RBS	A1	16	95.62±3.14	282.2	<0.001*
	A2	14	96.52±3.66
	B1	14	167.07±13.68
	B2	16	165.25±11.176

*Value of P is considered statistically significant P<0.05. RBS – Random blood sugar; n – Number of participants; SD – Standard deviation; ANOVA – Analysis of variance; F – Fisher in ANOVA test; P – Probability; T2DM – Type 2 diabetes mellitus; A1 – Periodontitis participants receiving scaling and root planing alone; A2 – Periodontitis participants receiving scaling and root planing with adjunctive low-level laser therapy; B1 – Periodontitis participants with T2DM receiving scaling and root planing alone; B2 – Periodontitis participants with T2DM receiving scaling and root planing with adjunctive low level-laser therapy

Intergroup comparison of Salivary TNF-α levels at 8 weeks after treatment showed a significant difference (P < 0.05), represented in [Table 4].

Table 4.

Intergroup comparison of salivary tumor necrosis factor-alpha levels at 8 weeks after treatment

Time interval	Study groups	n	Mean±SD	ANOVA
				F	P
8 weeks	A1	16	9.28±0.976	20.06	<0.001*
	A2	14	9.24±1.090
	B1	14	14.04±2.397
	B2	16	12.36±2.297

*Value of P is considered statistically significant P<0.05. P – Probability; n – Number of participants; SD – Standard Deviation; ANOVA – Analysis of variance; F – Fisher in ANOVA test; T2DM – Type 2 diabetes mellitus; A1 – Periodontitis participants receiving scaling and root planing alone; A2 – Periodontitis participants receiving scaling and root planing with adjunctive low-level laser therapy; B1 – Periodontitis participants with T2DM receiving scaling and root planing alone; B2 – Periodontitis participants with T2DM receiving scaling and root planing with adjunctive low level-laser therapy

Intergroup comparison of the change in clinical parameters, TNF-α, RBS levels from baseline to 8 weeks after treatment, is represented in [Table 5, 5a, 5b] respectively. PI, BI, salivary TNF-α, RBS showed a significant difference (P < 0.05), whereas PD, CAL did not show any difference.

Table 5.

Intergroup comparison of change in clinical parameters from baseline to 8 weeks after treatment

Parameter	Groups	Mean±SD	ANOVA
			F	P
PI	A1	1.04±0.54	7.68	<0.001*
	A2	1.11±0.52
	B1	1.62±0.61
	B2	1.83±0.51
BI	A1	1.46±0.59	7.05	<0.001*
	A2	1.17±0.46
	B1	1.54±0.66
	B2	1.39±0.56
PD	A1	3.16±0.70	0.24	0.87 (NS)
	A2	2.99±0.73
	B1	2.93±0.74
	B2	3.01±0.95
CAL	A1	3.18±0.72	0.13	0.94 (NS)
	A2	3.03±0.79
	B1	3.03±0.60
	B2	3.04±0.92

*Value of P is considered statistically significant P<0.05. P>0.05 statistically NS. SD – Standard deviation; ANOVA – Analysis of variance; F – Fisher in ANOVA test; P – Probability; NS – Nonsignificant. PI – Plaque index; BI – Bleeding index; PD – Probing depth; CAL – Clinical attachment level; T2DM – Type 2 diabetes mellitus; A1 – Periodontitis participants receiving scaling and root planing alone; A2 – Periodontitis participants receiving scaling and root planing with adjunctive low-level laser therapy; B1 – Periodontitis participants with T2DM receiving scaling and root planing alone; B2 – Periodontitis participants with T2DM receiving scaling and root planing with adjunctive low level-laser therapy

Table 5a.

Intergroup comparison of change in salivary Tumor necrosis factor-alpha levels from baseline to 8 weeks after treatment

Parameter	Groups	Mean±SD	ANOVA
			F	P
TNF-α	A1	5.18±1.98	12.06	<0.001*
	A2	4.39±2.10
	B1	6.88±1.60
	B2	9.23±2.11

*Value of P is considered statistically significant P<0.05. TNF-α – Tumor necrosis factor-alpha; SD – Standard deviation; ANOVA – Analysis of variance; F – Fisher in ANOVA test; P – Probability; T2DM – Type 2 diabetes mellitus; A1 – Periodontitis participants receiving scaling and root planing alone; A2 – Periodontitis participants receiving scaling and root planing with adjunctive low-level laser therapy; B1 – Periodontitis participants with T2DM receiving scaling and root planing alone; B2 – Periodontitis participants with T2DM receiving scaling and root planing with adjunctive low level-laser therapy

Table 5b.

Intergroup comparison of change in random blood sugar level from baseline to 8 weeks after treatment

Parameter	Groups	Mean±SD	ANOVA
			F	P
RBS	A1	93.62±4.33	282.9	<0.001*
	A2	93.64±3.69
	B1	164.28±13.68
	B2	162.68±11.17

RBS – Random blood sugar; SD – Standard Deviation; ANOVA – Analysis of variance; F – Fisher in ANOVA test; *Value of P is considered statistically significant P<0.05. P – Probability; T2DM – Type 2 diabetes mellitus; A1 – Periodontitis participants receiving scaling and root planing alone; A2 – Periodontitis participants receiving scaling and root planing with adjunctive low-level laser therapy; B1 – Periodontitis participants with T2DM receiving scaling and root planing alone; B2 – Periodontitis participants with T2DM receiving scaling and root planing with adjunctive low level-laser therapy

Correlation of clinical parameters, RBS and TNF-α levels at baseline and 8 weeks after treatment in different groups showed a significant difference in PI and RBS scores (P < 0.05), represented in [Table 6].

Table 6.

Represents correlation of clinical, random blood sugar and salivary tumor necrosis factor-alpha levels at baseline and 8 weeks after treatment in different groups

Time interval	Salivary parameter	Clinical parameters	Spearman’s corelation	P
Baseline	TNF-α levels	PI	0.15	0.24 (NS)
		BI	−0.17	0.19 (NS)
		PD	−0.11	0.40 (NS)
		CAL	−0.05	0.67 (NS)
		RBS	0.766	<0.001*
8 weeks	TNF-α levels	PI	−0.52	<0.01*
		BI	−0.13	0.29 (NS)
		PD	−0.07	0.54 (NS)
		CAL	0.007	0.38 (NS)
		RBS	0.630	<0.001*

*Value of P is considered statistically significant P<0.05. P>0.05 statistically NS. TNF-α – Tumor necrosis factor-alpha; PI – Plaque index; BI – Bleeding index; PD – Probing depth; CAL – Clinical attachment level; RBS – Random blood sugar; P – Probability; NS – Nonsignificant

DISCUSSION

Various efforts at eliminating infectious agents through periodontal treatment often do not represent a definitive therapy in periodontitis, warranting more sophisticated treatment modalities.[41]

In the present study, the demographic characteristics (age, gender, BMI) showed no significant difference between all the study groups indicating the proper random assignment of participants avoiding selection bias.

BMI within normal range (18.5–24.9) was taken into consideration because of its relevance to TNF-α. As the literature mentions that increased BMI (>24.9) leads to inflammatory state, that is characterised by the increase in production of proinflammatory cytokines (TNF-α), and its soluble receptors, leading to severity of periodontal disease and insulin resistance in T2DM when associated with periodontitis.[42,43,44]

In the present study glucometer was used as a chairside diagnostic device, for RBS evaluation,[37] in order to improve the patient's comfort. As the literature also mentions that in some cases, HbA1c cannot be trusted due to defect in glycation or severe anemia.[44]

The majority of investigations,[22,27] have involved GCF because of its proximity to the site and release of molecular markers, making it site-specific but, at the same time, limiting its role to express the status of other inactive sites. However, saliva sampling solved this problem because it represents pooled concentrations obtained from all the sites in the mouth, giving an overall assessment of disease status and severity.[45] Saliva is considered over serum for the estimation of TNF-α as it is noninvasive and has equal potency. The present study was conducted involving unstimulated saliva to assess the salivary TNF-α levels in periodontitis patients with and without T2DM before and after SRP alone and SRP with adjunctive LLLT.

In the present study FM SRP was performed in single sitting within 24 h, followed by maintenance phase. According to Apatzidou and Kinane[46] both FM-SRP and quadrant-SRP (Q-SRP) modalities are efficacious, and also FM-SRP shows less recolonization when compared to Q-SRP.[47]

Additionally, lasers of various wavelengths have been proposed as alternative treatments for nonsurgical periodontal therapy, because of considerable antibacterial potential by direct ablation, thermal denaturation, or other destruction of bacterial cells. The application of LLLT would reduce bacteria with fewer thermal side effects.[48,49]

LLLT was given within 1 week after SRP in groups A2 and B2. As LLLT showed reduction in migration of monocytes and neutrophils to the site of inflammation thus reducing the production of TNF-α, and other chemotactic factors, initiating the back-feeding of inflammatory process. Therefore, LLLT would contribute to the breakdown of positive feedback loop of inflammation, when it is applied after SRP.[27]

Clinical parameters PI, BI, PD, CAL and response of TNF-α levels were evaluated at 8 weeks after treatment. There was a time gap of 7 to 8 weeks after LLLT, before the final readings were taken. As the studies mention that there was precisely oriented collagen bundle fibers, between 4 and 8 weeks for the primary evaluation of nonsurgical therapy.[27,50,51]

No difference was observed in clinical parameters at baseline, indicating direct result of the intervention. A significance in TNF-α and RBS levels were observed as we had included participants with T2DM (Group B1, B2).[52]

Intra and intergroup comparisons of PI and BI from baseline to 8 weeks after treatment in all groups showed significant difference. This result can be attributed to reducing local factors like plaque and calculus by SRP and improving inflammatory conditions by SRP adjunct to LLLT. This was in accordance with the studies conducted by Calderín et al.,[27] Giannopoulou et al.,[53] Badersten et al.[54] Whereas, Nguyen et al.,[55] obtained similar results in 3 months period.

In the present study, intragroup comparison of PD and CAL from baseline to 8 weeks after treatment, all groups showed significant difference. This change can be due to reduced inflammation and the formation of reattachment following SRP and removal of pocket lining, hemostasis, and coagulation of periodontal inflamed soft tissues by SRP adjunct to LLLT. The results are in accordance with the studies conducted by Calderín et al.,[27] Reis et al.,[56] Giannopoulou et al.[53] Whereas Euzebio Alves et al.,[57] Morozumi et al.,[58] Chen et al.[17] obtained similar results in 6 weeks’ time period.

In the present study, the intergroup comparison of PD and CAL at 8 weeks after SRP showed no significant difference. May be due to mechanical therapy alone. It is not effective in eliminating pathogenic bacterial species in the soft tissue and areas inaccessible to periodontal instruments, such as deep pockets, furcation areas, and root depressions. The results are in agreement with studies conducted by Dukić et al.,[59] Calderín et al.,[27] Euzebio Alves et al.,[57] Giannopoulou et al.[53]

Intergroup comparison of PD and CAL at 8 weeks after SRP adjunct to LLLT showed no significant difference. The results are in accordance with study conducted by Calderín et al.[27] This may be due to the efficacy of LLLT in periodontal disease is still controversial.

In the present study, the intragroup comparison of salivary TNF-α levels from baseline to 8 weeks after treatment showed a significant difference in all the groups. This change may be due to mechanical therapy, and the anti-inflammatory effect of LLLT reduced the clinical inflammation. Whereas Giannopoulou et al.,[53] Calderín et al.,[27] observed similar results who evaluated in GCF, Chen et al.,[17] evaluated in serum, Pesevska et al.,[26] evaluated at the gingival tissue level. However, contradictory results were obtained by Morozumi et al.,[58] who evaluated TNF-α levels in GCF.

In the present study, intergroup Comparison at 8 weeks after SRP showed a significant difference in TNF-α levels. This change may be due to reduced clinical inflammation through mechanical therapy. Whereas Correa et al.,[19] and Kardesler et al.,[20] observed similar results who evaluated in serum. However, Chen et al.,[17] Morozumi et al.,[58] obtained contradictory results who evaluated in serum and in GCF respectively. This may be attributed to difference in sampling timings.

In the present study, intergroup comparison at 8 weeks after SRP adjunct to LLLT showed a significant difference in TNF-α levels; this change may be due to LLLT adjunct to SRP prevented the bacterial recolonisation. The results are in agreement with studies conducted by Giannopoulou et al.,[53] Chen et al.,[17] Reis et al.[56] However, contradictory results were observed by Morozumi et al.,[58] Lalla et al.,[60] D’Aiuto et al.,[14] Calderín et al.[27]

In the present study, both intra and intergroup comparison of RBS levels showed a significant difference from baseline to 8 weeks after treatment in all the groups. In the Diabetic group (B1 and B2), this significance difference may be indicative of the effects of nonsurgical periodontal therapy on diabetic patient's glycemic control. In comparison, similar results were observed in the study by Martínez-Aguilar et al.,[52] who evaluated by using HbA1c.

Comparison of RBS and salivary TNF-α levels at baseline and 8 weeks after treatment in different groups showed a significant correlation; this may be due to effective periodontal therapy that might have decreased RBS values by reducing TNF-α concentrations. Whereas Dağ et al.,[21] showed similar results by evaluating through HbA1c.

CONCLUSION

1. There is an association between salivary TNF-α levels in periodontitis and T2DM

2. SRP and SRP adjunct to LLLT showed significant improvement in clinical and salivary TNF-α levels

3. Further studies recommended with histological analysis and larger sample size.

Financial support and sponsorship

The study was registered at Clinical Trials Registry, India, with registration number CTRI/2019/02/017737.

Conflicts of interest

There are no conflicts of interest.