Chairside clinicomicrobiological assessment of subgingival boric acid irrigation in periodontitis patients: A randomized controlled trial

Abstract

Background:

Elemental boron has a positive role in many functions in the human body, such as immune regulation, wound healing, bone health maintenance, etc. A boron-containing molecule (AN0128) has shown additional antimicrobial properties against virulent red-complex bacteria. However, there is a dearth of human trials evaluating the effectiveness of readily available boronic compounds in periodontitis patients.

Materials and Methods:

Thirty-six patients with generalized periodontitis were included in this double-blind, placebo-controlled randomized clinical trial. Following scaling and root planing at baseline, each designated test site in the 18 treatment group subjects received subgingival 0.75% boric acid, whereas the remaining 18 subjects received placebo irrigation at the control site. At the 6^th week recall, test/control irrigations were repeated as per the group assigned. Clinical parameters were recorded at baseline, 6^th week and 12^th week, whereas chairside microbial assessment using benzoyl-DL-arginine-2-napthylamide (BANA) was done at baseline and 12^th week.

Results:

Comparatively, the test group demonstrated significant improvements (P < 0.05) in terms of the gingival index, bleeding on probing (BOP), probing pocket depth (PPD), and clinical attachment level (CAL) at the 12^th week. Improved mean BOP, PPD, and CAL differences were highly significant (P < 0.005) for the test group at the 6^th week. Microbial assessment using the BANA test did not observe a significant intergroup difference following therapy.

Conclusion:

Outcomes of the present trial demonstrated boron’s significant role in clinical improvements. Adjunctive subgingival boric acid irrigation can be effective in healing following phase-I therapy.

INTRODUCTION

Periodontitis derives its intricacy from a myriad of virulent bacterial microcolonies that cause immune hyper-response; leading to the periodontal inflammation.[1,2,3] Subgingival identification and quantification of the most significant red-complex bacterial group by utilizing various microscopic or culture techniques significantly correlates with the extent and progression of periodontitis; as well as response to treatment over a period of time.[4,5,6,7] Benzoyl-DL-arginine-2-napthylamide (BANA)-enzymatic chairside tool kit is one among such diagnostic tests with additional advantages of simplicity and exclusivity for the red-complex group.[8,9,10] It utilizes an enzyme highly specific to red complex; capable of hydrolyzing the synthetic trypsin substrate BANA.

Mechanical debridement with or without chemical plaque control adjuncts fails to completely disrupt the virulent subgingival ecosystem, leading to bacterial regrowth within months.[11,12] In this regard, localized subgingival drug delivery is effective in achieving a relatively higher therapeutic concentration for a longer duration of time without exhibiting any systemic side effects.[13,14]

Boric acid is an inorganic and weak monobasic acid, whose central atom boron illustrates its role in calcium homeostasis, Vitamin D utilization, immune regulation, oxidative stress reduction, and wound healing.[15,16,17,18,19] A newer boron-containing bioactive molecule (AN0128) has exhibited antibacterial and anti-inflammatory properties.[20,21] Furthermore, boron’s specific antagonism against the red-complex bacteria has also been noted.[22] However, there is a paucity of documented human trials evaluating the clinical effectiveness of boron in periodontitis; along with clinically assessing its antired-complex potency at the subgingival level.

The present study aims to determine the effect of adjunctive subgingival boric acid irrigation(s) on clinical and microbiological parameters using the chairside BANA test in patients with periodontitis.

MATERIALS AND METHODS

This randomized, placebo-controlled clinical trial was registered and planned as per Consolidated Standards of Reporting Trials 2010 directives [reg.no. CTRI/2021/01/030569, Figure 1]; at the institution’s Periodontology department and Biochemistry department with prior ethical approval from the university. Systemically healthy, 30–60 years age group subjects having generalized mild-to-moderate chronic periodontitis (corresponding to the different stages of periodontitis) but not undergone any periodontal therapy for at least 6 preceding months were included with additional criteria: At least 20 remaining natural teeth, along with minimum one site having a mean probing pocket depth (PPD) ≥5 mm, clinical attachment level (CAL) ≥4 mm, and noticeable bleeding on probing (BOP). Exclusion criteria comprised advanced periodontitis patients (PPD ≥9 mm), breastfeeding/lactating mothers, presence of necrotizing/ulcerative oral lesions, grade II/III mobility, and long-term chronic illnesses.

Figure 1.

Consolidated Standards of Reporting Trials flow chart for study phases. SRP – Scaling and root planing; SRP + BA – scaling and root planing + boric acid, BANA – Benzoyl-DL-arginine-2-napthylamide

The sample size was calculated based on a related previous study that considered 3-month changes in CAL as the primary outcome variable.[22] Keeping 5% significance level, 95% confidence interval, 10% maximum attrition, and ability to detect true intergroup mean differences, a total sample size of 36 patients divided into two groups was calculated in order to achieve 90% power with Type-I error (α) valuing 0.05. Secondary outcome measures included other clinical parameters (dependent variables): plaque index (PI), gingival index (GI), BOP, PPD and microbiological analysis: BANA test to detect red complex bacteria from subgingival plaque sample.

Randomization was done by assigning unique codes to each subject by the study coordinator (NL) in ascending order. This code list was obtained from a computer-generated randomization table prepared by a biostatistician (SK) using a balanced random permuted block approach. Within each block, the sequence of treatment assignments was predetermined.

The predetermined treatment sequencing warrants that the number of subjects assigned to each treatment group is balanced within each block.

Eligible subjects were sequentially recruited by NL without revealing the assigned codes. A Biochemistry lab assistant (VS) formulated nonlabeled coded and sealed opaque envelopes containing identical insulin syringes, which he filled with boric acid/placebo solution, corresponding to the randomization codes generated by SK. VS was not involved in any other trial component. He submitted these envelopes to NL. At scheduled intervention appointments, an investigator (JA) totally unacquainted with the allocated unique codes or treatment group assigned to subjects was provided with the boric acid/placebo-filled syringe by NL as per the sequence, which was subgingivally irrigated at predetermined sites in the patients by JA. Details of test/placebo groups, randomization codes, and associated subjects were revealed only after statistical analysis that helped in overall concealment of allocation and blinding.

For investigator calibration, 10 participants not included in the trial were clinically assessed by a single investigator (JA), twice daily, within 7 working days at definite time intervals. Assessments were repeated, and an intraclass kappa (correlation) coefficient of 0.94 was achieved.

For clinical measurements, all 6 sites at each tooth were examined using a manual periodontal probe (UNC-15 probe, Hu-Friedy). PI, GI, BOP, PPD and CAL scores were charted [Figure 2]. One tooth site per subject having the greatest average pocket depth (with the average values ranging between 5-8 mm; excluding molars) and fulfilling the inclusion criteria was selected as the test site.

Figure 2.

Clinical measurements at baseline using UNC-15 probe

For BANA analysis, following isolation and before pooled sampling from the designated test sites, the supragingival plaque was gently removed from the tooth surface using a cotton swab. Sterile Curettes (Hu-Friedy) were used to carefully extract the minimally disturbed subgingival plaque samples. These pooled plaque samples were directly applied on BANA-impregnated and raised lower portion of enzymatic test strips (Hexagon International [GB] Ltd., Berkhamsted, UK) and further processing was completed as per manufacturer’s guidelines [Figure 3]. Observations regarding color changes were noted and scored as per the manual.

Figure 3.

Microbial assessment using benzoyl-DL-arginine-2-napthylamide test at baseline

Boric acid solution was formulated by initially preparing a 12% stock solution and sequentially diluted to achieve 0.75% strength at the Biochemistry Department.[22] The solution was filtered, sterilized, and loaded into insulin syringes and submitted to the study coordinator.

The process of clinical interventions began by an initial briefing to the subjects about oral hygiene maintenance, including tooth brushing twice using a soft-bristle toothbrush and a fluoride-containing dentifrice. Scaling and root planing were performed on all participants using an ultrasonic scaler (Aceton Satelec Suprasson P5 Booster) and Universal Gracey curettes 2R/2 L and 4R/4L (Hu-Friedy).

Clinical parameters and BANA scores were recorded at baseline [Figures 2 and 3]. Trial sites were then subgingivally irrigated after single-sitting whole mouth scaling and root planing (SRP), and these sites were secured using periodontal dressing [Figures 4 and 5]. Irrigation was repeated again 6 weeks post-SRP and secured. Clinical measurements were done at baseline, 6 weeks, and 3 months, while the microbiological analysis was done twice at baseline and at the end of 3 months [Figures 6-10].

Figure 4.

Subgingival boric acid/placebo irrigation following scaling and root planing

Figure 5.

Securing trial site using periodontal dressing following irrigation

Figure 6.

Clinical assessment at 6^th week follow-up

Figure 10.

Benzoyl-DL-arginine-2-napthylamide test interpretation chart

Figure 7.

Microbial assessment using benzoyl-DL-arginine-2-napthylamide test at 6^th week follow-up

Figure 8.

Clinical assessment at 12^th week follow-up

Figure 9.

Microbial assessment using benzoyl-DL-arginine-2-napthylamide test at 12^th week follow up

Oral hygiene standards were thoroughly checked at the recall visits and reinforced whenever necessary. Patients were also instructed not to chew hard or sticky foods, or use any interdental aids and mouthwashes, antibiotics, or any antioxidant supplements during the study period. Subjects were kept under consideration for a comprehensive nonsurgical and supportive periodontal treatment necessary after completion of the trial.

For the statistical analysis of clinical variables, unpaired t-test and paired t-test were utilized for intra-group and inter-group comparisons, respectively. The diagnostic ability of the BANA test was assessed by taking receiver operating characteristic (ROC) analysis as a reference, where test sensitivity was plotted against [1 – specificity) for every evaluated clinical parameter Figure 10]. Analysis was done using IBM SPSS version 23.0 (SPSS, Chicago, IL, USA). For BANA enzymatic test results, Wilcoxon’s signed-rank test was used for intra-group comparison and the Mann-Whitney test for comparison among the two groups. A P < 0.05 and <0.001 were considered as significant and highly significant difference, respectively.

RESULTS

Comparison groups were showing nonsignificant demographic differences in terms of age and sex [Table 1]. With non-significant intergroup GI difference at baseline, significant improvements were observed in at the 6^th week and 12^th week [Table 2]. Intragroup comparison showed highly significant improvements from baseline to 6^th week, baseline to 12^th week in both the groups [Table 3]. Deterioration in mean values from the 6^th week to 12^th week was nearly nonsignificant in both the groups.

Table 1.

Demographic characteristics at baseline

Characteristics	SRP + boric acid group	SRP + placebo group	P
Age (mean±SD)	40.53±7.06	40.89±7.68	0.886
Sex (male/female)	10/7	10/8	0.845

Data are expressed as mean±SD. P<0.05 was considered statistically significant. SD – Standard deviation; SRP – Scaling and root planing; P – P-value

Table 2.

Intergroup comparison of clinical parameters

Parameters	Recall intervals	Treatment groups		t	P
		SRP + boric acid	SRP + placebo
PPD	Baseline	6.66±0.53	6.63±0.47	0.22	0.829
	6th weeks	4.58±0.43	4.98±0.35	−3.05	0.005
	12th weeks	4.49±0.48	5.12±0.42	−4.15	<0.001
CAL	Baseline	6.36±0.58	6.38±0.72	−0.06	0.953
	6th weeks	4.18±0.39	4.57±0.38	−2.97	0.005
	12th weeks	4.10±0.37	4.83±0.33	−6.15	<0.001
GI	Baseline	2.28±0.38	2.31±0.37	−0.21	0.839
	6th weeks	0.36±0.15	0.49±0.18	−2.15	0.039
	12th weeks	0.41±0.18	0.58±0.23	−2.49	0.018
PI	Baseline	2.15±0.43	2.31±0.34	−1.21	0.235
	6th weeks	0.50±0.20	0.47±0.17	0.45	0.657
	12th weeks	0.49±0.26	0.53±0.17	−0.58	0.565
BOP%	Baseline	91.18±15.16	86.11±19.60	0.85	0.401
	6th weeks	11.76±12.86	22.22±14.57	−2.25	0.032
	12th weeks	5.88±10.93	25.00±14.85	−4.32	<0.001

Paired t-test was used to compare the difference between the groups and unpaired t-test was used for intragroup comparisons. P<0.05 was considered statistically significant. P<0.001 was considered statistically highly significant. PPD – Probing pocket depth; CAL – Clinical attachment level; GI – Gingival index; PI – Plaque index; BOP – Bleeding on probing; SRP – Scaling and root planing; P – P-value; t – t-value

Table 3.

Intragroup comparison of clinical parameters

Para-meters	Recall intervals	Treatment groups
		SRP + boric acid			SRP + placebo
		Mean±SD	t	P	Mean±SD	t	P
PPD	Baseline versus 6th weeks	2.09±0.61	14.07	<0.001	1.65±0.58	12.16	<0.001
	Baseline versus 12th weeks	2.18±0.63	14.30	<0.001	1.51±0.65	9.85	<0.001
	6th weeks versus 12th weeks	0.09±0.10	3.46	0.003	−0.14±0.40	−1.49	0.155
CAL	Baseline versus 6th weeks	2.18±0.62	14.59	<0.001	1.80±0.57	13.32	<0.001
	Baseline versus 12th weeks	2.26±0.59	15.89	<0.001	1.55±0.82	8.00	<0.001
	6th weeks versus 12th weeks	0.08±0.13	2.71	0.015	−0.26±0.54	−2.02	0.060
GI	Baseline versus 6th weeks	1.91±0.29	27.09	<0.001	1.82±0.29	26.20	<0.001
	Baseline versus 12th weeks	1.87±0.28	27.39	<0.001	1.72±0.43	17.08	<0.001
	6th weeks versus 12th weeks	−0.05±0.09	−2.13	0.049	−0.10±0.23	−1.80	0.090
PI	Baseline versus 6th weeks	1.65±0.42	16.36	<0.001	1.83±0.34	22.68	<0.001
	Baseline versus 12th weeks	1.66±0.45	15.22	<0.001	1.78±0.27	27.97	<0.001
	6th weeks versus 12th weeks	0.01±0.21	0.29	0.773	−0.06±0.20	−1.17	0.260
BOP%	Baseline versus 6th weeks	79.41±18.19	18.00	<0.001	63.89±21.39	12.67	<0.001
	Baseline versus 12th weeks	85.29±15.46	22.75	<0.001	61.11±21.39	12.12	<0.001
	6th weeks versus 12th weeks	5.88±10.93	2.22	0.041	−2.78±14.57	−0.81	0.430

SRP – Scaling and root planing; PPD – Probing pocket depth; CAL – Clinical attachment level; GI – Gingival index; PI – Plaque index; BOP – Bleeding on probing; SD – Standard deviation; P – P-value; t – t-value

Specifically for BOP, no significant difference between the groups at baseline that improved to significant levels at the 6^th week and further improved to highly significant at the 12^th week in favor of the test group [Table 2]. Highly significant changes were observed both in the test group as well as BOP% from baseline to the 6^th week and baseline to the 12^th week. From the 6^th week to 12^th week, the change was significant [Table 3]. In the control group, highly significant changes were observed from baseline to the 6^th week and from baseline to the 12^th week, whereas from the 6^th week to 12^th week, the change was insignificant.

Intergroup comparison of PPD noted no significant difference between the groups at baseline but significant differences at 6^th week and highly significant at the 12^th week [Table 2]. Within both the groups, highly significant changes were observed in PPD from baseline to the 6^th week and baseline to the 12^th week. From the 6^th week to 12^th week, the change was significant in the test group but insignificant in the control group [Table 3].

With nonsignificant baseline CAL difference between the groups, significant differences were observed at 6^th week and highly significant at the 12^th week [Table 2]. Within both the groups, highly significant changes were observed in CAL from baseline to the 6^th week and from baseline to 12^th week, but only the test group showed significant change from 6^th week to 12^th week [Table 3]. In the control group, highly significant changes were observed from baseline to the 6^th week and from baseline to the 12^th week, while from the 6^th week to the 12^th week, the change was insignificant [Table 3].

Pre-as well as postoperative intergroup mean BANA score differences were nonsignificant, whereas intragroup changes were highly significant [Table 4].

Table 4.

Intergroup and intragroup comparison of benzoyl-DL-arginine-2-napthylamide scores

Time points	BANA scores (mean±SD)			Mann–Whitney test
	SRP + boric acid group	SRP + placebo group		U	P
Baseline	1.82±0.39	1.83±0.38		151.50	0.961
12th weeks	0.71±0.77	1.11±0.76		109.00	0.153
Time points	Test group			Control group
	Mean difference±SD (SRP + boric acid group)	Z	P	Mean difference±SD (SRP + placebo) group	Z	P
Baseline-12th weeks	1.12±0.70	6.61	<0.001	0.72±0.67	4.58	<0.001

Wilcoxon’s signed-rank test was used for intragroup comparison, and Mann–Whitney test was used for comparison between the two groups. P<0.001 was considered statistically highly significant. BANA – Benzoyl-DL-arginine-2-napthylamide; SRP – Scaling and root planing; SD – Standard deviation; Z – Z-value; P – P-value; U – U-value

DISCUSSION

Boric acid of 0.75% concentration was earlier found to be tolerable to periodontal fibroblast cells.[22] Inter-group analysis showed significant differences at the 6^th week and a highly significant difference at the 12^th week for both PPD and CAL, depicting better outcomes in the test group. Similar results were observed in earlier recent studies. Bashir and Krstic[23] conducted a systematic review that included four different randomized trials by Sağlam et al.,[22] Kanoriya et al.,[24] Singhal et al.,[25] and Mamajiwala et al., all evaluating the efficacy of boron/boric acid.[26] It had made similar observations in terms of pocket depth reduction and CAL gain at comparable trial periods. Such improvements are attributed to boric acid’s antioxidant behavior, antiproteolytic enzyme activity, and possibly its in vivo osteogenic ability. Glutathione acts as a potent oxygen scavenger in the human body. Boric acid minimizes the deleterious effects of reactive oxygen species at its site of action by facilitating an increase in the local glutathione concentration. Boric acid-mediated suppression of Serine proteolytic enzymes further improves clinical gains in terms of PPD and CAL. Osteogenic potential of boric acid has also been explored in recent years for its periodontal implications. As per Xu et al.[27] and Uysal et al.,[28] boron diet supplementation significantly helps in bone regeneration in rabbits. Hakki et al.[15] noted boron as having high therapeutic potential in regenerative therapy, as it influences the mechanism of bone metabolism through its osteoblastic transcription factor RunX2 and bone morphogenic proteins (BMPs). Therefore, the observations made in our trial in terms of localized GI, BOP, CAL, and PPD further consolidate the use of boric acid as an effective anti-inflammatory adjunctive to SRP, first given and proved by Sağlam et al.[22]

To the best of our knowledge, this is the first human clinical trial to use the BANA test to measure the antibacterial activity of boric acid and correlate it with clinical indicators. Multiple studies have demonstrated that BANA hydrolysis by plaque samples is a noninvasive, sophisticated, patient-compliant, cost-effective, cost and time-effective chair-side test that has the proven to be a marker of periodontal morbidity to assess antimicrobial medication efficiency, as well as diagnose and monitor therapy effectiveness in refractory individuals. It is positively connected with plaque levels in spirochete proportions, and a positive BANA test after initial treatment predicts future attachment loss during recall.

Mean BANA scores at baseline were 1.82 ± 0.39 and 1.83 ± 0.38 for the test and control group, respectively (P = 0.961). At 3 months follow-up, these scores decreased significantly for both the groups as shown in Table 4, underlining improvements in bacterial load after phase 1 periodontal therapy. Despite a greater reduction in mean BANA score for the test group, nonsignificant intergroup difference after 3 months suggests boric acid’s limited antimicrobial role at the subgingival level.

Our results are similar to the study conducted by Sağlam et al.[22] in which, drastic reduction in mean microbial levels in all groups was observed after SRP, but a nonsignificant intergroup difference at 1- and 3-month follow-ups. They concluded that the concentration and physical formulation of boric acid were insufficient to demonstrate appropriate antibacterial action in the pocket. More favorable results were observed in an animal study conducted by Luan et al.[21] Mamajiwala et al.[26] also had similar observations in which gel form of boric acid was applied, believed to impart a sustained release of drug at the target site, maintaining longer inhibitory effects over the microorganisms.

The ROC analysis, illustrated as Figure 11, was used to assess the validity of the BANA test, and sensitivity was determined to be highest for generalized PI (95.7%) and lowest for BOP (78.3%). Specificity was found to be maximum for BOP (91.7%) and minimum for generalized GI (50.0%). Overall accuracy was found to be maximum for Generalized PI(89.1%) and minimum for generalized GI (65.9%).

Figure 11.

Receiver operating characteristic analysis for benzoyl-DL-arginine-2-napthylamide enzymatic test. ROC – Receiver operating characteristic

In a 2010 research, Andrade et al.[10] observed that the BANA Test’s specificity rises following SRP, notably after 60 days, corresponding with an increased number of healthy sites. Sensitivity was the least for BOP%, primarily due to the DNA detection of the red complex species in BANA-negative samples. It was also noted that 5-min incubation time raises the BANA data’s specificity while lowering its sensitivity. As a result, it reduces false positives while increasing the number of real negatives. Our data supports the abovementioned results, as maximum specificity was observed for BOP%, whereas minimum for generalized GI, as it takes whole mouth GI values into account.

With significant improvements in GI, BOP, CAL, and PPD; the overall findings of this study further augment the innate therapeutic nature of boron. Boric acid’s anti-inflammatory nature and possible osteoblastic behavior further underlines its potential to act as an adjunct to the standard treatment in a chronic inflammatory disease such as periodontitis. Our study, for the first time, engaged a simple and accurate chairside BANA enzymatic test to quantify the anti-red complex-effectiveness of boric acid solution at the subgingival level. This was in line with the current trend to employ the most recent advances in diagnostics modalities and clinically utilize them for better patient compliance and improved treatment outcomes through real-time chairside assessment.

Future trials with larger sample sizes and longer follow-up periods can improve our understanding regarding boric acid’s adjunctive therapeutic effects.

CONCLUSION

Our trial findings suggest that boric acid, that has an inherent homeostatic potential for the human body, specifically helps in improving therapeutic response to standard periodontal therapy. The exact mechanism of such effects on different tissue types, as well as pathogenic microbiota, is yet to be fully understood.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

This study was partially supported by an intramural research grant for a postgraduate dissertation, King George’s Medical University, Lucknow, India (Grant No. 1248/R.Cell-2021 dated: February 22, 2021). No external funding, apart from the support of the institutional research grant, was available for this study.