A Novel Microencapsulated Probiotic Oral Rinse Improves Gingival Health and Influences Salivary Matrix Metalloproteinases 8 and 9 in Children with Down Syndrome: A Pilot Randomized Clinical Trial

doi:10.21203/rs.3.rs-4724602/v1

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A Novel Microencapsulated Probiotic Oral Rinse Improves Gingival Health and Influences Salivary Matrix Metalloproteinases 8 and 9 in Children with Down Syndrome: A Pilot Randomized Clinical Trial

https://doi.org/10.21203/rs.3.rs-4724602/v1

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Objectives

Children with Down Syndrome (DS) exhibit heightened oral health vulnerabilities owing to their unique physiological characteristics, necessitating the need for tailored interventions. The integration of natural MMP inhibitors such as probiotics has the potential to significantly improve oral health outcomes in this vulnerable population. In this double-blinded randomized controlled trial (CTRI/2023/04/051447), we investigated the effects of a novel sugar-free probiotic oral rinse compared to 0.2% chlorhexidine digluconate on salivary MMP-8 and MMP-9 levels in children with DS.

Materials and Methods

Thirty children, aged 6–14 years, participated in this study. Baseline assessments involved saliva collection for MMP-8 and MMP-9 quantification using an enzyme-linked immunosorbent assay (ELISA) and evaluation of gingival parameters. Following the baseline evaluations, individualized oral rehabilitative interventions were administered. Two weeks post-rehabilitation, the participants were randomized to receive either a probiotic (Group 1) or chlorhexidine rinse (Group 2). The oral rinses were used for two weeks, followed by repeated salivary MMP quantification and gingival parameter assessments after two weeks and six months.

Results

Both groups exhibited significant reductions in MMP levels, plaque index (PI), gingival index (GI), oral hygiene index, and bleeding on probing (BOP) (p < 0.05). The probiotic group demonstrated a significant reduction in BOP (p = 0.000).

Conclusion

This study emphasizes the importance of tailored oral care for children with DS and demonstrates significant post-treatment oral health improvements.

Matrix Metalloproteinase 8 (MMP-8)

Matrix Metalloproteinase 9 (MMP-9)

Down Syndrome

Probiotics

Chlorhexidine

Oral Rehabilitation

Down syndrome (DS), identified by John Langdon Down in 1866, is a genetic disorder that results from an extra copy of chromosome 21[1]. This anomaly, affecting approximately one in 732 infants in the United States and 10.1 per 10,000 live births in Europe, leads to significant physical and cognitive impairment[2]. Individuals with DS are at a higher risk of various health complications including cardiovascular defects, leukemia, and early onset Alzheimer's disease[3]. Among these, oral health disparities are particularly severe, with increased susceptibility to dental caries and periodontal disease[4, 5] due to systemic and local factors, such as immunological deficiencies[6] and oral morphological characteristics[7].

Matrix metalloproteinases (MMPs), particularly MMP-8 and MMP-9, are crucial in the degradation of extracellular matrix components and are key indicators in periodontal disease, dental caries, and other oral pathologies [8–11]. Elevated levels of these MMPs in individuals with DS suggest a heightened inflammatory response in the oral cavity, emphasizing the need for effective oral health interventions tailored to this population.[12–14]

This randomized clinical trial investigated the efficacy of chlorhexidine gluconate versus probiotic oral rinses in modulating salivary MMP-8 and MMP-9 levels in children with DS. Chlorhexidine gluconate is widely regarded as the gold standard for oral rinses, owing to its antimicrobial properties[15]. However, probiotic oral rinses offer a promising alternative, leveraging immunomodulatory and microbiota-modulating effects to potentially reduce MMP activity and improve oral health[16, 17].

This study aimed to provide a rigorous comparison between these two treatments and evaluate their impact on the critical biomarkers of periodontal disease and dental caries in children with DS. By focusing on localized probiotic interventions alongside comprehensive oral rehabilitation, this study seeks to determine whether probiotics can serve as an effective, targeted strategy to enhance oral health outcomes in this vulnerable population. These findings could inform clinical practices and contribute to closing the gap in healthcare disparities faced by children with DS, ultimately improving their quality of life.

The primary objective of this study was to evaluate changes in MMP-8 and MMP-9 levels before and after the use of a microencapsulated sugar-free probiotic oral rinse and 0.2% w/v chlorhexidine gluconate oral rinse. Measurements were taken at baseline, post-dental intervention, and oral health education, two weeks after using the oral rinse, and six months after usage to assess the effects of both rinses. Secondary objectives included evaluating changes in the plaque index (PI), gingival index (GI), simplified oral hygiene index (OHI-S), and bleeding on probing (BOP) at the following intervals: baseline, post-dental intervention and oral health education, 15 days post-usage, and 6 months post-usage.

Trial design

This study was a prospective, randomized, parallel, double-blind clinical trial, following the guidelines published by the Consolidated Standards of Reporting Trials (CONSORT) and approved by the Institutional Ethical Committee, A B Shetty Memorial Institute of Dental Sciences, Nitte (Deemed to be University) (ETHICS/ABSMIDS/227/2022). It was also registered in the Clinical Trials Registry India (CTRI) (CTRI/2022/10/046847). Written informed consent was obtained from all the enrolled subjects in accordance with the Declaration of Helsinki. It was conducted at the NITTE-Special Child Care Centre (N-SPECC), affiliated with the Department of Pediatric and Preventive Dentistry, A B Shetty Memorial Institute of Dental Sciences, NITTE (Deemed to be University), from May 2022 to September 2023.

Sample Size

The sample size was determined using a specialized statistical software (IBM SPSS Statistics 22.0; IBM Analytics, Armonk, NY, USA). Given the absence of similar studies involving children with DS, this calculation drew insights from a seminal article[18]. With the parameters set at a 5% significance level, 80% statistical power, 1% margin of error, an estimated proportion of 0.12%, and referencing a finite population of 213 individuals derived from local census data, the initial minimum sample size was established for 30 participants (15 in each group). To account for potential patient attrition owing to health vulnerabilities and adherence issues, an additional 70% of the total sample size was incorporated. The final sample size included 169 participants.

Participants

Patients were selected according to the following inclusion criteria: children diagnosed with DS aged 6–14 years, with mild intellectual disability, possessing good compliance, and having guardians who exhibited strong motivation and provided informed consent. Conversely, the exclusion criteria were children diagnosed with DS who had known allergies to probiotic drugs/products, xylitol, sorbitol, maltodextrin, or natural vanilla flavoring. Additionally, those with underlying systemic and metabolic diseases requiring medication, undergoing specialized treatments, such as orthodontic procedures or wearing dental appliances, or whose parents or guardians were unwilling to participate or failed to provide consent were excluded from the study. Each participant received verbal information regarding the products, study objectives, and protocols. Participants were also informed of their right to withdraw from the study at any point. After the participant recruitment process, 51 of the initial 169 individuals were excluded, leaving 118 participants for the study (Fig. 1).

Randomization and Allocation

Two investigators conducted the allocation process, with the first investigator responsible for sequence generation. The allocation concealment method utilized was the envelope method, wherein codes representing each mouthwash (Group 1 or Group 2) were randomly inscribed on 30 sheets of paper (15 for each group), which were then sealed within opaque envelopes labeled serially. Subsequently, the first investigator enrolled the participants and recorded their information without awareness of their respective group assignments. The second investigator, who was also unaware of participant details, assigned mouthwashes to the participants, thereby ensuring objectivity and minimizing potential bias. Consequently, the study was conducted in a double-blind manner, as both investigators and data analysts remained unaware of group assignments. This trial followed a double-blind, parallel-group design with an allocation ratio of 1:1. After participant selection, the 118 individuals were evenly divided into two groups, each comprising 59 participants (Fig. 1).

Interventions

At the baseline stage, pre-screened participants were directed to the dental clinic for complete oral examinations. A dentist conducted these examinations to confirm the eligibility criteria. Subsequently, the participants were randomly allocated to one of the two groups.

Group 1: Children with DS who were given the probiotic oral rinse

Group 2: Children with DS who were given chlorhexidine gluconate oral rinse

The detailed composition of the mouthwashes used in this study is listed in Table 1.

Table 1

Composition of the oral rinses
	Composition	Instructions
Group 1 (Probiotic group)	A lyophilized powdered probiotic suspension (Cipla ActivKids Unobiotics Junior, Cipla Pvt. Ld, India) containing approximately 10⁸ colony-forming units (CFU) of L. rhamnosus GG, weighing 30 mg, and reconstituted with phosphate-buffered saline (PBS).	• Mix the powder with 5 ml of phosphate-buffered saline (PBS) for 30 seconds. • Swish the mixture in the mouth for 60 seconds and then expectorate. • Use the oral rinse twice daily, 30 minutes after brushing teeth for optimal effectiveness.
Group 2 (0.2% w/v chlorhexidine gluconate)	Chlorhexidine gluconate (0.2% w/v) (Hexidine, ICPA Products Ltd., India)	• Swish 5 ml of the oral rinse in the mouth for 60 seconds and then expectorate. • Use the oral rinse twice daily, 30 minutes after brushing teeth for optimal effectiveness.

Following the oral examination of the children with DS, an informative pre-prescription session was conducted to raise awareness. Subsequently, a comprehensive oral rehabilitation programme was implemented. This program included diet counselling and modification, reinforcement of oral hygiene practices (such as tooth brushing using the scrub method, flossing, and tongue scraping) for both the child and their caregiver, as well as oral prophylaxis and topical fluoride application, and the application of pit and fissure sealants as necessary. Restorative treatments for carious lesions, extractions, pulp therapy, periodontal therapy, and prosthetic rehabilitation were performed when required (Fig. 2). Ultimately, 40 children completed full mouth rehabilitation: 20 in the probiotic group and 20 in the chlorhexidine group. However, 78 children were excluded from the study because they did not complete the entire oral rehabilitation process.

After dental rehabilitation, participants attended an initial visit to the outpatient clinic, where they received detailed instructions on how to use mouthwashes, as specified in Table 1. They also underwent supervised trial rinse by experts to ensure compliance with the oral rinse protocol. All mouthwashes were dispensed in 5 ml opaque plastic bottles labeled with unique identification numbers corresponding to each participant, guaranteeing precise allocation of the assigned oral rinse. These bottles were freshly prepared and delivered twice daily to the participants in special schools for their use. Additionally, participants were advised to refrain from oral hygiene practices for a minimum of 8 hours and a maximum of 18 hours before the oral examinations. Special care instructors at special schools underwent training on the proper use of oral rinses to effectively guide the children. They monitored the children's adherence to the designated oral rinse twice daily and promptly reported any deviation from the instructions. This protocol was implemented to ensure blindness for both the participants and investigators regarding the specific type of oral rinse administered. At the conclusion of the trial for the oral rinse protocol, ten children (five in the Chlorhexidine group and five in the Probiotic group) were excluded from the study due to non-compliance with the oral rinse protocol. (Fig. 1).

Assessment:

The study evaluated various parameters at four distinct intervals: Baseline (T₀), two weeks post-completion of dental rehabilitation (T₁), two weeks after using the respective oral rinses (T₂) and following a 6-month washout period (T₃). These assessments aimed to ensure homogeneous oral conditions and evaluate the durability of the post-supplementation effects (Fig. 3).

• Assessment of MMP-8 and MMP-9 Levels:

Unstimulated saliva samples (1.5 ml) were collected from children between 8–11 am in a passive dripping position[19]. The samples were collected after a 2-hour fasting period. After collection, the samples were transported to the Central Research Laboratory, KSHEMA, where they were centrifuged at 3000 rpm for 10 min. The supernatant was stored for subsequent analysis using ELISA kits (Krishgen Biosystem, Mumbai, India) to quantify MMP-8 and MMP-9 levels[12, 20].

• Gingival Parameters:

A single examiner calibrated for consistency achieved a high reproducibility score (κ = 0.92).

Gingival health was evaluated using two metrics:

Dental plaque accumulation: The modified Quigley-Hein Plaque Index (PI) with GUM Red-Cote® chewable disclosing tablets was assessed on six surfaces of each tooth, excluding the third molars. Scores ranged from 0 (no plaque) to 5 (plaque covering two-thirds or more of the crown), with PI computed as the average score across all examined teeth.[21]

Gingival Health: Evaluated using the Gingival Index (GI) by Löe and Silness, which assessed inflammation levels on four surfaces of six index teeth (16, 12, 24, 36, 32, and 44). Scores ranged from 0 (absence of inflammation) to 3 (severe inflammation), and GI was calculated as the average score for these teeth[21].

• Oral Hygiene Index:

The Simplified Oral Hygiene Index (OHI-S), developed by Greene and Vermillion, was used for assessment. This method entails inspecting specific teeth in the oral cavity to evaluate the presence of dental plaque and calculus. The index comprises two main components: the Debris Index-Simplified (DI-S) and the Calculus Index-Simplified (CI-S). Each component was graded on a scale ranging from 0 to 3, with 0 indicating no debris or calculus and 3 indicating heavy accumulation. The scores for debris and calculus were then aggregated and averaged to determine the overall OHI-S score for each participant. [21]

• Bleeding on Probing (BOP):

Gingival bleeding was assessed within 30 s of probing to the full pocket depth using a calibrated Marquis CP-12 probe (Hu-Friedy Mfg.Co., Chicago, IL, USA), with a probing force of approximately 0.75 N (76 gram-force) [21].

Statistical analysis:

The data were entered into Microsoft Excel and analyzed using SPSS for Windows, Version 17 (SPSS Inc., Chicago, IL, USA). For within-group comparisons of MMP-8 (pg/ml), the Wilcoxon signed-rank test was used. Additionally, for within-group comparisons of MMP-9 (ng/ml) levels, the paired t-test was used. To assess the statistical significance of differences in PI, GI, OHI-S, and BOP before and after the intervention, Student’s paired t-test was applied. For between-group comparisons of the variables at the 6-month follow-up, an independent t-test was conducted for PI, OHIS, GI, BOP, and MMP-8, while the Mann-Whitney test was used for MMP-9. A significance level of less than 0.05 (p < 0.05) was considered statistically significant throughout the analysis.

Among the 15 participants in each group, 7 (46.7%) in group 1 (probiotic group) and 7 (46.7%) in group 2 (chlorhexidine group) were male. The mean age in Groups 1 and 2 was 10.53 ± 2.70 years and 10.80 ± 2.76 years, respectively. The differences in age and sex distributions between the two study groups were not statistically significant.

Group 1 (probiotic group) showed significant reductions in MMP-8 levels from baseline to post-dental rehabilitation (p = 0.001), 15 days follow-up (p = 0.001), and 6 months follow-up (p = 0.001), as well as beprobiotict-dental rehabilitation and 15 days follow-up (p = 0.001), post-dental rehabilitation and 6 months follow-up (p = 0.000), and 15 days follow-up and 6 months follow-up (p = 0.001). Group 2 (chlorhexidine group) exhibited similar significant reductions at all intervals ( p ≤ 0.001) (Table 2).

Table 2

Within-group Comparison of the Parameters
MMP − 8 (pg/ml)
		Probiotic group (Group 1)			Chlorhexidine group (Group 2)
	Interval	Mean	SD	p-value	Mean	SD	p-value
Pair 1	Baseline (T₀)	1797.57	466.05	0.001*	1746.45	265.44	0.001*
Pair 1	Post dental rehabilitation (T₁)	894.57	231.73	0.001*	923.26	252.70	0.001*
Pair 2	Baseline (T₀)	1797.57	466.05	0.001*	1746.45	265.44	0.001*
Pair 2	15 Days Follow-up (T₂)	241.81	166.41	0.001*	258.32	174.38	0.001*
Pair 3	Baseline (T₀)	1797.57	466.05	0.001*	1746.45	265.44	0.001*
Pair 3	6 Months Follow-up (T₃)	124.7	93.9	0.001*	189.02	152.07	0.001*
Pair 4	Post dental rehabilitation (T₁)	894.57	231.73	0.001*	923.26	252.70	0.001*
Pair 4	15 Days Follow-up (T₂)	241.81	166.41	0.001*	258.32	174.38	0.001*
Pair 5	Post dental rehabilitation (T₁)	894.57	231.73	0.000*	923.26	252.70	0.000*
Pair 5	6 Months Follow-up (T₃)	124.7	93.9	0.000*	189.02	152.07	0.000*
Pair 6	15 Days Follow-up (T₂)	241.81	166.41	0.001*	258.32	174.38	0.001*
Pair 6	6 Months Follow-up (T₃)	124.7	93.9	0.001*	189.02	152.07	0.001*
Wilcoxon signed rank test; indicates a significant difference at p ≤ 0.05*
MMP − 9 (ng/ml)
		Probiotic group (Group 1)			Chlorhexidine group (Group 2)
	Interval	Mean	SD	p-value	Mean	SD	p-value
Pair 1	Baseline (T₀)	14.76	6.17	0.003*	12.03	4.24	0.274
Pair 1	Post dental rehabilitation (T₁)	6.44	4.51	0.003*	8.89	9.12	0.274
Pair 2	Baseline (T₀)	14.76	6.17	0.001*	12.03	4.24	0.001*
Pair 2	15 Days Follow-up (T₂)	1.38	0.89	0.001*	1.78	1.33	0.001*
Pair 3	Baseline (T₀)	14.76	6.17	0.000*	12.03	4.24	0.000*
Pair 3	6 Months Follow-up (T₃)	1.08	0.83	0.000*	1.51	1.1	0.000*
Pair 4	Post dental rehabilitation (T₁)	6.44	4.51	0.001*	8.89	9.12	0.009*
Pair 4	15 Days Follow-up (T₂)	1.38	0.89	0.001*	1.78	1.33	0.009*
Pair 5	Post dental rehabilitation (T₁)	6.44	4.51	0.000*	8.89	9.12	0.000*
Pair 5	6 Months Follow-up (T₃)	1.08	0.83	0.000*	1.51	1.1	0.000*
Pair 6	15 Days Follow-up (T₂)	1.38	0.89	0.001*	1.78	1.33	0.119
Pair 6	6 Months Follow-up (T₃)	1.08	0.83	0.001*	1.51	1.1	0.119
Paired t test; indicates a significant difference at p ≤ 0.05*
Silness and Loe plaque index (PI)
		Probiotic group (Group 1)			Chlorhexidine group (Group 2)
	Interval	Mean	SD	p-value	Mean	SD	p-value
Pair 1	Baseline (T₀)	2.58	0.25	0.001*	2.67	0.24	0.001*
Pair 1	Post dental rehabilitation (T₁)	1.53	0.28	0.001*	1.53	0.28	0.001*
Pair 2	Baseline (T₀)	2.58	0.25	0.001*	2.67	0.24	0.001*
Pair 2	15 Days Follow-up (T₂)	0.45	0.30	0.001*	0.47	0.29	0.001*
Pair 3	Baseline (T₀)	2.58	0.25	0.000*	2.67	0.24	0.000*
Pair 3	6 Months Follow-up (T₃)	0.37	0.31	0.000*	0.42	0.29	0.000*
Pair 4	Post dental rehabilitation (T₁)	1.53	0.28	0.001*	1.53	0.28	0.001*
Pair 4	15 Days Follow-up (T₂)	0.45	0.30	0.001*	0.47	0.29	0.001*
Pair 5	Post dental rehabilitation (T₁)	1.53	0.28	0.000	1.53	0.28	0.000*
Pair 5	6 Months Follow-up (T₃)	0.37	0.31	0.000	0.42	0.29	0.000*
Pair 6	15 Days Follow-up (T₂)	0.45	0.30	0.041	0.47	0.29	0.001*
Pair 6	6 Months Follow-up (T₃)	0.37	0.31	0.041	0.42	0.29	0.001*
Paired t test; indicates a significant difference at p ≤ 0.05*
Gingival index (GI)
		Probiotic group (Group 1)			Chlorhexidine group (Group 2)
	Interval	Mean	SD	p-value	Mean	SD	p-value
Pair 1	Baseline (T₀)	2.29	0.18	0.001*	2.37	0.17	0.001*
Pair 1	Post dental rehabilitation (T₁)	1.28	0.20	0.001*	1.39	0.22	0.001*
Pair 2	Baseline (T₀)	2.29	0.18	0.001*	2.37	0.17	0.001*
Pair 2	15 Days Follow-up (T₂)	0.43	0.24	0.001*	0.43	0.23	0.001*
Pair 3	Baseline (T₀)	2.29	0.19	0.000*	2.37	0.17	0.001*
Pair 3	6 Months Follow-up (T₃)	0.33	0.23	0.000*	0.33	0.21	0.001*
Pair 4	Post dental rehabilitation (T₁)	1.28	0.20	0.001*	1.39	0.22	0.001*
Pair 4	15 Days Follow-up (T₂)	0.43	0.24	0.001*	0.43	0.23	0.001*
Pair 5	Post dental rehabilitation (T₁)	1.28	0.20	0.000*	1.39	0.22	0.000*
Pair 5	6 Months Follow-up (T₃)	0.33	0.23	0.000*	0.33	0.21	0.000*
Pair 6	15 Days Follow-up (T₂)	0.43	0.24	0.001*	0.43	0.23	0.001*
Pair 6	6 Months Follow-up (T₃)	0.32	0.23	0.001*	0.33	0.21	0.001*
Paired t test; indicates a significant difference at p ≤ 0.05*
Oral Hygiene Index Simplified (OHI-S)
		Probiotic group (Group 1)			Chlorhexidine group (Group 2)
	Interval	Mean	SD	p-value	Mean	SD	p-value
Pair 1	Baseline (T₀)	4.33	0.95	0.001*	4.45	0.90	0.001*
Pair 1	Post dental rehabilitation (T₁)	1.17	0.19	0.001*	1.20	0.19	0.001*
Pair 2	Baseline (T₀)	4.33	0.95	0.001*	4.45	0.90	0.001*
Pair 2	15 Days Follow-up (T₂)	0.49	0.22	0.001*	0.50	0.21	0.001*
Pair 3	Baseline (T₀)	4.33	0.95	0.000*	1.20	0.19	0.000*
Pair 3	6 Months Follow-up (T₃)	0.29	0.21	0.000*	0.30	0.2	0.000*
Pair 4	Post dental rehabilitation (T₁)	1.17	0.19	0.001*	1.20	0.19	0.001*
Pair 4	15 Days Follow-up (T₂)	0.49	0.22	0.001*	0.50	0.21	0.001*
Pair 5	Post dental rehabilitation (T₁)	1.17	0.19	0.001*	1.20	0.19	0.001*
Pair 5	6 Months Follow-up (T₃)	0.29	0.21	0.001*	0.30	0.2	0.001*
Pair 6	15 Days Follow-up (T₂)	0.49	0.22	0.041*	0.50	0.21	0.001*
Pair 6	6 Months Follow-up (T₃)	0.29	0.21	0.041*	0.30	0.2	0.001*
Paired t test; indicates a significant difference at p ≤ 0.05*
Bleeding on probing (BOP)
		Probiotic group (Group 1)			Chlorhexidine group (Group 2)
	Interval	Mean	SD	p-value	Mean	SD	p-value
Pair 1	Baseline (T₀)	47.67	4.01	0.001*	50.47	2.48	0.001*
Pair 1	Post dental rehabilitation (T₁)	31.40	6.17	0.001*	31.87	5.62	0.001*
Pair 2	Baseline (T₀)	47.67	4.01	0.001*	50.47	2.48	0.001*
Pair 2	15 Days Follow-up (T₂)	19.00	1.51	0.001*	18.47	1.89	0.001*
Pair 3	Baseline (T₀)	47.67	4.01	0.000*	50.47	2.47	0.000*
Pair 3	6 Months Follow-up (T₃)	13.47	2.72	0.000*	17.6	1.4	0.000*
Pair 4	Post dental rehabilitation (T₁)	31.40	6.17	0.001*	31.87	5.62	0.001*
Pair 4	15 Days Follow-up (T₂)	19.00	1.51	0.001*	18.47	1.89	0.001*
Pair 5	Post dental rehabilitation (T₁)	31.40	6.17	0.000*	31.87	5.62	0.000*
Pair 5	6 Months Follow-up (T₃)	13.47	2.72	0.000*	17.6	1.4	0.000*
Pair 6	15 Days Follow-up (T₂)	19.00	1.51	0.001*	18.47	1.89	0.001*
Pair 6	6 Months Follow-up (T₃)	13.47	2.72	0.001*	17.6	1.4	0.001*
Paired t test; indicates a significant difference at p ≤ 0.05*

Group 1 (Probiotic group) had significant reductions in MMP-9 levels from baseline to post-dental rehabilitation (p = 0.003), 15 days follow-up (p = 0.001), and 6 months follow-up (p = 0.000), as well as between post-dental rehabilitation and 15 days follow-up (p = 0.001), post-dental rehabilitation and 6 months follow-up (p = 0.000), and 15 days follow-up and 6 months follow-up (p = 0.001). In contrast, group 2 (chlorhexidine group) showed significant reductions, except from baseline to post-dental rehabilitation (p = 0.274) and from 15 days follow-up to 6 months follow-up (p = 0.119) (Table 2).

Group 1 (probiotic group) showed significant reductions in the PI at all intervals: baseline to post dental rehabilitation (p = 0.001), 15 days follow-up (p = 0.001), and 6 months follow-up (p = 0.000), as well as between post-dental rehabilitation and 15 days follow-up (p = 0.001), post-dental rehabilitation and 6 months follow-up (p = 0.000), and 15 days follow-up and 6 months follow-up (p = 0.041). Group 2 (chlorhexidine group) also exhibited a significant reduction across all intervals ( p ≤ 0.001) (Table 2).

Both groups demonstrated significant reductions in GI at all intervals: baseline to post-dental rehabilitation (group 1: p = 0.001; group 2: p = 0.001), baseline to 15 days follow-up (group 1: p = 0.001; group 2: p = 0.001), and baseline to 6 months follow-up (group 1: p = 0.000; group 2: p = 0.001). Significant reductions were also observed between post-dental rehabilitation and 15 days follow-up, post-dental rehabilitation and 6 months follow-up, and 15 days follow-up and 6 months follow-up (all p ≤ 0.001 for both groups) (Table 2).

Group 1 (probiotic group) showed significant reductions in OHI-S from baseline to post-dental rehabilitation (p = 0.001), 15 days follow-up (p = 0.001), and 6 months follow-up (p = 0.000), as well as between post-dental rehabilitation and 15 days follow-up (p = 0.001), post-dental rehabilitation and 6 months follow-up (p = 0.001), and 15 days follow-up and 6 months follow-up (p = 0.041). Group 2 (chlorhexidine group) also exhibited significant reduction at all intervals (all p ≤ 0.001) (Table 2).

Both groups showed significant reductions in BOP at all intervals: baseline to post-dental rehabilitation (group 1: p = 0.001; group 2: p = 0.001), baseline to 15 days follow-up (group 1: p = 0.001; group 2: p = 0.001), and baseline to 6 months follow-up (group 1: p = 0.000; group 2: p = 0.000). Reductions were also significant between post-dental rehabilitation and 15 days follow-up, post-dental rehabilitation and 6 months follow-up, and 15 days follow-up and 6 months follow-up (all p ≤ 0.001 for both groups) (Table 2).

At the 6-month follow-up, group 1 (probiotic group) had significantly lower BOP (p = 0.000) than group 2 (chlorhexidine group). No significant differences were observed between the groups for the PI, OHI-S, GI, MMP-8, and MMP-9 levels, indicating that while both interventions were effective in maintaining oral hygiene, Group 1 (probiotic group) was superior in reducing BOP (Table 3).

Table 3

Between Group Comparison of the variables at 6-months follow-up
Variable	Group	Mean	Std. Deviation	Std. Error Mean	p-value
PI	1	0.37	0.3	0.1	0.677^†
PI	2	0.42	0.3	0.1	0.677^†
OHIS	1	0.29	0.2	0.1	0.933^†
OHIS	2	0.30	0.2	0.1	0.933^†
GI	1	0.33	0.2	0.1	1.000^†
GI	2	0.33	0.2	0.1	1.000^†
BOP	1	13.47	2.7	0.7	0.000^†*
BOP	2	17.60	1.4	0.4	0.000^†*
MMP-8	1	125.59	94.0	24.3	0.182^‡
MMP-8	2	189.02	152.1	39.3	0.182^‡
MMP-9	1	1.08	0.8	0.2	0.239^†
MMP-9	2	1.51	1.1	0.3	0.239^†
† Independent t-test; ‡ Mann Whitney test

Children with DS are especially vulnerable to oral diseases because of their distinct immune profiles and the complex orofacial anomalies associated with their genetic condition. Immune dysregulation in these individuals, characterized by alterations in immune cell populations and cytokine dynamics[22, 23], significantly affects oral health by promoting chronic inflammation. Additionally, individuals with DS exhibit elevated levels of MMP – 8 and MMP − 9[12], which are also found in high concentrations in conditions such as dental caries, periodontitis, peri-implantitis, and oral cancer[24, 25]. This parallel highlights the urgent need for targeted interventions to address these specific oral health challenges.

MMPs are enzymatic regulators crucial for tissue remodeling and degradation of extracellular matrix components, such as collagen and proteoglycans[26]. In the context of periodontal degradation, dysregulation of MMPs, notably MMP-8 and MMP-9, significantly contributes to the excessive breakdown of periodontal tissues, thereby exacerbating disease progression[24]. Evidence consistently indicates elevated MMP-8 levels in individuals with periodontitis compared with their periodontally healthy counterparts[27], suggesting its potential utility as a diagnostic biomarker for periodontal disease[28]. Upregulation of MMP expression primarily stems from the inflammatory response and subsequent tissue destruction, which is characteristic of periodontitis. Consequently, targeted therapeutic interventions aimed at modulating MMP dysregulation hold promise in attenuating periodontal tissue breakdown and preserving periodontal health[10].

In the present study, a specialized oral care program was specifically designed to cater to the unique oral health needs of children diagnosed with DS. This programme aimed to address the distinct oral health challenges encountered by this demographic group. Existing literature underscores the significant positive impact of oral health education (OHE) on periodontal health. Research findings suggest that ongoing OHE initiatives have the potential to sustain the long-term benefits of periodontal disease treatment, alleviate gingival inflammation, and enhance periodontal conditions[29]. Additionally, comprehensive full-mouth rehabilitation (FMR) plays a pivotal role in augmenting oral health-related quality of life (OHRQoL) in children with special healthcare needs (CSHCN). Consistent findings from multiple studies highlight the substantial improvements in OHRQoL resulting from FMR interventions, with significant positive changes observed across various quality of life and dental indices, and enduring benefits over time [30–32]. These findings underscore the critical role of tailored oral care interventions, exemplified by the program implemented in our study, in promoting optimal oral health outcomes for vulnerable populations, particularly for children diagnosed with DS.

Oral rehabilitation, specifically through non-surgical periodontal therapy such as scaling and root planing, aims to restore periodontal health while exerting a broader impact on oral inflammatory markers. Following such interventions, a discernible reduction in salivary MMPs was noted, indicating the efficacy of the treatment in reducing periodontal inflammation and tissue degradation[20]. Meschiari et al. (2013) highlighted this phenomenon, reporting a significant decrease in salivary MMP-8 and tissue inhibitor of metalloproteinase-2 (TIMP-2) concentrations, as well as gelatinolytic activity, following non-surgical periodontal therapy[33]. These findings underscore the potential of oral rehabilitation procedures to not only address localized periodontal issues, but also exert systemic effects by modulating inflammatory markers in saliva associated with periodontal disease.

After oral rehabilitation, the children were assigned to two groups: group 1 received a probiotic oral rinse, and group 2 received chlorhexidine oral rinse. Probiotics offer multiple oral health benefits including plaque reduction, immune system modulation, and inhibition of periodontal pathogens. Certain strains, such as Streptococcus thermophilus and Lactococcus lactis, inhibit plaque formation by adhering to saliva-coated hydroxyapatite beads. They also stabilize the oral microbiota, prevent bacterial re-colonization, and reduce inflammation and plaque accumulation. Specific Lactobacillus strains target periodontitis-causing pathogens, thereby improving periodontal health. Additionally, probiotics enhance traditional treatments, such as scaling and root planing, and help prevent antibiotic resistance. Therefore, incorporating probiotics into periodontal care holds promise in enhancing oral health outcomes[34].

Probiotics can also potentially influence salivary MMPs indirectly via their effects on the immune system and inflammation. They are known to modulate gut microbiota and immune responses, affecting cytokines and growth factors that regulate MMP expression and activity[35, 36]. Since MMPs are involved in tissue remodeling and inflammation, processes that are influenced by the gut microbiota and its metabolic products[37], it is plausible that probiotics may indirectly impact MMP activity through their immunomodulatory effects. Further studies are required to elucidate these mechanisms.

In contrast, chlorhexidine improves plaque and periodontal health through its potent antibacterial properties, offering both immediate bactericidal and long-term bacteriostatic effects by adsorption onto the enamel surface[38]. Clinical trials have demonstrated that chlorhexidine mouthwashes effectively reduce dental plaque and enhance periodontal health indices such as PI, GI, and BOP [39, 40]. Despite potential side effects, such as tooth staining and oral mucosal erosion, chlorhexidine remains the gold standard in dental care owing to its substantivity and broad antimicrobial spectrum[41]. Furthermore, Chlorhexidine (CHX) inhibits MMPs by binding to calcium and zinc ions, which are essential for MMP enzymatic activity[42].

The present study revealed promising results, demonstrating that both chlorhexidine and probiotic oral rinses significantly reduced MMP-8 and MMP-9 levels over a 6-month period. Notably, the two-week usage of both oral rinses showed comparable effectiveness, suggesting that probiotics could serve as a viable alternative with similar efficacy, considering the potential gastrointestinal symptoms associated with chlorhexidine ingestion and compromised gut function in individuals with DS[43]. Additionally, it was observed that Group 1 (probiotic group) exhibited a superior reduction in BOP, indicating a more favorable outcome in terms of periodontal health compared to Group 2 (chlorhexidine group).

The probiotic oral rinse proposed for children with DS has several advantages over chlorhexidine. It promotes a balanced oral microbiome by fostering beneficial bacteria without disrupting existing microbial communities, unlike chlorhexidine which can cause tooth staining, altered taste, and mucosal irritation[41]. Additionally, probiotics are associated with minimal side effects and offer immunomodulatory benefits, potentially reducing inflammation, and enhancing oral health. The formulation is sugar-free and microencapsulated, ensuring controlled release of active ingredients that improve stability and efficacy compared to conventional rinses [44–46]. This innovative approach not only enhances safety but also increases user compliance owing to its pleasant usage experience, thereby promising better long-term oral care outcomes for children with DS compared to traditional chlorhexidine treatments.

Our findings contradict those of Jäsberg et al., who demonstrated that probiotic intervention increased rather than decreased salivary MMP-9 levels in healthy adults [16, 17]. However, these changes were associated with improved gingival health, suggesting that probiotics may exert beneficial immunomodulatory effects in the oral cavity. In contrast, Twetman et al. found no influence of probiotic supplements containing L. reuteri on the concentrations of selected matrix metalloproteinases and interferons in mucosal wounds within one week after a standardized punch biopsy[47].

The present study observed a significant dropout rate for several reasons. First, many parents were unwilling to participate because such an intervention had never been performed in children with DS, and they were concerned about the consequences of accidental ingestion of oral rinses, particularly chlorhexidine. Second, many of the children did not meet the inclusion criteria because of systemic or metabolic problems. Another hurdle was the extensive oral care requirements of the children, many of whom had severe oral health issues that necessitated multiple treatments in the dental operatory, with some requiring procedures under general anesthesia. During this process, many children could not complete their oral rehabilitation due to logistical, transportation, or health issues during appointments. Compliance with performing the oral rinse was particularly challenging for children with DS because of their intellectual disability and taste preferences. This issue was notably more significant in the chlorhexidine group, as most patients showed taste aversion to chlorhexidine, whereas they preferred the taste of probiotic oral rinse. Finally, collecting salivary samples was difficult because children with intellectual disabilities found it challenging to maintain a passive dripping position and often swallowed their saliva, making the collection process extremely challenging.

However, this study has several limitations that must be acknowledged. The exclusion of children with moderate-to-severe intellectual disabilities could affect the generalizability of the findings[48]. Reliance on salivary samples limits the comprehensive assessment of MMP forms and individual variations in response to oral rehabilitation and rinses introduce potential variability in study outcomes[49]. Moreover, concerns regarding the potential of probiotics to induce demineralization highlight the need for further research and caution regarding their application[50].

Future research endeavors should focus on validating these findings in larger cohorts, exploring additional biomarkers, and assessing long-term effects and cost-effectiveness. Additionally, safety and tolerability assessments, evaluation of quality of life, adherence, behavioral changes, and cultural factors are essential for holistic care approaches tailored to the unique needs of children with DS.

Funding Information:

This study received financial support from the NU Faculty Short-term Research Grant by NITTE (Deemed to be University), sanctioned under order number N/RG/NUFR2/ABSMIDS/2022/05. We extend our sincere thanks to Cipla Ltd. and ICPA Health Products Limited for providing Activkids UnoBiotics Junior Probiotics Sachet and Hexidine Mouth Wash, respectively, as a generous contribution towards promoting oral care research for children with Down syndrome.

Author Contribution

S.S, M.R, K.R, S.K.N, P.P.N1 conceived the ideas; S.S, P.B, S.K.P, C.V.D collected the data; P.P.N2 and A.D.S analysed the data; S.S, M.R, K.R, P.P.N1 led the writing.

Acknowledgement

We extend our heartfelt gratitude to the postgraduates, teaching and non-teaching faculty of A B Shetty Memorial Institute of Dental Sciences, NITTE (Deemed to be University), Deralakatte, Mangaluru for their unwavering support and encouragement. We are also deeply thankful to Dr. Amarshree A Shetty, President of NITTE-Special Child Care Centre (N-SPECC), and Dr. Shanthi Clara Dsouza, N-SPECC coordinator, for their invaluable support and guidance through N-SPECC. Furthermore, we sincerely appreciate the patients and parents of the children with Down syndrome for their active participation in the study.

CONFLICTS OF INTEREST:

The authors declare that they have no conflict of interest.

Data Availability Statement:

The data supporting the findings of this study are available upon request from the corresponding author.

Ethics Statement:

The Ethics Committee of the AB Shetty Memorial Institute of Dental Sciences, Mangalore, approved this case report (ETHICS/ABSMIDS/333/2023).

Permission To Reproduce Material From Other Sources:

No copyrighted material was used to create this study. All content was original or used with proper authorisation, and no permissions for reproduction were required.

Patient Consent

The legal guardians of the patients provided consent for their participation in the study, and the patients provided assent.

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No competing interests reported.

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A Novel Microencapsulated Probiotic Oral Rinse Improves Gingival Health and Influences Salivary Matrix Metalloproteinases 8 and 9 in Children with Down Syndrome: A Pilot Randomized Clinical Trial

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Introduction

Methodology

Trial design

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Participants

Randomization and Allocation

Interventions

Assessment:

• Assessment of MMP-8 and MMP-9 Levels:

• Gingival Parameters:

• Oral Hygiene Index:

• Bleeding on Probing (BOP):

Statistical analysis:

Results

Discussion

Declarations

Funding Information:

Author Contribution

Acknowledgement

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