Effect of Bifidobacterium Probiotic Supplementation on Interleukin‐6 Levels and Osteoclast Count in Wistar Rats During Orthodontic Tooth Movement

ABSTRACT

Background

Probiotics may offer benefits in mitigating bone loss and related conditions. Bone resorption is essential for bone remodelling, particularly during orthodontic tooth movement induced by mechanical forces. This study investigates the effects of Bifidobacterium probiotic supplementation on interleukin‐6 (IL‐6) levels and osteoclast counts.

Methods and Materials

This study involved 48 healthy male Wistar rats, randomly assigned to six groups: control groups at 3 days (C3), 7 days (C7) and 14 days (C14), and probiotic groups at 3 days (P3), 7 days (P7) and 14 days (P14). The probiotic group received a daily gavage of Bifidobacterium at a dose of 1.5 × 10⁸ CFU/g. Orthodontic tooth movement was induced using a coil spring applying a force of 20 g. Osteoclast counts were assessed via light microscopy, while IL‐6 levels were measured using the ELISA method.

Results

Probiotic supplementation resulted in a significant reduction in IL‐6 levels and osteoclast counts compared to the control group. Peak IL‐6 levels and osteoclast counts in the control group were observed on Day 14, whereas the lowest levels were recorded in the probiotic group on the same day.

Conclusion

Despite certain limitations, this study suggests that Bifidobacterium animalis subsp. lactis supplementation effectively reduces IL‐6 levels and osteoclast counts during orthodontic tooth movement over a 14‐day period when compared to controls.

This study evaluates the effect of Bifidobacterium probiotic supplementation on interleukin‐6 (IL‐6) levels and osteoclast counts during orthodontic tooth movement in Wistar rats. Probiotic treatment significantly reduced IL‐6, an inflammatory cytokine involved in bone remodelling, and decreased osteoclast numbers associated with bone resorption. These findings suggest the potential of probiotics to modulate inflammation and bone metabolism that should be considered during orthodontic treatments.

1. Introduction

Probiotics are live microorganisms that provide health benefits to the host when consumed in adequate amounts. Primarily residing in the gut, they positively influence the microbiota, intestinal barrier function and immune system, resulting in systemic effects including improved bone health. Probiotics support bone growth, density and structure, particularly in conditions associated with gut dysbiosis, leaky gut syndrome and inflammation—factors known to contribute to bone loss and osteoporosis (McCabe and Parameswaran 2018).

The beneficial impact of probiotics on bone health is mediated through several mechanisms. Probiotics enhance mineral solubility by producing short‐chain fatty acids (SCFAs), which improve mineral absorption. Additionally, they produce phytase enzymes that reduce the inhibitory effects of phytate on mineral uptake. Probiotics also contribute to decreasing intestinal inflammation, a known factor that can increase bone mineral density by reducing inflammatory mediators. Furthermore, species such as Lactobacillus and Bifidobacterium improve the bioavailability of essential minerals by breaking down glycosidic bonds in foods, facilitating better nutrient assimilation (Parvaneh et al. 2014).

In orthodontics, mechanical forces applied to teeth induce an inflammatory response in the periodontal ligament (PDL) and alveolar bone. Cytokines such as interleukin‐6 (IL‐6) play a pivotal role in this process, being associated with increased osteoclast activity and subsequent bone resorption (Afshar et al. 2020; Karimi‐Afshar et al. 2021). Elevated IL‐6 levels have been observed in orthodontic patients and correlate with enhanced tooth movement (Jayaprakash et al. 2019).

Probiotics may modulate immune responses by reducing pro‐inflammatory cytokines and promoting anti‐inflammatory cytokines, potentially mitigating chronic inflammation (Duffles et al. 2022; Maslowski et al. 2009). Recent studies suggest that specific probiotic strains benefit oral health, especially in orthodontic patients, by decreasing cariogenic and periodontal pathogens (Mazziotta et al. 2023). For example, Duffles et al. (2022) reported changes in microbial parameters after 33 days of probiotic treatment; however, no significant changes were observed in bone architecture. While Bifidobacterium lactis positively influences bone remodelling, it does not disrupt overall bone homeostasis. Notably, osteoclast counts in probiotic‐treated animals were similar to controls, yet orthodontic tooth movement was significantly inhibited by 62% in the probiotic group (Duffles et al. 2022).

Furthermore, Triwardhani et al. (2021) investigated the effects of Bifidobacterium bifidum on heat shock protein 70 (HSP70) levels and osteoclast numbers during tooth movement in rats. On Day 14, in the compression area, the probiotic group exhibited lower HSP70 levels and fewer osteoclasts compared to controls (Triwardhani et al. 2021).

Despite growing interest in probiotics for oral health management, further research is necessary to clarify their effects on inflammatory factors influencing tooth movement, including osteoclast counts in orthodontic patients. Given the limited number of studies on this topic, the present study aims to investigate the effects of probiotic supplementation on IL‐6 levels and osteoclast numbers during orthodontic tooth movement in Wistar rats.

2. Methods and Materials

This experimental animal study involved 48 healthy male Wistar rats aged 16–20 weeks, weighing between 200 and 250 g. The sample size was calculated using G*Power software with an alpha level of 0.05, power of 80% and effect size of 0.25, resulting in eight rats per group. The rats were housed in polycarbonate cages in groups at the Laboratory Animal House of the Veterinary Faculty, Shahid Bahonar University of Kerman, Iran. They had ad libitum access to water and standard commercial laboratory chow (pellet form; Javaneh Khorasan Co., Mashhad, Iran). The ambient temperature was maintained at 25°C with 50% humidity, under a 12‐h light–dark cycle. Body weights were monitored throughout the study, and any rat losing more than 15% of its initial weight was excluded from the analysis.

After a 1‐week acclimatization period, the rats were randomly assigned into six groups: control groups at 3 days (C3), 7 days (C7) and 14 days (C14), and probiotic groups at 3 days (P3), 7 days (P7) and 14 days (P14). The probiotic groups received a daily oral gavage of Bifidobacterium animalis subsp. lactis, prepared by dissolving 1 g of probiotic powder in 10 mL of drinking water, containing 1.5 × 10⁸ CFU/g bacteria. Administration was performed daily at noon for 33 days, starting 21 days before coil spring installation and continuing until 14 days after force application (Duffles et al. 2022).

Orthodontic tooth movement was induced using a coil spring measuring 10 × 13 mm, fabricated from 0.012‐inch wire (G & H Orthodontics). The spring was designed to exert a constant force of 20 g when its internal components were in contact (Figure 1).

FIGURE 1.

The inactive form of the orthodontic appliance. Orthodontic coil spring measuring 10  × 13 mm, fabricated from 0.012‐in. wire. The spring is shown on a millimetre grid to illustrate its size and dimensions precisely.

Rats were sedated with an intraperitoneal injection of ketamine and xylazine (0.2 mL per rat). The coil spring was bonded to the maxillary incisors using light‐cured orthodontic adhesive (Sci‐Pharm CuRAY‐ECLIPSE). According to the protocol, no reactivation of the appliance was performed during the experiment (Figures 2 and 3).

FIGURE 2.

Installation of the orthodontic appliance. The coil spring bonded to the maxillary incisors using light‐cured orthodontic adhesive. The image demonstrates the clinical setup of the appliance with no reactivation during the experiment.

FIGURE 3.

Tooth movement observed on Day 14.

To prevent appliance debonding, powdered rodent food was provided. Despite this, five rats experienced debonding and were replaced. Additionally, seven rats died during the study and were substituted with new animals. Previous research suggests that a 14‐day period is optimal for evaluating tooth movement (Duffles et al. 2022).

Blood samples were collected directly from the hearts of rats on Days 3, 7 and 14 post mechanical loading to measure IL‐6 levels. After blood collection, rats were euthanized using a lethal dose of ketamine and xylazine (0.1 mg/kg). Maxillae, including incisors, were fixed in 10% neutral buffered formalin for 48 h, followed by decalcification in 14% EDTA (pH 7.4). Samples were embedded in paraffin for 21 days according to standard protocols. Sagittal sections (5 µm thick) were prepared from paraffin blocks containing incisor teeth and alveolar bone. Sections were stained by von Kossa method. Osteoclast numbers were counted under light microscopy in five consecutive microscopic fields across five sections per animal (total 25 fields), specifically focusing on the distal alveolar bone adjacent to the right incisor. Counting was performed by a trained observer blinded to the experimental groups. Mean osteoclast counts per animal were calculated. Statistical analysis was performed to compare counts between groups.

IL‐6 blood levels were quantified by ELISA. Blood samples were centrifuged at 6000 rpm for 4 min, and serum was distributed into wells coated with IL‐6 antibody according to the kit instructions. Absorbance was read at 450 nm to determine IL‐6 concentration.

3. Data Analysis

Data were analysed using SPSS version 26. Statistical comparisons were performed using t‐tests and ANOVA, with a significance level set at p < 0.05.

4. Results

The changes in interleukin‐6 in the control and probiotic groups, as well as the comparison of interleukin‐6 changes between the two groups, are presented in Tables 1, 2, 3. The changes in osteoclast numbers in the control group on days 3, 7, and 14 are presented in Table 4.

TABLE 1.

Mean interleukin‐6 levels in the control group at 3, 7 and 14 days after orthodontic force application.

IL‐6 Studied group	Mean (pg/µL)	Standard deviation	p‐value
C3	61.45	7.96	0.006 a
C7	70.43	7.06
C14	84.16	8.36

^aA statistically significant increasing trend in IL‐6 levels over time was observed in the control group.

TABLE 2.

Mean interleukin‐6 levels in the probiotic group at 3, 7 and 14 days after orthodontic force application.

IL‐6 Studied group	Mean (pg/µL)	Standard deviation	p‐value
P3	56.41	4.05	0.031 a
P7	48.37	5.68
P14	42.18	6.65

^aA significant decreasing trend in IL‐6 levels over time was observed in this group.

TABLE 3.

Comparison of interleukin‐6 levels (pg/µL) in the control and probiotic groups at 3, 7 and 14 days post‐force application.

IL‐6Studied group	Probiotic group (mean ± SD)	Control group (mean ± SD)	p‐value
Day 3	56.41 ± 4.05	61.45 ± 7.96	0.0001 a
Day 7	48.37 ± 5.68	70.43 ± 7.06
Day 14	42.18 ± 6.65	84.16 ± 8.36

^aSignificant differences were observed in all comparisons.

TABLE 4.

Average number of osteoclasts in the control group on Days 3, 7 and 14 following force application.

Osteoclasts number Studied group	Mean	Standard deviation	p‐value
C3	16.62	2.30	0.015 a
C7	19.32	3.06
C14	23.87	2.83

^aA consistent increase in osteoclast numbers over time was observed in the control group, with statistically significant differences.

Osteoclast counts in the alveolar bone adjacent to the right incisor were quantified using light microscopy following von Kossa staining. In the probiotic group, the average number of osteoclasts decreased over time after force application (Table 5).

TABLE 5.

Average number of osteoclasts in the probiotic group on Days 3, 7 and 14 following force application.

Osteoclasts number Studied group	Mean	Standard deviation	p‐value
P3	12.52	2.41	0.027 a
P7	9.87	2.95
P14	7.75	1.34

^aA decreasing trend in osteoclast numbers was observed over time in the probiotic group, with statistically significant differences.

The comparison of osteoclast numbers between the two groups at different time points after force application is presented in Table 6.

TABLE 6.

Comparison of osteoclast numbers in the control and probiotic groups at 3, 7 and 14 days post‐force application.

Osteoclasts number Studied group	Probiotic group (mean ± SD)	Control group (mean ± SD)	p‐value
Day 3	12.52 ± 2.41	16.62 ± 2.30	0.0001 a
Day 7	9.87 ± 2.95	19.32 ± 3.06
Day 14	7.75 ± 1.34	23.87 ± 2.83

^aPairwise comparisons revealed significant differences between all groups.

5. Discussion

The present study demonstrated a significant rise in mean IL‐6 levels, a key pro‐inflammatory cytokine, during orthodontic force application and tooth movement in Wistar rats within the control group. Conversely, the probiotic group manifested a notable attenuation in IL‐6 levels, exhibiting significantly lower concentrations compared to controls. Notably, IL‐6 reached its peak on Day 14 in the control group, whereas the probiotic group exhibited the lowest levels on the same day, indicating a potential immunomodulatory role of probiotics through downregulation of pro‐inflammatory cytokines (Seidel et al. 2022; Toyama et al. 2023). Specifically, probiotic intake substantially suppressed IL‐6 expression (Gao et al. 2022; Zhou et al. 2023).

These findings align with previous reports documenting probiotics' capacity to mitigate inflammatory processes. Triwardhani et al. (2021) demonstrated that Bifidobacterium bifidum significantly reduced HSP‐70 levels, a biomarker linked to inflammation triggered by oxidative stress in the PDL during orthodontic force application. Similarly, Duffles et al. (2022) observed increased production of SCFAs in probiotic‐treated groups, which inhibit osteoclast differentiation and bone resorption. Furthermore, probiotics were shown to enhance fibroblast growth factor expression, critical for osteoblast function and bone remodelling (Triwardhani et al. 2021).

Our study also revealed an elevation in osteoclast numbers in the control group and a decrease in the probiotic group during orthodontic tooth movement. The probiotic administration resulted in a significant decline in osteoclast count compared to controls, with the highest osteoclast numbers on Day 14 in the control group and the lowest in the probiotic group. This concurs with Pazzini et al.'s (2017) findings, which reported decreased osteoclast numbers following probiotic administration during orthodontic treatment. Despite the reduction in osteoclastic activity, bone resorption and tooth movement persisted, with comparable rates of movement observed between groups. Triwardhani et al.'s (2021) findings reinforce these observations, showing fewer osteoclasts in the probiotic‐treated compression area. Likewise, Parvaneh et al. (2015) noted increased osteoblasts coupled with decreased osteoclast numbers following probiotic supplementation in rats. However, Duffles et al. (2022) found no significant difference in osteoclast counts between groups, possibly due to methodological differences such as the use of TRAP staining versus von Kossa staining in our study.

While probiotics are broadly recognized for their health‐promoting properties, our findings suggest that probiotic supplementation may modulate orthodontic treatment outcomes by reducing IL‐6 levels and osteoclast activity. Clinicians should interpret these results with caution, and further research is warranted to explore the effects of different probiotic strains, dosages and treatment durations on orthodontic tooth movement.

Despite promising findings regarding the immunomodulatory and bone metabolism effects of probiotics, research specifically examining their influence on orthodontic tooth movement remains scarce. Thus, further well‐designed studies are warranted to elucidate the mechanisms involved and to determine optimal probiotic strains and treatment regimens in orthodontic practice.

6. Limitations

The following are the limitations:

1. The relatively small sample size may limit the generalizability of our findings.

2. The exclusive focus on Bifidobacterium strain limits insights into other potentially effective probiotic strains or combinations.

3. The 14‐day observation period restricts understanding of long‐term effects of probiotic supplementation on bone remodelling.

7. Suggestions for Future Research

Future studies should examine varying probiotic doses and combinations to optimize orthodontic treatment outcomes. Longitudinal research is needed to assess the clinical significance of probiotic‐induced modulation of tooth movement and bone remodelling. Investigations into the mechanisms underlying probiotic effects on inflammatory and bone remodelling pathways would further enhance clinical applications.

8. Conclusion

Within the constraints of this study, Bifidobacterium probiotic supplementation significantly reduced IL‐6 levels and osteoclast numbers during orthodontic tooth movement compared to controls over a 14‐day period. These findings highlight the potential of probiotics to modulate inflammatory and bone remodelling responses in orthodontics. Further comprehensive studies are essential to validate and expand upon these results.

Author Contributions

Data curation, investigation, writing – original draft and project administration: Somayeh Sarani. Conceptualization and methodology, funding acquisition and writing – review and editing: Marzieh Karimi Afshar. Software, validation and formal analysis: Amir Saeed Samimi. Supervision, visualization and resources: Mehrnaz Karimi Afshar.

Funding

The authors have nothing to report.

Ethics Statement

All procedures complied with the Guiding Principles for the Care and Use of Research Animals. The study protocol was reviewed and approved by the Ethics Committee of Kerman University of Medical Sciences (Ethics code: IR.KMU.REC.1401.196).

Conflicts of Interest

The authors declare no conflicts of interest.

Sarani, S. , Afshar M. K., Samimi A. S., and Afshar M. K.. 2026. “Effect of Bifidobacterium Probiotic Supplementation on Interleukin‐6 Levels and Osteoclast Count in Wistar Rats During Orthodontic Tooth Movement.” Veterinary Medicine and Science 12, no. 2: e70848. 10.1002/vms3.70848

Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the article and its Supporting Information.