Introduction
Adult orthodontic patients are often exposed to various stressors, which can affect orthodontic treatment. Therefore, this study investigated the effects of chronic restraint stress on orthodontic tooth movement and alveolar bone remodeling in vivo.
Methods
Ten 8-week-old male Wistar rats were randomly divided into sham-stress orthodontic (CO) and stress orthodontic (SO) groups. Restraint stress was applied for 21 days. The orthodontic intervention involved mesial traction of the maxillary first molar from days 8 to 21. Serum inflammatory cytokine levels were measured, and micro-computed tomography scanning was performed to analyze tooth movement and alveolar bone parameters of the first molar. Osteogenic and osteoclastic activities and macrophage polarization in periodontal tissues were assessed by histological and immunohistochemical staining.
Results
Tooth movement was significantly greater in the SO group than in the CO group, as were the serum interleukin-1β and-10 levels. The SO group had increased trabecular spacing, reduced bone density, and wider periodontal ligament spaces on the force-applied side of the alveolar bone. Enhanced osteogenic and osteoclastic activities were observed in both groups under orthodontic force, but significantly more osteoclasts were observed in the SO group than in the CO group. The inducible nitric oxide synthase to arginase 1 expression ratio was also significantly higher in the SO group than in the CO group.
Conclusions
Restraint stress may exacerbate orthodontic tooth movement and alveolar bone resorption, potentially mediated by systemic inflammatory responses, as well as enhance classically activated macrophage polarization in the alveolar bone.
Highlights
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Stress worsens alveolar bone resorption.
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Stress raises systemic inflammation.
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Stress shifts alveolar bone macrophages to proinflammatory phenotype.
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Histopathology shows severe root resorption lacunae and widened periodontal ligament spaces.
Orthodontic tooth movement (OTM) is biologically rooted in periodontal tissue remodeling. As a highly adaptable hard tissue, alveolar bone undergoes adaptive remodeling under orthodontic forces. The stress applied to the teeth is transmitted to the surrounding periodontal tissues, leading to bone formation in the tension zones and bone resorption in the compression zones, enabling tooth movement. Advancements in orthodontic techniques and evolving patient demographics have resulted in a considerably higher proportion of adult orthodontic patients. In today’s competitive and high-pressure societal environment, stressors, such as academic, occupational, lifestyle, and emotional challenges, increasingly affect orthodontic patients. Therefore, the effects of stress on the efficacy of orthodontic treatment and tooth movement have become critical areas of concern.
Stress refers to a nonspecific adaptive response of the body to internal, external, or psychosocial stimuli. Its systemic effects are extensive and complex, potentially modulating multiple biological processes, including alveolar bone remodeling under mechanical loading. Clinical studies have suggested that maternal emotional support significantly correlates with favorable orthodontic outcomes in stress-exposed patients. Experimental evidence further highlights stress-mediated alterations in bone remodeling. Gameiro et al , demonstrated that chronic restraint stress increases OTM and osteoclast numbers on the compression side of rat molars under identical force conditions. Conversely, Mirzakouchaki et al reported that 28-day crowding stress reduced OTM and osteoclast counts in rats, whereas Vandevska-Radunovic and Murison observed diminished tooth movement in rats subjected to daily foot shock stress. These discrepancies may stem from variations in stress paradigms (eg, type and duration). Nevertheless, cumulative evidence has confirmed that stress influences alveolar bone remodeling during orthodontic treatment, primarily by modulating osteoclast quantity and activity.
Bone homeostasis relies on the dynamic balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. T cells, B cells, cytokines, and immune signaling regulate osteoclast differentiation from monocyte-macrophage precursors. Notably, the gut microbiota has been implicated in enhancing osteoclast precursor populations in the bone marrow and modulating osteoclastogenesis via immune pathways involving activated T cells. Alterations in the gut microbiota composition may promote immune cell infiltration (eg, B cells and neutrophils) and dysregulate inflammatory mediators (eg, tumor necrosis factor-α [TNF-α], interleukin [IL]-1β, and IL-10), which have been linked to osteoclast precursor recruitment and differentiation in vitro. These mechanisms suggest a potential interplay between microbiota, immune responses, and osteoclast activity.
We previously established chronic restraint stress and OTM rat models, finding that both interventions adversely affected body weight, psychological status, colonic epithelial integrity, barrier function, the gut microbiota composition, and metabolites, with possible additive effects. Building on this foundation, the current study investigated the effects of chronic stress and orthodontic intervention on alveolar bone remodeling and OTM. By elucidating the systemic consequences of chronic stress during orthodontic treatment, this study aimed to advance the clinical strategies for managing stress-exposed orthodontic patients.
Material And Methods
Ten 8-week-old male, specific pathogen-free grade Wistar rats (200 ± 10 g) were housed with the same basal diet and water. After 1 week of acclimatization, the rats were randomly divided into SO (stress intervention group, n = 5) and CO (sham stress control group, n = 5) groups.
The stress or sham stress treatment began on day 0 (D0) and continued for 21 days (D21), as previously described ( Fig 1 , A and B ). On D8, all rats underwent unilateral orthodontic treatment to move the maxillary first molar mesially, as previously described ( Fig 1 , C ). , All rats were sacrificed on D21 for sample collection. The specimens from the orthodontic-force and nonorthodontic-force sides of the CO group were labeled CO1 and CO2, respectively. Similarly, the specimens from the orthodontic-force and nonorthodontic-force sides of the SO group were labeled SO1 and SO2, respectively. Our experiment complied with the Animal Research: Reporting In Vivo Experiments guidelines and was carried out in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals. Our institution’s animal ethics committee approved the animal experiments and related procedures (No. PZ2023038).
A, Experimental timeline; B, Restraint stress intervention: rats were placed in sterilized polyethylene glycol terephthalate cylindrical tubes with air holes for ventilation; C, Orthodontic treatment: mesial traction of unilateral maxillary first molar with initial force of 40 g.
On D21, after anesthesia, the rats were euthanized by cervical dislocation, and blood was collected from the abdominal aorta. The blood samples were incubated at room temperature for 2 hours and then centrifuged at 3000 rpm for 15 minutes at 4°C. The supernatant was collected, and enzyme-linked immunosorbent assays were performed according to the kit instructions to quantitatively detect the serum expression levels of 4 inflammatory cytokines (IL-1β, IL-10, IL-6, and interferon-γ [IFN-γ]). A standard curve was plotted based on the concentrations and optical density values of the standards, and the sample concentrations were calculated using the standard curve equation.
After euthanasia on D21, the bilateral maxillae containing complete dentition were carefully dissected and fixed in 4% paraformaldehyde for 48 hours. The specimens were placed in a micro-computed tomography (micro-CT) scanning tube and fixed onto the stage of a micro-CT scanner (Scanco Medical, Wangen-Brüttisellen, Switzerland). The scanning parameters were as follows: voltage 90 kV, current 50 μA, 450 projections, and resolution 10 μm. The scanned maxillae and maxillary dentition were reconstructed and analyzed using VG Studio Max software (Volume Graphics, Heidelberg, Germany).
Planes A and B were measured in a plane parallel to the long axis of the first molar and perpendicular to the line connecting the central fossae of the first and second molars and the occlusal plane of the first and second molars. Plane A was tangential to the distal contact point of the first molar at point a, and plane B was tangential to the mesial contact point of the second molar at point b. The horizontal distance between planes A and B was measured as the mesial movement of the maxillary first molars ( Fig 2 , A ).
Schematic diagram of the micro-CT measurements: A, First molar movement; B, Region of interest in the coronal plane; C-F, Region of interest selection in the (C) mesial root (cross-section), (D) mesial root (sagittal section), (E) distal root (cross-section), and (F) distal root (sagittal section) ( a , distal contact point of the first molar; b , mesial contact point of the second molar; red , distal buccal root of the first molar; blue , region of interest).
Micro-CT scan data were used for periodontal bone analyses. In the mesial and distal regions of the distal buccal root of the first molar, a 0.5 × 0.2 × 1.5 mm cubic region of interest was selected above the apical plane for the measurement and analysis. Then, the bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (TbTh), trabecular separation (TbSp), and trabecular number (TbN) in this region were calculated ( Fig 2 , B-F ).
After fixation, the maxillary specimens were washed and placed in 10% ethylenediaminetetraacetic acid decalcification solution for 4-5 weeks at 37°C. The specimens were washed, dehydrated, and embedded in paraffin. Within the range of the maxillary first and second molars, the tissue blocks were sectioned parallel to the long axis of the first molar and the line connecting the central fossae of the first and second molars, and perpendicular to the occlusal plane of the first and second molars, to obtain 3-μm–thick continuous sections.
Maxillary tissue sections were stained with hematoxylin and eosin to observe the morphological changes and inflammatory cell infiltration in the distal buccal root of the maxillary first molar, periodontal ligament, and surrounding alveolar bone.
The maxillary tissue sections were subjected to immunohistochemical staining. After routine antigen retrieval, blocking of endogenous peroxidase, serum blocking, and incubation with primary and secondary antibodies, the sections were developed with 3,3’-diaminobenzidine and counterstained with hematoxylin to detect the osteocalcin (OCN), cathepsin K (CTSK), arginase 1 (Arg-1), and inducible nitric oxide synthase (iNOS) expression in the alveolar bone around the mesial and distal regions of the distal buccal root of the maxillary first molar. The following primary antibodies were used: OCN (GB11233, 1:100; Servicebio, Wuhan, China), CTSK (GB111276, 1:2000, Servicebio), Arg-1 (GB11285, 1:1000; Servicebio), and iNOS (GB11119, 1:100; Servicebio). The stained sections were scanned using a digital scanner to capture images. Positive cell counts were statistically analyzed for CTSK-stained sections, and semiquantitative analyses of the percentage of positively stained areas were performed for other indicators. The measurement regions selected were 0.2 × 1.5 mm rectangular areas in the alveolar bone above the apical plane of the mesial and distal roots of the first molar, consistent with the region of interest selection in micro-CT ( Fig 2 , D and F ).
Statistical analysis
Experimental data are expressed as means ± standard deviations. Statistical analyses and graphing were performed using GraphPad Prism (version 8.0; GraphPad Software, La Jolla, Calif). Comparisons between 2 groups were performed using independent-samples t tests, and comparisons among multiple groups were analyzed by one-way analysis of variance and Tukey honest significant difference test. Statistical significance was set at P <0.05.
Results
The mesial movement of the first molars was significantly greater in the SO group than in the CO group ( Fig 3 , A ; Table I ).
Micro-CT analyses of the alveolar bone: A, First molar movement (independent-samples t test); B, BMD (one-way ANOVA); C, BV/TV (one-way ANOVA); D, TbTh (one-way ANOVA); E, TbSp (one-way ANOVA); F, TbN (one-way ANOVA). CO1, orthodontic-force side of sham stress intervention group; CO2 , nonorthodontic-force side of sham stress intervention group; SO1, orthodontic-force side of stress intervention group; SO2 , nonorthodontic-force side of stress intervention group. Error bars represent standard deviation; ∗ P <0.05.
Table 1
Results of micro-CT analyses
| Tooth movement (mm) | CO | SO | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Means | Standard deviations | Means | Standard deviations | ||||||
| 0.22 | 0.06 | 0.37 | 0.08 | ||||||
| P value for independent-samples t test between groups: 0.03 | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| CO1 | CO2 | SO1 | SO2 | ||||||
| Means | Standard deviations | Means | Standard deviations | Means | Standard deviations | Means | Standard deviations | ||
| BMD (g/cm 3) | Mesial | 1.79 | 0.05 | 1.73 | 0.05 | 1.78 | 0.04 | 1.81 | 0.06 |
| Distal | 1.70 | 0.06 | 1.78 | 0.07 | 1.58 | 0.06 | 1.72 | 0.05 | |
| BV/TV | Mesial | 0.72 | 0.12 | 0.76 | 0.06 | 0.75 | 0.15 | 0.82 | 0.04 |
| Distal | 0.76 | 0.04 | 0.80 | 0.07 | 0.63 | 0.10 | 0.73 | 0.06 | |
| TbTh (mm) | Mesial | 0.13 | 0.02 | 0.11 | 0.04 | 0.11 | 0.02 | 0.13 | 0.03 |
| Distal | 0.13 | 0.01 | 0.14 | 0.03 | 0.11 | 0.02 | 0.14 | 0.01 | |
| TbSp (mm) | Mesial | 0.07 | 0.01 | 0.06 | 0.02 | 0.07 | 0.02 | 0.05 | 0.01 |
| Distal | 0.09 | 0.01 | 0.06 | 0.01 | 0.09 | 0.01 | 0.07 | 0.01 | |
| TbN | Mesial | 5.25 | 0.72 | 6.16 | 0.89 | 5.67 | 0.57 | 5.42 | 0.82 |
| Distal | 4.87 | 0.33 | 5.03 | 0.50 | 5.32 | 0.76 | 4.88 | 0.27 | |
Note. CO, sham stress intervention group; SO, stress intervention group; CO1, orthodontic-force side of sham stress intervention group; CO2, nonorthodontic-force side of sham stress intervention group; SO1, orthodontic-force side of stress intervention group; SO2, nonorthodontic-force side of stress intervention group.
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