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Orthodontic tooth movement (OTM) depends on an inflammatory response and coordinated remodeling of the periodontal ligament and alveolar bone. Salivary mediators might offer a non-invasive indicator of these processes, but their relationship to measured tooth displacement is uncertain. Objective: To illustrate how the association between salivary interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), soluble receptor activator of nuclear factor kappa-B ligand (sRANKL), osteoprotegerin (OPG), and orthodontic tooth movement could be investigated. Methods: In this 28-day prospective cohort study, 48 patients aged 16–25 years undergoing standardized maxillary canine retraction would provide unstimulated whole-saliva samples at baseline and on days 1, 7, 14, and 28. Biomarkers would be quantified by enzyme-linked immunosorbent assays. Tooth displacement would be measured from serial digital dental models. A mixed-effects model would evaluate longitudinal biomarker changes; regression would examine whether early biomarker response was associated with displacement, adjusting for age, sex, baseline periodontal status, and force. Results: Mean canine movement over 28 days was 0.82 ± 0.24 mm. Median salivary IL-1β rose from 12.0 pg/mL at baseline to 19.5 pg/mL on day 1 and approached baseline by day 28. The sRANKL/OPG ratio increased from 0.42 to 0.66 on day 7. Greater day-1 IL-1β change was moderately associated with 28-day tooth displacement (Spearman ρ = 0.43; p = 0.002). In an adjusted model, each 10-pg/mL increase in day-1 IL-1β was associated with 0.09 mm greater displacement (95% CI 0.02–0.16 mm). These figures are invented solely to demonstrate reporting and interpretation. Conclusion: The early salivary IL-1β response to orthodontic force may be associated with later canine displacement, while TNF-α and the sRANKL/OPG balance may provide complementary information about inflammation and bone remodeling.
Orthodontic treatment applies controlled mechanical force to a tooth. Force changes the mechanical environment of the periodontal ligament, stimulating cell signaling and remodeling of adjacent alveolar bone.1 Osteoclast-mediated bone resorption is generally emphasized on the pressure side, while osteoblast-associated bone formation contributes to adaptation on the tension side. These events overlap in time, and the simplified pressure tension model should not be mistaken for two completely separate biological compartments.2
Inflammatory mediators help coordinate this response. IL-1β and TNF-α participate in early inflammatory signaling and may promote pathways favoring osteoclast differentiation.3 RANKL binds its receptor RANK on osteoclast precursors and supports osteoclast formation; OPG functions as a decoy receptor that limits RANKL activity. Consequently, the RANKL/OPG balance provides a mechanistic way to think about bone resorption, although a salivary ratio cannot directly measure osteoclast activity at a particular tooth.4
Saliva is attractive because collection is painless, inexpensive, and repeatable. Unlike gingival crevicular fluid (GCF)5, however, whole saliva combines contributions from salivary glands, oral mucosa, microorganisms, gingival exudate, and possibly blood contamination.6 Biomarkers may therefore reflect generalized oral inflammation as well as local orthodontic remodeling. Systematic reviews have described temporal changes in salivary inflammatory and bone-related biomarkers while emphasizing substantial heterogeneity and limited clinical validation.7,8
The primary aim of this study was to demonstrate a research approach for testing the association between early changes in salivary IL-1β and the amount of canine movement over 28 days. Secondary objectives are to illustrate changes in TNF-α, sRANKL, OPG and the sRANKL/OPG ratio, and to explore whether a multi-marker model adds explanatory value beyond conventional clinical factors.
The study was carried out after obtaining ethical approval. A sample of 48 patients (24 females and 24 males; mean age 19.8 ± 2.6 years). The proposed inclusion criteria are fixed-appliance patients requiring maxillary first-premolar extraction and unilateral or bilateral canine retraction, with clinically healthy periodontal tissues. Exclusion criteria would include active periodontitis, untreated gingivitis, systemic inflammatory disease, smoking or vaping, recent antibiotic use, medications materially affecting bone turnover, and recent periodontal intervention. A true study would require a formal power calculation based on a prespecified minimum clinically meaningful association and expected repeated-measures correlation.
Orthodontic intervention and movement measurement
After leveling and alignment, a standardized, calibrated retraction force of approximately 150 g per canine would be applied with a nickel-titanium closed-coil spring. Force would be checked at visits and recorded for each participant. Movement of one prespecified index canine per participant would be calculated from registered intraoral digital scans obtained immediately before retraction and on day 28. Stable palatal structures would be used for model superimposition where appropriate. A blinded examiner would measure the change along the intended direction of retraction, and repeatability would be assessed with duplicate measurements. Anchorage loss would be documented because uncorrected movement of reference teeth may distort the estimate of true canine displacement.
Saliva collection and laboratory analysis
Unstimulated whole saliva would be collected between 9:00 and 11:00 a.m. at baseline (T0), 24 hours (T1), 7 days (T2), 14 days (T3), and 28 days (T4). Participants would be instructed to avoid food, caffeine, vigorous exercise, toothbrushing, and mouthwash for at least 90 minutes beforehand, consistent with a prespecified protocol. After a gentle water rinse and rest period, saliva would be collected by passive drooling into sterile tubes for 5 minutes. The collected volume and time would permit estimation of flow rate.
Samples would be chilled promptly, centrifuged under a validated protocol, aliquoted, and stored at −80 °C until batch analysis. Hemorrhagic or visibly contaminated samples would be flagged. IL-1β, TNF-α, sRANKL, and OPG concentrations would be quantified using commercial ELISA kits validated for saliva; duplicate assays, standard curves, limits of detection, and intra-/inter-assay coefficients of variation would be reported. Because sRANKL and OPG may be near detection limits, a predefined plan for below-limit observations would be essential. The ratio would be calculated only when the components could be quantified reliably and in compatible units.
Clinical covariates and outcomes
The primary outcome would be 28-day canine displacement in millimeters. The primary predictor would be change in salivary IL-1β from baseline to day 1. Secondary predictors would be changes in TNF-α and the sRANKL/OPG ratio. Covariates would include age, sex, plaque and gingival indices, salivary flow rate, baseline biomarker concentration, and recorded orthodontic force. Oral hygiene and periodontal inflammation would be reassessed during follow-up because appliance-induced gingival inflammation can confound the association.
Statistical analysis
Biomarker distributions were first inspected for skewness; medians and interquartile ranges were used where appropriate, with log transformation for modeling if warranted. Repeated measures were examined using patient-level random-intercept mixed-effects models with time as a categorical predictor. Spearman correlations were described as unadjusted biomarker–movement associations. A prespecified multivariable linear regression were estimated as the adjusted association between day-1 IL-1β change and day-28 movement, reporting regression coefficients, 95% confidence intervals and model diagnostics. Secondary comparisons were designated exploratory and interpreted with multiplicity in mind. Missing samples were tabulated with reasons; a two-sided alpha of 0.05 was used for the single primary hypothesis.
Participant characteristics and tooth movement
In the cohort, 48 participants completed the 28-day follow-up and provided analyzable baseline and day-1 saliva samples. The mean age was 19.8 ± 2.6 years, and half of the participants were female. Mean canine movement was 0.82 ± 0.24 mm (range 0.40–1.35 mm). The mean applied retraction force at activation was assumed to be 151 ± 8 g.
Longitudinal biomarker concentrations
Table 1. Salivary biomarker concentrations
|
Biomarker |
Baseline |
Day 1 |
Day 7 |
Day 14 |
Day 28 |
|
IL-1β (pg/mL) |
12.0 (9.0–15.0) |
19.5 (15.0–24.0) |
16.3 (12.4–20.0) |
13.5 (10.4–17.0) |
12.4 (9.3–16.0) |
|
TNF-α (pg/mL) |
4.8 (3.6–6.1) |
6.9 (5.2–8.5) |
6.1 (4.7–7.6) |
5.1 (3.9–6.5) |
4.9 (3.7–6.2) |
|
sRANKL (pg/mL) |
18.0 (14–22) |
22.0 (17–27) |
25.0 (20–30) |
22.0 (17–27) |
19.0 (15–23) |
|
OPG (pg/mL) |
43.0 (36–51) |
41.0 (34–49) |
38.0 (31–46) |
40.0 (33–48) |
42.0 (35–50) |
|
sRANKL/OPG ratio |
0.42 (0.33–0.53) |
0.54 (0.42–0.66) |
0.66 (0.51–0.80) |
0.55 (0.43–0.68) |
0.45 (0.35–0.56) |
In this invented scenario, IL-1β and TNF-α display an early transient increase, whereas the sRANKL/OPG ratio reaches its highest descriptive value at day 7. The pattern illustrates the distinction between early signaling and ongoing remodeling; it is not evidence that this precise timing occurs consistently across patients or studies.
Association with tooth movement
Table 2. biomarker–movement associations
|
Predictor |
Spearman ρ with 28-day movement |
p value |
Interpretation of association |
|
Day-1 change in IL-1β |
0.43 |
0.002 |
Moderate positive association |
|
Day-1 change in TNF-α |
0.28 |
0.051 |
Weak positive trend; uncertain |
|
Day-7 sRANKL/OPG ratio |
0.36 |
0.012 |
Modest positive association |
|
Baseline IL-1β |
0.10 |
0.50 |
Little apparent association |
Each additional 10 pg/mL in day-1 IL-1β change corresponded to 0.09 mm greater canine movement over 28 days (95% CI 0.02–0.16 mm; p = 0.013), after adjustment for prespecified covariates. An exploratory multi-marker model appeared to explain more variation than a clinical-only model (adjusted R² 0.31 versus 0.18).
The results demonstrate how a mechanistic association might be translated into a testable clinical research question. IL-1β and TNF-α can participate in early orthodontic inflammatory signaling, while the RANKL/OPG axis relates more directly to osteoclast regulation. A transient early salivary rise is biologically plausible, and its correlation with later measured movement would be worth studying.
The principal issue is specificity. Whole saliva samples the entire oral environment. Increased IL-1β could arise from poor plaque control, gingival inflammation, recent tissue trauma, oral lesions, or other inflammatory influences rather than the intended canine retraction. Salivary flow and time of day can also alter measured concentrations. Thus, repeated standardized sampling and periodontal indices are necessary to separate the orthodontic signal from oral-health noise. Research that relies only on a single biomarker measurement risks confusing correlation with mechanism.9
The moderate correlation (ρ = 0.43) is educationally useful because it is neither zero nor determinative, it not mean that an IL-1β rise causes faster tooth movement. Unmeasured variation in tissue biology, force system, age, bone morphology, and adherence could influence both the biomarker and displacement. Moreover, a statistically significant coefficient may correspond to a small absolute difference here, only 0.09 mm per 10 pg/mL that may not improve treatment planning.10
The RANKL/OPG ratio is an appealing mechanistic candidate. However, saliva does not directly report the localized RANKL activity on the compression side. Assay variability, detection limits, and ratio instability can magnify measurement error. Investigators should assess absolute sRANKL and OPG concentrations alongside ratios, and ideally compare saliva with site-specific GCF in a prespecified subset. The distinction matters because much orthodontic biomarker research has used GCF, which is related to but not interchangeable with whole saliva.11,12
Existing work supports the general research question while advising caution. A systematic review of salivary protein biomarkers reported heterogeneous candidate proteins and measurement techniques.13 A more recent systematic review reported fluctuations in markers including sRANKL, OPG, IL-1β and prostaglandin E2 across orthodontic treatment stages, but clinical interpretation remains unsettled.14 A prospective study of salivary stress and pain biomarkers found no correlation between its measured salivary markers and patient-reported pain, reinforcing that not every salivary biological response is a valid surrogate for a clinical outcome.15
Limitations
Long-term treatment efficiency, root resorption and periodontal safety would remain unanswered. Additional studies would need robust preanalytical standardization and independent validation before saliva-guided force adjustment could be considered.
The early salivary IL-1β response to orthodontic force may be associated with later canine displacement, while TNF-α and the sRANKL/OPG balance may provide complementary information about inflammation and bone remodeling.
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