The null hypothesis of the study, stating that there is no change in the cytokine expression and no correlation with the degree of OIIRR secondary to orthodontic force application, was rejected.
Pro-resorptive cytokines
In the present study, the concentration of IL-1β in the TS teeth increased initially, peaked at days 1 and 7 and then decreased. This result indicates that IL-1β is expressed in the GCF during orthodontic tooth movement similar to that in the previous findings [31, 32]. Though no statistically significant differences were noted in IL-1β levels between the TS and CS teeth at any time points, this indicates that the increase in IL-1β level might be associated with inflammatory response secondary to orthodontic mechanical stress [33].
Similarly, the change in the IL-6 level was statistically insignificant in agreement with Basaran and colleagues [34]. Ren et al. [11] reported an opposite finding; they observed an increase in levels of IL-6 at early hours of tooth movement which peaked after hour 24. Moreover, the concentration of IL-8 in TS fluctuated during the experimental phase, but there was no considerable change between the TS and CS. This finding is parallel to the outcomes of Tuncer et al. [8] but contradicting with those by Basaran et al. [34] and Ren et al. [11]. In fact, the latter two studies reported a significant decrease in the IL-8 level at days 7 and 30 of force application respectively. These dissimilarities, in the detected levels of IL-6 and IL-8, in comparison to other studies may be due to the variation in the used appliance systems, force levels, small sample size and/or some individual variations.
On the other hand, there was a statistically significant difference (p = 0.01) in the level of IL-7 and gingival TNF-α between TS and CS that peaked after day 28, similar to that in another study [35]. The increase in the level of TNF-α at day 28 indicates a presence of localised inflammation secondary to force application; this correlates with the levels of IL-6 and IL-7 that also peaked at day 28.
The boost in the pro-resorptive cytokines signifies their crucial roles in stress-induced inflammation through stimulating precursor scavenger cells that required for the removal of hyalinized tissue [33]. Also, the increase in the levels of pro-resorptive cytokines, IL-6, 7 and TNF α, might indicate a continuous periodontal remodelling during the lag phase of tooth movement and a cellular prohibiting mechanism.
Anti-resorptive cytokines
The time-dependent change of IL-4 concentration followed that of IL-1β, i.e. peaked during the period from day 1 to day 3 but then declined. There were also no significant differences between the TS and CS teeth concerning IL-4 concentration. Similarly, the change in the level of IFN-γ among the TS and CS teeth was insignificant. IFN-γ concentration in the TS teeth fluctuated, increasing to its peak at 72 h. The increase in the levels of IL-4 and IFN-γ closely follows that in IL-1β. This unique style of cytokine expression is the result of combined active periodontal remodelling during initial stages of tooth movement and the cellular prohibiting mechanism that prevents additional differentiation and activation of osteoclastic cells.
With regard to GM-CSF level secondary to orthodontic force application, Ren and colleagues identified a significant elevation of anti-resorptive cytokine GM-CSF in juveniles after application of orthodontic force [36]. However, in the present study, the amounts of GM-CSF for the TS teeth dropped immediately after the start of the experimental phase, increased at day 7 and then reached its peak at day 28, but there was no significant difference between the TS and CS teeth at different time scale.
Cytokines in high and low OIIRR group
Comparing cytokine profile for participants with high OIIRR (>0.35 mm3) and those with low OIIRR (<0.15 mm3), GM-CSF was increased in low OIIRR cases (p = 0.03), while other cytokines showed no significant differences (p > 0.05). These results confirm the link between the high levels of anti-resorptive cytokines such as GM-CSF and the reduced root osteoclastic differentiation.
Micro-CT assessments of OIIRR at different root surface and positions
In this study, the amounts of OIIRR lacunae on the lingual and buccal surface and at apical and cervical third were statistically insignificant; however, it was significant at the pure tension site (mesial and distal surfaces and middle third region) in the TS teeth. These findings are similar to some of the previous studies [37, 38] but contradicting with many others [39,40,41,42]. It will be expected that the amount of OIIRR at the compression sites (bucco-cervical and palate-apical regions) would be higher as in the other study [39], however, this was not confirmed in the current study. The differences may be attributed to two reasons. The first reason is called masking effect which is due to the fact that the measurement of the OIIRR in our study was not surface-region-specific, i.e. the whole surface (mesial, distal, buccal or palatal) and the whole radicular regions (cervical, middle or apical third) were assessed. As each of the root regions and surfaces included an overlapped compression and tension site, theoretically, the metabolic changes between the compression and tension sides masked each other and were not distinguishable. Secondly, the differences in the adopted appliance systems, force levels, small sample size and/or some individual variations may explain these dissimilarities.
Clinical implications, limitations and future considerations
In this study, two of the pro-resorptive cytokines, namely, IL-7 and TNF-α, increased significantly in the TS teeth secondary to heavy force application. This trend might represent the crucial roles of these cytokines in active OIIRR following heavy orthodontic forces. Moreover, as the teeth with low OIIRR demonstrated significantly high level of GM-CSF (anti-resorptive cytokines) secondary to heavy force application, gingival GM-CSF measurement used in our project might be considered in the future as a non-invasive and useful biomarker to identify participants’ susceptibility to severe root resorption.
However, the findings of this study should be taken as an intriguing hypothesis, not as evidence for some reasons. Firstly, the sensitivity of cytokine detections had shown a wide range of variability (approximately ranged from 1 to 96%). Secondly, the lack of method error analysis in our small sample size study indicates that GCF sampling and quantifications of biomarkers are subjected to comparably significant errors [43]. Furthermore, it should be acknowledged that there were some gender imbalance and age heterogeneity in our study which could be considered as one of the main confounders. While it would be ideal to assess the correlation between cytokine expression and OIIRR on various parts of the tooth root, the gingival sulcus is a pool for CGF, and it would be impossible to identify cytokine expression on each surface.
Based on the findings of this study, authors recommend a long-term future randomised clinical trial with a large sample size taking in consideration general factors such as gender, age and growth pattern distribution as well as local factors such as habits, traumatised teeth, pre-existing root resorption and hypo-functional periodontium.