The present longitudinal pilot study used saliva as a research material to monitor changes in the levels of OPG and RANKL during OTM initiated by orthodontic forces. To the best of our knowledge, this is the first in vivo study in humans using saliva to monitor OPG and RANKL during the whole orthodontic treatment period. The results of the study showed that the levels of OPG increased already at alignment and, after a slight decrease at space closure, reached its peak level at finishing. The OPG levels showed higher values for all treatment stages compared with baseline. However, RANKL was below the detection level in Elisa analyses of unstimulated and stimulated saliva.
It is challenging to reduce the duration of orthodontic treatment, which ordinarily takes two years. There has been strong interest in shortening the treatment time; however, clinicians are ultimately limited by the biological response during the OTM. Understanding the biology behind orthodontic tooth movement is therefore of great clinical importance. Orthodontic force applied to the teeth causes biological changes in the PDL cells due to blood flow reduction, leading to the secretion of inflammatory mediators [12]. In vitro studies indicate that PDL can upregulate either RANKL or OPG, depending on the type of force applied. [13]. When PDL cells were subjected to compression forces, RANKL expression was upregulated and OPG downregulated [9]. In the current study, the low OPG levels observed during space closure reflects the increased osteoclast activation at this stage. The tension forces that occur on the opposite side of the tooth cause an increase in OPG expression and a decrease in RANKL expression [14]. In the present study, the peak levels of OPG found at the finishing stage are likely due to increased osteoblast activity as the intensity of the force is reduced, whereby osteoclast activation at this stage is likely reduced.
Gingival crevicular fluid, present in the gingival sulcus, is continuously secreted by the underlying connective tissue into the oral cavity [15]. Increased levels of RANKL in the gingival crevicular fluid (GCF) during OTM have been shown [9]. The use of GCF in clinical research has a few limitations. Excessive time is needed in the clinic and it is difficult to analyse due to only small amounts of sample can be collected by filter paper [16]. In the present study, the RANKL levels in saliva were too low to be measured. According to the manufacturer, the Elisa assay (R&D systems) has a sensitivity of 78.1 pg/mL minimum detectable concentration for sRANKL. This finding is in accordance with previous studies [7, 10]. Possible reasons for the low levels of RANKL in our study might be (1) the dilution effect in saliva, or (2) the saliva sampling intervals used in the study design was not likely to reflect the highest osteoclast activity during orthodontic treatment where the RANKL levels supposed to reach a detectable amount.
The balance between RANKL and OPG is essential for the human skeleton in bone remodelling. Increased RANKL or decreased OPG expression may cause bone resorption but, on the other hand, increased OPG expression or decreased RANKL may enhance bone formation. The dysregulation of RANKL-OPG levels has been shown in several pathological conditions, including multiple myeloma, rheumatoid arthritis and osteoporosis [17]. Postmenopausal women with osteoporosis have increased OPG levels in serum in response to high osteoclast activity to limit rapid bone loss [18]. Bone turnover is increased during OTM [14], which correlates with our study showing increased OPG levels. According to our results, we can speculate that upregulation of OPG limits unnecessary bone loss during OTM [19]. In the present study, only healthy young female patients who had their first menstrual period two years ago were included, to exclude confounding factors such as gender and hormonal differences in oestrogen levels.
Previous studies have highlighted that the maintenance of bone mass is predominantly provided by the presence of OPG in serum. OPG has been investigated in clinical use as an antiresorptive agent for the treatment of bone disorders with high osteoclast activity. In the current study, OPG levels remained high in all treatment stages of OTM, except for space closure when osteoclast activity was high. Accordingly, OPG seems to play a vital role in bone remodelling as a key regulator of osteoclast activity, which in turn helps to preserve bone mass during OTM [13].
In periodontitis, RANKL is increased, whereas OPG is decreased compared with health or gingivitis. The increased RANKL/OPG ratio is indicative of periodontitis [7]. The patients included in the present study showed optimal oral hygiene with a healthy periodontium before starting treatment but also during the entire treatment period.
The limitation of this pilot study is that a small number of subjects were included. However, the longitudinal nature of the study design allowed continuous collection of saliva during two years of orthodontic treatment, restricting the size of the study population. However, the results of this novel approach provide indications for longitudinal future studies on how and when to sample saliva during orthodontic treatment. This is beneficial for future studies that may include larger study populations but with fewer saliva samples, and important to improve the understanding of the biological processes that occur during orthodontic tooth movement.