Description of studies
In total, 1051 studies were identified from the electronic searches as relevant. After exclusion of all duplicates and assessment of the title and abstract of the reports, 64 studies were considered eligible for inclusion in this review. Out of the 64 studies, another 49 studies were excluded after full-text assessment, leaving 15 studies fulfilling the inclusion criteria (Additional file 5). Five were RCTs [16,17,18,19,20], 9 were prospective, non-randomized studies [21,22,23,24,25,26,27,28,29] and 1 was of cross-sectional design  (Table 1). The process of final study inclusion in this review is presented in Fig. 1.
The summary of methodological quality of the 5 included RCTs assessed on the basis of the Cochrane risk of bias tool is shown in Table 2. All were evaluated to be at high risk of bias [16,17,18,19,20]. This was mainly attributed to bias arising from the randomization process and bias arising in measurement of the outcome. Blinding of the clinicians, patients, and assessors was not universally possible due to the nature of the interventions, but the possibility of bias could not be excluded. Losses to follow-up were appropriately described, and there was no evidence of selective outcome reporting and other biases (Table 2).
Nine non-RCTs were identified. None was rated at low risk of bias. Five of the included studies were rated at moderate risk of bias [21, 22, 24, 27, 28]. Two studies were rated at serious risk of bias [23, 29] and another two at critical risk of bias [25, 26]. Detailed assessment of their risk of bias is depicted in Table 3.
Quantitative synthesis of the included studies
Due to the great heterogeneity between the interventions, the number of participants, the biomarkers assessed, and the follow-up period among studies, a meta-analysis was not feasible. The bias within studies and the fact that design of included studies has been diverse, have precluded, thus, a valid interpretation of the results through pooled estimates. Only qualitative assessment as a narrative review has been performed and reported (Table 5). The overall quality of evidence according to GRADE system was rated as low for NTX and TRAP or very low for the OPN, ALP, and OC (Table 6).
Qualitative synthesis of the included studies
Type of orthodontic intervention
Most of the studies evaluated the GCF of an upper canine prior, during, and after distalization. The other maxillary canine served as control [17, 18, 20, 23,24,25, 27, 29]. Several studies detected the biomarkers in various teeth under orthodontic treatment with fixed appliances [21, 22, 26, 28] or after the placement of separators . One study evaluated the GCF of patients with aligners . A headgear and a Bionator were the intervention in one study .
The following biomarkers for bone formation were assessed: bone alcaline phosphatase (BALP), alcaline phosphatase (ALP), and osteocalcin (OC).
The following biomarkers for bone resorption were assessed: deoxypyridinoline (DPD) and pyridinoline (PYD), N-terminal telopeptide (NTX), osteopontin (OPN), and tartrate-resistant acid phosphatase (TRAP). The follow-up period ranged mainly from baseline to 45 days. One study had an expanded follow-up period of up to 16 months  (Table 1).
Biomarkers of bone formation
Bone alcaline phosphatase (BALP) and alcaline phosphatase (ALP)
ΒALP was examined in one study . Although BALP values showed a descending character after activation visits, no statistically significant difference was reported overall. ALP was examined in 7 studies [17, 18, 20, 25, 27, 28, 30]. One study found no statistically significant differences in ALP levels compared with baseline . Alswafeeri et al. compared two groups during maxillary canine distalization with constant continuous vs. gradually increasing retraction forces. They found a specific pattern of the ALP activity in the constant force group . This pattern included an initial rise from baseline to the 1st week, then a peak in the 2nd week. This peak was followed by a reduction in enzymatic activity in the 3rd week. Overall increases in enzymatic activity in the constant force group were lower than in the gradually increasing force group. Besides, the use of a gradually increasing orthodontic force could induce an increase in osteoblastic activity during the initial stage of OTM compared with that induced by a relatively constant orthodontic force . Kalha et al. compared two groups of patients during space closure with Hycon-screw vs. active-tie backs. Increased levels were found in both groups; however, ALP increased more in the Hycon-screw group, due to the rapid initial force decay of the elastomeric modules. For the same reason, they concluded that the sequential repetitive loading of the periodontal ligament with the small and controlled activations of the screw was more effective for space closure . Batra et al. detected significant differences in ALP on days 7, 14, and 21. On days 7 and 14, ALP was increased whereas on day 21 declined . In the study of Perinetti et al., ALP levels during molar distalization were significantly higher from day 7 until the end of the treatment. The ALP levels were significantly higher in contralateral teeth, too . In another study of Perinetti et al., the GCF ALP activity significantly increased over time in both the mesial and the distal sites of the experimental teeth and the mesial sites of the contralateral. In the distal sites of contralateral teeth, there was an ALP activity increase, although not significant . Finally, in the antagonist teeth, this enzymatic activity was stable throughout the study, without any statistically significant changes. On day 28, enzymatic activity was significantly greater in the experimental teeth, as compared with the contralateral teeth . Both studies of Perinetti et al. revealed that ALP levels were higher at tension sites than in sites of compression. Insoft et al. stated that ALP levels peaked between the 1st and 3rd week after initiation of tooth movement. Additionally, ALP increased with inflammation in treated groups .
OC was assessed in 4 out of 55 studies [23, 24, 26, 29]. During canine retraction for a follow-up period of 28 days, Alfaqeeh et al. found the peak levels of OC on days 14 and 21 . Yang et al. found that OC levels in teeth under orthodontic movement were significantly higher in women in the ovulation period than in the menstrual period . Isik et al. observed a descending character of OC levels, with the exception of a slight rise on the 7th day. The aforementioned changes were not statistically significant . Griffiths et al. evaluated OC levels prior, during and after canine retraction and identified a higher concentration of OCN after fixed appliance fit, but no specific conclusion could be drawn due to the great variety between the findings of the sample .
Biomarkers of bone resorption
Deoxypyridinoline (DPD) and pyridinoline (PYD)
DPD was evaluated in two studies [26, 29]. According to Isik et al., DPD values showed a decreasing trend during tooth intrusion from 1 h to 28 days. That decrease was statistically significant at 22 and 28 days after force application . On the other hand, Griffiths et al. could not detect DPD in GCF prior, during, or after canine retraction .
N-terminal telopeptide (NTX)
NTX was investigated in 2 out of 5 studies [24, 26]. Alfaqeeh et al. demonstrated that NTX levels increased steadily during canine retraction. Significant differences between experimental and control sites were observed on day 14 and 21 after the initiation of the treatment with maximum NTX levels at the end of the experiment, on the 21st day .
However, in the Isik et al. study, NTX values were found to be below the detection limit with a few readings which showed large variations between subjects and stages of tooth movement .
OPN was investigated in 4 studies [16, 19, 21, 22]. Castroflorio et al. reported that the kinetics of OPN was characterized by a significant increase at the tension sites of the test teeth after 3 weeks from the application of orthodontic force . Barbieri et al. found that the concentration of OPN significantly decreased at the compression site 24 h after initiation of tooth movement with elastic separators . The other two studies came to the same conclusion (i.e. that there is no difference in the response to orthodontic activation between premenopausal and postmenopausal, as long as OPN is concerned) [21, 22].
Tartrate-resistant acid phosphatase (TRAP)
TRAP was detected only in one study . In the group of 100-g force, the TRAP levels were significantly elevated in the 5th week after force application compared with baseline. In contrast, the levels of TRAP in the group of 150-g force remained the same during the observational period. This finding indicated that light force has the ability to evoke frontal resorption of the bone .
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