## 4 Discussion  
Prolonged duration of orthodontic treatment is a crucial factor influencing outcome, quality of care, and patients' satisfaction. The implementation of pharmacological modalities to stimulate and control OTM could potentially enhance the predictability of orthodontic therapy and be a valuable adjunctive tool, especially when used together with fixed orthodontic appliances. An ideal pharmacological agent that would promote and stimulate tooth position alterations is expected to be a valuable adjunctive tool in orthodontic treatment using especially fixed orthodontic appliances. The PROSPERO protocol revealed that the current review is the first that aimed to assess the impact of CTL on tooth movement caused by orthodontic forces. Although the assessment of quality, grading, and determination of the level of recommendation of the included studies were approached using validated and reliable tools, a possibility of subjective bias exists. Additionally, one important study was not included as this systematic review focused only on articles written in the English language, while that study was written in Spanish [19]. Figure 4: Results of meta-analysis (std mean difference) and forest plot. Figure 3: Results of meta-analysis (mean difference) and forest plot. The literature search identified 13 animal studies, two in vitro studies, eight reviews, and two clinical trials and two cross-sectional studies, which all demonstrated a significant variability in the level of evidence, quality, and methodology. The GRADE-based evidence was low for all animal studies, however in single cases received high level of recommendation was received. A cumulative extrapolation of the data from the animal studies was complicated by differences in the research protocol, the route of CTL administration, the clinical orthodontic method used, and the period of observation. All the studies used rats in their experiments, with the exception of one study, where cats were used [28]. In most of the studies, CTL was administered locally, except for two studies, in which a systemic route was used [23; 25]. Moreover, three different techniques were used to mobilize teeth; some studies used coil springs [22; 23; 27], others elastomeric chains [18; 48], and the third group of studies applied helical stainless steel loops [17; 24; 27]. The period of observation also varied as the rate of tooth movement was, in some studies, measured for two weeks [17; 18; 22] and, in others, for 21 days or 28 days [23]. The majority of animal studies on vitamin D\({}_{3}\) injected 20 microliters (uL) of calcitriol locally in the site of tooth movement [24; 27; 28]; others used a low dose of vitamin D\({}_{3}\) diet [25]. Differences between the study protocols used in animal studies can have a significant impact on study outcomes and conclusions, also making it difficult to compare and pool the results cumulatively. Although a number of animal studies showed CTL as positive regulator of bone metabolism and tooth movement, it is evident that these results cannot be directly applied to humans, due to obvious differences between species. Four clinical studies were found that involved human participants and concentrated on investigating the effect of local CTL injections. Among these, two studies looked at the effect of CTL on external root resorption and the other two on tooth movement [43; 44]. Both orthodontic tooth movement studies had weaknesses, including lack of randomization and undefined method of sample size calculation. In addition, canine distalization was only measured for a short period of time, either for three weeks [43] or for one month [44]; considering such a short duration, it would have been impossible to detect significant differences in the tooth movement discrepancy. This is in contrast to other studies concentrating on tooth movement that chose to measure canine traction for the minimum of three months [49; 50; 51]. Both human studies also lacked justification and rational evaluation for the dose of CTL injections.