1. Introduction

2. Materials and Methods

3. Results

3.1. Human Studies

3.1.1. Biofunctional Molecules Involved in Enhancing OTM

The studies included in this systematic review assessed the efficacy of various biomolecules in accelerating orthodontic tooth movement (OTM) through local administration. The biofunctional molecules examined included Calcitriol—a vitamin D3 metabolite [17,18,19,20], Prostaglandin E1 [21,22], Vitamin C [23], and Recombinant human Relaxin [24].

The results were variable across substances and studies, as well as the study design, demographic details, sample size, study duration, intervention specifics (including the substance used and its administration pathway), applied force magnitude, tooth movement type, administration frequency, dosage, and outcome measures. The specific details can be found in Table 3 and Table 4.

3.1.1.1. Calcitriol

Calcitriol emerged as a prominent biofunctional molecule capable to improve OTM rate in four original studies; in two studies [18,20] it produced statistically significant outcomes in enhancing OTM, while the remaining two showed acceleration of OTM, although not all reached statistical significance [17,19]. The action of Calcitriol is multifaceted, with a powerful influence on the process of bone metabolism, contributing to both bone resorption and deposition, significantly enhancing the rate of OTM compared to control groups. More in depth, the stage of osteoblast development determines how much Calcitriol affects bone turnover. Normal vitamin D levels have been shown to reduce the receptor activator of nuclear factor kappa-B ligand/osteoprotegerin (RANKL/OPG) ratio and reduce osteoclastic bone resorption through vitamin D receptors in mature osteoblasts [9,25,26]. Likewise, Calcitriol acts on fully developed osteoblasts by enhancing the bone apposition rate. However, higher concentrations of Calcitriol stimulate osteoclastic bone resorption by increasing the RANKL/OPG ratio in less-developed osteoblasts. The impact of Calcitriol is associated with the augmentation of RANKL expression by nearby cells, thus leading to the stimulation of osteoclasts [26,27,28].

In line with these biological pathways, AlHasani et al. [17] investigated the efficiency of Vitamin D3 (25 pg/0.2 ml) injections to accelerate maxillary canine retraction over 3-6 months in a 17-patient trial. The intervention demonstrated no additional root resorption or adverse alveolar bone effects, confirming its safety. Remarkably, it achieved a 51% acceleration in tooth movement on the experimental sides, illustrating a favorable periodontal response and validating a prior human trial [20], where 15 patients received weekly Vitamin D3 injections in varying dosages over six months. The study found a dose-dependent effectiveness, with a 25 pg dose leading to a 51% faster canine movement rate. The absence of detrimental effects on the PDL emphasizes the safety and efficiency of Calcitriol in orthodontic applications.

Varughese et al. [18] administered monthly local periodontal injections of calcitriol (50 pg/0.2 ml), noting a statistically significant increase in the amount of canine distalization over three months​​ while IosubCiur et al. [19] investigated patients that received weekly Vitamin D3 injections over three months. The intervention showed no root resorption and resulted in 70% more tooth movement on the experimental side.

3.1.1.2. Prostaglandins

Prostaglandin E1 (PGE1) was consistently effective, with both studies reporting a statistically significant increase in OTM. This biofunctional molecule's role in accelerating OTM is likely through its impact on bone remodeling, particularly by enhancing bone resorption necessary for tooth movement [5]. PGE1 can act as an intermediary in transmitting mechanical stress during orthodontic tooth movement. It can enhance bone resorption, reduce collagen synthesis, and elevate cAMP (cyclic adenosine monophosphate) levels. Bone resorption is accentuated by promoting osteoclastogenesis and activating pre-existing osteoclasts. PGE1 also increased the levels of matrix metalloproteinase-8 (MMP-8), resulting in a reduction of procollagen, necessary for the remodeling of bone and the PDL [8,9,29].

The study conducted by Patil et al. [21] used PGE1 injections, revealing no adverse macroscopic or radiographic effects. It demonstrated a substantial enhancement in tooth displacement, with the intervention side moving 1.7 times faster than the control side. This significant acceleration highlights PGE1's potential as a safe and effective orthodontic adjuvant, results in line with Yamasaki’s et al. [22] PGE1 study, who observed that the rate of canine movement was almost 1.6-fold higher on the PGE1 injected side compared to the vehicle-injected side, with no significant side effects reported.

3.1.1.3. Vitamin C

Vitamin C, although studied in a single trial, presented a statistically significant outcome, enhancing the rate of impacted maxillary canine movement. Vitamin C's role in collagen synthesis and its influence on bone and soft tissue healing may account for its efficacy in accelerating OTM [30]. Vitamin C affects osteoclastic activity, osteogenesis, tissue repair, and PDL organization. It boosts collagen type I synthesis, a key component of bone matrix and PDL, as well as bone mineralization, collagen type X production, alkaline phosphatase expression, osteoblast development and differentiation, leading to bone regeneration [30,31].

The trial conducted by Yussif et al. [23] investigated the use of Vitamin C submucosal injections over 12 months in the traction and alignment of impacted canines. The intervention showed no evidence of bone or root resorption and led to clinically significant improvement in movement rate. These findings indicate Vitamin C as a potent catalyst for tooth eruption, maintaining PDL integrity.

3.1.1.4. Recombinant human Relaxin

Conversely, Recombinant human Relaxin did not demonstrate a significant change in OTM under the tested conditions. Despite its known influence on collagen turnover and potential effects on the periodontal ligament, the dosages or methods of administration used in the study may not have been sufficient to alter tooth movement rates [24].

Table 4. Side effects and outcomes of the analyzed biofunctional molecules.

Table 4. Side effects and outcomes of the analyzed biofunctional molecules.

Authors, year	Biofunctional molecules	Description of groups, dosage	Side effects	Outcome
1.AlHasani et al., 2021	Calcitriol	EG: OTM + 25pg/0.2ml Vitamin D3 diluted in DMSO CG: OTM + no intervention	The intervention did not result in additional root resorption and did not adversely affect the integrity of the alveolar bone (p > 0.05 in canine root resorption evaluations between the experimental and control sides)	The local injection of vitamin D3 improved canine retraction and led to a more favorable periodontal response. The study validated and referenced previous human studies demonstrating a 51% acceleration in tooth movement on the experimental sides.
2.Varughese et al., 2019	Calcitriol	EG: OTM + 50pg per 0.2mL of calcitriol (1,25 DHC) gel CG: OTM + 0.2mL placebo gel	The findings indicated a statistically significant decline in cancellous bone density on the experimental side in comparison to the control side. There also was a significant overall decline in bone density of the cortical bones during OTM, on both experimental and control sides.	Quantitative measurement of canine distalization revealed a highly significant difference (p < 0.001) between the experimental and control sides over a period of three months. During the second and third months, there was a notable increase in the rate of canine movement on the experimental side, which was statistically significant.
3.IosubCiur et al., 2016	Calcitriol	EG: OTM + 42 pg/mL - 0.2 mL of vitamin D3 diluted in DMSO CG: OTM + no intervention	There was no evidence of root resorption observed three months after the initial treatment with vitamin D3, as assessed by cone-beam CT examination	The mean rate of tooth displacement was higher in the experimental side compared to the control side. The experimental teeth exhibited an average of 70% more pronounced tooth movement compared to the control teeth. The disparities between the two quadrants (control and experimental) exhibited statistical significance (p<0.0313).
4.AlHasani et al., 2011	Calcitriol	EG1: OTM + 15 pg calcitriol diluted in 0.2 mL DMSO EG2: OTM + 25 pg calcitriol diluted in 0.2 mL DMSO EG3: OTM + 40 pg calcitriol diluted in 0.2 mL DMSO CG: OTM + placebo (0.2 mL DMSO)	The periapical radiographs revealed no detrimental impact of calcitriol on the periodontium.	The study found that, when calcitriol is injected locally, its effectiveness and cost efficiency in humans followed a dose-dependent pattern. Specifically, a dose of 25 pg of calcitriol resulted in a 51% faster rate of experimental canine movement compared to the control group. Additionally, doses of 15 pg and 40 pg each led to approximately a 10% acceleration in orthodontic tooth movement.
5.Patil et al., 2005	Prostaglandin E1	EG: OTM + 1g PGE1/ 1ml lignocaine CG: OTM + placebo (lignocaine only)	No adverse effects were noticed, neither macroscopically nor through X-ray imaging, in the area where PGE1 was injected during the experimental study and the two-year follow-up period of active orthodontic treatment.	The observed data clearly demonstrated a substantial enhancement in orthodontic tooth displacement on the side injected with PGE1, compared to the control side. The ratio of the intervention side to the control side was 1.7/1, as seen during a period of one month.
6.Yamasaki et al., 1984	Prostaglandin E1	EG: OTM + 10 µg PGE1 CG: OTM + placebo (lidocaine only)	No adverse effects were observed in the surrounding tissues when examined macroscopically and radiographically.	Local administration of 10 µg of PGE1, in the gingiva adjacent to the orthodontically treated teeth resulted in a significant increase in the rate of tooth movement compared to the control side. The distal canine movement rate was nearly 1.6 times higher on the side where PGE1 injections were administered compared to the side where the vehicle was injected. This difference was statistically significant (p< 0.05).
7.Yussif et al., 2018	Vitamin C	EG: OTM + vitamin C with a dosage calculated for a single tooth* CG: OTM + no intervention	There was no evidence of bone or root resorption in the teeth that were treated after receiving a vitamin C injection.	The intervention group showed a clinically significant improvement in movement rate (2-2.5 mm), while the control group had a lower rate (0.5-1.5 mm). The p-value was found to be p<0.003. The results implied that locally administered vitamin C is a powerful catalyst for eruption that has the benefit of preserving the integrity of the periodontium.
8.McGorray et al., 2012	Recombinant human Relaxin	EG: OTM + 50 mg Relaxin divided in 2 injections of 0.1 mL CG: OTM + placebo vehicle	None of the subjects exhibited any presence of anti-Relaxin antibodies at any given period and no side effects were noted.	There were no statistically significant differences in tooth displacement between the experimental and the control group.

OTM - orthodontic tooth movement; DMSO - Dimethyl sulfoxide; PGE1 - Prostaglandin E1. * Dosage calculated according to the author’s previous study [34].

3.1.2. Risk of Bias Assessments

Risk of Bias Assessments across the studies varied, with several studies demonstrating moderate risk due to small sample sizes, lack of detailed randomization, blinding processes, and potential confounding factors not being thoroughly controlled. The studies that provided statistically significant results offered promising evidence towards the efficacy of pharmacological agents in accelerating OTM. These factors necessitate a cautious interpretation of the results and underscore the need for further research with larger sample sizes and more rigorous methodologies​​​​.

More in detail, the results of the RoB assessment can be found in Figure 2.

The collective findings of these human studies suggest that certain pharmacological agents, particularly those influencing bone metabolism, can enhance the rate of OTM. However, the studies' limitations, particularly related to their design and execution, indicate that even though the evidence is promising, it is not definitive. Further investigation with more robust clinical trials is warranted to confirm these agents' efficacy and safety in clinical practice.

3.2. Animal Studies

In vivo animal studies evaluated numerous substances that could accelerate OTM (n = 28), out of which 27 had a local (injection – submucosal or intraligamentary) administration route and only one was applied topically (transmucosal absorption).

Prostaglandin E1 and E2 [32,33,34,35,36,37], calcitriol [28,34,38], RANKL and RANKL expression plasmid [39,40,41], growth factors such as epidermal growth factor [42,43], recombinant human insulin-like growth factor-1 [44], basic fibroblast growth factor (bFGF) [45], macrophage colony-stimulating factor (M-CSF) [46], PTH [47,48], osteocalcin [49,50], vitamin C and E [51], biocompatible reduced graphene oxide [52], exogenous thyroxine [53], sclerostin protein [54], a specific EP4 agonist (ONO-AE1-329) [55], carrageenan (CGN) [56] and herbal extracts [57,58] were evaluated in the researched studies.

Species, sample, OTM and force, frequency of administration, and outcomes vary between studies, but most studies show statistically significant OTM acceleration in experimental groups compared to control groups. Table 5 contains a more detailed breakdown of the stated aspects.

3.2.1. Prostaglandin E1 and E2

Prostaglandins alter bone metabolism and promote bone formation under mechanical stimulation by promoting osteoblast proliferation, both progenitor and mature. Applying mechanical strain to bone can increase bone resorption through increasing the osteoclastic population. Integrating orthodontic force with exogenous prostaglandin E2 has a combined effect on alveolar bone, enhancing both resorption and deposition [34,59].

Caǧlaroǧlu et al. [33] compared the effects of prostaglandin E2 (PGE2) on orthodontic tooth movement and bone metabolism in New Zealand rabbits (n = 45). PGE2 was administered via intravenous, submucosal, or intraligamentous routes after appliance insertion and on days 1, 3, 7, and 14 with a total dose of 1.2 μg. Results showed tooth movement in both experimental and positive control groups, but the intraligamentous PGE2 group had the highest values of all analyzed parameters, including serum calcium and phosphorus levels and osteoclastic and osteoblastic populations, concluding that submucosal and intraligamentous PGE2 administration significantly increased orthodontic tooth movement and bone metabolism.

Kale et al. [34] investigated the effects of calcitriol (1,25 DHCC) administration on days 0, 3 and 6 and PGE2 once, immediately after establishing the rat OTM model (n = 37), with a 1,25 DHCC dosage of 20 μL of 10⁻10mol/L and a PGE2 dosage of 0.1 μg/0.1 mL. Although tooth movement was relatively similar, the PGE2 group had much more osteoclastic activity than the 1,25-DHCC group. In contrast, the 1,25-DHCC group had a much higher number of osteoblasts on the alveolar surface. This research demonstrates that 1,25-DHCC promotes bone formation more effectively than PGE2, facilitating the relationship between formation and resorption in alveolar bone reconfiguration during OTM.

Seifi et al. [35] investigated the effects of local injections of prostaglandin E2 (PGE2) alone and with calcium gluconate (Ca) on orthodontic tooth movement (OTM) and root resorption. The rodents (n = 24), divided into experimental and control groups, received 0.1 ml of 1 mg/ml PGE2 submucosal injections, with or without an intraperitoneal injection of 200 mg/kg Ca (10%) in addition to PGE2. All injections were performed on days 0 and 7. The results showed a significant acceleration in OTM after PGE2 injection compared to the control group. Although Ca lowered OTM, a substantial increase (p < 0.05) was still seen. Root resorption differed significantly (p < 0.05) between the PGE2 and normal groups. The PGE2 group showed a considerable increase in root resorption compared to the normal group. There were no statistically significant changes in root resorption between the PGE2 + Ca and the normal or control groups. The findings demonstrate the role of calcium ions functioning in conjunction with PGE2 in regulating root resorption while considerably enhancing OTM.

Cui et al. [32] investigated the effect of injecting akebiasaponin D (ASD) at different doses locally on the speed of orthodontic tooth movement in rats (n = 40) compared to the effectiveness of PGE2 administration. Both substances were administered once, immediately after establishing the OTM model. The ASD solution was administered at varying concentrations, with a dosage of 5 mg/kg in the ASD1 group and 10 mg/kg in the ASD2 group. A solution of PGE2 was administered at a dosage of 25 μg/kg in the PGE2 group. The results indicated a progressive increase in the distance between the first and second molars, as compared to the control group. Nevertheless, there was no notable disparity observed between the ASD2 group and the PGE2 group. Osteoclast numbers exhibited an increase on the side experiencing tension, reaching their highest point on the 21st day, and thereafter declined. The study determined that the local administration of ASD solution can accelerate the process of orthodontic tooth movement. It was found that a dosage of 10 mg/kg of ASD solution is as effective as the PGE2 solution.

These studies confirm the findings of Leiker et al. [36] which evaluated the long-term effects of varying concentrations and frequencies of injectable, exogenous prostaglandin E2 (PGE2) on tooth movement and root resorption. In his study, 132 rats were divided into two experimental time periods of 2 and 4 weeks, and divided into four subgroups based on concentration levels of PGE2 injections. Half of the dosage subgroup received a single injection at appliance placement, while the other half received weekly injections. The results showed that injections of exogenous PGE2 over an extended period enhanced orthodontic tooth movement in rats. However, there was no statistically significant difference in tooth movement between the single and multiple injection groups or among the four concentration levels used in either the 2- or 4-week time periods. The amount of root resorption increased with the use of prostaglandin injections, specifically with increased numbers of injections and concentrations of PGE2. The study determined that a lower dosage of PGE 2 (0.1-1.0 ug) is enough to improve OTM. Similar results were also found by Yamasaki et al. [37], who demonstrated that combining local prostaglandin E1 or E2 with orthodontic tooth movement can nearly double the rate in monkeys compared to a control group. There were no macroscopical negative effects seen in the gingiva or related structures, after 40 ug PGE1 or PGE2 submucosal injections on days 0, 1, 5, 9, 12 and 15.

3.2.2. Growth Factors

In the context of OTM growth factors are involved in angiogenesis and therefore play an important role in increasing the rate of activation of osteoclasts. Peng et al. [44] explored the effects of Recombinant human insulin-like growth factor-1 (rhIGF-1) in bone remodeling and orthodontic tooth movement using submucosal injections every two days in a rodent model (n = 80). The results demonstrated that the rhIGF-1 group had more bone resorption and lacunae on the alveolar bone than the control group, with considerably higher number of osteoclasts in the compression side of the periodontal ligament than the control group. The distance of tooth movement in the rhIGF-1 group increased significantly during the experiment, indicating that rhIGF-1 enhanced OTM.

Similarly, the effect of basic fibroblast growth factor (bFGF) was investigated by Seifi et al. [45], who found out that a submucosal injection of either 0.02 cc of 10 ng, 100 ng or 1000 ng was effective in a dose-dependent manner, with rats (n = 50) receiving the most effective injection at 1000 ng. The current study found that local application of angiogenic factors such as bFGF can shorten the time of orthodontic therapy.

Alves et al. [42] and Saddi et al. [43] in their rodent model (n = 96, respectively, n = 32) both evaluated the influence of epidermal growth factors (EGF) injected submucosally once, immediately after establishing the mouse OTM model, in different concentrations, with the aim of accelerating orthodontic tooth movement. The findings confirm that EGF-liposome treatment can enhance tooth mobility and osteoclast numbers with statistically significant results compared to controls. This was associated with high RANKL expression, inducing osteoclast genesis and accelerating OTM [42].

On the other hand, Brooks et al. [46] determined that a 14% increase in tooth movement rate can be achieved in a mouse model (n = 48) with a single subperiosteally injection of macrophage colony-stimulating factor - an early osteoclast recruitment/differentiation factor (M-CSF), suggesting it may be a viable choice in therapeutic settings. M-CSF could be injected during appliance activation or reactivation, thus reducing treatment duration without increasing appointments with the orthodontist. Additionally, administering M-CSF may allow for lower force values, reducing the risk of undesired root resorption.

3.2.3. RANKL and RANKL expression plasmid

Rankl and Rankl expression plasmids play a role in orthodontic tooth movement through their ability to enhance osteoclast activation and bone degradation by attracting cytokines. Chang et al. [39] investigated the effect of receptor activator of nuclear factor kappa-B ligand (RANKL) formulated in microspheres (RANKL formulation with PLGA-aqueous hydroxyethyl cellulose microspheres – 1.5 mg/4.5 μl), injected through flapless osteoperforations in a rat model (n = 24), on orthodontic tooth movement. The study discovered that injectable RANKL formulations were successful in accelerating bone resorption in comparison to other control groups. There was a notable increase in tooth movement (129.2% more tooth movement than without formulation and 71.8% more than with placebo formulation) and a statistically significant increase in TRAP activity (an increase of the amount of osteoclast activity determined by using tartrate-resistant acid phosphatase staining). The groups did not show any noticeable differences in root resorption. The study's findings indicate that injectable RANKL formulations have a substantial positive impact on (OTM).

Similarly, Li et al. [40] used RANKL, injected daily subperiosteally for 2 weeks in a larger rodent model (n = 60) in order to assess its ability to enhance OTM. The study found out that the experimental group exhibited a statistically significant increase in tooth movement and reduction in bone volume on days 14 and 21. Additionally, the number of osteoclasts increased on days 3, 7, 14, and 21 but there was no notable difference at the end of the experimental period. Significantly elevated levels of RANKL expression were detected on days 7 and 14, accompanied by notable alkaline phosphatase activity. No substantial systemic alterations were detected.

Kanzaki et al. [41] evaluated the efficacity of RANKL expression plasmid (5-μl) in a hemagglutinating virus of Japan (HVJ) envelope vector, injected subperiosteally in a rat tooth movement model (n = 25). Local transfer of the RANKL gene increased the expression of RANKL and the formation of osteoclasts in the periodontal tissue in a statistically significant manner, without causing any systemic side effects.

3.2.4. Calcitriol

1.25 DHC (calcitriol – a Vitamin D3 derivate) supports alveolar bone remodeling by enhancing the proliferation and function of the osteoblasts, with a higher rate of recruitment and stimulation of mononuclear osteoclasts, leading to enhanced bone resorption on the pressure side of the periodontal ligament [28]. Takano Yamamoto’s [38] study on Wistar Rats (n = 40), as well as the experiment conducted by Collins et al. [28] on 10 young adult cats determined its role in enhancing orthodontic tooth movement with an increase varying form 60% (weekly intraligamentar injection of 0.1 ml of DMSO containing 50 ug/ml of 1,25D) [28] up to 245% (submucosal injections every 3 days of 1,25-(OH)2D3 20 μL of 10⁻10mol/L) [38], their findings being consolidated by the human studies previously mentioned [17,18,19,20].

3.2.5. PTH

The parathyroid hormone, when applied directly to the areas of compression of the PDL, has the potential to enhance the activity of osteoblasts and indirectly affect osteoclasts. Lu et al. [47] attempted to determine the effects of local administration (injections into a micro-osteoperforation) of parathyroid hormone (PTH) or parathyroid hormone-related protein (PTHrP) carried by PECE (thermosensitive PEG-PCL-PEG) hydrogel on osteogenesis and osteoclastogenesis during orthodontic tooth movement on a rodent model (n = 40). At 14 days, the total release of PTH or PTHrP from PECE hydrogels exceeded 75% on a sustained basis. The distance of OTM in the PTH or PTHrP group statistically significant compared to the control groups. PTH injections were also able to lower the rate of orthodontic relapse concluding that local injection of either PTH or PTHrP delivered via controlled release enhances OTM by controlling periodontal bone remodeling.

Soma et al. [48] also evaluated the potential of a slow-release formula of PTH injected subperiosteally every other day through the rat (n = 56) experiment in order to accelerate OTM, with statistically significant results. The experiment determined that PTH dissolved in methyl cellulose gel (MC gel) injections at 1 μg/400 g body weight caused a 1.6-fold increase in the rate of tooth movement.

3.2.6. Other Biosubstances

Osteocalcin [19,20], vitamin C and E [21], biocompatible reduced graphene oxide [22], exogenous thyroxine [23], sclerostin protein [24], a specific EP4 agonist (ONO-AE1-329) [25], carrageenan (CGN) [26] and herbal extracts [27,28] were also investigated.

The proteins examined demonstrated their capacity to recruit and differentiate osteoclasts, hence playing a role in bone remodeling. Lu et al. [54] evaluated the ability of sclerostin protein to accelerate orthodontic tooth movement in Wistar Rats (n = 48), showing that injecting sclerostin protein submucosally into the alveolar bone on the compression side enhances the formation of osteoclasts via stimulating osteoclastogenesis. The results demonstrated a significant increase in tooth movement in the groups that received sclerostin injections of 4 μg/kg and 20 μg/kg. Additionally, the group receiving a dosage of 20 μg/kg experienced a notable reduction in BV/TV (bone volume fraction) and an increase in the number of TRAP-positive osteoclasts at the site of compression. Animal studies (rodent models, n = 88 and n = 33) on the effects of calcitonin (- a major noncollagenous bone matrix protein) local injections were conducted by Hashimoto et al. [49] and respectively by Kobayashi et al. [50], both studies showing statistically significant results compared to the control groups both in amount and rate of tooth movement. Additionally, histological analysis demonstrated that the injections significantly enhanced the presence of osteoclasts on the compressed side of the alveolar bone surface. The findings indicate that osteocalcin has a supplementary impact on the speed of orthodontic tooth movement by promoting the formation of osteoclasts on the side experiencing pressure [50].

Jiao et al. [52] assessed the impact of biocompatible gelatin reduced graphene oxide (GOG) on the process of bone remodeling, osteoclastogenesis, and angiogenesis, as well as its ability to speed up orthodontic tooth movement. The mouse model experiment (n = 12) showed that local injections of GOG resulted in an increased rate of tooth movement, along with more bone resorption by osteoclasts and the formation of new blood vessels, as compared to control groups. Furthermore, when locally injected around teeth, the buildup of GOG in the spleen did not have any adverse effects on animal survival and lifespan. The results illustrate the effectiveness of GOG in promoting bone remodeling and OTM while ensuring biosecurity and biological functionality.

In a 4 weeks dog OTM model (n = 8), Jung et al. [53] tested a transmucosal administration (via tablets bonded on the orthodontic appliance and replaced once a week) of exogenous thyroxine, and found that although the experiment did not have a statistically significant effect on the rate of OTM, microscopic evaluation at the end of the experimental period revealed an increase in the osteoclasts number.

Chung et al. [55] aimed to examine the combined enhancement of tooth mobility and bone growth in vivo by stimulating the prostaglandin receptor EP4. The rat experiment (n = 25) investigated the local administration of a particular EP4 agonist (ONO-AE1-329) via injection to replicate the effects of ligand binding. To assess tooth movement and bone volume, researchers employed soft x-ray and micro-computed tomography. The results indicated that the administration of the EP4 agonist alone did not have a significant impact on body weight, macro-structures, or bone volume when compared to the group treated with only vehicle or control side. Nevertheless, the administration of the injection, when combined with tooth movement, enhanced both tooth movement and the volume of bone in the alveolar tension side, obtaining statistically significant results.

Lastly, Carrageenan (CGN), a seaweed-derived common polysaccharide food additive and two different herbal extracts (Asperosaponin VI and dried root aqueous extract of Salvia miltiorrhiza) were evaluated in order to assess their ability to influence OTM.

Kavoli et al. [56], in their rat model experiment (n = 28), revealed that CGN has the ability to trigger inflammation (through increased osteoclastic activity), which could potentially accelerate OTM. The findings indicate that carrageenan injected locally can induce inflammation and increase the rate of OTM, which may enhance the efficacy of orthodontic treatment, with a statistically significant difference between the experimental and the control groups. The authors state that carrageenan shows promise as a viable aid in OTM, nevertheless mentioning that additional research is necessary to comprehensively grasp its potential advantages. Similarly, Xiao et al. [58] had statistically significant results when evaluating the potential of a Chinese herbal aqueous root extract (Salvia miltiorrhiza - ESM) as well as of its main component – Danshensu. The rat experimental model (n = 150) also observed an increase in the levels of RANKL and OPG in the treatment groups as compared to the control group. Nevertheless, the rate of OPG expression increased at a slower rate compared to that of RANKL. The levels of RANKL dramatically dropped when no orthodontic pressure was applied, particularly in the ESM groups, which indicates that ESM might accelerate periodontal changes by osteoclasts proliferation. Another natural biomolecule, Asperosaponin VI (ASA VI) was evaluated by Ma et al. [57] in order to assess its effects on OTM. The study conducted on a rat model (n = 64) found that ASA VI enhanced tooth movement in the experimental group, increased the count of osteoclasts, and improved the expression of osteoclast differentiation factors in comparison to the control group. The research team's conclusion states that ASA VI local administration could accelerate tooth movement by enhancing the activity of osteoclasts, which in turn promotes bone resorption on the compression side, while exerting at the same time a beneficial impact on bone formation on the tension side.

3.2.7. Risk of Bias Assessment

The SYRCLE's risk of bias assessment [62] for the included 28 animal studies revealed that while the studies differed in certain methodological areas, the majority had comparable risk profiles across specific bias categories.

Figure 3. Risk of Bias assessment.

Figure 3. Risk of Bias assessment.

The majority of the animal studies had a low risk of randomization bias, indicating that the separation of animals into treatment and control groups was often performed correctly. Most studies verified that the animals used had comparable baseline characteristics, resulting in a minimal risk of bias in this area. Allocation concealment was a consistent source of confusion throughout the trials, posing a medium to high risk of bias. The majority of the studies were classified as low risk in random housing, implying stable and regulated living conditions among experimental groups. Most studies did not provide appropriate information on whether treatment delivery and outcome assessment were blinded, raising concerns about data interpretation. The techniques of outcome measurement were typically consistent and methodical across trials, resulting in a low risk of bias in this area. Blinding during therapy administration and the lack of blinding in outcome assessment were common, indicating a significant risk of bias that might impair the reliability of the findings. Most studies provided detailed data on animal follow-up and tracked all animals throughout the experimental period, indicating a minimal likelihood of attrition bias.

Despite the need for more robust and standardized study designs, particularly in terms of improving blinding procedures and increasing transparency in allocation concealment and outcome reporting, the majority of the studies evaluated show a low risk of bias in critical areas such as randomization, baseline characteristic uniformity, and outcome data completeness. This conclusion confirms the dependability of the animal study findings in this sample, while the highlighted areas for improvement indicate that more severe methodological requirements are required to further strengthen the scientific rigor of future research in this subject.

4. Discussion and Perspectives for Enhancing OTM

5. Conclusions

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