1 Introduction

Botulinum toxin type A (BTX-A) reduces muscular contractions by temporarily inhibiting acetylcholine release at the neuromuscular junction. Since its initial use in the treatment of strabismus (Scott et al., 1989), BTX-A has been shown to be effective in treating disorders characterized by local muscle hyperactivity. In past decades, the use of BTX-A as a therapeutic agent has expanded widely. It has been used to treat adult patients with severe primary axillary hyperhidrosis, cervical dystonia, strabismus, blepharospasm, and other conditions. Among pediatric patients, even though all uses are off-label, BTX-A is used in cerebral palsy to reduce spasticity, leading to slight improvements in gait patterns (da Fonseca and Casamassimo, 2011).

The use of BTX-A in the orofacial region has emerged in the field of dentistry. It is used to treat primary and secondary masticatory and facial muscle spasms, facial tics, orofacial dyskinesias, dystonias, as well as pain disorders without a clear-cut motor hyperactivity basis (Clark et al., 2007). It is also often used in patients with masseter muscle hypertrophy (Ahn and Kim, 2007). BTX-A reduces muscle thickness, leading to changes in the facial contour (Kim et al., 2010). It is well known that a change in muscle activity not only has an esthetic effect, but also may relieve severe bruxism-related neurological disorders.

This study aimed to investigate the effects of BTX-A beyond the treatment options described above. We focused on the effects of reducing volume and altering the function of muscle hypertrophy and skeletal development. According to the functional matrix theory (Moss and Rankow, 1968), craniofacial growth and development are not intrinsically regulated by bone or cartilage, but by the surrounding muscle. Thus, the induction of muscle hypofunction may influence facial growth. Recent studies using BTX-A (Kim et al., 2008; Tsai et al., 2010) have supported the theory. These studies included animal experiments designed to investigate changes in masticatory muscle function due to the effects of BTX-A on skeletal development. They have shown that BTX-A can have inhibitory effects in the developing rat mandible.

In a study of rat mandibles injected with BTX-A in bilateral masseter muscles, mandibular dimensions were reduced compared with those of saline-injected rats (Kim et al., 2008). However, a limitation of that study was that the comparison was made between different individuals. To overcome this limitation, our study was designed to evaluate the effects of BTX-A in the same individual—one side of the mandible was injected with BTX-A and compared to the other side, which served as a control, injected with saline.

Therefore, this study investigated the effects of unilateral injection of BTX-A into the masseter muscle in growing rat mandibles.

2 Materials and methods

2.1 Subjects and study design

A total of 63 four-week-old (T0) male Sprague-Dawley rats were randomly divided into three groups, with 21 subjects in each group: Group 1, control group; Group 2, unilateral BTX-A group; and, Group 3, bilateral BTX-A group. The body weight of each rat was measured. The BTX-A used in this study was Botox® (Allergan Inc., Irvine, CA, USA). One vial of Botox® (100 U) was diluted with saline to make 3 U of Botox® in a 0.05-ml solution.

The masseter muscle of each rat was injected with either saline or BTX-A solution, depending on the group to which it was assigned. (1) Control group (Group 1): 0.05 ml of saline was administered to both sides of the superficial portion of the masseter muscle. (2) Unilateral BTX-A group (Group 2): one masseter muscle of each rat was injected with 0.05 ml saline, whereas the masseter muscle on the other side was injected with 3 U (0.05 ml) of BTX-A solution. The saline side was designated as the control side (Group 2-1), and the BTX-A side was designated as the experimental side (Group 2-2). Rats were randomly assigned to groups. Half were injected with BTX-A on the left side and half were injected with BTX-A on the right side. (3) Bilateral BTX-A group (Group 3): 3 U (0.05 ml) of BTX-A solution was administered to bilateral masseter muscles.

Experimental animals were fed ground rat pellets along with standard pellets to compensate for the possible adverse effect of a reduced incisive function in BTX-A-injected animals. Pellets that were fed to each subject were weighed weekly. All subjects were also weighed weekly (from T0 to T1, T2, T3, and T4) until sacrifice at four weeks (T4).

This study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Samsung Biomedical Research Institute (SBRI), Korea.

2.2 Dry mandibles

The mandibles were dissected from sacrificed animals and sectioned into two halves. Photographs of dry mandibles were taken using a digital camera, which was held at a constant distance from the mandible. Photos were transferred to computer images. The mandibular measurements used in this study (Fig. 1) were described by Asano (1986). All measurements were performed by three investigators.

Fig. 1
figure 1

Landmarks and measurements of the mandible used in this study

Me, the most inferior point of mental protuberance; Me′, the most inferior point of anterior alveolar bone; Me-Me″, the tangent to the bottom of the angular process through Me; Go, the most posterior tip of the angular process; Cd, the central point of the condyle; Cd′, the crossing point on Me-Me″ perpendicular to Me-Me″ from Cd; Co, the tip of the coronoid process; Co′, the crossing point on Me-Me″ perpendicular to Me-Me″ from Co; Me-Go, mandibular body length; Me-Cd, condylar length; Me-Co, coronoid process length; Me-Me′, anterior region height; Co-Co′, coronoid process height; Cd-Cd′, condylar height. The figure has been adopted from the study of Asano (1986)

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2.3 Statistical analysis

The overall body weights at T0 were evaluated for inter-group variability using the Kruskal-Wallis test with Bonferroni’s correction. Differences between specific groups were assessed using multiple comparison tests. The rate of weight increase was estimated using weights from T0 to T4 and was evaluated between groups using the Kruskal-Wallis test and multiple comparison tests. Mandibular measurements are described by means and standard deviations (SDs). The repeated-measures analysis of variance (ANOVA) was used for the evaluation of each mandibular measurement for all groups. A P value of <0.05 was considered to be statistically significant. The reliability of the three investigators was evaluated using the intraclass correlation test.

3 Results

Sixty of 63 animals completed the study protocol. Some mandibles were excluded from the measurement due to fractures during processing. The intraclass correlation test for reliability gave a mean coefficient of 0.6165, demonstrating good reliability between investigators.

3.1 Amount of pellets

The total amounts of pellets fed to the animals did not differ significantly between groups. However, when the amounts of ground pellets and standard pellets were analyzed separately, significant differences were observed. At T1, T2, and T3, rats in Group 3 ingested fewer standard pellets than those in Group 1. In contrast, when comparing amounts of ground pellets, Group 3 rats ingested more than Group 1 rats at T1 and T3.

3.2 Weight measurements

Although the experimental animals were randomly divided into three groups, there was a statistical difference in body weight among the three groups at the start of the trial. Average body weight was highest in the control group ((91.19±5.72) g), with the lowest weight in the unilateral BTX-A group ((83.19±1.65) g), as shown in Table 1. The rate of weight increase was estimated in each group ((W4-W0)/W0×100%, where W4 and W0 are the weights at T4 and T0, respectively) and statistically analyzed. The rate was significantly higher in Group 1 ((273.48± 22.14)%) and Group 2 ((270.26±21.67)%) than in Group 3 ((249.97±24.37)%), but there was no significant difference between Groups 1 and 2.

Table 1
figure Tab1

Mean body weight of rats in the three groups measured weekly during the experimental period

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3.3 Mandibular measurements

Mandibular measurements for all groups are shown in Table 2, and comparisons between groups are shown in Table 3 (P<0.05). For all six mandibular measurements in Group 2, the experimental side (Group 2-2) had significantly smaller dimensions than the control side (Group 2-1). Differences in measurements between the control group (Group 1) and the control side of Group 2 (Group 2-1) were not statistically significant, except for condylar height. In contrast, most mandibular measurements of the experimental side of Group 2 (Group 2-2) had smaller dimensions than those of the control group (Group 1), with the exception of condylar length, which was not significantly different between these two groups. Comparison between the control group and the bilateral BTX-A group (Group 3) showed shorter lengths in Group 3, with the exception of the condylar and coronoid process lengths. Differences between the experimental side of Group 2 (Group 2-2) and Group 3 were not statistically significant.

Table 2
figure Tab2

Mean mandibular measurements at the time of sacrifice ( T 4 , 8 weeks of age) for each group

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Table 3
figure Tab3

Significant differences in mandibular measurements between groups when compared pairwise

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4 Discussion

It is essential to understand the process of craniofacial growth and development, especially in the field of pediatric dentistry. Growth deficiencies or overgrowth of the mandible may result in malocclusion, which can influence masticatory function as well as esthetics among growing children. Many studies have attempted to control skeletal growth with the aim of restoring normal function and facial appearance by altering masticatory muscle function. Horowitz and Shapiro (1955) and Moore (1973) altered masseter muscles by myectomy, resulting in vertically-directed growth patterns of the mandible. In another study, denervation of the masseter muscle resulted in decreased mandibular height and length (Sato et al., 1986).

In this study, we used a non-invasive method involving BTX-A injection to alter masseter muscle function. Surgical denervation and myectomy may cause scar tissue or damage adjacent structures that can further alter growth patterns (Gardner et al., 1980). This study was designed to evaluate the influence of BTX-A on mandibular development by comparing changes in dimensions between BTX-Ainjected and saline-injected subjects. Since both sides of the mandible function as one unit, it is possible to experience a unilateral BTX-A injection effect leading to decreased bilateral movement of mandible. for this reason, growth of the control side of the unilateral group (Group 2-1) was compared to that of the control group (Group 1), and growth of the experimental side of the unilateral group (Group 2-2) was compared to that of the bilateral BTX-A-injected group (Group 3) to see whether BTX-A was effective, even though the mandible functions as a single unit.

4.1 Changes in mandibular measurements

Mandibular measurements were statistically smaller in Group 3 than in Group 1, with the exception of the condylar and coronoid process lengths. The lack of statistical differences for these two measurements was presumably due to observational errors stemming from the difficulty in locating the condylar point and the tip of the coronoid process. Our finding of statistically smaller dimensions in Group 3 coincides with that of a previous study (Kim et al., 2008). In previous studies, histological findings revealed an inhibitory action of BTX-A due to induced apoptosis at the proliferation stage of the reserve zone of condylar cartilage in developing mandibles. The current study included unilateral injection because it enabled the measurement of differences in dimensions between treatments in a single rat, thereby minimizing subject-dependent variability.

When control and experimental sides of the unilateral BTX-A group were compared, all measurements showed statistically significant differences. Similar results were reported in another experiment using rabbits (Kwon et al., 2007). They found that BTX-A induces site-specific muscular hypofunction and influences morphology at a local skeletal site. In the current study, BTX-A also induced localized craniofacial growth changes with unilateral effects.

There were some limitations to this study. Despite the fact that 63 rats were randomly divided into three groups, the average weight of the control group (Group 1) was significantly higher than those of the other two groups. Therefore, the statistical difference in mandibular measurements between Groups 1 and 3 may have been influenced by original weight differences at T0, as well as by the presence of BTX-A. However, BTX-A injection seemed to have an effect on skeletal development, as comparisons between Group 2-1 and Group 3, and between Group 2-2 and Group 3, showed no significant weight differences.

4.2 Overall body weights and amounts of pellets consumed

When the weight increase in each group was compared, there were no statistically significant differences between Groups 1 and 2, even though Group 1 started out with a significantly higher average weight. These findings suggest that unilateral BTX-A injection did not have systemic effects, but rather localized effects on mandibular development at the injected site. This result coincides with those of other studies where the overall growth of rabbits and rats was not altered by local injections of BTX-A into the masticatory muscles (Matic et al., 2007b; Tsai et al., 2011). Reduced masticatory function due to the toxin did not alter the systemic functions of the rats.

However, weight gain was significantly higher in Groups 1 and 2 compared with Group 3. This finding coincides with that of our previous study (Kim et al., 2008), in which we concluded that weight differences may be the result of reduced muscle tonicity and mass induced by bilateral BTX-A injection.

The results pertaining to weight increase also coincided with our findings regarding pellet weight measurements. Group 3 rats ate more ground pellets and fewer standard pellets. Even though the overall pellet weight was not significantly different between groups, Group 3 rats ingested increased amounts of softer ground pellets than standard pellets. This finding was related to decreased masticatory function in Group 3 compared with other groups, in keeping with the findings of Kiliaridis et al. (1985). They found that consuming ground pellets may alter masticatory function in rats.

4.3 Limitations of this study

Measuring muscle mass and monitoring electromyography (EMG) to improve our understanding of muscle activity should be attempted in future studies, to improve results and statistical analysis. Research on masticatory muscles other than the masseter muscle, including the temporal muscle and digastric muscles, will help to improve our knowledge of craniofacial morphology and the specific influence of masticatory muscles (Ueda et al., 1998).

This study demonstrated that BTX-A could be used to inhibit masticatory muscle contraction and to influence mandible growth. Even though many other studies have also shown that BTX-A can affect masticatory muscles and influence skeletal development, the specific actions of BTX-A remain unknown (Matic et al., 2007a). Therefore, further studies investigating the influence of BTX-A on muscle change and skeletal development must be performed. Clinical applications of results from these studies may offer increased possibilities for treating mandibular problems via non-invasive, non-surgical methods. However, it is important to remember that BTX-A is not widely used in the pediatric population, and safe dosages have yet to be established. Unusual systemic effects have been reported with repeated injections (Howell et al., 2007). Therefore, additional studies are needed to determine the safety profile of BTX-A.

5 Conclusions

This study demonstrated that unilateral injection of BTX-A was associated with reduced mandibular growth on the BTX-A-injected side compared with the non-injected side. Localized BTX-A injection may induce craniofacial growth changes. Although both sides of the mandible function as one unit, the skeletal effects might be unilateral.

Compliance with ethics guidelines

Chanyoung PARK, Kitae PARK, and Jiyeon KIM declare that they have no conflict of interest.

All institutional and national guidelines for the care and use of laboratory animals were followed.