*p* < 0.05, vs. control group.

#### **3. Discussion**

The stimulatory effect of BoNT/A HC on neurite outgrowth has previously been verified in vitro. In this work, the intermittent administration of BoNT/A HC through intrathecal injection on a lumbar unilateral SCI rat model was used to determine the role of BoNT/A HC in the local expression of growth-associated proteins. It was then explored whether, in vitro, BoNT/A HC was able to promote re-growth by upregulating the expression of growth-associated proteins after an SCI. Unlike the peripheral nervous system (PNS), the central nervous system (CNS) is deficient in its ability to regenerate after injury [34,35]. Based on previous research, the CNS has difficulty regenerating due to the abundance of myelin inhibitory factors and the shortage of macrophages [6]. Another possible explanation is the limited or arrested initiation of intrinsic neuron growth programs in the injured CNS [7]. Because the spine is part of the CNS, most patients suffer from severe spinal dysfunction and lifelong disability due to various pathogeneses when SCIs develop. Therefore, one of the principal foci of regenerative medicine after an SCI is to explore strategies and methods which can influence the improvement of the spinal microenvironment (e.g., the exclusion of myelin inhibitory factors) or revive the intrinsic regenerative ability of neuron itself [36].

The human recombinant BoNT/A HC used in this study is a non-toxic peptide, unlike the original potential holotoxin. The results evident after treatment with BoNT/A HC can be explained by its ability to bind to membranes and the activation of related intracellular signals upon the attachment of the heavy chain to the receptor. A previous study from the same research team has demonstrated that the BoNT/A HC had a stimulatory role in promoting neurite outgrowth in Neuro-2a cell cultures. It is known that the basis for neurogenesis and neuritogenesis is the synthesis of proteins; even the myelin inhibitory factors that accumulate after SCI can be regarded as a secondary phospholipoprotein. Therefore, it can be assumed that there is a significant modification in protein expression after an SCI, and the administration of the BoNT/A HC following an SCI should have an input in protein expression. Some definite results were obtained from this study. First, the application of BoNT/A HC reversed the alterations caused by the SCI, such as the increased expression of some proteins when the SCI developed. When the BoNT/A HC was given at the same time as the injury established, the increase dropped towards the levels of the controls, which is consistent with our previous study (published in Chinese). Briefly, the main difference in the previous study are as foolows: (1) the SCI model was established with a modified needle inserted into the lumbar spinal cord at the same anatomical location as this study; and (2) different doses of BoNT/A heavy chain (2 μg, 4 μg, 6 μg and 8 μg) was injected one-time into the spinal cord cavity while the injury made. The second result observed in this study is that BoNT/A HC treatment magnified the expression of some proteins evident from the SCI, for example, the expression of GAP43 and SCG10 were obviously increased after BoNT/A HC treatment. In this study, the selection of GAP43 and SCG10 as markers (to detect the efficiency of BoNT/A HC on stimulating protein regeneration) is based on the changes of protein molecular weight on the two-dimensional gel and the Western blot. In fact, the levels of both GAP43 and SCG10 displayed a certain elevation shortly after the SCI (two days post-injury), but these declined as the post-injury period progressed. However, when the SCI model was treated with BoNT/A HC, both proteins exhibited a continuous increased expression until the end of the experiment.

The increased expression of GAP43 after injury has been considered by most researchers to be actively involved in axonal regeneration [37–41]. GAP43 is mainly involved in the sprouting and regeneration of mature axons in their phosphorylated form. Its expression was upregulated following nerve injury. SCG10, also known as stathmin-like 2 (STMN2) protein, is another regenerative protein [42]. It is mainly involved in axonal microtubule dynamics and protein transport [43–45]. The initial increase in GAP43 and SCG10 expression after the SCI, without the application of BoNT/A HC, can be explained as a limited regenerative response after spinal cord injuries, however the response ceased when the injury continued to exist. The mechanism might be related to the intervention of myelin inhibitory factors binding to their specific receptors. The continuous enhancement of GAP43 and SCG10 expression when BoNT/A HC is applied is evidence of the recombinant peptide's involvement in promoting nerve regeneration after injury. The study also gave an outline of the role BoNT/A HC plays in stimulating the in vivo sprouting of neuronal processes. When BoNT/A HC treatment was administered during the SCI's development, the length and the number of neuronal process around injury site (in both its rostral and caudal parts) increased compared to measurements in the SCI-only group. The percentage of neurons with processes was also greater than that in the SCI-only group. Additionally, it was found that the spinal motor function at the ipsilateral hindlimb was improved due to the efficiency of BoNT/A HC in the upregulation of growth-associated proteins and in the promotion of neuronal process re-growth. The ineffectiveness of BoNT/A HC on improving spinal sensory function might be attributed to the difference in the expression of the heavy chain binding receptor in sensory neurons. Besides, direct damage of the sensory neurons in injury location might contribute to the difference of sensory and motor after SCI with or without BoNT/A HC treatment. Therefore, more detailed information about these should be explored after all in the future.

#### **4. Conclusions**

The local injection with intermittent intrathecal administration of BoNT/A heavy chain to rats with SCI increased the local expression of GAP-43 and SCG 10, which might be affiliated with the regeneration of neuronal processes surrounding the injury, and might also be favorable to the relief of spinal motor dysfunction. The exact role and mechanism in vivo of BoNT/A heavy after nervous injury need to be verified in the future.

#### **5. Materials and Methods**

#### *5.1. Establishing the Rat Spinal Cord Hemi-Section Injury Model*

All experimental procedures were done in accordance with the National Institute of Health's Guide for the Care and Use of Laboratory Animals, and were approved by the Ethics Committee of Animal Research at Shanxi Medical University (IACUC2017-001) on 20 January 2017. Every effort was made to minimize animal suffering and the number of animals sacrificed.

Sprague-Dawley rats (6–7 weeks old, and weighing between 200 and 220 g) were provided by Beijing Haidian Experimental Animal Farm (No. SCXK (Beijing) 2014-0013). As there is no difference regarding spinal cord injury study, male rats were used in this research. The rats were deeply anaesthetized with 1% pentobarbital sodium (at a dose of 4 mL/kg body weight) and given a laminectomy at vertebral level T9/10 to expose the spinal cord. Briefly, the T9 and T10 vertebrae were first orientated using the iliac crest as an anatomical landmark. The lumbar spinal cord was exposed by removing the T9 vertebra dorsally. A unilateral (left) lumbar spinal injury was achieved by nipping against the left side of the dorsal median vein with the tip of a pair of fine forceps. Left hindlimb paralysis was regarded as the sign of success in achieving a unilateral SCI. Meanwhile, the loss of motor function (based on hindlimb grasp power measurement) and the abnormality of sensory function (based on an assay of thermal hyperalgesia) after surgery were used to evaluate the severity of spinal cord function impact. The animals were placed in a temperature-controlled (24 ◦C ± 1 ◦C) chamber under a 12 h light/dark cycle. Standard amounts of food and tap water were given daily.

#### *5.2. BoNT/A Heavy Chain Administration and Groups*

Recombinant BoNT-A HC was purchased from List Biological Laboratories Inc. (Campbell, CA, USA). Intermittent administration of BoNT/A HC applied in the study was done via two routes: (1) local application of the BoNT/A HC directly onto injury site (4 μg/μL in 16 μL saline); and (2) administration of BoNT/A HC via a lumbar intrathecal catheter [46]. After establishing the rat model, 2 μg/μL of BoNT/A HC were administered every week to the SCI in the BoNT/A HC treatment groups. Animals were divided into the following groups, with six rats in each group: (1) the control (or pseudo surgery) group for which the skin incisions were made, the laminae were cut, and the spinal cords were exposed but not injured; (2) the SCI-only group for which unilateral (left side) lumbar spinal cord injuries were made (to prevent the vehicle intervention on BoNT/A heavy chain, the same volume of sterile saline was applied in SCI-only animal with same method and period); and (3) the SCI with BoNT/A HC treatment, in which BoNT/A HC was administered every week, and the injury was made. This group was further divided into four groups (with six rats each) based on different periods post-injury, i.e., two days, one week, two weeks and four weeks. The groups of animals are summarized in Table 2.


#### *5.3. Two-Dimensional Gel Electrophoresis*

The spinal cords were fully exposed when the experiment's period was completed. Then, the segment of the cord containing the spinal lesion center and the rostral and caudal area (6 mm in total) was collected. Spinal protein was extracted using a pre-cooled lysis buffer (urea: 21 g, thiourea: 7.9 g, DTE: 0.5 g, Tris: 0.5 g, in 50 mL of DD H2O). The protein concentration was determined using the Bradford protein assay.

For the two-dimensional gel, two 150 mg protein samples from each group were loaded onto IPG strips (NL) for first-dimension isoelectric focusing (IEF). The IPG strips were then washed and equilibrated in a 2D equilibration buffer, and then they underwent a 12.5% sodium dodecyl sulfate polyacrylamide gel vertical electrophoresis. After that, the gel was stained with 0.1% silver nitrate. The gel was imaged and analyzed with the Bio-Rad gel imaging system. Some interesting protein dots from different group were chosen and compared upon their changes on size and density at the same level of molecular weight and the isoelectric point (pI). For statistical analysis, two gels were prepared. The selective dots were circled and assessed by the integrated optional density (IOD) using ImageJ software in a double-blinded way and two people who were not related to the study were asked to do the assessment. In this way, four values four each dot were obtained and analyzed.

#### *5.4. SDS-PAGE and Western Blot*

Twenty micrograms of protein homogenates were subjected to 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Subsequently, the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes for Western blotting. The PVDF membranes were blocked with 5% non-fat milk powder in a TBST wash buffer (Tris buffered saline containing 1% Tween-20) for 1 h at room temperature. The selective primary antibodies were applied onto the membrane and incubated at 4 ◦C overnight. These antibodies were the following: rabbit anti-phosphorylated GAP43 antibody (1:3000, Thermo Fisher, Waltham, MA, USA); rabbit anti-SCG10 polyclonal antibody (1:1000, Thermo Fisher, Waltham, MA, USA); and rabbit anti-GAPDH polyclonal antibody (1:5000, Bioword, Nanjing, China). The following day, the membranes were incubated with HRP-conjugated secondary antibodies (goat anti-rabbit IgG, 1:1000, Bioword, Nanjing, China) for 1 h at room temperature. The membranes were then immersed into the EasySee Western blot kit (DW101, TRAN, Beijing, China; A solution: B solution = 1; 1 plus 2 μL C solution) and made to undergo the reaction for 3 min in dark. X-ray film which corresponded to the membrane was developed in a darkroom. The protein bands were imaged and analyzed for the variation in integrated optional density (IOD), while GAPDH bands as a loading control, with Bio-Rad gel imaging system.

#### *5.5. Immunofluorescence Staining*

At the set time point, rats from each group were anesthetized lethally with 1% pentobarbital sodium. They then underwent a 30 min transcardial perfusion with ice-cold 4% paraformaldehyde for fixation purposes. A 1 cm segment of spinal cord containing the lesion center was dissected out. The samples were post-fixed in the same fixatives for another hour and subsequently immersed in a 30% sucrose solution at 4 ◦C overnight. After being embedded onto an OCT compound, a section of spinal cord 16 μm in thickness across the lesion site was cut dorsal-coronary-longitudinally by cryostat (1950-Cryostat, Leca, Nussloch, Germany).

The sections were penetrated with 0.1% Triton-X100 for 10 min, washed three times with 0.1 mol/L PBS buffer, and blocked with a blocking buffer (10% goat or donkey serum and 0.1% Triton X-100 in PBS) for 1 h. Primary antibodies (as previously listed) were incubated with the section at 4 ◦C overnight. The following day, the sections were incubated with Alexa Fluoro-594-labeled goat anti-rabbit IgG (1:500; Life Technologies, Shanghai, China) and Alexa Fluoro-488-labeled donkey anti-rabbit IgG (1:500; Life Technologies, Shanghai, China) in the dark for 1 h, at room temperature. The sections were then mounted with an antifade mountant with DAPI (2-(4-amidinophenyl)-1H-indole-6-carboxamidine, Life Technologies, Grand Island, NY, USA). These were then checked and photographed under a fluorescence microscope (Olympus IX71, Tokyo, Japan).

#### *5.6. Measurement of Neuronal Processes*

The immunofluorescence staining of SCG 10 was applied to 6–8 sections each samples. Approximately 5–7 of images were captured of each section. The imaging area was mainly at the periphery of the spinal cord lesion. The measurements included the total length of neurites, the number

of neurites, and percentage of cells with neurites in all the immunofluorescence positive cells in the image. These were taken using ImageJ software. The identification of neurites was determined and confirmed according to literature [47]. Neurites were traced from the cell body to the end of the process, and the total length was calculated for each process. Neurites were distinguished according to their soma diameter; this meant that only neurites with a length greater than the maximum diameter of the cell would be considered a real neurite.

#### *5.7. Behavioral Evaluation*

Six rats in each group were used for assessment of motor and sensory function of ipsilateral hindlimb. Alteration in sensory and motor function at the ipsilateral hindlimb of rats with an SCI or an SCI with BoNT/A HC treatment were evaluated using the thermal hyperalgesia test and the grip power test, respectively. The thermal hyperalgesia test was completed using thermal stimulation of the PWTL by the paw thermal radiation stimulation tester (SERIES 8; RWD Life Science). The response time (in seconds) for the ipsilateral paw to withdraw because of the thermal stimulus was recorded. In fact, the response time represented the latency of the stimulation of the rat's foot (accurate to 0.1 s). To avoid tissue damage caused by long time thermal stimulation, 25 s was regarded as the maximum time of PWTL. At every time point, the average value was obtained using at least three measurements. A 10 min interval was suggested between taking these measurements. The values from three or more assessments was applied to calculate the average ± SD at a certain time point.

The grip test was performed using the rats grip tester based on the manufacturer's instructions (YLS-13A, Zhishuduobao, Baoding, China). First, wrap two fore-limbs and one counter-lateral hindlimb with tape, leaving the ipsilateral hind-limb free. Then, the rat was placed gently on the grasping force tester, the rat's tail was grabbed and dragged backwards. The power (in grams) of the ipsilateral hind paw in holding the grip was automatically recorded by the instrument as the maximum rat grip power; this stands for its motor function. The value was the average of three measurements, with 15 min intervals between each measurement.

#### *5.8. Statistical Analysis*

The data were expressed as mean and standard error (SEM). The protein bands or protein spots were quantified by ImageJ software. Data were analyzed using GraphPad Prism 5.0 software and one-way ANOVA was used to analyze the significance. *p* < 0.05 was considered statistically significant.

**Acknowledgments:** We are grateful for the financial support by National Natural Science Foundation of China (81171178) and the grant for returned overseas of Shanxi Province of China (2012047). We would like to express our thanks to all of the graduates who were involved in the projects. In addition, we sincerely appreciate Julie A. Coffield at the University of Georgia for the knowledge introduction of BoNTs.

**Author Contributions:** Y.-F.W. wrote the main manuscript. Y.W. and J.L. performed experiments and collected samples. Y.W., J.B. and F.L. analyzed data and prepared figures. X.-Q.L. and Y.W. designed experiment. X.-Q.L. offered the financial support to the project. All authors approved the final version of the paper.

**Conflicts of Interest:** All of the authors declare no conflict of interest.

#### **References**


© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Correction of Malocclusion by Botulinum Neurotoxin Injection into Masticatory Muscles**

**Hyun Seok <sup>1</sup> and Seong-Gon Kim 2,\***


Received: 1 December 2017; Accepted: 31 December 2017; Published: 2 January 2018

**Abstract:** Botulinum toxin (BTX) is a neurotoxin, and its injection in masticatory muscles induces muscle weakness and paralysis. This paralytic effect of BTX induces growth retardation of the maxillofacial bones, changes in dental eruption and occlusion state, and facial asymmetry. Using masticatory muscle paralysis and its effect via BTX, BTX can be used for the correction of malocclusion after orthognathic surgery and mandible fracture. The paralysis of specific masticatory muscles by BTX injection reduces the tensional force to the mandible and prevents relapse and changes in dental occlusion. BTX injection in the anterior belly of digastric and mylohyoid muscle prevents the open-bite and deep bite of dental occlusion and contributes to mandible stability after orthognathic surgery. The effect of BTX injection in masticatory muscles for maxillofacial bone growth and dental occlusion is reviewed in this article. The clinical application of BTX is also discussed for the correction of dental malocclusion and suppression of post-operative relapse after mandibular surgery.

**Keywords:** botulinum neurotoxin; masticatory system; maxillofacial bone; dental occlusion; orthognathic surgery

**Key contribution:** BTX injection into masticatory muscles affects the maxillofacial bony growth and dental occlusion. In clinical practices, BTX injection has been used for reducing post-operative relapse after mandibular surgery.

#### **1. Introduction**

For the correction of malocclusion, the understanding of growth and development is a key component. Broken harmony between the maxilla and the mandible during growth influences dental occlusion [1]. Most discrepancies in growth are under genetic control [2]. However, the interaction between muscles and the skeleton is the front line in the battlefield of growth. For these reasons, many orthodontists have used orthopedic appliances to correct abnormal jaw bone growth.

Dental occlusion is located in the border between the buccal shelf and the tongue. A patient with a narrow dental arch is treated using the appliance, which shields the action of the buccinator muscles. To prevent tongue thrust habit, an appliance can be used for treating anterior open-bite. In the case of pediatric mandibular prognathism, a chin cap, high-pull headgear, and other types of functional appliances have been used to restrain forward growth of the mandible. However, these efforts have often not achieved their treatment goals because most patients have shown relapse [3]. Compared to young patients, the results of orthodontic treatment are poor in aged patients because of slow bony remodeling and periodontal problems [4]. In the case of maxillary molar area, 20–30% of relapse has been reported between one and three years after treatment [5,6]. As the reasons of malocclusion are many, such as genetics, environmental and habitual factors, clinicians should consider all contributing

factors [7]. Generally, relapse after treatment is associated with the severity of disease. Accordingly, intensive maintenance is required for patients needing large corrections [8].

If certain treatments can strengthen or weaken the power of specific muscles, they may replace unpleasant long-term usage of orthopedic appliances. Botulinum toxin is produced by bacterium and can weaken the power of specific muscles. If the rationale of the orthopedic appliance treatment is applied, BTX can be a highly effective treatment option for the correction of malocclusion-associated problems. Accordingly, recent knowledge regarding BTX on facial bone growth is reviewed in this article. Additionally, several frontier clinical applications of BTX are discussed.

Botulinum toxin-A (BTX) is a family of BTX and is most commonly used in clinical practice [9]. BTX reversibly reduces muscle activity and induces muscle paralysis by inhibiting the release of acetylcholine in presynaptic membrane of nerve terminal [10]. This treatment degrades synaptosomal-associated protein of 25 kDa (SNAP-25), which is required for acetylcholine secretion and release [11]. This paralytic effect of BTX has been used in various fields of oral and maxillofacial regions for the treatment of facial muscle spasms, muscle myalgia, temporomandibular disorder, masticatory muscle hypertrophy, and cosmetic purposes [12,13]. A therapeutic dose of BTX can be safely used in masticatory muscles with few complications [14].

The BTX injection in masticatory muscles influences balanced masticatory activity, including food mastication, swallowing, and breathing [15]. The BTX injection can disturb the balanced masticatory muscle activity and lead to masticatory muscle weakness and decreased mastication activity [16]. The paralytic effect of BTX in masticatory muscles influences maxillofacial bone growth when administered in the growth phase [17]. In animal studies, BTX injection in masticatory muscles has an effect on various portions of maxillofacial bone growth that are muscle-related areas and significantly decreased size and morphology [18]. The unilateral masticatory muscle weakness by BTX injection induces the maxillofacial bone hypoplasia and facial asymmetry [19]. The decrease of masticatory muscle function and bite force contributes to the changes of molar tooth eruption state and potentially affects dental occlusion [19,20].

In this review, we introduced the balanced masticatory muscle function in normal masticatory activity and the role of masticatory muscles in the masticatory system. We investigated the effect of the BTX injection in masticatory muscles on the changes of maxillofacial bone growth, dental eruption, and occlusion. We also suggested the therapeutic application of BTX for the recovery and correction of dental occlusion.

#### **2. Balanced Muscle Power in the Masticatory System**

The masticatory system is a complex functional unit composed of the maxillofacial bones, masticatory muscles, teeth, tongue, and temporomandibular joint (TMJ) [21]. This system is supplied by vascular and neuromuscular supports for function and activity [22], is controlled by the neurological system and cooperatively interacts with head and neck musculatures, ligaments, salivary gland, lips, palate, and cheek [23]. Mastication activity is highly organized and controlled neuromuscular activity that is integrated with the various masticatory system components [21]. The neurologically coordinated mastication system effectively regulates mandible movement and contracture of surrounding tissue [24]. The balanced muscle function and jaw movement contributes to normal masticatory activities, such as food intake, digestion, mastication, speech, and swallowing [15].

Mastication is a highly complex and organized neuromuscular activity involved with bone, muscles, teeth, and surrounding structure [22]. This activity requires the movement of jaw and TMJ, and masticatory muscle activity [21]. Balanced masticatory muscle activity is effectively regulated by the central nervous system [24]. The sensory input from the receptors in teeth, periodontal ligament, and TMJ is received to the brain stem and cortex through the afferent nerve [25]. The brain stem and cortex organizes this sensory input and provides motor activity output through efferent nerve fiber in masticatory muscles [24]. The integrated muscle functions are possible by the control of the central pattern generator (CPG) in the brain stem [24]. The masticatory CPG is located between rostral poles of

trigeminal and facial motor nucleus and composed of several nuclei, such as nucleus ambigus, nucleus tracti solitary [26]. A neural signal from the hypothalamus activates neurons in the medulla oblongata through the nucleus tracti solitari and elicits masticatory activity [27]. The motor activity of CPG regulates the contracture of certain muscles and the relaxation of others [28]. The CPG regulates the rhythm and timing of muscle activity such that the activities of chewing, swallowing, and breathing can be effectively performed [23].

Mastication is rhythmic and repetitive chewing action and the beginning of digestion [24]. The muscles activated during mastication are temporalis, masseter, medial and lateral pterygoid, and suprahyoid musculatures [15]. These masticatory muscles are innervated with the trigeminal nerve and receive motor activity through the trigeminal motor nucleus [15]. Each of the masticatory muscles is attached on both sides of mandible and correspondingly activated according to the jaw movement and chewing phase [29]. In the opening phase of mandible, the inferior head of lateral pterygoid and digastric muscle are activated, and the digastric muscle acts in the rotation of mandible [30]. The temporalis, masseter, and medial pterygoid muscles involve the closing phase of mandible and act on clenching and chewing the food bolus [15]. Food mastication activity is also supported by the function of the lip, tongue, and buccinators [22]. The lip and perioral muscles involve the intake of food to the oral cavity, and maintain the sealing of mouth during mastication [31]. The tongue and buccinators contribute to effective chewing by repositioning food on the occlusal surface of teeth [31].

Normal mastication activity and masticatory muscle function can influence the maxillofacial bone morphology [32]. Mandible has symmetrical bone morphology, being a mirror image [27]. This bone connects with the cranial bone by the articulation of TMJ [29]. In addition, mandible can be moved by the symmetric movement of TMJ and activation of both sides of masticatory muscles [33]. Disruption of this harmonious masticatory muscle function and movement of mandible affects the symmetric growth of maxillofacial bones [34]. In functional matrix theory, maxillofacial mandibular bone growth can be affected by attached muscle activity and surrounding soft tissue [35]. The balancing of masticatory muscle function and activity can affect harmonious maxillofacial bone morphology, jaw movement, proper dental occlusion, and TMJ function [33,36].

#### **3. Broken Balance Muscle Function by BTX Injection and Its Effects on Maxillofacial Growth**

Balanced masticatory muscle function is closely related with maxillofacial bone growth and development [32,35]. Impaired masticatory muscle activity affects the reduced growth of the craniofacial bone structure [37,38]. Animal studies that have masticatory muscle hypofunction by soft food diet, muscle myotomy, and motor nerve denervation show reduced growth of maxillofacial bone [37,39–41]. Masticatory muscle hypofunction affects the bone mass, size, and length [42,43], and also affects the composition of the trabecular bone and thickness of the cortical bone [44]. The maxillofacial bone growth can be affected by the paralytic effect of BTX when it is administered in masticatory muscles [45]. BTX is a neurotoxin that reversibly reduces muscle activity without tissue damage [12]. BTX injection in masticatory muscle can disturb the balance and symmetric growth of maxillofacial bone in growing rats [36], and affects the change of craniofacial bone dimension and composition [18,46].

#### *3.1. The Changes of Maxillofacial Bone Growth by BTX Injection in Masticatory Muscles*

BTX injection in masseter muscles decreases muscle activity and affects the maxillofacial bone growth in animal studies [16,45]. Masseter muscle is attached to the zygomatic arch and inserted to the ramus and angle of mandible [47]. With the use of unilateral BTX injection in rabbit masseter muscle, the bone volumes of zygomatic and mandibular bone are significantly reduced [43]. In addition, with BTX injection in the masseter of growing rat, the mandibular length and ramus height are also significantly reduced (Figures 1 and 2) [17]. The unilateral injection of BTX in masseter muscle induces the growth retardation of mandible (Figure 2) [17,18,48] and causes mandible deviation and facial asymmetry in adult rats (Figure 2c,d) [36]. The BTX injection in temporalis muscle also affects

craniofacial bone growth. The temporalis muscle extends from the temporal bone and to the coronoid process of mandible [18]. Rats that received BTX in unilateral temporalis muscles had a significantly reduced skull base dimension [18], and the premaxilla, maxilla, and zygomatic arch dimensions were also decreased [18]. These previous animal studies show that the hypofunction of masticatory muscle by BTX injection affects the growth potential of the involved craniofacial bone and induces morphological changes in facial bone growth [17,43].

**Figure 1.** Anthropometric measurement of ramus height and mandible length. (**a**) Ramus height II is the distance between the zygomatic arch and inferior point of antegonial notch; ramus height III is the distance between the temporozygomatic suture of zygomatic arch and inferior point of mandible; (**b**) Mandible length is the distance between posterior point of mandible condyle and anterior point of mandible crest in mandible incisor.

**Figure 2.** Unilateral Botulinum toxin (BTX) injection to the right masseter muscle induces the retardation of mandible and facial asymmetry. (**a**,**b**) The control group with saline injection to the right masseter muscle; (**c**,**d**) the experimental group with BTX injection to the right masseter muscle (red arrow; the deviation of the mandible midline to the BTX injection side).

Masticatory muscle function has an important role in maintaining the bone density and quality of skeleton [49]. Decreased muscle function affects the bone metabolism and remodeling [50], and increases osteoclastic activity and accelerates bone resorption [51]. Muscle paralysis contributes to the disruption of bone homeostasis and leads to bone degradation and morphological changes [50]. Masticatory hypofunction with a soft food diet affects the internal bone structure of mandible in growing rats [44] and shows the thinner cortical bone and reduced bone density in the mandible ramus region [44]. These changes are also observed in the BTX application in masticatory muscle. The paralytic effect of BTX in masseter muscle influences the structural changes of mandible in rat [19]. The BTX-injected side of the mandible shows the significantly reduced bone mineral content and cortical bone thickness [19,52], and the proportion of the trabecular bone area is also reduced [19]. The rats that were BTX-injected in both masseter and temporalis show significantly reduced the trabecular bone in the alveolar and condylar bone region [53]. BTX injection in temporalis muscle of rat reduces the bone mineral density in the bones associated with temporalis muscle [46].

#### *3.2. The Effect of BTX on the Growth of the Mandibular Condyle and Condylar Cartilage*

BTX injection into the masticatory muscle may influence the growth of the mandibular condyle [54]. BTX injection into the masseter muscle in mice shows the significantly reduced condylar head width [55], and the distance between the medial and lateral margins of the condylar head is also significantly reduced in growing rats [43]. In BTX injection into the masseter and temporalis muscles, the bone volume is reduced in the BTX-injected side of the condyle [53]. The BTX injection into the masticatory muscle is also related to the bone density and quality of the condyle [53,54].

In animal research, trabecular bone density and condyle thickness are significantly reduced by the BTX injection into the masseter muscle [18], and the marrow cavity and trabecular spacing area are significantly increased [53,55]. The osteoclast activity and bone turnover are decreased in the subchondral area of the condyle [55], which suggests that BTX has sufficient paralytic effects to affect condyle development and induce bony hypoplasia or mandible deviation [17,18,43]. Masticatory muscle hypofunction induced by BTX injection not only negatively affects condylar growth, size, and volume, but also bone density and quality [55].

The detailed mechanism for the change of the condylar cartilage has been unclear. BTX injection of the masticatory muscle influences the temporomandibular joint via altering masticatory loading and causes structural and cellular changes in the condylar cartilage [48]. The growing rat, after receiving a BTX injection into the masseter muscle, shows significantly thinner condylar cartilage compared to the non-injected side [46]. This structural change of the condylar cartilage is associated with a decrease in cellular proliferation and division in the proliferative zone of the cartilage [55]. Unilateral BTX injection in the masseter muscle leads to an increase in the apoptotic process and a decrease in cellular proliferation in the proliferative zone of the cartilage [48]. Decreases in chondrocyte proliferation and proteoglycan secretion are observed in the BTX injection side of the cartilage [55]. Additionally, this change is a cellular response to the decrease in loading on the condylar surface [55]. This result indicates that the muscle paralysis caused by BTX injection may have an inhibitory effect on condylar cartilage proliferation [56].

#### *3.3. The Effect of Masticatory Hypofunction by BTX on Dental Occlusion*

Masticatory muscle hypofunction caused by BTX injection decreases the bite force and affects the dental occlusion and tooth eruption [19,20]. The masseter muscle volume and weight are significantly reduced by BTX injection [19], and the maximum bite force is also decreased [57]. The weakness of the bite force is directly related to the loading on the occlusal surface and the eruption state of the tooth [45]. In animal research, rats receiving BTX injection in the masseter muscle show decreased masseter muscle size and weight and overeruptions of the lower molars and incisors [19]. Furthermore, the maxilla and mandibular molar height are also increased after BTX injection into the masseter muscle [17]. Masticatory muscle weakness caused by BTX injection affects the tooth eruption state [19,20], and this tooth overeruption can affect the facial morphological changes, such as anterior open-bite, increased anterior facial height, and dolichofacial morphology [58,59].

#### **4. Clinical Application of BTX**

The application of BTX on the perioral area has been performed for cosmetic purposes or for the treatment of temporomandibular disorder [12]. Recently, BTX application has been tried to prevent post-operative relapse after orthognathic surgery. Post-operative relapse has been reported after orthognathic surgery [60]. The main reason for post-operative relapse is the memory of masticatory muscles in their preoperative position [61]. When muscles and connective tissues are extended by jaw bone movement, the stretch receptor will be activated and attempt to restore its original length [62]. Accordingly, the prevention of postoperative relapse has been designed to resist muscular tension.

Considering that post-operative relapse after orthognathic surgery is induced by the muscular tension, the strategy for reducing muscular tension can be an effective treatment option. In this aspect, BTX injection can be a solution for the postoperative relapse. Though the literature on this issue is scarce, there are several articles on the correction of open-bite after the treatment of trauma. The open-bite can be frequently found in bilateral mandibular angle fractures and the chin is depressed by the contracture of the digastric muscles [63]. Most patients can be corrected by open reduction and intermaxillary fixation. When patients do not receive the open reduction in time, reduced segments might be unstable due to the tensional force of the digastric muscles. Similar to BTX injection, radiofrequency therapy is also effective for reducing muscular power and volume. Targeting the anterior belly of the digastric muscle, the application of radiofrequency therapy is effective for correcting post-traumatic open-bite [64]. Based on this finding, similar cases have been treated by 20-unit BTX injections into the anterior belly of the digastric muscle [65]. When the patient is in the state of open-bite, the anterior belly of the digastric muscle receives the tensional force according to the counterclockwise rotation of the mandible in the course of treatment (Figure 3). Accordingly, the mandible has a tendency of clockwise rotation after reduction, and this mechanism will contribute to relapse after treatment. In fact, BTX injection into the anterior belly of the digastric muscle has been shown to be successful, and there has been no relapse after injection (Figure 4) [65]. As improper injection of BTX in the neck may induce such complications as dysphagia, the precise localization of injection site may be important to avoid these complications [66].

**Figure 3.** Schematic illustration of relapse mechanism after open-bite correction. During the correction of anterior open-bite, the mandible was rotated in a counterclockwise direction and the anterior belly of the digastric muscle was lengthened. Accordingly, the tensional force was generated and the relapse of the open-bite could have occurred during relieving the tensional force.

In the case of malocclusion patients, the anterior open bite has been frequently observed, and this protocol can be applied for these patients. The treatment of the open bite has been challenging because it has multiple etiological factors [67]. The open bite can be caused by the imbalance of the growth between the mandible and the maxilla, airway obstruction, para-functional habits, and trauma [68]. In the case of mandibular prognathism, approximately 30% of patients show an open bite [69]. These skeletal open-bite patients show clockwise rotation of the mandible and higher anterior facial height [70]. The patients with mandibular prognathism and open bite can be corrected by surgical treatment, and the mandible is moved backward and counterclockwise after the operation [71]. Postoperative anterior open bite is caused by unstable condylar position and muscular pull [72]. Postoperative anterior open bite after orthognathic surgery is a kind of relapse, and its rate has been reported at 10 to 15% [73,74]. Many kinds of modifications have been introduced for minimizing postoperative relapse. Overcorrection is overtreatment, rather than therapeutic movement, considering repositioning of jaw bones. Distal cutting of the mandibular proximal segment has been done to reduce the tension applied on the pterygomasseteric sling after the posterior movement of the mandible. If the surgeon modifies the design of osteotomy, the amount and the type of muscles attached to each sectioned bony segment can be changed. By adapting vertical ramus osteotomy design, postoperative relapse may be reduced [75]. Myotomy, as a preventive measure of the postoperative relapse, is an aggressive approach that targets the muscle directly. Most literature claims that these modifications have been successful in reducing postoperative relapse. However, cutting additional bone and myotomy have higher rates of complications, such as bleeding and nerve damage. A number of clinicians are concerned that the duration of the therapeutic effects of BTX is temporary. However, BTX application for the prevention of postoperative relapse can be promising, considering that the greatest amount of relapse (47.8%) has been observed during the early postoperative period [76].

**Figure 4.** Schematic illustration of relapse mechanism after deep bite correction. During the correction of deep bite, the mandible was moved in a downward direction and the mylohyoid muscle was lengthened. Accordingly, the tensional force was generated and the relapse of the deep bite could have occurred while relieving the tensional force.

"Deep bite" is the opposite of open-bite. The status of malocclusion has been frequently found in mandibular retrognathism [77]. For the surgical correction of this malocclusion, the position of the mandible usually moves downward and the myohyoid muscle receives tension [78] (Figure 4). Accordingly, relapse after treatment occurs at a high frequency, regardless of treatment protocol [79,80]. There has been comparative study on this issue. BTX has been given to the myohyoid muscle to reduce tension after surgery [60]. When compared to untreated control, the BTX application group has shown significantly higher positional stability. Myotomy for the suprahyoid muscles also showed an increase in stability after the mandibular advancement, and these findings can be interpreted in that the tensional force of the suprahyoid muscles is a contributing factor for skeletal relapse [61]. Considering the complications of suprahyoid muscle myotomy [81], BTX injection is a relatively simple and effective treatment.

In the course of orthognathic surgery, patients usually prefer the intra-oral approach to the trans-buccal approach. However, compared to bi-cortical screws fixation, single plate fixation is less rigid fixation [82]. Patients who received bi-cortical screw fixation may open their mouth immediately after operation. In the case of single plate fixation, patients may be asked about intermaxillary fixation for three to four weeks. In our preliminary study [83], the patients (*n* = 7) received BTX-A injection into their masseter muscles along with two weeks of intermaxillary fixation. This group was compared to the patients (*n* = 11) who did not receive BTX treatment and the same period of intermaxillary fixation. The incidence of plate fractures was 14.3% in the BTX-injected group and 31.8% in the untreated control group (Figure 5). As the plate fracture is largely a fatigue type of fracture induced by the action of the masticatory muscles, reduced muscle power by BTX application may prevent the plate fracture. Though postoperative relapse has not been assessed, it may be reduced by BTX injection. To draw definite conclusions, further follow-up studies will be required.

**Figure 5.** BTX treatment after orthognathic surgery. (**a**) Single plate fixation group after orthognathic surgery without BTX treatment (plate fracture in red arrow); (**b**) BTX injection group in both masseter muscles after orthognathic surgery.

The application of BTX in pediatric patients has been rare. In the progress of growth, the size of muscle fibers increases [84]. In experimental research, BTX prevents the exercise-induced increase in muscle fiber size of young rats via reduction in contractile activity [85]. When the upward movement of the maxillary posterior teeth is affected by posterior impaction, overbite in the anterior teeth can be increased, and anterior open bite can be corrected [86]. Accordingly, posterior bite block can be considered nonsurgical treatment of open bite [87]. If the open bite is caused by tongue thrust habit, tongue spurs can be used to control the force generated by tongue muscles [88]. When the parafunctional habit is intervened in the early stage of growth, irreversible open bite can be prevented [89]. The correction of open bite in the children is mainly composed of functional appliance that can shield the action of perioral musculatures [89]. Though many types of functional appliances have been introduced, their therapeutic effects have not been promising due to study design limitations [90].

BTX injection into perioral muscle has been considered a relatively safe treatment. Except for periocular injection, complications related to BTX injection have been rarely reported. Recently, a case of temporary blindness has been reported after BTX injection into the masseter muscle [91]. The blindness after BTX injection may be induced by intravascular introduction of BTX [92]. When BTX is introduced into the vessel, it may induce myocardial infarction and pulmonary embolism via pro-thrombotic effect [93]. When an ocular event is observed after BTX injection, early injection of steroid may be helpful for relieving retinal pressure [94]. To avoid intravascular introduction of BTX, BTX should not be diluted too much and a small-sized needle should be used. Deep injection may increase the probability of intravascular introduction of BTX. There is no difference in the therapeutic effect between intradermal and intramuscular injection of BTX [95]. To prevent systemic effects of BTX injection, the clinician should make every effort to minimize diffusion and vascular introduction of BTX after injection.

#### **5. Conclusions**

Balanced masticatory muscle function is a key component of maxillofacial bone growth and development. The dysfunction of masticatory muscle influences the retardation of facial bone growth and disruption of dental occlusion. The BTX injection to the masticatory muscle induces reversible paralysis and weakness of muscle power. The injection of BTX in masticatory muscle disrupts the balanced function of mastication and can influence maxillofacial bone growth and dental occlusion when administered during the growth phase. The weakness of masticatory muscle activity by BTX induces the hypoplasia of maxillofacial bone in the zygoma, temporal bone, mandible, and condyle area, and the alteration of the tooth occlusion state.

Clinically, the paralysis of masticatory muscle by BTX has an effect on maintaining mandible stability and preventing changes in dental occlusion after orthognathic and mandible fracture surgery. BTX injection in digastric muscle reduces the tensional force of mandible and prevents the counterclockwise rotation of mandible and open-bite of teeth. The BTX injection in mylohyoid muscle also prevents the deep bite of teeth and postoperative relapse after orthognathic surgery. Compared with a surgery-only patient, the patient in our clinic who received BTX in both masseter muscles after orthognathic surgery showed more stable dental occlusion. The BTX injection is an effective method for the correction of dental occlusion by inducing specific masticatory muscle paralysis without major complications. In an animal growth study, the injection of BTX in masticatory muscle has an effect on the growth potential of the maxillofacial bones. Additionally, this treatment could be an effective tool for the correction of facial bone and dental occlusion in the pediatric patient. Further study will be necessary for the therapeutic use of BTX in orthopedic treatment to correct abnormal jaw bone growth and malocclusion.

**Acknowledgments:** This work was carried out with the support of the "Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01121404)" Rural Development Administration, Republic of Korea.

**Author Contributions:** Seong-Gon Kim conceived and designed the review; Seong-Gon Kim and Hyun Seok write the first draft and reviewed and wrote the paper. All of the authors read and approved the final version of the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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