Unilateral injection of botulinum toxin type A into the masseter muscle induces mandibular asymmetry in adolescent rats by suppressing the angular process growth

Introduction

Mandibular asymmetry has negative impacts on maxillofacial aesthetics and psychological well-being. This study investigated the effects of unilateral injection of botulinum toxin type A (BTX-A) into the masseter muscle on mandibular symmetry.

Methods

Forty Wistar rats (4-week-old) were divided into 4 groups (n = 10): control, group 1 (1U BTX-A), group 2 (3U BTX-A), and group 3 (1U BTX-A for 3 times). BTX-A was injected into the right masseter of treatment groups. Cone-beam computerized tomography scans were taken before the injection and then at 2 weeks, 4 weeks, and 6 weeks after injection. Histologic and immunohistochemical staining were done for the condylar cartilage. RNA sequencing and quantitative reverse transcription polymerase chain reaction were used to detect gene expression in the angular process.

Results

In Groups 2 and 3, the right angular process length and the ramus height were reduced 4 weeks after injection, resulting in the mandibular midline deviating to the right side; the right condylar cartilage had reduced thickness and decreased expression of RUNX2 , SOX9 , and COL II ( P <0.05). Two hundred sixty-one genes were differentially expressed (256 downregulated) in the angular process at 3 days post-BTX-A injection, and the calcium signaling pathway was unveiled through the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Furthermore, TRPC1 , Wnt5a , CaMKII , Ctnnb1 , and RUNX2 expression were significantly downregulated at 1 and 3 days postinjection.

Conclusions

Unilateral injection of BTX-A into the masseter muscle in adolescent rats induces mandibular asymmetry by suppressing the angular process growth on the injected side.

Highlights

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Unilateral BTX-A injection in the masseter muscle results in mandibular asymmetry.
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The angular process growth and condylar growth are suppressed on the injection side.
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Unilateral mastication in adolescents might lead to mandibular asymmetry.
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Unilateral BTX-A injection might have the potential for improving mandibular asymmetry.

Mandibular asymmetry can negatively impact facial esthetics, social psychology, and quality of life. It has been found that a skeletal deviation of >4 mm will make asymmetry visible on the human face, and the mandible is the main factor causing facial asymmetry. The clinical incidence of mandibular asymmetry has been reported to be 18% in the United States, 11% in South Korea, 23% in Belgium, 21% in Hong Kong, and 17.4% in Brazil.

It has been found that muscle activities, such as unilateral chewing habits, persistent sleep on one side, deleterious oral habits, and unilateral crossbite, can affect mandibular asymmetry. Animal experiments and clinical studies have demonstrated that masticatory muscle function could affect craniofacial development and jaw shape. An impairment of masticatory muscle activity could induce bone loss and shape changes. Bone structure of the mandibular condyle of rabbits has been found to respond to variations in mechanical loading throughout life, and masticatory load is one of the major environmental stimuli that cause craniofacial changes.

Botulinum toxin (BTX), a neurotoxin protein from the bacterium Clostridium botulinum, has been widely used in medical and dental practices. Seven serotypes (A-G) of BTX have been identified; the most effective serotype is botulinum toxin type A (BTX-A), which is also the most commonly used clinic. Synaptic vesicles store the neurotransmitter acetylcholine in the presynaptic membrane at the normal neuromuscular junction. The vesicles bind to the proteins on the surface of the nerve cell membrane and fuse through the vesicles to allow the cell to release acetylcholine into the synaptic clefts through exocytosis and eventually bind to the receptors on the muscle to cause muscle contraction. After injection of BTX-A, the cleavage of soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins, which play a key role in membrane vesicle fusion, blocks the release of acetylcholine from the presynaptic membrane and severs the connection among nerves and muscles. Because BTX-A does not damage nerve and muscle structures and is completely reversible after a few months, injection of BTX-A has been considered a safe and minimally invasive treatment.

Injection of BTX-A has been used to treat facial wrinkles and pain caused by temporomandibular joint disorders. BTX-A is also widely used for face thinning and facial symmetry restoration after it was reported to be effective in treating masseter hypertrophy. BTX-A injection into the lateral masseter muscle on the prominent side of the patients with lower facial asymmetry reduced the volume and bulkiest height on the injected side. The BTX-A-induced masticatory muscle atrophy could decrease the mandibular cortical bone quality. In an animal study, BTX-A was injected in the masseter muscle on one side of the growing rats, leading to muscle atrophy and alterations of craniofacial bone growth; however, only gross anthropometric measurements at the endpoint were done. An interesting case report showed that the facial asymmetry of a growing patient was improved in a short period with BTX-A injection and orthodontic treatment by altering the mandibular ramus height.

Thus, the effects of BTX-A on mandibular symmetry in growing patients require further clarification, and this study was to observe the longitudinal morphologic alterations on unilateral BTX-A injection in the masseter muscle of adolescent rats, followed by a preliminary exploration of the potential mechanisms.

Material and methods

The animal experiment protocol was approved by the Medical Ethics Committee of West China Hospital of Stomatology, Sichuan University (WCHSIRB-D-2022-618). Forty 3-week-old male Wistar rats were kept in quarantine for a week at the Animal Laboratory of Sichuan University, Chengdu, China. All animals were allowed free access to water and food.

BTX-A (Botox, Allergan Pharmaceuticals, Dublin, Ireland) was diluted with 0.9% saline solution and used in this research. The rats at the age of postnatal 4 weeks were randomly divided into 4 groups: (1) group 1 (n = 10): 1U BTX-A was injected into the right masseter muscle; (2) group 2 (n = 10): 3U BTX-A was injected into the right masseter muscle; (3) group 3 (n = 10): 3U BTX-A was injected into the right masseter muscle in 3 doses, 1 unit for each with 2 weeks apart; and control (n = 10): Matching volumes of saline (100 μL) was injected into the right masseter muscle.

Forty rats were killed with pentobarbital sodium and placed on the foam rack for fixation. All rats received cone-beam computerized tomography (CBCT) scanning (3D Accuitomo; J. Morita Corp, Kyoto, Japan) with the following settings: 86 kVp, 5 mA, a field of view of 80 × 80 mm, and a voxel size of 0.16 mm ³ . Three-dimensional (3D) image files were stored in the digital image and communication in medicine format. The Mimics software (version 21; Materialise, Leuven, Belgium) was used for 3D reconstruction of the mandible, and then the images were imported into the Geomagic studio software (version 13; 3D Systems, Rock Hill, SC) in stereolithography format for measurement.

BTX-A was injected into the right masseter muscle immediately after the first CBCT scan (T0), and then CBCT scans were performed at 2 weeks (T1), 4 weeks (T2), and 6 weeks (T3) postinjection ( Fig 1 ).

Twenty reference points and 2 reference planes were used for the measurements ( Table I ). Internasal point (In), basion (Ba), and prosthion point (Pr) were the reference points of the craniomaxillary complex ( Fig 2 , A ), and the rest were the reference points of the mandible with bilateral symmetry ( Fig 2 , B ). Three reference points of cranial-maxillary complex form the midsagittal plane (MSP) and the mandibular inferior border plane (MP) were formed through menton (Me) and gonion tangent (Go[t]) and perpendicular to the plane over condyle (Con), Me, and Go(t).

Table I

Reference points and reference planes

Points/planes	Name	Definition
In	Internasal point	Most anterior point of the internasal suture in the MSP
Pr	Prosthion point	Most inferior and anterior points on the alveolar bone between the 2 maxillary incisors
Ba	Basion	The midpoint on the anterior margin of the foramen magnum on the occipital bone
Con	Condyle	The intersection of the long axis of the condyle passing the Maf point with the surface of the condyle head
Cor	Coronoid	The most distal point of the coronoid process
Go	Gonion	The most distal point of the angular process
Go(t)	Gonion tangent	The most inferior point of the angular process
Maf	Mandibular foramen	The most superior point of mandibular foramen
Mef	Mental foramen	The most superior point of mental foramen
Me	Menton	The most inferior point of the anterior mandible
L1	Lingual alveolar point of mandibular incisor	The most anterior point of lingual alveolar point of mandibular incisor
L1M	Mandibular midline	The midpoint of bilateral L1
MSP	Midsagittal plane	A plane passing through In, Ba, and Pr
MP	Mandibular inferior border plane	A plane through Me and Go(t), perpendicular to the plane over Con, Me, Go(t)

The distance between L1M, the midpoint of the bilateral lingual alveolar point of the mandibular incisor (L1), and MSP was measured ( Fig 2 , C ). If L1M was located on the right side of MSP, it was defined as a positive value; if L1M was located on the left side of MSP, it was defined as a negative value; if L1M was located on the MSP, it was denoted as 0.

Based on the literature, the mandible was divided into 5 skeletal units (condylar process, coronoid process, angular process, mandibular body, and symphyseal region) because of differences in development, growth, and function. Because the mandibular foramen (Maf) and mental foramen (Mef) are relatively stable anatomic markers during mandibular growth and development, the reference points of each skeletal unit connected to them can be used to evaluate the growth of their respective regions ( Table II ; Fig 2 , D ).

Table II

Measurements of skeletal units, vertical height, and anteroposterior length

Measurements	Definition
Skeletal unit
Maf-Con	The condylar process length
Maf-Cor	The coronoid process length
Maf-Go	The angular process length
Maf-Mef	The mandibular body length
Mef-L1	The symphyseal region length
Vertical height
Con-MP	The ramus height
Maf-MP	The mandibular body height
Mef-MP	The symphyseal region height
Anteroposterior length
L1-Con	The length from L1 to Con
L1-Go	The length from L1 to Go

The vertical growth of the mandible was evaluated by measuring the distance from MP to Con, Maf, and Mef ( Fig 2 , E ). The anteroposterior growth of the mandible was evaluated by measuring the distance from L1 to Con and Go ( Fig 2 , F ).

All the rats were killed after the fourth CBCT at T3. Bilateral masseter muscles were separated by surgical scissors and weighed ( Fig 3 , A ). The condyles were dissected carefully, fixed in 4% paraformaldehyde for 24 hours, and then decalcified with 10% ethylene diamine tetraacetic. The decalcifying solution was changed once every 3 days. After 2 months of decalcification, the condyle specimens were embedded in paraffin and sectioned into 5 μm thick slices prepared in the sagittal direction. We selected the consecutive sections with the largest cross-sectional area of the condylar cartilage for the subsequent staining.