Introduction
Nonsurgical treatments that can prevent or reduce the extent of the mandibular excess at an early stage are desirable. A single botulinum toxin (BTX) injection into the unilateral and bilateral masseter can regulate mandibular contour and condylar cartilage. However, BTX injection is frequency dependent when used in facelifts. This study aimed to evaluate the effect of BTX injection into the bilateral masseter at different frequencies on the mandibular contour and condylar cartilage.
Methods
In the present study, 24 female Sprague Dawley rats (4 weeks old) were divided into 3 groups: control, single injection, and triple injection. Contour measurement of the mandible was carried out by radiographic imaging. Microcomputerized tomography was performed to determine the change in bone volume in the subchondral bone. Hematoxylin and eosin staining was used to observe the morphologic changes of condylar cartilage. Immunohistochemistry was performed to detect the expression level of biomechanically sensitive factors, including transforming growth factor-β1, parathyroid hormone-related protein, SRY-box 9, and type II collagen.
Results
Bone volume and/or total volume, trabecular number, and trabecular thickness of the mineralized cartilage and subchondral bone significantly decreased in the triple injection group when compared with the single injection group. Mandibular contour also diminished after increased BTX injection frequencies. Chondrocyte proliferation ability and the expression levels of transforming growth factor-β1, parathyroid hormone-related protein, SRY-box 9, and type II collagen significantly decreased in all BTX injection groups and more in the triple injection group.
Conclusions
Morphologic changes of the mandible and condylar cartilage become more obvious after increased BTX injection frequencies, suggesting that multiple BTX injections into the masseter of patients may relieve the severity of mandibular deformity at an early stage.
Highlights
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The mandible contour is smaller after increased botulinum toxin injection frequencies.
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The proliferative level of condylar cartilage decreased after botulinum toxin injection.
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Mechanical-sensitive molecules may participate in the regulation of mandible contour.
Asymmetric mandibular deformity—often manifests as laterognathism of the mandible and hemimandibular hypertrophy , —is one of the most common dentomaxillofacial deformities. Currently, combining orthognathic surgery with orthodontic treatment is the main method to correct maxillofacial deformities, including mandibular excess. However, the optimal time for orthognathic surgery is in adulthood, when development is mostly completed. Otherwise, patients may suffer relapses because of subsequent growth potential. More importantly, soaring costs leave millions of families shackled by debt in developing counties with inadequate health care. Accordingly, searching for a nonsurgical treatment that can prevent or reduce the extent of the asymmetry at an early stage is imperative.
According to Moss’s functional matrix hypothesis, the mandible is a composited structure formed of the functional matrix (soft tissue) and skeletal units (skeletal tissue). The growth of the several functional matrices occurs first, followed by the growth of the related skeletal units in a secondary compensatory and mechanically obligatory fashion. Therefore, mandibular growth is affected by the surrounding muscles attached to it. Botulinum toxin (BTX), widely used in medical cosmetology, can lead to muscle atrophy. , Existing studies have explored the effects of masticatory muscle atrophy on maxillofacial growth by injecting BTX into animal models without surgical intervention. , Dutra et al reported that unilateral single injection of BTX in murine masseter muscle significantly reduced the bone mass and condylar width compared with the contralateral mandible without BTX treatment. A similar study demonstrated that degeneration of condylar cartilage occurred and the degree of mineralization of the cartilage and subchondral bone was decreased in rabbit mandibular condyles after a single injection of BTX.
Considering that the left and right mandibles are a combined functional unit, unilateral injection of BTX may cause deviation of the mandible. Therefore, in our previous study, 2-unit BTX solutions were injected intramuscularly into both sides of the rat masseter muscle to avoid the compensatory effect in the contralateral masseter muscle. The results showed that condylar cartilage contour was diminished because of BTX treatments. However, the effects of a single BTX injection are temporary. Clinically, in order to achieve the “face-lift” purpose, injecting BTX into the masseter muscle approximately every 6 months is generally necessary. In the present study, we explored the effects of different BTX injection frequencies into the bilateral masseter muscle on mandibular contour and condylar cartilage. Our results showed that mandibular contour diminished after increased BTX injection frequencies, suggesting that multiple injections of BTX into the masseter muscle can prevent mandibular excess.
Material and methods
This study was conducted in Sprague Dawley rats. The animals were kept in special feeding facilities under the supervision of veterinarians from the specific pathogen free Animal Laboratory of the School of Stomatology, Wuhan University. Food and water were available ad libitum. All the animal experimental protocols were approved by the Medical Ethics Committee of the Hospital of Stomatology, Wuhan University. Twenty-four 4-week-old female rats were randomly divided into 3 groups: single injection group (3 units BTX per side) injected once into the bilateral masseter, triple injection group (3 units BTX per side) injected 3 times (at 2-week intervals) into the bilateral masseter, and controls group sham injected with matching volumes of saline. Botox (Allergan, Irvine, Calif) solution was diluted with 1 unit for every 0.1 ml saline. There were no accidental deaths or loss of rats during the whole experiment. All the animals were injected intraperitoneally with sodium pentobarbital (AS1090; Aspen Technology, Bedford, Mass) at a dose of 40 mg/kg body weight for anesthesia. At 10 weeks, an overdose was administered to kill the rats.
Radiographs of the mandibles were taken with a radiography system (DXS Pro 4000; Bruker, Kontich, Belgium) at a 35 kV for 150 seconds. We determined the marker points and measured distances on mandibular radiographic images with reference to Hyun’s method. Specifically, measurement points contain coronoid notch (Co), condylion (Cd), gonion (Go), menton (Me), and gnathion (Gn). Co refers to the most inferior point of the coronoid notch. Cd refers to the most posterior and superior point on the mandibular condyle. Go refers to the most posterior point of bony contour of the gonial angle of the mandible. Me refers to the most inferior point of the mandibular symphysis. Gn refers to the most inferior point of the bony contour of the gonial angle of the mandible. Mandibular plane refers to the line from Me to Gn. Measurement distances consist of mandibular heights and mandibular lengths. Mandibular heights include CoH, CdH, and GoH. CoH, CdH, and GoH refer to the vertical distance from Co, Cd, and Go to mandibular plane, respectively. Mandibular lengths include CoL, CdL, and GoL. CoL, CdL, and GoL refer to the distance from Co, Cd, and Go to Me, respectively. The distances were measured using Adobe Photoshop CS6 (version 7.0; Adobe Systems, San Jose, Calif). All animal samples were measured 3 times for statistical analysis by researchers who were blinded as to the treatment the rats received.
To examine mineralized cartilage and subchondral bone, we scanned the condylar process by microcomputerized tomography (microCT) (Skyscan 1176; Bruker) equipped with an x-ray tube operating at 58 kV and 431 μA. The tube was fixed on a brass stub with tape and analyzed with an image pixel size of 9 μm and a rotation step of 0.3°. A 0.5-mm aluminum filter was used to lessen beam hardening effects and to decrease noise in the images. With reference to Souza’s method, to encompass only the condylar head, 354 consecutive slices of each sample were chosen, resulting in a cylinder (height, 3.186 mm; bottom diameter, 6.000 mm), namely, the volume of interest. NRecon software (version 1.7.4.6; Bruker) was used to reconstruct, and CTAn (version 1.18.8.0+; Bruker) was used to analyze structural parameters including cortical bone volume (BV), bone volume and/or total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular space (Tb.Sp).
The right and the left mandibles were dissected from the killed animals. The specimens were fixed in 4% paraformaldehyde solution at room temperature for 24 hours and were then decalcified in 10% ethylene diamine tetraacetic acid for 5 weeks. After gradient dehydration in alcohol, the specimens were embedded in paraffin. Six-μm sagittal sections were made using cross-sectional sections (Leica RM2245, Wetzlar, Germany). To assess morphologic and histologic differences in condylar cartilage among the groups, we performed hematoxylin and eosin (HE) staining and immunohistochemistry staining according to our previous protocols. The primary antibodies used for immunohistochemistry staining are as follows: ki67 (A2094; Abclonal Biotechnology, Wuhan, China; 1:200), TGFβ-1 (21898-1-AP; Proteintech, Rosemont, Ill; 1:200), PTHrP (SC-20728; Santa Cruz Biotechnology, Santa Cruz, Calif), Sox9 (A2479; ABclonal Biotechnology; 1:200), Col2a1 (ab34712, Abcam, Cambridge, Mass; 1:200), and muscle RING-finger protein-1 (MuRF-1) (IMX-3924; Novus, Centennial, Colo; 1:100). Then sections were visualized using an Olympus DP72 microscope (Tokyo, Japan). For the HE staining and immunohistochemical analysis, 6 sections were selected from each rat, and under light microscopy, 4 regions were randomly selected from each section for data acquisition under blinded conditions. The overall thickness of cellular zones and thickness of the proliferative zone of condylar cartilage were measured by Photoshop CS6. Immunohistochemical images were processed using the Image-Pro Plus software (version 6.0; Media Cybernetics, Bethesda, Md) according to a previous protocol: (1) defining the area of interest, (2) setting the reference value that identifies the positive staining, (3) counting the objects automatically and generating the sum integrated optical density and sum area (area), and (4) expressing the results as a mean density (integrated optical density/area).
Statistical analysis
GraphPad Prism software (GraphPad Software, San Diego, Calif) was used for all analyses. Data were expressed as mean ± standard deviation and were analyzed by 1-way analysis of variance. A P <0.05 was considered to be statistically significant.
Results
To observe the effects of BTX injection into the bilateral masseter muscle at different frequencies on mandible contour dimensions, we examined radiographic images to measure the morphologic parameters of the mandible ( Figs 1 , A and B ). In line with our previous study, mandibular heights (CoH and CdH) in both single and triple injection BTX groups were lower than those in the control groups ( Fig 1 , C ). Notably, CdH in the triple injection group was lower than in the single injection group ( Fig 1 , D ). Mandibular lengths (CoL, CdL, and GoL) in both single and triple injection BTX groups were shorter than those in the control groups ( Figs 1 , F-H ). Similarly, CoL and CdL in the triple injection group were lower than those in the single injection group ( Figs 1 , F and G ). Triple BTX injections lead to a greater decrease in mandibular contour compared with single injections.
The morphologic changes in condylar processes were further analyzed because the condylar process is a growth site of the mandible. The microCT analysis indicated that cortical and trabecular BV in the BTX-injected groups significantly decreased compared with the control groups. Specifically, results from the single injection group showed that (1) cortical bone volume of the condylar neck lost an average of 4.65% volume ( P >0.05) ( Fig 2 , A ); (2) BV/TV decreased nearly 10.44% ( P <0.05) ( Fig 2 , B ); (3) Tb.N decreased almost 41.01% ( P <0.05) ( Fig 2 , C ); (4) Tb.Th decreased 12.8% ( P <0.05) ( Fig 2 , D ); and (5) Tb.Sp increased 5.51% ( P <0.05) ( Fig 2 , E ). Results from the triple injection group showed that (1) cortical bone volume of the condylar neck decreased an average of 24.42% ( P <0.05) ( Fig 2 , A ); (2) BV/TV decreased nearly 27.71% ( P <0.05) ( Fig 2 , B ); (3) Tb.N decreased almost 50.77% ( P <0.05) ( Fig 2 , C ); (4) Tb.Th decreased 23.38% ( P <0.05) ( Fig 2 , D ); and (5) Tb.Sp increased 7.77% ( P <0.05) ( Fig 2 , E ). To easily observe the morphologic change in the condylar process, 3-dimensional reconstruction by a microCT scan of the condyle was performed. The posterior surface of the condyle was rougher in the single injection group than in the control group ( Figs 2 , F , G , I , J , L , and M ), whereas the surface in the triple injection group had obvious crater resorption ( Figs 2 , H , K , and N ). Triple injections of BTX lead to decreased cortical and trabecular BV in the condylar process.
