Does Botulinum Toxin Injection into Masseter Muscles Affect Subcutaneous Thickness?

Does Botulinum Toxin Injection into Masseter Muscles Affect Subcutaneous Thickness? Abstract Background Botulinum toxin (BoNT) is widely used to treat masseter muscle hypertrophy. Changes in the muscle thickness have been found in many studies, but there has been no report on changes in the thickness from the skin surface to the masseter muscle. Objectives We aimed to use ultrasonography to measure not only changes in the muscle thickness but also changes in subcutaneous thickness. Methods This study enrolled 20 volunteer patients: 10 were assigned to an experimental group (injected with each side 25 U of botulinum toxin into both masseter muscles) and 10 to a control group (injected with normal saline). The thicknesses were measured before the injection and at 4, 8, and 12 weeks after the injection both at rest and during maximum muscle contraction. Results The subcutaneous thickness did not differ significantly over time either at rest (P = 0.063) or during maximal contraction (P = 0.392), or between the experimental and control groups at rest (P = 0.392) or during maximum contraction (P = 0.259). The muscle thickness in the experimental group differed significantly over time. Conclusions Botulinum toxin injection only changes the muscle thickness and does not affect the subcutaneous thickness from the skin surface to the masseter muscle. Level of Evidence: 2 Masseter muscle hypertrophy is an abnormal hypertrophy that occurs either unilaterally or bilaterally. It was first reported by Legg, since when it has been reported several times, but its etiology remains controversial.1 Surgical methods such as masseter resection have been applied as a treatment, but reversible and preservative treatment methods are required due to postoperative complications and the high prevalence of patients not accepting invasive treatments. Botulinum toxin (BoNT) is a substance made by Clostridium botulinum that causes temporary chemodenervation by suppressing acetylcholine secretion in neuromuscular junctions.2,3 This mechanism causes muscle paresis and atrophy in striated muscles. BoNT is currently used not only in FDA-approved fields such as bladder dysfunction, chronic migraine, upper limb spasticity, cervical dystonia, primary axillary hyperhidrosis, blepharospasm, and strabismus, but also off-label in various neuromuscular disorders.4 The use of BoNT to treat masseter muscle hypertrophy was first introduced in 1994 in the field of plastic surgery.5 It is still considered a safer and simpler method than invasive surgical treatment, and hence is widely used in clinics. Previous investigations of the use of BoNT in masseter muscle hypertrophy have measured its effects using photography,6 computed tomography (CT),7-12 magnetic resonance imaging (MRI),13,14 3-dimensional (3D) imaging,15-17 and ultrasonography.18-21 All of these studies found that BoNT injection causes masseter muscle atrophy that results in a decrease in the volume of the lower face. BoNT is generally thought to only affect muscle tissue, but recently there have been suggestions that it could also have effects on other tissues. Tsai et al reported decreases in cortical thickness, trabecular thickness, and bone mineral content in the mandible of adult rats after injecting BoNT unilaterally into the masseter muscle.22 Rafferty et al reported condylar bone loss in adult rabbits after injecting BoNT unilaterally into the masseter muscle.23 Kun-Darbois et al reported alveolar bone and condylar bone loss in adult rats after injecting BoNT unilaterally into the masseter and temporalis muscles.24 BoNT injection may cause changes in the thickness from the skin surface to the masseter muscle (S-M) by diffusing into adjacent tissue. However, changes in S-M thickness after BoNT injection have not been studied previously. The purpose of this study was to measure the S-M and masseter muscle thicknesses after BoNT injection to detect differences and determine if changes in S-M thickness occur. METHODS Study Design With the approval of the Yonsei University Institutional Review Board (IRB), from October 2014 to September 2015 a total of 20 patients between the ages of 20 and 40 who requested treatment for masseter muscle hypertrophy were prospectively enrolled in this study. Three researchers participated in the study: (1) the investigator assigned patients into 2 different groups and diluted BoNT and saline; (2) the injector who performed the BoNT injection; and (3) the evaluator who measured ultrasonography. The patients were randomly divided into 2 groups by the investigator, who randomly allocated patients using a computer-generated randomization scheme (Microsoft Excel, Microsoft Corp., Redmond, WA). Group assignment was kept in sealed envelopes accessible only to the investigator. The investigator who was not involved in patient assessment prepared the appropriate solutions so that the study patients, injector, and evaluator remained blinded to group assignment. The exclusion criteria were pregnancy, a history of any serious medical illnesses including drug allergy, and receiving a BoNT injection, orthodontic treatment, or plastic surgery within the previous 1 year. The taking of several drugs that can affect muscles was prohibited during the experimental period, including muscle relaxants, benzodiazepines, and anticholinergic drugs. Injection of Botulinum Toxin Type A BoNT (Meditoxin, Medytox, Ochang, Korea) was supplied as a freeze-dried powder and reconstituted to a concentration of 50 U/mL (100 U in 2 mL of sterile saline) and used immediately. A volume of 25 U of BoNT was injected into the masseter muscle bilaterally using a 1-mL syringe with a 29-G ½-inch-long needle. Injections were performed at 2 points separated by 1 cm at the center of the middle one third of the masseter muscle (Figure 1). Figure 1. View largeDownload slide Blue points indicate the BoNT injection sites. Figure 1. View largeDownload slide Blue points indicate the BoNT injection sites. Ultrasonography Procedure All scans were performed using a diagnostic ultrasound system (E-cube9, Alpinion, Seoul, Korea) with a broadband linear transducer (3.0-12.0 MHz; L3-12H, Seoul, Korea). The system was operated using the following parameters: frequency, 12.0 MHz; dynamic range, 70; gain, 50; frame rate, 59; and depth, 3.0. Ultrasound transmission gel was applied to the probe before performing the imaging procedure. The masseter muscle was scanned bilaterally along a line from the cheilion to the otobasion inferius (Figure 2). The transducer was held perpendicular to the surface of the skin throughout the imaging process, with care taken to avoid exerting excessive pressure against the skin. The transducer was kept perpendicular to the ramus by tilting it until the ramus appeared on the screen as a sharp white line. The thickness was defined as the largest distance between the ramus and surface of the masseter muscle perpendicular to the underlying ramus. Figure 2. View largeDownload slide Photograph of a 28-year-old man with reference line for ultrasonography spanning from the cheilion, which is a cephalometric point located at the corner of the mouth, to the otobasion inferius (Obi), which is a cephalometric point of attachment of the earlobe to the cheek. Figure 2. View largeDownload slide Photograph of a 28-year-old man with reference line for ultrasonography spanning from the cheilion, which is a cephalometric point located at the corner of the mouth, to the otobasion inferius (Obi), which is a cephalometric point of attachment of the earlobe to the cheek. The imaging and measurements were performed with the subjects in an upright position under the following 2 conditions (Figure 3): (1) with the teeth not clenched, and hence with the masseter muscle in a relaxed position (the physiologic resting position); and (2) with the teeth clenched, so as to elicit the maximum contraction of the masseter muscle. Figure 3. View largeDownload slide (A) Ultrasound image obtained at rest. (B) Ultrasound image obtained during maximum contraction. The arrows indicate the thicknesses of the S-M and masseter muscle (M). Figure 3. View largeDownload slide (A) Ultrasound image obtained at rest. (B) Ultrasound image obtained during maximum contraction. The arrows indicate the thicknesses of the S-M and masseter muscle (M). The measurements were made directly on the images obtained at the time of scanning. The S-M and masseter muscle thicknesses were measured four times using ultrasonography: before the injection and at 4, 8, and 12 weeks after the injection. Statistical Analysis Data were analyzed using SPSS software (version 20, SPSS, Chicago, IL). The thicknesses of the S-M and the muscle were measured both at rest and during maximum contraction as functions of time. Two-way repeated-measures analysis of variance was used to evaluate the statistical significance of changes in thickness over time and by group. Mauchly’s sphericity test was used to verify the independent variable. When the probability value obtained in Mauchly’s sphericity test was less than 0.05, Greenhouse-Geisser (epsilon < 0.75) and Huynh-Feldt (epsilon > 0.75) corrections were used to modify the degrees of freedom. Probability values were considered to be indicative of statistical significance when they were less than 0.05. Data are presented as mean and SD values. RESULTS A total of 20 patients were randomly divided into 2 groups and received BoNT and saline injections. 10 patients (3 men and 7 women) aged 21 to 40 years (mean age, 29.8 years) received a single BoNT injection (experimental group), whereas the remaining 10 patients (4 men and 6 women) aged 27 to 40 years (mean age, 31.9 years) received saline injections (control group). Of the 20 study patients, 3 patients in the control group formally withdrew refusing further participation in the study because of loss to follow up. There had been no reports of side effects during this study. The masseter muscle thickness was consistently decreased at rest (14.19 ± 2.13 mm at preinjection, 12.51 ± 3.23 mm at 4 weeks, 11.76 ± 2.67 mm at 8 weeks, 11.27 ± 2.85 mm at 12 weeks and total decrease was 2.92 mm) and during maximum contraction (16.58 ± 2.17 mm at preinjection, 14.04 ± 3.48 mm at 4 weeks, 13.72 ± 2.87 mm at 8 weeks, 13.24 ± 2.97 mm at 12 weeks and total decrease was 3.34 mm) in the experimental group. Table 1 lists the changes in the S-M and masseter muscle thicknesses at rest. The S-M thickness did not differ significantly over time (P = 0.063) or by group (P = 0.392), and there was no significant interaction between time and group (P = 0.823). In contrast, there was a significant interaction between time and group for the masseter muscle thickness (P = 0.024), and a large decrease in its thickness was seen in the experimental group compared to the control group (Figure 4). Table 1. S-M and Masseter Muscle Thicknesses and Differences Therein at Resta   Preinjection  4 weeks  8 weeks  12 weeks  Time  Group  Time group  Mean ± SD  Mean ± SD  Mean ± SD  Mean ± SD  P  P  P  S-Mb  Exp  5.79 ± 0.97  5.55 ± 1.12  5.73 ± 1.10  5.91 ± 1.43  0.063  0.392  0.823  Con  5.41 ± 0.68  5.20 ± 0.80  5.15 ± 0.62  5.55 ± 0.92  Mc  Exp  14.19 ± 2.13  12.51 ± 3.23  11.76 ± 2.67  11.27 ± 2.85  0.001*  0.121  0.024*  Con  14.84 ± 2.93  14.60 ± 2.75  14.28 ± 2.31  14.30 ± 2.50    Preinjection  4 weeks  8 weeks  12 weeks  Time  Group  Time group  Mean ± SD  Mean ± SD  Mean ± SD  Mean ± SD  P  P  P  S-Mb  Exp  5.79 ± 0.97  5.55 ± 1.12  5.73 ± 1.10  5.91 ± 1.43  0.063  0.392  0.823  Con  5.41 ± 0.68  5.20 ± 0.80  5.15 ± 0.62  5.55 ± 0.92  Mc  Exp  14.19 ± 2.13  12.51 ± 3.23  11.76 ± 2.67  11.27 ± 2.85  0.001*  0.121  0.024*  Con  14.84 ± 2.93  14.60 ± 2.75  14.28 ± 2.31  14.30 ± 2.50  Con, control group; Exp, experimental group; M: thickness of masseter muscle (mm); S-M: thickness from skin surface to masseter muscle (mm). aMain effects and interactions were tested by two-way repeated-measures analysis of variance (ANOVA). bNo significant interaction and main effect of time and group. cSignificant interaction of time and group (with Greenhouse-Geisser correction, epsilon = 0.650, F = 4.279, P = 0.024) and significant main effect of time (with Greenhouse-Geisser correction, epsilon = 0.650, F = 9.677, P = 0.001). No significant main effect of group. *P < 0.05. View Large Figure 4. View largeDownload slide Mean S-M and masseter muscle thicknesses at rest. Exp, experimental group; Con, control group. Figure 4. View largeDownload slide Mean S-M and masseter muscle thicknesses at rest. Exp, experimental group; Con, control group. Table 2 lists the S-M and masseter muscle thicknesses and the differences therein over time and by group during maximum contraction. The S-M thickness did not differ significantly over time (P = 0.166) or by group (P = 0.259), and there was no significant interaction between time and group (P = 0.115). In contrast, there was a significant interaction between time and group for the masseter muscle thickness (P = 0.025), and a large decrease in its thickness was seen in the experimental group compared to the control group (Figure 5). Table 2. S-M and Masseter Muscle Thicknesses and Differences Therein During Maximum Contractiona   Preinjection  4 weeks  8 weeks  12 weeks  Time  Group  Time group  Mean ± SD  Mean ± SD  Mean ± SD  Mean ± SD  P  P  P  S-Mb  Exp  4.84 ± 0.92  5.42 ± 1.19  5.14 ± 1.12  5.30 ± 1.24  0.166  0.259  0.115  Con  4.75 ± 0.66  4.69 ± 0.70  4.45 ± 0.58  4.70 ± 0.86  Mc  Exp  16.58 ± 2.17  14.04 ± 3.48  13.72 ± 2.87  13.24 ± 2.97  0.002*  0.082  0.025*  Con  17.01 ± 2.25  16.56 ± 2.09  14.46 ± 2.13  16.47 ± 1.84    Preinjection  4 weeks  8 weeks  12 weeks  Time  Group  Time group  Mean ± SD  Mean ± SD  Mean ± SD  Mean ± SD  P  P  P  S-Mb  Exp  4.84 ± 0.92  5.42 ± 1.19  5.14 ± 1.12  5.30 ± 1.24  0.166  0.259  0.115  Con  4.75 ± 0.66  4.69 ± 0.70  4.45 ± 0.58  4.70 ± 0.86  Mc  Exp  16.58 ± 2.17  14.04 ± 3.48  13.72 ± 2.87  13.24 ± 2.97  0.002*  0.082  0.025*  Con  17.01 ± 2.25  16.56 ± 2.09  14.46 ± 2.13  16.47 ± 1.84  aMain effects and interactions were tested by two-way repeated-measures ANOVA. bNo significant interaction and main effect of time and group. cSignificant interaction of time and group (with Greenhouse-Geisser correction, epsilon = 0.484, F = 4.956, P = 0.025) and significant main effect of time (with Greenhouse-Geisser correction, epsilon = 0.650, F = 9.907, P = 0.002). No significant main effect of group. * P < 0.05. View Large Figure 5. View largeDownload slide Mean S-M and masseter muscle thicknesses during maximum contraction. Figure 5. View largeDownload slide Mean S-M and masseter muscle thicknesses during maximum contraction. DISCUSSION A square face is caused by a complex mechanism involving protrusion of the mandibular angle and hypertrophy of the masseter muscle.25 Many Koreans have a wide mandible that is typical of Mongolian races. Moreover, traditional Korean food is mostly rough and hard, which promotes the development of the masseter muscle. However, a common standard of beauty in Asian women is having a slim face and soft-looking features, which can result in a square face being considered a problem that needs to be treated.26,27 Masseter muscle hypertrophy is a common cause of a square face, and it is mostly due to parafunction such as bruxism or clenching, as well as dietary habits. Various treatments have been introduced for reducing the masseter muscle volume. This can be achieved surgically, but such invasive interventions are associated with side effects such as postoperative pain, delayed healing time, bleeding, and nerve injury.28 BoNT is one of the neurotoxin proteins (A, B, C1, C2, D, E, F, and G) produced by Clostridium botulinum, which is an anaerobe that causes food poisoning. Clostridium botulinum was first extracted in 1897 by Van Ermengen from a dead body and salted pork, and BoNT was separated and refined in 1946 by Edward J. Schantz. In 1949, Burgen et al reported that BoNT suppressed muscle contraction by depressing acetylcholine secretion at cholinergic nerve terminals.2,29 These actions of BoNT mean that injecting it into the masseter muscle results in reversible decreases in the contraction force and volume of the muscle. BoNT started to replace surgical methods in many clinics during the 1990s.30 Previous studies utilizing photography, CT, MRI, 3D imaging, and ultrasonography to detect changes have demonstrated that BoNT injection is effective in decreasing the size of the masseter muscle.6-21 CT, MRI, and ultrasonography can also be used to measure the S-M thickness. Performing repeated CT-based measurements will increase the patient’s exposure to ionizing radiation. While MRI does not have this problem, it is expensive and difficult to acquire subdivided images, hindering the acquisition of exact values. In the clinic, ultrasonography is favored since it is fast, easy, and harmless. Previous studies that used ultrasonography to measure the thickness of the masseter muscle employed different measuring references. For example, Volk et al placed the probe transversely at the middle of the zygomatic bone and mandibular angle, while other studies simply performed measurements at the point of maximum thickness or no reference point was reported. Emshoff et al reported that the masseter muscle was thickest in its middle area when it was divided into 5 sections from its origin to insertion point.31 Raadsheer et al reported strong correlations between measurements of the masseter muscle using MRI and ultrasonography, with the strongest correlations for measurements at the middle of the masseter muscle.14 Suh et al reported that the maximum thickness of the masseter muscle from the mandibular angle in Koreans was 27.77 mm in males and 25.79 mm in females, which occurred 53% and 59% from the mandibular angle, respectively.32 Therefore, in the present study we attempted to minimize errors due to changes in the reference point in repeated measurements of the maximum thicknesses of S-M and masseter muscle by using the line from the cheilion to the otobasion inferius as a reference. BoNT injection can also directly affect adjacent tissue other than the masseter muscle due to drug diffusion or indirectly affect the S-M thickness by weakening the chewing force. The S-M layer consists of epidermis, dermis, and subcutaneous tissue. The subcutaneous tissue is mainly composed of fat tissue, which is very closely related to body weight.33 However, there was no subject who reported changes in body weight in this study. The BoNT diffusion potency has been studied by several authors. Studies in experimental animals found that injecting 5 to 10 U of BoNT resulted in biologic activity at 4.5 cm from the injection site within the targeted muscle,34 that the muscle fascia did not act as a barrier to diffusion of the toxin,35 and that doses of 10 U had biologic effects that spread across the fascial planes to adjacent, noninjected muscles for 2.5 to 4.5 cm from the injection site.34,36 However, muscles are not present in the S-M layer, and so even if BoNT diffuses into this layer, it may still not exert any direct effects. In this study we measured the thicknesses in snapshots captured from videos recorded when using ultrasonography. A reference point was determined at rest, and then the subject made a clenching motion to contract the muscle. The thicknesses of the thickest points of the muscle and S-M were measured during the maximum contraction, and this was thicker than the same reference area measured at rest. Ideally the pressure applied to the skin by the ultrasound probe should have been the same when making the measurement in the maximum contraction and rest states. Moreover, it was especially difficult when repositioning the probe to apply the same pressure at the same measuring point. Also, the patient could maintain the maximum contraction force for only a few seconds, which made it technically impossible to reposition the probe at the correct time. However, the technique used in this study can be considered to accurate reflect the situations encountered in clinical applications. Clenching the masseter muscle tends to push the ultrasound probe away due to the contraction of the muscle, which will cause an increase in thickness and volume. Therefore, considerable skill is required from the investigator to ensure that an even pressure is maintained. The average S-M thickness before BoNT injection was 5.63 mm at rest and 4.80 mm during the maximum contraction. This decrease during the maximum contraction compared to the rest state may have been due to the masseter muscle pressing on the subcutaneous tissue during contraction or stretching of subcutaneous tissue during clenching. We did not find a statistically significant relationship between the changes in the masseter muscle thickness and the S-M thickness. However, this could have been due to the small number of subjects in the experimental group, and so this is worth studying in future experiments. The possibility of the S-M thickness decreasing due to pressure from the thickened muscle when it is contracting cannot be excluded. CT and MRI do not involve applying pressure on the muscle, and so these methods would be the best choice for minimizing measurement errors. However, maintaining a consistent maximum contraction state during CT or MRI is likely to be difficult to achieve in the clinical situation. This should also be taken into consideration in future studies. Suh et al found that the depth of the masseter muscle from the skin surface when using CT was significantly greater in females (7.37 ± 1.93 mm) than in males (6.15 ± 2.90 mm).32 This is probably related to differences in body fat between males and females. In our study, the average subcutaneous tissue thickness at rest before the injection was 5.63 mm, with a maximum of 8.04 mm. It is therefore obvious that the needle needs to be longer than 8 mm when injecting BoNT into the masseter muscle, and so using a 29-G ½-inch-long needle is recommended. Recent studies have raised the possibility that BoNT may affect not only muscles but other adjacent tissues.22-24 The results of this study suggest that BoNT injection does not affect the S-M thickness, but the limitation of this study is the small sample size. Future studies should include a larger sample size. CONCLUSION This study measured the S-M and masseter muscle thicknesses before and after BoNT injection, and analyzed differences between the experimental and control groups over time. The masseter muscle thickness in the experimental group decreased significantly over time, whereas the S-M thickness did not differ significantly between the experimental and control groups. It can therefore be concluded that BoNT only affects muscles, and does not affect the surrounding subcutaneous tissues. Disclosures The authors declared no potential conflicts of interest with respect to the research, authorship, and publication of this article. Funding The authors received no financial support for the research, authorship, and publication of this article. REFERENCES 1. Legg J. Enlargement of the temporal and masseter muscles on both sides. Trans Pathol Soc London . 1880; 31: 361. 2. Burgen AS, Dickens F, Zatman LJ. The action of botulinum toxin on the neuro-muscular junction. J Physiol . 1949; 109( 1-2): 10- 24. Google Scholar CrossRef Search ADS PubMed  3. Wheeler A, Smith HS. Botulinum toxins: mechanisms of action, antinociception and clinical applications. Toxicology . 2013; 306: 124- 146. Google Scholar CrossRef Search ADS PubMed  4. Bigalke H. Botulinum toxin: application, safety, and limitations. Curr Top Microbiol Immunol . 2013; 364: 307- 317. Google Scholar PubMed  5. Moore AP, Wood GD. The medical management of masseteric hypertrophy with botulinum toxin type A. Br J Oral Maxillofac Surg . 1994; 32( 1): 26- 28. Google Scholar CrossRef Search ADS PubMed  6. Castro WH, Gomez RS, Da Silva Oliveira J, Moura MD, Gomez RS. Botulinum toxin type A in the management of masseter muscle hypertrophy. J Oral Maxillofac Surg . 2005; 63( 1): 20- 24. Google Scholar CrossRef Search ADS PubMed  7. Park MY, Ahn KY, Jung DS. Botulinum toxin type A treatment for contouring of the lower face. Dermatol Surg . 2003; 29( 5): 477- 83; discussion 483. Google Scholar PubMed  8. Kim JH, Shin JH, Kim ST, Kim CY. Effects of two different units of botulinum toxin type a evaluated by computed tomography and electromyographic measurements of human masseter muscle. Plast Reconstr Surg . 2007; 119( 2): 711- 717. Google Scholar CrossRef Search ADS PubMed  9. No YA, Ahn BH, Kim BJ, Kim MN, Hong CK. Three dimensional CT might be a potential evaluation modality in correction of asymmetrical masseter muscle hypertrophy by botulinum toxin injection. J Cosmet Laser Ther . 2016; 18( 2): 1- 12. Google Scholar CrossRef Search ADS PubMed  10. Chang CS, Bergeron L, Yu CC, Chen PK, Chen YR. Mandible changes evaluated by computed tomography following Botulinum Toxin A injections in square-faced patients. Aesthetic Plast Surg . 2011; 35( 4): 452- 455. Google Scholar CrossRef Search ADS PubMed  11. Yu CC, Chen PK, Chen YR. Botulinum toxin a for lower facial contouring: a prospective study. Aesthetic Plast Surg . 2007; 31( 5): 445- 51; discussion 452. 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[Analysis of the complications following correction of mandibular angle prominence]. Zhonghua Zheng Xing Wai Ke Za Zhi . 2003; 19( 5): 364- 366. Google Scholar PubMed  29. Jankovic J, Brin MF. Botulinum toxin: historical perspective and potential new indications. Muscle Nerve Suppl . 1997; 6: S129- S145. Google Scholar CrossRef Search ADS PubMed  30. Jankovic J, Brin MF. Therapeutic uses of botulinum toxin. N Engl J Med . 1991; 324( 17): 1186- 1194. Google Scholar CrossRef Search ADS PubMed  31. Emshoff R, Emshoff I, Rudisch A, Bertram S. Reliability and temporal variation of masseter muscle thickness measurements utilizing ultrasonography. J Oral Rehabil . 2003; 30( 12): 1168- 1172. Google Scholar CrossRef Search ADS PubMed  32. Suh HW, Kim HS, Ha KYet al.  . Anatomical Measurement of the Masseter Muscle and Surface Mapping of the Maximal Thickness Point Using Computed Tomography Analysis. J Korean Soc Plast Reconstr Surg . 2011; 38( 2): 173- 181. 33. 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Does Botulinum Toxin Injection into Masseter Muscles Affect Subcutaneous Thickness?

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Abstract

Abstract Background Botulinum toxin (BoNT) is widely used to treat masseter muscle hypertrophy. Changes in the muscle thickness have been found in many studies, but there has been no report on changes in the thickness from the skin surface to the masseter muscle. Objectives We aimed to use ultrasonography to measure not only changes in the muscle thickness but also changes in subcutaneous thickness. Methods This study enrolled 20 volunteer patients: 10 were assigned to an experimental group (injected with each side 25 U of botulinum toxin into both masseter muscles) and 10 to a control group (injected with normal saline). The thicknesses were measured before the injection and at 4, 8, and 12 weeks after the injection both at rest and during maximum muscle contraction. Results The subcutaneous thickness did not differ significantly over time either at rest (P = 0.063) or during maximal contraction (P = 0.392), or between the experimental and control groups at rest (P = 0.392) or during maximum contraction (P = 0.259). The muscle thickness in the experimental group differed significantly over time. Conclusions Botulinum toxin injection only changes the muscle thickness and does not affect the subcutaneous thickness from the skin surface to the masseter muscle. Level of Evidence: 2 Masseter muscle hypertrophy is an abnormal hypertrophy that occurs either unilaterally or bilaterally. It was first reported by Legg, since when it has been reported several times, but its etiology remains controversial.1 Surgical methods such as masseter resection have been applied as a treatment, but reversible and preservative treatment methods are required due to postoperative complications and the high prevalence of patients not accepting invasive treatments. Botulinum toxin (BoNT) is a substance made by Clostridium botulinum that causes temporary chemodenervation by suppressing acetylcholine secretion in neuromuscular junctions.2,3 This mechanism causes muscle paresis and atrophy in striated muscles. BoNT is currently used not only in FDA-approved fields such as bladder dysfunction, chronic migraine, upper limb spasticity, cervical dystonia, primary axillary hyperhidrosis, blepharospasm, and strabismus, but also off-label in various neuromuscular disorders.4 The use of BoNT to treat masseter muscle hypertrophy was first introduced in 1994 in the field of plastic surgery.5 It is still considered a safer and simpler method than invasive surgical treatment, and hence is widely used in clinics. Previous investigations of the use of BoNT in masseter muscle hypertrophy have measured its effects using photography,6 computed tomography (CT),7-12 magnetic resonance imaging (MRI),13,14 3-dimensional (3D) imaging,15-17 and ultrasonography.18-21 All of these studies found that BoNT injection causes masseter muscle atrophy that results in a decrease in the volume of the lower face. BoNT is generally thought to only affect muscle tissue, but recently there have been suggestions that it could also have effects on other tissues. Tsai et al reported decreases in cortical thickness, trabecular thickness, and bone mineral content in the mandible of adult rats after injecting BoNT unilaterally into the masseter muscle.22 Rafferty et al reported condylar bone loss in adult rabbits after injecting BoNT unilaterally into the masseter muscle.23 Kun-Darbois et al reported alveolar bone and condylar bone loss in adult rats after injecting BoNT unilaterally into the masseter and temporalis muscles.24 BoNT injection may cause changes in the thickness from the skin surface to the masseter muscle (S-M) by diffusing into adjacent tissue. However, changes in S-M thickness after BoNT injection have not been studied previously. The purpose of this study was to measure the S-M and masseter muscle thicknesses after BoNT injection to detect differences and determine if changes in S-M thickness occur. METHODS Study Design With the approval of the Yonsei University Institutional Review Board (IRB), from October 2014 to September 2015 a total of 20 patients between the ages of 20 and 40 who requested treatment for masseter muscle hypertrophy were prospectively enrolled in this study. Three researchers participated in the study: (1) the investigator assigned patients into 2 different groups and diluted BoNT and saline; (2) the injector who performed the BoNT injection; and (3) the evaluator who measured ultrasonography. The patients were randomly divided into 2 groups by the investigator, who randomly allocated patients using a computer-generated randomization scheme (Microsoft Excel, Microsoft Corp., Redmond, WA). Group assignment was kept in sealed envelopes accessible only to the investigator. The investigator who was not involved in patient assessment prepared the appropriate solutions so that the study patients, injector, and evaluator remained blinded to group assignment. The exclusion criteria were pregnancy, a history of any serious medical illnesses including drug allergy, and receiving a BoNT injection, orthodontic treatment, or plastic surgery within the previous 1 year. The taking of several drugs that can affect muscles was prohibited during the experimental period, including muscle relaxants, benzodiazepines, and anticholinergic drugs. Injection of Botulinum Toxin Type A BoNT (Meditoxin, Medytox, Ochang, Korea) was supplied as a freeze-dried powder and reconstituted to a concentration of 50 U/mL (100 U in 2 mL of sterile saline) and used immediately. A volume of 25 U of BoNT was injected into the masseter muscle bilaterally using a 1-mL syringe with a 29-G ½-inch-long needle. Injections were performed at 2 points separated by 1 cm at the center of the middle one third of the masseter muscle (Figure 1). Figure 1. View largeDownload slide Blue points indicate the BoNT injection sites. Figure 1. View largeDownload slide Blue points indicate the BoNT injection sites. Ultrasonography Procedure All scans were performed using a diagnostic ultrasound system (E-cube9, Alpinion, Seoul, Korea) with a broadband linear transducer (3.0-12.0 MHz; L3-12H, Seoul, Korea). The system was operated using the following parameters: frequency, 12.0 MHz; dynamic range, 70; gain, 50; frame rate, 59; and depth, 3.0. Ultrasound transmission gel was applied to the probe before performing the imaging procedure. The masseter muscle was scanned bilaterally along a line from the cheilion to the otobasion inferius (Figure 2). The transducer was held perpendicular to the surface of the skin throughout the imaging process, with care taken to avoid exerting excessive pressure against the skin. The transducer was kept perpendicular to the ramus by tilting it until the ramus appeared on the screen as a sharp white line. The thickness was defined as the largest distance between the ramus and surface of the masseter muscle perpendicular to the underlying ramus. Figure 2. View largeDownload slide Photograph of a 28-year-old man with reference line for ultrasonography spanning from the cheilion, which is a cephalometric point located at the corner of the mouth, to the otobasion inferius (Obi), which is a cephalometric point of attachment of the earlobe to the cheek. Figure 2. View largeDownload slide Photograph of a 28-year-old man with reference line for ultrasonography spanning from the cheilion, which is a cephalometric point located at the corner of the mouth, to the otobasion inferius (Obi), which is a cephalometric point of attachment of the earlobe to the cheek. The imaging and measurements were performed with the subjects in an upright position under the following 2 conditions (Figure 3): (1) with the teeth not clenched, and hence with the masseter muscle in a relaxed position (the physiologic resting position); and (2) with the teeth clenched, so as to elicit the maximum contraction of the masseter muscle. Figure 3. View largeDownload slide (A) Ultrasound image obtained at rest. (B) Ultrasound image obtained during maximum contraction. The arrows indicate the thicknesses of the S-M and masseter muscle (M). Figure 3. View largeDownload slide (A) Ultrasound image obtained at rest. (B) Ultrasound image obtained during maximum contraction. The arrows indicate the thicknesses of the S-M and masseter muscle (M). The measurements were made directly on the images obtained at the time of scanning. The S-M and masseter muscle thicknesses were measured four times using ultrasonography: before the injection and at 4, 8, and 12 weeks after the injection. Statistical Analysis Data were analyzed using SPSS software (version 20, SPSS, Chicago, IL). The thicknesses of the S-M and the muscle were measured both at rest and during maximum contraction as functions of time. Two-way repeated-measures analysis of variance was used to evaluate the statistical significance of changes in thickness over time and by group. Mauchly’s sphericity test was used to verify the independent variable. When the probability value obtained in Mauchly’s sphericity test was less than 0.05, Greenhouse-Geisser (epsilon < 0.75) and Huynh-Feldt (epsilon > 0.75) corrections were used to modify the degrees of freedom. Probability values were considered to be indicative of statistical significance when they were less than 0.05. Data are presented as mean and SD values. RESULTS A total of 20 patients were randomly divided into 2 groups and received BoNT and saline injections. 10 patients (3 men and 7 women) aged 21 to 40 years (mean age, 29.8 years) received a single BoNT injection (experimental group), whereas the remaining 10 patients (4 men and 6 women) aged 27 to 40 years (mean age, 31.9 years) received saline injections (control group). Of the 20 study patients, 3 patients in the control group formally withdrew refusing further participation in the study because of loss to follow up. There had been no reports of side effects during this study. The masseter muscle thickness was consistently decreased at rest (14.19 ± 2.13 mm at preinjection, 12.51 ± 3.23 mm at 4 weeks, 11.76 ± 2.67 mm at 8 weeks, 11.27 ± 2.85 mm at 12 weeks and total decrease was 2.92 mm) and during maximum contraction (16.58 ± 2.17 mm at preinjection, 14.04 ± 3.48 mm at 4 weeks, 13.72 ± 2.87 mm at 8 weeks, 13.24 ± 2.97 mm at 12 weeks and total decrease was 3.34 mm) in the experimental group. Table 1 lists the changes in the S-M and masseter muscle thicknesses at rest. The S-M thickness did not differ significantly over time (P = 0.063) or by group (P = 0.392), and there was no significant interaction between time and group (P = 0.823). In contrast, there was a significant interaction between time and group for the masseter muscle thickness (P = 0.024), and a large decrease in its thickness was seen in the experimental group compared to the control group (Figure 4). Table 1. S-M and Masseter Muscle Thicknesses and Differences Therein at Resta   Preinjection  4 weeks  8 weeks  12 weeks  Time  Group  Time group  Mean ± SD  Mean ± SD  Mean ± SD  Mean ± SD  P  P  P  S-Mb  Exp  5.79 ± 0.97  5.55 ± 1.12  5.73 ± 1.10  5.91 ± 1.43  0.063  0.392  0.823  Con  5.41 ± 0.68  5.20 ± 0.80  5.15 ± 0.62  5.55 ± 0.92  Mc  Exp  14.19 ± 2.13  12.51 ± 3.23  11.76 ± 2.67  11.27 ± 2.85  0.001*  0.121  0.024*  Con  14.84 ± 2.93  14.60 ± 2.75  14.28 ± 2.31  14.30 ± 2.50    Preinjection  4 weeks  8 weeks  12 weeks  Time  Group  Time group  Mean ± SD  Mean ± SD  Mean ± SD  Mean ± SD  P  P  P  S-Mb  Exp  5.79 ± 0.97  5.55 ± 1.12  5.73 ± 1.10  5.91 ± 1.43  0.063  0.392  0.823  Con  5.41 ± 0.68  5.20 ± 0.80  5.15 ± 0.62  5.55 ± 0.92  Mc  Exp  14.19 ± 2.13  12.51 ± 3.23  11.76 ± 2.67  11.27 ± 2.85  0.001*  0.121  0.024*  Con  14.84 ± 2.93  14.60 ± 2.75  14.28 ± 2.31  14.30 ± 2.50  Con, control group; Exp, experimental group; M: thickness of masseter muscle (mm); S-M: thickness from skin surface to masseter muscle (mm). aMain effects and interactions were tested by two-way repeated-measures analysis of variance (ANOVA). bNo significant interaction and main effect of time and group. cSignificant interaction of time and group (with Greenhouse-Geisser correction, epsilon = 0.650, F = 4.279, P = 0.024) and significant main effect of time (with Greenhouse-Geisser correction, epsilon = 0.650, F = 9.677, P = 0.001). No significant main effect of group. *P < 0.05. View Large Figure 4. View largeDownload slide Mean S-M and masseter muscle thicknesses at rest. Exp, experimental group; Con, control group. Figure 4. View largeDownload slide Mean S-M and masseter muscle thicknesses at rest. Exp, experimental group; Con, control group. Table 2 lists the S-M and masseter muscle thicknesses and the differences therein over time and by group during maximum contraction. The S-M thickness did not differ significantly over time (P = 0.166) or by group (P = 0.259), and there was no significant interaction between time and group (P = 0.115). In contrast, there was a significant interaction between time and group for the masseter muscle thickness (P = 0.025), and a large decrease in its thickness was seen in the experimental group compared to the control group (Figure 5). Table 2. S-M and Masseter Muscle Thicknesses and Differences Therein During Maximum Contractiona   Preinjection  4 weeks  8 weeks  12 weeks  Time  Group  Time group  Mean ± SD  Mean ± SD  Mean ± SD  Mean ± SD  P  P  P  S-Mb  Exp  4.84 ± 0.92  5.42 ± 1.19  5.14 ± 1.12  5.30 ± 1.24  0.166  0.259  0.115  Con  4.75 ± 0.66  4.69 ± 0.70  4.45 ± 0.58  4.70 ± 0.86  Mc  Exp  16.58 ± 2.17  14.04 ± 3.48  13.72 ± 2.87  13.24 ± 2.97  0.002*  0.082  0.025*  Con  17.01 ± 2.25  16.56 ± 2.09  14.46 ± 2.13  16.47 ± 1.84    Preinjection  4 weeks  8 weeks  12 weeks  Time  Group  Time group  Mean ± SD  Mean ± SD  Mean ± SD  Mean ± SD  P  P  P  S-Mb  Exp  4.84 ± 0.92  5.42 ± 1.19  5.14 ± 1.12  5.30 ± 1.24  0.166  0.259  0.115  Con  4.75 ± 0.66  4.69 ± 0.70  4.45 ± 0.58  4.70 ± 0.86  Mc  Exp  16.58 ± 2.17  14.04 ± 3.48  13.72 ± 2.87  13.24 ± 2.97  0.002*  0.082  0.025*  Con  17.01 ± 2.25  16.56 ± 2.09  14.46 ± 2.13  16.47 ± 1.84  aMain effects and interactions were tested by two-way repeated-measures ANOVA. bNo significant interaction and main effect of time and group. cSignificant interaction of time and group (with Greenhouse-Geisser correction, epsilon = 0.484, F = 4.956, P = 0.025) and significant main effect of time (with Greenhouse-Geisser correction, epsilon = 0.650, F = 9.907, P = 0.002). No significant main effect of group. * P < 0.05. View Large Figure 5. View largeDownload slide Mean S-M and masseter muscle thicknesses during maximum contraction. Figure 5. View largeDownload slide Mean S-M and masseter muscle thicknesses during maximum contraction. DISCUSSION A square face is caused by a complex mechanism involving protrusion of the mandibular angle and hypertrophy of the masseter muscle.25 Many Koreans have a wide mandible that is typical of Mongolian races. Moreover, traditional Korean food is mostly rough and hard, which promotes the development of the masseter muscle. However, a common standard of beauty in Asian women is having a slim face and soft-looking features, which can result in a square face being considered a problem that needs to be treated.26,27 Masseter muscle hypertrophy is a common cause of a square face, and it is mostly due to parafunction such as bruxism or clenching, as well as dietary habits. Various treatments have been introduced for reducing the masseter muscle volume. This can be achieved surgically, but such invasive interventions are associated with side effects such as postoperative pain, delayed healing time, bleeding, and nerve injury.28 BoNT is one of the neurotoxin proteins (A, B, C1, C2, D, E, F, and G) produced by Clostridium botulinum, which is an anaerobe that causes food poisoning. Clostridium botulinum was first extracted in 1897 by Van Ermengen from a dead body and salted pork, and BoNT was separated and refined in 1946 by Edward J. Schantz. In 1949, Burgen et al reported that BoNT suppressed muscle contraction by depressing acetylcholine secretion at cholinergic nerve terminals.2,29 These actions of BoNT mean that injecting it into the masseter muscle results in reversible decreases in the contraction force and volume of the muscle. BoNT started to replace surgical methods in many clinics during the 1990s.30 Previous studies utilizing photography, CT, MRI, 3D imaging, and ultrasonography to detect changes have demonstrated that BoNT injection is effective in decreasing the size of the masseter muscle.6-21 CT, MRI, and ultrasonography can also be used to measure the S-M thickness. Performing repeated CT-based measurements will increase the patient’s exposure to ionizing radiation. While MRI does not have this problem, it is expensive and difficult to acquire subdivided images, hindering the acquisition of exact values. In the clinic, ultrasonography is favored since it is fast, easy, and harmless. Previous studies that used ultrasonography to measure the thickness of the masseter muscle employed different measuring references. For example, Volk et al placed the probe transversely at the middle of the zygomatic bone and mandibular angle, while other studies simply performed measurements at the point of maximum thickness or no reference point was reported. Emshoff et al reported that the masseter muscle was thickest in its middle area when it was divided into 5 sections from its origin to insertion point.31 Raadsheer et al reported strong correlations between measurements of the masseter muscle using MRI and ultrasonography, with the strongest correlations for measurements at the middle of the masseter muscle.14 Suh et al reported that the maximum thickness of the masseter muscle from the mandibular angle in Koreans was 27.77 mm in males and 25.79 mm in females, which occurred 53% and 59% from the mandibular angle, respectively.32 Therefore, in the present study we attempted to minimize errors due to changes in the reference point in repeated measurements of the maximum thicknesses of S-M and masseter muscle by using the line from the cheilion to the otobasion inferius as a reference. BoNT injection can also directly affect adjacent tissue other than the masseter muscle due to drug diffusion or indirectly affect the S-M thickness by weakening the chewing force. The S-M layer consists of epidermis, dermis, and subcutaneous tissue. The subcutaneous tissue is mainly composed of fat tissue, which is very closely related to body weight.33 However, there was no subject who reported changes in body weight in this study. The BoNT diffusion potency has been studied by several authors. Studies in experimental animals found that injecting 5 to 10 U of BoNT resulted in biologic activity at 4.5 cm from the injection site within the targeted muscle,34 that the muscle fascia did not act as a barrier to diffusion of the toxin,35 and that doses of 10 U had biologic effects that spread across the fascial planes to adjacent, noninjected muscles for 2.5 to 4.5 cm from the injection site.34,36 However, muscles are not present in the S-M layer, and so even if BoNT diffuses into this layer, it may still not exert any direct effects. In this study we measured the thicknesses in snapshots captured from videos recorded when using ultrasonography. A reference point was determined at rest, and then the subject made a clenching motion to contract the muscle. The thicknesses of the thickest points of the muscle and S-M were measured during the maximum contraction, and this was thicker than the same reference area measured at rest. Ideally the pressure applied to the skin by the ultrasound probe should have been the same when making the measurement in the maximum contraction and rest states. Moreover, it was especially difficult when repositioning the probe to apply the same pressure at the same measuring point. Also, the patient could maintain the maximum contraction force for only a few seconds, which made it technically impossible to reposition the probe at the correct time. However, the technique used in this study can be considered to accurate reflect the situations encountered in clinical applications. Clenching the masseter muscle tends to push the ultrasound probe away due to the contraction of the muscle, which will cause an increase in thickness and volume. Therefore, considerable skill is required from the investigator to ensure that an even pressure is maintained. The average S-M thickness before BoNT injection was 5.63 mm at rest and 4.80 mm during the maximum contraction. This decrease during the maximum contraction compared to the rest state may have been due to the masseter muscle pressing on the subcutaneous tissue during contraction or stretching of subcutaneous tissue during clenching. We did not find a statistically significant relationship between the changes in the masseter muscle thickness and the S-M thickness. However, this could have been due to the small number of subjects in the experimental group, and so this is worth studying in future experiments. The possibility of the S-M thickness decreasing due to pressure from the thickened muscle when it is contracting cannot be excluded. CT and MRI do not involve applying pressure on the muscle, and so these methods would be the best choice for minimizing measurement errors. However, maintaining a consistent maximum contraction state during CT or MRI is likely to be difficult to achieve in the clinical situation. This should also be taken into consideration in future studies. Suh et al found that the depth of the masseter muscle from the skin surface when using CT was significantly greater in females (7.37 ± 1.93 mm) than in males (6.15 ± 2.90 mm).32 This is probably related to differences in body fat between males and females. In our study, the average subcutaneous tissue thickness at rest before the injection was 5.63 mm, with a maximum of 8.04 mm. It is therefore obvious that the needle needs to be longer than 8 mm when injecting BoNT into the masseter muscle, and so using a 29-G ½-inch-long needle is recommended. Recent studies have raised the possibility that BoNT may affect not only muscles but other adjacent tissues.22-24 The results of this study suggest that BoNT injection does not affect the S-M thickness, but the limitation of this study is the small sample size. Future studies should include a larger sample size. CONCLUSION This study measured the S-M and masseter muscle thicknesses before and after BoNT injection, and analyzed differences between the experimental and control groups over time. The masseter muscle thickness in the experimental group decreased significantly over time, whereas the S-M thickness did not differ significantly between the experimental and control groups. It can therefore be concluded that BoNT only affects muscles, and does not affect the surrounding subcutaneous tissues. Disclosures The authors declared no potential conflicts of interest with respect to the research, authorship, and publication of this article. Funding The authors received no financial support for the research, authorship, and publication of this article. REFERENCES 1. Legg J. Enlargement of the temporal and masseter muscles on both sides. Trans Pathol Soc London . 1880; 31: 361. 2. Burgen AS, Dickens F, Zatman LJ. The action of botulinum toxin on the neuro-muscular junction. 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Aesthetic Surgery JournalOxford University Press

Published: Feb 1, 2018

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