The purpose of this study was to use botulinum neurotoxin type A (BoNT/A) selectively to evaluate the influence of localized masticatory atrophy and paresis on craniofacial growth and development. 60 growing rats, 4 weeks old, weighing approximately 120 g, were randomly divided according as follows (Long-Evans, N = 15 per group): I (Mb + Tns); II (Mns + Tb); III (Mb + Tb); IV (Mns + Tns), where Mb or Tb is the BoNT/A-injected masseter or temporalis muscles (1.0 U/muscle, 2.5 ml) and Mns or Tns is the saline-injected muscles (2.5 ml). After 7 weeks, the mature rats were killed, the muscles dissected and mean muscle mass recorded. Anthropometric cranial, maxillary and mandibular measurements were taken from the dried skulls. Changes in animal weight during the growth period were not statistically significant. The mean masticatory muscle mass was smaller for the BoNT/A-injected muscles of Mb and Tb. Anthropometric measurements of bony structures inserted by masseter and temporalis muscles revealed a significant treatment effect. The measurements showed a facial morphology typical of a dolichofacial profile: short upper face accompanied by a long lower face with an extended mandibular length and ramus height and constricted bicoronoidal and bigonial widths. The results suggest that induction of localized masticatory muscle atrophy with BoNT/A alters craniofacial growth and development.
Moss’s functional matrix theory states that facial muscles play a central role in bone growth . The surrounding muscular environment directs the cells concerned with the morphology, orientation and spatial relationship of bone growth, while other tissues (embryonic functional matrices) guide differentiation at appropriate times and locations . Hypothetically, therefore, induction of muscle dysfunction and hypoactivity would alter craniofacial growth and local development around muscle attachment and insertion sites.
Optimal dental treatment involves understanding the effects of masticatory muscle function on craniofacial bone growth to achieve a desirable outcome. In addition to appropriate application of biomechanical principles, optimal treatment planning requires an understanding of the craniofacial muscular environment of each patient. The facial muscles play an important role in the etiology and treatment of malocclusions and jaw deformities and are crucial for the stability of dental treatment .
Previous investigations of the effect of muscle function on facial bone growth have been limited by major shortcomings in the experimental models. For example, changes in diet consistency such as eating a soft or comminuted diet, can indirectly induce masticatory hypofunction, but do not reflect the actual influences of muscle activity on function. Myotomy or myoectomy decrease the blood supply, change the loading of the entire skeleton, and often introduce biomechanical force. Denervation is likely to cause a loss of sensation or damage to the motor nucleus , which could interfere with normal growth of bone .
Botulinum neurotoxins (BoNTs) have been used widely in medical applications due to their rapid and safe effects. BoNTs are fermentation products of the spore-forming bacterium Clostridium botulinum . Seven neurotoxins have been identified (type A–G) , which inhibit the release of acetylcholine from the presynaptic nerve terminal of cholinergic nerve endings and induce muscle paralysis . The onset and duration of muscle paralysis are influenced by the origin of presynaptic nerve terminal and the type of toxin used. At the neuromuscular junction in humans, toxin recovery takes 2–4 months while recovery of the autonomic nervous system is more prolonged . Among the neurotoxins, the action of botulinum neurotoxin type A (BoNT/A) has the longest recovery time in animal studies with a reversible, temporary inhibition of acetylcholine release .
Clinical evaluation of the effects of masseter muscle function following BoNT/A injections have mainly been focused on enhancing cosmetics (volumetric reduction) or diminishing excessive muscle activity (bruxism decrease). Basic scientific research with BoNT/A is limited , and its full anatomical effects are not known, particularly with regard to the mandible and relevant anthropometric measurements. The purpose of this study was to evaluate the influence of the musculature on the craniofacial skeleton in a growing animal model by inducing localized masticatory atrophy and paresis.
Materials and Methods
This was an open-label study over 7 weeks. Prior to the start of the study, full protocol and ethics approval were obtained from the animal centre of Taipei Medical University. 60 male growing Long-Evans rats, weighing approximately 120 g, were enrolled in the study. All study animals were male to limit sexual dimorphism. Study animals were housed in separate cages in the same room under light and climate-controlled conditions, and were fed a standard diet of hard pellets and water ad libitum . Mean weights (accuracy to nearest 0.01 g) were measured during the 7-week experimental period (T1–T7) with an electronic digital scale.
At age 4 weeks (T0), weaned study animals were injected with BoNT/A (Botox ® , Allergan Pharmaceuticals, Irvine, CA, USA). BoNT/A was diluted with 4.0 ml sterile, non-preserved 0.9% saline to yield 25 U/ml preparation. Study animals received a 2.5-ml intramuscular injection of BoNT/A (1.0 U/muscle mass) as follows. Group I (Mb + Tns): injection into bilateral masseter muscles (bilateral temporalis muscles received equivalent amounts of 0.9% sterile, non-preserved saline). Group II (Mns + Tb): injection into bilateral temporalis muscles (bilateral masseter muscles received equivalent amounts of 0.9% sterile, non-preserved saline). Group III (Mb + Tb): injection into bilateral masseter and temporalis muscles. Group IV (control, Mns + Tns): injection of 0.9% sterile, non-preserved saline into bilateral masseter and temporalis muscles.
A single dose of 50 mg/kg ketamine (Zoletil ® , Virbac Inc., CA, USA) and 10 mg/kg xylazine (Rompum ® , Bayer Inc., Toronto, Canada) were administered intraperitoneally to achieve initial anaesthesia for injections of BoNT/A and saline. The bilateral injection sites for the temporalis muscles were located one-third and two-thirds along the line connecting the orbitale and outer meatus. Masseter muscles received superficial and deep muscle layer injections. The superficial muscle site was located halfway along the line connecting the orbitale and outer meatus, perpendicular to the line adjoining the mandibular plane angle and outer oral commisure. The deep muscle site was posterior to the orbitale on the line connecting the outer meatus and orbitale. Every temporalis injection site received 1.0 U and every masseter injection site received 0.5 U of BoNT/A or the equivalent amount of saline. At the end of the 7-week experimental period, the rats were perfused and killed.
The masseter and temporalis muscles were dissected carefully and harvested by the same operator (W.C.C.). The mean muscle mass was recorded with a precision balance (model ZSA80, Scientech, Denver, USA) and differences were compared among Groups I, II, III and the control. Dried skulls were prepared for direct cranial, maxillary and mandibular anthropometric measurements. 40 anthropometric parameters were analyzed ( Fig. 1 a and b ) as described by L evrini et al. , Y amamoto , U lgen et al. , T sai and L iao , and M atic et al. .
Animal weight data were compared using repeated measures of analysis of variance (ANOVA) followed by post hoc tests using least square deviation. For description of data, mean values and standard deviations were calculated and presented as mean ± SD. Changes in muscle mass and anthropometric measurements were analyzed using one-way ANOVA followed by the Mann–Whitney U -test for comparison between experimental groups and control. Statistical significance was defined at the 5% level. All statistical analyses were performed using SPSS version 13.0 software (SPSS Inc., Chicago, IL, USA).
Changes in animal weight
Rats were enrolled 4 weeks after weaning (T0) with a mean weight of 123.6 ± 29.7 g for the experimental groups (Groups I, II, III) and 119.0 ± 17.4 g for the control group. At age 8 weeks (T4), rats reached maturity and had a mean weight of 323.3 ± 43.8 g in the experimental groups and 327.5 ± 37.9 g in the control group. Thereafter, weight increased at a slower rate. At the end of the study (T7), rats were age 11 weeks and had a mean weight of 391.3 ± 46.1 g in the experimental groups and 388.2 ± 29.7 g in the control group. No statistically significant differences were found in animal weights as shown in Fig. 2 . Weight gain remained stable throughout the study. Neither disturbances in growth nor clinically evident differences in size or shape of the animals were seen.
Changes in muscle mass
Data showed that BoNT/A-treated masseter muscles were smaller than saline-injected masseter muscles with a significant difference between Group III and the control ( Fig. 3 ). BoNT/A-treated temporalis muscles showed a similar result with a significance difference between Groups II and III ( p < 0.001).
Comparisons of direct anthropometric measurements
Only statistically significant parameters compared with control are listed and displayed in Table 1 and discussed.
|Group/parameters||Group I||Group II||Group III||Group IV|
|Sagittal cranial measurements|
|1. Maximum skull height (7)||15.88||0.60||15.59||0.38||14.90 *||1.41||15.92||0.43|
|2. Upper anterior facial height (8)||7.10||0.32||6.6 *||0.71||6.53 *||0.73||7.43||0.86|
|3. Lower anterior facial height (9)||12.51 *||0.75||12.21||0.77||13.09 *||0.97||12.07||0.58|
|4. Total anterior facial height (10)||17.84||0.63||16.73 *||0.79||18.01||0.79||17.73||0.63|
|Maxillary dental measurements|
|5. U8 intermolar distance (15)||9.13 *||0.19||8.98||0.18||9.19 *||0.16||8.91||0.21|
|Sagittal Mandibular measurements|
|6. Mandibular length I (21)||9.69||0.51||9.47||0.84||10.21 *||0.76||9.41||0.52|
|7. Mandibular length II (22)||10.16 *||0.55||9.70 *||0.86||10.97||0.55||10.10||0.58|
|8. Mandibular length III (23)||15.85||0.56||15.27||1.25||16.54 *||0.41||15.25||1.18|
|9. Corpus length (24)||25.16||0.75||25.00||1.85||24.75 *||1.01||26.01||0.73|
|Vertical mandibular measurements|
|10. Ramus height I (25)||11.67||0.57||11.04||0.29||11.34 *||0.30||10.49||0.68|
|11. Ramus height II (26)||11.79||0.31||11.44||0.32||11.66 *||0.28||10.91||0.62|
|12. Ramus height III (27)||12.06||0.48||11.39 *||0.29||11.95 *||0.58||11.24||0.77|
|13. Ramus height IV (28)||14.41||0.35||13.93||0.45||14.32 *||0.49||13.66||0.74|
|14. Mandibular plane angle (30)||22.67 *||3.16||23.41 *||3.51||25.01 *||1.92||19.66||2.24|
|Transverse mandibular measurements|
|15. Bicoronoidal width (31)||17.22||1.16||17.35||0.81||16.37 *||0.80||17.63||0.77|
|16. Bigonial width (33)||17.10||0.57||17.88||1.36||16.16 *||0.87||17.88||1.32|