Botulinum toxin in masticatory muscles: short- and long-term effects on muscle, bone, and craniofacial function in adult rabbits

Abstract

Paralysis of the masticatory muscles using botulinum toxin (BTX) is a common treatment for cosmetic reduction of the masseters as well as for conditions involving muscle spasm and pain. The effects of this treatment on mastication have not been evaluated, and claims that the treatment unloads the jaw joint and mandible have not been validated. If BTX treatment does decrease mandibular loading, osteopenia might ensue as an adverse result. Rabbits received a single dose of BTX or saline into one randomly chosen masseter muscle and were followed for 4 or 12 weeks. Masticatory muscle activity was assessed weekly, and incisor bite force elicited by stimulation of each masseter was measured periodically. At the endpoint, strain gages were installed on the neck of the mandibular condyle and on the molar area of the mandible for in vivo bone strain recording during mastication and muscle stimulation. After termination, muscles were weighed and mandibular segments were scanned with micro CT. BTX paralysis of one masseter did not alter chewing side or rate, in part because of compensation by the medial pterygoid muscle. Masseter-induced bite force was dramatically decreased. Analysis of bone strain data suggested that at 4 weeks, the mandibular condyle of the BTX-injected side was underloaded, as were both sides of the molar area. Bone quantity and quality were severely decreased specifically at these underloaded locations, especially the injection-side condylar head. At 12 weeks, most functional parameters were near their pre-injection levels, but the injected masseter still exhibited atrophy and percent bone area was still low in the condylar head. In conclusion, although the performance of mastication was only minimally harmed by BTX paralysis of the masseter, the resulting underloading was sufficient to cause notable and persistent bone loss, particularly at the temporomandibular joint.

Figure 1

Experimental design.

Figure 2

A. Superficial masseter muscle of the rabbit. The three locations shown as dots within circles are the points where BTX or saline was injected. These same three locations were the sites recorded during wire EMG sessions (anterior, middle and posterior superficial masseter). The x's within squares on the molar region and the condylar neck are the sites where strain gages were bonded to mandible for recording bone strain. Note partial removal of the deep masseter muscle over the neck of the condyle. B. Rabbit mandible. The shaded molar region and condylar process were removed for micro CT scanning.

Figure 3

Average EMG amplitude of chewing bursts over time, measured using surface electrodes over the masseter muscle (data in Supplement A). Working (left chart) and balancing (right chart) cycles refer to whether the masseter was on the same side as the bolus or the opposite side, respectively. BL – baseline recording before injection of one masseter with either BTX or saline. The week 0 recording was made a few days after injection. Surface EMG was not performed during week 4. The uninjected masseters and the saline-injected masseters demonstrated similar EMG amplitudes throughout the entire period. EMG from the BTX-injected muscles dropped precipitously after injection and reached its lowest point during week 2. Levels remained low at least through week 10 when the injected muscle was on the working side but were normalized by week 5 when the bolus was on the opposite side. The asterisks indicate likely differences (p < 0.05) between the BTX-injected and the saline-injected muscles, and the double daggers indicate likely differences (p < 0.05) between the injected and uninjected sides of the BTX animals.

Figure 4

Fine-wire EMG from BTX injected-side muscle locations during chewing at baseline (before injection, small-dotted horizontal line at 100%) and various post-injection time points. EMG amplitude is shown as a percentage of baseline (calculated from data in Supplement B). ASM (heavy solid line), MSM (medium solid line) and PSM (thin solid line) are the anterior, middle and posterior locations in the superficial masseter, all of which received BTX during week 0. The deep masseter (DM, dashed line) and medial pterygoid (MPT, heavy dotted line) muscles were not injected but may have received some BTX through diffusion. The error bars indicate one SE (not SD as elsewhere in this paper). All muscles tended to have reduced EMG 1-2 weeks after injection (p < 0.05 vs. baseline or controls for ASM and MSM). The injected ASM, MSM, and PSM remained low at 5-6 weeks (p < 0.05 for all three). No other differences reached p < 0.05.

Figure 5

Raw recordings of fine-wire EMG from the same BTX group rabbit during working side chewing before (Baseline) and after (Week 1) BTX administration to the right superficial masseter muscle. Right (R) side muscles shown are the middle superficial masseter (R. MSM) and the medial pterygoid (R. MPT). On the uninjected side the left middle superficial masseter (L. MSM), posterior superficial masseter (L. PSM) and medial pterygoid (L. MPT) are shown. The dotted vertical lines show the onset of activity in the reference muscle (L. PSM) for each chewing cycle. The arrows indicate the onset of activity in the injected-side medial pterygoid (R. MPT). The loss of activity in the R. MSM after BTX injection is obvious. In addition, there was a change in the timing of contraction for the injected-side medial pterygoid. Before BTX, the R-MPT became active in synchrony with the other muscles, but after BTX it consistently started earlier. This change, seen consistently in the BTX animals, persisted until weeks 5-6 after BTX administration to the masseter.

Figure 6

Relative bite force produced by BTX vs. saline-injected masseters. Each masseter was separately tetanized to produce a bite force at the incisors. The values shown are bite force produced by the injected muscle as a percentage of the uninjected muscle of the same animal (see Supplement C for absolute values). BTX or saline was injected after the baseline values were measured. The error bars represent one SD. Double asterisks denote p ≤ 0.002.

Figure 7

Compressive strains on the molar region and condylar neck during mastication. The upper figure shows averages from the saline controls, and the lower figure shows the injected side of BTX animals 4 weeks after the toxin was administered to the masseter muscle (data from Table 2). The length of the arrows is proportional to strain magnitude (note calibration arrow labeled -400 με). Compared to saline controls and regardless of the side of chewing, strain was reduced and reoriented at both locations in the BTX sample.

Figure 8

Coronal sections (reconstructed from micro CT scans) of pairs of mandibular condylar processes from representative rabbits. The injected-side condyle is shown on the left of each pair regardless of the actual side of injection. A. Four weeks after injection of saline into one masseter muscle. The dashed white outline shows the trabecular area analyzed as the condylar head for the lower part of Table 4 and for Table 5. The two condyles are similar in percent bone area. B. Four weeks after injection of BTX into one masseter. The injected-side condyle has less bone than the uninjected-side condyle. C. Twelve weeks after injection of BTX into one masseter. A differential between the two condyles in percent bone area is still apparent, particularly with regard to the greater trabecular separation on the BTX-injected side.