Botulinum Toxin-induced Muscle Paralysis Inhibits Heterotopic Bone Formation

Abstract

Background: Short-term muscle atrophy induced by botulinum toxin A (BTxA) has been observed to impair osteogenesis in a rat closed femur fracture model. However, it is unclear whether the underlying mechanism is a direct effect of BTxA on muscle-bone interactions or an indirect effect that is driven by skeletal unloading. Because skeletal trauma in the closed fracture model also leads to disuse atrophy, we sought to mitigate this confounding variable by examining BTxA effects on muscle-bone interactions in two complementary in vivo models in which osteogenesis is induced in the absence of skeletal unloading. The overall aim of this study was to identify a potential strategy to inhibit pathological bone formation and heterotopic ossification (HO).

Questions/purposes: (1) Does muscle paralysis inhibit periosteal osteogenesis induced by a transcortical defect? (2) Does muscle paralysis inhibit heterotopic bone formation stimulated by intramuscular bone morphogenetic protein (BMP) injection?

Methods: Focal osteogenesis was induced in the right hindlimb of mice through surgical initiation of a small transcortical defect in the tibia (fracture callus; n = 7/group) or intramuscular injection of BMP-2 (HO lesion; n = 6/group), both in the presence/absence of adjacent calf paralysis. High-resolution micro-CT images were obtained in all experimental groups 21 days postinduction and total volume (ie, perimeter of periosteal callus or HO lesion) and bone volume (calcified tissue within the total volume) were quantified as primary outcome measures. Finally, these outcome measures were compared to determine the effect of muscle paralysis on inhibition of local osteogenesis in both studies.

Results: After a transcortical defect, BTxA-treated mice showed profound inhibition of osteogenesis in the periosteal fracture callus 21 days postsurgery compared with saline-treated mice (total volume: 0.08 ± 0.06 versus 0.42 ± 0.11 mm(3), p < 0.001; bone volume: 0.07 ± 0.05 versus 0.32 ± 0.07 mm(3), p < 0.001). Similarly, BMP-2-induced HO formation was inhibited by adjacent muscle paralysis at the same time point (total volume: 1.42 ± 0.31 versus 3.42 ± 2.11 mm(3), p = 0.034; bone volume: 0.68 ± 0.18 versus 1.36 ± 0.79 mm(3), p = 0.045).

Conclusions: Our data indicate that BTxA-induced neuromuscular inhibition mitigated osteogenesis associated with both a transcortical defect and BMP-2-induced HO.

Clinical relevance: Focal neuromuscular inhibition represents a promising new approach that may lead to a new clinical intervention to mitigate trauma-induced HO, a healthcare challenge that is severely debilitating for civilian and war-wounded populations, is costly to both the patient and the healthcare system, and currently lacks effective treatments.

Fig. 1

Whole bone micro-CT scan shows the location of the transcortical defect. The surgically induced defect was 0.6 mm in diameter and was drilled approximately 1 mm proximal to the tibiofibular junction.

Fig. 2

Serial micro-CT images along a 2-mm region of the tibial diaphysis in a saline-treated mouse (“saline,” top) demonstrates exuberant osteogenesis both distal and proximal to the surgically created bone defect. Furthermore, it can be seen that the cortical defect is being repaired by calcifying tissues (white arrow in the middiaphyseal image). In contrast, transient paralysis of the calf muscle induced by BTX (“BTxA,” bottom) inhibited osteogenesis along the entire diaphyseal length without affecting calcifying tissues adjacent to or within the injury itself.

Fig. 3

Total fracture callus (mean ± SD) and callus bone volume 21 days after a transcortical defect was significantly inhibited by BTxA-induced paralysis in the adjacent calf muscle group as compared with the saline group (*p < 0.001).

Fig. 4A–B

Three-dimensional volumetric reconstructions are shown of heterotopic bone nodules (red) 21 days after BMP implantation in the calf (A; saline). Transient muscle paralysis dramatically inhibits HO formation (B; BTxA) when compared with saline-injected controls.

Fig. 5

Total volume (mean ± SD) and bone volume of the heterotopic lesion 21 days after BMP-2/BME implantation were significantly inhibited by prophylactic BTxA-induced paralysis in the adjacent calf muscle group as compared with the saline group (*p = 0.034 and 0.045 for total volume and bone volume, respectively).