Elsevier

Bone

Volume 77, August 2015, Pages 75-82
Bone

Original Full Length Article
Botulinum toxin in masticatory muscles of the adult rat induces bone loss at the condyle and alveolar regions of the mandible associated with a bone proliferation at a muscle enthesis

https://doi.org/10.1016/j.bone.2015.03.023Get rights and content

Highlights

  • BTX injection in masticatory muscles is commonly used in human.

  • BTX injection in masticatory muscles of the rat leads to a major condylar and alveolar bone loss at the mandible.

  • A new technique was used for analysis of mandibular 3D porosity by a vector projection algorithm.

  • The occurrence of a hypertrophic bone metaplasia at the enthesis of the digastric muscle is of considerable interest.

  • This hyper muscle load can be an etiological factor for a torus mandibularis.

Abstract

In man, botulinum toxin type A (BTX) is injected in masticatory muscles for several indications such as trismus, bruxism, or masseter hypertrophy. Bone changes in the mandible following BTX injections in adult animal have therefore became a subject of interest. The aim of this study was to analyze condylar and alveolar bone changes following BTX unilateral injections in masseter and temporal muscles in adult rats. Mature male rats (n = 15) were randomized into 2 groups: control (CTRL; n = 6) and BTX group (n = 9). Rats of the BTX group received a single injection of BTX into right masseter and temporal muscles. Rats of the CTRL group were similarly injected with saline solution. Rats were sacrificed 4 weeks after injections. Masticatory muscles examination and microcomputed tomography (microCT) were performed. A significant difference of weight was found between the 2 groups at weeks 2, 3 and 4 (p < 0.05). Atrophy of the right masseter and temporal muscles was observed in all BTX rats. MicroCT analysis showed significant bone loss in the right alveolar and condylar areas in BTX rats. Decrease in bone volume reached − 20% for right alveolar bone and − 35% for right condylar bone. A hypertrophic bone metaplasia at the digastric muscle enthesis was found on every right hemimandible in the BTX group and none in the CTRL group. BTX injection in masticatory muscles leads to a significant and major mandible bone loss. These alterations can represent a risk factor for fractures in human. The occurrence of a hypertrophic bone metaplasia at the Mus Digastricus enthesis may constitute an etiological factor for tori.

Introduction

Type A botulinum toxin (BTX) is a bacterial metalloprotease produced by Clostridium botulinum. It is a neurotoxin that causes specific inhibition of the neurotransmitter release in cholinergic nerve terminals. Acetylcholine vesicles are blocked at the presynaptic membrane of neuromuscular junctions because BTX degrades the SNAP-25 protein required for vesicle fusion and release of acetylcholine at the axon end [1]. This leads to a transient muscle paralysis which is fully reversible in a few months [1], [2], [3]. BTX injection in the Mus Quadriceps femoris of rodents is now well-known to create a model of tibial and femoral bone loss by immobilization which is easily quantified by reduction of the trabecular bone volume and cortical thickness [4], [5], [6].

In human, BTX injections are used in masticatory muscles (Mus Masseter and/or Mus Temporalis) for several indications such as trismus, bruxism, masticatory myalgia, temporomandibular joint disorders or masseter hypertrophy [7], [8], [9], [10]. It is also used in facial injections (subcutaneous, intraglandular or intramuscular); the main indications being sialorrhoea, blepharospasm, hemifacial spasm and aesthetic use [7], [8], [11]. There are level 1 or 2 evidences supporting the efficacy of BTX in many of those treatments [7], [12]. Repeated injections are needed in many indications. Side effects are rare and reversible: non-desirable focal facial muscle paralysis by toxin diffusion and complications at the injection site (bruising, pain, and edema) [13], [14], [15], [16].

The mandible is a non-weight bearing bone which is stimulated by masticatory muscles. It is composed of alveolar and basal bone. Teeth roots are anchored into alveolar bone by the periodontal ligament. Alveolar bone has a high plasticity and is remodeled at a high rate. Its mechanical stimulation during mastication is essential in keeping the teeth and underlying bone healthy. Loss of teeth leads to an irreversible alveolar bone resorption [17], [18]. Alveolar bone loss is also found in several metabolic bone diseases or due to glucocorticoid treatment [19], [20], [21]. However, trabecular bone is also present in other parts of the mandible such as the condylar process. Fractures of this area are also observed in clinical practice and may occur in osteoporosis [22], [23].

The aim of the present study was to evaluate bone changes following a unilateral BTX injection in the right masseter and temporalis muscles in adult rats. Two areas were selected and analyzed by microcomputed tomography (condylar and alveolar bone) 4 weeks after injection.

Section snippets

Animals and experimental procedure

Eighteen week-old male Sprague–Dawley rats (n = 15), weighing 595 ± 41 g, were used for the study (Janvier-Labs, Le Genest-Saint-Isle, France). They were acclimated for two weeks to the local vivarium conditions (24 °C and 12 h/12 h light dark cycle) where they were given standard laboratory food (UAR, Villemoison-sur-Orge, France) and water ad libitum. Rats were randomized into 2 groups: control group (CTRL, n = 6) and BTX-injected group (BTX, n = 9). Rats from the BTX group were anesthetized with

Body weight and anatomic muscle examination

No significant difference in body weight was found within each group during the time course of the study. However, a significant difference was found between the 2 groups at weeks 2, 3 and 4 (p < 0.05) (Fig. 2). The weight loss was − 15 ± 11.17 g (i.e. − 2.5% of initial weight) in the BTX group. All rats of the BTX group presented a masseter and temporal amyotrophy at the injected side (Fig. 3). No differences were noticed in the CTRL group.

MicroCT analysis of bone effects of BTX injection

Bone loss was clearly seen on the right side of alveolar bone

Discussion

The present study was designed to determine the effect of motor denervation induced by Clostridium botulinum toxin serotype A on the mandibular bone of the rat. In our study, amyotrophy was anatomically evidenced by the naked eye and no attempt was done to quantify muscle loss after dissection. Amyotrophy after BTX injection has been reported in a large number of papers [4], [27], [28], [29], [30], [31]. It is due to a complete motor denervation starting between 2 and 5 days after injection;

Conflicts of interest

The authors declare that they have no conflicts of interest concerning this article.

Acknowledgments

Authors are greatly indebted to the SCAHU (Service commun d'animalerie hospitalo-universitaire) of Angers, especially to P. Legras and J. Roux for their help with the animal care. Our vector analysis software (VECTOPOR) is now licensed by the APP (Agence de Protection des Programmes) JDKD IDDN.FR.001.150014.000.S.P.2015.000.10000 received a scholarship from the ARS (Agence Régionale de Santé) “Pays de la Loire”.

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