A single injection of botulinum toxin injected in masticatory muscles induces alveolar bone loss associated with muscle enthesis hypertrophic bone proliferation. The tissular and cellular mechanisms involved in this phenomenon are unknown. Because new bone formation due to inflammation at enthesis is observed in articular disorder like spondyloarthritis, we hypothesize that an inflammatory process could explain the development of hypertrophic bone proliferation in the BTX model. The aims of the study were to test this hypothesis and to determine whether IL-17A was involved.Mature Sprague-Dawley male rats (n = 36) were randomized into 4 groups. Three groups received BTX injections in right masseter and temporalis muscle. One BTX group was treated with an anti-IL17A antibody for 31 days (BTX31+antiIL17A). The two other groups were respectively sacrificed at 21 (BTX21) and 31 (BTX31) days post BTX injection. The remaining non-BTX group received anti-IL17A treatment for 31 days (control + anti-IL-17A). Alveolar bone loss and hypertrophic bone proliferation at enthesis were studied using microcomputed tomography. The presence of inflammatory process and the implication of IL17A was assessed by histology and immunohistochemistry on decalcified right side hemimandibles.Quantitative measurement of the hypertrophic bone (bone volume and thickness) showed no significant differences between the 3 BTX groups. Histology revealed the presence of chondrocytes in large hypertrophic area but no inflammatory infiltrated cells. Immunochemistry confirmed the absence of inflammatory process but positive reaction for Ki67 which is in favor of actively growing and proliferative chondrocytes.BTX-related muscle atrophy of masticatory muscle induced chondrocyte proliferation at enthesis without inflammation process.

A single injection of botulinum toxin injected in masticatory muscles induces alveolar bone loss associated with muscle enthesis hypertrophic bone proliferation. The tissular and cellular mechanisms involved in this phenomenon are unknown. Because new bone formation due to inflammation at enthesis is observed in articular disorder like spondyloarthritis, we hypothesize that an inflammatory process could explain the development of hypertrophic bone proliferation in the BTX model. The aims of the study were to test this hypothesis and to determine whether IL-17A was involved. Mature Sprague-Dawley male rats (n=36) were randomized into 4 groups. Three groups received BTX injections in right masseter and temporalis muscle. One BTX group was treated with an anti-IL17A antibody for 31 days (BTX31+antiIL17A). The two other groups were respectively sacrificed at 21 (BTX21) and 31 (BTX31) days post BTX injection. The remaining non-BTX group received anti-IL17A treatment for 31 days (control+anti-IL-17A). Alveolar bone loss and hypertrophic bone proliferation at enthesis were studied using microcomputed tomography. The presence of inflammatory process and the implication of IL17A was assessed by histology and immunohistochemistry on decalcified right side hemimandibles. Quantitative measurement of the hypertrophic bone (bone volume and thickness) showed no significant differences between the 3 BTX groups. Histology revealed the presence of chondrocytes in large hypertrophic area but no inflammatory infiltrated cells. Immunochemistry confirmed the absence of inflammatory process but positive reaction for Ki67 which is in favor of actively growing and proliferative chondrocytes. BTX-related muscle atrophy of masticatory muscle induced chondrocyte proliferation at enthesis without inflammation process. ### Competing Interest Statement The authors have declared no competing interest. Novartis (France), https://ror.org/00geypp27

Objectives Botulinum toxin (BTX) induces muscle paralysis. It is used in human in masticatory muscles injections performed often repeatedly. A single BTX injection in masticatory muscles in animal induces mandibular bone loss (alveolar and condylar) with a muscle enthesis hypertrophic metaplasia. Our aim was to evaluate mandibular bone changes after unilateral repeated injections of BTX in temporal and masseter muscles in adult rats. Materials and Methods Mature male rats were randomized into 3 groups: one, two or three injections. Each injection was performed 4 weeks after the prior injection. Each rat received injections in right masseter and temporalis muscles. The left side was the control side. Microcomputed tomography was used to perform 2D and 3D analyses. Results Bone loss was evidenced on the right sides of alveolar and condylar bone. Alveolar bone volume increased in both control left side and injected right side whereas condylar bone volume remained constant in all groups, for both left and right sides. Enthesis bone hypertrophic metaplasias were evidenced on the BTX injected sides without any modification with the number of injections. Conclusions BTX repeated injections in masticatory muscles lead to major mandibular condylar and alveolar bone loss that does not worsen. They lead to the occurrence of an enthesis bone proliferation that is not dependent on the number of injections. Clinical relevance These results are an argument for the safety of BTX injections in masticatory muscles in human.

Supplementary Data from Pharmacologic inhibitors of IκB kinase suppress growth and migration of mammary carcinosarcoma cells <i>in vitro</i> and prevent osteolytic bone metastasis <i>in vivo</i>

The use of resorbable devices for osteosynthesis has become a subject of interest. Nacre has been proposed as a resorbable and osteoconductive material favoring bone apposition without triggering an inflammatory reaction. We compared the in vivo osseointegration and erosion of nacre screws in an animal model with titanium screws. Implantation of similar nacre and titanium screws was performed in the femoral condyles of adult rats. Animals (n = 41) were randomized in four groups sacrificed at day one, 1, 6, and 12 months. Microcomputed tomography (microCT) allowed 3D morphometry of erosion of nacre. Osseointegration was measured as the volume of trabecular bone bone volume/tissue volume (BV/TV) in a standardized volume of interest around each screw. Undecalcified bone histology was also done. Gross examination revealed a similar clinical osseointegration for titanium and nacre screws. A progressive erosion of nacre screws, but no erosion of titanium screws, was observed in microCT. The volume of nacre screws progressively decreased over time whereas no modification occurred for titanium. For titanium screws, BV/TV remained stable throughout the study. For nacre screws, the BV/TV decrease was not statistically different. A significant difference was found between nacre and titanium screws at 6 months but not at 12 months. The screw heads, outside the bone shaft, were not eroded even after 12 months. Erosion of nacre occurred during the entire study period, only within the bone shaft in direct contact with bone marrow. Bone apposition was observed on nacre surfaces without signs of erosion. Nacre is a promising biomaterial in maxillofacial surgery.

McDonald and colleagues reported osteoclast-related dynamic mecha-nisms that lead, by fission, to osteo-morphs; motile, fusion-competent cells capable of forming bone-resorbing osteoclasts. scRNA-seq analyses revealed that osteomorphs are transcriptionally distinct from osteoclasts and macrophages and might be implicated in rare and common bone diseases in humans.

•Electrospinning was used to produce Gelatine bases fibers for maxillofacial surgery.•Raman spectroscopy and SEM were used to characterize the generated nanofibers.•Gelatin and gelatin/sodium hyaluronate nanofibers were successfully electrospun.•The addition of β-TCP formed agglomerate outside the fibers.•The fibers lacked of sufficient mechanical resistance to be implanted in vivo.

β-TCP is a resorbable bony biomaterial but its biodegradation mechanisms in vivo remains unclear. Osteoclast can resorb β-TCP but a role for macrophages has also been suggested by in vivo studies. However no in vitro study has clearly evidenced the action of macrophages in the resorption process. We prepared flat β-TCP tablets with a smooth surface to investigate the in vitro capability of murine (RAW 264.7) and human macrophage cells (PBMCs) to resorb the biomaterial. In parallel, these cells were differentiated into multinucleated osteoclasts with M-CSF and RANK-L. The action of these cells was evaluated by scanning electron microscopy and Raman microspectroscopy after a 21 day culture on the tablets. Human macrophages and osteoclasts derived from PBMCs appeared able to resorb β-TCP by forming resorption pits at the surface of the flat tablets. RAW macrophages were unable to resorb β-TCP but they exhibited this possibility when they have been differentiated into osteoclasts. These cells can engulf β-TCP grains in their cytoplasm as evidenced by light and TEM microscopy with production of carbonic anhydrase (revealed by the immunogold technique in TEM). The resorbed areas were characterized by severe degradation of the grains showing speckled and stick-like aspects indicating a chemical corrosion. The effect was maximal at the grain boundaries which have a slightly different chemical composition. Changes in the Raman spectrum were observed between the resorbed and un-resorbed β-TCP suggesting crystal modifications. In contrast, un-differentiated murine macrophages were not able to chemically attack β-TCP and no resorption pit was observed. RAW cell is not a representative model of the macrophage-biomaterial interactions that occur in human. This in vitro study evidences that both human osteoclasts and macrophages represent active cell populations capable to resorb β-TCP.

Sinus elevation is a common procedure to increase bone volume in the atrophic maxilla to allow placement of dental implants. Autogenous bone is the gold standard but is limited in quantity and causes morbidity at the donor site. β-TCP is a synthetic biomaterial commonly used in that purpose. It appears to induce a poor inflammatory response. This study aimed to evaluate the degree of edema of the sinus mucosa after sinus lift surgery according to the type of biomaterial. Forty sinuses (20 patients) were included retrospectively and divided into 2 groups according to the biomaterial that was used: synthetic biomaterial (BTCP group), natural bone (BONE group). A control group (CTRL group) was constituted by the non-grafted maxillary sinuses. Twelve measurements per sinus were realized on pre- and post-operative computed tomography and averaged to provide the sinus membrane thickness value (SM.Th). SM.Th was thicker post-operatively in the BTCP and BONE groups in comparison with the CTRL group and in comparison with pre-operative measurements. No difference was found post operatively between the BTCP and BONE groups. We found that a synthetic biomaterial (β-TCP) induced the same degree of edema, and thus of inflammation, as natural bone. It constitutes therefore an interesting alternative to autogenous bone for maxillary sinus lifts.

Using multi-slice computed tomography (MSCT), Barrier et al. described the disappearance at the posterior auricular surface of a “central line” (CL) and “juxtalinear cells” (JLCs) belonging to a trabecular bundle, and a trabecular density gradient around the CL that decreased with age. The aim of our study was to use micro-CT to test these findings, referring to the concept of Ascadi and Nemeskeri.

Noninvasive diagnostic by imaging combined with a contrast agent (CA) is by now the most used technique to get insight into human bodies. X-ray and magnetic resonance imaging (MRI) are widely used technologies providing complementary results. Nowadays, it seems clear that bimodal CAs could be an emerging approach to increase the patient compliance, accessing different imaging modalities with a single CA injection. Owing to versatile designs, targeting properties, and high payload capacity, nanocarriers are considered as a viable solution to reach this goal. In this study, we investigated efficient superparamagnetic iron oxide nanoparticle (SPION)-loaded iodinated nano-emulsions (NEs) as dual modal injectable CAs for X-ray imaging and MRI. The strength of this new CA lies not only in its dual modal contrasting properties and biocompatibility, but also in the simplicity of the nanoparticulate assembling: iodinated oily core was synthesized by the triiodo-benzene group grafting on vitamin E (41.7% of iodine) via esterification, and SPIONs were produced by thermal decomposition during 2, 4, and 6 h to generate SPIONs with different morphologies and magnetic properties. SPIONs with most anisotropic shape and characterized by the highest r2/ r1 ratio once encapsulated into iodinated NE were used for animal experimentation. The in vivo investigation showed an excellent contrast modification because of the presence of the selected NEs, for both imaging techniques explored, that is, MRI and X-ray imaging. This work provides the description and in vivo application of a simple and efficient nanoparticulate system capable of enhancing contrast for both preclinical imaging modalities, MRI, and computed tomography.

Différentes anomalies de la fonction musculaire caractérisées par une atrophie musculaire ont des répercussions sur la masse et l’architecture osseuse. Une perte osseuse est observée dans la dystrophie musculaire de Duchenne (DMD) humaine et dans des modèles animaux de DMD. Cependant les interactions entre os et muscle dans cette pathologie sont encore mal connues. L’objectif de cette étude était d’évaluer chez la souris mdx la perte osseuse corticale et trabéculaire en fonction des différents stades d’évolution de l’atteinte musculaire. La perte osseuse a été analysée ex vivo en microtomographie-X sur les fémurs et tibias gauches et droits de souris âgés de 24 semaines : mdx (n = 6), contrôle C54Bl/6J (n = 4). La masse osseuse corticale 3D a été déterminée sur la diaphyse fémorale ; la masse osseuse trabéculaire 3D a été déterminée sur la métaphyse tibiale. La masse osseuse corticale est significativement plus faible dans le groupe mdx comparativement aux souris contrôle (49,9 ± 1,1 % vs 53,6 ± 0,3 %, p < 0,05). Le volume osseux trabéculaire est diminué dans le groupe mdx sans atteindre la significativité (20,1 ± 1,7 % vs 24,0 ± 1,9 %). Les paramètres de microarchitecture du réseau trabéculaire, nombre de travées (Tb.N : 2,58 ± 0,21 vs 3,26 ± 0,17, p < 0,05) et espacement intertrabéculaire (Tb.Sp : 242 ± 16 μm vs 186 ± 4 μm, p < 0,05) sont significativement altérés traduisant une désorganisation de l’architecture et une fragilisation du tissu osseux trabéculaire. Cette étude préliminaire a montré une perte osseuse chez des souris de 24 semaines présentant une DMD. L’analyse osseuse chez des souris mdx, âgées de 3 semaines et de 12 semaines, permettra de mieux comprendre les interactions os/muscles en fonction du stade de développement de la DMD.

Pathogenesis of bisphosphonate-related osteonecrosis of the jaws (BRONJ) is not fully explained. An antiangiogenic effect of bisphosphonates (BPs) or an altered bone quality have been advocated. The aims of the present study were to analyze alveolar mandibular vascularization and bone quality in rats with BRONJ.

Beta-tricalcium phosphate (β-TCP) is a synthetic ceramic used for filling bone defects. It is a good alternative to autologous grafts since it is biocompatible, resorbable and osteoconductive. Previous in vivo studies have shown that macrophages are one of the first cells coming in contact with the biomaterial followed by osteoclasts and osteoblasts that will elaborate new bone packets. Studies have focused on osteoclast morphology and very few of them have investigated the role of macrophages. The aims of this study were to characterize (i) the biomaterial surface; (ii) the in vitro behavior of macrophages (J774.2 and Raw264.7 cells) using the description of cell morphology by scanning electron microscopy (SEM) at 7 and 14 days; (iii) the behavior of osteoblasts (SaOs-2 and MC3T3-E1 cells) seeded at the surface of the biomaterial 24, 48 and 72hours by SEM and confocal microscopy. Cell proliferation was analyzed by MTT assays. Viability and affinity of the macrophages for β-TCP were found significantly increased after 7 and 14d. MC3T3-E1 cells were anchored and stretched onto the β-TCP surface as early as 24h with a high proliferation rate (+190%) when compared to the surface of a well plate. SaOs-2 exhibited the same morphological profile at 72h. Proliferation became significantly higher compared to the plastic surface at only 72h (+129%). This study emphasises the importance of choice of the cell line used in exploring the osteoconductive and osteoinductive properties of a biomaterial. Additional studies are needed to analyze differentiation of macrophages into giant multinucleated cells and how the biomaterial surface influences osteoblast differentiation.

Le microenvironnement médullaire et osseux joue un rôle fondamental dans la progression tumorale du myélome multiple (MM). L’objectif de cette étude était d’évaluer dans le modèle murin 5T2MM, les effets d’une altération du remodelage osseux par une carence calcique alimentaire. Deux groupes de souris ont reçu une injection IV de cellules 5T2MM puis un régime alimentaire normal ou appauvri en calcium (Ca+/Ca−MM+) pendant 6, 8 et 10 semaines (s). Deux groupes témoins ont reçu ou non un régime appauvri en calcium (Ca+/Ca−MM−). La croissance tumorale a été évaluée par dosage de la protéine Monoclonale sérique. Les effets sur l’ostéolyse ont été analysés par radiographie et microtomographie. L’expression médullaire des gènes de la voie Wnt, de RANKL et de MIP-1α a été analysée par PCR quantitative. Dans le groupe Ca-MM-, la microtomographie a révélé une perte osseuse trabéculaire et corticale associée à une augmentation du taux de PTH, reflétant une hyperparathyroïdie secondaire. Dans le groupe Ca-MM+, le taux de protéine M était plus élevé à 10s et 4 souris ont développé une paralysie à 8/9s. De nombreuses lacunes ostéolytiques ont été observées sur les os longs dans les 2 groupes à 8s avec un amincissement plus sévère des corticales dans le groupe Ca−MM+. À 6s, le nombre d’ostéoclastes à la surface des corticales internes était significativement plus élevé. Une ostéolyse vertébrale sévère était observée dans le groupe Ca−MM+. Des différences significatives dans l’expression de gènes ont été observées à 6s avec une augmentation de MIP-1α et une diminution de Lrp5 dans le groupe Ca−MM+ vs Ca+MM+. Cette étude montre l’impact du microenvironnement sur le développement tumoral et l’explication possible d’un passage brutal d’un MM indolent à un MM agressif chez les patients.

Sinus lift elevation restores bone mass at the maxilla in edentulate patients before the placement of dental implants. It consists of opening the lateral side of the sinus and grafting beta-tricalcium phosphate granules (β-TCP) under the olfactory membrane. Bone biopsies were obtained in five patients after 60 weeks. They were embedded undecalcified in poly(methyl methacrylate) (pMMA); blocks were analyzed by nanocomputed tomography (nanoCT); specific areas were studied by Raman microspectroscopy. Remnants of β-TCP were osseointegrated and covered with mineralized bone; osteoid tissue was also filling the inner porosity. Macrophages having engulfed numerous β-TCP grains were observed in marrow spaces. β-TCP was identified by nanoCT as osseointegrated particles and as granules in the cytoplasm of macrophages. Raman microspectroscopy permitted to compare the spectra of β-TCP and bone in different areas. The ratio of the ~820 cm−1 band of pMMA (–CH2 groups) on the ν1 phosphate band at 960 cm−1 reflected tissue hydration because water was substituted by MMA during histological processing. In bone, the ratio of the ~960 cm−1 phosphate to the amide 1 band and the ratio ν2 phosphate band by the 1240–1250 amide III band reflect the mineralization degree. Specific bands of β-TCP were found in osseointegrated β-TCP granules and in the grains phagocytized by the macrophages. The hydration degree was maximal for β-TCP phagocytized by macrophages. Raman microspectroscopy associated with nanoCT is a powerful tool in the analysis of the biomaterial degradation and osseointegration.

It is not clear why patients with an indolent form of multiple myeloma (MM) develop into an aggressive form with poor prognostic. We investigated the effect of a dietary calcium deficiency on tumor growth, osteolysis and gene expression in the 5T2MM murine model. Two groups of C57BL/KaLwRij mice received 5T2MM cells and started a diet with normal (0.8%; "normal-Ca-MM") or low calcium content (0.05%; "low-Ca-MM"). Two control groups (without 5T2MM cells) received either a normal or low calcium diet (normal-Ca and low-Ca groups). Tumor growth, osteolysis and marrow gene expression of the Wnt pathway, RANKL and MIP-1α were monitored at 6, 8 and 10 weeks (w) after cell injection. In low-Ca mice, serum level of PTH was higher after 10w; microCT showed trabecular bone loss and decrease of cortical thickness at the tibia. A higher M-protein level was evidenced at 10w and 4 mice developed paraplegia at 8/9w in low-Ca-MM group only. Numerous cortical perforations of the tibia were observed in MM groups with a marked decrease in cortical thickness in low-Ca-MM. At 6w, osteoclast number from the endosteum was significantly higher in low-Ca-MM compared to normal-Ca MM. This observation was not found at 8 and 10w. MicroCT of the lumbar vertebrae showed dramatic bone destruction in the low-Ca-MM group. qPCR revealed no difference in RANKL expression whereas differences were obtained in the expression of Lrp5/Lrp6 and MIP-1α from 6w. A low calcium diet induced higher bone destruction in the tibia and vertebra associated with an earlier decrease in bone formation level and a higher increase in bone resorption level at early time in the MM development.

Muscle and bone masses are highly correlated and muscles impose large loads on bone. Muscle wasting that accompanies bone loss has been poorly investigated. 21 female mice were spread into seven groups. At day 0, 18 mice received Botulinum toxin (BTX) injection in the quadriceps muscle to induce paralysis of the right hind limb; the left contralateral side was used as control. Mice were sacrificed at 7, 14, 21, 28, 56 and 90 days post-injection. A remaining group was sacrificed at day 0. Trabecular bone volume was determined by microcomputed tomography (microCT) at the distal femur and tibia proximal metaphyses on both sides. Limbs were immersed in an HgCl2 solution allowing muscle visualization by microCT. On 2D sections, the cross-sectional areas and form-factors were measured for the quadriceps at mid-thigh and gastrocnemius at mid-leg and these muscles were dissected and weighed. Bone volume decreased in the paralysed side. Bone loss was maximal at 56 days followed by recuperation at 90 days. The cross-sectional areas of gastrocnemius and quadriceps were significantly lower in the paralysed limb from 7 days; the decrease was maximum at 21 days for the gastrocnemius and 28 days for the quadriceps. No difference in form-factors was found between the two limbs. Similar results were obtained with the anatomical method and significant correlations were obtained between the two methods. Quantitative analysis of muscle loss and recovery was possible by microCT after using a metallic contrast agent. Loss of bone secondary to muscle wastage induced by BTX and recovery showed a parallel evolution for bone and muscles.

Introduction: Botulinum toxin (BTX) injected in a muscle causes paralysis with a subsequent bone loss. It represents a model of disuse osteoporosis. Although bone loss has been regularly evaluated at the metaphysis of long bones, little is known concerning the bone changes occurring in the epiphysis.Material and methods: Ten Copenhagen male rats received a single BTX injection in the Mus quadriceps femoris on the right side and unilateral paralysis developed in the following days. Animals were euthanized after 28 days; femur and tibia were harvested and analyzed by microCT. Vector analysis of porosity was applied to the 2D sections and produced a frontal image with mapping in pseudo-colors. This allows quantitative analysis at the epiphysis and metaphysis. "Hot spot'' were evidenced and indicated bone loss. Quantitative analysis of these images was done by decomposition of the R, G and B planes and deriving the ratio of R + G pixels on the whole pixel number.Results: At the metaphysis, this ratio was correlated with measurement of the bone volume obtained by microCT. At the epiphysis, which has a complex shape in 3D, the method easily identified the bone loss.Discussion: Paralysis of a unilateral quadriceps induces bone loss at the metaphysis of the long bones. However, the epiphysis, having a reduced bone remodeling is also concerned by disuse. MicroCT analysis of this part of the bones is difficult due to its complex shape in 3D. Vector analysis is a new and robust method to quantify bone loss in such complex areas. (C) 2016 Anatomical Society of India. Published by Elsevier, a division of Reed Elsevier India, Pvt. Ltd. All rights reserved.