Abrasion of 6 dentifrices measured by vertical scanning interference microscopy

OBJECTIVES: The abrasion of dentifrices is well recognized to eliminate the dental plaque. The aims of this study were to characterize the abrasive powders of 6 dentifrices (3 toothpastes and 3 toothpowders) and to measure the abrasion on a test surface by Vertical Scanning Interference microscopy (VSI). MATERIAL AND METHODS: Bright field and polarization microscopy were used to identify the abrasive particles on the crude dentifrices and after prolonged washes. Scanning electron microscopy and microanalysis characterized the shape and nature of the particles. Standardized and polished blocks of poly(methylmethacrylate) were brushed with a commercial electric toothbrush with the dentifrices. VSI quantified the mean roughness (Ra) and illustrated in 3D the abraded areas. RESULTS: Toothpastes induced a limited abrasion. Toothpowders induced a significantly higher roughness linked to the size of the abrasive particles. One powder (Gencix® produced a high abrasion when used with a standard testing weight. However, the powder is based on pumice particles covered by a plant homogenate that readily dissolves in water. When used in the same volume, or after dispersion in water, Ra was markedly reduced. CONCLUSION: Light and electron microscopy characterize the abrasive particles and VSI is a new tool allowing the analysis of large surface of abraded materials

Osteoclast Cytomorphometry and Scanning Electron Microscopy of Bone Eroded Surfaces During Leukemic Disorders

Tartrate resistant acid phosphatase (TRAP) is a reliable histochemical marker of osteoclasts when used on tissue sections of undecalcified bone. This paper presents an original morphometric analysis which can be done after histochemical identification of osteoclasts. These bone resorbing cells were demonstrated on undecalcified bone biopsies from control subjects and patients presenting a malignant disease of the lymphocyte B lineage. Computerized analysis of the osteoclastic population revealed that: (1) all TRAP positive cells along bone trabeculae belong to a osteoclastic population; (2) that B cell malignancies had an increased bone resorption. At the scanning electron microscopic level small resorption bays (about 10 μm in diameter) were observed either associated or separated from eroded surfaces presenting a normal appearance; TRAP staining of histological sections of undecalcified bone, coupled with morphometric studies, may help in the understanding of bone disease pathobiology

Pharmacologic inhibitors of IκB kinase suppress growth and migration of mammary carcinosarcoma cells in vitro and prevent osteolytic bone metastasis in vivo

The NF-κB signaling pathway is known to play an important role in the regulation of osteoclastic bone resorption and cancer cell growth. Previous studies have shown that genetic inactivation of IκB kinase (IKK), a key component of NF-κB signaling, inhibits osteoclastogenesis, but the effects of pharmacologic IKK inhibitors on osteolytic bone metastasis are unknown. Here, we studied the effects of the IKK inhibitors celastrol, BMS-345541, parthenolide, and wedelolactone on the proliferation and migration of W256 cells in vitro and osteolytic bone destruction in vivo. All compounds tested inhibited the growth and induced apoptosis of W256 cells as evidenced by caspase-3 activation and nuclear morphology. Celastrol, BMS-345541, and parthenolide abolished IL1β and tumor necrosis factor α–induced IκB phosphorylation and prevented nuclear translocation of NF-κB and DNA binding. Celastrol and parthenolide but not BMS-345541 prevented the activation of both IKKα and IKKβ, and celastrol inhibited IKKα/β activation by preventing the phosphorylation of TAK1, a key receptor–associated factor upstream of IKK. Celastrol and parthenolide markedly reduced the mRNA expression of matrix metalloproteinase 9 and urinary plasminogen activator, and inhibited W256 migration. Administration of celastrol or parthenolide at a dose of 1 mg/kg/day suppressed trabecular bone loss and reduced the number and size of osteolytic bone lesions following W256 injection in rats. Histomorphometric analysis showed that both compounds decreased osteoclast number and inhibited bone resorption. In conclusion, pharmacologic inhibitors of IKK are effective in preventing osteolytic bone metastasis in this model and might represent a promising class of agents to the prevention and treatment of metastatic bone disease associated with breast cancer

Le risque d ostéoporose au cours des gammapathies monoclonales de signification indéterminée (étude prospective portant sur 201 patients)

Introduction : Les gammapathies monoclonales de signification indéterminée (MGUS) sont définies par une absence d'atteinte osseuse. Néanmoins, plusieurs études rétrospectives tendent à montrer une augmentation du risque d'ostéoporose fracturaire ou densitométrique dans cette population. L objectif de notre étude était de décrire le statut osseux des patients porteurs d une MGUS et d en déterminer les facteurs associés Patients et Méthodes : au cours d'une étude prospective réalisée entre 2008 et 2013, les patients porteurs d une gammapathie monoclonale de découverte fortuite sans antécédent fracturaire ou ostéoporotique connu ont tous bénéficiés des examens suivants : recueil des facteurs de risque d'ostéoporose, radiographies du rachis thoraco-lombaire, dosage des paramètres phosphocalciques et hématologiques, densitométrie osseuse par absorbtiométrie biphotonique à rayons X sur le site lombaire, col fémoral et extrémité supérieure du fémur, typage de la MGUS, prélèvement médullaire si le contingent monoclonal le justifiait. Ceux chez qui les résultats concluaient au diagnostic de maladie de waldenström asymptomatique ou symptomatique ou de myélome multiple asymptomatique ou symptomatique ont été exclus. Résultats : 201 patients porteurs d une MGUS ont été analysés : âge moyen 66,63 +- 12,49 ans; 48,3% de femmes, 104 IgG (51,7%), 67 IgM (33,3%), 21 IgA (10,4%), 9 double isotype (4,5%). 127 patients (63,2%) avaient une chaîne légère kappa, 63 (31,3%) une chaîne légère lambda et 9 (4,5%) un double contingent de chaînes légères. Le pic monoclonal moyen était de 5,98 g/l et la plasmocytose moyenne de 3,3%. 59 (29,4%) patients étaient ostéoporotiques (fracture vertébrale et/ou T-Score <= -2.5 DS), dont 37 (18,4%) présentaient une ou plusieurs fractures vertébrales thoraco-lombaires ostéoporotiques. Les patients fracturés étaient significativement plus âgés, avaient une densitométrie significativement plus basse aux 3 sites et étaient plus fréquemment d'isotype de chaîne légère lambda. Le risque relatif de fracture vertébrale chez les MGUS avec isotype lambda comparé à l isotype kappa était de 2,5 (IC 95 % 1,21-5,24). En analyse multivariée en tenant compte de l âge, du sexe et de la densité osseuse, le risque de fracture associé à la chaîne lambda restait significatif (p<0,01). Discussion : nous ne retrouvons pas dans cette étude de lien entre l isotype de la chaine lourde et le risque de fracture vertébrale mais une augmentation du risque associée à la présence de la chaîne légère lambda. Ce lien n a jamais été décrit dans la littérature et le mécanisme physiopathologique est inconnu. Ce résultat nécessite d être confirmé sur une plus large cohorte. Conclusion : dans cette cohorte de patients porteurs d une MGUS, nous décrivons pour la première fois une augmentation du risque de fracture vertébrale ostéoporotique associée à la chaîne légère lambda.Introduction: Monoclonal gammopathy of undetermined significance (MGUS) is defined by the absence of bone involvement. However, several retrospective studies suggest an increased risk of fracture or BMD osteoporosis in this population. The aim of our study was to describe the bone status of MGUS patients and to determine the associated factors with osteoporosis in MGUS. Patients and Methods: In a prospective study between 2008 and 2013, the holders of a monoclonal gammopathy of fortuitous discovery, without a history of fracture or osteoporosis, benefited all of the following tests: a collection of risk factors for osteoporosis, radiographs of the thoracolumbar spine, dosage of calcium, phosphate and haematological parameters, bone densitometry by dual-energy X-ray on lumbar site, femoral neck and total hip, typing MGUS, marrow sampling if warranted by the monoclonal quota. Patients diagnosed with smoldering or symptomatic Waldenstrom or smoldering or symptomatic multiple myeloma were excluded. Results: 201 holders of MGUS patients were analyzed: mean age 66.63 +- 12.49 years, 48.3 % women, 104 IgG (51.7% ), 67 IgM ( 33.3% ), 21 IgA (10.4%), 9 dual heavy chain isotype (4.5%). 127 patients had a kappa light chain (63.2 %), 63 had a lambda light chain (31.3%), 9 dual light chain isotype (4.5%). The average monoclonal peak was 5.98 g/l and the average plasma cells was 3.3%. 59 (29.4 %) patients had osteoporosis (vertebral fracture and/or T- score <= -2.5 SD), 37 (18.4%) had one or more osteoporotic vertebral fracture. Fractured patients were significantly older, had a significantly lower densitometry on the three sites and were more frequently lambda light chain isotype. The relative risk of vertebral fracture in MGUS with isotype lambda compared to isotype kappa was 2.52 (95% CI 1.21 to 5.24). In multivariate analysis taking into account age, sex, and bone density, the risk of fracture associated with the lambda light chain remained significant (p <0.01). Discussion : We did not find in this study link between heavy chain isotype and vertebral fracture risk but an increased risk associated with the presence of the lambda light chain isotype. This link has never been described in the literature and the pathophysiologic mechanism is unknown. This result needs to be confirmed on a larger cohort. Conclusion : In this cohort of MGUS patients, we described for the first time an increased risk of osteoporotic vertebral fracture associated with lambda light chain.ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF

Three-Dimensional Characterization of the Vascular Bed in Bone Metastasis of the Rat by Microcomputed Tomography (MicroCT)

BackgroundAngiogenesis contributes to proliferation and metastatic dissemination of cancer cells. Anatomy of blood vessels in tumors has been characterized with 2D techniques (histology or angiography). They are not fully representative of the trajectories of vessels throughout the tissues and are not adapted to analyze changes occurring inside the bone marrow cavities. Methodology/Principal Findings We have characterized the vasculature of bone metastases in 3D at different times of evolution of the disease. Metastases were induced in the femur of Wistar rats by a local injection of Walker 256/B cells. Microfil®, (a silicone-based polymer) was injected at euthanasia in the aorta 12, 19 and 26 days after injection of tumor cells. Undecalcified bones (containing the radio opaque vascular casts) were analyzed by microCT, and a first 3D model was reconstructed. Bones were then decalcified and reanalyzed by microCT; a second model (comprising only the vessels) was obtained and overimposed on the former, thus providing a clear visualization of vessel trajectories in the invaded metaphysic allowing quantitative evaluation of the vascular volume and vessel diameter. Histological analysis of the marrow was possible on the decalcified specimens. Walker 256/B cells induced a marked osteolysis with cortical perforations. The metaphysis of invaded bones became progressively hypervascular. New vessels replaced the major central medullar artery coming from the diaphyseal shaft. They sprouted from the periosteum and extended into the metastatic area. The newly formed vessels were irregular in diameter, tortuous with a disorganized architecture. A quantitative analysis of vascular volume indicated that neoangiogenesis increased with the development of the tumor with the appearance of vessels with a larger diameter. Conclusion This new method evidenced the tumor angiogenesis in 3D at different development times of the metastasis growth. Bone and the vascular bed can be identified by a double reconstruction and allowed a quantitative evaluation of angiogenesis upon time

Synthesis and use of pHEMA microbeads with human EA.hy 926 endothelial cells

Cancer has become a major problem in public health and the resulting bone metastases a worsening factor. Facing it, different strategies have been proposed and mechanisms involved in tumor angiogenesis are being studied. Enhanced permeability retention (EPR) effect is a key step in designing new anticancer drugs. We have prepared poly 2-hydroxyethyl methacrylate (pHEMA) microbeads to target human endothelial EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells. Microbeads were synthesized by emulsion precipitation method and carried positive or negative charges. EA.hy 926 cells were cultured in 24-well plates and microbeads were deposited on cells at various times. Scanning and transmission electron microscopy, flow cytometry, confocal microscopy, and three-dimensional (3D) reconstruction were used to characterize microbeads and their location outside and inside cells. Microbeads were uptaken by endothelial cells with a better internalization for negatively charged microbeads. 3D reconstruction of confocal optical sections clearly evidenced the uptake and internalization of microbeads by endothelial cells. pHEMA microbeads could represent potential drug carrier in tumor model of metastases

Modèles animaux d'hyper-résorption osseuse (méthodes d'étude et physiopathologie)

Plusieurs modèles animaux d'hyper-résorption osseuse ont été étudiés : modèle du rat orchidectomisé (ORX), modèle murin de myélome (5T2MM) combiné ou non à une ovariectomie (OVX). Nous avons évalué la performance et la sensibilité de différentes méthodes d'étude de la perte osseuse, et caractérisé la physiopathologie de l'hyper-résorption. Des mesures densitométriques (DXA) du contenu minéral osseux (CMO) d'os de rats contrôles, ont été obtenues avec une bonne reproductibilité et une bonne exactitude sur 3 générations de densitomètres, avec cependant une influence du poids de l'os sur le CMO. Des mesures de CMO, effectuées sur des os de rats ORX et ORX traités par bisphosphonate, ont montré qu' une large distribution du CMO n'altère pas l'exactitude des mesures de DXA. Cette méthode est cependant moins sensible que l'histomorphométrie pour apprécier le degré de perte osseuse dans le modèle du rat ORX. Chez le rat ORX, l'histomorphométrie a mis en évidence une altération précoce de la microarchitecture osseuse précédent la perte osseuse. Dans le modèle murin de myélome 5T2MM, l'hyper-résorption se traduit par une disparition de l'os trabéculaire et la présence de nombreuses perforations corticales. Nous avons élaboré un modèle associant OVX au modèle 5T2MM. L'OVX induit un hyper-remodelage associé à une augmentation de la progression tumorale et une apparition plus précoce des lésions ostéolytiques. Ce résultat pourrait expliquer le passage brutal d'un myélome indolent à un myélome agressif chez l'homme lorsqu'une modification du niveau de remodelage survient.Several animal models, with a high bone resorption level, were studied : the orchidectomized (ORX) rat model, the 5T2MM murine myeloma model with or without ovariectomy (OVX). We have first examined reproducibility, accuracy and sensibility of several methods used to evaluate bone loss. Then, we have studied the physiopathology of high remodeling rate in these animal models. Densitometric measurements (DXA) of bone mineral content (BMC) were done in control rats. Precise and accurate BMC measurements were obtained on 3 different generations of densitometer. However, discrepancy of BMC was dependent on the net weight of the bone. BMC measurements were performed on bone of ORX rat and ORX treated with a bisphosphonate. Accuracy was not affected by a large distribution of BMC values. DXA appeared to be less sensitive than bone histomorphometry to appreciate bone loss in the ORX rat model. In the ORX rat, histomorphometry evidenced alteration of trabecular bone architecture before bone loss occured. In the 5T2MM murine myeloma model, the increase of bone resorption induced disaparition of trabecular bone and numerous cortical perforations. We have proposed a combined animal model in which OVX was performed in mice prior inoculation of 5T2MM cells. OXV induced an increase bone remodeling which was associated with an increase of tumor growth and earlier development of osteolytic lesions. This result could explain some sudden burden of indolent MM into aggressive MM in man when a modification of mode remodeling happens.ANGERS-BU Médecine-Pharmacie (490072105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Modèles animaux de perte osseuse bénigne et maligne utilisable pour l évaluation de biomatériaux

Plusieurs modèles animaux de perte osseuse ont été développés : le modèle du rat orchidectomisé (ORX) et paralysé par la toxine botulique (BTX), le modèle du rat injecté avec des cellules Walker induisant des métastases ostéolytiques, le modèle du rat injecté avec des cellules MAT-Ly-Lu induisant des métastases ostéocondensantes. Nous avons montré que la combinaison ORX-BTX avait des effets cumulatifs ; elle induit des changements microarchitecturaux et une perte osseuse massive et rapide due à une hausse rapide et transitoire du remodelage osseux. Le modèle ORX-BTX a été utilisé pour l'implantation d'un biomatériau injectable qui a été transitoirement remplacé par de l'os non mature résorbé ensuite sans être substitué par de l'os lamellaire mature. Des mesures densitométrique, histomorphométrique et microtomographique chez le rat ORX-BTX ont montré que le risédronate a un effet protecteur contre la perte osseuse trabéculaire et la modification de la microarchitecture et un effet plus modéré sur la perte osseuse corticale. La testostérone ne prévient pas la perte osseuse mais préserve la masse maigre corporelle. L'analyse de texture et la microtomographie ont permis de détecter et de différencier les altérations osseuses induites par métastases ostéolytiques ou ostéocondensantes. Nous avons également montré par études histologique et immunohistochimique que les interactions cellules cancéreuses-cellules stromales provoquent une production stromale de cytokines aboutissant à l'ostéoclastogénèse uniquement dans le microenvironnement osseux. Le microenvironnement osseux peut donc avoir un rôle important dans l'établissement et la prolifération métastatique.Several animal models of bone loss were designed: the orchidectomized (ORX) and paralysed with botulinum toxin (BTX) rat model, the rat model injected with Walker cells inducing osteolytic metastases, the rat model injected with MAT-Ly-Lu cells inducing osteoblastic metastases. The combination of ORX and BTX had cumulative effects; it induced microarchitectural changes; a fast and severe bone loss due to a transient increase of bone remodeling. The ORX-BTX model was used for the implantation of an injectable biomaterial. The biomaterial was transiently replaced by woven bone and then resorbed without substitution by lamellar bone. Densitometric, histomorphometric and microtomographic measurements in the ORX-BTX rat showed a protective effect of risedronate against the trabecular bone loss and the microarchitectural changes, but the effects were moderate on the cortical bone loss. Testosterone did not prevent the bone loss but preserved the body lean mass. Texture analysis and microtomography were used to detect and differentiate bone alterations due to osteolytic or osteoblastic metastases. We have also shown, by histological methods and immunohistochemistry, that interactions between cancer and stromal cells induced the production of cytokines leading to osteoclastogenesis only in bone microenvironment. The bone microenvironment appears to have an important role in the homing and proliferation of cancer cells.ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF

Biomimétisme, cytocompatibilité et cytodynamique

L'influence d'un revêtement de type RGD sur du Ticp sur l'adhésion, l'étalement et la minéralisation des ostéoblastes a été étudié. Ce revêtement accélérait la vitesse de minéralisation et augmentait la surface des zones de minéralisation. Des CNTs fonctionnalisés ont été utilisés pour induire la calcification dans le but de mimer in vitro la minéralisation des fibres de collagène dans le tissu osseux. Après 14 jours d'incubation dans un liquide biosynthétique, les CNTs fonctionnalisés ont induit la formation de calcosphérites, similaires à ceux formés dans le woven bone. Ce modèle de calcification in vitro biomimétique, mis au point sur des polymères dans notre laboratoire, a permis d'étudier l'incorporation du Sr2+ dans le minéral osseux et sa désorption. Sr2+ a été incorporé dans le minéral lors de la calcification sans induire de changement des paramètres du cristal ou de la cristallinité quelque soit la concentration initiale en Sr2+. La désorption du Sr2+ a été rapide initialement avec libération de 20 à 25 % en 16 jours, puis plus lente pour atteindre 30 % après 61 jours. Enfin, les observations en cytodynamique ont permis de voir les premières étapes d'adhésion cellulaire et de colonisation du b-TCP. Les séquences vidéo réalisées sur 8 jours de culture, ont permis d'observer la prolifération des ostéoblastes (SaOs-2) qui sembla se produire en direction des particules de b-TCP pour venir en contact direct avec les particules. Les macrophages (J774-2 et macrophages péritonéaux de souris) ne proliféraient pas mais venaient au contact direct des particules pour les dégrader. Les observations MEB ont permis de constater que les cellules émettaient des prolongements cytoplasmiques et s'étiraient pour venir se fixer et ensuite s'étaler à la surface du biomatériau. Les données obtenues grâce aux observations faites avec la vidéomicroscopie sur long terme nous ont permis de confirmer la cytocompatibilité et la biodégradabilité du b-TCP.The influence of RDG coating on Ti was tested on osteoblasts behaviors. This coating allowed an earlier mineralization and an increase of surface mineralized. Functionalized CNTs have been used to promote calcification to mimic the in vivo mineralization of collagen fibers. After 14 days of incubation, functionalized CNTs promoted the calcospherites formation, which were similar to those in woven bone. This in vitro calcification model developed in our laboratory was used to study the Sr2+ incorporation in mineral and its released. Sr2+ was incorporated in mineral during calcification without inducing any change in crystal parameters or crystallinity. Sr2+ released was rapid at the begging and then slower to reach 30% of Sr2+ released after 61 days. In a last study, cell behaviors in direct contact with b-TCP particles using long term culture under videomicroscopy were observed. Video sequences realized after 8 days of culture showed an active proliferation of SaOs-2 occurring in the direction of the particles. J774.2 did not proliferate but came in direct contact with the particles. Peritoneal macrophages did not proliferate but came in direct contact with the particles and actively eroded them. SEM showed that cells were stretched and fixed onto the surface and seemed to climb from the coverslip to the particles. The long term culture under videomicroscopy allowed a better understanding of the colonization process of b-TCP particles by osteoblast-like cells and macrophages. Data obtained from long term videomicroscopy are in agreement with in vivo observations confirming the interest of b-TCP to promote osteogenesis.ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF