Extra-skeletal manifestations in mice affected by Clcn7-dependent autosomal dominant osteopetrosis type 2 clinical and therapeutic implications.

Autosomal dominant osteopetrosis type 2 (ADO2) is a high-density brittle bone disease characterized by bone pain, multiple fractures and skeletal-related events, including nerve compression syndrome and hematological failure. We demonstrated that in mice carrying the heterozygous Clcn7 G213R mutation, whose human mutant homolog CLCN7 G215R affects patients, the clinical impacts of ADO2 extend beyond the skeleton, affecting several other organs. The hallmark of the extra-skeletal alterations is a consistent perivascular fibrosis, associated with high numbers of macrophages and lymphoid infiltrates. Fragmented clinical information in a small cohort of patients confirms extra-skeletal alterations consistent with a systemic disease, in line with the observation that the CLCN7 gene is expressed in many organs. ADO2 mice also show anxiety and depression and their brains exhibit not only perivascular fibrosis but also ß-amyloid accumulation and astrogliosis, suggesting the involvement of the nervous system in the pathogenesis of the ADO2 extra-skeletal alterations. Extra-skeletal organs share a similar cellular pathology, confirmed also in vitro in bone marrow mononuclear cells and osteoclasts, characterized by an impairment of the exit pathway of the Clcn7 protein product, ClC7, through the Golgi, with consequent reduced ClC7 expression in late endosomes and lysosomes, associated with high vesicular pH and accumulation of autophagosome markers. Finally, an experimental siRNA therapy, previously proven to counteract the bone phenotype, also improves the extra-skeletal alterations. These results could have important clinical implications, supporting the notion that a systematic evaluation of ADO2 patients for extra-skeletal symptoms could help improve their diagnosis, clinical management, and therapeutic options.

Assessment of predictivity of volatile organic compounds carcinogenicity and mutagenicity by freeware in silico models.

The application of in silico methods is increasing on toxicological risk prediction for human and environmental health. This work aimed to evaluate the performance of three in silico freeware models (OSIRIS v.2.0, LAZAR, and Toxtree) on the prediction of carcinogenicity and mutagenicity of thirty-eight volatile organic compounds (VOC) related to chemical risk assessment for occupational exposure. Theoretical data were compared with assessments available in international databases. Confusion matrices and ROC curves were used to evaluate the sensitivity, specificity, and accuracy of each model. All three models (OSIRIS, LAZAR and Toxtree) were able to identify VOC with a potential carcinogenicity or mutagenicity risk for humans, however presenting differences concerning the specificity, sensitivity, and accuracy. The best predictive performances were found for OSIRIS and LAZAR for carcinogenicity and OSIRIS for mutagenicity, as these softwares presented a combination of negative predictive power and lower risk of false positives (high specificity) for those endpoints. The heterogeneity of results found with different softwares reinforce the importance of using a combination of in silico models to occupational toxicological risk assessment.

Citocompatibilidade do cimento reparador ceramicrete em cultura primária de osteoblastos humanos / Cytocompatibility of the ceramicrete repair cement in primary culture of human osteoblasts

Ainda são muito raras as informações sobre a resposta de osteoblastos humanos ao Ceramicrete (CC), um material proposto para ser usado como cimento reparador que apresenta características bastante promissoras. Assim, o objetivo do presente estudo foi verificar o grau de citocompatibilidade do CC em culturas primárias de osteoblastos humanos. Para isso, foi utilizado um modelo experimental baseado na quantidade de material usado em uma retro-obturação, criando condições mais próximas a situação clínica real. Retrocavidades obturadas com CC foram colocados em poços de cultura contendo meio de cultura (α-MEM), por 24h (n=4). Meios de cultura contendo dentes retro-obturados com ProRoot MTA e OZE foram usados como controle negativo e positivo, respectivamente. Todos os meios foram armazenados. Posteriormente, foram cultivados osteoblastos humanos (2 x 104 células/amostra), por 24h, em poços com os meios armazenados dos 3 materiais. Foi avaliada a citocompatibilidade utilizando um Kit específico (Cytotox Kit, Xenometrix, Alemanha), que possibilita o estudo de 3 parâmetros (XTT, NR e CVDE) utilizando a mesma amostra. Foi encontrada uma diferença estatisticamente significativa entre os 3 materiais para todos os 3 parâmetros avaliados (ANOVA, p< 0,05). O CC e o MTA foram sempre estatisticamente superiores ao OZE (Duncan, p<0,05). Entretanto, não foram encontradas diferenças entre os 2 cimentos reparadores, CC e MTA (Duncan, p> 0,05). Portanto, pode-se concluir que o Ceramicrete expressa um padrão de citocompatibilidade similar a do MTA, em cultura primária de osteoblastos humanos, quando usado como material retro-obturador.

Ceramicrete is an endodontic material with very promising characteristics. However, information about the response of human osteoblasts to CC is very rare. So, the purpose of this study was to assess the cytocompatibility of CC in primary cultures of human osteoblasts. For this, we used a modelo f retro-obturation, creating conditions close to real clinical situation. The apical portions of canines were retro-obturated with CC or control materials. The, the teeth were placed in Wells containing culture médium (α-MEM) for 24h (n=4). ProRoot MTA and ZOE were used as negative and positive controls, respectively. All media were stored. Subsequently, human osteoblasts were cultured (2x104 cells) for 24 h in Wells with the stored medium. The cytocompatibility was evaluated using a specific kit (Cytotoxic, Xenometrix, Germany), which enables the study of three parameters of cell viability using the same sample (CTT, NR and CVDE). It was found significant difference among the three materials Fo all parameters (ANOVA, p> 0.05). The CC and the MTA were always statistically superior to ZOE (Duncan, p> 0.05). However, no differentes were found between the two cements, CC and MTA (Duncan, p> 0.05). It can be concluded that CC is an endodontic cement as cytocompatible as MTA when used as a retro-obturation material in primary cultures of human osteoblasts.