A three-dimensional cell culture approach to investigate cytotoxic effects and production of inflammatory mediators by epoxy resin-based and calcium silicate-based endodontic sealer.

OBJECTIVES: The aim of the present study was to assess the cytocompatibility of epoxy resin-based AH Plus Jet (Dentsply De Trey, Konstanz, Germany), Sealer Plus (MK Life, Porto Alegre, Brazil), calcium silicate-based Bio-C Sealer (Angelus, Londrina, PR, Brazil), Sealer Plus BC (MK Life) and AH Plus BC (Dentsply) through a tridimensional (3D) culture model of human osteoblast-like cells. METHODS: Spheroids of MG-63 cells were produced and exposed to fresh root canal sealers extracts by 24 h, and the cytotoxicity was assessed by the Lactate Dehydrogenase assay (LDH). The distribution of dead cells within the microtissue was assessed by fluorescence microscopy, and morphological effects were investigated by histological analysis. The secreted inflammatory mediators were detected in cell supernatants through flow luminometry (XMap Luminex). RESULTS: Cells incubated with AH Plus Jet, AH Plus BC, Sealer Plus BC and Bio-C Sealer extracts showed high rates of cell viability, while the Sealer Plus induced a significant reduction of cell viability, causing reduction on the spheroid structure. Sealer Plus and Seaker Plus BC caused alterations on 3D microtissue morphology. The AH Plus BC extract was associated with the downregulation of secretion of pro-inflammatory cytokines IL-5, IL-7, IP-10 and RANTES. CONCLUSIONS: The new AH Plus BC calcium silicate-based endodontic sealer did not reduce cell viability in vitro, while led to the downregulation of pro-inflammatory cytokines. CLINICAL SIGNIFICANCE: Choosing the appropriate endodontic sealer is a crucial step. AH Plus BC demonstrated high cell viability and downregulation of pro-inflammatory cytokines, appearing reliable for clinical use, while Sealer Plus presented lower cytocompatibility.

In vivo evaluation of the biocompatibility and biodegradation of a new denatured plasma membrane combined with liquid PRF (Alb-PRF).

Guided bone regeneration (GBR) is a process that involves the regeneration of bone defects through the application of occlusive membranes that mechanically exclude the population of non-osteogenic cells from the surrounding soft tissue. Interestingly, platelet-rich fibrin (PRF) has previously been proposed as an autologous GBR membrane despite its short-term resorption period of 2-3 weeks. Recent clinical observations have demonstrated that, by heating a liquid platelet-poor plasma (PPP) layer and mixing the cell-rich buffy coat zone, the resorption properties of heated albumin gel with liquid-PRF (Alb-PRF) can be significantly improved. The aim of this study was to evaluate the inflammatory reaction, biocompatibility, and extended degradation properties of a new autologous Alb-PRF membrane in comparison to commonly utilized standard PRF after nude mice implantation, according to ISO 10993-6/2016. Two standard preparations of PRF (L-PRF and H-PRF) were compared to novel Alb-PRF following subcutaneous implantation at 7, 14, and 21 days. All groups demonstrated excellent biocompatibility owing to their autologous sources. However, it is worth noting that, while both L-PRF and H-PRF membranes demonstrated significant or complete resorption by 21 days, the Alb-PRF membrane remained volume-stable throughout the duration of the study. This study demonstrates-for the first time, to the best of our knowledge-a marked improvement in the membrane stability of Alb-PRF. This indicates its future potential for use as a biological barrier membrane for GBR procedures with a long-lasting half-life, or as a biological filler material in esthetic medicine applications. Thus, further studies are warranted to explore future clinical applications in various fields of medicine.

New Law of Brazilian Biodiversity: Legal Aspects and Impact in the Field of Biotechnology.

Access to genetic resources (GR) and/or traditional knowledge associated with genetic resources (ATK) has been regulated in Brazil since 2001. The law 13,123 / 2015 determined a significant change in the theme, mainly on the rules of distribution of benefits obtained for conservation and sustainable use of biodiversity, the access to technology and technology transfer, the exploitation of products or reproductive material from the GR or ATK and consignment to the outside of part or all the living or dead organism shipped for GR. The implementation of international treaties on GR and ATK for research, biotechnological development and bioprospecting have been causing difficulties for Brazilian researchers, mainly due to the lack of information and dissemination available for compliance with the legislation. In this work, the members of the Committee for Access to Genetic Resources and Associated Traditional Knowledge of the Federal Fluminense University (UFFGEN) - Brazil, and collaborators performed a critical reflection on the new law, helping Brazilian researchers with information necessary to understand the changes made by the new legislation, especially in the field of Biotechnology associated with Brazilian Biodiversity.

Cytocompatibility of filling pastes by primary teeth root simulating model.

Evaluate the cytocompatibility of Calen®/ZO, Calcicur®, Vitapex®, Endoflas®, and zinc oxide/eugenol-based (ZOE) root canal pastes (RCP) to human primary osteoblasts (HPO) through a simplified model for primary teeth. The model employed pipette tips filled with 0.037 g of paste, exposed to 185 µL of culture medium for 24 h (n = 6). Release of components was analysed by Proton Nuclear Magnetic Resonance Spectroscopy (1H-NMR). HPO were exposed to conditioned media for 24 h. Cell viability was assessed by cell density and metabolic activity, and release of interleukin 6 (IL-6), vascular endothelial growth factor (VEGF) and fibroblast growth factor (bFGF) by immunological assay. Physicochemical properties and antimicrobial efficacy were also evaluated. 1H-NMR spectra analysis showed similarity between ZOE, Endoflas®, Calcicur®, and Vitapex® compared to Calen®/ZO and positive control, which showed distinct released components. Calen®/ZO and Calcicur® exhibited high alkaline pH in all periods and showed similar solubility. Calen®/ZO, ZOE, and Vitapex® showed similar flow rate. Calen®/ZO, Calcicur®, and Vitapex® did not exhibit antimicrobial efficacy. Calen®/ZO presented cytotoxicity (p < 0.05). Pastes did not increase IL-6 release compared to control. Apart from Vitapex®, all pastes significantly induced VEGF/bFGF release. Interactive effects among released products may affect biological response to filling pastes. Calcicur®, ZOE, Endoflas® and Calen®/ZO presented good to moderate cytocompatibility, with low impact on pro-inflammatory cytokine release and induction of growth factors of interest to tissue repair. This simplified model, specific for the evaluation of the cytocompatibility of RCPs on primary teeth, suggests how these pastes might contribute to bone repair in clinical situations of apical periodontitis in children.

Apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) contributes to osteoblast differentiation and osteogenesis.

The role of apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) in bone healing remains to be understood. To address this issue, we investigated the requirement of inflammasome-related genes in response to bone morphogenetic protein 7 (BMP7)-induced osteoblast differentiation in vitro. To validate the importance of ASC on osteogenesis, we subjected wild-type (WT) and ASC knockout C57BL/6 mice (ASC KO) to tibia defect to evaluate the bone healing process (up to 28 days). Our in vitro data showed that there is an involvement of ASC during BMP7-induced osteoblast differentiation, concomitant to osteogenic biomarker expression. Indeed, primary osteogenic cells from ASC KO presented a lower osteogenic profile than those obtained from WT mice. To validate this hypothesis, we evaluated the bone healing process of tibia defects on both WT and ASC KO mice genotypes and the ASC KO mice were not able to fully heal tibia defects up to 28 days, whereas WT tibia defects presented a higher bone de novo volume at this stage, evidencing ASC as an important molecule during osteogenic phenotype. In addition, we have shown a higher involvement of runt-related transcription factor 2 in WT sections during bone repair, as well as circulating bone alkaline phosphatase isoform when both were compared with ASC KO mice behavior. Altogether, our results showed for the first time the involvement of inflammasome during osteoblast differentiation and osteogenesis, which opens new avenues to understand the pathways involved in bone healing.

Advances and potential application of gold nanoparticles in nanomedicine.

Nanomedicine is an emerging research area which has brought new possibilities and promising applications in image, diagnosis, and treatment. Nanoparticles (NPs) for medicinal purposes can be made of several material types such as silica, carbon, different polymers, and metals as silver, copper, titanium, and gold. Gold NPs (AuNPs) are the most studied and used, mostly due to their characteristics including simple preparation, controllable size and distribution, biocompatibility, good acceptance of surface modifications, and specific surface plasmon resonance (SPR). This study reviews the scientific literature regarding the potential applications of AuNPs in the development of new diagnostic and therapeutic strategies for nanomedicine, including their biomedical use as a drug carrier, as an agent in radio and phototherapy, and bioimaging for image diagnosis. While it becomes clear that much research remains to be done to improve the use of these nanoparticles, with particular concern for safety issues, the evidence from the literature already points to the great potential of AuNPs in nanomedicine.

Does the association of blood-derived growth factors to nanostructured carbonated hydroxyapatite contributes to the maxillary sinus floor elevation? A randomized clinical trial.

OBJECTIVE: The combination of calcium phosphate with blood-derived growth factors (BDGF) has been widely used in bone regeneration procedures although its benefits are still unclear. The purpose of this study was to evaluate whether or not BDGF improves the efficacy of a modified carbonated calcium phosphate biomaterial in sinus floor augmentation. MATERIAL AND METHODS: Ten patients underwent 20 sinus floor augmentation procedures using nanostructured carbonated hydroxyapatite (cHA) microspheres alone or associated with BDGF in a randomized controlled clinical trial. The in vitro release of growth factors was assessed by an elution assay. Bone grafts were randomly implanted in the right and left maxillary sinuses of each participant, associated either with a 0.9% saline solution or BDGF. Bone gain was evaluated through cone beam tomography after 180 days. RESULTS: Nine women and one man composed the sample. The blood-derived concentrates were able to release high levels of growth factors and cytokines. A significant clinical advantage was observed in the use of the BDGF after fibrin polymerization around the biomaterial microspheres, optimizing the surgical procedures, thereby reducing the time and displacement, and improving the adaptation of the biomaterial in the maxillary sinus. No synergistic effect was observed in bone formation when cHA was associated with BDGF (p > 0.05). CONCLUSION: Equivalent new bone formation was observed for cHA in the presence or absence of the BDGF concentrate in bilateral sinus floor elevation after 6 months. Blood-derived growth factors did not improve bone repair when associated with calcium phosphate in sinus lift procedures.

Brazil starts to ban animal use in higher education: A positive and progressive development.

The Brazilian government has published a resolution that bans animal use in some practical classes within undergraduate and high school technical education from April 2019. Resolution No. 38/2018, issued by the National Council for the Control of Animal Experimentation (CONCEA), bans the killing of animals for dissection purposes and animal experiments in practical classes that do not involve the acquisition of new skills. The initial call for the ban was by the Brazilian Network for Humane Education (RedEH), an independent body comprising Brazilian professors and international collaborators dedicated to the implementation of replacement alternatives in education. The call was supported by InterNICHE, and many professors and other international organisations. The Brazilian Council of Veterinary Medicine, which is responsible for regulating the veterinary profession in Brazil, also stated its support for humane education and for the ban. The call was the first formal request, and it eventually led to the first legal resolution for the replacement of animal use in education in Brazil. This represents an important historic landmark in the advancement of science education.

Genetic Polymorphisms in DEFB1 and miRNA202 Are Involved in Salivary Human ß-Defensin 1 Levels and Caries Experience in Children.

The antimicrobial peptides human ß-defensins (hBDs) are encoded by ß-defensin genes (DEFBs) and are possibly involved in caries susceptibility. In this study we aimed (1) to investigate the relationship between salivary hBDs and caries and (2) to evaluate the association of genetic polymorphisms in DEFB1 and microRNA202 (miRNA202) with salivary levels of hBDs and caries experience. Two data sets were available for this study, totalizing 678 Brazilian children. Dental examination and saliva collection were performed in all included children. The salivary level for hDB1, hBD2, and hBD4 was assessed by ELISA sandwich technique in 168 children. The DNA was extracted from saliva, and polymorphisms in DEFB1 and miRNA202 were analyzed by real-time PCR. Statistical analysis was performed to investigate the associations between caries experience, hBD salivary level, genotype, and allele distribution, with an alpha of 0.05. The hBD1 level was significantly higher in caries-free children (p < 0.0001). The miRNA202 was associated with a lower level of salivary hBD1 (p < 0.05). Also, the polymorphic distribution of miRNA202 was associated with caries (p = 0.006). The polymorphisms in DEFB1 were not associated with hBD salivary level and caries experience (p > 0.05). In conclusion, our results indicate that genetic polymorphism in miRNA202 is involved in hBD1 salivary level as well as caries experience in children.

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.

On the journey toward humane education in Brazil: First request for a total ban of harmful animal use in professional and higher education.

The Brazilian Network for Humane Education (RedEH( is an independent and self-managed group comprised of academics from ten different Brazilian states and a number of international collaborators. In 2016, in a concerted effort to change the educational field in Brazil and propagate humane education, RedEH sent a request to the Brazilian National Council for the Control of Animal Experimentation (CONCEA(, asking that harmful animal use in education in professional and undergraduate courses be banned. This was the first formal request for a total replacement of harmful animal use in education in Brazil, and represented a major historic landmark in the advancement of Brazilian science education. This paper presents the full text of the request, as well as outlining its national and international repercussions. The request was supported by InterNICHE and representatives of 18 other international organisations. A major national impact of the request was its recognition by the Federal Council of Veterinary Medicine. With this action, academics and researchers took a potentially revolutionary step in the Brazilian education arena, with regard to advancing and supporting a higher quality, ethical and democratic educational system.

In vitro Analysis of Cytotoxicity of Temporary Resilient Relining Materials.

AIMS: The aim of this study is to evaluate the in vitro response of human gingival fibroblasts in primary cultures to two materials for temporary relining of dentures: Temporary Soft (TDV, Brazil) and Trusoft (Bosworth, USA) for 24 hours, 7 and 30 days by using a multi-parametric analysis. MATERIALS AND METHODS: Each material sample (TDV, TS, Polystyrene, Latex) was prepared and incubated in a culture medium for 1, 7, and 30 days at 37°C. Human gingival fibroblasts were exposed to the extracts and cell viability was evaluated by a multi-parametric assay, which allowed sequential analysis of mitochondrial activity (XTT), membrane integrity [neutral red (NR)], and cell density [crystal violet dye exclusion (CVDE)] in the same cells. Analysis of variance (ANOVA) was used to test the interactions of the three sources of variation (material, test method, and time) with the proportions of viable cells for each relining material. RESULTS: Both evaluated materials (TDV and TS) had low cytotoxic effects during 1, 7, and 30 days after manipulation of the material, as assessed by all three methods used. A statistical difference was found when comparing the negative control group (latex fragments) with the other groups, which showed high toxicity and low percentage of cell viability in all tests used. There was no significant difference among other materials (p > 0.05). CONCLUSION: Low cytotoxicity levels were detected by representatives of the major groups of temporary prosthetic relining materials, as evaluated by multiple cellular viability parameters in human fibroblasts. CLINICAL SIGNIFICANCE: There are various soft materials on the market for relining prostheses; however, the effects of these materials on tissues need to be clarified to avoid problems for patients.

Evaluation of Inflammatory Response to Endodontic Sealers in a Bone Defect Animal Model.

AIM: The aim of this study was to evaluate the inflammatory response to MTA Fillapex, AH Plus, and Pulp Canal Sealer Extensive Work Time (EWT), in a murine bone defect grafting model. MATERIALS AND METHODS: Bilateral mandibular critical defects were produced in 45 Wistar rats with a trephine bur#2 and filled with the endodontic sealers. After 7, 14, and 28 days, the rats were euthanized and their jaws were histologically prepared. RESULTS: For the 7-day group, no statistical significance was observed among all studied groups (p > 0.05), and high levels of inflammatory infiltrate were detected. After 14 and 28 days, Pulp Canal Sealer EWT showed statistically lower inflammatory response in comparison to other sealers (p < 0.05) except for the control group (no sealers). CONCLUSION: Pulp Canal Sealer EWT presented the lowest levels of inflammatory response. The critical defect grafting model was an effective method to detect differences among differences on the biological response to endodontic sealers. CLINICAL SIGNIFICANCE: Knowing the biocompatibility of endodontics sealers that will be used in filling the root canal.

Genetic influences on dental enamel that impact caries differ between the primary and permanent dentitions.

Clinically, primary and permanent teeth are distinct anatomically and the presentation of caries lesions differs between the two dentitions. Hence, the possibility exists that genetic contributions to tooth formation of the two dentitions are different. The purpose of this study was to test the hypothesis that genetic associations with an artificial caries model will not be the same between primary and permanent dentitions. Enamel samples from primary and permanent teeth were tested for microhardness at baseline, after carious lesion creation, and after fluoride application to verify association with genetic variants of selected genes. Associations were found between genetic variants of ameloblastin, amelogenin, enamelin, tuftelin, tuftelin interactive protein 11, and matrix metallopeptidase 20 and enamel from permanent teeth but not with enamel from primary teeth. In conclusion, our data continue to support that genetic variation may impact enamel development and consequently individual caries susceptibility. These effects may be distinct between primary and permanent dentitions.

BMP2 Is Associated with Caries Experience in Primary Teeth.

Bone morphogenetic proteins (BMPs) play an important role during the initial process of enamel development and therefore may play a role in caries susceptibility. The purpose of this study was to evaluate the association between the polymorphisms in the BMP2, BMP4 and BMP7 genes and their association with caries experience and primary enamel microhardness characteristics. DNA from buccal cells as well as clinical and demographic information from 1,731 subjects from three different data sets from Brazil were included. Polymorphisms in BMP2, BMP4 and BMP7 were analyzed by real-time polymerase chain reaction from genomic DNA. Association between caries experience, genotype, and allele distribution in both cohorts was evaluated using χ(2) and logistic regression analyses. In the family-based set, the association between caries experience and alleles was tested using the transmission disequilibrium test. In the Rio de Janeiro cohort, microhardness data on 108 exfoliated primary teeth before and after demineralization and remineralization challenges was included. Associations between microhardness values and genotype and allele distribution were evaluated using χ(2) and logistic regression analyses. Differences between caries experience and some risk factors were statistically significant. In the cohort from Nova Friburgo, BMP2 was associated with caries experience in primary dentition during logistic regression analysis (p = 0.023; OR = 2.58; 95% CI 1.13-5.86). There was no association between genotype and allele distribution for BMP polymorphisms and primary enamel microhardness alterations. Our result suggests that BMP2 may be involved in caries experience in primary dentition from a Nova Friburgo cohort.

Humane Education in Brazil: Organisation, Challenges and Opportunities.

Humane education and the debate on alternatives to harmful animal use for training is a relatively recent issue in Brazil. While animal use in secondary education has been illegal since the late 1970s, animal use in higher science education is widespread. However, alternatives to animal experiments in research and testing have recently received attention from the Government, especially after the first legislation on animal experiments was passed, in 2008. This article proposes that higher science education should be based on a critical and humane approach. It outlines the recent establishment of the Brazilian Network for Humane Education (RedEH), as a result of the project, Mapping Animal Use for Undergraduate Education in Brazil, which was recognised by the 2014 Lush Prize. The network aims to create a platform to promote change in science education in Brazil, starting by quantitatively and qualitatively understanding animal use, developing new approaches adapted to the current needs in Brazil and Latin America, and communicating these initiatives nationally. This paper explores the trajectory of alternatives and replacement methods to harmful animal use in training and education, as well as the status of humane education in Brazil, from the point of view of educators and researchers engaged with the network.

Bone Morphogenetic Proteins: structure, biological function and therapeutic applications.

Bone Morphogenetic Proteins (BMPs) are multifunctional secreted cytokines, which belong to the TGF-ß superfamily. These glycoproteins act as a disulfide-linked homo- or heterodimers, being potent regulators of bone and cartilage formation and repair, cell proliferation during embryonic development and bone homeostasis in the adult. BMPs are promising molecules for tissue engineering and bone therapy. The present review discusses this family of proteins, their structure and biological function, their therapeutic applications and drawbacks, their effects on mesenchymal stem cells differentiation, and the cell signaling pathways involved in this process.

Effects of plasma rich in growth factors on wound healing in patients with venous ulcers.

Introduction: Significant evidence suggests that plasma-rich in growth factors (PRGF) favor the repair of chronic wounds, enabling a rapid return to functionality. However, components of PRGF and their effects on persistent ulcers and epithelial tissues are not well characterized. The goals of this research were to analyze the biological properties of platelet-derived factors, to examine their effectiveness on healing of venous ulcers, and to establish a correlation with clinical and sociodemographic data. Methods: For the preparation of PRGF, the centrifugation technique was used, obtaining a 100 % autologous and biocompatible blood sample that was treated with sodium citrate and calcium chloride. The patients were attended weekly at the outpatient clinic for nursing consultation and wound dressing changes, with PRGF application every 15 days. The treatment protocols are described, and follow-up results are reported. Results: Initially, the patients' ulcers ranged in sizes from 4 to 84 cm2. After 12 weeks of treatment, there was a significant mean reduction of 46.2 % in ulcer area. At baseline, epithelial tissue was absent in all venous ulcers, but its presence grew significantly by the treatment period. However, the reduction of the area of the ulcers did not show significant correlation with the concentrations of the patient's growth factors. Conclusions: Using the established protocol for PRGF isolating, it was possible to obtain a product with the presence of the six growth factors related to tissue regeneration and observed a positive response on wound healing following treatment of venous ulcers, with capacity to accelerate re-epithelialization and restore the skin functional integrity.

The Association of Nanostructured Carbonated Hydroxyapatite with Denatured Albumin and Platelet-Rich Fibrin: Impacts on Growth Factors Release and Osteoblast Behavior.

Platelet-rich Fibrin (PRF), a second-generation blood concentrate, offers a versatile structure for bone regeneration due to its composition of fibrin, growth factors, and cytokines, with adaptations like denatured albumin-enriched with liquid PRF (Alb-PRF), showing potential for enhanced stability and growth factor dynamics. Researchers have also explored the combination of PRF with other biomaterials, aiming to create a three-dimensional framework for enhanced cell recruitment, proliferation, and differentiation in bone repair studies. This study aimed to evaluate a combination of Alb-PRF with nanostructured carbonated hydroxyapatite microspheres (Alb-ncHA-PRF), and how this association affects the release capacity of growth factors and immunomodulatory molecules, and its impact on the behavior of MG63 human osteoblast-like cells. Alb-PRF membranes were prepared and associated with nanocarboapatite (ncHA) microspheres during polymerization. MG63 cells were exposed to eluates of both membranes to assess cell viability, proliferation, mineralization, and alkaline phosphatase (ALP) activity. The ultrastructural analysis has shown that the spheres were shattered, and fragments were incorporated into both the fibrin mesh and the albumin gel of Alb-PRF. Alb-ncHA-PRF presented a reduced release of growth factors and cytokines when compared to Alb-PRF (p < 0.05). Alb-ncHA-PRF was able to stimulate osteoblast proliferation and ALP activity at lower levels than those observed by Alb-PRF and was unable to positively affect in vitro mineralization by MG63 cells. These findings indicate that the addition of ncHA spheres reduces the biological activity of Alb-PRF, impairing its initial effects on osteoblast behavior.