Although the discipline of cell biology (CB) is part of the curricula of predoctoral dental schools, students often fail to recognize its practical relevance. The aim of this study was to assess the effectiveness of a practical-theoretical project-based course in closing the gaps among CB, scientific research, and dentistry for dental students. A project-based learning course was developed with nine sequential lessons to evaluate 108 undergraduate dental students enrolled in CB classes of a Brazilian school of dentistry during 2013-16. To highlight the relevance of in vitro studies in the preclinical evaluation of dental materials at the cellular level, the students were challenged to complete the process of drafting a protocol and performing a cytocompatibility assay for a bone substitute used in dentistry. Class activities included small group discussions, scientific database search and article presentations, protocol development, lab experimentation, and writing of a final scientific report. A control group of 31 students attended only one laboratory class on the same theme, and the final reports were compared between the two groups. The results showed that the project-based learning students had superior outcomes in acknowledging the relevance of in vitro methods during biocompatibility testing. Moreover, they produced scientifically sound reports with more content on methodological issues, the relationship with dentistry, and the scientific literature than the control group (p<0.05). The project-based learning students also recognized a higher relevance of scientific research and CB to dental practice. These results suggest that a project-based approach can help contextualize scientific research in dental curricula.

Several tests for the biological evaluation of bioceramic materials and medical devices are provided in specific international standards, where in vitro tests have a major role. Tests involving exposure of cells in culture require the use of validated positive controls, which, in the same preparation and treatment conditions, present a substantial and well-known cytotoxicity. The present work aimed to test and validate 3 different sources of low cost, commercially available latex, as positive controls in cytotoxicity tests for bioceramic materials performed by indirect exposure. The tested origins for latex samples were: surgical gloves without powder, 100% pure amber latex hospital-grade tourniquets and 60 % latex White tubing. MC3T3-E1 murine pre-osteoblasts in culture were exposed to conditioned media (extracts) of each material tested, along with sintered stoichiometric hydroxyapatite bioceramics, and polystyrene beads as negative control. Cell viability was determined by XTT and Crystal Violet Exclusion tests. Concentration curves of the extracts were performed to obtain the DC50. Only the 100% pure amber latex tubing was proven to be cytotoxic, with cell survival less than 5%. This material did not affected neighboring groups at the same experimental system. Moreover, latex samples showed great repeatability in different tests against latex and biomaterials, with consistent toxicity under 20% cell survival as shown in 3 different cell viability parameters. We conclude that fragments of latex ambar tubing are suited as effective positive controls in tests of medical bioceramic materials.

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Synthetic hydroxyapatite (HA) is a widely used ceramic biomaterial due to its well described biocompatibility. Some modifications in HA surface can be made to increase surface porosity. Likewise, HA can be modified by the coating with proteins, which may impact on biocompatibility. In this work, we aimed to evaluate the impact of two surface modifications – coating with albumin, a major serum protein, and augmented porosity - over osteoblast adhesion on stoichiometric HA discs. Dense HA discs were obtained by pressing HA powder at 30 KN and sinterization at 1000°C, while porous HA was molded after the addition of alginate (15:1), followed by thermal treatment. Protein adsorption was attained by incubation on 0.5mg/mL bovine serum albumin (BSA) for 24 h at 37°C. MC3T3 mouse preosteoblasts were seeded over both protein-coated and uncoated dense or porous tablets, and cell viability after 24 h was estimated by XTT and Neutral Red assays. Cell density was quantified by fluorescence microscopy. While both dense and porous discs presented altered surfaces after protein treatment, as observed by scanning electron microscopy, porous HA tablets presented significantly higher levels of adsorbed protein. There was a decrease in the concentration of calcium ions in all samples analyzed. Porous HA treated with protein presented significant higher mitochondrial dehydrogenase activity (XTT) than non treated tablets (p<0.001). Although the BSA adsorption didn`t affect cell adhesion, the results obtained in fluorescence quantification suggests that de dense surface was best for cellular adhesion and spread than the porous one. We conclude that differences in the topography of a biomaterial can directly influence their ability to adsorb proteins, while the dense surface was more favorable for both the adhesion and the spreading of pre-osteoblasts.

Cell adhesion, proliferation and differentiation are important specific parameters to be evaluated on biocompatibility studies of candidate biomaterials for clinical applications. Several different methodologies have been employed to study, both qualitative and quantitatively, the direct interactions of ceramic materials with cultured mammal and human cells. However, while quantitatively evaluating cell density, viability and metabolic responses to test materials, several methodological challenges may arise, either by impairing the use of some widely applied techniques, or by generating false or conflicting results. In this work, we tested the inherent interference of different representative calcium phosphate ceramic surfaces (stoichiometric dense and porous hydroxyapatite (HA) and cation-substituted apatite tablets) on different tests for quantitative evaluation of osteoblast adhesion and metabolism, either based on direct cell counting after trypsinization, colorimetric assays (XTT, Neutral Red and Crystal Violet) and fluorescence microscopy. Cell adhesion estimation after trypsinization was highly dependent on the time of treatment, and the group with the highest level of estimated adhesion was inverted from 5 to 20 minutes of exposition to trypsin. Both dense and porous HA samples presented high levels of background adsorption of the Crystal Violet dye, impairing cell detection. HA surfaces also were able to adsorb high levels of fluorescent dyes (DAPI and phalloidin-TRITC), generating backgrounds which, in the case of porous HA, impaired cell detection and counting by image processing software (Image Pro Plus 6.0). We conclude that the choice for the most suitable method for cell detection and estimation is highly dependent on very specific characteristics of the studied material, and methodological adaptations on well established protocols must always be carefully taken on consideration.