Adriana Brandão Ribeiro Linhares

Optimal cytocompatibility of a bioceramic nanoparticulate cement in primary human mesenchymal cells.

INTRODUCTION The aim of this study was to evaluate and compare the cytotoxic effects of BioAggregate (Innovative BioCaramix Inc, Vancouver, BC, Canada), a novel bioceramic nanoparticulate cement, on human mesenchymal cells. White Pro-Root MTA (Dentsply, Tulsa Dental, Tulsa, OK) was used as a reference for comparison. METHODS Fifty-six human maxillary incisor teeth were submitted to a step-back flaring technique and prepared for cytotoxicity assay in an in situ root-end filling experimental model. After retro filling, each root containing MTA, BioAggregate, or empty root canals (control) was exposed to culture media for 24, 48, or 72 hours, providing several extraction media. Mesenchymal cells were incubated with each extract medium for 24 hours, and toxicity was evaluated by three different parameters of cell survival and integrity on the same sample: XTT, neutral red, and crystal violet dye elution. RESULTS No statistically significant differences between MTA and BioAggregate were found in all the experimental periods (p > 0.05). CONCLUSION DiaRoot BioAggregate displayed in vitro compatibility similar to MTA.

A multiparametric assay to compare the cytotoxicity of endodontic sealers with primary human osteoblasts.

AIM   To compare the cytotoxicity of four endodontic sealers (Sealapex, Pulp Canal Sealer EWT, Real Seal and MTA Fillapex) either 1 or 7 days after mixing, when assessed through a multiparametric analysis employing human primary cells closely related to periapical tissues. METHODOLOGY   Extracts of each sealer were prepared following 24-h exposure to culture media, at either 24 h or 7 days after mixing. Primary human osteoblasts were exposed to extracts for 24 h, at 37 °C with 5% CO(2) , and cell viability was evaluated by a multiparametric assay assessing sequentially, on the same cells, mitochondrial activity (XTT), membrane integrity (neutral red test) and total cell density (crystal violet dye exclusion test). Results from each test and experimental time were compared by 2-way analysis of variance (anova). RESULTS   All endodontic sealers had strong cytotoxicity 24 h after mixing, according to all parameters evaluated. At a longer setting period (7 days), viability for Sealapex was significantly increased (P < 0.05) and Pulp Canal Sealer achieved levels of cytocompatibility similar to the control group. The anova indicated a general correlation between the cytotoxicity of the materials and the time after mixing, with some level of dependence on the cell viability assay employed. CONCLUSIONS   All materials had high cytotoxic levels for human primary cells, mostly on a time-dependent basis, as shown by three different cell viability tests.

Cytocompatibility of the ready-to-use bioceramic putty repair cement iRoot BP Plus with primary human osteoblasts

AIM To verify the in vitro cytocompatibility of iRoot BP Plus (iRoot) and to compare it with White ProRoot MTA (MTA). METHODOLOGY Thirty-six human maxillary incisor root canals were prepared using a step-back flaring technique. The apical 3 mm was resected perpendicular to the long axis at the roots, and root-end cavities were prepared with the aid of an ultrasonic device plus a diamond retrotip with continuous irrigation using water, producing standardized preparations. After that, the root-end cavities were filled with iRoot or MTA, and each root was exposed to cell culture media for 24 or 48 h. Human osteoblast cells were exposed to the extracts thus obtained, and a multiparametric cell viability assay was performed, evaluating mitochondrial activity, membrane integrity and cell density. The results were analysed by one-way analysis of variance, complemented with the Duncan post-test (P < 0.05). RESULTS Cells exposed to MTA revealed a cytocompatibility pattern similar to the untreated cells (negative control), at both experimental times (P > 0.05). iRoot, however, promoted a significantly poorer viability than MTA and the control, after 48 h of exposure (P < 0.001). Nevertheless, iRoot did not induce critical cytotoxic effects because cell viability remained higher than 70% of the control group in most tests performed. CONCLUSION iRoot and MTA were biocompatible and did not induce critical cytotoxic effects.

Short‐Term Response of Human Osteoblast‐Like Cells on Titanium Surfaces With Micro‐ and Nano‐Sized Features

Since the way that human bone cells behave on contact with different surfaces topographies seems to be crucial to osseointegration, the aim of the present study is to evaluate the participation of some micro- and nanosized features of Ti surfaces in the short-term response of primary human osteoblast-like cells (HOC). Surfaces were prepared as ground (G-Ti), hydrofluoric acid etched (HF-Ti), and sandblasted/HF-etched (SLA-Ti), and analyzed using both three-dimensional (3D) profilometer and atomic force microscope (AFM). Cell morphology was assessed using scanning electron microscopy (SEM) after 4 and 24 h in culture. Cell viability, adhesion, and spreading were also evaluated 4 and 24 h after seeding over each surface. Data were compared by analysis of variance (ANOVA) complemented by Duncan test. Cell morphology, cell counting, and membrane integrity (Neutral Red, NR) were not affected by surface treatment at any time. However, HF-Ti presented the smallest surface area and did not increase tetrazolium hydroxide (XTT) reduction from 4 to 24 h. On the other hand, a higher level of spreading was only found on the rougher and isotropic SLA-Ti at 4 h. In conclusion, although all evaluated Ti surfaces allowed HOC short-term adhesion, the finer topography introduced by HF as single treatment did not favor HOC mitochondrial activity and spreading. The rougher and more complex SLA surface seems to provide a better substrate for HOC short-term response.

Evaluation of Commercial Latex as a Positive Control for In Vitro Testing of Bioceramics

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.

Evaluation of Cytocompatibility of Bioglass-Niobium Granules with Human Primary Osteoblasts: A Multiparametric Approach

The pursuit for an ideal bone substitute remains the main focus of many tissue engineering researchers. Among the myriad types of grafts available, synthetic bone grafts are of special importance, because it is available in large amounts, reduce the surgical trauma and eliminate the risk of diseases’ transmission. In this context, bioactive glasses have received attention mostly due to its described biocompatibility and rapid rate of surface reactivity when compared with other materials, allowing for faster interactions with the local tissue. The addition of niobium to this material has been shown as increasing the chemical resistance of the compound and providing greater stability. However, alterations on the chemical composition of biomaterials may impact on its biocompatibility. Therefore, the aim of this study was to evaluate the in vitro biocompatibility of bioglass-Niobium (BgNb) granules, in comparison with standard commercial bioglass (Biogran®) throughout an interesting multiparametrical approach, employing Phenol 2% and dense polystyrene beads as positive and negative controls, respectively. Extracts from each material were prepared by 24 hours incubation in culture medium (DMEM). Human primary osteoblasts were then exposed for 24 hours to each extract and cell viability was evaluated by three parameters: mitochondrial activity (XTT method), membrane integrity (neutral red dye uptake) and cell density (crystal violet dye exclusion test). BgNb extracts were highly compatible, since the levels of viable cells were similar to the control group (unexposed cells), on all parameters studied. The mean cell density on the Biogran® group was slightly lower than BgNb, even though this material was also non-cytotoxic. The excellent in vitro response for BgNb granules indicates the suitability of this material to future studies on its biological and physical properties when applied in vivo.

Cytocompatibility of a self-adhesive gutta-percha root-filling material

Context: A novel root-filling material based on the incorporation of ultrafine alkaline bioactive glass particles (bioactive gutta-percha, [BGP]) was developed to work without sealer. Aim: In the present study, the objective was to verify the in vitro biological response to this material by assessing its cytocompatibility. Materials and Methods: Prototypes of BGP were compared to conventional gutta-percha (GP), dense polystyrene beads as a negative control and fragments of latex as a positive control. Extracts of each material were prepared according to ISO 10993-5:2009, and human osteoblast-like cells in primary culture were exposed to all extracts for 24 h. Cell viability was assayed sequentially for three different parameters: mitochondrial activity, membrane integrity, and cell density. Statistical Analysis Used: Nonparametric analysis (using Kruskal–Wallis test combined with post hoc Dunns test) was performed for comparison among groups, with significance established at 5%. Results: BGP reduced mitochondrial activity to 62% of control, but presented no toxicity on membrane integrity and proliferation assays. BGP effect on metabolism was dose-dependent and reduced to acceptable levels with dilution. Conclusion: The novel GP material presented slight dose-dependent effects on cell metabolism but did not affect cell survival.

A 3D Osteoblast In Vitro Model for the Evaluation of Biomedical Materials

Biomedical materials for bone therapy are usually assessed for their biocompatibility and safety employing animal models or in vitro monolayer cell culture assays. However, alternative in vitro models may offer controlled conditions closer to physiological responses and reduce animal testing. In this work, we developed a 3D spheroidal cell culture with potential to evaluate simultaneously material-cell and cell-cell interactions. Different cell densities of murine MC3T3-E1 preosteoblasts or human primary osteoblasts (HOb) were used to determine the ideal procedure of spheroidal cultures and their adequacy to material testing. Cells were seeded on 96-well plates coated with agar and incubated in agitation from 1 to 7 days. Aggregate morphology was qualitatively evaluated considering the shape, size, repeatability, handling, and stability of spheroids. Higher cell densities induced more stable spheroids, and handling was considered appropriate starting from 2 × 104 cells. Confocal microscopy and Scanning Electron Microscopy indicate that most cells within the aggregate core are viable. Exposure to positive controls has shown a dose dependent cell death as measured by XTT assay. Aggregates were stable and presented good viability when employed on standardized testing of metallic and polymer-based biomaterials. Therefore, osteoblast spheroids may provide a promising tool for material screening and biocompatibility testing.

Multiparametric In Vitro Evaluation of Cytocompatibility of 1% Strontium-Containing Nanostructured Hydroxyapatite

Hydroxiapatite (HA), one of the most widely employed bioceramic bone substitutes, when applied on its nanostructured form (nHA) may contribute to achieve a crystalline structure which is closer to the size and morphology of biological apatite. Furthermore, HA might also be doped with several different cations with biological effects including Sr2+. Therefore, a biomaterial based on nanostructured HA containing 1% Strontium (nSrHA) could present interesting biological properties, as strontium is described as a modulator of both osteoblast and osteoclast activities, presenting an important regulatory role on bone resorption. However, such modifications may also affect the biocompatibility of this material, which should be accessed initially by in vitro methods. Therefore, the present work aimed to evaluate the in vitro biocompatibility of 1% nSrHA discs with human primary osteoblasts through a multiparametric assay which assesses simultaneously metabolic activity (XTT assay), membrane integrity (NR test) and cell density (CVDE). Extracts of nSrHA, latex fragments (positive control), polystyrene beads (negative control) and nHA (for comparison) were prepared and exposed to 104 cells for 24h at 37°C/5% CO2 on test plates, according to ISO 10993-5:2009, on quintuplicates. Cells exposed to unconditioned media were used as experimental control. After exposure, cells were tested for viability with a commercial multiparametric kit (In Cytotox, Xenometrix, Germany). The positive and negative controls presented the expected results, validating the assay. Both nHA and SrnHA were considered biocompatible, since the presented a cell viability after exposure statistically similar to the experimental control. In conclusion, the synthesized nSrHA discs are cytocompatible and, consequently, adequate for further in vitro tests on cell adhesion, proliferation and differentiation.