Microfluidic-based skin-on-chip systems for safety assessment of nanomaterials.

The skin is the body's largest organ, continuously exposed to and affected by natural and anthropogenic nanomaterials (materials with external and internal dimensions in the nanoscale range). This broad spectrum of insults gives rise to irreversible health effects (from skin corrosion to cancer). Organ-on-chip systems can recapitulate skin physiology with high fidelity and potentially revolutionize the safety assessment of nanomaterials. Here, we review current advances in skin-on-chip models and their potential to elucidate biological mechanisms. Further, strategies are discussed to recapitulate skin physiology on-chip, improving control over nanomaterials exposure and transport across cells. Finally, we highlight future opportunities and challenges from design and fabrication to acceptance by regulatory bodies and industry.

Evaluation of genetic risk related to catechol-O-methyltransferase (COMT) and ß2-adrenergic receptor (ADRB2) activity in different diagnostic subgroups of temporomandibular disorder in Brazilian patients.

The aim of this study was to evaluate the possible association between polymorphisms in the catechol-O-methyltransferase (COMT) and ß2-adrenergic receptor (ADRB2) genes and muscular temporomandibular disorders (TMD). This was a case-control study. Individuals were evaluated using the Research Diagnostic Criteria for Temporomandibular Disorders and were divided into three groups: unaffected (no TMD) (n=154); exclusively muscular TMD (n=49); exclusively articular TMD (n=49). Genomic DNA was obtained from saliva samples, and single nucleotide polymorphisms in the COMT (rs165774, rs6269, rs9332377) and ADRB2 (rs2053044, rs1042713, rs1042714) genes were investigated. The TT genotype for the COMT rs9332377 gene was highly associated with the presence of muscular TMD (P= 0.03). With respect to the ADRB2 gene, the non-polymorphic AA genotype in the rs1042713 region was more prevalent in the articular TMD group than in the muscular TMD group (P= 0.05). This study supports the hypothesis that alterations in the COMT and ADRB2 genes influence the muscular pathophysiology.

The two faces of titanium dioxide nanoparticles bio-camouflage in 3D bone spheroids.

Titanium (Ti) and its alloys are widely used in dental implants and hip-prostheses due to their excellent biocompatibility. Growing evidence support that surface degradation due to corrosion and wear processes, contribute to implant failure, since the release of metallic ions and wear particles generate local tissue reactions (peri-implant inflammatory reactions). The generated ions and wear debris (particles at the micron and nanoscale) stay, in a first moment, at the interface implant-bone. However, depending on their size, they can enter blood circulation possibly contributing to systemic reactions and toxicities. Most of the nanotoxicological studies with titanium dioxide nanoparticles (TiO2 NPs) use conventional two-dimensional cell culture monolayers to explore macrophage and monocyte activation, where limited information regarding bone cells is available. Recently three-dimensional models have been gaining prominence since they present a greater anatomical and physiological relevance. Taking this into consideration, in this work we developed a human osteoblast-like spheroid model, which closely mimics bone cell-cell interactions, providing a more realistic scenario for nanotoxicological studies. The treatment of spheroids with different concentrations of TiO2 NPs during 72 h did not change their viability significantly. Though, higher concentrations of TiO2 NPs influenced osteoblast cell cycle without interfering in their ability to differentiate and mineralize. For higher concentration of TiO2 NPs, collagen deposition and pro-inflammatory cytokine, chemokine and growth factor secretion (involved in osteolysis and bone homeostasis) increased. These results raise the possible use of this model in nanotoxicological studies of osseointegrated devices and demonstrate a possible therapeutic potential of this TiO2 NPs to prevent or reverse bone resorption.

Rutile nano-bio-interactions mediate dissimilar intracellular destiny in human skin cells.

The use of nanoparticles (NPs) in the healthcare market is growing exponentially, due to their unique physicochemical properties. Titanium dioxide nanoparticles (TiO2 NPs) are used in the formulation of sunscreens, due to their photoprotective capacity, but interactions of these particles with skin cells on the nanoscale are still unexplored. In the present study we aimed to determine whether the initial nano-biological interactions, namely the formation of a nano-bio-complex (other than the protein corona), can predict rutile internalization and intracellular trafficking in primary human fibroblasts and keratinocytes. Results showed no significant effect of NPs on fibroblast and keratinocyte viability, but cell proliferation was possibly compromised due to nano-bio-interactions. The bio-complex formation is dependent upon the chemistry of the biological media and NPs' physicochemical properties, facilitating NP internalization and triggering autophagy in both cell types. For the first time, we observed that the intracellular traffic of NPs is different when comparing the two skin cell models, and we detected NPs within multivesicular bodies (MVBs) of keratinocytes. These structures grant selected input of molecules involved in the biogenesis of exosomes, responsible for cell communication and, potentially, structural equilibrium in human tissues. Nanoparticle-mediated alterations of exosome quality, quantity and function can be another major source of nanotoxicity.

Impact of bisphosphonate therapy on dental implant outcomes: An overview of systematic review evidence.

The purpose of this overview was to assess the methods, quality, and outcomes of systematic reviews conducted to evaluate the impact of bisphosphonates on dental implants and the risk of developing bisphosphonate-related osteonecrosis of the jaw after dental implant surgery. An electronic search without date or language restriction was performed in the PubMed/MEDLINE, Cochrane CENTRAL, Web of Science, and LILACS databases (to January 2018). Eligibility criteria included systematic reviews that evaluated the impact of bisphosphonates on implant outcomes. The quality assessment of the included reviews was done using AMSTAR 2 guidelines. The protocol of this overview was registered in PROSPERO (CRD42018089617). The search and selection process yielded seven reviews, published between 2009 and 2017. None of the systematic reviews included in this study obtained the maximum score in the quality assessment. The scientific evidence available demonstrates that patients with a history of bisphosphonate use do not present a higher risk of dental implant failure or marginal bone loss compared to patients who have not used bisphosphonates. The literature also suggests that patients who undergo surgical trauma during the installation of dental implants may be more susceptible to bisphosphonate-related osteonecrosis of the jaw.

Evaluation of the effects of the solution used for electrochemical dissolution of nickel-titanium endodontic files on dentine structure, microhardness and cell viability.

AIM: To evaluate the effects of the [NaF 12 g L-1  + NaCl 1 g L-1 ] solution used in the electrochemical dissolution process of fractured endodontic files, as well as its NiTi-containing product, on dentine hardness, topography and human fibroblast viability. METHODOLOGY: Sixty single-rooted human teeth were evaluated for dentine microhardness using the Vickers hardness test and the area and number of dentinal tubules by scanning electron microscopy. The samples were divided according to the dentine surface treatment: distilled water; 17% EDTA; [NaF 12 g L-1  + NaCl 1 g L-1 ]; and 17% EDTA + [NaF 12 g L-1  + NaCl 1 g L-1 ]. Thirty-six single-rooted human teeth were divided according to the irrigation protocol: Dulbecco's Modified Eagle's Medium + 10% foetal bovine serum; 5.25% NaOCl; [NaF 12 g L-1  + NaCl 1 g L-1 ]; and [NaF 12 g L-1  + NaCl 1 g L-1  + NiTi]. The extracts in contact with the apical foramen were used in the MTT assay to evaluate human fibroblast viability, with dilutions of 100%, 50%, 25% and 12.5%. Statistical tests used were paired t-tests, one-way anova, Tukey's test, Kruskal-Wallis test and Dunn's post-test. RESULTS: The [NaF 12 g L-1  + NaCl 1 g L-1 ] solution did not modify dentine microhardness or the average dentinal tubule area. However, EDTA induced changes in dentine structure and microhardness (P < 0.05). The [NaF 12 g L-1  + NaCl 1 g L-1 ] solution, and its NiTi-containing product had lower cytotoxicity than NaOCl at dilutions of 25% and 50% (P < 0.01). CONCLUSIONS: The [NaF 12 g L-1  + NaCl 1 g L-1 ] solution did not alter dentine microhardness or damage the dentine structure. It also demonstrated lower cytotoxicity than NaOCl.

Haplotypes of the RANK and OPG genes are associated with chronic arthralgia in individuals with and without temporomandibular disorders.

The aim of this study was to evaluate the association between genetic polymorphisms and the comorbid presence of chronic systemic arthralgia in patients with articular temporomandibular disorders (TMD). Subjects were evaluated for the presence of TMD and asked about the presence of chronic joint pain. Four groups were included in the study: articular TMD and systemic arthralgia (n=85), no articular TMD and systemic arthralgia (n=82), articular TMD and no systemic arthralgia (n=21), no articular TMD and no systemic arthralgia (control, n=72). A total of 14 single nucleotide polymorphisms in the OPG, RANK, and RANKL genes were investigated. In the statistical analysis, a P-value of <0.05 was considered significant. For the OPG gene, an association was observed between the group with chronic arthralgia and joint TMD and the control group (P=0.04). There was also a tendency towards an association of the haplotype CGCCAA with an increased risk of developing chronic joint pain, even in the absence of TMD (P=0.06). For the RANK gene, the AGTGC haplotype was associated with the lowest risk of presenting chronic joint pain in individuals without TMD (P=0.03). This study supports the hypothesis that changes in the OPG and RANK genes influence the presence of chronic joint pain in individuals with and without TMD.

High throughput toxicity screening and intracellular detection of nanomaterials.

With the growing numbers of nanomaterials (NMs), there is a great demand for rapid and reliable ways of testing NM safety-preferably using in vitro approaches, to avoid the ethical dilemmas associated with animal research. Data are needed for developing intelligent testing strategies for risk assessment of NMs, based on grouping and read-across approaches. The adoption of high throughput screening (HTS) and high content analysis (HCA) for NM toxicity testing allows the testing of numerous materials at different concentrations and on different types of cells, reduces the effect of inter-experimental variation, and makes substantial savings in time and cost. HTS/HCA approaches facilitate the classification of key biological indicators of NM-cell interactions. Validation of in vitro HTS tests is required, taking account of relevance to in vivo results. HTS/HCA approaches are needed to assess dose- and time-dependent toxicity, allowing prediction of in vivo adverse effects. Several HTS/HCA methods are being validated and applied for NM testing in the FP7 project NANoREG, including Label-free cellular screening of NM uptake, HCA, High throughput flow cytometry, Impedance-based monitoring, Multiplex analysis of secreted products, and genotoxicity methods-namely High throughput comet assay, High throughput in vitro micronucleus assay, and γH2AX assay. There are several technical challenges with HTS/HCA for NM testing, as toxicity screening needs to be coupled with characterization of NMs in exposure medium prior to the test; possible interference of NMs with HTS/HCA techniques is another concern. Advantages and challenges of HTS/HCA approaches in NM safety are discussed. WIREs Nanomed Nanobiotechnol 2017, 9:e1413. doi: 10.1002/wnan.1413 For further resources related to this article, please visit the WIREs website.

Trojan-Like Internalization of Anatase Titanium Dioxide Nanoparticles by Human Osteoblast Cells.

Dentistry and orthopedics are undergoing a revolution in order to provide more reliable, comfortable and long-lasting implants to patients. Titanium (Ti) and titanium alloys have been used in dental implants and total hip arthroplasty due to their excellent biocompatibility. However, Ti-based implants in human body suffer surface degradation (corrosion and wear) resulting in the release of metallic ions and solid wear debris (mainly titanium dioxide) leading to peri-implant inflammatory reactions. Unfortunately, our current understanding of the biological interactions with titanium dioxide nanoparticles is still very limited. Taking this into consideration, this study focuses on the internalization of titanium dioxide nanoparticles on primary bone cells, exploring the events occurring at the nano-bio interface. For the first time, we report the selective binding of calcium (Ca), phosphorous (P) and proteins from cell culture medium to anatase nanoparticles that are extremely important for nanoparticle internalization and bone cells survival. In the intricate biological environment, anatase nanoparticles form bio-complexes (mixture of proteins and ions) which act as a kind of 'Trojan-horse' internalization by cells. Furthermore, anatase nanoparticles-induced modifications on cell behavior (viability and internalization) could be understand in detail. The results presented in this report can inspire new strategies for the use of titanium dioxide nanoparticles in several regeneration therapies.

ESRRB polymorphisms are associated with comorbidity of temporomandibular disorders and rotator cuff disease.

Temporomandibular disorders (TMD) are associated with comorbidity. Shoulder pain is among the symptoms associated with TMD. The purpose of this study was to investigate the association between TMD and rotator cuff disease (RCD) and related genetic aspects. All subjects underwent orofacial and shoulder examinations. The control group comprised 30 subjects with no pain. Affected subjects were divided into three groups: RCD (TMD-free, n=16), TMD (RCD-free, n=13), and TMD/RCD (patients with both RCD and TMD, n=49). A total of eight single nucleotide polymorphisms in the ESRRB gene were investigated. A chemiluminescent immunoassay was used to measure estradiol levels. Surface electromyography recorded head and cervical muscle activity. The χ(2) test and Student t-test/Mann-Whitney test were used to assess the significance of nominal and continuous variables. A P-value of <0.05 was considered significant. TMD subjects were seven times more susceptible to RCD than controls. The rs1676303 TT (P=0.02) and rs6574293 GG (P=0.04) genotypes were associated with RCD and TMD, respectively. TMD/RCD subjects showed associations with rs4903399 (P=0.02), rs10132091 (P=0.02), and CTTCTTAG/CCTCTCAG (P=0.01) haplotypes and lower muscle activity. Estradiol levels were similar among groups. This study supports TMD as a risk factor for RCD. ESRRB haplotypes and low muscle activity are common biomechanical characteristics in subjects with both diseases.

Micro-arc oxidation as a tool to develop multifunctional calcium-rich surfaces for dental implant applications.

Titanium (Ti) is commonly used in dental implant applications. Surface modification strategies are being followed in last years in order to build Ti oxide-based surfaces that can fulfill, simultaneously, the following requirements: induced cell attachment and adhesion, while providing a superior corrosion and tribocorrosion performance. In this work micro-arc oxidation (MAO) was used as a tool for the growth of a nanostructured bioactive titanium oxide layer aimed to enhance cell attachment and adhesion for dental implant applications. Characterization of the surfaces was performed, in terms of morphology, topography, chemical composition and crystalline structure. Primary human osteoblast adhesion on the developed surfaces was investigated in detail by electronic and atomic force microscopy as well as immunocytochemistry. Also an investigation on the early cytokine production was performed. Results show that a relatively thick hybrid and graded oxide layer was produced on the Ti surface, being constituted by a mixture of anatase, rutile and amorphous phases where calcium (Ca) and phosphorous (P) were incorporated. An outermost nanometric-thick amorphous oxide layer rich in Ca was present in the film. This amorphous layer, rich in Ca, improved fibroblast viability and metabolic activity as well as osteoblast adhesion. High-resolution techniques allowed to understand that osteoblasts adhered less in the crystalline-rich regions while they preferentially adhere and spread over in the Ca-rich amorphous oxide layer. Also, these surfaces induce higher amounts of IFN-γ cytokine secretion, which is known to regulate inflammatory responses, bone microarchitecture as well as cytoskeleton reorganization and cellular spreading. These surfaces are promising in the context of dental implants, since they might lead to faster osseointegration.

Association between the epidermal growth factor gene polymorphism and endometriosis in women from Brazil.

The aim of this study was to verify the association between the epidermal growth factor (EGF) +61 G/A polymorphism and the susceptibility to endometriosis using a case-control design study. The control group included fertile women without endometriosis and the case group included endometriosis patients. Polymerase chain reaction-restriction fragment length polymorphism analysis was used to genotype the EGF +61 G/A polymorphism. Initially, a total of 184 individuals were analyzed. After matching by ethnicity, the control group was composed of 57 individuals, while the endometriosis group was composed of 57 patients. No statistically significant associations were observed between EGF +61 variants and the risk of endometriosis development (P>0.05). This is the first study correlating the EFG +61 G/A polymorphism and endometriosis in women from Brazil, and demonstrates that EFG +61 G/A is not associated with endometriosis susceptibility in Brazilian women.

Bone morphogenetic proteins: facts, challenges, and future perspectives.

Bone morphogenetic proteins (BMPs) are members of the TGF-ß superfamily, acting as potent regulators during embryogenesis and bone and cartilage formation and repair. Cell and molecular biology approaches have unveiled the great complexity of BMP action, later confirmed by transgenic animal studies. Genetic engineering allows for the production of large amounts of BMPs for clinical use, but they have systematically been associated with a delivery system, such as type I collagen and calcium phosphate ceramics, to ensure controlled release and to maximize their biological activity at the surgical site, avoiding systemic diffusion. Clinical orthopedic studies have shown the benefits of FDA-approved recombinant human BMPs (rhBMPs) 2 and 7, but side effects, such as swelling, seroma, and increased cancer risk, have been reported, probably due to high BMP dosage. Several studies have supported the use of BMPs in periodontal regeneration, sinus lift bone-grafting, and non-unions in oral surgery. However, the clinical use of BMPs is growing mainly in off-label applications, with robust evidence to ascertain rhBMPs' safety and efficacy through well-designed, randomized, and double-blind clinical trials. Here we review and discuss the critical data on BMP structure, mechanisms of action, and possible clinical applications.

Measuring the impact of quality of life of children treated for orofacial clefts: a case-control study.

OBJECTIVE: The aim of this study was to assess the quality of life (QoL) of children previously treated for cleft lip and/or palate (CL/P) and compare with non-cleft children. METHOD: A case-control study with 70 children between 5 and 12 years old was carried out. The case group consisted of 35 individuals previously treated for non-syndromic CL/P and presently receiving assessment at a rehabilitation hospital in Brazil. The children had received primary surgical treatment for CL/P reconstruction during early childhood. The control group consisted of 35 healthy children selected to ensure close similarity to the cleft group in age, gender and socioeconomic status. QoL was measured using the AUQEI questionnaire. RESULTS: Cleft lip and palate had no significant influence on the QoL in children (p = 0.44). A higher percentage of the cleft lip and palate group of children reported a lower QoL than the cleft lip or cleft palate groups. Gender had no significant difference on the quality of life in CL/P children (p = 0.2) and in control group (p = 1.0). CONCLUSION: The QoL in children with CL/P was found to be similar to the non-cleft group. Our results confirm that clefts repaired during earlier childhood associated with a health care program, including psychological support, is beneficial for CL/P children.

Candidate gene studies in hypodontia suggest role for FGF3.

INTRODUCTION: The majority of tooth agenesis cases are mild (hypodontia) and typically not associated with the gene mutations linked to oligodontia. From this, we hypothesise that most cases of tooth agenesis fit a polygenic mode of inheritance, where several genes with small effects cause a variety of varying phenotypes. MATERIALS AND METHODS: In this study, we looked at 18 not typically studied genes in this condition, to ascertain their contribution to hypodontia. Our study subjects consisted of 167 patients with hypodontia and their parents from two cohorts (one from Brazil and one from Turkey). An additional 465 DNA samples (93 cases with hypodontia and 372 controls without family history for tooth agenesis or oral clefts) from Brazil were also available for this study. Ninety-three single nucleotide polymorphisms that maximally represent the linkage disequilibrium structure of the genes for the 18 genes were selected and genotyped using Taqman chemistry. Chi square was used to test if genotype distributions were in Hardy-Weinberg equilibrium, and 24 markers that were in Hardy-Weinberg equilibrium and had allele frequencies higher than 5 % in a panel of 50 CEPH samples were further tested. Association between hypodontia and genetic variants was tested with the transmission disequilibrium test within the programme Family-Based Association Test (FBAT) and by using Chi square and Fisher's exact tests. Alpha at a level of 0.05 was used to report results. RESULTS: Results suggest possible associations between several genes and hypodontia in the three populations. In the Turkish cohort (n = 51 parent-affected child trios) the most significant results were as follows: FGF3 rs1893047, p = 0.08; GLI3 rs929387, p = 0.03; GLI3 haplotype rs929387-rs846266, p = 0.002; and PAX9 rs2073242, p = 0.03. In the Brazilian cohort (n = 116 parent-affected child trios), the results were as follows: DLX1 rs788173, p = 0.07; FGF3 rs12574452, p = 0.03; GLI2 rs1992901, p = 0.03; and PITX2 rs2595110, p = 0.01. The second Brazilian cohort also suggested that FGF3 (rs12574452, p = 0.01) is associated with hypodontia and added EDAR (rs17269487, p = 0.04), LHX6 (rs989798, p = 0.02), and MSX1 (rs12532, p = 0.003). CONCLUSION: Our results suggest that several genes are potentially associated with hypodontia and their individual contributions may be modest. Hence, these cases may not be explained by inactivating mutations such as many oligodontia cases segregating in a Mendelian fashion but rather are influenced by one or more susceptibility alleles in multiple small effect genes.

Fine-mapping of 5q12.1-13.3 unveils new genetic contributors to caries.

Caries is a multifactorial disease and little is still known about the host genetic factors influencing susceptibility. Our previous genome-wide linkage scan has identified the interval 5q12.1-5q13.3 as linked to low caries susceptibility in Filipino families. Here we fine-mapped this region in order to identify genetic contributors to caries susceptibility. Four hundred and seventy-seven subjects from 72 pedigrees with similar cultural and behavioral habits and limited access to dental care living in the Philippines were studied. DMFT scores and genotype data of 75 single-nucleotide polymorphisms were evaluated in the Filipino families with the Family-Based Association Test. For replication purposes, a total 1,467 independent subjects from five different populations were analyzed in a case-control format. In the Filipino cohort, statistically significant and borderline associations were found between low caries experience and four genes spanning 13 million base pairs (PART1, ZSWIM6, CCNB1, and BTF3). We were able to replicate these results in some of the populations studied. We detected PART1 and BTF3 expression in whole saliva, and the expression of BTF3 was associated with caries experience. Our results suggest BTF3 may have a functional role in protecting against caries.

Tooth agenesis association with self-reported family history of cancer.

It has been proposed that tooth agenesis and cancer development share common molecular pathways. We performed a cross-sectional study to investigate the epidemiological and molecular association between tooth agenesis and self-reported family history of cancer. Eighty-two individuals with tooth agenesis and 328 individuals with no birth defect were recruited from the same institution. Tooth agenesis was assessed in permanent teeth and was defined based on the age of the participants and when initial tooth formation should be radiographically visible. We also investigated the role of genes involved in dental development that have been implicated in tumorigenesis, and 14 markers in AXIN2, FGF3, FGF10, and FGFR2 were genotyped. Individuals with tooth agenesis had an increased risk of having a family history of cancer (p = 0.00006; OR = 2.7; 95% C.I., 1.6-4.4). There were associations between AXIN2, FGF3, FGF10, and FGFR2 with tooth agenesis [i.e., individuals who carried the polymorphic allele of FGFR2 (rs1219648) presented higher risk for having premolar agenesis (p = 0.02; OR = 1.8; 95% C.I., 1.1-3.0)]. In conclusion, tooth agenesis was associated with positive self-reported family history of cancer and with variants in AXIN2, FGF3, FGF10, and FGFR2. Prospective studies are needed to confirm if tooth agenesis can be used as a risk marker for cancer.

MMP13 polymorphism decreases risk for dental caries.

Recent evidence suggests that genetic studies may contribute to a better understanding of individual susceptibility to caries. Matrix metalloproteinases (MMPs) and their tissue inhibitors have been suggested to be involved in the caries process. The purpose of this study was to determine if polymorphisms in MMP2 (rs243865), MMP9 (rs17576), MMP13 (rs2252070), and TIMP2 (rs7501477) were associated with caries. Eligible unrelated children and adolescents were evaluated using a cross-sectional design. Data on oral health habits was obtained through a questionnaire and caries data was collected by clinical examination. Genotyping of the selected polymorphisms was carried out by real-time PCR. Allele and genotype frequencies were compared between individuals with and without caries experience. Of 505 subjects, 212 were caries-free and most subjects (61.2%) had mixed dentition. Allele frequency of MMP2, MMP13 and TIMP2 was different between caries-affected and caries-free individuals, with significant association for MMP13 (p = 0.004). Mutant allele carriers for MMP13 demonstrated a significantly decreased risk for caries (OR = 0.538, 95% CI 0.313-0.926); this result remained significant after adjustment for candidate genes, type of dentition and dietary factors. Allelic and genotype frequencies of the polymorphism in MMP9 were similar in caries-affected and caries-free individuals. Genetic variations in MMP13 may contribute to individual differences in caries susceptibility. Our findings reinforce that susceptibility to caries results from gene-environment interactions.

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.

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.