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The repair and homeostasis of peri-implant tissues depend on several factors such as the local presence of pathogenic bacteria and their products. Among other events, peri-implant tissue response is also related to the implant material used, which interferes with cells and extracellular matrix interactions, affecting the osseointegration process. In this study, the influence of zirconia (Zr) and titanium (Ti) substrates on the response of preosteoblasts (MC3T3) and murine macrophages (RAW 264.7) exposed to lipopolysaccharide (LPS, P. gingivalis) was evaluated. Zr and Ti disks were obtained and subjected to surface roughness standardization, which was analyzed by scanning electronic microscopy (SEM). The cells were subsequently cultured on Zr and Ti surfaces in AlphaMEM culture medium for 24 h, followed by LPS stimulus for 4 h. The production of reactive oxygen species (ROS) and gene expression of inflammatory markers were determined. SEM images showed that Ti disks exhibited higher surface roughness than that of Zr disks. Cells that seeded onto Ti and Zr had increased expression of inflammatory mediators and ROS production in the presence of LPS; however, such cell responses were more evident for Ti disks. These data indicate that contact of cells with Zr surfaces may lead to a lower inflammatory potential than Ti surfaces. Elucidation of the inflammatory response triggered by LPS for cells in contact with titanium and zirconia may contribute to the selection of materials for installation of osseointegrated implants.

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OBJECTIVES: The present study aimed to assess the oxidative stress and the viability of dental pulp cells stimulated by lipopolysaccharide (LPS) and submitted to photobiomodulation (PBM) with infrared light-emitting diode (LED, 850 nm). DESIGN: Three healthy primary teeth (n = 3) were collected and seeded in 24-well plates with 10 µg/mL of LPS to induce inflammatory mediator formation. The cells were irradiated (850 nm, 40 mW/cm2 and 80 mW/cm2) at the proposed radiant exposures of 0 (control), 4, 15, and 30 J/cm2 shortly after LPS supplementation. The tests were performed 24 h after irradiation to assess mitochondrial activity (MTT assay), the number of viable cells (Trypan Blue), cell morphology (Scanning Electron Microscopy - SEM), and the quantification of Nitric Oxide (NO) and Reactive Oxygen Species (ROS). The data were analyzed using Kruskal-Wallis and Dunn's tests (p < 0.05). RESULTS: The irradiated groups showed larger viable cells number than the non-irradiated group with LPS (p < 0.0001). All irradiation parameters decreased ROS concentrations after LPS application compared to the non-irradiated group (p < 0.05). All irradiation parameters enhanced the NO values compared to those of the control group (p < 0.05). The SEM images showed cells with regular morphology that adhered to the substrate. CONCLUSIONS: According to the parameters used in this study, the radiant exposure of 15 J/cm2 and irradiance of 40 mW/cm2 were the most effective irradiation parameters to stimulate and modulate oxidative stress in the primary teeth-derived dental pulp cells.

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Among other factors, types of bisphosphonates and treatment regimens seem to be strongly associated with the success or failure of installation of osseointegrated implants. This study investigated the influence of two bisphosphonates, sodium alendronate (SA) and zoledronic acid (ZA), on the metabolism of osteoblasts. Human osteoblasts (Saos-2) were seeded onto machined or acid-treated titanium discs previously placed on 24-well plates in complete culture medium. After 24 h, cells were exposed to bisphosphonates at 0.5, 1 or 5 µM for 24 h, 48 h or 7 days. The effects of SA and ZA on osteoblasts were assessed based on the adhesion of these cells to the titanium surfaces by direct fluorescence, cell viability, total protein and collagen synthesis. Alkaline phosphatase activity and mineral nodule deposition by these cells were also evaluated. Data were evaluated by ANOVA and Tukey tests (α=0.05). Decreased adhesion of cells to the titanium discs was observed when exposed to both bisphosphonates; however, this lack of cell adhesion was more evident for ZA-treated cells. In addition, the exposure of osteoblasts to ZA decreased the viability, ALP activity and mineral nodule deposition, which may be related to poor osseointegration after implant installation.

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Abstract Among other factors, types of bisphosphonates and treatment regimens seem to be strongly associated with the success or failure of installation of osseointegrated implants. This study investigated the influence of two bisphosphonates, sodium alendronate (SA) and zoledronic acid (ZA), on the metabolism of osteoblasts. Human osteoblasts (Saos-2) were seeded onto machined or acid-treated titanium discs previously placed on 24-well plates in complete culture medium. After 24 h, cells were exposed to bisphosphonates at 0.5, 1 or 5 µM for 24 h, 48 h or 7 days. The effects of SA and ZA on osteoblasts were assessed based on the adhesion of these cells to the titanium surfaces by direct fluorescence, cell viability, total protein and collagen synthesis. Alkaline phosphatase activity and mineral nodule deposition by these cells were also evaluated. Data were evaluated by ANOVA and Tukey tests (α=0.05). Decreased adhesion of cells to the titanium discs was observed when exposed to both bisphosphonates; however, this lack of cell adhesion was more evident for ZA-treated cells. In addition, the exposure of osteoblasts to ZA decreased the viability, ALP activity and mineral nodule deposition, which may be related to poor osseointegration after implant installation.

Resumo Entre outros fatores, os tipos de bisfosfonatos bem como os regimes de tratamento parecem estar diretamente associados com o sucesso ou falhas na instalação de implantes osseointegrados. Este estudo avaliou a influência de dois bisfosfonatos, o alendronato de sódio (AS) e o ácido zoledrônico (AZ), no metabolismo de osteoblastos. Osteoblastos humanos (Saos-2) foram cultivados sobre discos de titânio polidos ou submetidos a tratamento ácido superficial, previamente alocados em placas de 24 compartimentos, utilizando meio de cultura completo. Após 24 horas, as células foram expostas aos bisfosfonatos, nas concentrações de 0,5, 1 ou 5 µM, por 24 h, 48 h, ou 7 dias. Os efeitos do AZ e AZ sobre os osteoblastos foram determinados considerando a adesão destas células às superfícies de titânio, por meio de fluorescência direta, a viabilidade celular, produção de proteína total e síntese de colágeno. A atividade de fosfatase alcalina e a deposição de nódulos mineralizados também foram avaliadas. Os dados foram analisados por meio do teste ANOVA complementado por Tukey (α = 0.05). Menor adesão dos osteoblastos foi observada quando estas células foram expostas a ambos os bisfosfonatos, porém, esta falha na adesão foi mais evidente para as células tratadas com AZ. Além disso, a exposição dos osteoblastos ao AZ também resultou em diminuição da viabilidade, atividade de ALP e deposição de nódulos mineralizados, o que pode estar relacionado a uma pobre osseointegração após a instalação do implante.

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STATEMENT OF PROBLEM: If the components in the acrylic resins used to fabricate interim crows are cytotoxic, they can interfere with the integrity of the adjacent periodontal tissue and the dentin-pulp complex. PURPOSE: The purpose of this in vitro study was to assess the cytotoxicity of resin-based materials used to prepare interim crowns. MATERIAL AND METHODS: The following materials were used in this study: CAR, conventional acrylic resin powder and liquid; BR, bis-acrylic resin; and PAR, pressed acrylic resin of the CAD-CAM type. Glass disks were used as the control (Co). Oral epithelial cells (NOK) were seeded on glass disks and standardized disks prepared with the resins under study. After incubation for 24 hours, the cells were analyzed for viability (Alamar Blue and Live or Dead), adhesion, and morphology (SEM and fluorescence), as well as epidermal growth factor synthesis (EGF-ELISA). The surface roughness (Ra) of test specimens was evaluated under a confocal microscope. The data were submitted to ANOVA and the Tukey HSD statistical tests (α=.05). RESULTS: The highest Ra value was observed in BR in comparison with CAR, PAR, and Co (P<.05). The highest viability, adhesion, and EGF synthesis values were determined for the cells in contact with PAR (P<.001). CONCLUSIONS: The computer-aided design and computer-aided manufacturing (CAD-CAM)-type resin favored adhesion, metabolism, and epithelial cell proliferation, and it was therefore considered cytocompatible.

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BACKGROUND: Antimicrobial photodynamic therapy (aPDT) has been considered an alternative therapeutic modality for the treatment of Candida infections. However, most studies are focused mainly on microorganism's inactivation efficiency. Here, we evaluated the efficacy of aPDT mediated by chloro-aluminum phthalocyanine encapsulated in cationic nanoemulsions (ClAlP-NE) to treat oral candidiasis in vivo and its effect on the adhesion and biofilm formation of Candida albicans. METHODS: For this, mice were immunosuppressed and inoculated with C. albicans to produce oral candidiasis. aPDT and Nystatin were applied for 5 successive sessions. Next, the microbiological evaluation was determined (CFU/ml) and the analyses of virulence factors (adhesion capacity and biofilm formation) were performed. Data were analyzed by Two-way ANOVA (α = 0.05). RESULTS: aPDT was as effective as Nystatin reducing 1.4 and 2.0 log10 of the cell viability (p ≤ 0.0001), respectively. Both treatments reduced the adhesion capacity and biofilm formation of C. albicans (p ≤ 0.0001) CONCLUSION: : ClAlP-NE-mediated aPDT was effective in reducing the virulence factors of C. albicans and also to treat induced oral candidiasis in mice.

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OBJECTIVES: To evaluate the effect of analogues of cationic peptides on the viability and the expression of phenotypic and genotypic markers of dentin mineralization in MDPC-23 odontoblast-like cells. MATERIALS AND METHODS: Cells were exposed to serial dilutions of analogues of cationic peptides hBD-3-1CV and KR-12-a5 compared to peptide LL-37 and their viability was assessed by methyltetrazolium assay. Next, peptides (0.78-62.5 µg/mL) were applied on the MDPC-23 cells for evaluating the total protein (TP) production, alkaline phosphatase (ALP) activity and mineralized nodule deposition. Gene expression of mineralization markers (DSPP and DMP-1) was also determined by quantitative PCR. RESULTS: LL-37 and hBD-3-1CV treatment did not affect cellular viability at concentrations below 62.5 µg/mL. KR-12-a5 reduced cell viability above 31.25 µg/mL. TP production was similar for all groups compared with the control group, except by hBD-3-1CV (at 15.62 µg/mL). LL-37 (at 62.5 µg/mL) induced higher ALP activity than control and other experimental groups. LL-37 and hBD-3-1CV, at 62.5 µg/mL and KR-12-a5 at 31.25 µg/mL stimulated the highest deposition of mineralized nodule. Overall, no statistical differences were observed between the groups for DSPP-1 and DMP-1 expressions. CONCLUSIONS: LL-37 was the only peptide that induced both ALP activity and mineralized nodules deposition, without affecting cell viability. None of peptides tested induced the expression of DSPP or DMP-1, genes commonly involved in active dentin mineralization.

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INTRODUCTION: The improvement of biomaterials capable of driving the regeneration of the pulp-dentin complex mediated by resident cells is the goal of regenerative dentistry. In the present investigation, a chitosan scaffold (CHSC) that released bioactive concentrations of simvastatin (SIM) was tested, aimed at the development of a cell-free tissue engineering system. METHODS: First, we performed a dose-response assay to select the bioactive dose of SIM capable of inducing an odontoblastic phenotype in dental pulp cells (DPCs); after which we evaluated the synergistic effect of this dosage with the CHSC/DPC construct. SIM at 1.0 µmol/L (CHSC-SIM1.0) and 0.5 µmol/L were incorporated into the CHSC, and cell viability, adhesion, and calcium deposition were evaluated. Finally, we assessed the biomaterials in an artificial pulp chamber/3-dimensional culture model to simulate the cell-free approach in vitro. RESULTS: SIM at 0.1 µmol/L was selected as the bioactive dose. This drug was capable of strongly inducing an odontoblastic phenotype on the DPC/CHSC construct. The incorporation of SIM into CHSC had no deleterious effect on cell viability and adhesion to the scaffold structure. CHSC-SIM1.0 led to significantly higher calcium-rich matrix deposition on scaffold/dentin disc assay compared with the control (CHSC). This biomaterial induced the migration of DPCs from a 3-dimensional culture to its surface as well as stimulated significantly higher expressions of alkaline phosphatase, collagen type 1 alpha 1, dentin matrix acidic phosphoprotein 1, and dentin sialophosphoprotein on 3-dimensional-cultured DPCs than on those in contact with CHSC. CONCLUSIONS: CHSC-SIM1.0 scaffold was capable of increasing the chemotaxis and regenerative potential of DPCs.

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STATEMENT OF PROBLEM: Improved stability of the adhesive interface can be obtained using crosslinkers. However, research on the use of crosslinkers in root dentin is lacking. PURPOSE: The purpose of this in vitro study was to evaluate the effect of crosslinkers on the proteolytic activity of root dentin and on the bond strength of resin-cemented fiber posts. MATERIAL AND METHODS: Single root canals were obtained from premolars (n=48) and endodontically treated before being divided into 4 groups: deionized water (control), 0.5 mol/L carbodiimide, 5% proanthocyanidin, or 5% glutaraldehyde. After removing the canal sealer, the dentin was etched with phosphoric acid, followed by water rinsing and the application of the crosslinkers for 60 seconds. Fiber posts were cemented using an adhesive (Single Bond 2) and resin cement (RelyX ARC). The roots were then transversally sectioned to obtain 1 mm thick specimens from the cervical, middle, and apical thirds and then aged for 24 hours or 9 months. Nine roots per group were used for the push-out test and 3 for determining the proteolytic activity of the root dentin by in situ zymography. Bond strength data were submitted to a mixed-model ANOVA and Bonferroni tests (α=.05). RESULTS: Only proanthocyanidin negatively affected the 24-hour bond strength. After 9 months, a significant decrease in bond strength was seen for all groups, except for the crosslinked treated specimens from the cervical third of the root canal. Intense gelatinolytic activity was detected in the control group after 24 hours but was inhibited in the crosslinker-treated groups. Proteolytic activity was also not detected after 9 months for the groups treated with the crosslinkers, irrespective of the root canal third. Conversely, proteolytic activity increased for the specimens from the control group. CONCLUSIONS: Although no proteolytic activity was detected in the hybrid layers along the entire root canal, dentin biomodification with crosslinkers was effective in preventing bond strength loss only in the cervical third.

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Several in vitro studies evaluated the cellular and molecular events related to interactions between phototherapy and target tissues, including oral keratinocytes and fibroblasts, providing elucidative data about phototherapy-induced healing. However, these interactions were limited to the application of a bidimensional cell culture model of oral mucosal cells. Thus, thisstudy evaluated the use of an organotypic oral epithelium model to elucidate the morphological and phenotypic responses of cells subjected to low-level laser therapy (LLLT). Oral keratinocytes were seeded in the ex vivo-produced oral mucosal equivalent (EVPOME) model, with a porcine acellular dermal matrix. LLLT was applied by means of the LaserTABLE device (780 nm, 25 mW) at 0.5, 1.5 and 3 J cm-2 . After three irradiations, morphology, proliferation and gene expression of growth factors were assessed. LLLT and control groups presented similar morphological features, characterized by the formation of a stratified, differentiated and keratinized epithelium. LLLT enhanced the cell proliferation and gene expression of keratinocytes (hKGF) as well as epidermal (hEGF) growth factors. In general, analysis of these data shows that the three-dimensional cell culture model can be applied for phototherapy studies and that the positive effects of LLLT were confirmed by the use of an organotypic model.

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