Cytotoxicity of acrylic resin-based materials used to fabricate interim crowns.

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.

Effect of crosslinkers on bond strength stability of fiber posts to root canal dentin and in situ proteolytic activity.

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.

Proliferation, migration, and expression of oral-mucosal-healing-related genes by oral fibroblasts receiving low-level laser therapy after inflammatory cytokines challenge.

BACKGROUND AND OBJECTIVES: Increased expression of inflammatory cytokines in the oral cavity has been related to the etiopathogenesis of oral mucositis and to delayed oral mucosal repair. Low-level laser therapy (LLLT) stimulates proliferation and migration of gingival fibroblasts, but the effects of specific inflammatory cytokines on oral mucosal cells and the modulation of these effects by LLLT have not been fully investigated. Therefore, this study investigated the effects of LLLT on oral fibroblasts after being challenged by oral-mucositis-related inflammatory cytokines. METHODS: Human gingival fibroblasts were seeded in plain culture medium (DMEM) containing 10% fetal bovine serum (FBS) for 24 hours. Then, cells were kept in contact with inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-8) in serum-free DMEM for 24 hours. After this period, cells were subjected to LLLT with a diode laser device (LaserTABLE, InGaAsP, 780 nm, 25 mW) delivering energy doses from 0.5 to 3 J/cm2 . Irradiation was repeated for 3 consecutive days. Twenty-four hours after the last irradiation, cell migration (wound-healing and transwell migration assays), cell proliferation (BrdU), gene expression of COL-I and growth factors (real-time PCR), and synthesis of COL-I (Sirius Red assay) and VEGF (ELISA) were assessed. Data were subjected to two-way ANOVA and Tukey's tests or Kruskall-Walis and Mann-Whitney tests (P < 0.05). RESULTS: The inflammatory cytokines decreased the migration capacity of gingival fibroblasts. However, a statistically significant difference was observed only for IL-6, detected by transwell assay, where 30% less cells migrated through the pores (P < 0.05) and IL-8, with an increased wound area (116%; P < 0.05), detected by the wound healing method. Cell proliferation was not affected by contact with cytokines, while growth factors and COL-I expression (approximately 80%; P < 0.05), as well as VEGF synthesis (approximately 20%; P < 0.05), were decreased after contact to all tested cytokines. The opposite was seen for total collagen synthesis. LLLT promoted an acceleration of fibroblast migration (30%; P < 0.05) and proliferation (112%; P < 0.05) when delivering 0.5 J/cm2 to the cells previously in contact with the inflammatory cytokines. Gene expression of VEGF (approximately 30%; P < 0.05), and EGF (17%; P < 0.05), was stimulated by LLLT after contact with TNF-α and IL-6. CONCLUSION: LLLT can counteract the negative effects of high concentrations of inflammatory cytokines, especially IL-6 and IL-8 on gingival fibroblast functions directly related to the wound-healing process. Lasers Surg. Med. 48:1006-1014, 2016. © 2016 Wiley Periodicals, Inc.

Low-level laser therapy in 3D cell culture model using gingival fibroblasts.

Besides extensive data about the effects of low-level laser therapy (LLLT) on different cell types, so far, these results were obtained from monolayer cell culture models, which have limitations in terms of cell morphology and phenotype expression. Therefore, for better in vitro evaluation of the effects of LLLT, this study was performed with a 3D cell culture model, where gingival fibroblasts were seeded in collagen matrix. Cells isolated from a healthy patient were seeded in wells of 24-well plates with culture medium (DMEM) supplemented with 10 % fetal bovine serum and collagen type I solution. After 5 days, a serum-free DMEM was added to the matrices with cells that were subjected or not to three consecutive irradiations of LLLT by means of the LaserTABLE diode device (780 nm, 25 mW) at 0.5, 1.5, and 3 J/cm(2). Twenty-four hours after the last irradiation, cell viability and morphology as well as gene expression of growth factors were assessed. Histological evaluation of matrices demonstrated uniform distribution and morphology of gingival fibroblasts within the collagen matrix. LLLT at 3 J/cm(2) increased gingival fibroblast viability. Enhanced gene expression of hCOL-I and hEGF was observed for 0.5 J/cm(2), while no significant changes were detected for the other irradiation densities tested. In conclusion, LLLT promoted biostimulation of gingival fibroblasts seeded in a 3D cell culture model, demonstrating that this model can be applied for phototherapy studies and that LLLT could penetrate the collagen matrix to increase cell functions related to tissue repair.

Uninfiltrated Collagen in Hybrid Layers produced after Reduced Acid-etching Time on Primary and Permanent Dentin.

AIM: This study evaluated the influence of acid-etching time on collagen exposure in adhesive interfaces established on primary and permanent dentin. MATERIALS AND METHODS: Flat dentin surfaces were produced on sound primary molars and premolars (n = 8). The surfaces were divided into mesial and distal halves, and each half was etched with phosphoric acid for 5 or 15 seconds. The teeth were randomly allocated into two groups according to the adhesive system applied: Prime & Bond NT or Prime & Bond 2.1. After the adhesive application, the specimens were processed for Goldner's trichrome staining. The thickness of the uninfiltrated collagen zone (UCZ) in the hybrid layer was measured under optical microscopy. Data were analyzed by analysis of variance and Tukey tests (α = 0.05). RESULTS: The thickness of UCZ was adhesive dependent. Within the same substrate, the specimens treated with Prime & Bond 2.1 presented thicker UCZ when etched for 15 seconds. Collagen exposure was significantly higher for the primary teeth etched for 5 seconds and treated with Prime & Bond 2.1. CONCLUSION: The thickness of UCZ in hybrid layers is directly affected by acid-etching time and by the adhesive system applied. Primary dentin seems to be more susceptible to collagen exposure than is permanent dentin. CLINICAL SIGNIFICANCE: Both acid-etching time and adhesive system can influence the amount of exposed collagen interfering on resin-dentin bond quality, especially on primary dentin.

Bifunctional naringenin-laden gelatin methacryloyl scaffolds with osteogenic and anti-inflammatory properties.

OBJECTIVE: To fabricate and characterize an innovative gelatin methacryloyl/GelMA electrospun scaffold containing the citrus flavonoid naringenin/NA with osteogenic and anti-inflammatory properties. METHODS: GelMA scaffolds (15 % w/v) containing 0/Control, 5, 10, or 20 % of NA w/w were obtained via electrospinning. The chemical composition, fiber morphology/diameter, swelling/degradation profile, and NA release were investigated. Cytotoxicity, cell proliferation, adhesion and spreading, total protein/TP production, alkaline phosphatase/ALP activity, osteogenic genes expression (OCN, OPN, RUNX2), and mineralized nodules deposition/MND with human alveolar bone-derived mesenchymal stem cells (aBMSCs) seeded on the scaffolds were assessed. Moreover, aBMSCs seeded on the scaffolds and stimulated with tumor necrosis factor-alpha/TNF-α were submitted to collagen, nitric oxide/NO, interleukin/IL-1α, and IL-6 production assessment. Data were analyzed using ANOVA and t-student/post-hoc tests (α = 5 %). RESULTS: NA-laden scaffolds presented increased fiber diameter, lower swelling capacity, and faster degradation profile over 28 days (p < 0.05). NA release was detected over time. Cell adhesion and spreading, and TP production were similar between GelMA and GelMA+NA5 % scaffolds, while cell proliferation, ALP activity, OCN/OPN/RUNX2 gene expression, and MND were higher for GelMA+NA5 % scaffolds (p < 0.05). Cells seeded on control scaffolds and TNF-α-stimulated presented higher levels of NO, IL-1α/IL-6, and lower levels of collagen (p < 0.05). In contrast, cells seeded on GelMA+NA5 % scaffolds showed downregulation of inflammatory markers and higher collagen synthesis (p < 0.05). SIGNIFICANCE: GelMA+NA5 % scaffold was cytocompatible, stimulated aBMSCs proliferation and differentiation, and downregulated inflammatory mediators' synthesis, suggesting its therapeutic effect as a multi-target bifunctional scaffold with osteogenic and anti-inflammatory properties for bone tissue engineering.

Zoledronic acid inhibits human osteoblast activities.

BACKGROUND: Bisphosphonates are potent inhibitors of bone resorption. These kinds of drugs, which are used for the treatment of osteolytic diseases, have been associated with the occurrence of oral osteonecrosis, especially in patients over 60 years old. Current studies have demonstrated that the cytotoxic effects of bisphosphonates on osteoblasts play an important role in oral osteonecrosis development. OBJECTIVE: The aim of this study was to evaluate the effect of long-term application of a highly potent bisphosphonate - zoledronic acid (ZA) - on human osteoblasts in vitro. METHODS: Human osteoblasts (MG63 cell line) were seeded for 72 h in wells of 24-well plates. The Dulbecco's modified Eagle's medium (DMEM) was then replaced by culture medium without fetal bovine serum (FBS), and the cells were incubated for an additional 24 h, after which ZA was added to the DMEM without FBS and incubated in contact with osteoblasts for 7, 14 or 21 days. Cell viability (CV), total protein production (TPP), alkaline phosphatase (ALP) activity, mineral nodule formation (MNF), and gene expression of ALP and osteocalcin (OCN), as well as cell morphology by scanning electronic microscopy, were evaluated. Data were statistically analyzed by Kruskal-Wallis and Mann-Whitney tests, with a significance level of 5%. RESULTS: The cytotoxic effects of ZA on osteoblasts were characterized by reduction of CV, TPP, ALP and MNF production. In addition, ZA MNF caused a decrease in gene expression of ALP and OCN, as well as intense cell morphology alterations. All these negative effects of ZA were concentration and period dependent. CONCLUSION: Both concentrations of ZA (1 and 5 µM) caused cytotoxic effects to osteoblasts which reduced the production and expression of proteins that play an important role in bone matrix synthesis and mineralization.

Biostimulatory effect of low-level laser therapy on keratinocytes in vitro.

Epithelial cells play an important role in reparative events. Therefore, therapies that can stimulate the proliferation and metabolism of these cells could accelerate the healing process. To evaluate the effects of low-level laser therapy (LLLT), human keratinocytes were irradiated with an InGaAsP diode laser prototype (LASERTable; 780 ± 3 nm; 40 mW) using 0.5, 1.5, 3, 5, and 7 J/cm2 energy doses. Irradiations were done every 24 h totaling three applications. Evaluation of cell metabolism (MTT assay) showed that LLLT with all energy doses promoted an increase of cell metabolism, being more effective for 0.5, 1.5, and 3 J/cm2. The highest cell counts (Trypan blue assay) were observed with 0.5, 3, and 5 J/cm2. No statistically significant difference for total protein (TP) production was observed and cell morphology analysis by scanning electron microscopy revealed that LLLT did not promote morphological alterations on the keratinocytes. Real-time polymerase chain reaction (qPCR) revealed that LLLT also promoted an increase of type I collagen (Col-I) and vascular endothelial growth factor (VEGF) gene expression, especially for 1.5 J/cm2, but no change on fibroblast growth factor-2 (FGF-2) expression was observed. LLLT at energy doses ranging from 0.5 to 3 J/cm2 promoted the most significant biostimulatory effects on cultured keratinocytes.

LED light attenuation through human dentin: a first step toward pulp photobiomodulation after cavity preparation.

PURPOSE: To evaluate the transdentinal light attenuation of LED at three wavelengths through different dentin thicknesses, simulating cavity preparations of different depths. METHODS: Forty-two dentin discs of three thicknesses (0.2, 0.5 and 1 mm; n = 14) were prepared from the coronal dentin of extracted sound human molars. The discs were illuminated with a LED light at three wavelengths (450+/-10 nm, 630 +/-10 nm and 850 +/-10 nm) to determine light attenuation. Light transmittance was also measured by spectrophotometry. RESULTS: In terms of minimum (0.2 mm) and maximum (1.0 mm) dentin thicknesses, the percentage of light attenuation varied from 49.3% to 69.9% for blue light, 42.9% to 58.5% for red light and 39.3% to 46.8% for infrared. For transmittance values, an increase was observed for all thicknesses according to greater wavelengths, and the largest variation occurred for the 0.2 mm thickness. All three wavelengths were able to pass through the dentin barrier at different thicknesses. Furthermore, the LED power loss and transmittance showed wide variations, depending on dentin thickness and wavelength.

Influence of titanium and zirconia substrates on the synthesis of inflammatory mediators.

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.

Specific parameters of infrared LED irradiation promote the inhibition of oxidative stress in dental pulp cells.

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.

Influence of Bisphosphonates on the Behavior of Osteoblasts Seeded Onto Titanium Discs.

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.

Antimicrobial photodynamic therapy reduces adhesion capacity and biofilm formation of Candida albicans from induced oral candidiasis in mice.

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.

Effect of analogues of cationic peptides on dentin mineralization markers in odontoblast-like cells.

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.

LLLT Effects on Oral Keratinocytes in an Organotypic 3D Model.

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.

Phenotypic markers of oral keratinocytes seeded on two distinct 3D oral mucosa models.

This study validates the use of a full-thickness oral mucosa model for in vitro studies with a collagen type I matrix, by comparison of this model with two other 3D oral mucosa models: human-sourced and porcine acellular dermal matrices (AlloDerm®/Strattice®, respectively). For the collagen matrix model, gingival fibroblasts were seeded either onto the dermal side of the AlloDerm® and Strattice® matrices or within the collagen matrices in complete culture medium (DMEM). For all scaffolds, DMEM was replaced every 24 h up to 72 h. For the full-thickness oral mucosa models, 72 h after fibroblast seeding, oral keratinocytes were seeded on the epidermal sides of AlloDerm® and Strattice® matrices or collagen matrices. All matrices and models were subjected to histological analysis, complementing phenotypic characterization by evaluation of glucose consumption, cell proliferation, gene expression and synthesis of growth factors. A higher fibroblast ratio was observed for the collagen matrix, in which the distribution of gingival fibroblasts was also more homogeneous. Metabolism, proliferation, and gene expression and synthesis of VEGF of these cells were also increased for the collagen matrix. All matrices provided a suitable substrate for oral keratinocytes adhesion, proliferation, and phenotypic expression; however, higher proliferation, stratification, and differentiation were noted when oral keratinocytes were seeded on the dermal matrices.

Biological Analysis of Simvastatin-releasing Chitosan Scaffold as a Cell-free System for Pulp-dentin Regeneration.

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.

Effect of different implant abutment surfaces on OBA-09 epithelial cell adhesion.

For the long-term success of implants, it is necessary to achieve a direct contact between the implant and the subjacent bone. To avoid bacterial penetration that could adversely affect the initial wound healing as well as the long-term behavior of the implants, an early tissue barrier must form that is able to protect the biological peri-implant structures. Given the need of an effective tissue early barrier around dental implants, the present study evaluated, in vitro, the influence of physical and chemical characteristics of two implant abutment surfaces on gingival epithelial cells (OBA-9) adhesion. To this end, titanium (Ti) and zirconia (ZrO2 ) disk-shaped specimens were used mimicking the abutment components surfaces, while bovine enamel (BE) and glass cover slips (GCS) disks served as positive and negative controls, respectively. Roughness and surface free energy (SFE) of all materials were evaluated previously to cellular adhesion step. In sequence, the effect of each material on cells morphology and viability was analyzed after 1 and 24 hr. The results showed that roughness and SFE had no effect on the cell viability data or on their interaction (p = .559), independent of a post-contact analysis of 1 or 24 hr. However, cells attachment and spreading increased after 24 hr on Ti and ZrO2 than BE, corresponding to the highest SFE values. SFE appears to be an important property interfering on the quality of the soft tissue surrounding dental implants. These data can be considered a trigger point for developing new material surfaces.

Analyses of Biofilm on Implant Abutment Surfaces Coating with Diamond-Like Carbon and Biocompatibility.

The aim of this study was to evaluate the surface free energy (SFE), wetting and surface properties as well as antimicrobial, adhesion and biocompatibility properties of diamond-like carbon (DLC)-coated surfaces. In addition, the leakage of Escherichia coli through the abutment-dental implant interface was also calculated. SFE was calculated from contact angle values; R a was measured before and after DLC coating. Antimicrobial and adhesion properties against E. coli and cytotoxicity of DLC with human keratinocytes (HaCaT) were evaluated. Further, the ability of DLC-coated surfaces to prevent the migration of E. coli into the external hexagonal implant interface was also evaluated. A sterile technique was used for the semi-quantitative polymerase chain reaction (semi-quantitative PCR). The surfaces showed slight decreases in cell viability (p<0.05), while the SFE, R a, bacterial adhesion, antimicrobial, and bacterial infiltration tests showed no statistically significant differences (p>0.05). It was concluded that DLC was shown to be a biocompatible material with mild cytotoxicity that did not show changes in R a, SFE, bacterial adhesion or antimicrobial properties and did not inhibit the infiltration of E. coli into the abutment-dental implant interface.

Tumor Necrosis Factor-α and Interleukin (IL)-1ß, IL-6, and IL-8 Impair In Vitro Migration and Induce Apoptosis of Gingival Fibroblasts and Epithelial Cells, Delaying Wound Healing.

BACKGROUND: Multiple factors affect oral mucosal healing, such as the persistence of an inflammatory reaction. The present study evaluates effects of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß, IL-6, and IL-8 on epithelial cells (ECs) and human gingival fibroblasts (GFs) in vitro. METHODS: GFs and ECs were seeded in 96-well plates (1 × 10(4) cells/well) in plain culture medium (Dulbecco's modified Eagle's medium [DMEM]) containing 1% antibiotic/antimycotic solution and 10% fetal bovine serum, and incubated for 24 hours. Both cell lines were exposed for 24 hours to the following cytokines: 1) TNF-α (100 ng/mL); 2) IL-1ß (1 ng/mL); 3) IL-6 (10 ng/mL); and 4) IL-8 (10 ng/mL). All cytokines were diluted in serum-free DMEM. Control cultures were exposed only to serum-free DMEM. Effects of exposure to inflammatory cytokines were determined by means of: 1) apoptosis (anexin V); 2) cell migration (wound healing assay); 3) inflammatory cytokine synthesis (enzyme-linked immunosorbent assay). Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests (α = 0.05). RESULTS: Increased apoptosis rates were noted when cells were exposed to inflammatory cytokines, except ECs exposed to IL-1ß. Cell migration was negatively affected by all inflammatory cytokines for both cell lines. ECs and GFs exposed to IL-6 and IL-8 significantly increased synthesis of TNF-α and IL-1ß. CONCLUSIONS: Demonstrated results indicate negative effects of tested inflammatory cytokines on ECs and GFs, inducing apoptosis and impairing cell migration. These results can justify delayed oral mucosa healing in the presence of inflammatory reaction.