Transdentinal effects of S-PRG fillers on odontoblast-like cells.

OBJECTIVES: To investigate the transdentinal effects of surface reaction-type pre-reacted glass-ionomer (S-PRG) fillers on odontoblast-like cells. METHODS: An eluate of S-PRG fillers was obtained by dissolving the particles in distilled water (1:1 m/v). Dentin discs with similar permeability were mounted into artificial pulp chambers and MDPC-23 cells were seeded on their pulpal surface. The occlusal surface was treated with (n = 10): ultrapure water (negative control - NC), hydrogen peroxide (positive control - PC), S-PRG eluate exposure for 1 min (S-PRG 1 min), or S-PRG filler eluate exposure for 30 min (S-PRG 30 min). After 24 h, cell viability (alamarBlue) and morphology (SEM) were evaluated. The extract obtained from transdentinal diffusion was applied to MDPC-23 pre-cultured in plates for another 24 h to evaluate viability (alamarBlue, 1, 3, and 7 days), gene expression of Col1a1, Alpl, Dspp, and Dmp1 (RT-qPCR, 1 and 7 days), and mineralization (Alizarin Red, 7 days). Data were analyzed with ANOVA (α = 5 %). RESULTS: While S-PRG 1 min did not differ from NC, S-PRG 30 min reduced 17.9 % viability of cells from discs. S-PRG treatments resulted in low cell detaching from dentin, and the remaining cells exhibited typical morphology or minor cytoplasmic contraction. S-PRG 30 min slightly increased cell viability (6 %) 1 day after contact with the extract. S-PRG treatments upregulated the expression of the investigated genes, especially after 1 day. S-PRG 30 min stimulated mineralization activity by 39.7 %. CONCLUSIONS: S-PRG filler eluate does not cause transdentinal cytotoxicity on odontoblast-like cells, and long-term exposure can stimulate their dentinogenic-related mineralization activity. SIGNIFICANCE: The transdentinal elution of ions from S-PRG fillers is not expected to be harmful to the dental pulp and may exert bioactive effects by inducing dentin matrix deposition through the metabolism of underlying odontoblasts.

Functionalization of PCL-Based Fiber Scaffolds with Different Sources of Calcium and Phosphate and Odontogenic Potential on Human Dental Pulp Cells.

This study investigated the incorporation of sources of calcium, phosphate, or both into electrospun scaffolds and evaluated their bioactivity on human dental pulp cells (HDPCs). Additionally, scaffolds incorporated with calcium hydroxide (CH) were characterized for degradation, calcium release, and odontogenic differentiation by HDPCs. Polycaprolactone (PCL) was electrospun with or without 0.5% w/v of calcium hydroxide (PCL + CH), nano-hydroxyapatite (PCL + nHA), or ß-glycerophosphate (PCL + ßGP). SEM/EDS analysis confirmed fibrillar morphology and particle incorporation. HDPCs were cultured on the scaffolds to assess cell viability, adhesion, spreading, and mineralized matrix formation. PCL + CH was also evaluated for gene expression of odontogenic markers (RT-qPCR). Data were submitted to ANOVA and Student's t-test (α = 5%). Added CH increased fiber diameter and interfibrillar spacing, whereas ßGP decreased both. PCL + CH and PCL + nHA improved HDPC viability, adhesion, and proliferation. Mineralization was increased eightfold with PCL + CH. Scaffolds containing CH gradually degraded over six months, with calcium release within the first 140 days. CH incorporation upregulated DSPP and DMP1 expression after 7 and 14 days. In conclusion, CH- and nHA-laden PCL fiber scaffolds were cytocompatible and promoted HDPC adhesion, proliferation, and mineralized matrix deposition. PCL + CH scaffolds exhibit a slow degradation profile, providing sustained calcium release and stimulating HDPCs to upregulate odontogenesis marker genes.

Influence of coating dental enamel with a TiF4-loaded polymeric primer on the adverse effects caused by a bleaching gel with 35% H2O2.

OBJECTIVE: To evaluate whether coating enamel with a polymeric primer (PPol) containing titanium tetrafluoride (TiF4) before applying a bleaching gel with 35% H2O2 (35% BG) increases esthetic efficacy, prevents changes in morphology and hardness of enamel, as well as reduces the cytotoxicity from conventional in-office bleaching. MATERIALS AND METHODS: Standardized enamel/dentin discs were stained and bleached for 45 min (one session) with 35% BG. Groups 2TiF4, 6TiF4, and 10TiF4 received the gel on the enamel previously coated with PPol containing 2 mg/mL, 6 mg/mL, or 10 mg/mL, respectively. No treatment or application of 35% BG directly on enamel were used as negative control (NC), and positive control (PC), respectively. UV-reflectance spectrophotometry (CIE L*a*b* system, ΔE00, and ΔWI, n = 8) determined the bleaching efficacy of treatments. Enamel microhardness (Knoop, n = 8), morphology, and composition (SEM/EDS, n = 4) were also evaluated. Enamel/dentin discs adapted to artificial pulp chambers (n = 8) were used for trans-amelodentinal cytotoxicity tests. Following the treatments, the extracts (culture medium + bleaching gel components diffused through the discs) were collected and applied to odontoblast-like MDPC-23 cells, which were assessed concerning their viability (alamarBlue, n = 8; Live/Dead, n = 4), oxidative stress (n = 8), and morphology (SEM). The amount of H2O2 in the extracts was also determined (leuco crystal violet/peroxidase, n = 8). The numerical data underwent one-criterion variance analysis (one-way ANOVA), followed by Tukey's test, at a 5% significance level. RESULTS: Regarding the ΔE00, no difference was observed among groups 2TiF4, 6TiF4, and PC (p > 0.05). The ΔWI was similar between groups 2TiF4 and PC (p > 0.05). The ΔWI of group 6TiF4 was superior to PC (p < 0.05), and group 10TiF4 achieved the highest ΔE00 and ΔWI values (p < 0.05). Besides limiting enamel microstructural changes compared to PC, group 10TiF4 significantly increased the hardness of this mineralized dental tissue. The highest cellular viability occurred in 10TiF4 compared to the other bleached groups (p < 0.05). Trans-amelodentinal H2O2 diffusion decreased in groups 2TiF4, 6TiF4, and 10TiF4 in comparison with PC (p < 0.05). CONCLUSION: Coating enamel with a PPol containing TiF4 before applying a 35% BG may increase enamel microhardness and esthetic efficacy and reduce the trans-amelodentinal cytotoxicity of conventional in-office tooth bleaching. The PPol containing 10 mg/mL of TiF4 promoted the best outcomes.

Pulp cell response to the application of silver diamine fluoride and potassium iodide on caries-like demineralized dentin.

OBJECTIVES: To investigate the response of pulp cells to the application of silver diamine fluoride (SDF) and potassium iodide (KI) on demineralized dentin. MATERIALS AND METHODS: The occlusal surfaces of human dentin discs (0.4 mm thick) with similar permeability were subjected to an artificial caries protocol, and then the discs were adapted into artificial pulp chambers. MDPC-23 cells were seeded on the healthy pulp dentin surface, while the demineralized surface was treated with SDF, KI, SDF + KI, or hydrogen peroxide (positive control-PC) (n = 8). The negative control (NC) received ultrapure water. After 24 h, cell viability (alamarBlue) and morphology (SEM) were evaluated. The extracts were then applied to new MDPC-23 cells seeded in culture plates to assess their viability and the formation of mineralized nodules (MN; Alizarin Red) after seven days. The data were analyzed using one-way analysis of variance/Tukey or Games-Howell tests (α = 5%). RESULTS: SDF and PC significantly reduced the viability of cells seeded on discs (45.6% and 71.0%, respectively). Only cells treated with SDF or PC detached from the dentin substrate, while the remaining cells showed altered morphology. Cells in contact with extracts showed less reduction in viability, but it was still more toxic compared to NC. Only PC reduced MN deposition. SDF + KI or KI alone did not affect the cell response. CONCLUSIONS: SDF applied alone showed a mild to moderate transdentinal cytotoxic effect on pulp cells. However, the combination of SDF + KI reduced the cytotoxic effects. Both materials used alone or in combination did not affect the mineralization ability of pulp cells. CLINICAL RELEVANCE: Besides improving esthetic results, associating potassium iodide with silver diamine fluoride may reduce the transdentinal cytotoxic effects of this cariostatic agent on pulp cells.

Chronic exposure to lipopolysaccharides as an in vitro model to simulate the impaired odontogenic potential of dental pulp cells under pulpitis conditions.

BACKGROUND: Simulating a bacterial-induced pulpitis environment in vitro may contribute to exploring mechanisms and bioactive molecules to counteract these adverse effects. OBJECTIVE: To investigate the chronic exposure of human dental pulp cells (HDPCs) to lipopolysaccharides (LPS) aiming to establish a cell culture protocol to simulate the impaired odontogenic potential under pulpitis conditions. METHODOLOGY: HDPCs were isolated from four healthy molars of different donors and seeded in culture plates in a growth medium. After 24 h, the medium was changed to an odontogenic differentiation medium (DM) supplemented or not with E. coli LPS (0 - control, 0.1, 1, or 10 µg/mL) (n=8). The medium was renewed every two days for up to seven days, then replaced with LPS-free DM for up to 21 days. The activation of NF-κB and F-actin expression were assessed (immunofluorescence) after one and seven days. On day 7, cells were evaluated for both the gene expression (RT-qPCR) of odontogenic markers (COL1A1, ALPL, DSPP, and DMP1) and cytokines (TNF, IL1B, IL8, and IL6) and the production of reactive nitrogen (Griess) and oxygen species (Carboxy-H2DCFDA). Cell viability (alamarBlue) was evaluated weekly, and mineralization was assessed (Alizarin Red) at 14 and 21 days. Data were analyzed with ANOVA and post-hoc tests (α=5%). RESULTS: After one and seven days of exposure to LPS, NF-κB was activated in a dose-dependent fashion. LPS at 1 and 10 µg/mL concentrations down-regulated the gene expression of odontogenic markers and up-regulated cytokines. LPS at 10 µg/mL increased both the production of reactive nitrogen and oxygen species. LPS decreased cell viability seven days after the end of exposure. LPS at 1 and 10 µg/mL decreased hDPCs mineralization in a dose-dependent fashion. CONCLUSION: The exposure to 10 µg/mL LPS for seven days creates an inflammatory environment that is able to impair by more than half the odontogenic potential of HDPCs in vitro, simulating a pulpitis-like condition.

Chronic exposure to lipopolysaccharides as an in vitro model to simulate the impaired odontogenic potential of dental pulp cells under pulpitis conditions

Abstract Simulating a bacterial-induced pulpitis environment in vitro may contribute to exploring mechanisms and bioactive molecules to counteract these adverse effects. Objective To investigate the chronic exposure of human dental pulp cells (HDPCs) to lipopolysaccharides (LPS) aiming to establish a cell culture protocol to simulate the impaired odontogenic potential under pulpitis conditions. Methodology HDPCs were isolated from four healthy molars of different donors and seeded in culture plates in a growth medium. After 24 h, the medium was changed to an odontogenic differentiation medium (DM) supplemented or not with E. coli LPS (0 - control, 0.1, 1, or 10 µg/mL) (n=8). The medium was renewed every two days for up to seven days, then replaced with LPS-free DM for up to 21 days. The activation of NF-κB and F-actin expression were assessed (immunofluorescence) after one and seven days. On day 7, cells were evaluated for both the gene expression (RT-qPCR) of odontogenic markers (COL1A1, ALPL, DSPP, and DMP1) and cytokines (TNF, IL1B, IL8, and IL6) and the production of reactive nitrogen (Griess) and oxygen species (Carboxy-H2DCFDA). Cell viability (alamarBlue) was evaluated weekly, and mineralization was assessed (Alizarin Red) at 14 and 21 days. Data were analyzed with ANOVA and post-hoc tests (α=5%). Results After one and seven days of exposure to LPS, NF-κB was activated in a dose-dependent fashion. LPS at 1 and 10 µg/mL concentrations down-regulated the gene expression of odontogenic markers and up-regulated cytokines. LPS at 10 µg/mL increased both the production of reactive nitrogen and oxygen species. LPS decreased cell viability seven days after the end of exposure. LPS at 1 and 10 µg/mL decreased hDPCs mineralization in a dose-dependent fashion. Conclusion The exposure to 10 µg/mL LPS for seven days creates an inflammatory environment that is able to impair by more than half the odontogenic potential of HDPCs in vitro, simulating a pulpitis-like condition.

Inhibitory activity of S-PRG filler on collagen-bound MMPs and dentin matrix degradation.

OBJECTIVES: To evaluate the inhibitory activity of an ion-releasing filler (S-PRG) eluate on dentin collagen-bound metalloproteinases (MMPs) and dentin matrix degradation. METHODS: Dentin beams (5 × 2 × 0.5 mm) from human molars were completely demineralized to produce dentin matrix specimens. The dry mass was measured, and a colorimetric assay (Sensolyte) determined the initial total MMP activity to allocate the beams into four treatment groups (n = 10/group): 1) water for 1 min (negative control); 2) 2% chlorhexidine digluconate (CHX - inhibitor control) for 1 min; 3) S-PRG eluate for 1 min; 4) S-PRG eluate for 30 min. After the treatments, the total MMP activity was reassessed. The specimens were stored in simulated body fluid (SBF) at 37 °C for up to 21 days. The dry mass was reassessed weekly. On day 7, the dentin matrix degradation was analyzed for the presence of collagen fragments (CF; Sirius Red) and hydroxyproline (Hyp) in the SBF. Statistical analyses were performed with ANOVA/Tukey, paired t-tests, and RM-ANOVA/Sidak (α = 5%). RESULTS: S-PRG eluate exposure for 1 and 30 min reduced (p < 0.0001) MMP activity. S-PRG exposure for 30 min presented MMP activity inhibition equivalent to CHX (p = 0.061). S-PRG and CHX decreased CF (p ≤ 0.007) and Hyp (p < 0.046) release. After 21 days of storage, S-PRG-treated beams, regardless of exposure time, presented a reduced (p ≤ 0.017) mass loss, intermediate between CHX and control. CONCLUSION: Treating demineralized dentin with S-PRG eluate for 1 or 30 min reduced matrix-bound MMP activity and dentin matrix degradation for up to 21 days. CLINICAL SIGNIFICANCE: S-PRG filler may hinder the progression of dentin carious/erosive lesions and enhance the stabilization of dentin bonding interfaces.

Nano-hydroxyapatite-incorporated polycaprolactone nanofibrous scaffold as a dentin tissue engineering-based strategy for vital pulp therapy.

OBJECTIVES: Targeting a tissue engineering-based vital pulp therapy (VPT), this study investigated the incorporation of nano-hydroxyapatite (nHA) into polycaprolactone (PCL) nanofibers, and the metabolism of human dental pulp cells (HDPCs) seeded on the scaffolds. METHODS: PCL-based solutions (10% w/v) containing nHA (0 - control; 0.5; 1.0; or 2.0% w/v) were electrospun into nanofibrous scaffolds. The scaffolds were characterized for morphology and composition (MEV/EDS), solubility, the release of calcium/phosphate (C/P), and modulation of medium pH. Then, HDPCs were seeded on the scaffolds and evaluated for cell viability (alamarBlue and live/dead), adhesion and spreading (F-actin), total protein (TP; Lowry), alkaline phosphatase activity (ALP; thymolphthalein assay), expression of odontogenic genes (RT-qPCR), and formation of a mineralized matrix (Alizarin Red). Data were analyzed with ANOVA and post-hocs (α = 5%). RESULTS: Higher nHA concentrations roughened fiber surfaces, whereas PCL+ 2%nHA increased the interfibrillar spaces. PCL+ 1%nHA or PCL+ 2%nHA significantly released more C/P but the medium pH was maintained below 8.0. HDPCs viability was not affected by nHA, while cell adhesion/spreading was favored, especially for PCL+ 2%nHA. Higher protein content and ALP activity were seen for scaffolds incorporated with nHA, after 21 days. PCL+ 1%nHA and PCL+ 2%nHA upregulated the expression of DSPP and DMP1 in 14 days, and COL1A1, ALPL, and DMP1 in 21 days. The formation of a mineralized matrix was nHA concentration-dependent, and it was about 9 × higher for PCL+ 2%nHA. SIGNIFICANCE: nHA-incorporated PCL nanofibrous scaffolds are cytocompatible and can stimulate the adhesion and odontogenic potential of HDPCs. PCL+ 2%nHA formulation is a bioactive tissue engineering-based cell-homing strategy for VPT.

Cytocompatibility and bioactivity of calcium hydroxide-containing nanofiber scaffolds loaded with fibronectin for dentin tissue engineering.

OBJECTIVES: The aim of this study was to characterize polycaprolactone-based nanofiber scaffolds (PCL) incorporated with calcium hydroxide (CH) and evaluate their bioactivity on human dental pulp cells (HDPCs) when loaded with fibronectin (FN). MATERIALS AND METHODS: CH (0.1%; 0.2%; 0.4% w/v; or 0%) was incorporated into PCL (10% w/v) scaffolds prepared by electrospinning. Morphology and composition were characterized using SEM/EDS. HDPCs were seeded on the scaffolds and evaluated for viability (alamarBlue; Live/Dead), and adhesion/spreading (F-actin). Next, scaffolds containing 0.4% CH were loaded with FN (20 µg/mL). HDPCs were evaluated for viability, adhesion/spreading, migration (Trans-well), gene expression (RT-qPCR), alkaline phosphatase activity (ALP), and mineralization nodules (Alizarin Red). Data were submitted to ANOVA and post-hoc tests (α = 5%). RESULTS: Nanofibers with larger diameter were seen as CH concentration increased, while there was no effect on interfibrillar spaces. An increase in cell viability was seen for 0.4% CH, in all periods. Incorporation of CH and FN into the scaffolds increased cellular migration, spread, and viability, all intensified when CH and FN were combined. ALPL and DSPP expression, and ALP activity were not affected by CH and FN. COL1A1 was downregulated in all groups, while DMP1 was upregulated in the presence of CH, with no differences for the groups loaded with FN. CH increased the formation of mineralized matrix, which was not influenced by FN. CONCLUSIONS: In conclusion, the incorporation of CH enhanced the odontogenic potential of HDPCs, irrespective of the presence of FN. The PCL + 0.4% CH formulation may be a useful strategy for use in dentin tissue engineering. CLINICAL RELEVANCE: A change in the form of presentation of calcium hydroxide-based materials used for direct pulp capping can increase biocompatibility and prolong the vitality of dental pulp.

Response of pulp cells to resin infiltration of enamel white spot-like lesions.

OBJECTIVES: To investigate the trans-enamel and trans-dentinal biological effects of treating enamel white spot-like lesions (EWSLs) with resin infiltration components (RICs) on odontoblast-like cells (MDPC-23) and human dental pulp cells (HDPCs). METHODS: EWSLs were induced in 60 enamel/dentin discs (4.0 ± 0.2 mm thick) using S. mutans. The discs were adapted into artificial pulp chambers and MDPC-23 were seeded on the dentin surface. The components of a resin infiltration system (Icon) were applied individually or in combination on the enamel surface as following (n = 10/treatment): Etch, Infiltrant, Etch+Infiltrant, or Etch+Dry+Infiltrant. The application of water or hydrogen peroxide served as negative and positive controls, respectively. After 72 h, MDPC-23 viability was evaluated. The extracts were exposed for 72 h to pre-cultured MDPC-23 and HDPCs in 96-well plates to evaluate cell viability, alkaline phosphatase activity (ALP), mineralized nodule formation (MN), and the expression of inflammatory cytokines (ICs) and mineralization-related genes (MRs). Data were analyzed by ANOVA complemented with Tukey or Games-Howell post-hocs (α = 5%). RESULTS: Cell viability, ALP activity, and MN formation were significantly reduced in response to the RICs, presenting intermediate values compared to positive and negative controls. Likewise, ICs were upregulated, whereas MRs were downregulated. Among the RICs, the Etch component caused the most notorious detrimental effects. SIGNIFICANCE: Resin infiltration of EWSLs negatively affected the metabolism of pulp cells in vitro. Therefore, even though resin infiltration is a micro-invasive therapy for non-cavitated caries in enamel, it should be closely followed up seen that components may diffuse and unbalance pulp homeostasis.

Structural integrity of MIH-affected teeth after treatment with fluoride varnish or resin infiltration: An 18-Month randomized clinical trial.

OBJECTIVE: To evaluate the influence of fluoride varnish (FV) therapies or resin infiltration (RI) to maintain the structural integrity of Molar Incisor Hypomineralization (MIH) -affected teeth. METHODS: Fifty-one children aged 6-12 years with at least one incisor and one first permanent molar with yellow/brown MIH opacities were included. Patients were randomly allocated into three groups: FV - Fluoride Varnish (Duraphat); FV+etch - Fluoride Varnish (Duraphat) after enamel etching with 37% phosphoric acid; or RI - Resin Infiltration system (Icon). Opacities were monitored for 18 months. The primary outcome was the loss of integrity due to post-eruptive enamel breakdown (PEB). Covariables included sex, age, DMFT index, opacity colour, plaque index, number of MIH-affected teeth, and number of MIH-affected surfaces. Fisher's Exact was used to test the association of treatments with PEB, the Kaplan-Meyer method analysed the survival rates and Cox-regression determined which covariables would predict failure (α=0.05). RESULTS: From a total of 235 teeth, the PEB rate for RI (6.1%) was significantly lower (p<0.05) than FV (17.9%; OR 3.0, 95%CI 1.07, 8.48) and FV+etch (17.3%; OR 3.1, 95%CI 1.13, 8.73). DMFT index >3, brown opacities, cusp involvement, and age between 6-8 years predicted PEB (p<0.05). CONCLUSIONS: Resin infiltration positively influenced the structural integrity maintenance of MIH-affected teeth by decreasing the risk of enamel breakdown over18 months follow-up. Registry of Clinical Trials (RBR-8wwk3n). CLINICAL RELEVANCE: Resin infiltration proved to be a more efficacious intervention to maintain the structural integrity of MIH-affected teeth than fluoride varnish therapies.

Development of fibronectin-loaded nanofiber scaffolds for guided pulp tissue regeneration.

Fibronectin (FN)-loaded nanofiber scaffolds were developed and assessed concerning their bioactive potential on human apical papilla cells (hAPCs). First, random (NR) and aligned (NA) nanofiber scaffolds of polycaprolactone (PCL) were obtained by electrospinning technique and their biological properties were evaluated. The best formulations of NR and NA were loaded with 0, 5, or 10 µg/ml of FN and their bioactivity was assessed. Finally, FN-loaded NR and NA tubular scaffolds were prepared and their chemotactic potential was analyzed using an in vitro model to mimic the pulp regeneration of teeth with incomplete root formation. All scaffolds tested were cytocompatible. However, NR and NA based on 10% PCL promoted the highest hAPCs proliferation, adhesion and spreading. Polygonal and elongated cells were observed on NR and NA, respectively. The higher the concentration of FN added to the scaffolds, greater cell migration, viability, proliferation, adhesion and spreading, as well as collagen synthesis and gene expression (ITGA5, ITGAV, COL1A1, COL3A1). In addition, tubular scaffolds with NA loaded with FN (10 µg/ml) showed the highest chemotactic potential on hAPCs. It was concluded that FN-loaded NA scaffolds may be an interesting biomaterial to promote hAPCs-mediated pulp regeneration of endodontically compromised teeth with incomplete root formation.