Indirect cytotoxicity of a 35% hydrogen peroxide bleaching gel on cultured odontoblast-like cells

The aim of this study was to evaluate the trans-enamel and trans-dentinal effects of a 35% hydrogen peroxide (H2O2) bleaching gel on odontoblast-like cells. Enamel/dentin discs obtained from bovine incisors were mounted in artificial pulp chambers (APCs). Three groups were formed: G1- 35% H2O2; G2- 35% H2O2 + halogen light application; G3- control. The treatments were repeated 5 times and the APCs were incubated for 12 h. Then, the extract was collected and applied for 24 h on the cells. Cell metabolism, total protein dosage and cell morphology were evaluated. Cell metabolism decreased by 62.09% and 61.83% in G1 and G2, respectively. The depression of cell metabolism was statistically significant when G1 and G2 were compared to G3. Total protein dosage decreased by 93.13% and 91.80% in G1 and G2, respectively. The cells in G1 and G2 exhibited significant morphological alterations after contact with the extracts. Regardless of halogen light application, the extracts caused significantly more intense cytopathic effects compared to the control group. After 5 consecutive applications of a 35% H2O2 bleaching agent, either catalyzed or not by halogen light, products of gel degradation were capable to diffuse through enamel and dentin causing toxic effects to the cells.

Cytotoxic effects of white-MTA and MTA-Bio cements on odontoblast-like cells (MDPC-23)

This study evaluated the cytotoxic effects of 2 mineral trioxide aggregate (MTA) cements - White-MTA-Angelus and a new formulation, MTA-Bio - on odontoblast-like cell (MDPC-23) cultures. Twenty-four disc-shaped (2 mm diameter x 2 mm thick) specimens were fabricated from each material and immersed individually in wells containing 1 mL of DMEM culture medium for either 24 h or 7 days to obtain extracts, giving rise to 4 groups of 12 specimens each: G1 - White-MTA/24 h; G2 - White-MTA/7 days; G3 - MTA-Bio/24 h; and G4 - MTA-Bio/7 days. Plain culture medium (DMEM) was used as a negative control (G5). Cells at 30,000 cells/cm 2 concentration were seeded in the wells of 24-well plates and incubated in a humidified incubator with 5% CO 2 and 95% air at 37°C for 72 h. After this period, the culture medium of each well was replaced by 1 mL of extract (or plain DMEM in the control group) and the cells were incubated for additional 2 h. Cell metabolism was evaluated by the MTT assay and the data were analyzed statistically by ANOVA and Tukey's test (α=0.05). Cell morphology and the surface of representative MTA specimens of each group were examined by scanning electron microscopy. There was no statistically significant difference (p>0.05) between G1 and G2 or between G3 and G4. No significant difference (p>0.05) was found between the experimental and control groups either. Similar cell organization and morphology were observed in all groups, regardless of the storage periods. However, the number of cells observed in the experimental groups decreased compared to the control group. MTA-Bio presented irregular surface with more porosities than White-MTA. In conclusion, White-MTA and MTA-Bio presented low cytotoxic effects on odontoblast-like cell (MDPC-23) cultures.

Effect of low-level laser therapy on odontoblast-like cells exposed to bleaching agent

The aim of the present study was to evaluate the effect of low-level laser therapy (LLLT) on odontoblast-like MDPC-23 cells exposed to carbamide peroxide (CP 0.01 %-2.21 μg/mL of H2O2). The cells were seeded in sterile 24-well plates for 72 h. Eight groups were established according to the exposure or not to the bleaching agents and the laser energy doses tested (0, 4, 10, and 15 J/cm2). After exposing the cells to 0.01 % CP for 1 h, this bleaching solution was replaced by fresh culture medium. The cells were then irradiated (three sections) with a near-infrared diode laser (InGaAsP-780 ± 3 nm, 40 mW), with intervals of 24 h. The 0.01 % CP solution caused statistically significant reductions in cell metabolism and alkaline phosphate (ALP) activity when compared with those of the groups not exposed to the bleaching agent. The LLLT did not modulate cell metabolism; however, the dose of 4 J/cm2 increased the ALP activity. It was concluded that 0.01 % CP reduces the MDPC-23 cell metabolism and ALP activity. The LLLT in the parameters tested did not influence the cell metabolism of the cultured cells; nevertheless, the laser dose of 4 J/cm2 increases the ALP activity in groups both with and without exposure to the bleaching agent. © 2013 Springer-Verlag London.

Effects of zoledronic acid on odontoblast-like cells

The aim of the study was to evaluate the effects of a highly potent bisphosphonate, zoledronic acid (ZOL), on cultured odontoblast-like cells MDPC-23. The cells (1.5 × 104 cells/cm2) were seeded for 48 h in wells of 24-well dished. Then, the plain culture medium (DMEM) was replaced by fresh medium without fetal bovine serum. After 24 h, ZOL (1 or 5 μM) was added to the medium and maintained in contact with the cells for 24 h. After this period, the succinic dehydrogenase (SDH) enzyme production (cell viability-MTT assay), total protein (TP) production, alkaline phosphatase (ALP) activity, and gene expression (qPCR) of collagen type I (Col-I) and ALP were evaluated. Cell morphology was assessed by SEM. Five μM ZOL caused a significant decrease in SDH production. Both ZOL concentrations caused a dose-dependent significant decrease in TP production and ALP activity. ZOL also produced discret morphological alterations in the MDPC-23 cells. Regarding gene expression, 1 μM ZOL caused a significant increase in Col-I expression. Although 5 μM ZOL did not affect Col-I expression, it caused a significant alteration in ALP expression (ANOVA and Tukey's test, p < 0.05). ZOL presented a dose-dependent cytotoxic effect on the odontoblast-like cells, suggesting that under clinical conditions the release of this drug from dentin could cause damage to the pulpo-dentin complex. © 2012 Elsevier Ltd.

Cytotoxicity of adhesive systems of different hydrophilicities on cultured odontoblast-like cells

This study evaluated the cytotoxicity of experimental adhesive systems (EASs) on odontoblast-like cells. Paper discs (n=132) were impregnated with 10 μL of each EAS-R1, R2, R3, R4, and R5 (in an ascending order of hydrophilicity), followed by photoactivation. R1 and R2 are nonsolvated hydrophobic blends, R3 represents a simplified etch-and-rinse adhesive system, and R4 and R5 represent simplified self-etch adhesive systems. Discs were immersed in Dulbecco's modified Eagle's medium for 24 h to obtain eluates applied on MDPC-23 cell cultures. No material was applied on discs used as control (R0). Cell viability [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay], total protein (TP) production, alkaline phosphatase (ALP) activity, type of cell death, and degree of monomer conversion Fourier transform infrared (%DC-FTIR) were evaluated. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (α=0.05). Considering R0 (control) as having 100% of cell viability, R1, R2, R3, R4, and R5 reduced the metabolic activity of cells by 36.4, 3.1, 0.2, 21.5, and 65.7%, respectively, but only R1 and R5 differed from R0. Comparing with R0, lower TP production was observed for R1, R4, and R5, while ALP activity decreased for R1 and R5. Necrotic cell death was predominant for all EASs, but only R1, R4, and R5 differed from R0. Only R5 presented a different apoptotic cell death ratio from R0. R1 presented the lowest %DC (ca. 37%), whereas R4 and R5 presented the highest (ca. 56%). In conclusion, R2 and R3 were not toxic to the MDPC-23 cells, suggesting that the degree of hydrophilicity or %DC of the EASs alone were not responsible for their cytopathic effects. © 2013 Wiley Periodicals, Inc.

Cytotoxicity of dimethyl sulfoxide (DMSO) in direct contact with odontoblast-like cells

To evaluate the cytotoxicity of dimethyl sulfoxide (DMSO) on the repair-related activity of cultured odontoblast-like MDPC-23 cells. Methods Solutions with different concentrations of DMSO (0.05, 0.1, 0.3, 0.5 and 1.0 mM), diluted in culture medium (DMEM), were placed in contact with MDPC-23 cells (5 × 104 cells/cm2) for 24 h. Eight replicates (n = 8) were prepared for each solutions for the following methods of analysis: violet crystal dye for cell adhesion (CA), quantification of total protein (TP), alizarin red for mineralization nodules formation (MN) and cell death by necrosis (flow cytometry); while twelve replicates (n = 12) were prepared for viable cell number (Trypan Blue) and cell viability (MTT assay). Data were analyzed by ANOVA and Tukey or Kruskal–Wallis and Mann–Whitney's tests (p < 0.05). Results Cell viability, adhesion and percentage of cell death by necrosis were not affected by DMSO at any concentration, with no statistical significant difference among the groups. A significant reduction in total protein production was observed for 0.5 and 1.0 mM of DMSO compared to the control while increased mineralized nodules formation was seen only for 1.0 mM DMSO. Significance: DMSO caused no or minor cytotoxic effects on the pulp tissue repair-related activity of odontoblast-like cells.

Protective effect of sodium ascorbate on odontoblast-like cells MDPC-23 exposed to a bleaching agent

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Indirect cytocompatibility of a low-concentrated hydrogen peroxide bleaching gel to odontoblast-like cells

Aim To assess the initial cytotoxicity and the late phenotype marker expression of odontoblast-like cells (MDPC-23) subjected to less aggressive in-office bleaching therapies. Methodology A 17.5% hydrogen peroxide (H2O2) gel was applied for 45, 15 or 5 min to enamel/dentine discs adapted to trans-wells positioned over cultured MDPC-23 cells. No treatment was performed on the negative control. Immediately after bleaching, the cell viability, gene expression of inflammatory mediators and quantification of H2O2 diffusion were evaluated. The ALP activity, DSPP and DMP-1 gene expression and mineralized nodule deposition (MND) were assessed at 7, 14 or 21 days post-bleaching and analysed statistically with Mann–Whitney U-tests (α = 5%). Results H2O2 diffusion, proportional to treatment time, was observed in all bleached groups. Reductions of approximately 31%, 21% and 13% in cell viability were observed for the 45-, 15- and 5-min groups, respectively. This reduction was significant (P < 0.05) for the 45- and 15-min groups, which also presented significant (P < 0.05) over-expression of inflammatory mediators. The 45-min group was associated with significant (P < 0.05) reductions in DMP-1/DSPP expression at all periods, relative to control. The ALP activity and MND were reduced only in initial periods. The 15-min group had less intense reduction of all markers, with no difference to control at 21 days. Conclusions The 17.5% H2O2 applied to tooth specimens for 5 min caused no alteration in the odontoblast-like cells. When this gel was applied for 45 or 15 min, a slight cytotoxicity, associated with alterations in phenotypic markers, was observed. However, cells were able to recover their functions up to 21 days post-bleaching.

Transdentinal cytotoxicity of carbodiimide (EDC) and glutaraldehyde on odontoblast-like cells

Objective: To evaluate the transdentinal cytotoxicity of three different concentrations of carbodiimide (EDC) or 5% glutaraldehyde (GA) on MDPC-23 cells. Methods: Seventy 0.4-mm-thick dentin disks obtained from human molars were adapted to artificial pulp chambers. MDPC-23 cells were seeded on the pulpal surface of the disks. After 48 hours, the occlusal dentin was acid-etched and treated for 60 seconds with one of the following solutions (n=10): no treatment (negative control); 0.1 M, 0.3 M, or 0.5 M EDC; 5% GA; Sorensen buffer; or 29% hydrogen peroxide (positive control). Cell viability and morphology were assessed by methyltetrazolium assay and scanning electron microscopy (SEM), respectively. The eluates were collected after the treatments and applied on MDPC-23 seeded in a 24-well plate to analyze cell death, total protein (TP), and collagen production. The last two tests were performed 24 hours and seven days after the challenge. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (p<0.05). Results: EDC at all test concentrations did not reduce cell viability, while 5% GA did increase cell metabolism. Cell death by necrosis was not elicited by EDC or 5% GA. At the 24-hour period, 0.3 M and 0.5 M EDC reduced TP production by 18% and 36.8%, respectively. At seven days, increased TP production was observed in all groups. Collagen production at the 24-hour period was reduced when 0.5 M EDC was used. After seven days, no difference was observed among the groups. SEM showed no alteration in cell morphology or number, except in the hydrogen peroxide group. Conclusions: Treatment of acid-etched dentin with EDC or GA did not cause transdentinal cytotoxic effects on odontoblast-like cells.

Transdentinal cytotoxicity of glutaraldehyde on odontoblast-like cells

This study investigated the transdentinal cytotoxicity of glutahaldehyde-containing solutions/materials on odontoblast-like cells. Dentin discs were adapted to artificial pulp chambers. MDPC-23 cells were seeded on the pulpal side of the discs and the occlusal surface was treated with the following solutions: water, 2% glutaraldehyde (GA), 5% GA, 10% GA, Gluma Comfort Bond+Desensitizer (GCB+De) or Gluma Desensitizer (GDe). Cell viability and morphology were assessed by the Alamar Blue assay and SEM. The eluates were collected and applied on cells seeded in 24-well plates. After 7 or 14 days the total protein (TP) production, alkaline phosphatase activity (ALP) and deposition of mineralized nodules (MN) were evaluated. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (p<0.05). GA solutions were not cytotoxic against MDPC-23. GCB+De (85.1%) and GDe (77.2%) reduced cell viability as well as TP production and ALP activity at both periods. After 14 days, GCB+De and GDe groups produced less MN. Affected MDPC-23 presented deformation of the cytoskeleton and reduction of cellular projections. The treatment with 2.5%, 5% and 10% GA was not harmful to odontoblast-like cells. Conversely, when GA was combined with other components like HEMA, the final material became cytotoxic. Glutaraldehyde has been used to decrease dentin hypersensitivity. This substance is also capable of preventing resin-dentin bond degradation by cross-linking collagen and MMPs. This study showed that GA might be safe when applied on acid etched dentin. However, when combined with HEMA the product becomes cytotoxic.

Differentiation of embryonic mesenchymal cells to odontoblast-like cells by overexpression of dentin matrix protein 1

Cells of the craniofacial skeleton are derived from a common mesenchymal progenitor. The regulatory factors that control their differentiation into various cell lineages are unknown. To investigate the biological function of dentin matrix protein 1 (DMP1), an extracellular matrix gene involved in calcified tissue formation, stable transgenic cell lines and adenovirally infected cells overexpressing DMP1 were generated. The findings in this paper demonstrate that overexpression of DMP1 in pluripotent and mesenchyme-derived cells such as C3H10T1/2, MC3T3-E1, and RPC-C2A can induce these cells to differentiate and form functional odontoblast-like cells. Functional differentiation of odontoblasts requires unique sets of genes being turned on and off in a growth- and differentiation-specific manner. The genes studied include transcription factors like core binding factor 1 (Cbfa1), bone morphogenetic protein 2 (BMP2), and BMP4; early markers for extracellular matrix deposition like alkaline phosphatase (ALP), osteopontin, osteonectin, and osteocalcin; and late markers like DMP2 and dentin sialoprotein (DSP) that are expressed by terminally differentiated odontoblasts and are responsible for the formation of tissue-specific dentin matrix. However, this differentiation pathway was limited to mesenchyme-derived cells only. Other cell lines tested by the adenoviral expression system failed to express odontoblast-phenotypic specific genes. An in vitro mineralized nodule formation assay demonstrated that overexpressed cells could differentiate and form a mineralized matrix. Furthermore, we also demonstrate that phosphorylation of Cbfa1 (osteoblast-specific transcription factor) was not required for the expression of odontoblast-specific genes, indicating the involvement of other unidentified odontoblast-specific transcription factors or coactivators. Cell lines that differentiate into odontoblast-like cells are useful tools for studying the mechanism involved in the terminal differentiation process of these postmitotic cells.

Transenamel and transdentinal cytotoxicity of carbamide peroxide bleaching gels on odontoblast-like MDPC-23 cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Toxic effects of daily applications of 10% carbamide peroxide on odontoblast-like MDPC-23 cells

Background. Tooth bleaching has been widely studied, mainly due to the possible undesirable effects that can be caused by this esthetic procedure. The cytotoxicity of the bleaching agents and its components to pulp cells has been demonstrated in several researches. The aim of this study was to evaluate the toxic effects of successive applications of 10% carbamide peroxide (CP) gel on odontoblast-like cells. Materials and methods. Enamel-dentin discs obtained from bovine incisors were adapted to artificial pulp chambers (APCs). The groups were formed as follows: G1: Without treatment (control group); G2: 10% carbamide peroxide, CP (five applications/one per day); G3: 10% CP (one unique application); and G4: 35% hydrogen peroxide, HP (three applications of 15 min each). After treatment, cell metabolism (MTT), alkaline phosphatase (ALP) activity and plasma membrane damage (flow cytometry) were analyzed. Results. Reductions in cell metabolism and alkaline phosphatase activity along with severe damage of the cytoplasmic membrane were noted in G2. In G3, no damage was observed, compared to the control group. Intermediary values of toxicity were obtained after 35% HP application. Conclusion. It can be concluded that one application of 10% CP did not cause toxic effects in odontoblast-like cells, but the successive application of this product promoted severe cytotoxic effects. The daily application of the bleaching agents, such as used in the at-home bleaching technique, can increase the damages caused by this treatment to the dental pulp cells. © 2013 Informa Healthcare.

Low-Level Laser Therapy Influences Mouse Odontoblast-Like Cell Response in Vitro

Objective: The purpose of this study was to analyze the influence of two different irradiation times with 85mW/cm(2) 830nm laser on the behavior of mouse odontoblast-like cells. Background data: The use of low-level laser therapy (LLLT) to stimulate pulp tissue is a reality, but few reports relate odontoblastic responses to irradiation in in vitro models. Methods: Odontoblast-like cells (MDPC-23) were cultivated and divided into three groups: control/nonirradiated (group 1); or irradiated with 85mW/cm(2), 830nm laser for 10 sec (0.8 J/cm(2)) (group 2); or for 50 sec (4.2 J/cm(2)) (group 3) with a wavelength of 830 nm. After 3, 7, and 10 days, it was analyzed: growth curve and cell viability, total protein content, alkaline phosphatase (ALP) activity, calcified nodules detection and quantification, collagen immunolocalization, vascular endothelial growth factor (VEGF) expression, and real-time polymerase chain reaction (PCR) for DMP1 gene. Data were analyzed by Kruskall-Wallis test (alpha = 0.05). Results: Cell growth was smaller in group 2 (p < 0.01), whereas viability was similar in all groups and at all periods. Total protein content and ALP activity increased on the 10th day with 0.8 J/cm(2) (p < 0.01), as well as the detection and quantification of mineralization nodules (p < 0.05), collagen, and VEGF expression (p < 0.01). The expression of DMP1 increased in all groups (p < 0.05) compared with control at 3 days, except for 0.8 J/cm(2) at 3 days and control at 10 days. Conclusions: LLLT influenced the behavior of odontoblast-like cells; the shorter time/smallest energy density promoted the expression of odontoblastic phenotype in a more significant way.

Differentially Expressed Protein Profile of Human Dental Pulp Cells in the Early Process of Odontoblast-like Differentiation In Vitro

Dental pulp cells (DPCs) are capable of differentiating into odontoblasts that secrete reparative dentin after pulp injury. The molecular mechanisms governing reparative dentinogenesis are yet to be fully understood. Here we investigated the differential protein profile of human DPCs undergoing odontogenic induction for 7 days. Using two-dimensional differential gel electrophoresis coupled with matrix-assisted laser adsorption ionization time of flight mass spectrometry, 2 3 protein spots related to the early odontogenic differentiation were identified. These proteins included cytoskeleton proteins, nuclear proteins, cell membrane-bound molecules, proteins involved in matrix synthesis, and metabolic enzymes. The expression of four identified proteins, which were heteronuclear ribonuclear proteins C, annexin VI, collagen type VI, and matrilin-2, was confirmed by Western blot and real-time realtime polymerase chain reaction analyses. This study generated a proteome reference map during odontoblast- like differentiation of human DPCs, which will be valuable to better understand the underlying molecular mechanisms in odontoblast-like differentiation.