Table 2. Main characteristics and effects of protocols on cell viability/cytotoxicity, morphology, and mineralization.
| Author | Experimental model | Groups | Bleaching gel protocol | Additional protocol | Period of analysis | Methods for outcome assessment | Main results | |
|---|---|---|---|---|---|---|---|---|
| In vitro studies using enamel/dentin discs and MSCs | ||||||||
| Dias et al. 2023 [62] | 5.6 × 2.3 mm enamel/dentin discs, and MDPC-23 cells | Negative control, PCP, 10% HP, 10% HP + PCP, 20% HP, 20% HP + PCP, 35% HP, 35% HP + PCP | 10%, 20% and 35% HP for 45 min | 10 μL of PCP (topically): before HP | 4 h for cell viability, 1 h for cell morphology | Cell viability: Alamar Blue and fluorescence; Cell morphology: SEM | Coating enamel with PCP before the bleaching protocols minimized the cytotoxic effects and morphological changes caused by HP, independent of its concentration. | |
| de Oliveira Ribeiro et al. 2022 [60] | 5.6 × 2.3 mm enamel/dentin discs, and MDPC-23 cells | Negative control, 35% HP, 10% HP, 10% HP + 2 mg/mL MnO2, 10% HP + 6 mg/mL MnO2, 10% HP + 10 mg/mL MnO2 | 10% HP for 45 min | 20 μL of 10% HP mixed with 2-10 mg/mL MnO2 (topically): 45 min (3 applications of 15 min) | 1 h | Cell viability: MTT and live/dead | Higher concentrations of MnO2 applied to the gel reduced cytotoxicity, especially 10 mg/mL of MnO2. | |
| Ribeiro et al. 2022 [59] | 5.6 × 2.3 mm bovine enamel/dentin discs, and MDPC-23 cells | CG: untreated; G1: 35% HP; G2: 35% HP + 2 mg/mL MnO2; G3: 35% HP + 6 mg/mL MnO2; G4: 35% HP + 10 mg/mL MnO2 | 35% HP for 45 min (3 × 15 min) | 2, 6, and 10 mg/mL of MnO2 incorporated into the bleaching gel: for 45 min (3 × 15 min) | 1 h | Cell viability: MTT; Cytotoxicity: live/dead assay; Cell morphology: SEM | 6 and 10 mg/mL of MnO2 incorporated into the bleaching gel increased cell viability. | |
| Ortecho-Zuta et al. 2019 [57] | 5.6 × 2.3 mm enamel/dentin discs, and MDPC-23 cells | Untreated, HP: 35% HP, 35% HP + HRP | 35% HP for 45 min (3 applications of 15 min) | 1 mL of 35% HP mixed with 10 mg of HRP (topically): for 45 min (3 applications of 15 min) | 1 h | Cell viability: MTT; cell morphology: SEM; cytotoxicity: live/dead assay | HRP associated with HP increased cell viability compared to 35% HP alone, in addition to showing less impact on morphology. | |
| Soares et al. 2019 [23] | 5.6 ± 3.5 mm enamel/dentin discs, and MDPC-23 cells | Untreated, HP: 35% HP, 35% HP + FS, 35% HP + MC, 35% HP + PR, 35% HP + CT | 35% HP for 45 min (3 applications of 15 min) | 40 mL of 35% HP mixed with 1 mg of FS, MC, PR, or CT (topically): for 45 min (3 applications of 15 min) | 1 and 24 h | Cell viability: MTT | All chemically activated groups increased cell viability, mainly in the HP + PR group. | |
| de Oliveira Duque et al. 2014 [45] | 5.6 ± 3.5 mm enamel/dentin discs, HDPCs and MDPC-23 cells | Untreated, 10% CP, 35% HP, 35% HP + FS | 10% CP for 4 h, and 35% HP for 45 min (3 applications of 15 min) | 0.004 g FS mixed with 35% HP (topically): 45 min (3 applications of 15 min) | 1 h | Cell viability: MTT | Chemical activation of HP by FS had no significant protective effect against cytotoxicity, decreasing cell viability compared to CP. | |
| Soares et al. 2013 [34] | 5.6 × 3.5 mm enamel/dentin discs, and MDPC-23 cells | Control: DW, 1 d 16% CP, 7 d 16% CP, 14 d 16% CP, 7 d 16% CP + 0,05% fluoride, 14 d 16% CP + 0.05% fluoride, 7 d 16% CP + 0.2% fluoride, 14 d 16% CP + 0.2% fluoride | 16% CP for 8 h/d | 0.05 % or 0.2% fluoride (topically): for 1 min after 16% CP | 1, 7 and 14 d | Cell viability: MTT; ALP activity: colorimetric endpoint assay; cell membrane damage: flow cytometry | Fluoride solutions cannot prevent the toxic effects of a 16% CP bleaching applied on enamel, in addition to having no impact on ALP activity. | |
| Lima et al. 2010 [47] | 0.5 mm dentin discs and MDPC-23 cells | Control: untreated, 10% sodium ascorbate, 10% CP, 10% sodium ascorbate + 10% CP, 16% CP, 10% sodium ascorbate + 16% CP | 10% and 16% CP for 6 h | Sodium ascorbate 10% (topically): for 6 h, before CP | 6 h | Cell viability: MTT; cell morphology: SEM | 10% sodium ascorbate on the dentin discs before the use of the CP reduced the cytotoxic effects of these products on cells. | |
| In vitro studies using MSCs | ||||||||
| Huang et al. 2019 [56] | HDPCs | NC: negative control, HP (50, 150, 250, 350 μM), HP+NAC: HP (250 μM) + NAC (2.5 mM), HP+CsA: HP (250 μM) + CsA (2 μM), siRNA-CypD: CypD siRNA targeting human PPIF, siRNA-CypD+HP: CypD siRNA targeting human PPIF + HP (250 μM) | 250 μM HP for 24 h | NAC or CsA reagents for 24 h; CypD siRNA-PPIF for 24 h | 1, 3, 6, 12, 24 and 48 h for MTT, 24 h for the other analyses. | Cell viability: MTT; measurement of cell death: FITC and TUNEL assays; intercellular ATP level: ATP detection kit; detection of Ca2+: fluorescence microscope | NAC, CsA and CypD siRNA-PPIF were able to preserve the cell viability, mitigate cell death, decrease the intracellular Ca2+ and enhance the ATP level. | |
| Kim et al. 2017 [54] | HDPCs | Control: untreated, 180 μM HP, HP + 50 μM IAA, HP + 100 μM IAA, HP + 150 μM IAA, HP + 200 μM IAA, HP + 250 μM IAA, HP + 300 μM IAA | 180 μM HP for 24 h | IAA: ranging from 1 to 300 μM | 24 h | Cell viability: MTS | IAA treatment increased cell viability. | |
| Kim et al. 2017 [55] | HDPCs | Control: untreated, HP: 300 µM HP, CA: 20 µM CA, CA + HP: 20 µM CA and then 300 µM HP, CoPP: 20 µM CoPP, CoPP+HP: 20 µM CoPP and then 300 µM HP | 300 µM HP for 24 h | 20 µM CA: for 24 h, before HP | 24 h | Cell viability: MTT; cytotoxicity: LDH activity assay | Pre-treatment with CA effectively prevented HP-induced cell death. | |
| Vargas et al. 2014 [53] | MDPC-23 cells | Negative control: DMEM + 5% DMSO, positive control: 0.018% HP, 1 mM α-T, 3 mM α-T, 5 mM α-T: 10 mM α-T, 1mM α-T + 0.018% HP, 3 mM α-T + 0.018% HP, G9: 5 mM α-T followed by 0.018% HP, G10: 10 mM α-T followed by 0.018% HP | 0.018% HP for 30 min | 1, 3, 5, or 10 mM α-T: for 60 min before HP | 60 min | Cell viability: MTT | Pretreatment with vitamin E α-T isomer increased cell viability of MDPC-23 pulp cells, especially using 5 and 10 mM α-T. | |
| Vargas et al. 2014 [52] | MDPC-23 cells | 1 mM α-T, 1 mM α-T + 0.018% HP, 3 mM α-T, 3 mM α-T + 0.018% HP, 5 mM α-T, 5 mM α-T +, 0.018% HP, 10 mM α-T, 10 mM α-T + 0.018% HP, negative control: DMEM + 5% DMSO, positive control: 0.018% HP | 0.018 % HP for 30 min | 1, 3, 5, or 10 mM α-T: for 1, 4, 8 and 24 h before HP | 1, 4, 8 and 24 h | Cell viability: MTT | Vitamin E alpha-tocopherol isomer showed a protective effect against HP cytotoxicity, especially using 1 and 3 mM α-T for 24 h. | |
| Jeong et al. 2010 [46] | HDPCs | 1 mM HP, HP + 5 μM sappanchalcone, HP + 10 μM sappanchalcone, HP + 20 μM sappanchalcone, HP + 40 μM sappanchalcone, HP + 40 μM sappanchalcone + 100 μM SnPP, 100 μM SnPP, HP + 20 μM CoPP, positive control: 20 μM | 1 mM HP for 12 h | 5–40 μM sappanchalcone: 12 h | 12 h | Cell viability: MTT | Copp and 20 and 40 μM sappanchalcone reduced HP-induced cytotoxicity. | |
| Lee et al. 2013 [24] | HDPCs | Control: untreated, 1mM HP, 1mM HP + 2.5 μM butein, 1mM HP + 5 μM butein, 1mM HP + 10 μM butein, 1mM HP + 20 μM butein, 1mM HP + 20 μM CoPP, 1mM HP+ 100 μM SnPP | 1 mM HP for 12 h | Butein, CoPP and SnPP: for 8 h or until 24 h | 8 h | Cell viability: MTT | Butein inhibited HP-induced cytotoxicity. | |
| Lee et al. 2013 [49] | HDPCs | HP+Ad/PPARγ: HP+Ad PPARγ, HP+Ad/LacZ: control, HP | 150 μmol HP for 12 d | Ad/PPARγ virus: a dose of 100 MOI for 24 h | 12 d | Cell viability: MTT; dentin mineralization: alizarin red stain and ALP activity assay. | PPARγ in pulp cells increased cell viability, odontoblastic differentiation and dentin mineralization. | |
| Lee et al. 2013 [50] | HDPCs | Control: untreated, HP: 500 µM HP, 500 µM HP + 5 µM sulfuretin, 500 µM HP + 10 µM sulfuretin, 500 µM HP + 20 µM sulfuretin, 500 µM HP + 40 µM sulfuretin, sulfuretin: 5-40 µM sulfuretin, HP+CoPP: 500 µM HP + 20 µM CoPP, positive control: 20 µM CoPP | 500 µM HP for 12 h | 5-40 µM sulfuretin: for 12 h, before HP | 12 h | Cell viability: MTT | Pre-treatment with sulfuretin increased cell viability, presumably through HO-1 expression. | |
| Lee et al. 2013 [51] | HDPCs | HP: 150 µM HP, 150 µM HP + 15 µM pachymic acid, 150 µM HP + 5 µM pachymic acid, untreated cells | 150 µM HP for 1, 3, 5, 7 and 12 d | 15 µM pachymic acid: before 1 h prior to incubation with HP | 1, 3, 5, 7, and 12 d | Cell viability: MTT; odontoblast differentiation level: ALP activity and alizarin red S staining | Pachymic acid increased cell viability and mineralization. | |
| Choi et al. 2012 [48] | hDPSCs | HP: 200 µM HP, 200 µM HP + 2 µM SOD1, 200 µM HP + 2 µM LMWP-SOD1, 2 µM SOD1, 2 µM LMWP-SOD1, untreated | 200 µM HP for 2 h | 2 µM LMWP-SOD1: for 3 h, before HP | 3 and 28 d | Cell viability: MTT; matrix mineralization: alizarin red S staining; expression of odontogenic markers: PCR | LMWP-SOD1 did not influence cell viability and the calcified area. However, it reverses HP inhibition of osteogenic markers. | |
α-T, alpha-tocopherol; Ad, adenovirus; ALP, alkaline phosphatase; ATP, adenosine triphosphate; CA, cinnamaldehyde; CG, control group; CoPP, cobalt protoporphyrin; CP, carbamide peroxide; CsA, cyclosporine A; CT, catalase; DMEM, Dulbecco’s Modified Eagle’s Medium; DMSO, dimethyl sulfoxide; DW, deionized water; FITC, fluorescein isothiocyanate; FS, ferrous sulfate; G, group; HDPCs, human dental pulp cells; hDPSCs, human dental pulp stem cells; HO-1, heme oxygenase 1; HP, hydrogen peroxide; HRP, horseradish peroxidase; IAA, indole-3-acetic acid; LDH, lactate dehydrogenase; LMWP, low-molecular weight protamine; MC, manganese chloride; MDPC-23, mouse dental papilla Cell-23; MnO2, manganese dioxide; MOI, multiplicity of infection; MSCs, mesenchymal stem cells; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAC, N-acetylcysteine; NC, negative control; PCP, polymeric catalyst primer; PCR, polymerase chain reaction; PPARγ, proliferator-activated receptor gamma; PPIF, peptidylprolyl isomerase F; PR, peroxidase; SEM, scanning electron microscopy; siRNA-CypD, cyclophilin D small interfering ribonucleic acid; SnPP, tin protoporphyrin; SOD1, superoxide dismutase 1; TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling.