Abstract
Objectives
To develop an enhanced, reproducible and discriminatory biocompatibility testing model for non-precious dental casting alloys, prepared to a clinically relevant surface finishing condition, using TR146 oral keratinocyte cells.
Methods
Comparative biocompatibility was determined following direct and indirect exposure of TR146 cells to two nickel–chromium (Ni–Cr) and a cobalt–chromium (Co–Cr) alloy-discs. The surface roughness of the discs was determined using a contact stylus profilometer and the elemental ion release by inductively coupled plasma mass spectrometry (ICP-MS). Subsequent biocompatibility analysis included cell morphology, cell density measurements with Trypan blue exclusion assay, inflammatory cytokine expression with ELISAs, cellular metabolic activity using XTT and cellular toxicity using lactate dehydrogenase (LDH) release assay.
Results
TR146 cell morphology was altered following direct and indirect exposure to the Ni–Cr alloys but not the Co–Cr alloy. Significant reductions (all P < 0.001) in viable cell density measurements, cellular metabolic activity, significant increases inflammatory cytokine expression and cellular toxicity were observed when TR146 cells were exposed to the Ni–Cr alloys. Significant decreases in cell density measurements, cellular metabolic activity, significant increases inflammatory cytokine expression and cellular toxicity for the Ni–Cr d.Sign ® 15 alloy compared with d.Sign ® 10 alloy were identifiable (all P < 0.001). Cellular toxicity was attributed to nickel ion release levels in solution detected by ICP-MS analysis.
Discussion
Nickel ions from the Ni–Cr alloys permeated the epithelial cells and activated a proinflammatory response, namely IL-1a, IL-8 and PGE2 expression. Further evidence of nickel ioninduced cell death was supported by the decreased biocompatibility of the highest nickel ion releasing alloy (d.Sign ® 15 compared with d.Sign ® 10) and the increased biocompatibility of the Co–Cr (d.Sign ® 30) alloy where nickel ions were absent.
1
Introduction
Since the first commercially successful dental gold alloy was patented in 1962 , attempts have been made to develop technically superior alternatives. As the price of gold rose from $35 an ounce in 1968, researchers were forced to develop an alternative cost-effective alloy, and the nickel–chrome–beryllium (Ni–Cr–Be) system was introduced in 1974 . The Rand Survey in 1981 highlighted the majority of metal–ceramic restorations employed in the USA were fabricated from dental casting alloys with no gold content and today, Ni-based dental casting alloys account for approximately 80% of fixed prosthodontic restorations . Nickel allergic reactions have been associated with nickel-containing dental appliances which can remain in situ adjacent to the oral mucosa for substantial periods of time. Schmalz and Garhammer suggested that the comparatively high incidence of nickel allergy should provide an impetus to replace Ni-based alloys when a suitable alloy alternative was available.
The majority of comparative biocompatibility studies reported in the dental literature on Ni–Cr alloys have focused on the reaction of connective tissue to simple metal salts (nickel chloride, chromium chloride and molybdenum oxide) . However, mass spectrometry studies performed on Ni–Cr alloys identified the metal ion release profiles were not representative of the bulk alloy composition emphasizing the biocompatibility of Ni–Cr alloys cannot be accurately replicated by simple metal salts . In addition to tissue type (epithelial ) or connective tissue ), the analysis methodology employed for biocompatibility determination of Ni–Cr alloys is variously reported as cell morphology , cell density measurements , inflammatory cytokine expression , cellular metabolic activity or cellular toxicity analyses . However, oral keratinocytes are the primary tissue target of nickel and are associated with the inflammatory response of the oral mucosa during nickel sensitivity in vivo . Gazel et al. suggested keratinocytes were the initiator cells for nickel allergic contact sensitivity reactions which further adds to the argument that oral keratinocytes are the most appropriate tissue type for comparative biocompatibility analysis. Additionally, inflammatory cytokines have been reported to be inducible from keratinocytes in response to nickel ions.
The aim of the study was to develop an enhanced, reproducible and discriminatory biocompatibility testing model for non-precious dental casting alloys, prepared to a clinically relevant surface finishing condition , on a TR146 oral keratinocyte cell line. The biocompatibility testing model involved cell morphology, cell density measurements, inflammatory cytokine expression, cellular metabolic activity and cellular toxicity analyses following direct and indirect exposure to two Ni–Cr and a cobalt–chromium (Co–Cr) alloy.
2
Materials and methods
2.1
Materials
Two Ni–Cr (d.Sign ® 10 and d.Sign ® 15, Ivoclar-Vivadent, Schaan, Liechtenstein) and a Co–Cr (d.Sign ® 30, Ivoclar-Vivadent) dental casting alloy ( Table 1 ) were employed. Alloy discs (15 mm diameter and 1.0 mm thickness and n = 20 for each alloy tested) were cast in a carbon-free phosphate-bonded casting investment and divested to remove the residual investment material in accordance with the manufacturer’s recommendations . The discs were polished to achieve a surface finishing condition equivalent to that employed clinically using polishing discs in accordance with the polishing regime outlined previously . The discs were washed using sterilized Milli-Q ® Biocel-purified water (Millipore™, Cork, Ireland), air-dried at room temperature prior to being placed aseptically into Defend ® Self-Sealing Sterilization Pouches (Carl Parker Associates, Mydent Corporation, New York, USA) and sterilized at 115 °C for 15 min in an LTE Touchclave-LAB autoclave (LTE Scientific Ltd., Oldham, UK).
| Ni | Cr | Mo | Co | Al | Nb | Si | Fe | Ga | |
|---|---|---|---|---|---|---|---|---|---|
| d.Sign ® 10 | 75.4 | 12.6 | 8.0 | – | 3.3 | – | <1.0 | – | – |
| d.Sign ® 15 | 58.7 | 25.0 | 12.1 | <0.1 | – | – | 1.7 | 1.9 | – |
| d.Sign ® 30 | – | 30.1 | <1.0 | 60.2 | <1.0 | 3.2 | <1.0 | <1.0 | 3.98 |
2.2
Profilometry
The Ra value (surface roughness) of three discs for each alloy investigated was determined using a contact stylus profilometer (Talysurf CLI 2000 Taylor-Hobson Precision, Leicester, UK) employing a 90° conisphere stylus tip (2 μm radius) across a 100 mm 2 area. Data points were recorded every 5 μm in both the x – and y -directions, at a scanning velocity of 1 mm/s under an applied load of 0.75 mN, with an 8.6 nm resolution in the z -direction. The Ra value was determined by employing a 0.25 mm cut-off Gaussian roughness filter to the data from the 2001 traces ( x -direction) and 2001 traces ( y -direction) in accordance with ISO 4287 .
2.3
Cell culture and biocompatibility testing
The TR146 oral keratinocyte immortal squamous cell carcinoma line was used and the chemicals and antibiotics used throughout were of cell culture-grade (Sigma–Aldrich Ltd., Dublin, Ireland). The TR146 cells were maintained in Complete Medium (CM) [Dulbeccos’ Modified Eagle’s medium (DMEM, pH 7.0) supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 units/mL) and streptomycin (10 mg/mL)] under normal incubation conditions (95% relative humidity in a 5% carbon dioxide (CO 2 ) atmosphere at 37 °C). For cellular metabolic activity and cellular toxicity assays, CM was substituted with a bovine serum-free medium (SFM) [DMEM (4500 mg/L of glucose and sodium bicarbonate without phenol red) supplemented with 4 mM/L l -glutamine, penicillin (10,000 units/mL) and streptomycin (10 mg/mL)]. Filter sterilized (0.22 μm) Dulbecco’s Phosphate Buffer Saline (DPBS) was also used at pH 7.4.
2.4
Assay format
Confluent TR146 cells grown in CM were trypsinized using sterile-filtered 0.25% (w/v) trypsin–edetate disodium (trypsin–EDTA) for 10 min at 37 °C. Detached cells were spun at 250 × g for 10 min in an Eppendorf Centrifuge 5804 (Davidson & Hardy Ltd., Belfast, UK). Then, 2 × 10 3 cells were re-suspended in 2 mL CM or SFM and incubated for 24 h under normal conditions. Sterilized Ni–Cr and Co–Cr alloy-discs were placed directly onto confluent TR146 cells and incubated for up to 72 h for biocompatibility analysis. In addition, confluent TR146 cells were indirectly exposed to the Ni–Cr and Co–Cr alloy-disc immersion solutions following immersion of the sterilized discs into 50 mL of SFM. Cells were then exposed to aliquots of the alloy-disc immersion solutions removed after 1, 5, 9 and 14 days (immersion duration) for biocompatibility analysis.
2.5
ICP-MS analysis
Aseptic discs of each alloy investigated (d.Sign ® 10, d.Sign ® 15 and d.Sign ® 30) were incubated in 50 mL of SFM at room temperature for 1, 7 and 14 days, prior to ICP-MS analysis using an Agilent 7500a Series ® ICP-MS (Agilent Technologies, Dublin, Ireland). To enable quantification a sample dilution in deionized water of 1:10 (v/v) and a pH adjustment to 2.0 was required.
2.6
Analysis of cell morphology
To assess cellular distortion or loss of attachment, the TR146 cell morphology following direct exposure (to the discs) or indirect exposure (to the 1, 5, 9 and 14 day immersion solutions) was recorded at 48 h using a Nikon™ Eclipse TS100 inverted microscope (Micron Optical Co. Ltd., Dublin, Ireland) with a Nikon™ COOLPIX CP990 digital camera. The experiment was performed in triplicate on at least three separate occasions.
2.7
Assessment of cell density measurements
TR146 cell density measurements were performed following direct or indirect alloy exposure using a trypsin-free Trypan blue dye exclusion assay. The TR146 cells were treated with Trypan blue dye and counted at 2, 24, 48 and 72 h using the field of vision and then for the entire well. All experiments were performed in triplicate on at least three occasions with the untreated TR146 cells used as the control.
2.8
Assessment of inflammatory cytokine expression
Sterilized alloy-discs were placed directly onto confluent TR146 cells seeded at 2 × 10 3 cells in 2 mL CM and previously incubated for 24 h under normal conditions. Indirect alloy exposure involved the addition of 1 mL aliquots of the 1 day immersion solutions being placed onto the confluent TR146 cells and incubated for 24 h in a 6-well dish. Treated cell suspensions were removed for specific inflammatory cytokine expression analyses (Interleukin-1α (IL-1α), Interleukin-8 (IL-8), Prostaglandin E 2 (PGE 2 ) and Tumor Necrosis Factor-α (TNF-α)) and quantified using the Quantikine ® Immunoassay Systems (RnD Systems Ltd., Abingdon, UK) in accordance with the manufacturers instructions. The ELISAs were performed in triplicate on at least three separate occasions.
2.9
Assessment of cellular metabolic activity and cellular toxicity
TR146 cells seeded at 2 × 10 2 in 0.2 mL SFM in a 96 well cell culture dish (Cellstar ® , Greiner Bio-One, Sarstedt Ltd.) were employed to assess cellular metabolic activity using an XTT reduction assay and cellular toxicity using LDH quantification (CytoTox 96 ® Non-Radioactive Cytotoxicity Assay, Promega, Dublin, Ireland). For the analyses, the TR146 cells were allowed to attach for 24 h (under normal incubation conditions) and 100 μL of the 1, 5, 9 and 14 day alloy immersion solutions were added. At 2, 24, 48 and 72 h (exposure time) the XTT reduction assay and LDH release assays were performed. The untreated TR146 cells served as a control for the XTT reduction assay while a 1% Triton X-100 solution was added to the TR146 cells to act as a control for the LDH release assay. The cellular metabolic and cellular toxicity experiments were performed in triplicate on at least three separate occasions.
2.10
Statistical analysis
Linear regression analyses of the cell density measurements for the untreated control TR146 cells and following direct alloy-disc exposure to the TR146 cells for 2, 24, 48 and 72 h (exposure times) was employed using statistical software (SPSS 12.0.1, SPSS Inc., Chicago, IL, USA). Further regression analyses of the cell density measurements were used following indirect alloy exposure for the 1, 5, 9 and 14 day (immersion durations) and exposure times. All indirect exposure cell density measurements were pooled for each alloy and additional linear regression analyses used to determine the influence of increasing immersion duration. A one-way analysis of variance (ANOVA) was used to compare the pooled mean cell density data for the three alloys investigated ( P < 0.05) and a Student’s t -test comparison on the pooled mean cell density data was employed to highlight significant differences ( P < 0.05) between the alloy exposure methods. Four two-way ANOVAs (alloy × exposure method) were used ( P < 0.05) to determine inflammatory cytokine expression (IL-1α, IL-8, PGE 2 and TNF-α) for the untreated controls and following direct and indirect alloy exposure to the TR146 cells at 24 h. Tukey’s post hoc tests were used ( P < 0.05) to show inflammatory cytokine expression for the untreated controls and individual alloy treated cells with a Student’s t -test ( P < 0.05) used to compare the inflammatory cytokine expression with alloy exposure method. Linear regression analyses of the cellular metabolic activity and cellular toxicity data were used independently for the control and following indirect alloy exposure for the immersion durations and exposure times investigated ( P < 0.05). The indirect exposure cellular metabolic activity and cellular toxicity data were independently pooled for the alloys tested and further linear regression analyses used to establish the influence of increasing immersion duration ( P < 0.05). A one-way ANOVA (alloy) was used for the pooled mean XTT data for the untreated control and alloy treated TR146 cells. A further one-way ANOVA (alloy) was employed for the pooled mean LDH data for the Triton-X treated control and the alloy treated TR146 cells ( P < 0.05).
2
Materials and methods
2.1
Materials
Two Ni–Cr (d.Sign ® 10 and d.Sign ® 15, Ivoclar-Vivadent, Schaan, Liechtenstein) and a Co–Cr (d.Sign ® 30, Ivoclar-Vivadent) dental casting alloy ( Table 1 ) were employed. Alloy discs (15 mm diameter and 1.0 mm thickness and n = 20 for each alloy tested) were cast in a carbon-free phosphate-bonded casting investment and divested to remove the residual investment material in accordance with the manufacturer’s recommendations . The discs were polished to achieve a surface finishing condition equivalent to that employed clinically using polishing discs in accordance with the polishing regime outlined previously . The discs were washed using sterilized Milli-Q ® Biocel-purified water (Millipore™, Cork, Ireland), air-dried at room temperature prior to being placed aseptically into Defend ® Self-Sealing Sterilization Pouches (Carl Parker Associates, Mydent Corporation, New York, USA) and sterilized at 115 °C for 15 min in an LTE Touchclave-LAB autoclave (LTE Scientific Ltd., Oldham, UK).
| Ni | Cr | Mo | Co | Al | Nb | Si | Fe | Ga | |
|---|---|---|---|---|---|---|---|---|---|
| d.Sign ® 10 | 75.4 | 12.6 | 8.0 | – | 3.3 | – | <1.0 | – | – |
| d.Sign ® 15 | 58.7 | 25.0 | 12.1 | <0.1 | – | – | 1.7 | 1.9 | – |
| d.Sign ® 30 | – | 30.1 | <1.0 | 60.2 | <1.0 | 3.2 | <1.0 | <1.0 | 3.98 |
2.2
Profilometry
The Ra value (surface roughness) of three discs for each alloy investigated was determined using a contact stylus profilometer (Talysurf CLI 2000 Taylor-Hobson Precision, Leicester, UK) employing a 90° conisphere stylus tip (2 μm radius) across a 100 mm 2 area. Data points were recorded every 5 μm in both the x – and y -directions, at a scanning velocity of 1 mm/s under an applied load of 0.75 mN, with an 8.6 nm resolution in the z -direction. The Ra value was determined by employing a 0.25 mm cut-off Gaussian roughness filter to the data from the 2001 traces ( x -direction) and 2001 traces ( y -direction) in accordance with ISO 4287 .
2.3
Cell culture and biocompatibility testing
The TR146 oral keratinocyte immortal squamous cell carcinoma line was used and the chemicals and antibiotics used throughout were of cell culture-grade (Sigma–Aldrich Ltd., Dublin, Ireland). The TR146 cells were maintained in Complete Medium (CM) [Dulbeccos’ Modified Eagle’s medium (DMEM, pH 7.0) supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 units/mL) and streptomycin (10 mg/mL)] under normal incubation conditions (95% relative humidity in a 5% carbon dioxide (CO 2 ) atmosphere at 37 °C). For cellular metabolic activity and cellular toxicity assays, CM was substituted with a bovine serum-free medium (SFM) [DMEM (4500 mg/L of glucose and sodium bicarbonate without phenol red) supplemented with 4 mM/L l -glutamine, penicillin (10,000 units/mL) and streptomycin (10 mg/mL)]. Filter sterilized (0.22 μm) Dulbecco’s Phosphate Buffer Saline (DPBS) was also used at pH 7.4.
2.4
Assay format
Confluent TR146 cells grown in CM were trypsinized using sterile-filtered 0.25% (w/v) trypsin–edetate disodium (trypsin–EDTA) for 10 min at 37 °C. Detached cells were spun at 250 × g for 10 min in an Eppendorf Centrifuge 5804 (Davidson & Hardy Ltd., Belfast, UK). Then, 2 × 10 3 cells were re-suspended in 2 mL CM or SFM and incubated for 24 h under normal conditions. Sterilized Ni–Cr and Co–Cr alloy-discs were placed directly onto confluent TR146 cells and incubated for up to 72 h for biocompatibility analysis. In addition, confluent TR146 cells were indirectly exposed to the Ni–Cr and Co–Cr alloy-disc immersion solutions following immersion of the sterilized discs into 50 mL of SFM. Cells were then exposed to aliquots of the alloy-disc immersion solutions removed after 1, 5, 9 and 14 days (immersion duration) for biocompatibility analysis.
2.5
ICP-MS analysis
Aseptic discs of each alloy investigated (d.Sign ® 10, d.Sign ® 15 and d.Sign ® 30) were incubated in 50 mL of SFM at room temperature for 1, 7 and 14 days, prior to ICP-MS analysis using an Agilent 7500a Series ® ICP-MS (Agilent Technologies, Dublin, Ireland). To enable quantification a sample dilution in deionized water of 1:10 (v/v) and a pH adjustment to 2.0 was required.
2.6
Analysis of cell morphology
To assess cellular distortion or loss of attachment, the TR146 cell morphology following direct exposure (to the discs) or indirect exposure (to the 1, 5, 9 and 14 day immersion solutions) was recorded at 48 h using a Nikon™ Eclipse TS100 inverted microscope (Micron Optical Co. Ltd., Dublin, Ireland) with a Nikon™ COOLPIX CP990 digital camera. The experiment was performed in triplicate on at least three separate occasions.
2.7
Assessment of cell density measurements
TR146 cell density measurements were performed following direct or indirect alloy exposure using a trypsin-free Trypan blue dye exclusion assay. The TR146 cells were treated with Trypan blue dye and counted at 2, 24, 48 and 72 h using the field of vision and then for the entire well. All experiments were performed in triplicate on at least three occasions with the untreated TR146 cells used as the control.
2.8
Assessment of inflammatory cytokine expression
Sterilized alloy-discs were placed directly onto confluent TR146 cells seeded at 2 × 10 3 cells in 2 mL CM and previously incubated for 24 h under normal conditions. Indirect alloy exposure involved the addition of 1 mL aliquots of the 1 day immersion solutions being placed onto the confluent TR146 cells and incubated for 24 h in a 6-well dish. Treated cell suspensions were removed for specific inflammatory cytokine expression analyses (Interleukin-1α (IL-1α), Interleukin-8 (IL-8), Prostaglandin E 2 (PGE 2 ) and Tumor Necrosis Factor-α (TNF-α)) and quantified using the Quantikine ® Immunoassay Systems (RnD Systems Ltd., Abingdon, UK) in accordance with the manufacturers instructions. The ELISAs were performed in triplicate on at least three separate occasions.
2.9
Assessment of cellular metabolic activity and cellular toxicity
TR146 cells seeded at 2 × 10 2 in 0.2 mL SFM in a 96 well cell culture dish (Cellstar ® , Greiner Bio-One, Sarstedt Ltd.) were employed to assess cellular metabolic activity using an XTT reduction assay and cellular toxicity using LDH quantification (CytoTox 96 ® Non-Radioactive Cytotoxicity Assay, Promega, Dublin, Ireland). For the analyses, the TR146 cells were allowed to attach for 24 h (under normal incubation conditions) and 100 μL of the 1, 5, 9 and 14 day alloy immersion solutions were added. At 2, 24, 48 and 72 h (exposure time) the XTT reduction assay and LDH release assays were performed. The untreated TR146 cells served as a control for the XTT reduction assay while a 1% Triton X-100 solution was added to the TR146 cells to act as a control for the LDH release assay. The cellular metabolic and cellular toxicity experiments were performed in triplicate on at least three separate occasions.
2.10
Statistical analysis
Linear regression analyses of the cell density measurements for the untreated control TR146 cells and following direct alloy-disc exposure to the TR146 cells for 2, 24, 48 and 72 h (exposure times) was employed using statistical software (SPSS 12.0.1, SPSS Inc., Chicago, IL, USA). Further regression analyses of the cell density measurements were used following indirect alloy exposure for the 1, 5, 9 and 14 day (immersion durations) and exposure times. All indirect exposure cell density measurements were pooled for each alloy and additional linear regression analyses used to determine the influence of increasing immersion duration. A one-way analysis of variance (ANOVA) was used to compare the pooled mean cell density data for the three alloys investigated ( P < 0.05) and a Student’s t -test comparison on the pooled mean cell density data was employed to highlight significant differences ( P < 0.05) between the alloy exposure methods. Four two-way ANOVAs (alloy × exposure method) were used ( P < 0.05) to determine inflammatory cytokine expression (IL-1α, IL-8, PGE 2 and TNF-α) for the untreated controls and following direct and indirect alloy exposure to the TR146 cells at 24 h. Tukey’s post hoc tests were used ( P < 0.05) to show inflammatory cytokine expression for the untreated controls and individual alloy treated cells with a Student’s t -test ( P < 0.05) used to compare the inflammatory cytokine expression with alloy exposure method. Linear regression analyses of the cellular metabolic activity and cellular toxicity data were used independently for the control and following indirect alloy exposure for the immersion durations and exposure times investigated ( P < 0.05). The indirect exposure cellular metabolic activity and cellular toxicity data were independently pooled for the alloys tested and further linear regression analyses used to establish the influence of increasing immersion duration ( P < 0.05). A one-way ANOVA (alloy) was used for the pooled mean XTT data for the untreated control and alloy treated TR146 cells. A further one-way ANOVA (alloy) was employed for the pooled mean LDH data for the Triton-X treated control and the alloy treated TR146 cells ( P < 0.05).
3
Results
3.1
Profilometry
The mean Ra value of the polished d.Sign ® 15 alloy-discs ( Fig. 1 a) was 0.089 ± 0.023 μm with minimum and maximum peak heights of 0.053 and 0.203 μm, respectively. The polished d.Sign ® 30 alloy-discs ( Fig. 1 b) resulted in a mean Ra value of 0.152 ± 0.037 μm with minimum and maximum Ra values of 0.0908 and 0.350 μm, respectively. Previously the authors reported the mean Ra value (and associated standard deviation) of polished d.Sign ® 10 alloy-discs as 0.053 ± 0.023 μm with minimum and maximum peak heights of 0.026 and 0.170 μm when analyzed under the same experimental conditions. The one-way ANOVA of the mean Ra values for alloy type showed significant differences were evident ( P = 0.0080) and the Tukey’s post hoc tests showed significantly higher Ra values for d.Sign ® 30 compared with d.Sign ® 15 ( P = 0.0020) and d.Sign ® 10 ( P = 0.0004) alloy-discs. The Ra value for d.Sign ® 15 was significantly increased ( P = 0.0010) compared with d.Sign ® 10 alloy-discs.
3.2
ICP-MS analysis
The Ni–Cr (d.Sign ® 10 and d.Sign ® 15) alloy-discs released high levels of nickel ions over the 14 day immersion period with maximum levels detected of 361.8 and 417.5 μg/L, respectively ( Table 2 ). The Co–Cr (d.Sign ® 30) alloy-discs, when immersed for up to a maximum of 14 days, released chromium, iron and cobalt at 57.3, 27.2 and 17.3 μg/L, respectively, with no nickel ions evident ( Table 2 ).
| Alloy | Day | Ni | Cr | Mo | Fe | Co | Cu |
|---|---|---|---|---|---|---|---|
| d.Sign ® 10 | 1 | 68.4 | 11.5 | 17.4 | 52.4 | – | 240.7 |
| 7 | 204.5 | 13.7 | 27.6 | 87.5 | – | 245.1 | |
| 14 | 361.8 | 13.4 | 86.7 | 106.5 | – | 240.4 | |
| d.Sign ® 15 | 1 | 140.8 | 14.5 | 25.4 | 67.1 | – | 267.1 |
| 7 | 289.1 | 22.6 | 35.9 | 83.4 | – | 271.6 | |
| 14 | 417.5 | 29.7 | 44.9 | 114.7 | – | 270.9 | |
| d.Sign ® 30 | 1 | – | 37.4 | – | 12.8 | 25.1 | – |
| 7 | – | 44.1 | – | 20.1 | 22.7 | 1.8 | |
| 14 | – | 57.3 | – | 27.2 | 17.4 | 5.7 |
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