- Email: [email protected]
Effect of dental adhesives on the cell cycle of fibroblasts
e7
d e n t a l m a t e r i a l s 2 5 ( 2 0 0 9 ) e5–e46
Proprietary products
24 h
48 h
72 h
1. Rockle’s 4 2. Rockle’s caustic 3. Rockle’s essential 4. Tempophore 5. Calcium hydroxide Control
11.40 10.70 12.40 8.55 8.06 1.55
12.37 11.32 13.65 9.68 8.13 2.60
14.60 12.47 17.58 10.24 9.90 3.42
96 h 15.50 13.05 18.45 12.34 11.67 4.56
Cell mortality (%) induced in the primary culture of gingival fibroblasts Proprietary products
24 h
48 h
72 h
1. Rockle’s 4 2. Rockle’s caustic 3. Rockle’s essential 4. Tempophore 5. Calcium hydroxide Control
8.01 6.25 8.40 7.15 7.84 2.23
9.75 7.89 9.62 8.55 8.33 2.80
12.82 8.65 12.39 11.20 10.69 3.71
96 h 14.40 10.56 13.85 13.27 13.43 4.44
Conclusions: Rockle’s 4 and Rockle’s essential, which contain formaldehyde, had the highest toxicity in both cell cultures. Tempophore and Calcium hydroxide were more cytotoxic in primary culture of gingival fibroblasts than in ICP-23 cell line. These last products had also a higher cytotoxicity in primary culture of gingival fibroblasts even than Rockle’s caustic which contains phenol but no formaldehyde. Further studies have to elucidate which type of cell line culture is more relevant. doi:10.1016/j.dental.2009.01.011 A non-destructive method to determine critical flaw size of dental veneering ceramics K. Tsuchiya 1,∗ , B. Taskonak 1 , M. Ito 2 1 2
Indiana University, Indianapolis, IN, USA Matsumoto Dental College, Nagano, Japan
Objectives: Dental ceramic prostheses lack mechanical reliability because of their brittle nature. Their failure stress values can be determined with destructive testing, but there is no established non-destructive method. However, microcomputed tomography (CT) can detect and measure the size of the critical flaw. Then stress at failure can be computed using a fracture mechanics equation, f =
Kc √ Y c
where f (MPa) is the critical stress for crack propagation, Kc (MPa m1/2 ) is the fracture toughness, Y is the geometrical factor, and c (m) is the critical flaw size. The objective of this study was to test the hypothesis that there is no statistically significant difference between the flaw sizes determined using CT vs. established fractographic analysis. Materials and methods: Ten ceramic bar specimens (1.2 mm × 4.0 mm × 25 mm) were fabricated via slurry preparation and sintering of dental ceramic powders (Lava Ceram Veneer ceramic; 3 M ESPE, St. Paul, MN). After polishing, prior to four-point bending test, the specimens were scanned and
analyzed using a CT instrument (Skyscan® 1072; Skyscan, Aartselaar, Belgium). The sizes and locations of porosities within the area corresponding to the tensile surface of specimens in four-point bending were determined. Specimens were annealed to relieve residual stresses and they were subjected to a four-point bending test using an inner span size of 10 mm and an outer span size of 20 mm (ASTM C1161). Fracture origins were identified using fractography and the size of the fracture initiating porosity was measured. The sizes of the flaws, measured using the CT scanner and fractography, were summarized and comparisons between the two methods of flaw size measurement were made using paired t-tests, plots, Pearson correlation coefficients, and intraclass correlation coefficients. Results: The critical flaw sizes (m) of the specimens, measured using the CT scanner and fractography, are summarized in the table below. There was not a statistically significant difference (p > 0.05) between the fractography and CT measurements of the critical flaw sizes.AxisMethodMeanS.D.MinMaxp-ValueFlaw size (m)Fractography66.79.65877CT67.76.56174Fractography–CT− 1.03.5−330.6667 Conclusions: Microcomputed tomography (CT) can be used to evaluate the flaw distribution and therefore, the strength, of ceramic fixed partial dentures. doi:10.1016/j.dental.2009.01.012 Effect of dental adhesives on the cell cycle of fibroblasts E.A. Koulaouzidou 1,∗ , K.T. Helvatjoglu-Antoniades 1 1 2
Papazisis 2 , G.
Palaghias 1 , M.
Aristotle University, Thessaloniki, Greece Theagenion Cancer Institute, Thessaloniki, Greece
Objectives: Previous studies have shown that dental adhesives contain monomers that are capable of diffusion through dentin and interaction with pulp. The aim of this study was to assess the effect of newly marked dentin bonding agents on the cell proliferation and on the cell cycle of normal fibroblasts. Materials and methods: The tested adhesives were: Adhese (Vivadent), Clearfil tri-S bond (Kuraray), G-Bond (GC Corporation) and XP bond (Dentsply). The cell lines used for the experiments were: MRC5 (human lung fibroblasts), RPCC2A (rat pulp cells) and L929 (mouse skin fibroblasts). Cells were grown as monolayer cultures at 37 ◦ C in an atmosphere containing 5% CO2 and 100% relative humidity. The culture medium was Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. The adhesives were prepared in a volume of 200 l, were light cured with an Astralis unit (Vivadent). Thereafter, culture medium was added and left at 37 ◦ C for 24 h. The extract was sterile filtered and used as a starting material to prepare serial dilutions in culture medium. Negative controls with DMEM were used. Antiproliferative effect was evaluated by the sulphorhodamine B (SRB) assay, effect on DNA synthesis by the bromodeoxyuridine (BrdU) assay and effect on the cell cycle by flow cytometry.
e8
d e n t a l m a t e r i a l s 2 5 ( 2 0 0 9 ) e5–e46
Table 1 – Mean (S.D.) values of color change (E). Superscript letters denote statistical significances (different letters), and non-significant differences (symmetrical letters). Group
Time (months) 1
Time passage [control] Sebum solution aging Artificial day-light aging
2.34(1.12) 3.74(1.25) 5.85(2.14)
2 2.65(1.42) 3.50(2.00) 7.65(1.54)
Results: All tested materials exhibited a dose- and timedependent cytotoxicity. Higher doses and longer exposure times resulted in reduced cell survival, as was shown by the SRB method. XP bond expressed the higher cytotoxic activity of all tested substances (p < 0.05). The other three agents exhibited a similar antiproliferative pattern, with G-Bond however being more cytotoxic than Adhese or Clearfil tri-S bond. XP bond exposure resulted in an inhibition of DNA synthesis in the three cell lines whilst Adhese, G-Bond and Clearfil tri-S bond inhibited DNA synthesis to a far lesser extend. From all the tested bonding agents XP bond resulted in the most potent effect on cell cycle distribution. A dilution down to 2.5% (0.025) was high enough to decrease cell numbers by 50%, leaving the remaining cells in G2 /M phase arrest. Cells exposed to XP bond exhibited very early apoptotic morphology when observed under the inverted microscope. The other three bonding agents (G-Bond, Clearfil tri-S bond and Adhese) did not have any major impact on cell cycle distribution, and their modest anti-proliferative activity can be explained by induction of cell death (apoptosis or necrosis) in high concentrations. Conclusions: The effect of dental adhesives on cell cycle may have clinical significance. Acknowledgement: The study was supported by Procter and Gable Ltd. (AUTh Research Committee project #81990).
3 3.18(1.38) 3.19(2.23) 10.18(1.43)
4 3.10(1.23) 3.09(1.43) 10.31(1.70)
5 3.16(1.75) 3.10(1.98) 11.20(2.39)
6 3.2a (1.55) 3.10a (1.60) 11.43b (1.82)
ated artificial day-light aging using an environmental chamber for 180 h. Aging cycle included alternating wet aging (18 min) and dry aging (102 min). Color measurements were recorded every 30 h of aging. Color measurements were performed with a colorimeter according to the CIE-Lab (L*, a*, b*) system. Color change (E) was calculated using the following equation: E = [(L*)2 + (a*)2 + (b*)2 ]1/2 , where L*, a* and b* are the differences in the respective values before and after aging. Results: One-way ANOVA and Bonferroni tests on data obtained after 6 months showed statistical significance between all groups (p < 0.05) except between time passage and sebum groups (p > 0.05) (Table 1). Artificial day-light aging exhibited the greatest color change (E) after 6 months. However, among the individual color parameters, there was statistically significant decrease in a* and b* values between all groups. Conclusions: Maxillofacial silicone elastomer exhibit color changes as a result of surrounding environmental factors and human sebaceous secretions. doi:10.1016/j.dental.2009.01.014 Release of monomers from different core build-up materials O. Polydorou 1,∗ , A. König 2 , K. Kümmerer 2 , E. Hellwig 1 1
doi:10.1016/j.dental.2009.01.013 Color changes of maxillofacial silicone elastomer due to extra-oral aging M.M. Hatamleh ∗ , D.C. Watts School of Dentistry, The University of Manchester, Manchester, UK Objectives: To investigate the effect of artificial day-light aging (with and without moisture) and sebaceous oil secretions (Sebum) on color stability of maxillofacial silicone elastomer within a 6-month simulated period of clinical service. Materials and methods: Twenty-four disk-shaped specimens were prepared (8 mm diameter, 3 mm thickness) using Marfa silicone elastomer and were colored with intrinsic skin pigments (skin tone). Specimens were randomly allocated into three groups (n = 8) as follows: Group 1 (time passage) specimens were stored in a dark chamber away from light (control group), and color measurements were recorded every 30 days. Group 2: specimens were immersed in sebum solution (10% palmitic acid, 2% tri-palmitin dissolved in 88% linoleic acid) for 3 months. Color of specimens was recorded every 15 days of conditioning. Group 3: specimens were exposed to acceler-
Dental School and Hospital, University Medical Center, Freiburg, Germany 2 University Medical Center, Freiburg, Germany Objectives: The aim of the present study was to evaluate and compare the elution of monomers from three different core build-up composite materials. Materials and methods: Three different core build-up composite materials (a chemically cured, a photo-cured, and a dual-cured) were tested. Ten samples (diameter: 4.5 mm, thickness: 2 mm) of each material were fabricated. The photocured samples were polymerized for 40 s and the dual-cured samples for 20 s. The samples remained for 10 minutes undisturbed and then were stored in 1 ml of 75% ethanol at room temperature. The storage medium was renewed after 24 h, 7 days, and 28 days. From the storage medium that was removed, samples were prepared and analyzed by LC–MS/MS. Results: The three composite materials differed significantly concerning the elution of monomers (BisGMA: p < 0.0001; TEGDMA: p < 0.0001; and Bisphenol A: p < 0.0001). A significantly higher amount of BisGMA and TEGDMA was released from the chemically cured composite compared to the other two materials. The dual-cured material eluted significantly higher amounts of BisGMA and TEGDMA compared to the light-cured composite, whereas the photo-cured mate-
d e n t a l m a t e r i a l s 2 5 ( 2 0 0 9 ) e5–e46
Proprietary products
24 h
48 h
72 h
1. Rockle’s 4 2. Rockle’s caustic 3. Rockle’s essential 4. Tempophore 5. Calcium hydroxide Control
11.40 10.70 12.40 8.55 8.06 1.55
12.37 11.32 13.65 9.68 8.13 2.60
14.60 12.47 17.58 10.24 9.90 3.42
96 h 15.50 13.05 18.45 12.34 11.67 4.56
Cell mortality (%) induced in the primary culture of gingival fibroblasts Proprietary products
24 h
48 h
72 h
1. Rockle’s 4 2. Rockle’s caustic 3. Rockle’s essential 4. Tempophore 5. Calcium hydroxide Control
8.01 6.25 8.40 7.15 7.84 2.23
9.75 7.89 9.62 8.55 8.33 2.80
12.82 8.65 12.39 11.20 10.69 3.71
96 h 14.40 10.56 13.85 13.27 13.43 4.44
Conclusions: Rockle’s 4 and Rockle’s essential, which contain formaldehyde, had the highest toxicity in both cell cultures. Tempophore and Calcium hydroxide were more cytotoxic in primary culture of gingival fibroblasts than in ICP-23 cell line. These last products had also a higher cytotoxicity in primary culture of gingival fibroblasts even than Rockle’s caustic which contains phenol but no formaldehyde. Further studies have to elucidate which type of cell line culture is more relevant. doi:10.1016/j.dental.2009.01.011 A non-destructive method to determine critical flaw size of dental veneering ceramics K. Tsuchiya 1,∗ , B. Taskonak 1 , M. Ito 2 1 2
Indiana University, Indianapolis, IN, USA Matsumoto Dental College, Nagano, Japan
Objectives: Dental ceramic prostheses lack mechanical reliability because of their brittle nature. Their failure stress values can be determined with destructive testing, but there is no established non-destructive method. However, microcomputed tomography (CT) can detect and measure the size of the critical flaw. Then stress at failure can be computed using a fracture mechanics equation, f =
Kc √ Y c
where f (MPa) is the critical stress for crack propagation, Kc (MPa m1/2 ) is the fracture toughness, Y is the geometrical factor, and c (m) is the critical flaw size. The objective of this study was to test the hypothesis that there is no statistically significant difference between the flaw sizes determined using CT vs. established fractographic analysis. Materials and methods: Ten ceramic bar specimens (1.2 mm × 4.0 mm × 25 mm) were fabricated via slurry preparation and sintering of dental ceramic powders (Lava Ceram Veneer ceramic; 3 M ESPE, St. Paul, MN). After polishing, prior to four-point bending test, the specimens were scanned and
analyzed using a CT instrument (Skyscan® 1072; Skyscan, Aartselaar, Belgium). The sizes and locations of porosities within the area corresponding to the tensile surface of specimens in four-point bending were determined. Specimens were annealed to relieve residual stresses and they were subjected to a four-point bending test using an inner span size of 10 mm and an outer span size of 20 mm (ASTM C1161). Fracture origins were identified using fractography and the size of the fracture initiating porosity was measured. The sizes of the flaws, measured using the CT scanner and fractography, were summarized and comparisons between the two methods of flaw size measurement were made using paired t-tests, plots, Pearson correlation coefficients, and intraclass correlation coefficients. Results: The critical flaw sizes (m) of the specimens, measured using the CT scanner and fractography, are summarized in the table below. There was not a statistically significant difference (p > 0.05) between the fractography and CT measurements of the critical flaw sizes.AxisMethodMeanS.D.MinMaxp-ValueFlaw size (m)Fractography66.79.65877CT67.76.56174Fractography–CT− 1.03.5−330.6667 Conclusions: Microcomputed tomography (CT) can be used to evaluate the flaw distribution and therefore, the strength, of ceramic fixed partial dentures. doi:10.1016/j.dental.2009.01.012 Effect of dental adhesives on the cell cycle of fibroblasts E.A. Koulaouzidou 1,∗ , K.T. Helvatjoglu-Antoniades 1 1 2
Papazisis 2 , G.
Palaghias 1 , M.
Aristotle University, Thessaloniki, Greece Theagenion Cancer Institute, Thessaloniki, Greece
Objectives: Previous studies have shown that dental adhesives contain monomers that are capable of diffusion through dentin and interaction with pulp. The aim of this study was to assess the effect of newly marked dentin bonding agents on the cell proliferation and on the cell cycle of normal fibroblasts. Materials and methods: The tested adhesives were: Adhese (Vivadent), Clearfil tri-S bond (Kuraray), G-Bond (GC Corporation) and XP bond (Dentsply). The cell lines used for the experiments were: MRC5 (human lung fibroblasts), RPCC2A (rat pulp cells) and L929 (mouse skin fibroblasts). Cells were grown as monolayer cultures at 37 ◦ C in an atmosphere containing 5% CO2 and 100% relative humidity. The culture medium was Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. The adhesives were prepared in a volume of 200 l, were light cured with an Astralis unit (Vivadent). Thereafter, culture medium was added and left at 37 ◦ C for 24 h. The extract was sterile filtered and used as a starting material to prepare serial dilutions in culture medium. Negative controls with DMEM were used. Antiproliferative effect was evaluated by the sulphorhodamine B (SRB) assay, effect on DNA synthesis by the bromodeoxyuridine (BrdU) assay and effect on the cell cycle by flow cytometry.
e8
d e n t a l m a t e r i a l s 2 5 ( 2 0 0 9 ) e5–e46
Table 1 – Mean (S.D.) values of color change (E). Superscript letters denote statistical significances (different letters), and non-significant differences (symmetrical letters). Group
Time (months) 1
Time passage [control] Sebum solution aging Artificial day-light aging
2.34(1.12) 3.74(1.25) 5.85(2.14)
2 2.65(1.42) 3.50(2.00) 7.65(1.54)
Results: All tested materials exhibited a dose- and timedependent cytotoxicity. Higher doses and longer exposure times resulted in reduced cell survival, as was shown by the SRB method. XP bond expressed the higher cytotoxic activity of all tested substances (p < 0.05). The other three agents exhibited a similar antiproliferative pattern, with G-Bond however being more cytotoxic than Adhese or Clearfil tri-S bond. XP bond exposure resulted in an inhibition of DNA synthesis in the three cell lines whilst Adhese, G-Bond and Clearfil tri-S bond inhibited DNA synthesis to a far lesser extend. From all the tested bonding agents XP bond resulted in the most potent effect on cell cycle distribution. A dilution down to 2.5% (0.025) was high enough to decrease cell numbers by 50%, leaving the remaining cells in G2 /M phase arrest. Cells exposed to XP bond exhibited very early apoptotic morphology when observed under the inverted microscope. The other three bonding agents (G-Bond, Clearfil tri-S bond and Adhese) did not have any major impact on cell cycle distribution, and their modest anti-proliferative activity can be explained by induction of cell death (apoptosis or necrosis) in high concentrations. Conclusions: The effect of dental adhesives on cell cycle may have clinical significance. Acknowledgement: The study was supported by Procter and Gable Ltd. (AUTh Research Committee project #81990).
3 3.18(1.38) 3.19(2.23) 10.18(1.43)
4 3.10(1.23) 3.09(1.43) 10.31(1.70)
5 3.16(1.75) 3.10(1.98) 11.20(2.39)
6 3.2a (1.55) 3.10a (1.60) 11.43b (1.82)
ated artificial day-light aging using an environmental chamber for 180 h. Aging cycle included alternating wet aging (18 min) and dry aging (102 min). Color measurements were recorded every 30 h of aging. Color measurements were performed with a colorimeter according to the CIE-Lab (L*, a*, b*) system. Color change (E) was calculated using the following equation: E = [(L*)2 + (a*)2 + (b*)2 ]1/2 , where L*, a* and b* are the differences in the respective values before and after aging. Results: One-way ANOVA and Bonferroni tests on data obtained after 6 months showed statistical significance between all groups (p < 0.05) except between time passage and sebum groups (p > 0.05) (Table 1). Artificial day-light aging exhibited the greatest color change (E) after 6 months. However, among the individual color parameters, there was statistically significant decrease in a* and b* values between all groups. Conclusions: Maxillofacial silicone elastomer exhibit color changes as a result of surrounding environmental factors and human sebaceous secretions. doi:10.1016/j.dental.2009.01.014 Release of monomers from different core build-up materials O. Polydorou 1,∗ , A. König 2 , K. Kümmerer 2 , E. Hellwig 1 1
doi:10.1016/j.dental.2009.01.013 Color changes of maxillofacial silicone elastomer due to extra-oral aging M.M. Hatamleh ∗ , D.C. Watts School of Dentistry, The University of Manchester, Manchester, UK Objectives: To investigate the effect of artificial day-light aging (with and without moisture) and sebaceous oil secretions (Sebum) on color stability of maxillofacial silicone elastomer within a 6-month simulated period of clinical service. Materials and methods: Twenty-four disk-shaped specimens were prepared (8 mm diameter, 3 mm thickness) using Marfa silicone elastomer and were colored with intrinsic skin pigments (skin tone). Specimens were randomly allocated into three groups (n = 8) as follows: Group 1 (time passage) specimens were stored in a dark chamber away from light (control group), and color measurements were recorded every 30 days. Group 2: specimens were immersed in sebum solution (10% palmitic acid, 2% tri-palmitin dissolved in 88% linoleic acid) for 3 months. Color of specimens was recorded every 15 days of conditioning. Group 3: specimens were exposed to acceler-
Dental School and Hospital, University Medical Center, Freiburg, Germany 2 University Medical Center, Freiburg, Germany Objectives: The aim of the present study was to evaluate and compare the elution of monomers from three different core build-up composite materials. Materials and methods: Three different core build-up composite materials (a chemically cured, a photo-cured, and a dual-cured) were tested. Ten samples (diameter: 4.5 mm, thickness: 2 mm) of each material were fabricated. The photocured samples were polymerized for 40 s and the dual-cured samples for 20 s. The samples remained for 10 minutes undisturbed and then were stored in 1 ml of 75% ethanol at room temperature. The storage medium was renewed after 24 h, 7 days, and 28 days. From the storage medium that was removed, samples were prepared and analyzed by LC–MS/MS. Results: The three composite materials differed significantly concerning the elution of monomers (BisGMA: p < 0.0001; TEGDMA: p < 0.0001; and Bisphenol A: p < 0.0001). A significantly higher amount of BisGMA and TEGDMA was released from the chemically cured composite compared to the other two materials. The dual-cured material eluted significantly higher amounts of BisGMA and TEGDMA compared to the light-cured composite, whereas the photo-cured mate-