Reaction of cultured pulp cells to eight different cements based on glass ionomers J. MOiler G. Bruckner E. Kraft W. H6rz*
Department of Prosthodontics Poliklinik for Prothetik Goethestr. 70 8000 ML~nchen2, West Germany *Department of Physiological Chemistry Institut fL~rPhysiologische Chemie Physikalische Biochemie und Zellbiologie Schillerstr. 22 8000 M0nchen 2, West Germany Received August 22, 1989 Accepted March 2, 1990 This investigation was supported in part by the editorial board of the M#nchener Medizinische Wochenschrift, Munich, West Germany. Dent Mater 6:172-177, July, 1990
Abstract-A comparative study was carried out to determine the cytotoxicity of eight different glass-ionomer cements by means of cell culture. Only fibrobiastlike cells from the pulp of the same subcultures were used. Factors for the evaluation of the biological tolerance were, first, adhesion of cells to the slides as well as to the material, second, relative growth rates of cultures, and, in addition, cytomorphology. The cultured cells reacted quite differently to the glassionomer cements tested. The results imply that cytotoxic substances are eluted from some products even after a hardening period of 48 hours. In contrast, three of the glass-ionomer cements tested had only slight cytotoxic effects on the cultured pulp cells.
he introduction of glass-ionomer cements (gic's) as a dental material was first reported by Wilson and Kent (1972). Due to their chemical nature, these materials are able to adhere to the enamel and to dentin substances. Because of additional advantages, such as mechanical strength and translucency, glassionomer cements are often used by dentists, especially as filling, luting, and sealing materials. Nevertheless, due to rather different results obtained with cell cultures, animal experiments, and clinical studies, their biocompatibility is still controversial (Cooper, 1980; Paterson and Watts, 1981; Plant et al., 1984; Ucok, 1986; Council on Dental Materials, 1984; Walls, 1986). Most of these studies, however, examined only one or a few cements based on glass ionomers. The aim of this study was to examine the reaction of cultured fibroblast-like pulp cells when they are in contact with eight different cements based on glass ionomers. For comparison, a commonly used liner based on calcium hydroxide was examined in the same way.
T
MATERIALSAND METHODS (1) Ceil Culture.-The cells used in the experiments were fibroblast-like pulpal cells of rabbits (White New Zealand, body weight 400-600 g). Culture characteristics and morphology of the cells were described in a separate study (Mtiller et al., 1990). For each experimental series, cells of the same subculture were seeded to one dish of every material tested. Only those series were selected for the biocompatibility evaluation which gave g r o w t h of unaltered cells in all control dishes.
(2) Preparation of the Materials to be Examined.-The products tested in this study are listed in Table 1 (four gic's of type II and four additional prod172 MULLER et al./BIOCOMPATIBILITY OF GLASS-IONOMER CEMENTS
ucts recommended for underfilling). The materials were dispensed and mixed according to the specifications of the manufacturers except for Ketac®-Cem. This product was dosed in a ratio for a filling material of high viscosity. One-half (180 mm 2) of the surface of specific slides (TCCS-tissue culture cover slips, Lux® Thermanox, Miles Laboratories, Inc., Naperville, IL) was covered with the freshly mixed material and stored for 12 h in an atmosphere of 50% humidity to facilitate setting. From 8 to 25 ~g of mixed material was used for every cover slip. After the cements hardened, slides were attached to the b o t t o m s of tissue culture dishes (Falcon ® 3001 F, Becton Dickinson Labware, Lincoln Park, N J) with one drop of a collagen mixture (TD 268, Ethicon GmbH, Hamburg, FRG) diluted with a-MEM (Gibco, Grand Island, NY) at a ratio of 1:10. After sterilization (15% ethylene oxide, 85% carbon dioxide at 5.5 × 105 Pa for 3.5 h), the dishes were de-gassed for 32.5 h in an atmosphere of 50% humidity. Then, 2 mL of tissue culture medium (a-MEM without nucleosides) supplemented with 15% FCS (fetal calf serum, Gibco), 100 I.U./mL penicillin, and 0.t ~L/L streptomycin (Seromed, Berlin, FRG) were added to every culture dish containing the slides with the materials and to controls with TCCS only. The culture medium was equilibrated with carbon dioxide in the incubator for 24 h, thus stabilizing the pH value. After this period, the pH value was determined inside the incubator by use of an electrode (pH-Einstabmesskette Lot 403-M8, Bachofer GmbH, Reutlingen, FRG). Three days after being mixed, the materials 50000 to 70000 suspended fibroblast-like pulp cells of the second subculture were added to every dish. Cells originating from the same subcultures were dispensed to dishes with the eight materials tested and
AdheSion to TGC:5 In • 30, t • 3d]
I% ~ l t u r e 4 ]
TABLE 1 MATERIALS USED IN STUDY SO
Material Fuji Ionomer Type II F
Abbreviation "F II F"
Ionobond
Ionobond
Ketac®-Cem
"KetCem"
Ketac®-Silver
"KS"
Meron
Meron
Glaslonomer~ Base Cement
"SBC"
Glaslonomer~ Cement Type II Core Zionomer~ Kit
"SGC I1"
"ZIO Core"
Manufacturer GC Dental Industrial Corp., Tokyo, Japan VOCO Chemie, Cuxhaven, FRG ESPE, Seefeld, FRG ESPE, Seefeld, FRG VOCO Chemie, Cuxhaven, FRG Shofu Dental Corp., Bohannon, CA, USA Shofu Dental Corp., Bohannon, CA, USA Den-Mat Corp., Santa Maria, CA, USA
Batch No. powder: 310361 liquid: 260880
powder: 7122 liquid: 6538 powder: 0050 liquid: 0005 B 0129
,c
4o
43
~~
/ o
:
• A Kof,P ~
F II-F I ~ M
•
•
," : KB
kkl~
K
K
n ZlO Coro
Fig. 1. Adhesion of vital cells to material-free surfaces of slides (TCCS): More than 96% of the cultures with
Ketac®-Cem,Ketac®-Silver,and
Glaslonomer~ Base Cement showed vital cells after
powder: 7212 liquid: 6538 powder: 018609 liquid: 088610
the three-day culturing are listed in Table 1.)
period. (Abbreviations used
Adhesion to TCCS It - 9dl
I% c u l t u r u ]
O7
loo
powder: 018507 liquid: 678507
ao
eo
powder: 276010 liquid: 272010
~"
•
40
3
F ~F kmo~md I~tCem
TABLE 2 oH VALUES OF THE MATERIALSTESTEDAFTEREQUILIBRATIONWITH 5% CARBONDIOXIDE FOR 24 H pH Value Material Mean Min Max Fuii Ionomer Type II F 7.33 7.28 3.37 Ionobond 7.35 7.30 7.39 Ketac®-Cem 7.42 7.37 7.47 Ketac®-Silver 7.49 7.45 7.53 Meron 7.26 7.22 7.31 Glaslonomer~9 Base Cement 7.22 7.17 7.25 Glaslonomer~ Cement Type II 7.40 7.36 7.44 Core Zionomer~ Kit 7.44 7.39 7.48 Mean values were calculated from five measurements inside the incubator. The pH values were allowed to stabilize for one hour•
I~
Uero¢
~
K
II ZlO G m
Fig. 2. Adhesion of vital ceils to material-free surfaces of slides (TCCS): Except for cultures with Ketac®-Cem, Ketac®-Silver, and Glaslonomer~ Base Cement, there are only small percentages with vital cells adhering to the slides after incubation for nine days. (Abbreviations used are listed in Table 1.) to material In • ao, t . ad) IOO
Im
dm
4O /
F II-F I 4 N I N I ~
Ir.etlC4m
Kill
Im¢
l l ~ : II Z I O G G ~
Fig. 3. Adhesion of vital cells to the materials
to controls (dishes with and without slides and cover slips with Kerr ®Life) during every series of experimerits. All cultures were incubated in a humidified atmosphere of 5% carbon dioxide (CQ) and 95% air at 37°C for nine days. During the culturing period, the pH of the medium was determined for every material in five dishes and for the controls as described above. Every thh'd day, the cultures were analyzed by use of an inverse phasecontrast microscope (ICM 405, Zeiss, Oberkochen, FRG), and the culture medium was completely renewed. For every material tested as well as for the controls, 30 cultures in ten series of experiments that used dif-
ferent primary cultures (i.e., different animals) were analyzed. Evaluation of the materials tested was done in terms of the following parameters: (1) adhesion of cells to the material-free surfaces of the tissue culture cover slips, (2) adhesion of cells to the material tested, (3) relative growth rate of the cultures on the material-free surfaces of the slides, and (4) morphology of the cells.
tested: After incubation for three days, more than 96% of the cultures with Ketac®-Cem and Ketac®-
Silver exhibited contact of unchanged cells with these materials. (Abbreviations used are listed in
Table 1 .) [% ¢ u l l u r l i ]
A d h e s i o n to material It - g
Department of Prosthodontics Poliklinik for Prothetik Goethestr. 70 8000 ML~nchen2, West Germany *Department of Physiological Chemistry Institut fL~rPhysiologische Chemie Physikalische Biochemie und Zellbiologie Schillerstr. 22 8000 M0nchen 2, West Germany Received August 22, 1989 Accepted March 2, 1990 This investigation was supported in part by the editorial board of the M#nchener Medizinische Wochenschrift, Munich, West Germany. Dent Mater 6:172-177, July, 1990
Abstract-A comparative study was carried out to determine the cytotoxicity of eight different glass-ionomer cements by means of cell culture. Only fibrobiastlike cells from the pulp of the same subcultures were used. Factors for the evaluation of the biological tolerance were, first, adhesion of cells to the slides as well as to the material, second, relative growth rates of cultures, and, in addition, cytomorphology. The cultured cells reacted quite differently to the glassionomer cements tested. The results imply that cytotoxic substances are eluted from some products even after a hardening period of 48 hours. In contrast, three of the glass-ionomer cements tested had only slight cytotoxic effects on the cultured pulp cells.
he introduction of glass-ionomer cements (gic's) as a dental material was first reported by Wilson and Kent (1972). Due to their chemical nature, these materials are able to adhere to the enamel and to dentin substances. Because of additional advantages, such as mechanical strength and translucency, glassionomer cements are often used by dentists, especially as filling, luting, and sealing materials. Nevertheless, due to rather different results obtained with cell cultures, animal experiments, and clinical studies, their biocompatibility is still controversial (Cooper, 1980; Paterson and Watts, 1981; Plant et al., 1984; Ucok, 1986; Council on Dental Materials, 1984; Walls, 1986). Most of these studies, however, examined only one or a few cements based on glass ionomers. The aim of this study was to examine the reaction of cultured fibroblast-like pulp cells when they are in contact with eight different cements based on glass ionomers. For comparison, a commonly used liner based on calcium hydroxide was examined in the same way.
T
MATERIALSAND METHODS (1) Ceil Culture.-The cells used in the experiments were fibroblast-like pulpal cells of rabbits (White New Zealand, body weight 400-600 g). Culture characteristics and morphology of the cells were described in a separate study (Mtiller et al., 1990). For each experimental series, cells of the same subculture were seeded to one dish of every material tested. Only those series were selected for the biocompatibility evaluation which gave g r o w t h of unaltered cells in all control dishes.
(2) Preparation of the Materials to be Examined.-The products tested in this study are listed in Table 1 (four gic's of type II and four additional prod172 MULLER et al./BIOCOMPATIBILITY OF GLASS-IONOMER CEMENTS
ucts recommended for underfilling). The materials were dispensed and mixed according to the specifications of the manufacturers except for Ketac®-Cem. This product was dosed in a ratio for a filling material of high viscosity. One-half (180 mm 2) of the surface of specific slides (TCCS-tissue culture cover slips, Lux® Thermanox, Miles Laboratories, Inc., Naperville, IL) was covered with the freshly mixed material and stored for 12 h in an atmosphere of 50% humidity to facilitate setting. From 8 to 25 ~g of mixed material was used for every cover slip. After the cements hardened, slides were attached to the b o t t o m s of tissue culture dishes (Falcon ® 3001 F, Becton Dickinson Labware, Lincoln Park, N J) with one drop of a collagen mixture (TD 268, Ethicon GmbH, Hamburg, FRG) diluted with a-MEM (Gibco, Grand Island, NY) at a ratio of 1:10. After sterilization (15% ethylene oxide, 85% carbon dioxide at 5.5 × 105 Pa for 3.5 h), the dishes were de-gassed for 32.5 h in an atmosphere of 50% humidity. Then, 2 mL of tissue culture medium (a-MEM without nucleosides) supplemented with 15% FCS (fetal calf serum, Gibco), 100 I.U./mL penicillin, and 0.t ~L/L streptomycin (Seromed, Berlin, FRG) were added to every culture dish containing the slides with the materials and to controls with TCCS only. The culture medium was equilibrated with carbon dioxide in the incubator for 24 h, thus stabilizing the pH value. After this period, the pH value was determined inside the incubator by use of an electrode (pH-Einstabmesskette Lot 403-M8, Bachofer GmbH, Reutlingen, FRG). Three days after being mixed, the materials 50000 to 70000 suspended fibroblast-like pulp cells of the second subculture were added to every dish. Cells originating from the same subcultures were dispensed to dishes with the eight materials tested and
AdheSion to TGC:5 In • 30, t • 3d]
I% ~ l t u r e 4 ]
TABLE 1 MATERIALS USED IN STUDY SO
Material Fuji Ionomer Type II F
Abbreviation "F II F"
Ionobond
Ionobond
Ketac®-Cem
"KetCem"
Ketac®-Silver
"KS"
Meron
Meron
Glaslonomer~ Base Cement
"SBC"
Glaslonomer~ Cement Type II Core Zionomer~ Kit
"SGC I1"
"ZIO Core"
Manufacturer GC Dental Industrial Corp., Tokyo, Japan VOCO Chemie, Cuxhaven, FRG ESPE, Seefeld, FRG ESPE, Seefeld, FRG VOCO Chemie, Cuxhaven, FRG Shofu Dental Corp., Bohannon, CA, USA Shofu Dental Corp., Bohannon, CA, USA Den-Mat Corp., Santa Maria, CA, USA
Batch No. powder: 310361 liquid: 260880
powder: 7122 liquid: 6538 powder: 0050 liquid: 0005 B 0129
,c
4o
43
~~
/ o
:
• A Kof,P ~
F II-F I ~ M
•
•
," : KB
kkl~
K
K
n ZlO Coro
Fig. 1. Adhesion of vital cells to material-free surfaces of slides (TCCS): More than 96% of the cultures with
Ketac®-Cem,Ketac®-Silver,and
Glaslonomer~ Base Cement showed vital cells after
powder: 7212 liquid: 6538 powder: 018609 liquid: 088610
the three-day culturing are listed in Table 1.)
period. (Abbreviations used
Adhesion to TCCS It - 9dl
I% c u l t u r u ]
O7
loo
powder: 018507 liquid: 678507
ao
eo
powder: 276010 liquid: 272010
~"
•
40
3
F ~F kmo~md I~tCem
TABLE 2 oH VALUES OF THE MATERIALSTESTEDAFTEREQUILIBRATIONWITH 5% CARBONDIOXIDE FOR 24 H pH Value Material Mean Min Max Fuii Ionomer Type II F 7.33 7.28 3.37 Ionobond 7.35 7.30 7.39 Ketac®-Cem 7.42 7.37 7.47 Ketac®-Silver 7.49 7.45 7.53 Meron 7.26 7.22 7.31 Glaslonomer~9 Base Cement 7.22 7.17 7.25 Glaslonomer~ Cement Type II 7.40 7.36 7.44 Core Zionomer~ Kit 7.44 7.39 7.48 Mean values were calculated from five measurements inside the incubator. The pH values were allowed to stabilize for one hour•
I~
Uero¢
~
K
II ZlO G m
Fig. 2. Adhesion of vital ceils to material-free surfaces of slides (TCCS): Except for cultures with Ketac®-Cem, Ketac®-Silver, and Glaslonomer~ Base Cement, there are only small percentages with vital cells adhering to the slides after incubation for nine days. (Abbreviations used are listed in Table 1.) to material In • ao, t . ad) IOO
Im
dm
4O /
F II-F I 4 N I N I ~
Ir.etlC4m
Kill
Im¢
l l ~ : II Z I O G G ~
Fig. 3. Adhesion of vital cells to the materials
to controls (dishes with and without slides and cover slips with Kerr ®Life) during every series of experimerits. All cultures were incubated in a humidified atmosphere of 5% carbon dioxide (CQ) and 95% air at 37°C for nine days. During the culturing period, the pH of the medium was determined for every material in five dishes and for the controls as described above. Every thh'd day, the cultures were analyzed by use of an inverse phasecontrast microscope (ICM 405, Zeiss, Oberkochen, FRG), and the culture medium was completely renewed. For every material tested as well as for the controls, 30 cultures in ten series of experiments that used dif-
ferent primary cultures (i.e., different animals) were analyzed. Evaluation of the materials tested was done in terms of the following parameters: (1) adhesion of cells to the material-free surfaces of the tissue culture cover slips, (2) adhesion of cells to the material tested, (3) relative growth rate of the cultures on the material-free surfaces of the slides, and (4) morphology of the cells.
tested: After incubation for three days, more than 96% of the cultures with Ketac®-Cem and Ketac®-
Silver exhibited contact of unchanged cells with these materials. (Abbreviations used are listed in
Table 1 .) [% ¢ u l l u r l i ]
A d h e s i o n to material It - g
