0099-2399/92/1812-0589/$03.00/0 JOURNALOF ENDODONTICS Copyright © 1992 by The American Association of Endodontists
Printed in U.S,A,
VOL. 18, NO, 12, DECEMBER1992
Delineation of Cytotoxic Concentrations of Two Dentin Bonding Agents In Vitro C. T. Hanks, DDS, PhD, J. C. Wataha, DMD, PhD, R. R. Parsell, and S. E. Strawn, BSE
5), it is also of interest to know whether extracts from certain materials in contact with the floor of the cavity preparation are sufficiently concentrated to cause pulpal damage. During placement of the restoration, even if the adhesion of a bonding agent was incomplete, the presence of the bonding agent should cause reduction in conductance of fluids because of partial blockage of the dentinal tubules. After placement of the restoration, the mechanism for introduction of either bacterial products or extracts of restorative materials into the pulp would be diffusional permeability of the dentin. Aqueous continuity between the floor of the cavity and the pulp would allow establishment of a diffusion gradient between the restoration and the pulp (6). There have been relatively few articles on the biocompatibility of bonding agents in the literature. A recent review of dentin bonding systems indicates that these agents have little or no effect on pulpal tissues (7). Only two articles have focused on the cytotoxicity of bonding agents in tissue culture (8, 9). The present report focuses on the effects of two dentin bonding systems, GLUMA and SB2, and two ingredients, glutaraldehyde and 2-hydroxyethylmethacrylate, on cultured cells, both directly and indirectly through dentin, in an attempt to estimate whether these agents might partially contribute to pulpal injury in vivo.
Until adhesiveness of dentin bonding agents and other restorative materials to dental structures can be assured, microleakage into resulting "gaps" and dentin permeability will remain major concerns in cases of pulpal irritation. The objectives of the present study were to (a) delineate the kinds and levels of metabolic cytotoxicity of the GLUMA and Scotchbond 2 systems as well as glutaraldehyde and 2hydroxyethylmethacrylate, and (b) compare the effects of these same materials after diffusion through dentin discs approximately 0.5-ram thick. In monolayer cultures, glutaraldehyde was much more cytotoxic than 2-hydroxyethylmethacrylate. However, GLUMA sealer and Scotchbond 2 adhesive exhibited similar cytotoxicity in monolayer cultures. After diffusion through dentin, glutaraldehyde and 2-hydroxyethylmethacrylate effects were diluted 14.7 and 26.7 times, respectively. The postdiffusional effects of the GLUMA and Scotchbond 2 systems were not significantly different and less than those effects in monolayer cultures. This study should help in the evaluation of possible causes of pulpal irritation following restorative procedures.
MATERIALS AND METHODS Two dentin bonding systems and two ingredients of these systems were studied. These materials, their abbreviations, and their composition in moles per liter are given in Table 1. The cytotoxicity of these materials and components was compared in monolayer cultures of BALB/c 3T3 cells (clone A31; American Type Culture Collection, Rockville, MD) before and after diffusion through dentin in a modified "in vitro pulp chamber" (IVPC; Fig. 1). The cells were maintained and passaged in 75-cm 2 flasks in Dulbecco's modified Eagle's medium (with 4.5 g/liter dextrose; Flow Laboratories, McLean, VA) with 3 % donor calf serum and supplemented with glutamine and penicillin-streptomycin (Gibco Laboratories, Grand Island, NY).
One of the major rationales for development of dentin bonding agents is to eliminate the gap between the composite restoration and tooth structure. Two of the newer dentin bonding systems, GLUMA and Scotchbond 2 (SB2), call for the removal of the smear layer from the cavity preparation before placing the bonding agent. Tao and Pashley (1) have shown that after removal of the smear layer relatively low hydraulic pressure (32 cm of phosphate-buffered saline), representing dentin perfusion from pulpal tissue fluid pressure, significantly reduces shear bond strengths up to 48 h after initial polymerization. The lack of effective bonding of any restorative material to tooth structure in situ leads to numerous gaps between restoration and tooth structure (2). These gaps are responsible for microleakage by capillary action of various solutions and suspensions under restorations (3). Although bacteria and bacterial products have been implicated in pulpitis because of microleakage into contraction gaps (4,
Monolayer Tests For unpolymerized components of the bonding agents, monolayer cultures were plated at approximately 30,000 cells/
589
590
Hanks el al.
Journal of Endodontics
TABLE 1. Dentin bonding agents and components, manufacturers, control numbers and moles/liter
System
Material
Scotchbond 2 Batch P891208 (3M, St. Paul, MN)
GLUMA (Columbus Dental Division, Miles Inc., St. Louis, MO)
%
mol/liter
Scotchprep Dentin Primer
SP primer
Abbrev
HEMA
Component*
55.0
4.09t
Scotchbond 2 Light Cure Dental Adhesive
SB2 adhesive
Maleic acid HEMA
2.5 37.0
0.22~t 2.75t
bis-GMA
63.0
1.231:
GLUMA 3 Primer Batch 16MAR88
GLUMA primer
GLUT
5.0
0.49:~
GLUMA 4 Sealer Batch 29MAR88
GLUMA sealer
HEMA TEGDMA
35.0 45.0
2.601" 1.57:~
55.0
1.071:
Stock (concentrate) Batch 767
GLUT
70.0
6.99t
Stock (concentrate) Batch 404194
HEMA
97.0
7.21 t
bis-GMA Glutaraldehyde (Ladd Research Industries, Inc., Burlington, VT) 2-Hydroxyethylmethacrylate (Polysciences, Inc., Warrington, PA)
* Components of bonding agents from Erickson (20). t mol/liter calculated on basis of weight percent, i.e. g/liter divided by g/tool. mol/liter for HEMA was determined by correcting volume for density of HEMA (1,034 g/ml) and then calculated as above.
{: ,.,o, Nylon ~ Spacer ~ .
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nlilli IIIIII, Illlillll
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FIG 1. This apparatus consists of a dentin disc attached to the bottom of a one-quarter-inch in diameter nylon cylinder (RSN-8/8; Small Parts, Inc.) by means of epoxy long-setting cement (Ross Epoxy Glue). The apparatus was dried for 2 h at 60°C and extracted with 70% ethanol for 12 h to remove unreacted reagents. The apparatus was then rehydrated with medium and set on top of a nylon washer (WN-3/8) in the bottom of a well of a 24-well dish that had been plated 24 h earlier with 30,000 BALB/c 3T3 cells in 200 pl of complete medium. The apparatus was used with the insert for testing diffusion from polymerized bonding agents as well as the active ingredients, GLUT and HEMA (see text for explanation).
cm 2 in wells (16 mm in diameter) of 24-well dishes (#3524; Costar, Cambridge, MA) and allowed to grow for 24 h before introducing the test materials. At that time (time 0), either
glutaraldehyde (GLUT) or 2-hydroxyethyl methacrylate (HEMA) was added to the media so that the final concentrations for GLUT ranged from 0.005 to 2.5 ~mol/liter, and the final concentration for HEMA ranged from 0.16 to 80 umol/ liter in the experimental wells. These ranges varied from concentrations which had no toxic effect to those which depressed all measurable metabolic activity. For each time period after the addition of the test substances (0, 8, and 24 h), six wells for each concentration of each test substance plus six wells for untreated controls were harvested. At the end of the 24-h test period, medium was changed to fresh medium in six additional wells for each concentration of component, as well as six additional control wells, to determine whether cell metabolism could recover in the next 24 h (reversibility of cytotoxic effects). After harvesting, the cultures were assessed for DNA synthesis, protein synthesis, succinyl dehydrogenase activity, and total protein content of the wells. Either [3H]leucine or [3H] thymidine was added at 90 and 45 min, respectively, prior to harvest as 20-#1 aliquots in simple medium to "pulse" label the cultures (10). The final concentration of each isotope was 5 uCi/ml in each well. Stock radioisotope solutions had specific activities of 64 Ci/mmol for [3H]leucine or 6.7 Ci/ mmol for [3H]thymidine (ICN Radiochemicals, Irvine, CA). To assure that the lack of thymidine did not limit the rate of DNA synthesis, the total thymidine including radioisotopelabeled thymidine was brought to 5 pmol/liter with cold thymidine for the 90-rain pulse. The protein synthetic assays and total protein content were performed on the same wells. The DNA synthetic assays and the succinyl dehydrogenase assay utilizing 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) as the tetrazolium salt were performed on separate but parallel wells because the processing sequences for the assays were different. When harvesting cells, the medium was suctioned off and the wells were processed as described previously (10). Discs (2.95 mm in diameter; 3.36-mm thickness) of GLUMA sealer and SB2 adhesive were prepared by curing
Vol. 18, No. 12, December 1992
layers of approximately 1-mm thickness sequentially. The discs were placed in the center of each well (3.8 cm in diameter) of 6-well polystyrene dishes (#3406; Costar). Ethanol-washed nylon discs of the same size were used as control samples. After placement of the discs, BALB/c 3T3 cells were plated in a monolayer around the discs at 25,000 cells/cm 2 in 3 ml of complete medium. At the time of harvest, the discs of bonding agents were removed and the MTT assay for succinic dehydrogenase activity was run on each well. Finally, in a separate experiment, GLUMA sealer and SB2 adhesive were spread on the bottom of wells of 24-well dishes and polymerized and were then washed for 18 h with 60% ethanol and tested for growth of cells on the surfaces of the agents. Diffusion Studies Freshly extracted human molar teeth without carious lesions were cleaned of debris and periodontal ligament tissue and placed in 70% ethanol for 24 h before their use. Dentin discs were cut in cross-section from the widest part of the crowns and as close to the pulp as possible without cutting through pulp horns. The discs were cut at 230 rpm with a low-speed wheelsaw using 10.2-cm diameter diamond wafering blades (CO-153, 320 grit; Mager Scientific Inc., Dexter, MI), followed by hand sanding with 400 and then 600 grit silicon carbide paper each for approximately 20 figure-eight rotations. The discs were treated for 2 min with 0.5 M EDTA (pH 7.4) and rinsed with double distilled H20 before hydraulic conductance (Lp) values were determined as described previously (11). The modified IVPC (Fig. 1) consisted of a 0.49-cm inside diameter nylon spacer (RSN-10/8; Small Parts, Inc., Miami, FL) which was attached to the coronal surface of the dentin disc by means of epoxy long-setting cement (Ross Epoxy Glue; Ross Adhesives/Control Corp., Detroit, MI). This apparatus was dried for 2 h at 60"C and extracted with 70% ethanol for 12 h to remove unreacted epoxy ingredients. Each apparatus was rehydrated with medium and set on top of a nylon washer (WN-3/8; Small Parts, Inc.) in the bottom of a well of a 24-well dish which had been plated 24 h earlier with 30,000 BALB/c 3T3 cells in 200 #1 of complete medium. After 24 h of exposure to the test materials, the ceils in the bottom of the well were tested for succinyl dehydrogenase (MTT) activity. In the first diffusion experiment, each of the two dentin bonding systems was utilized with a separate set of six dentin discs in the modified IVPC (Table 2). Dentin discs used for the GLUMA system had an average (SD) thickness of 0.485 (0.043) mm and the average (SD) Lp of 0.016 (0.006 ~1 cm -2 rain -l cm H20 -l. Dentin discs for the SB2 system had an average thickness of 0.485 (0.037) mm and an average (SD) Lp of 0.014 (0.006) ~1 cm -2 min -1 cm H20 -l. In both cases, the bottom of cylindrical nylon spacers (RSN-4/2; Small Parts, Inc.) were filled with Silux composite (3M, Minneapolis, MN). These spacers, which have an outside diameter of 0.47 cm and an inside diameter of 0.29 cm, are shown as an insert for the larger spacers in Fig. 1 and were used as carriers for the bonding/adhesive systems. In the case of the SB2 system, the Silux was polymerized as described by the manufacturer. Then, the SP primer was painted onto the bottom
Dentin Bonding Agents
591
of the insert, allowed to set for 30 s, and blown dry before application and polymerization of the SB2 adhesive. For the GLUMA system, Silux (1-mm thick) was placed in the insert and left unpolymerized prior to placement of the GLUMA primer and sealer. GLUMA primer was applied to the bottom of the insert and gently dried before application of GLUMA sealer. The GLUMA/Silux systems were then polymerized together. Both bonding systems were polymerized with a Prismetics Lite (Caulk/Dentsply, Milford, DE) for 1 min at a distance of approximately 4 mm. Finally, 20 ul of medium were added to the top of each dentin disc inside the larger nylon spacer just before placing the insert into position. The insert was placed inside the larger spacer against the dentin disc (Fig. 1) for the first 24 h immediately after polymerization. After this period, each apparatus with the insert in place was moved to a fresh well for a second 24 h. After each exposure period, the cells in each experimental well (six for each agent) and control wells (six) were tested for MTT activity and compared with untreated controls. The results were reported as a percentage of negative controls. Paired Student's t tests were used to compare the cytotoxicity data for each bonding procedure to the other bonding procedure at both time periods as well as each bonding procedure to itself at 1 and 2 days after polymerization. In a second experiment, either 80 ~mol/liter HEMA or 2.5 umol/liter GLUT were placed in the top reservoir of the modified IVPC (Fig. 1) on top of each of four dentin discs with an average (SD) thickness of 0.5 (0.04) mm for each experimental material. No inserts were used in this experiment. An estimation of the diffusion of these reagents was interpolated by the comparison of the level of metabolic responses (MTT) of the cell test system to the response curve for various concentrations of the same substances by cells in monolayer after 24 h of exposure. RESULTS GLUT in culture medium was tested in monolayer cultures at final concentrations between 0.005 and 2.5 umol/liter. As compared with the control wells, it gradually and increasingly inhibited DNA and protein synthesis, as well as mitochondrial respiration and total protein solubility as the concentration increased (Figs. 2 and 3). The effects of concentrations through 0.02 #mol/liter were reversible for all four parameters measured when fresh medium was added between 24 and 48 h. When the cellular response data were graphed as optical density of the MTT response plotted against GLUT concentration, 2.5 umol/liter GLUT depressed the mitochondrial enzyme response by 99.9%, and the dose of GLUT which was toxic to half the cell monolayer (TCs0) was 0.04 #tool/ liter (Fig. 4). On the other hand, when 2.5 umol/liter GLUT was placed on the 0.5-mm dentin disc (Fig. 1) opposite 200 ~1 of medium which in turn nourished a cell monolayer, the inhibitory effect on MTT activity was reduced (Fig. 4). After diffusion through dentin, 2.5 umol/liter GLUT inhibited MTT activity by 88%. Because we had not developed an assay for concentrations of these chemicals, we interpolated the average concentration of GLUT in the diffusates from the GLUT dose-response curve for monolayer cultures. Therefore, the 88% MTT activity of the diffusate matched the GLUT concentration of about 0.17 #mol/liter, which was a
592
Journal of Endodontics
Hanks et al.
TABLE 2. Testing of GLUMA and SB2 in in vitro pulp chamber
Disc
Thickness
Lp
(mm) GLUMA A1 A2 A3 A4 A5 A6
5~(IO
% of MTT Activity First 24 h
Second 24 h
0.469 0.946 0.760 0,471 0.771 0.634
0.358 0.510 0.348 0.450 0.335 0.321
4(I(1(I
'
0 02 .moles/L (11 pmolc~L
•
----6-- o 5~mote~/L 0.480 0.500 0.540 0.500 0.410 0.480
0.0200 0.0100 0.0100 0.0240 0.0120 0.0220
Average
0.485
0.0160
0.668
0.387
SD
0.043
0.0060
0.118
0.076
SB2 B1 B2 B3 B4 B5 B6
0.540 0.460 0.480 0.520 0.450 0.460
0.0180 0.0100 0.0180 0.0035 0.0150 0.0210
0.634 0.406 1.160 0.779 0.777 0.571
0.563 0.389 0.377 0.399 0.368 0.606
Average SD
0.485 0.037
0.0140 0.0060
0.721 0.256
0.450 0.105
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dilution of 14.7 times when compared with the concentration applied at the top of the gradient. For the first 24 h in culture, increasing concentrations of HEMA had increasingly inhibitory effects on each metabolic parameter (Figs. 5 and 6). From 24 to 48 h, even after HEMAcontaining medium was replaced by fresh medium, HEMA concentrations of 0.8/~mol/liter and above allowed no recovery of DNA synthesis and 4.0 ~mol/liter HEMA irreversibly inhibited both protein synthesis and mitochondria activity. The cell mass (measured by total protein) failed to show substantial recovery in cells treated with 4.0 ~mol/liter or more HEMA. When the cellular response data were graphed as optical density of MTT response plotted against HEMA concentration, 16 pmol/liter HEMA depressed MTT activity by about 99.1% during the first 24 h of incubation and 80 ~mol/liter HEMA depressed MTT activity by 99.3%. The TCs0 value for HEMA with this cell test system was 1.1 ~mol/ liter (Fig. 7). When 80 #mol/liter HEMA was placed on the 0.5-mm dentin disc rather than directly into the monolayer culture medium, again there was less inhibition of MTT activity in the cell test system. After diffusion through dentin, the HEMA diffusate inhibited only 84.3% of the MTT activity and the interpolated concentration was 3.0 #mol/liter or a dilution of 26.7 times when compared with the concentration applied to the top of the gradient. When pellets of GLUMA sealer and SB2 adhesive were centered in 3.8-cm diameter wells with monolayer cultures, each material produced a ring of inhibition of approximately 1 cm around the test material. Within this ring, a large number of cells were missing and most cells which remained exhibited rounding and other cytopathic effects. The ceils on the periphery of the well appeared to be as healthy as those in the control dish. A sharp transitional zone was present between the ring of inhibition and the healthy peripheral cells. The average outside diameter (n = 6) representing the MTT activity of the remaining cells in each well was 47.8% of the negative controls for SB2 adhesive and 49.4% of controls for
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FtG 2. The effects of GLUT concentrations (0.005 to 2.5 ~mol/liter), in comparison to those of untreated controls (0 ;Lmol/liter), on (a) DNA synthesis ([3H]thymidine incorporation into DNA) or (b) protein synthesis ([3H]leucine incorporation into protein) in monolayer cultures of BALB/c 3T3 cells. At time 0, medium with or without GLUT was added to each well. Each point represents the mean concentration and standard error of the mean (vertical bars) of six wells. At 24 h (arrow), fresh medium without GLUT was placed into sets of treated wells for the next 24 h to determine whether the effects were reversible.
GLUMA sealer. This difference was not statistically significant. In experiments which tested diffusion of materials from freshly polymerized dentin bonding systems through dentin, the same set of dentin discs was used to measure diffusion of GLUMA-related materials through dentin for the first and second 24-h periods. Likewise, the same set of discs were used to measure diffusion of SB2-related materials during both time periods. As shown in Table 2, there was an attempt to match average thickness and Lp for the two sets of discs. Two observations were made concerning these comparisons. First, the GLUMA system depressed MTT activity more than did the SB2 system during both time periods. Thus, for the first 24 h, the G L U M A system and the SB2 system allowed about 66.8% and 72%, respectively, of the MTT activity found in the control wells. However, there was no significant difference (p > 0.05) between the two values. For both the GLUMA system and the SB2 system, MTT activity was significantly lower (p < 0.05) for the second 24 h. Again, there was no significant difference (p < 0.05) between the effects of these two agents.
Dentin Bonding Agents
Vol. 18, No. 12, December 1992
593
2.3
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for 18 h. However, they could not be cultured on surfaces of the unwashed resin controls.
120 ~-O--
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FIG 3. The effects of GLUT concentrations, in comparison to those of untreated controls, on (a) succinyl dehydrogenase activity (MTT) and (b) total protein in BALB/c 3T3 cell cultures under conditions similar to those used in Fig. 2.
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FtG 4. The 24-h effects of GLUT concentrations (circles; 0.005 to 2.5 #mol/liter), in comparison to effects of 2.5 umol/liter GLUT (squares) after diffusion through 0.5-mm dentin, on MTT activity in BALB/c 3T3 cell cultures. The TCso dose for 24-h exposure to GLUT is indicated by the dashed line.
Finally, the observation was made that ceils could be plated and cultured on sheets of.either GLUMA sealer and SB2 adhesive after these sheets had been washed in 70% ethanol
Animal usage studies and microleakage studies (4, 5) suggest that dentin permeability and lack of marginal integrity of restorations are responsible for pulpal reactions, and that the presence of bacteria or their products between the composite and the walt of the cavity preparation is a larger problem than leachables from restorative materials. With ample evidence for bacterial mieroleakage, why should we be concerned with the cytotoxicity of dentin bonding agents? There are two reasons for this concern. First, there is some in vivo evidence of cytotoxicity of the GLUMA bonding system. Second, there is evidence that dentin thickness (length of the diffusion gradient) is important. In a study by H6rsted-Bindslev (12), it was reported that if the dentin was less than 0.1mm thick, a "toxic" reaction could be observed in the pulp of a cavity which had been pretreated with the GLUMA system. This suggested that a pulpal wall of greater than 0.1 mm was necessary for dentinal tubules to adsorb GLUMA system-associated leachables present in tubular fluid. Meryon and Brook (9) used 0.1- and 0.5-mm-thick dentin slices to test cytotoxicity (by cell number) of three dentin bonding systems: SB2, GLUMA, and Tripton (ICI Dental) on baby
594
Journal of Endodontics
Hanks et al. 2.5
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CONCENTRATION (Hmoles/L) FiG 7. The 24-h effects of HEMA (circles; 0.16 to 80.0 #mol/liter), in comparison to effects of 80 #mol/liter HEMA (squares) after diffusion through 0.5-mm dentin, on MTT activity in BALB/c 3T3 cell cultures. The TCso dose for 24-h exposure to HEMA is indicated by the dashed
line.
hamster kidney fibroblasts after diffusion through dentin. They found that all the materials were significantly cytotoxic, as compared with controls, through both thicknesses of dentin. With 0.1-mm discs, GLUMA was most cytotoxic, with
only 26.9% viable cells remaining in the tissue culture dish as compared with untreated controls. SB2 was next most toxic, with 54.2% of controls and Tripton was least toxic (68.8% of controls). However, with 0.5-mm discs, the rankings of GLUMA and SB2 were reversed. Tripton, GLUMA and SB2 gave 70.8%, 66.6%, and 52.6% of the negative controls, respectively. In the present study, cytotoxicity tests of two ingredients of the two bonding systems in monolayer cultures identified the concentrations above which connective tissue cells were irreversibly damaged. GLUT, at a concentration of 2.5 pmol/ liter and above, was irreversibly inhibitory to all four metabolic parameters tested (Figs. 2 and 3). For HEMA, 16 umol/ liter and above were irreversibly inhibitory for all four metabolic parameters (Figs. 5 and 6). With this information, it is possible to predict which concentrations of these materials, if they reach the pulp, are capable of causing metabolic depression or death of exposed pulpal cells. In the experiments which tested diffusion of the dentin bonding systems, each agent was applied to the insert (Fig. 1), not to the dentin. This was done to keep the dentinal tubules patent for diffusion. The experiments were not designed to test how the dentin bonding systems reduced dentin permeability, but rather to test the extent of diffusability of GLUT, HEMA, and unidentified leachables from the GLUMA and SB2 bonding systems. If the agents had been painted onto the dentin, the resins may have penetrated variable distances into the dentin, altering the diffusion gradients for any leachable components. This present system may simulate areas of bonded dentin which have just become debonded because of forces of polymerization contraction under physiological pulpal pressures (13, 14). From Table 1, it can be seen that GLUT is an ingredient only of GLUMA primer, but that HEMA is found in SP primer, SB2 adhesive, and GLUMA primer. The molecular weight (mol wt) of GLUT (100.12) is approximately the same as that of phenol (94.11). Hanks et al. (15) reported that when 0.009 M phenol was applied to 0.5-mm thick dentin discs, phenol was diluted approximately 500 times. In this previous study, the cell chamber had a volume of 1.87 ml, in contrast to 0.2 ml in the present cell chamber. If we assume similar diffusion characteristics for GLUT and phenol, then we would expect the present cell chamber to have diluted GLUT only about 50-fold (0.2/1.87 = 0.107; 500 x 0.107 = 53.5). Assuming this dilution factor, the concentration of GLUT which was painted on the dentin (5% or 0.49 mol/liter) should have been diluted to 9.8 × 10 -3 mol/liter in the cell chamber. This level is over 1000 times greater than the concentration of GLUT which will completely inhibit cell metabolism (Figs. 2 and 3). These assumptions should be verified by means of radioisotope experiments. Previous studies (16) have suggested that GLUT-containing bonding agents are desirable because they kill residual bacteria to which the pulp is exposed. These concentrations are also capable of killing mammalian cells and fixing pulpal tissue. Likewise, the concentrations of HEMA (mol wt = 130.14) in GLUMA primer (2.6 mol/liter), SP primer (4.09 mol/ liter), and SB2 adhesive (2.75 mol/liter), if diluted 50-fold by simple diffusion, would give HEMA concentrations of 5.2 x 10-2, 8.2 x 10-2, and 5.5 x 10 2 tool/liter, respectively. Figures 5 and 6 show that HEMA completely inhibits metabolic activity at 16 umol/liter, which is four orders of mag-
Vol. 18, No. 12, December 1992
nitude less than the potential concentrations of HEMA in the diffusates from 0.5-mm dentin. Maleic acid is also a component of SP primer. Using HEMA in the absence of maleic acid may even underestimate the cytotoxicity of SP primer. Finally, Hanks et al. (15) found a 2500-fold dilution of phenol across dentin discs 1.4-ram thick. Even a 2500-fold dilution would not have lowered the concentration of 35% HEMA to nontoxic levels. There is some suggestion of agreement between data from the present study and that of the phenol study (15). In the present study, Figs. 4 and 7 show that the GLUT concentration, measured by means of a biological assay (MTT), fell about 15-fold and the HEMA concentration fell about 27fold after diffusion through the 0.5-mm dentin. In this same chamber, if we correct for volume, phenol concentration would have fallen in the same order of magnitude, 53.5-fold. Several other components of dentin bonding systems are important. Bis-GMA (tool wt = 512.65) is present in SB2 adhesive (at 1.23 tool/liter) and GLUMA sealer (at 107 mol/ liter). Triethylene glycol dimethacrylate (TEGDMA) (tool wt = 286.36) is present in GLUMA sealer at 1.57 mol/liter. Hanks et al. (17) have shown that although the molecular weight of bis-GMA is almost twice as large as that of TEGDMA, the concentration of bis-glycidyl methacrylate (bis-GMA) (25 /~mol/liter) necessary for irreversible depression of DNA and protein synthesis was at least an order of magnitude less than the necessary concentration of TEGDMA (450 umol/liter). The following relationship holds for the relative cytotoxicities for all four molecules in these two bonding systems in terms of IDso: GLUT (0.04 umol/liter) > HEMA (1.1 tzmol/liter) > bis-GMA (13 to 16 ttmol/liter) > TEGDMA (70 to 100 ~zmol/liter). Diffusion characteristics of these molecules through dentin are being studied currently. Usage studies do not routinely report much pulpal reaction to dentin bonding agents containing GLUT and/or HEMA which cannot be attributed to bacteria (18). Why are there such discrepancies between diffusion data in vitro and usage studies? This may be either because certain variables have not been controlled in the in vitro experiments, or that in vivo conditions are different. Examples of uncontrolled variables may include (a) the time the primers or sealer/adhesives are allowed to stand on the patent dentin before drying or polymerization (this is diffusion time), (b) the depth of polymerization of HEMA, (c) the degree of conversion of HEMA to poly-HEMA and of bis-GMA and TEGDMA to polymerized resin, (d) the degree of total obstruction of the tubules because of polymerized materials, (e) the effect of the wet environment (tissue fluid or GLUT) upon polymerization, (f) the amount of adsorption of any of these agents to the walls of the tubules, and (g) the different time frames for in vitro studies and usage studies. In the present study, cells were found to grow on the resin surfaces of both GLUMA sealer and SB2 adhesive, which had been polymerized to the bottom of tissue culture wells, only if the surfaces had been washed for several hours with 70% ethanol. This suggested that the toxic material(s) were derived from the poorly polymerized oxygen-inhibited layer of the resins. Several aspects of the current study were designed to improve the measurement of cytotoxicity leachables following diffusion through dentin. The IVPC concept was adapted for a 24-well dish and utilized nylon washers and spacers (insert,
Dentin Bonding Agents
595
Fig. 1) which are readily available. This was done to make the procedure and results available for testing and duplication in other laboratories. As in previous studies (11), EDTA was used to open the dentin tubules to a maximum extent. A difference between this device and that used in the Meryon and Brook study (9) was the volume of medium into which the diffusates were diluted. They used an apparatus in which any extracts which diffused through the dentin would be further diluted by 4 ml of tissue culture medium, approximately 20 times the volume of the cell-culture chamber in the present study. In the present study, only 0.5-mm discs were used, and the variable was the time over which the study was performed. The rank order remained the same for both the first and second 24-h periods, GLUMA being more cytotoxic than SB2. The ironic finding was that the materials were even more toxic the second 24 h than the first. This indicated that both materials continued to leach out soluble materials in an aqueous environment for at least 48 h. Ferracane and Condon (19) showed that for a bis-GMA/TEGDMA resin composite system (Silux), most of the water-soluble leachables could be extracted by 24 h, but leaching did continue through 72 h in vitro. There have not been studies of aqueous leaching of dentin bonding agents in the presence and absence of composite restorations, but we believe a similar prolonged leaching may occur. Whereas the present study focused on the cytotoxicity of GLUMA and SB2, it also gave an indication of concentrations of materials after diffusion across dentin. These concentrations are important because they may be sufficient to activate immune or complement systems or other biological reactions in addition to their cytotoxic effects. Pashley (6) has suggested that the effects of SB2 and GLUMA on healthy pulpal tissue are minimal because of rapid blood exchange in pulpal capillaries. However, for odontoblasts and other cells directly adjacent to the dentin tubules, as well as cells in inflamed pulps, the rate of exchange of extracellular fluid may not be as rapid and this may allow toxic concentrations of the extracted materials to accumulate. Pashley (6) has stated that in vitro studies of this sort represent the maximum limits or sensitivities of living test systems. Thus, if a material is placed in an in vitro system such as this and it does not exhibit microleakage or cytotoxicity, then there is a high probability that it will not cause clinical difficulties. If, however, there is some diffusion or cytoxicity, then it would seem that clinical success is in question. We would add that the concentrations of the diffusates are most important both in vitro and in vivo, and that attempts should be made to measure both starting concentrations and concentrations of diffusates in order to more effectively correlate causes and effects of all diffusible materials. This investigation was supported in part by USPHS Research Grant DE09296 and USPHS Training Grant DE-07101. Dr. Hanks, Dr. Wataha, R. Parsell, and S. Strawn are affiliated with the University of Michigan School of Dentistry, Ann Arbor, MI. Address requests for reprints to Dr. Carl Hanks, 5223 School of Dentistry, University of Michigan, 1011 N. University Avenue, Ann Arbor, MI 48109-1078,
References
1. Tao L, Pashley DH. Dentin perfusion effects on the shear bond strengths of bonding agents to dentin. Dent Mater 1989;5:181-4.
596
Hanks et al.
2. Ben-Amur A, Cardash HS. The fluid-filled gap under amalgam and resin composite restorations. Am J Dent 1991 ;4:226-30. 3. O'Brien WJ. Dental materials: properties and selection. Chicago: Quintessence Publishing Co., Inc., 1989:79. 4. Brannstrom M, Nyborg H. Pulpal reaction to composite resin restorations. J Prosthet Dent 1972;27:181-9. 5. Bergenholtz G, Cox CF, Loesche WJ, Syed SA. Bacterial leakage around dental restorations: its effect on the dental pulp. J Oral Pathol 1982;11:43950. 6. Pashley DH. Clinical considerations of microleakage. J Endodon 1990;16:70-7. 7. Council on Dental Materials, Instruments, and Equipment. Dentin bonding systems: an update. J Am Dent Assoc 1987;114:91-5. 8. Dumsha TC, Sydiskis RJ. Cytotoxicity testing of a dentin bending system. Oral Surg 1985;59:637-41. 9. Meryon SD, Brook AM. In vitro cytotoxicity of three dentine bonding agents. J Dent 1989;17:279-83. 10. Wataha JC, Hanks CT, Craig RG. The in vitro effects of metal cations on eukaryotic cell metabolism. J Biomed Mater Res 1991 ;25:1133-49. 11. Hanks CT, Craig RG, Diehl ML, Pashley DH. Cytotoxicity of dental composites and other materials in a new in vitro device. J Oral Pathol 1988;17:396-403.
Journal of Endodontics 12. HOrsted-Bindslev P. Monkey pulp reactions to cavities treated with GLUMA Dentin Bond and restored with a microfilled composite. Scand J Dent Res 1987;95:347-55. 13. Terkla LG, Mitchern JC, Mahler DB. Bonding of dentin adhesives in clinically-simulated occlusal resin composite restorations. Am J Dent 1991 ;4:214-8. 14. Mitchem JC, Gronas DG. Adhesion to dentin with and without smear layer under varying degrees of wetness. J Prosthet Dent 1991 ;66:619-22. 15. Hanks CT, Diehl ML, Craig RG, Makinen P-L, Pashley DH. Characterization of the "in vitro pulp chamber" using the cytotoxicity of phenol. J Oral Pathol Med 1989;18:97-107. 16. Hill SD, Berry CW, Seale NS, Kaga M. Comparison of antimicrobial and cytotoxic effects of glutaraldehyde and formocresol. Oral Surg 1991 ;71:89-95. 17. Hanks CT, Strawn SE, Wataha JC, Craig RG. Cytotoxic effects of resin components on cultured mammalian fibroblasts. J Dent Res 1991 ;70:1450-5. 18. Felton D, Bergenholtz G, Cox CF. Inhibition of bacterial growth under composite restorations following GLUMA pretreatment. J Dent Res 1989;68:491-5. 19. Ferracane JL, Condon JR. Rate of elution of leachable components from composite. Dent Mater 1990;6:282-7. 20. Erickson R. Surface interactions of dentin adhesive materials. Oper Dent (in press).
A Word for the Wise A singular misapprehension in modern English usage is failure to recognize that the following words are plural: criteria, data, phenomena, strata, sequelae, algae.
David Bishop
Printed in U.S,A,
VOL. 18, NO, 12, DECEMBER1992
Delineation of Cytotoxic Concentrations of Two Dentin Bonding Agents In Vitro C. T. Hanks, DDS, PhD, J. C. Wataha, DMD, PhD, R. R. Parsell, and S. E. Strawn, BSE
5), it is also of interest to know whether extracts from certain materials in contact with the floor of the cavity preparation are sufficiently concentrated to cause pulpal damage. During placement of the restoration, even if the adhesion of a bonding agent was incomplete, the presence of the bonding agent should cause reduction in conductance of fluids because of partial blockage of the dentinal tubules. After placement of the restoration, the mechanism for introduction of either bacterial products or extracts of restorative materials into the pulp would be diffusional permeability of the dentin. Aqueous continuity between the floor of the cavity and the pulp would allow establishment of a diffusion gradient between the restoration and the pulp (6). There have been relatively few articles on the biocompatibility of bonding agents in the literature. A recent review of dentin bonding systems indicates that these agents have little or no effect on pulpal tissues (7). Only two articles have focused on the cytotoxicity of bonding agents in tissue culture (8, 9). The present report focuses on the effects of two dentin bonding systems, GLUMA and SB2, and two ingredients, glutaraldehyde and 2-hydroxyethylmethacrylate, on cultured cells, both directly and indirectly through dentin, in an attempt to estimate whether these agents might partially contribute to pulpal injury in vivo.
Until adhesiveness of dentin bonding agents and other restorative materials to dental structures can be assured, microleakage into resulting "gaps" and dentin permeability will remain major concerns in cases of pulpal irritation. The objectives of the present study were to (a) delineate the kinds and levels of metabolic cytotoxicity of the GLUMA and Scotchbond 2 systems as well as glutaraldehyde and 2hydroxyethylmethacrylate, and (b) compare the effects of these same materials after diffusion through dentin discs approximately 0.5-ram thick. In monolayer cultures, glutaraldehyde was much more cytotoxic than 2-hydroxyethylmethacrylate. However, GLUMA sealer and Scotchbond 2 adhesive exhibited similar cytotoxicity in monolayer cultures. After diffusion through dentin, glutaraldehyde and 2-hydroxyethylmethacrylate effects were diluted 14.7 and 26.7 times, respectively. The postdiffusional effects of the GLUMA and Scotchbond 2 systems were not significantly different and less than those effects in monolayer cultures. This study should help in the evaluation of possible causes of pulpal irritation following restorative procedures.
MATERIALS AND METHODS Two dentin bonding systems and two ingredients of these systems were studied. These materials, their abbreviations, and their composition in moles per liter are given in Table 1. The cytotoxicity of these materials and components was compared in monolayer cultures of BALB/c 3T3 cells (clone A31; American Type Culture Collection, Rockville, MD) before and after diffusion through dentin in a modified "in vitro pulp chamber" (IVPC; Fig. 1). The cells were maintained and passaged in 75-cm 2 flasks in Dulbecco's modified Eagle's medium (with 4.5 g/liter dextrose; Flow Laboratories, McLean, VA) with 3 % donor calf serum and supplemented with glutamine and penicillin-streptomycin (Gibco Laboratories, Grand Island, NY).
One of the major rationales for development of dentin bonding agents is to eliminate the gap between the composite restoration and tooth structure. Two of the newer dentin bonding systems, GLUMA and Scotchbond 2 (SB2), call for the removal of the smear layer from the cavity preparation before placing the bonding agent. Tao and Pashley (1) have shown that after removal of the smear layer relatively low hydraulic pressure (32 cm of phosphate-buffered saline), representing dentin perfusion from pulpal tissue fluid pressure, significantly reduces shear bond strengths up to 48 h after initial polymerization. The lack of effective bonding of any restorative material to tooth structure in situ leads to numerous gaps between restoration and tooth structure (2). These gaps are responsible for microleakage by capillary action of various solutions and suspensions under restorations (3). Although bacteria and bacterial products have been implicated in pulpitis because of microleakage into contraction gaps (4,
Monolayer Tests For unpolymerized components of the bonding agents, monolayer cultures were plated at approximately 30,000 cells/
589
590
Hanks el al.
Journal of Endodontics
TABLE 1. Dentin bonding agents and components, manufacturers, control numbers and moles/liter
System
Material
Scotchbond 2 Batch P891208 (3M, St. Paul, MN)
GLUMA (Columbus Dental Division, Miles Inc., St. Louis, MO)
%
mol/liter
Scotchprep Dentin Primer
SP primer
Abbrev
HEMA
Component*
55.0
4.09t
Scotchbond 2 Light Cure Dental Adhesive
SB2 adhesive
Maleic acid HEMA
2.5 37.0
0.22~t 2.75t
bis-GMA
63.0
1.231:
GLUMA 3 Primer Batch 16MAR88
GLUMA primer
GLUT
5.0
0.49:~
GLUMA 4 Sealer Batch 29MAR88
GLUMA sealer
HEMA TEGDMA
35.0 45.0
2.601" 1.57:~
55.0
1.071:
Stock (concentrate) Batch 767
GLUT
70.0
6.99t
Stock (concentrate) Batch 404194
HEMA
97.0
7.21 t
bis-GMA Glutaraldehyde (Ladd Research Industries, Inc., Burlington, VT) 2-Hydroxyethylmethacrylate (Polysciences, Inc., Warrington, PA)
* Components of bonding agents from Erickson (20). t mol/liter calculated on basis of weight percent, i.e. g/liter divided by g/tool. mol/liter for HEMA was determined by correcting volume for density of HEMA (1,034 g/ml) and then calculated as above.
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FIG 1. This apparatus consists of a dentin disc attached to the bottom of a one-quarter-inch in diameter nylon cylinder (RSN-8/8; Small Parts, Inc.) by means of epoxy long-setting cement (Ross Epoxy Glue). The apparatus was dried for 2 h at 60°C and extracted with 70% ethanol for 12 h to remove unreacted reagents. The apparatus was then rehydrated with medium and set on top of a nylon washer (WN-3/8) in the bottom of a well of a 24-well dish that had been plated 24 h earlier with 30,000 BALB/c 3T3 cells in 200 pl of complete medium. The apparatus was used with the insert for testing diffusion from polymerized bonding agents as well as the active ingredients, GLUT and HEMA (see text for explanation).
cm 2 in wells (16 mm in diameter) of 24-well dishes (#3524; Costar, Cambridge, MA) and allowed to grow for 24 h before introducing the test materials. At that time (time 0), either
glutaraldehyde (GLUT) or 2-hydroxyethyl methacrylate (HEMA) was added to the media so that the final concentrations for GLUT ranged from 0.005 to 2.5 ~mol/liter, and the final concentration for HEMA ranged from 0.16 to 80 umol/ liter in the experimental wells. These ranges varied from concentrations which had no toxic effect to those which depressed all measurable metabolic activity. For each time period after the addition of the test substances (0, 8, and 24 h), six wells for each concentration of each test substance plus six wells for untreated controls were harvested. At the end of the 24-h test period, medium was changed to fresh medium in six additional wells for each concentration of component, as well as six additional control wells, to determine whether cell metabolism could recover in the next 24 h (reversibility of cytotoxic effects). After harvesting, the cultures were assessed for DNA synthesis, protein synthesis, succinyl dehydrogenase activity, and total protein content of the wells. Either [3H]leucine or [3H] thymidine was added at 90 and 45 min, respectively, prior to harvest as 20-#1 aliquots in simple medium to "pulse" label the cultures (10). The final concentration of each isotope was 5 uCi/ml in each well. Stock radioisotope solutions had specific activities of 64 Ci/mmol for [3H]leucine or 6.7 Ci/ mmol for [3H]thymidine (ICN Radiochemicals, Irvine, CA). To assure that the lack of thymidine did not limit the rate of DNA synthesis, the total thymidine including radioisotopelabeled thymidine was brought to 5 pmol/liter with cold thymidine for the 90-rain pulse. The protein synthetic assays and total protein content were performed on the same wells. The DNA synthetic assays and the succinyl dehydrogenase assay utilizing 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) as the tetrazolium salt were performed on separate but parallel wells because the processing sequences for the assays were different. When harvesting cells, the medium was suctioned off and the wells were processed as described previously (10). Discs (2.95 mm in diameter; 3.36-mm thickness) of GLUMA sealer and SB2 adhesive were prepared by curing
Vol. 18, No. 12, December 1992
layers of approximately 1-mm thickness sequentially. The discs were placed in the center of each well (3.8 cm in diameter) of 6-well polystyrene dishes (#3406; Costar). Ethanol-washed nylon discs of the same size were used as control samples. After placement of the discs, BALB/c 3T3 cells were plated in a monolayer around the discs at 25,000 cells/cm 2 in 3 ml of complete medium. At the time of harvest, the discs of bonding agents were removed and the MTT assay for succinic dehydrogenase activity was run on each well. Finally, in a separate experiment, GLUMA sealer and SB2 adhesive were spread on the bottom of wells of 24-well dishes and polymerized and were then washed for 18 h with 60% ethanol and tested for growth of cells on the surfaces of the agents. Diffusion Studies Freshly extracted human molar teeth without carious lesions were cleaned of debris and periodontal ligament tissue and placed in 70% ethanol for 24 h before their use. Dentin discs were cut in cross-section from the widest part of the crowns and as close to the pulp as possible without cutting through pulp horns. The discs were cut at 230 rpm with a low-speed wheelsaw using 10.2-cm diameter diamond wafering blades (CO-153, 320 grit; Mager Scientific Inc., Dexter, MI), followed by hand sanding with 400 and then 600 grit silicon carbide paper each for approximately 20 figure-eight rotations. The discs were treated for 2 min with 0.5 M EDTA (pH 7.4) and rinsed with double distilled H20 before hydraulic conductance (Lp) values were determined as described previously (11). The modified IVPC (Fig. 1) consisted of a 0.49-cm inside diameter nylon spacer (RSN-10/8; Small Parts, Inc., Miami, FL) which was attached to the coronal surface of the dentin disc by means of epoxy long-setting cement (Ross Epoxy Glue; Ross Adhesives/Control Corp., Detroit, MI). This apparatus was dried for 2 h at 60"C and extracted with 70% ethanol for 12 h to remove unreacted epoxy ingredients. Each apparatus was rehydrated with medium and set on top of a nylon washer (WN-3/8; Small Parts, Inc.) in the bottom of a well of a 24-well dish which had been plated 24 h earlier with 30,000 BALB/c 3T3 cells in 200 #1 of complete medium. After 24 h of exposure to the test materials, the ceils in the bottom of the well were tested for succinyl dehydrogenase (MTT) activity. In the first diffusion experiment, each of the two dentin bonding systems was utilized with a separate set of six dentin discs in the modified IVPC (Table 2). Dentin discs used for the GLUMA system had an average (SD) thickness of 0.485 (0.043) mm and the average (SD) Lp of 0.016 (0.006 ~1 cm -2 rain -l cm H20 -l. Dentin discs for the SB2 system had an average thickness of 0.485 (0.037) mm and an average (SD) Lp of 0.014 (0.006) ~1 cm -2 min -1 cm H20 -l. In both cases, the bottom of cylindrical nylon spacers (RSN-4/2; Small Parts, Inc.) were filled with Silux composite (3M, Minneapolis, MN). These spacers, which have an outside diameter of 0.47 cm and an inside diameter of 0.29 cm, are shown as an insert for the larger spacers in Fig. 1 and were used as carriers for the bonding/adhesive systems. In the case of the SB2 system, the Silux was polymerized as described by the manufacturer. Then, the SP primer was painted onto the bottom
Dentin Bonding Agents
591
of the insert, allowed to set for 30 s, and blown dry before application and polymerization of the SB2 adhesive. For the GLUMA system, Silux (1-mm thick) was placed in the insert and left unpolymerized prior to placement of the GLUMA primer and sealer. GLUMA primer was applied to the bottom of the insert and gently dried before application of GLUMA sealer. The GLUMA/Silux systems were then polymerized together. Both bonding systems were polymerized with a Prismetics Lite (Caulk/Dentsply, Milford, DE) for 1 min at a distance of approximately 4 mm. Finally, 20 ul of medium were added to the top of each dentin disc inside the larger nylon spacer just before placing the insert into position. The insert was placed inside the larger spacer against the dentin disc (Fig. 1) for the first 24 h immediately after polymerization. After this period, each apparatus with the insert in place was moved to a fresh well for a second 24 h. After each exposure period, the cells in each experimental well (six for each agent) and control wells (six) were tested for MTT activity and compared with untreated controls. The results were reported as a percentage of negative controls. Paired Student's t tests were used to compare the cytotoxicity data for each bonding procedure to the other bonding procedure at both time periods as well as each bonding procedure to itself at 1 and 2 days after polymerization. In a second experiment, either 80 ~mol/liter HEMA or 2.5 umol/liter GLUT were placed in the top reservoir of the modified IVPC (Fig. 1) on top of each of four dentin discs with an average (SD) thickness of 0.5 (0.04) mm for each experimental material. No inserts were used in this experiment. An estimation of the diffusion of these reagents was interpolated by the comparison of the level of metabolic responses (MTT) of the cell test system to the response curve for various concentrations of the same substances by cells in monolayer after 24 h of exposure. RESULTS GLUT in culture medium was tested in monolayer cultures at final concentrations between 0.005 and 2.5 umol/liter. As compared with the control wells, it gradually and increasingly inhibited DNA and protein synthesis, as well as mitochondrial respiration and total protein solubility as the concentration increased (Figs. 2 and 3). The effects of concentrations through 0.02 #mol/liter were reversible for all four parameters measured when fresh medium was added between 24 and 48 h. When the cellular response data were graphed as optical density of the MTT response plotted against GLUT concentration, 2.5 umol/liter GLUT depressed the mitochondrial enzyme response by 99.9%, and the dose of GLUT which was toxic to half the cell monolayer (TCs0) was 0.04 #tool/ liter (Fig. 4). On the other hand, when 2.5 umol/liter GLUT was placed on the 0.5-mm dentin disc (Fig. 1) opposite 200 ~1 of medium which in turn nourished a cell monolayer, the inhibitory effect on MTT activity was reduced (Fig. 4). After diffusion through dentin, 2.5 umol/liter GLUT inhibited MTT activity by 88%. Because we had not developed an assay for concentrations of these chemicals, we interpolated the average concentration of GLUT in the diffusates from the GLUT dose-response curve for monolayer cultures. Therefore, the 88% MTT activity of the diffusate matched the GLUT concentration of about 0.17 #mol/liter, which was a
592
Journal of Endodontics
Hanks et al.
TABLE 2. Testing of GLUMA and SB2 in in vitro pulp chamber
Disc
Thickness
Lp
(mm) GLUMA A1 A2 A3 A4 A5 A6
5~(IO
% of MTT Activity First 24 h
Second 24 h
0.469 0.946 0.760 0,471 0.771 0.634
0.358 0.510 0.348 0.450 0.335 0.321
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0.0200 0.0100 0.0100 0.0240 0.0120 0.0220
Average
0.485
0.0160
0.668
0.387
SD
0.043
0.0060
0.118
0.076
SB2 B1 B2 B3 B4 B5 B6
0.540 0.460 0.480 0.520 0.450 0.460
0.0180 0.0100 0.0180 0.0035 0.0150 0.0210
0.634 0.406 1.160 0.779 0.777 0.571
0.563 0.389 0.377 0.399 0.368 0.606
Average SD
0.485 0.037
0.0140 0.0060
0.721 0.256
0.450 0.105
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dilution of 14.7 times when compared with the concentration applied at the top of the gradient. For the first 24 h in culture, increasing concentrations of HEMA had increasingly inhibitory effects on each metabolic parameter (Figs. 5 and 6). From 24 to 48 h, even after HEMAcontaining medium was replaced by fresh medium, HEMA concentrations of 0.8/~mol/liter and above allowed no recovery of DNA synthesis and 4.0 ~mol/liter HEMA irreversibly inhibited both protein synthesis and mitochondria activity. The cell mass (measured by total protein) failed to show substantial recovery in cells treated with 4.0 ~mol/liter or more HEMA. When the cellular response data were graphed as optical density of MTT response plotted against HEMA concentration, 16 pmol/liter HEMA depressed MTT activity by about 99.1% during the first 24 h of incubation and 80 ~mol/liter HEMA depressed MTT activity by 99.3%. The TCs0 value for HEMA with this cell test system was 1.1 ~mol/ liter (Fig. 7). When 80 #mol/liter HEMA was placed on the 0.5-mm dentin disc rather than directly into the monolayer culture medium, again there was less inhibition of MTT activity in the cell test system. After diffusion through dentin, the HEMA diffusate inhibited only 84.3% of the MTT activity and the interpolated concentration was 3.0 #mol/liter or a dilution of 26.7 times when compared with the concentration applied to the top of the gradient. When pellets of GLUMA sealer and SB2 adhesive were centered in 3.8-cm diameter wells with monolayer cultures, each material produced a ring of inhibition of approximately 1 cm around the test material. Within this ring, a large number of cells were missing and most cells which remained exhibited rounding and other cytopathic effects. The ceils on the periphery of the well appeared to be as healthy as those in the control dish. A sharp transitional zone was present between the ring of inhibition and the healthy peripheral cells. The average outside diameter (n = 6) representing the MTT activity of the remaining cells in each well was 47.8% of the negative controls for SB2 adhesive and 49.4% of controls for
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FtG 2. The effects of GLUT concentrations (0.005 to 2.5 ~mol/liter), in comparison to those of untreated controls (0 ;Lmol/liter), on (a) DNA synthesis ([3H]thymidine incorporation into DNA) or (b) protein synthesis ([3H]leucine incorporation into protein) in monolayer cultures of BALB/c 3T3 cells. At time 0, medium with or without GLUT was added to each well. Each point represents the mean concentration and standard error of the mean (vertical bars) of six wells. At 24 h (arrow), fresh medium without GLUT was placed into sets of treated wells for the next 24 h to determine whether the effects were reversible.
GLUMA sealer. This difference was not statistically significant. In experiments which tested diffusion of materials from freshly polymerized dentin bonding systems through dentin, the same set of dentin discs was used to measure diffusion of GLUMA-related materials through dentin for the first and second 24-h periods. Likewise, the same set of discs were used to measure diffusion of SB2-related materials during both time periods. As shown in Table 2, there was an attempt to match average thickness and Lp for the two sets of discs. Two observations were made concerning these comparisons. First, the GLUMA system depressed MTT activity more than did the SB2 system during both time periods. Thus, for the first 24 h, the G L U M A system and the SB2 system allowed about 66.8% and 72%, respectively, of the MTT activity found in the control wells. However, there was no significant difference (p > 0.05) between the two values. For both the GLUMA system and the SB2 system, MTT activity was significantly lower (p < 0.05) for the second 24 h. Again, there was no significant difference (p < 0.05) between the effects of these two agents.
Dentin Bonding Agents
Vol. 18, No. 12, December 1992
593
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for 18 h. However, they could not be cultured on surfaces of the unwashed resin controls.
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Finally, the observation was made that ceils could be plated and cultured on sheets of.either GLUMA sealer and SB2 adhesive after these sheets had been washed in 70% ethanol
Animal usage studies and microleakage studies (4, 5) suggest that dentin permeability and lack of marginal integrity of restorations are responsible for pulpal reactions, and that the presence of bacteria or their products between the composite and the walt of the cavity preparation is a larger problem than leachables from restorative materials. With ample evidence for bacterial mieroleakage, why should we be concerned with the cytotoxicity of dentin bonding agents? There are two reasons for this concern. First, there is some in vivo evidence of cytotoxicity of the GLUMA bonding system. Second, there is evidence that dentin thickness (length of the diffusion gradient) is important. In a study by H6rsted-Bindslev (12), it was reported that if the dentin was less than 0.1mm thick, a "toxic" reaction could be observed in the pulp of a cavity which had been pretreated with the GLUMA system. This suggested that a pulpal wall of greater than 0.1 mm was necessary for dentinal tubules to adsorb GLUMA system-associated leachables present in tubular fluid. Meryon and Brook (9) used 0.1- and 0.5-mm-thick dentin slices to test cytotoxicity (by cell number) of three dentin bonding systems: SB2, GLUMA, and Tripton (ICI Dental) on baby
594
Journal of Endodontics
Hanks et al. 2.5
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hamster kidney fibroblasts after diffusion through dentin. They found that all the materials were significantly cytotoxic, as compared with controls, through both thicknesses of dentin. With 0.1-mm discs, GLUMA was most cytotoxic, with
only 26.9% viable cells remaining in the tissue culture dish as compared with untreated controls. SB2 was next most toxic, with 54.2% of controls and Tripton was least toxic (68.8% of controls). However, with 0.5-mm discs, the rankings of GLUMA and SB2 were reversed. Tripton, GLUMA and SB2 gave 70.8%, 66.6%, and 52.6% of the negative controls, respectively. In the present study, cytotoxicity tests of two ingredients of the two bonding systems in monolayer cultures identified the concentrations above which connective tissue cells were irreversibly damaged. GLUT, at a concentration of 2.5 pmol/ liter and above, was irreversibly inhibitory to all four metabolic parameters tested (Figs. 2 and 3). For HEMA, 16 umol/ liter and above were irreversibly inhibitory for all four metabolic parameters (Figs. 5 and 6). With this information, it is possible to predict which concentrations of these materials, if they reach the pulp, are capable of causing metabolic depression or death of exposed pulpal cells. In the experiments which tested diffusion of the dentin bonding systems, each agent was applied to the insert (Fig. 1), not to the dentin. This was done to keep the dentinal tubules patent for diffusion. The experiments were not designed to test how the dentin bonding systems reduced dentin permeability, but rather to test the extent of diffusability of GLUT, HEMA, and unidentified leachables from the GLUMA and SB2 bonding systems. If the agents had been painted onto the dentin, the resins may have penetrated variable distances into the dentin, altering the diffusion gradients for any leachable components. This present system may simulate areas of bonded dentin which have just become debonded because of forces of polymerization contraction under physiological pulpal pressures (13, 14). From Table 1, it can be seen that GLUT is an ingredient only of GLUMA primer, but that HEMA is found in SP primer, SB2 adhesive, and GLUMA primer. The molecular weight (mol wt) of GLUT (100.12) is approximately the same as that of phenol (94.11). Hanks et al. (15) reported that when 0.009 M phenol was applied to 0.5-mm thick dentin discs, phenol was diluted approximately 500 times. In this previous study, the cell chamber had a volume of 1.87 ml, in contrast to 0.2 ml in the present cell chamber. If we assume similar diffusion characteristics for GLUT and phenol, then we would expect the present cell chamber to have diluted GLUT only about 50-fold (0.2/1.87 = 0.107; 500 x 0.107 = 53.5). Assuming this dilution factor, the concentration of GLUT which was painted on the dentin (5% or 0.49 mol/liter) should have been diluted to 9.8 × 10 -3 mol/liter in the cell chamber. This level is over 1000 times greater than the concentration of GLUT which will completely inhibit cell metabolism (Figs. 2 and 3). These assumptions should be verified by means of radioisotope experiments. Previous studies (16) have suggested that GLUT-containing bonding agents are desirable because they kill residual bacteria to which the pulp is exposed. These concentrations are also capable of killing mammalian cells and fixing pulpal tissue. Likewise, the concentrations of HEMA (mol wt = 130.14) in GLUMA primer (2.6 mol/liter), SP primer (4.09 mol/ liter), and SB2 adhesive (2.75 mol/liter), if diluted 50-fold by simple diffusion, would give HEMA concentrations of 5.2 x 10-2, 8.2 x 10-2, and 5.5 x 10 2 tool/liter, respectively. Figures 5 and 6 show that HEMA completely inhibits metabolic activity at 16 umol/liter, which is four orders of mag-
Vol. 18, No. 12, December 1992
nitude less than the potential concentrations of HEMA in the diffusates from 0.5-mm dentin. Maleic acid is also a component of SP primer. Using HEMA in the absence of maleic acid may even underestimate the cytotoxicity of SP primer. Finally, Hanks et al. (15) found a 2500-fold dilution of phenol across dentin discs 1.4-ram thick. Even a 2500-fold dilution would not have lowered the concentration of 35% HEMA to nontoxic levels. There is some suggestion of agreement between data from the present study and that of the phenol study (15). In the present study, Figs. 4 and 7 show that the GLUT concentration, measured by means of a biological assay (MTT), fell about 15-fold and the HEMA concentration fell about 27fold after diffusion through the 0.5-mm dentin. In this same chamber, if we correct for volume, phenol concentration would have fallen in the same order of magnitude, 53.5-fold. Several other components of dentin bonding systems are important. Bis-GMA (tool wt = 512.65) is present in SB2 adhesive (at 1.23 tool/liter) and GLUMA sealer (at 107 mol/ liter). Triethylene glycol dimethacrylate (TEGDMA) (tool wt = 286.36) is present in GLUMA sealer at 1.57 mol/liter. Hanks et al. (17) have shown that although the molecular weight of bis-GMA is almost twice as large as that of TEGDMA, the concentration of bis-glycidyl methacrylate (bis-GMA) (25 /~mol/liter) necessary for irreversible depression of DNA and protein synthesis was at least an order of magnitude less than the necessary concentration of TEGDMA (450 umol/liter). The following relationship holds for the relative cytotoxicities for all four molecules in these two bonding systems in terms of IDso: GLUT (0.04 umol/liter) > HEMA (1.1 tzmol/liter) > bis-GMA (13 to 16 ttmol/liter) > TEGDMA (70 to 100 ~zmol/liter). Diffusion characteristics of these molecules through dentin are being studied currently. Usage studies do not routinely report much pulpal reaction to dentin bonding agents containing GLUT and/or HEMA which cannot be attributed to bacteria (18). Why are there such discrepancies between diffusion data in vitro and usage studies? This may be either because certain variables have not been controlled in the in vitro experiments, or that in vivo conditions are different. Examples of uncontrolled variables may include (a) the time the primers or sealer/adhesives are allowed to stand on the patent dentin before drying or polymerization (this is diffusion time), (b) the depth of polymerization of HEMA, (c) the degree of conversion of HEMA to poly-HEMA and of bis-GMA and TEGDMA to polymerized resin, (d) the degree of total obstruction of the tubules because of polymerized materials, (e) the effect of the wet environment (tissue fluid or GLUT) upon polymerization, (f) the amount of adsorption of any of these agents to the walls of the tubules, and (g) the different time frames for in vitro studies and usage studies. In the present study, cells were found to grow on the resin surfaces of both GLUMA sealer and SB2 adhesive, which had been polymerized to the bottom of tissue culture wells, only if the surfaces had been washed for several hours with 70% ethanol. This suggested that the toxic material(s) were derived from the poorly polymerized oxygen-inhibited layer of the resins. Several aspects of the current study were designed to improve the measurement of cytotoxicity leachables following diffusion through dentin. The IVPC concept was adapted for a 24-well dish and utilized nylon washers and spacers (insert,
Dentin Bonding Agents
595
Fig. 1) which are readily available. This was done to make the procedure and results available for testing and duplication in other laboratories. As in previous studies (11), EDTA was used to open the dentin tubules to a maximum extent. A difference between this device and that used in the Meryon and Brook study (9) was the volume of medium into which the diffusates were diluted. They used an apparatus in which any extracts which diffused through the dentin would be further diluted by 4 ml of tissue culture medium, approximately 20 times the volume of the cell-culture chamber in the present study. In the present study, only 0.5-mm discs were used, and the variable was the time over which the study was performed. The rank order remained the same for both the first and second 24-h periods, GLUMA being more cytotoxic than SB2. The ironic finding was that the materials were even more toxic the second 24 h than the first. This indicated that both materials continued to leach out soluble materials in an aqueous environment for at least 48 h. Ferracane and Condon (19) showed that for a bis-GMA/TEGDMA resin composite system (Silux), most of the water-soluble leachables could be extracted by 24 h, but leaching did continue through 72 h in vitro. There have not been studies of aqueous leaching of dentin bonding agents in the presence and absence of composite restorations, but we believe a similar prolonged leaching may occur. Whereas the present study focused on the cytotoxicity of GLUMA and SB2, it also gave an indication of concentrations of materials after diffusion across dentin. These concentrations are important because they may be sufficient to activate immune or complement systems or other biological reactions in addition to their cytotoxic effects. Pashley (6) has suggested that the effects of SB2 and GLUMA on healthy pulpal tissue are minimal because of rapid blood exchange in pulpal capillaries. However, for odontoblasts and other cells directly adjacent to the dentin tubules, as well as cells in inflamed pulps, the rate of exchange of extracellular fluid may not be as rapid and this may allow toxic concentrations of the extracted materials to accumulate. Pashley (6) has stated that in vitro studies of this sort represent the maximum limits or sensitivities of living test systems. Thus, if a material is placed in an in vitro system such as this and it does not exhibit microleakage or cytotoxicity, then there is a high probability that it will not cause clinical difficulties. If, however, there is some diffusion or cytoxicity, then it would seem that clinical success is in question. We would add that the concentrations of the diffusates are most important both in vitro and in vivo, and that attempts should be made to measure both starting concentrations and concentrations of diffusates in order to more effectively correlate causes and effects of all diffusible materials. This investigation was supported in part by USPHS Research Grant DE09296 and USPHS Training Grant DE-07101. Dr. Hanks, Dr. Wataha, R. Parsell, and S. Strawn are affiliated with the University of Michigan School of Dentistry, Ann Arbor, MI. Address requests for reprints to Dr. Carl Hanks, 5223 School of Dentistry, University of Michigan, 1011 N. University Avenue, Ann Arbor, MI 48109-1078,
References
1. Tao L, Pashley DH. Dentin perfusion effects on the shear bond strengths of bonding agents to dentin. Dent Mater 1989;5:181-4.
596
Hanks et al.
2. Ben-Amur A, Cardash HS. The fluid-filled gap under amalgam and resin composite restorations. Am J Dent 1991 ;4:226-30. 3. O'Brien WJ. Dental materials: properties and selection. Chicago: Quintessence Publishing Co., Inc., 1989:79. 4. Brannstrom M, Nyborg H. Pulpal reaction to composite resin restorations. J Prosthet Dent 1972;27:181-9. 5. Bergenholtz G, Cox CF, Loesche WJ, Syed SA. Bacterial leakage around dental restorations: its effect on the dental pulp. J Oral Pathol 1982;11:43950. 6. Pashley DH. Clinical considerations of microleakage. J Endodon 1990;16:70-7. 7. Council on Dental Materials, Instruments, and Equipment. Dentin bonding systems: an update. J Am Dent Assoc 1987;114:91-5. 8. Dumsha TC, Sydiskis RJ. Cytotoxicity testing of a dentin bending system. Oral Surg 1985;59:637-41. 9. Meryon SD, Brook AM. In vitro cytotoxicity of three dentine bonding agents. J Dent 1989;17:279-83. 10. Wataha JC, Hanks CT, Craig RG. The in vitro effects of metal cations on eukaryotic cell metabolism. J Biomed Mater Res 1991 ;25:1133-49. 11. Hanks CT, Craig RG, Diehl ML, Pashley DH. Cytotoxicity of dental composites and other materials in a new in vitro device. J Oral Pathol 1988;17:396-403.
Journal of Endodontics 12. HOrsted-Bindslev P. Monkey pulp reactions to cavities treated with GLUMA Dentin Bond and restored with a microfilled composite. Scand J Dent Res 1987;95:347-55. 13. Terkla LG, Mitchern JC, Mahler DB. Bonding of dentin adhesives in clinically-simulated occlusal resin composite restorations. Am J Dent 1991 ;4:214-8. 14. Mitchem JC, Gronas DG. Adhesion to dentin with and without smear layer under varying degrees of wetness. J Prosthet Dent 1991 ;66:619-22. 15. Hanks CT, Diehl ML, Craig RG, Makinen P-L, Pashley DH. Characterization of the "in vitro pulp chamber" using the cytotoxicity of phenol. J Oral Pathol Med 1989;18:97-107. 16. Hill SD, Berry CW, Seale NS, Kaga M. Comparison of antimicrobial and cytotoxic effects of glutaraldehyde and formocresol. Oral Surg 1991 ;71:89-95. 17. Hanks CT, Strawn SE, Wataha JC, Craig RG. Cytotoxic effects of resin components on cultured mammalian fibroblasts. J Dent Res 1991 ;70:1450-5. 18. Felton D, Bergenholtz G, Cox CF. Inhibition of bacterial growth under composite restorations following GLUMA pretreatment. J Dent Res 1989;68:491-5. 19. Ferracane JL, Condon JR. Rate of elution of leachable components from composite. Dent Mater 1990;6:282-7. 20. Erickson R. Surface interactions of dentin adhesive materials. Oper Dent (in press).
A Word for the Wise A singular misapprehension in modern English usage is failure to recognize that the following words are plural: criteria, data, phenomena, strata, sequelae, algae.
David Bishop
