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JOURNAL OF ENDODONTICS Copyright 9 1991byThe American Associ~ionofEndodontists
VOL. 17, NO. 11, NOVEMBER1991
Root Canal Sealer Cytotoxicity on Human Gingival Fibroblasts: II. Silicone- and Resin-based Sealers Benjamin M Brisefio, Dr. Med. Dent., and Brita Willershausen, Dipl. Chem., Dr. Med. Dent.
reports (10, 14) regarding the cytotoxicity of Lee Endo-Fill, a silicone-based root canal sealer. The purpose of this study was to test the toxic effects of these types of filling materials on human gingival fibroblasts by measuring the capability of the fibroblasts to synthesize proteins after being in contact with the sealers for a period of up to 21 days.
The cytotoxicity of two resin (AH26 and Diaket)- and one silicone (Lee Endo-Fill)-based root canal sealers were tested over a period of 21 days. The incorporation rate of L-[l*C]leucine was measured to establish the potential of human gingival fibroblasts to synthesize protein after being in contact with the sealers. The cells were gained from biopsies from healthy patients between 20 and 30 years of age. The sealers were allowed to set for 24 and 48 h before dispensing the fibroblastic suspension into the culture dishes. AH26 showed in the 24-h group a severe cytotoxic reaction during the entire experimental period. Diaket showed a relative severe cytotoxic potential with slight cell recovery after 3 days of culturing. Lee Endo-Fill showed a significantly lower (p < 0.05) cytotoxic potential during the first 11 days of incubation. However, between the 15th and 21st days of culturing an ascending cytotoxic potential was registered. With Diaket and Lee Endo-Fill a relative lower cytotoxic response was observed in the 48-h group; however, cytotoxic patterns similar to the 24-h group prevailed.
MATERIALS AND METHODS
Source of Tissues Biopsies were gained under aseptic precautions from healthy male and female patients between 20 and 30 years of age from the attached gingiva at the lower molar region. No lesions or signs of inflammation were present in the tissues during the collection of the biopsies. The different cell lines were cultivated up to the 20th passage without being pooled.
Gingival Fibroblasts Culture The fibroblasts were explanted into cultures in 6-cm O Petri dishes (Falcon; Becton Dickinson Labware, Lincoln Park, N J) according to the method of Martin (15) and stored in 250-ml culture bottles (Nunc; Nunc GmbH, Wiesbaden, Germany). Between the fifth and sixth day of cultivation, a trypsin EDTA solution (0.05/0.02%), in phosphate-buffered saline without Ca-'+,Mg2§ (Seromed; Seromed Biochrom-Produkte, Wiesbaden, Germany), was added for 10 to 15 min at 37"C by transferring the fibroblasts from one culture bottle to another in a 1:2 relation. Eagle's basal medium (Grand Island Biological Co., Grand Island, NY) supplemented with 10% fetal calf serum (Grand Island Biological Co.), 50 IU of penicillin per ml (Seromed), and 50 #g of streptomycin per ml (Seromed) solution was used. The cells were cultured in 250-ml plastic flasks (Nunc) in a water vapor saturated atmosphere at 37"C in a mixture of 5% CO2 and 95% air (type B 5060 EC; Heraeus, Osterode, Germany). The culture medium was changed after 24 h. Mycoplasma type infection was prevemed by using the method of Cinatl and Tolar (16). Periodical observation of the cultures in a phase contrast microscope (MPS50; Leitz-Wild, Wetzler, Germany) showed no bacterial growth during the entire research period. The fibroblasts were subcultivated 3 to 4 days after they had achieved confluence. Only normal diploid fibroblasts from
The adequate combination of sealing ability and biocompatibility of a root canal sealer is important for a favorable prognosis in root canal treatment. Therefore, the chemical tissue irritation effect is most important in considering sealer properties before selecting one. The sealing qualities of resinbased sealers are described as equal or better than those of other types. However, it should be made clear that if a root canal has not been properly cleaned and shaped, the sealing properties of a root canal sealer cannot impro,Je the results of the treatment. Furthermore, another source of treatment failure can come from sealers containing toxic components included with the aim of counteracting the effects of poor root canal debridement. Such sealers might be only capable of masking objective treatment failure symptoms. Many researchers have tested the cytotoxic properties of resin-based sealers in animal implants (1-6) and with different types of cell cultures (6-12). Although Biggs et al. (13) used a silicone elastomer as implant control, there have been few
537
538
Journal of Endodontics
Brisefio and Willershausen
four cell lines between the fifth and seventh passage were used after having analyzed the form and number of the cells' chromosomes (17). Fresh culture medium was added two to three times a week. Research Materials The following materials were tested: (a) Lee Endo-Fill (Lee Pharmaceuticals, E1 Monte, CA) which is according to the manufacturer a self-curing medical silicone rubber with a high percentage of bismuth subnitrate. Its exact composition was not obtained from the manufacturers. (b) Diaket (Espe, Seefeld, Germany), a polyketone sealer. One gram of powder contains zinc oxide 0.7 g. One gram of solution contains dichlorophen (0.005 g), triethanolamine (0.002 g), and propionylacethophenone (0.76 g). (c) AH26 (De Trey, FreresZurich, Switzerland), an epoxy resin root canal sealer. The powder contains silver powder (10%), bismuth oxide (60%), hexamethylenetetramine (25%), and titanium oxide (5%). The fluid contains epoxybisphenol-resin 100%. Protein Synthesis The measurement of the incorporation rate of L-[~4C]leucine (55 tzCi/nmol; Amersham Buchler, Buckinghamshire, England) in an acid precipitation material was used to determine the fibroblasts' protein synthesis potential. After having cultured the fibroblasts along with the different sealers for different periods of time, they were marked with L-[~4C] leucine (0.2 #Ci/ml) for 2 h after being rinsed with a 3-ml buffered phosphate physiological saline solution (Biochrom, Berlin, Germany). Following the method of Mans and Novelli (18), the incorporation of L-[~4C]leucine was stopped by precipitation in a 3-ml 7.5% trichloroacetic acid solution. The fibroblasts were then passed through a glass fiber filter (Whatman GF/C O 2.5 cm; Whatman Ltd., Maidstone, England) after being loosened with a rubber policeman and rinsed subsequently with 2 ml of 7.5% trichloroacetic acid. The radioactive fibroblastic material that remained in the filter was rinsed twice with 7.5% trichloroacetic acid and 95% ethanol and air dried. The fibroblasts' protein content was determined according to the method of Lowry et at. (19) (27 ~g/dish). The radioactivity of the cell material was measured in a 10-ml toluene scintillation solution (Quickszint 501; Zinsser Analytic, Frankfurt, Germany) with a liquid scintillation spectometer (Tri-Carb 1500; Packard GmbH, Frankfurt, Germany). Research Procedures The root canal sealers were placed into 18-mm 0 multiwell tissue culture plates (Falcon 3040; Becton Dickinson Labware) after being mixed under aseptic conditions according to the manufacturer's instructions. The sealer amount was weighed (___0.1 mg) with an analytic balance (Mettler H3; Mettler, Zurich, Switzerland). The sealer amount could be accurately calculated by subtracting the total weight of the sample in the multiwell from the weight of the previously registered sample(s). The weight of the sealers varied between 2.2 and 2.6 mg in the 24-h group and 2.1 and 2.4 mg in the
48-h group. The appropriate amount was determined by a pilot study (7). Possible bacterial growth during setting was prevented by allowing the sealers to set respectively for 24 and 48 h under ultraviolet light at 37"C. Approximately 104 cells were dispensed into each dish after having rinsed the sealers three times with physiological saline. The cell suspension was stained with trypan blue in order to facilitate cell counting (Fuchs-Rosenthal counting chamber). The multiwells were incubated for a 21-day period under the conditions described previously. A diffusion contact was established between the materials and the fibroblasts after they sedimented and attached to the flee bottom surface of the dishes. The culture medium was changed every second or third day. Evaluation Parameters Measurements of the incorporation rate of L-[14C]leucine were made in a total of 140 samples of each sealer. Seventy of them were tested 24 h after having set on the 1st, 3rd, 5th, 10th, 12th, 14th, and 21st day of culturing (n = 10 each). The other 70 samples were distributed in the same manner in a 48-h group after having set. The results express the means and standard deviations. Three dishes (per multiwell) cultured with gutta-percha (12 mm; Vereinigte Dentalwerke 0 40, Munich, Germany) and another four dishes with only fibroblasts served as controls in the 24-h as well as in the 48-h groups. RESULTS Through periodical microscopic observation it could be ascertained that the fibroblasts were viable at all times. No significant differences were found between the L-[14C]leucine incorporation rates of the controls with gutta-percha and fibroblasts only. However, only cultures with fibroblasts without foreign materials were used as controls. The mean values of L-[14C]leucine incorporation into the controls was determined as 100% (Table 1). Statistically (Student's t test) significant differences (p < 0.05) were found between Lee Endo-Fill and Diaket and Lee Endo-Fill and AH26 from the 1st to the 1lth day of culturing in the 24- and 48-h groups. A statistically significant difference was also found between Diaket and AH26 in the 48-h group between the I st and 5th day of culturing. At the beginning AH26 showed a relative high cytotoxicity in the group that was covered with fibroblasts 24 h after TABLE 1. Incorporation rate values of [L-l*C]leucine (cpm/mg protein) at the different experiment testing periods
Day
Incorporation rates (cpm/mg protein _+ SD)
1 3 5 11 13 15 21
33,475 _+ 1,150 32,970 +_ 2,189 34,213 + 1,690 31,867 + 2,935 32,033 _+ 4,549 37,013 _+ 6,089 38,567 +_ 4,744
Vol. 17, No. 11, November 1991
Sealer Toxicity
allowing to set. At the 5th day slight recovery was observed. After the 15th day almost no fibroblast protein synthesis could be measured (Table 2). Up to the 1 lth day the fibroblasts showed a slightly higher protein synthesis potential with Diaket in comparison to AH26. After the 1 lth day an ascending cytotoxic potential tendency without recovery was measured (Table 2). Lee Endo-Fill showed moderate cytotoxicity until between the 5th and the 1 lth day. After the 1 lth day a decrease in the capability of the fibroblasts to synthesize protein was observed. After the 15th day almost no more fibroblast protein synthesis potential could be measured (Table 2). The group in which the fibroblastic suspension was dispensed 48 h after mixing showed in the case of AH26 a less cytotoxic potential in comparison to the 24-h group (Table 3). No statistically significant differences resulted, however, between both groups with this material. Diaket showed in the beginning phase a relative moderate cytotoxicity, but after the 5th day the cytotoxicity increased in a manner similar to that of the 24-h group (Table 3). Lee Endo-Fill also showed in this group a less toxic response (in comparison to AH26 and Diaket) up to the 1 lth day, but like the 24-h group a cytotoxic ascending tendency was measured after this time (Table 3). DISCUSSION In the beginning phase, Lee Endo-Fill demonstrated a moderate cytotoxicity, but a toxic increment tendency after the 1 lth day was observed in both trials. This reaction might be, as in the case of some zinc oxide-eugenol-based sealers (7), because the setting time of this sealer takes a longer period than estimated. Further studies concerning this point of view are necessary. Sp~ngberg and Pascon (10) report similar results. TABLE 2. Metabolism of gingival fibroblasts in contact with resin- and silicone-based sealers having set for 24 h* Day 1 3 5 11 13 15 21
AH26 2.7 1.9 9.6 8.4 6.2 6.8 5.2
+ 0.24 __-0.07 + 0.57 + 1.09 _ 0.74 --+ 0.20 _+ 0.72
Diaket 14.4 10.3 11.6 11.1 7.2 7.5 5.7
+ 0.28 __ 0.72 + 1.04 _+ 0.88 + 0.86 ___0.60 _+ 0.39
Lee Endo-Fill 44.3 34.7 43.0 9.4 8.7 7.9 4.7
-+ 5.31 ___4.85 -+ 4.30 -+ 0.28 + 0.34 ___0.15 _ 0.79
* The values express the mean _+ SD of the L-[14C]leucine incorporation in percentage of the respective controls (Table 1).
TASLE 3. Metabolism of gingival fibroblasts in contact with resin- and silicone-based sealers having set for 48 h* Day
AH26
Diaket
Lee Endo-Fill
1 3 5 11 13 15 21
8.7 _ 0.60 10.9 + 0.87 9.3 -+ 1.30 6.5 _+ 0.58 6.4 _+ 0.76 3.7+0.11 5.8 _+ 0.69
38.4 _+ 0.76 22.5 _ 1.57 11.7 + 1.05 5.7 _+ 0.45 7.2 ___0.86 6.4+0.51 6.8 _+ 0.47
69.2 _+ 5.53 58.5 __ 5.85 66.7 _+ 9.33 6.7 __+0.53 8.4 _+ 0.50 7.2+0.21 7.0 __ 1.12
* The values express the mean -+ SD of the L-[14C]leucine incorporation in percentage of the respective controls (Table 1).
539
In the case of AH26 our results do not correlate with the ones obtained by several investigators (11-13). Schroeder (20) observed on histological specimens, in human teeth that had to be extracted due to root fracture or in root apex specimens that were obtained from apicectomies, contact without inflammation between the sealing material and the mesenchym when AH26 was confined in the root canal, and an inflammation-free connective tissue around what he called in the case of an overfilling, "neutral implant material." In our experiments AH26 demonstrated severe toxicity against gingival fibroblasts in both trials at the beginning phase. These results correlate with those of other researchers (2, 4, 8, 10). The reported setting time of AH26 is supposed to be between 24 and 40 h at 37~ but the fact that the no flbroblast recovery could be measured in both groups indicates that the release of toxic products takes place for a longer period of time. Safavi et al. (9) using mouse fibroblasts reported similar results with AH26 at the beginning phase of their experiment, but in contrast to this report cell recovery occurred after 24 h of culturing. Diaket proved to be slightly better tolerated than AH26. The beginning phase proved to be not as toxic as that of AH26, especially in the 48-h group. Yesilosy and Feigal (11) observed similar results with a different type of research method. The setting time of Diaket varies from a couple of minutes to several hours, therefore we are of the opinion that the difference between the 24- and 48-h groups seems to be because the setting of Diaket is relatively sensitive to the environment conditions. The cytotoxicity of these types of sealers did not decrease proportionally to their setting time as does that of most zinc oxide-eugenol-based sealers tested under the same parameters (7); therefore, the possibility that these types of sealers could irritate the periapical tissues for a longer period of time should be considered. Sp~ngberg and Pascon (10) have demonstrated in cell cultures that the amounts of the components mixed into a root canal sealer are relevant to their cytotoxicity. The results of these types of experiments depend not only on the standardization of the proportions but many other variables. However, in daily practice it is quite impossible to standardize exactly the amounts of sealers' components to be mixed; therefore, the use of nonirritating components in root canal sealers is desirable. It is also desirable to develop an investigative method that could mimic the in vivo parameters in a more objective manner than either the cell culture or implant methods. The dynamic nature of human periapical tissues cannot easily be simulated in vitro. Nevertheless, results obtained from these types of studies can give practitioners the opportunity to form opinions regarding the toxic potential of different root canal sealers. We wish to thank Mrs. Cheryl Lee Butz and Mr. Larry Selesko for their valuable assistance in the preparation of this manuscript. Dr. Brisefio and Dr. Willershausen are members of the Department of Conservative Dentistry, University of Munich, Munich Germany.
References 1. Feiglin B. Effect of some endodontic sealers on cell migration in experimental granulomas. Oral Surg 1987;63:371-4. 2. Hensten-Pettersen A, Orstavik D, Wennberg A. Allergenic potential of root canal sealers. Endod Dent Traumato11985;1:61-5.
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Brise~o and Willershausen
3. Olsson B, Sliwkowski A, Langeland K. Subcutaneous implantation for the biological evaluation of endodontic materials. J Endodon 1981 ;7:355-69. 4. Olsson B, Wennberg A. Early tissue reaction to endodontic filling materials. Endod Dent Traumato11985;1:138-41. 5. Tagger M, Tagger E. Effect of implantation of AH26 silver-free in subcutaneous tissue of guinea pigs. Int Endod J 1986;19:90-7. 6. Wennberg A. Biological evaluation of root canal sealers using in vitro and in vivo models. J Endodon 1980;6:784-7. 7. Brisef~o MB, Willershausen B. Root canal sealers cytotoxicity with human gingival fibroblasts. I. Zinc oxide-eugenol basis sealers. J Endodon 1990;16:383-6. 8. Nakamura H, Sakakibara F, Matsumoto Y, et al. Study on the cytotoxicity of root canal filling materials. J Endodon 1986;12:156-60. 9. Safavi KE, Sp&ngberg LSW, Costa NS Jr, Sapounas G, An in vitro method for longitudinal evaluation of toxicity of endodontic sealers. J Endodon 1989; 15:484-6. 10. Sp~ngberg L, Pascon E. The importance of matedal preparation for the expression of cytotoxicity during in vitro evaluation of biomaterials. J Endodon 1988;14:247-50. 11, Yesilosy C, Feigal RJ. Effect of endodontic materials on cell viability across standard pore size filters. J Endodon 1985;11:401-7.
Journal of Endodontics 12. Zmener O, Cabrini RL. Adhesion of human blood monocytes and lymphocytes to different endodontic cements. A methodological in vitro study. J Endodon 1986;12:150-5. 13. Biggs JT, Kaminski EJ, Osetek E M Rat macrophage response to implanted sealer cements. J Endodon 1985;11:30-6. 14. Lee H, Teigler D. Review of biological safety testing of Endo-Fill root canal sealant and filling material. Lee Pharmaceuticals Research Report RR 84-108, 1984. 15. Martin G M Human skin fibroblasts. In: Kruse PF, Patterson MK, eds. Tissue culture methods and applications. New York: Academic Press, 1973:39. 16. Cinatl J, Tolar M. Technik tier Zetlkultivation. In: Mauersberger R, ed. Aktuelle probleme der Zellz(Jchtung. Stuttgart: Gustav Fischer, 1971:63. 17. Dutrillaux B, Couturier J. Praktikum der Chromosomenanalyse. Stuttgart: Enke Verlag, 1983. 18. Mans RJ, Novelli GD. A convenient, rapid and sensitive method for measuring the incorporation of radioactive aminoacids into protein. Biochem Biophys Res 1960;3:540-3. 19. Lowry OH, Rosebrough NJ, Farr AL, Randall RS. Protein measurement with Folin phenol reagent. J Biol Chem 1951 ;193:265-72. 20. Schroeder A. Gewebsvertr~.glichkeit des Wurzelf~llmittels AH26. (Histologische und klinische Pr0fung). Zahnarztl Welt Ref 1957;58:563-7.
Y o u M i g h t B e I n t e r e s t e d to K n o w There is a putative Chinese curse that runs something to the effect of, "May you be visited by great troubles and long life to suffer them." As to the sources of such longevity--for better or worse--diet and genes are currently in vogue. However, a study of 1500 centenarians (N Engl J Med 1951; 247:299) concluded the following: 1. Longevity is not inheritable; 2. Sexual activity enhances longevity; 3. The strain of rearing children does not shorten life; 4. An old person's offspring's loving support is irrelevant to longevity; 5. People should work hard all their lives. Most of us will take comfort that at least one factor affects us favorably. But, ah, which one?
Cosby Newell
JOURNAL OF ENDODONTICS Copyright 9 1991byThe American Associ~ionofEndodontists
VOL. 17, NO. 11, NOVEMBER1991
Root Canal Sealer Cytotoxicity on Human Gingival Fibroblasts: II. Silicone- and Resin-based Sealers Benjamin M Brisefio, Dr. Med. Dent., and Brita Willershausen, Dipl. Chem., Dr. Med. Dent.
reports (10, 14) regarding the cytotoxicity of Lee Endo-Fill, a silicone-based root canal sealer. The purpose of this study was to test the toxic effects of these types of filling materials on human gingival fibroblasts by measuring the capability of the fibroblasts to synthesize proteins after being in contact with the sealers for a period of up to 21 days.
The cytotoxicity of two resin (AH26 and Diaket)- and one silicone (Lee Endo-Fill)-based root canal sealers were tested over a period of 21 days. The incorporation rate of L-[l*C]leucine was measured to establish the potential of human gingival fibroblasts to synthesize protein after being in contact with the sealers. The cells were gained from biopsies from healthy patients between 20 and 30 years of age. The sealers were allowed to set for 24 and 48 h before dispensing the fibroblastic suspension into the culture dishes. AH26 showed in the 24-h group a severe cytotoxic reaction during the entire experimental period. Diaket showed a relative severe cytotoxic potential with slight cell recovery after 3 days of culturing. Lee Endo-Fill showed a significantly lower (p < 0.05) cytotoxic potential during the first 11 days of incubation. However, between the 15th and 21st days of culturing an ascending cytotoxic potential was registered. With Diaket and Lee Endo-Fill a relative lower cytotoxic response was observed in the 48-h group; however, cytotoxic patterns similar to the 24-h group prevailed.
MATERIALS AND METHODS
Source of Tissues Biopsies were gained under aseptic precautions from healthy male and female patients between 20 and 30 years of age from the attached gingiva at the lower molar region. No lesions or signs of inflammation were present in the tissues during the collection of the biopsies. The different cell lines were cultivated up to the 20th passage without being pooled.
Gingival Fibroblasts Culture The fibroblasts were explanted into cultures in 6-cm O Petri dishes (Falcon; Becton Dickinson Labware, Lincoln Park, N J) according to the method of Martin (15) and stored in 250-ml culture bottles (Nunc; Nunc GmbH, Wiesbaden, Germany). Between the fifth and sixth day of cultivation, a trypsin EDTA solution (0.05/0.02%), in phosphate-buffered saline without Ca-'+,Mg2§ (Seromed; Seromed Biochrom-Produkte, Wiesbaden, Germany), was added for 10 to 15 min at 37"C by transferring the fibroblasts from one culture bottle to another in a 1:2 relation. Eagle's basal medium (Grand Island Biological Co., Grand Island, NY) supplemented with 10% fetal calf serum (Grand Island Biological Co.), 50 IU of penicillin per ml (Seromed), and 50 #g of streptomycin per ml (Seromed) solution was used. The cells were cultured in 250-ml plastic flasks (Nunc) in a water vapor saturated atmosphere at 37"C in a mixture of 5% CO2 and 95% air (type B 5060 EC; Heraeus, Osterode, Germany). The culture medium was changed after 24 h. Mycoplasma type infection was prevemed by using the method of Cinatl and Tolar (16). Periodical observation of the cultures in a phase contrast microscope (MPS50; Leitz-Wild, Wetzler, Germany) showed no bacterial growth during the entire research period. The fibroblasts were subcultivated 3 to 4 days after they had achieved confluence. Only normal diploid fibroblasts from
The adequate combination of sealing ability and biocompatibility of a root canal sealer is important for a favorable prognosis in root canal treatment. Therefore, the chemical tissue irritation effect is most important in considering sealer properties before selecting one. The sealing qualities of resinbased sealers are described as equal or better than those of other types. However, it should be made clear that if a root canal has not been properly cleaned and shaped, the sealing properties of a root canal sealer cannot impro,Je the results of the treatment. Furthermore, another source of treatment failure can come from sealers containing toxic components included with the aim of counteracting the effects of poor root canal debridement. Such sealers might be only capable of masking objective treatment failure symptoms. Many researchers have tested the cytotoxic properties of resin-based sealers in animal implants (1-6) and with different types of cell cultures (6-12). Although Biggs et al. (13) used a silicone elastomer as implant control, there have been few
537
538
Journal of Endodontics
Brisefio and Willershausen
four cell lines between the fifth and seventh passage were used after having analyzed the form and number of the cells' chromosomes (17). Fresh culture medium was added two to three times a week. Research Materials The following materials were tested: (a) Lee Endo-Fill (Lee Pharmaceuticals, E1 Monte, CA) which is according to the manufacturer a self-curing medical silicone rubber with a high percentage of bismuth subnitrate. Its exact composition was not obtained from the manufacturers. (b) Diaket (Espe, Seefeld, Germany), a polyketone sealer. One gram of powder contains zinc oxide 0.7 g. One gram of solution contains dichlorophen (0.005 g), triethanolamine (0.002 g), and propionylacethophenone (0.76 g). (c) AH26 (De Trey, FreresZurich, Switzerland), an epoxy resin root canal sealer. The powder contains silver powder (10%), bismuth oxide (60%), hexamethylenetetramine (25%), and titanium oxide (5%). The fluid contains epoxybisphenol-resin 100%. Protein Synthesis The measurement of the incorporation rate of L-[~4C]leucine (55 tzCi/nmol; Amersham Buchler, Buckinghamshire, England) in an acid precipitation material was used to determine the fibroblasts' protein synthesis potential. After having cultured the fibroblasts along with the different sealers for different periods of time, they were marked with L-[~4C] leucine (0.2 #Ci/ml) for 2 h after being rinsed with a 3-ml buffered phosphate physiological saline solution (Biochrom, Berlin, Germany). Following the method of Mans and Novelli (18), the incorporation of L-[~4C]leucine was stopped by precipitation in a 3-ml 7.5% trichloroacetic acid solution. The fibroblasts were then passed through a glass fiber filter (Whatman GF/C O 2.5 cm; Whatman Ltd., Maidstone, England) after being loosened with a rubber policeman and rinsed subsequently with 2 ml of 7.5% trichloroacetic acid. The radioactive fibroblastic material that remained in the filter was rinsed twice with 7.5% trichloroacetic acid and 95% ethanol and air dried. The fibroblasts' protein content was determined according to the method of Lowry et at. (19) (27 ~g/dish). The radioactivity of the cell material was measured in a 10-ml toluene scintillation solution (Quickszint 501; Zinsser Analytic, Frankfurt, Germany) with a liquid scintillation spectometer (Tri-Carb 1500; Packard GmbH, Frankfurt, Germany). Research Procedures The root canal sealers were placed into 18-mm 0 multiwell tissue culture plates (Falcon 3040; Becton Dickinson Labware) after being mixed under aseptic conditions according to the manufacturer's instructions. The sealer amount was weighed (___0.1 mg) with an analytic balance (Mettler H3; Mettler, Zurich, Switzerland). The sealer amount could be accurately calculated by subtracting the total weight of the sample in the multiwell from the weight of the previously registered sample(s). The weight of the sealers varied between 2.2 and 2.6 mg in the 24-h group and 2.1 and 2.4 mg in the
48-h group. The appropriate amount was determined by a pilot study (7). Possible bacterial growth during setting was prevented by allowing the sealers to set respectively for 24 and 48 h under ultraviolet light at 37"C. Approximately 104 cells were dispensed into each dish after having rinsed the sealers three times with physiological saline. The cell suspension was stained with trypan blue in order to facilitate cell counting (Fuchs-Rosenthal counting chamber). The multiwells were incubated for a 21-day period under the conditions described previously. A diffusion contact was established between the materials and the fibroblasts after they sedimented and attached to the flee bottom surface of the dishes. The culture medium was changed every second or third day. Evaluation Parameters Measurements of the incorporation rate of L-[14C]leucine were made in a total of 140 samples of each sealer. Seventy of them were tested 24 h after having set on the 1st, 3rd, 5th, 10th, 12th, 14th, and 21st day of culturing (n = 10 each). The other 70 samples were distributed in the same manner in a 48-h group after having set. The results express the means and standard deviations. Three dishes (per multiwell) cultured with gutta-percha (12 mm; Vereinigte Dentalwerke 0 40, Munich, Germany) and another four dishes with only fibroblasts served as controls in the 24-h as well as in the 48-h groups. RESULTS Through periodical microscopic observation it could be ascertained that the fibroblasts were viable at all times. No significant differences were found between the L-[14C]leucine incorporation rates of the controls with gutta-percha and fibroblasts only. However, only cultures with fibroblasts without foreign materials were used as controls. The mean values of L-[14C]leucine incorporation into the controls was determined as 100% (Table 1). Statistically (Student's t test) significant differences (p < 0.05) were found between Lee Endo-Fill and Diaket and Lee Endo-Fill and AH26 from the 1st to the 1lth day of culturing in the 24- and 48-h groups. A statistically significant difference was also found between Diaket and AH26 in the 48-h group between the I st and 5th day of culturing. At the beginning AH26 showed a relative high cytotoxicity in the group that was covered with fibroblasts 24 h after TABLE 1. Incorporation rate values of [L-l*C]leucine (cpm/mg protein) at the different experiment testing periods
Day
Incorporation rates (cpm/mg protein _+ SD)
1 3 5 11 13 15 21
33,475 _+ 1,150 32,970 +_ 2,189 34,213 + 1,690 31,867 + 2,935 32,033 _+ 4,549 37,013 _+ 6,089 38,567 +_ 4,744
Vol. 17, No. 11, November 1991
Sealer Toxicity
allowing to set. At the 5th day slight recovery was observed. After the 15th day almost no fibroblast protein synthesis could be measured (Table 2). Up to the 1 lth day the fibroblasts showed a slightly higher protein synthesis potential with Diaket in comparison to AH26. After the 1 lth day an ascending cytotoxic potential tendency without recovery was measured (Table 2). Lee Endo-Fill showed moderate cytotoxicity until between the 5th and the 1 lth day. After the 1 lth day a decrease in the capability of the fibroblasts to synthesize protein was observed. After the 15th day almost no more fibroblast protein synthesis potential could be measured (Table 2). The group in which the fibroblastic suspension was dispensed 48 h after mixing showed in the case of AH26 a less cytotoxic potential in comparison to the 24-h group (Table 3). No statistically significant differences resulted, however, between both groups with this material. Diaket showed in the beginning phase a relative moderate cytotoxicity, but after the 5th day the cytotoxicity increased in a manner similar to that of the 24-h group (Table 3). Lee Endo-Fill also showed in this group a less toxic response (in comparison to AH26 and Diaket) up to the 1 lth day, but like the 24-h group a cytotoxic ascending tendency was measured after this time (Table 3). DISCUSSION In the beginning phase, Lee Endo-Fill demonstrated a moderate cytotoxicity, but a toxic increment tendency after the 1 lth day was observed in both trials. This reaction might be, as in the case of some zinc oxide-eugenol-based sealers (7), because the setting time of this sealer takes a longer period than estimated. Further studies concerning this point of view are necessary. Sp~ngberg and Pascon (10) report similar results. TABLE 2. Metabolism of gingival fibroblasts in contact with resin- and silicone-based sealers having set for 24 h* Day 1 3 5 11 13 15 21
AH26 2.7 1.9 9.6 8.4 6.2 6.8 5.2
+ 0.24 __-0.07 + 0.57 + 1.09 _ 0.74 --+ 0.20 _+ 0.72
Diaket 14.4 10.3 11.6 11.1 7.2 7.5 5.7
+ 0.28 __ 0.72 + 1.04 _+ 0.88 + 0.86 ___0.60 _+ 0.39
Lee Endo-Fill 44.3 34.7 43.0 9.4 8.7 7.9 4.7
-+ 5.31 ___4.85 -+ 4.30 -+ 0.28 + 0.34 ___0.15 _ 0.79
* The values express the mean _+ SD of the L-[14C]leucine incorporation in percentage of the respective controls (Table 1).
TASLE 3. Metabolism of gingival fibroblasts in contact with resin- and silicone-based sealers having set for 48 h* Day
AH26
Diaket
Lee Endo-Fill
1 3 5 11 13 15 21
8.7 _ 0.60 10.9 + 0.87 9.3 -+ 1.30 6.5 _+ 0.58 6.4 _+ 0.76 3.7+0.11 5.8 _+ 0.69
38.4 _+ 0.76 22.5 _ 1.57 11.7 + 1.05 5.7 _+ 0.45 7.2 ___0.86 6.4+0.51 6.8 _+ 0.47
69.2 _+ 5.53 58.5 __ 5.85 66.7 _+ 9.33 6.7 __+0.53 8.4 _+ 0.50 7.2+0.21 7.0 __ 1.12
* The values express the mean -+ SD of the L-[14C]leucine incorporation in percentage of the respective controls (Table 1).
539
In the case of AH26 our results do not correlate with the ones obtained by several investigators (11-13). Schroeder (20) observed on histological specimens, in human teeth that had to be extracted due to root fracture or in root apex specimens that were obtained from apicectomies, contact without inflammation between the sealing material and the mesenchym when AH26 was confined in the root canal, and an inflammation-free connective tissue around what he called in the case of an overfilling, "neutral implant material." In our experiments AH26 demonstrated severe toxicity against gingival fibroblasts in both trials at the beginning phase. These results correlate with those of other researchers (2, 4, 8, 10). The reported setting time of AH26 is supposed to be between 24 and 40 h at 37~ but the fact that the no flbroblast recovery could be measured in both groups indicates that the release of toxic products takes place for a longer period of time. Safavi et al. (9) using mouse fibroblasts reported similar results with AH26 at the beginning phase of their experiment, but in contrast to this report cell recovery occurred after 24 h of culturing. Diaket proved to be slightly better tolerated than AH26. The beginning phase proved to be not as toxic as that of AH26, especially in the 48-h group. Yesilosy and Feigal (11) observed similar results with a different type of research method. The setting time of Diaket varies from a couple of minutes to several hours, therefore we are of the opinion that the difference between the 24- and 48-h groups seems to be because the setting of Diaket is relatively sensitive to the environment conditions. The cytotoxicity of these types of sealers did not decrease proportionally to their setting time as does that of most zinc oxide-eugenol-based sealers tested under the same parameters (7); therefore, the possibility that these types of sealers could irritate the periapical tissues for a longer period of time should be considered. Sp~ngberg and Pascon (10) have demonstrated in cell cultures that the amounts of the components mixed into a root canal sealer are relevant to their cytotoxicity. The results of these types of experiments depend not only on the standardization of the proportions but many other variables. However, in daily practice it is quite impossible to standardize exactly the amounts of sealers' components to be mixed; therefore, the use of nonirritating components in root canal sealers is desirable. It is also desirable to develop an investigative method that could mimic the in vivo parameters in a more objective manner than either the cell culture or implant methods. The dynamic nature of human periapical tissues cannot easily be simulated in vitro. Nevertheless, results obtained from these types of studies can give practitioners the opportunity to form opinions regarding the toxic potential of different root canal sealers. We wish to thank Mrs. Cheryl Lee Butz and Mr. Larry Selesko for their valuable assistance in the preparation of this manuscript. Dr. Brisefio and Dr. Willershausen are members of the Department of Conservative Dentistry, University of Munich, Munich Germany.
References 1. Feiglin B. Effect of some endodontic sealers on cell migration in experimental granulomas. Oral Surg 1987;63:371-4. 2. Hensten-Pettersen A, Orstavik D, Wennberg A. Allergenic potential of root canal sealers. Endod Dent Traumato11985;1:61-5.
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3. Olsson B, Sliwkowski A, Langeland K. Subcutaneous implantation for the biological evaluation of endodontic materials. J Endodon 1981 ;7:355-69. 4. Olsson B, Wennberg A. Early tissue reaction to endodontic filling materials. Endod Dent Traumato11985;1:138-41. 5. Tagger M, Tagger E. Effect of implantation of AH26 silver-free in subcutaneous tissue of guinea pigs. Int Endod J 1986;19:90-7. 6. Wennberg A. Biological evaluation of root canal sealers using in vitro and in vivo models. J Endodon 1980;6:784-7. 7. Brisef~o MB, Willershausen B. Root canal sealers cytotoxicity with human gingival fibroblasts. I. Zinc oxide-eugenol basis sealers. J Endodon 1990;16:383-6. 8. Nakamura H, Sakakibara F, Matsumoto Y, et al. Study on the cytotoxicity of root canal filling materials. J Endodon 1986;12:156-60. 9. Safavi KE, Sp&ngberg LSW, Costa NS Jr, Sapounas G, An in vitro method for longitudinal evaluation of toxicity of endodontic sealers. J Endodon 1989; 15:484-6. 10. Sp~ngberg L, Pascon E. The importance of matedal preparation for the expression of cytotoxicity during in vitro evaluation of biomaterials. J Endodon 1988;14:247-50. 11, Yesilosy C, Feigal RJ. Effect of endodontic materials on cell viability across standard pore size filters. J Endodon 1985;11:401-7.
Journal of Endodontics 12. Zmener O, Cabrini RL. Adhesion of human blood monocytes and lymphocytes to different endodontic cements. A methodological in vitro study. J Endodon 1986;12:150-5. 13. Biggs JT, Kaminski EJ, Osetek E M Rat macrophage response to implanted sealer cements. J Endodon 1985;11:30-6. 14. Lee H, Teigler D. Review of biological safety testing of Endo-Fill root canal sealant and filling material. Lee Pharmaceuticals Research Report RR 84-108, 1984. 15. Martin G M Human skin fibroblasts. In: Kruse PF, Patterson MK, eds. Tissue culture methods and applications. New York: Academic Press, 1973:39. 16. Cinatl J, Tolar M. Technik tier Zetlkultivation. In: Mauersberger R, ed. Aktuelle probleme der Zellz(Jchtung. Stuttgart: Gustav Fischer, 1971:63. 17. Dutrillaux B, Couturier J. Praktikum der Chromosomenanalyse. Stuttgart: Enke Verlag, 1983. 18. Mans RJ, Novelli GD. A convenient, rapid and sensitive method for measuring the incorporation of radioactive aminoacids into protein. Biochem Biophys Res 1960;3:540-3. 19. Lowry OH, Rosebrough NJ, Farr AL, Randall RS. Protein measurement with Folin phenol reagent. J Biol Chem 1951 ;193:265-72. 20. Schroeder A. Gewebsvertr~.glichkeit des Wurzelf~llmittels AH26. (Histologische und klinische Pr0fung). Zahnarztl Welt Ref 1957;58:563-7.
Y o u M i g h t B e I n t e r e s t e d to K n o w There is a putative Chinese curse that runs something to the effect of, "May you be visited by great troubles and long life to suffer them." As to the sources of such longevity--for better or worse--diet and genes are currently in vogue. However, a study of 1500 centenarians (N Engl J Med 1951; 247:299) concluded the following: 1. Longevity is not inheritable; 2. Sexual activity enhances longevity; 3. The strain of rearing children does not shorten life; 4. An old person's offspring's loving support is irrelevant to longevity; 5. People should work hard all their lives. Most of us will take comfort that at least one factor affects us favorably. But, ah, which one?
Cosby Newell
