Root Canal Sealer Cytotoxicity with Human Gingival Fibroblasts. III. Calcium Hydroxide-based Sealers
Benjamin M. Brisefio, Dr. reed. dent., and Brita Willershausen, Priv. Doz. Dr. med. dent.
The cytotoxicity of four different calcium hydroxide- based root canal sealers (Sealapex, Apexit, CRCS, and Endoflas FS) was tested using human gingiva fibroblasts. The fibroblasts were acquired from the attached gingiva of apparently healthy patients. The sealers were allowed to set for 24 and 48 h before being covered with a fibroblastic suspension. The estimation of the incorporation rate of L-[14C]leucine in the fibroblasts after being in contact with the sealers was utilized to designate the fibroblasts' protein synthesis potential. The research was con- ducted over a period of 21 days. Endoflas FS in- duced a dramatic reduction in the protein synthesis potential of the fibroblasts in the 24-h group. In the 48-h group, Endoflas FS showed a slightly better response. Endofias FS, however, showed a signifi- cantly higher cytotoxicity with respect to other seal- ers in both trials (p < 0.05). Sealapex demonstrated a relatively low cytotoxicity after 3 days of culturing. Although CRCS showed a slightly higher cytotoxicity during the initial phase of the experiments, a declin- ing level of toxicity could be measured after 3 days of culturing. Apexit had a relatively high cytotoxicity in the beginning phase, but an ascending incorpo- ration rate of L-[14C]leucine in the fibroblasts could be distinguished after 3 days of culturing.
Although the pharmacological mechanism of calcium hy- droxide is not well known and its osteogenic potential is going through extensive reevaluation, its biocompatibility when in- corporated in a root canal sealer is well recognized (1-6). In 1950, Hermann (7) recommended the use of calcium hydrox- ide as a "biological" root canal filling material. Manhart (8) postulated in 1974 the use of a calcium hydroxide-based pulp capping agent as a permanent root canal sealer. Calcium hydroxide materials are not only soon absorbed in the peri- apical tissues but also in the root canal space (9); hence, calcium hydroxide's sealing properties are deficient. Different calcium hydroxide-based sealers that are supposedly not able to be absorbed--or at least are absorbed as slowly as any
110
other type of sealer--in the root canal space are recommended for use as permanent root canal sealers.
The incorporation of calcium hydroxide in permanent root canal sealers has gained the favor of many operators; however, reports (1-6, 10-13) concerning its cytotoxic potential are scarce when compared with other types of sealers. Calcium hydroxide itself, as well as root canal sealers containing cal- cium hydroxide, has been tested by different researchers uti- lizing different investigative methods: among others, in ani- mal implants (4-5, 10-13) and with different types of cell cultures (1-3, 6).
The purpose of this study was to ascertain, through obser- vation of protein synthesis, the degree of cytotoxicity of some calcium hydroxide-containing root canal filling materials through the use of human gingival fibroblasts over a 21-day period.
MATERIALS AND METHODS
Root Canal Sealers
Four root canal sealers containing calcium hydroxide were tested: (a) Apexit (Vivadent; Schaan, Lichtenstein) an exper- imental calcium hydroxide base sealer that, according to the manufacturer, is composed as follows--base: calcium hydrox- ide-phosphate (40 to 45%), inert texturizing agent/filler (10 to 20%), vehicle (35 to 40%). Accelerator: disalicylate (30 to 40%), opacifying agent (30 to 40%), inert texturizing agent/ filler (20 to 25 %), vehicle (4 to 7 %); (b) Sealapex (Kerr Sybron; Romulus, MI). An approximate composition of mixed sealer, according to the manufacturer, is calcium hydroxide (25%), barium sulfate (18.6%), zinc oxide (6.5 %), titanium dioxide (5.1%), and zinc stearate (1.0%) in a blend of ethyl toluene sulfonamide, polysalicylate, methyl salicylate, and a pigment; (c) CRCS (Calcibiotic Root Canal Sealer; Hygienic, Akron, OH). According to the manufacturer, CRCS is essentially a modified zinc oxide-eugenol composition in which about one third of the zinc oxide has been replaced with calcium hy- droxide. Barium sulfate was added as an opacifying agent at about 15% (wt/wt) and 15% (wt/wt) of inert texturizing agent/filler is added. Twenty percent (wt/wt) of the total is a modified wood resin which serves as a binder. The liquid portion contains eugenol and eucalyptol. Precise information was not available from the manufacturer. (d) The fourth sealer was Endoflas FS (Sanlor Laboratories Ltda; Cali, Colombia).
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Powder: zinc eugenolate, iodoform, calcium hydroxide and barium sulfate. Liquid: eugenol and paramonochlorophenol. The precise formulation was not available.
Tissue Sources
Biopsies were gained under aseptic precautions from the attached gingiva in the lower molar region from apparently healthy male and female patients between 20 and 30 yr of age. At the time the biopsies were taken, no lesions or signs of inflammation were present. Three different cell lines from three donors were cultivated separately.
The culture method has been described earlier in detail (14).
Experimental Procedures
The manufacturer's recommendations were strictly ad- hered to in the aseptic preparation of the root canal sealers. The sealers were placed at the junction between the base and wall of each multiwell cylinder (18 mm O, Falcon 3040; Becton Dickinson Labware, Lincoln Park, N J). In this man- ner, the sealers covered only a small surface area of the well. The materials were allowed to set for 24 and 48 h under ultraviolet light to prevent bacterial contamination. The seal- ers' amounts were determined according to preliminary ex- periments and calculated by weighing the sealers with an analytical balance (+0.01 mg, Mettler H3; Zurich, Switzer- land) (14). Subsequently, the sealers were rinsed three times with physiological saline before being covered with a suspen- sion of gingival fibroblasts at a concentration of 104 cells/ml. The cell number was determined after staining the culture with trypan blue in a Fuchs-Rosenthal counting chamber. Controls were included for each testing period. Four wells per multiwell tray were plated with fibroblasts to serve as controls. The multiwell plates were incubated at 37°C in 5% CO2 in air for various time periods up to 21 days. A diffusion contact was established between the sealers and the fibroblasts after they sedimented and attached to the free bottom surface of the wells. The culture medium was changed every second day.
A total of 20 samples was tested for each sealer and time period. Ten samples were covered with fibroblasts 24 h after setting, and the remaining 10 after 48 h of setting. Measure- ments of the incorporation rate of L-[~4C]leucine were done at the 1st, 3rd, 5th, llth, 13th, 15th, and 21st days of culturing. The amounts of sealers varied between 2.6 and 3. I mg in the two experiments.
Evaluation of Protein Synthesis
The protein synthesis was measured by determining the incorporation rate of L-[~4C]leucine (55 uCi/nmol; Amersham Buchler, Buckinghamshire, England) in an acid precipitation material. The fibroblasts were labeled with L-[~4C]leucine (0.2 uCi/ml) for 2 h after the cultures were rinsed with 3 ml of phosphate-buffered saline (Biochrom; Berlin, Germany). The method of Lowry et al. (15) was used to estimate the protein contents of the fibroblasts (control value = 27 ug/well; +0.45 SD). The incorporation of L-[~4C]leucine was stopped by precipitation in a 3 ml of 7.5% trichloroacetic acid solution.
Calcium Hydroxide Sealer Toxicity 111
After loosening the fibroblasts with a rubber policeman, they were filtered through a glass fiber filter (Whatman GF/C 0 2.5 cm; Whatman Ltd, Maidstone, England) (16) and rinsed with 2 ml of 7.5% trichloroacetic acid. The radioactive fibro- blastic material that remained in the filter was rinsed twice with 5 ml of 7.5% trichloroacetic acid, followed by 95% ethanol and air dried. The measurement of the radioactive cell material was accomplished using a Liquid Scintillation Spectometer (Tri-Carb 1500; Packard GmbH, Frankfurt, Ger- many) using a Quickszint 501 solution (Zinsser Analytic; Frankfurt, Germany).
RESULTS
No bacterial growth could be observed microscopically in any of the experiments. During these observations, the mor- phological changes observed in the fibroblasts seemed to be subjectively correlated to the incorporation rates of e-[14C] leucine. The incorporation of L-[~4C]leucine in the controls (n = 4/multiwell plate) was determined to be 100% (Table 1). Significant differences were found between Sealapex, CRCS, and Apexit (Student's t test, p < 0.05) in the 24-h and 48-h groups during the first days of the experiments. Highly significant differences were observed between Endoflas FS and the other three sealers in both groups.
In the 24-h group, the fibroblasts proved to have a constant increment in their capability to synthesize proteins with Seal- apex. This increment reached its highest point after the 1 lth day of culturing and remained this way until the end of the experiment (Table 2). CRCS and Apexit showed a similar pattern; however, they demonstrated a significantly higher toxicity than Sealapex between the beginning of the experi- ment and the third and fifth days, respectively. CRCS and Apexit reached their lowest cytotoxic points on the 5th and 1 lth days of culturing (Table 2). Endoflas FS proved to have a severe cytotoxicity during the entire research period (Table 2). A significant difference was found between Endoflas FS and all other sealers.
The group that was set for 48-h showed a similar cell metabolism pattern as the 24-h group. The beginning stages of Apexit, Sealapex, and CRCS were significantly lower in toxicity when compared with the 24-h group, reaching their lowest toxic level between the third and fifth day of culturing (Table 3). Endoflas FS showed no lower cytotoxic levels in comparison to the 24-h group (Table 3). No statistically significant differences were found when comparing the 24-h and 48-h groups of Endoflas FS. Significant differences,
TABLE 1. Incorporation rate values of L-[l*C]leucine (cpm/mg protein) for the controls at the different experiment testing
• Rows a express the L-[14C]leucine incorporation in percentage of the controls (Table 1) ± SD, Rows b represent the means of the protein concentrations (,ug/ml protein) ± SD.
TABLE 3. Protein synthesis of gingival fibroblasts in contact with calcium hydroxide-based sealers having Bet for 48 h
* Rows a express the L-[14C]leucine incorporation in percentage of the controls (Table 1) ± SD. Rows b represent the means of the protein concentrations (/zg]ml protein) ± SD.
though, were found between all of the other sealers and Endoflas FS in this group.
DISCUSSION
The results obtained with Endoflas FS suggest that the addition of bactericidal agents such as iodoform and para- monochlorophenol in the formula of root canal sealers can augment their cytotoxicity. Similar results (1, 2) have been reported with different sealers containing different antiseptic ingredients. The manufacturers of Endoflas FS claim that the paste is conceived to act in a septic field, possibly explaining the incorporation of the bactericidal agents, thereby rendering the disinfection of the root canal superfluous (17). However, it has been clinically and radiographically demonstrated that after proper cleaning, enlarging, and adequate sealing of the root canal space, healing occurs in most cases with the use of sealers that contain less irritating agents in their formulas (4, 5, 10-11). Therefore, the use of sealers containing unnecessary irritating components might disrupt healing of the periapical
Journal of Endodontics
tissues due to the irritation potential of such agents. It should always be remembered that the function of a root canal filling is only to seal and prevent further recontamination in the root canal in as inert a form as possible.
The results obtained with Apexit, Sealapex, and CRCS are encouraging. The cytotoxic potential of these three sealing materials against gingival fibroblasts in this experimental model was very small. These materials compare favorably when considering other types of sealing materials (14, 18). The protein synthesis of the cells was higher than that obtained in similar experiments with zinc oxide-eugenol sealers such as Endoseal, Hermetic, Procosol, Tubli-Seal, and Wach's Formula (14), and silicone- and resin-based sealers such as Diaket, AH26, and Lee Endo-Fill (18). Although the experi- mental conditions in this study differ from others, our obser- vations are similar to results reported previously (1-6). In contrast, other authors (10-13) found severe inflammatory reactions when implanting CRCS and Sealapex. However, Zmener et al. (13) found decreasing inflammation with time and normal tissue when implanting CRCS after a period of 90 days. The same authors (6, 10-13) report disintegration of the material in the tissues at the sites where these materials were implanted. Similar observations were made in this study in the culture medium surrounding the test material. The culture substrate surrounding Sealapex showed a much larger amount of disintegrating small particles in comparison to the other materials. This might be attributed to the capability of calcium hydroxyl ions to diffuse and precipitate from the set sample in the medium. Previous experiments done by other investigators (6, 10, 12, 14) report about the ability of calcium hydroxide ions to diffuse.
According to the manufacturer, the liquid portion of CRCS contains sufficient eugenol to react with the zinc oxide but is diluted with eucalyptol to prevent an eugenol excess after the reaction takes place. The initial toxic reaction of CRCS in both groups might be due to a eugenol excess. We are of the opinion that the dilution ofeugenol did not take place during mixing but some time during the setting of the material. This can explain the recovery of the fibroblasts between the firs{ and third day of culturing. Studies that could confirm this opinion are necessary.
The measurement of the incorporation rate of L-[~4C]leu- cine in the cell culture appears to be an objective and sensitive investigative method in which the fibroblasts' metabolic activ- ity can be measured. Tissue cultures are a valuable tool to observe the behavior of cells in vitro in the hope that isolated cells will reflect in vivo activity. The use of human-oral origin fibroblasts as means of evaluating the cytotoxicity of root canal filling materials is a recognized method due to the ease of their isolation, successful growth in culture, and method standardization (19). A problem remains in how to present the materials to the cells. Freshly mixed materials will diffuse more rapidly and thoroughly in the medium, and the use of filters to prevent diffusion impedes the release of toxic com- ponents from the materials. The changing of culture medium on a regular basis reduces the water-soluble toxic materials in the cell culture. However, this applies for all the materials that were tested. This suggests that the release of water soluble toxic materials, with the exemption of Endoflas FS, was markedly reduced at the third day with Sealapex and CRCS and at the 1 lth day with Apexit in the 24-h group. In the 48- h group, a more rapid reduction of the release of water-soluble
Vol. 18, No. 3, March 1992
toxic components was observed. Evaluation of toxicity of filling materials should always be conducted under standard- ized parameters; therefore, the development of a method that can reduce these problems to a minimum seems to be a desirable goal. Although the data gained with these types of research protocols are valuable to identify toxic filling mate- rials, the testing of root canal filling materials with cell cultures represents some problems, since such materials when clini- cally used are freshly placed in direct apposition with the periapex, and the dynamics of the periapical tissues cannot be reproduced in cell cultures. Therefore, when extrapolating data from in vitro to in vivo situations, caution should always be exercised.
We wish to thank Mrs. Cheryl Lee Butz, Mr. Larry Selesko, and Mr. Daniel Wolter 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. Address re- quests for reprints to Dr. Benjamin Brisefio, Riedemweg 10, 8089 Emmering, Federal Republic of Germany.
References
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Calcium Hydroxide Sealer Toxicity 113
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