0099-2399/90/1612-0566/$02.00/0 JOURNAL OF ENDODONTICS Copyright 9 1990 by The American Association of Endodontists
Printed in U.S.A.
VOL. 16, NO. 12, DECEMBER1990
In Vitro Bacterial Penetration of Coronally Unsealed Endodontically Treated Teeth Mahmoud Torabinejad, DMD, MS, Borasmy Ung, DDS, and James D. Kettering, PhD
In addition to dyes, radioisotopes have been used to study microleakage in alloy, resins, temporary filling substances, and root canal filling materials (4-9). Although isotopes may be a good tool for comparing relative leakage, they cannot give a true picture of the leakage which occurs clinically. This is because the ions used are much smaller than dye molecules and they diffuse much more rapidly than other small molecules (4). Isotopes are indicators of ion exchange, diffusion, or metabolism within the tissues rather than indicators of true leakage (10, 11). Mortensen et al. (12) and Krakow et al. (13) have stated that microorganism penetration might be more appropriate than dye or isotope penetration for studying leakage in vivo. Goldman et al. (14) have pointed out that bacteria performed better than dye in testing for leakage of hydrophilic materials and that dyes could give a false positive reading if their molecules were small enough. Because air bubbles can prevent dye leakage, the results of dye studies have been questioned (15; Goldman et al., personal communication). Because of inherent inadequacies in dye and radioisotope studies, it appears that bacterial leakage studies can provide more accurate information in clinical situations. We have found no reports on the length of time that passes before the entire obturated root canal is invaded by bacteria in obturated root canals without coronal seals. The purpose of this experiment was to determine the length of time needed for bacteria to penetrate a standardized length of obturated root canals which were intentionally exposed to one of two species of microorganisms.
Forty-five root canals were cleaned, shaped, and then obturated with gutta-percha and root canal sealer, using a lateral condensation technique. The coronal portions of the root filling materials were placed in contact with Staphylococcus epidermidis and Proteus vulgaris. The number of days required for these bacteria to penetrate the entire root canals was determined. Over 50% of the root canals were completely contaminated after 19-day exposure to S. epidermidis. Fifty percent of the root canals were also totally contaminated when the coronal surfaces of their fillings were exposed to P. vulgaris for 42 days.
Sealed root canals can be recontaminated under several circumstances: (a) if the patient has had endodontic treatment but has delayed placement of permanent restorations; (b) if the seal of the temporary filling material has broken down; or (c) if filling materials and/or tooth structures have fractured or been lost. When these situations occur, the coronal portion of the root canal system is exposed to oral flora. The question is how quickly the entire root canal system becomes contaminated again, to the point that retreatment of the canal may be necessary. Swanson and Madison (1) evaluated the length of time that the obturation material could be exposed to artificial saliva before compromising the integrity of the seal. They exposed the coronal portion of obturated root canals to artificial saliva for various time periods, and then immersed them in Pelikan ink for 48 h. They found that the dye penetrated from 79 to 85% of the root length in all exposed specimens. There was no leakage in the control group which was not exposed to artificial saliva, but was placed in contact with ink for 48 h. The follow-up study by Madison et al. (2) showed that in teeth exposed to artificial saliva for 7 days, the ink penetrated between 33 and 80% of the root length, depending on the type of sealer used. The latest findings by Madison and Wilcox (3) evaluating in vivo microleakage did not, however, confirm their in vitro studies. They found that some of the positive controls (no sealer) did not show microleakage, while some of the negative controls (temporary not removed) showed dye penetration.
MATERIALS AND METHODS To test bacterial penetration, a set-up similar to the one used by Goldman et al. (14) and Williams and Goldman (16) was used in this experiment.
Apparatus Set-up By using a high-speed handpiece and a #2 round bur, a small circular opening (about l mm in diameter) was made through the cap of a 20-ml scintillation flask. A paper clip was threaded through the opening and an alligator clip was hung on the inside. The outside end was bent to stabilize the alligator clip against the cap. The outside opening of the cap
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Vol. 16, No. 12, December 1990
was resealed with acrylic material. The flasks, with the alligator clips attached to their caps, were steam sterilized. After instrumentation and obturation of root canals, a 25m m length of latex tubing was placed over the coronal portion of each tooth and the edges were sealed with epoxy resin (Quick Gel, non-run super-glue; Loctite Corp., Cleveland,
OH). The tubes, with the teeth attached, were sterilized in 5.25% sodium hypochlorite for 15 rain (based on the results of a pilot study) and then were rinsed with approximately 300 ml of sterile water. The tubes were then fastened to the caps of the previously sterilized scintillation vials. Ten milliliters of sterile phenol red broth with 3% lactose was added to the bottom of a flask, and the length of the tubing was adjusted so that a m i n i m u m of 2 m m of the apical part of each tooth was immersed in the solution (Fig. 1).
In Vitro Bacterial Penetration
567
Monitoring of the Samples The results from a pilot study showed that viable P. vulgaris were present 3 wk after incubation, while the S. epidermidis organisms were viable for only 1 wk. Fresh overnight cultures of organisms and sterile artificial saliva were added to the tubes at 5-day intervals for S. epidermidis and at 5- to 10-day intervals for P. vulgaris. When the bacterial culture was replenished, the old culture was plated to confirm continued viability o f the microorganisms. The samples were monitored daily until the red indicator solution at the bottom of the flask turned yellow. After color change, a sample of the yellow medium was plated on blood agar to assure that it contained the same type o f bacteria as that placed in the tubing.
Preparation of Teeth Bacterial Preparations Two species of bacteria were used as contaminants for this experiment; Proteus vulgaris, which is highly motile, and Staphylococcus epidermidis, which is nonmotile. These were grown overnight in 30 ml of trypticase soy broth (approximately 4.7 x l08 per ml o f / ' . vulgaris and 7.5 x 1 0 6 per ml of S. epidermidis). Two milliliters of the bacterial suspension and 0.7 ml of sterile artificial saliva as specified by Swanson and Madison (1) (1 mM CaC12, 3 mM NAH2PO4, 20 mM NaHCO3) were placed into the tubing. Since both organisms are acid formers, we expected the phenol red indicator solution to change to a yellow color when the bacteria reached it (16, 17).
Forty-five maxillary incisors and cuspids with straight canals were used in this study. The teeth had been stored previously in 10% formalin and were kept moist at all times throughout the experiment. After initial radiographs, standard access cavities were prepared, and the coronal portions of the canals were enlarged with #2 to #4 Gates Glidden drills. In order to obtain a standardized diameter, the apical foramina of the teeth were enlarged and kept patent to a #40 file, using a step-back filing technique. Approximately 2 ml of 5.25% NaOC1 were used between each file size, to remove debris. The prepared teeth were divided into experimental and control groups.
Experimental Groups The root canals of 33 teeth were obturated with guttapercha and Roth's sealer (Roth Drug Co., Chicago, IL) using the lateral condensation technique. To obtain a standardized length of filling, the coronal portion of the gutta-percha was removed with hot pluggers until only l0 m m of the filling material remained in the canal. To prevent bacteria from penetrating the root surfaces, two layers of fingernail polish were applied to the outside of the root except for 1 m m at the apex. GROUP 1 The coronal portions of the root canals of 16 teeth in this group were placed in contact with 2 ml of P. vulgaris in trypticase soy broth and 0.7 ml of sterile artificial saliva as described above. The tubing was then suspended over the phenol broth so that approximately 2 m m of the apex of the tooth was immersed in it. GROUP 2
FiG 1. Diagram of a tooth attached to a tube and suspended in the flask by an alligator clip.
The coronal portions of the root canals of the rest of the teeth in the experimental group (17 teeth) were exposed to 2 ml ofS. epidermidis and 0.7 ml of artificial saliva. The apices of these teeth were also immersed in phenol red broth.
568
Torabinejad et al.
Joumal of Endodontics
Control Groups
TABLE 2. Rate of total recontamination of obturated root canals exposed to S. epidermidis
To test the reliability of our results, the rest of the prepared teeth were divided into two control groups. GROUP 3 The root canal in each of the eight teeth that were used as positive controls was filled with a single gutta-percha cone without sealer, simulating poorly obturated root canals. Each of the two species of bacteria were placed separately in the tubing to contaminate four root canals (per microorganism), as described in the experimental group. GROUP 4 To verify that no contamination developed during the experiments (negative controls), four instrumented root canals were filled with gutta-percha and sealer. After removing the coronal portion of the filling material and leaving 10 m m of it in the root canal, the coronal portions of the filling were exposed to sterile artificial saliva applied as described above.
No. of Samples
% of Total
Cumulative %
1 1 5 2 2 4 1 1
6 6 29 12 12 23 6 6
6 12 41 53 65 88 94 100
Total 17
100%
No. of Days 15 16 17 19 20 30 45 51
Average: 24.1 days 0.4 mm/day
t test of independent samples shows that the difference is statistically significant (t = 4.68), with p < 0.01. Except for one sample in the positive control group (group 3), the rest of the samples (seven of eight) caused a color change in the phenol red medium after 1 to 4 days. The culture medium did not change color in teeth whose coronal segments were in contact with only sterile saliva (group 4) throughout the experiment (over 90 days).
RESULTS DISCUSSION In group 1, contaminated with P. vulgaris, two of the samples became positive after 2 days. One of these may have been a sample from the positive control, whereas the other one was found to have leakage through the latex tubing. These two samples were discarded. Table 1 shows the time it took P. vulgaris to reach the apex through 10 m m of the filling material. It varied from 10 to 73 days. The average length of time for leakage was 48.6 days. The time periods required for S. epidermidis to reach the apex in group 2 are shown in Table 2. Compared with those obtained in group 1, the results were more consistent, i.e. most apical leakage occurred between 15 and 30 days, with a range between 15 and 51 days. The average length of time for total penetration was 24.1 days. A statistical analysis using a
TABLE 1. Rate of total recontamination of obturated root canals exposed to P, vulgaris
No. of Samples 2 discarded 1 1 1 2 2 2 1 1 1 1 1 Total 14
% of Total 7 7 7 14.3 14.3 14.3 7 7 7 7 7
99.9%
Cumulative % 7 14 21 36 50 64 71 79 86 93 100
No. of Days
10 29 31 39 42 57 63 64 66 68 73 Average: 48.6 days 0.2 mm/day
Most of the samples in the positive control group with poorly filled canals showed leakage by 1 to 4 days, except for one which did not show a change until the 22nd day. There are two possible technical errors which could have caused the latter; one is that this tooth may have been switched by mistake with one of the experimental samples, the other is that the space prepared may have been circular enough to provide a very tight seal. The results of the positive control group are a confirmation of studies by Marshall and Massler (4), Evans and Simon (5), and Skinner and Himel (17) who showed that sealers are needed to improve the apical seal. As none of the negative controls led to a color change in the phenol red medium, it appears that our set-up did provide a contamination-free chamber. Over 85% of the teeth inoculated with P. vulgaris became completely penetrated in 66 days, whereas most (88%) of those inoculated with S. epidermidis were totally infected in 30 days, suggesting that motility may not be a factor in rate of penetration to the apices. We found a significant variability in the time it took the bacteria to penetrate the entire root canal system. Similar results were reported by Swanson and Madison (1) and Madison et al. (2) when they studied dye penetration in obturated root canals. This might be due to the shape of the prepared canal, type of sealer used, or the nature of the solution to which the coronal portions of the root canal were exposed. Goldman et al. (14) studied bacterial penetration in root canals filled with poly-HEMA, a hydrophilic, plastic polymer. They found no bacterial penetration after 42 days. The main reasons for lack of bacterial penetration in their results could be that poly-HEMA does not support bacterial growth as reported by Kronman et al., (18) because the pores are substantially smaller than the bacteria, or that the polymers have
Vol. 16, No. 12, December 1990
the same adhesive properties as the acrylic nail polish which was used to cover the external surfaces of the teeth. Compared with clinical conditions, the model used in our study was static, its media for bacterial growth was not totally similar to saliva and for ease of management of experimental conditions and interpretations of the data, its bacterial contents were purposely limited to only two species. Because of these limitations and their possible effects on the results, in vitro models simulating clinical conditions are needed to investigate the rate of leakage in unsealed, obturated root canals.
We gratefully acknowledge the assistance of Dr. Junichi Ryu and William Keeler with the bacterial culture aspects of the experiment. Dr. Torabinejad is director, Postgraduate Endodontics, Dr. Ung is a former graduate student in endodontics, and Dr. Kettering is professor of microbiology, Loma Linda University School of Medicine, Loma Linda, CA.
References 1. Swanson KS, Madkson S. An evaluation of coronal microleakage in endodontically treated teeth. Part I. Time periods. J Endodon 1987;13:56-9. 2. Madison S, Swanson KL, Chile SA. An evaluation of coronal microleakage in endodontically treated teeth. Part I1. Sealer types. J Endodon 1987;13:109-12. 3. Madison S, Wilcox LR. An evaluation of coronal microleakage in endodontically treated teeth. Part III. In vivo study. J Endodon 1988;14:455-8. 4. Marshall FJ, Massler M. The sealing of pulpless teeth evaluated with
In Vitro Bacterial Penetration
569
radio-isotopes. J Dent Med 1961 ;16:172-84. 5. Evans JT, Simon JHS. Evaluation of the apical seal produced by injected thermoplasticized gutta-percha in the absence of smear layer and root canal sealer. J Endodon 1986;12:101-7. 6. Kapsimalis P, Evans R. Sealing properties of endodontic filling materials using radioactive polar and nonpolar isotopes. Oral Surg Oral Med Oral Pathol 1966;22:355-8. 7. Higginbotham TL. A comparative study of the physical properties of five commonly used root canal sealers. Oral Surg Oral Med Oral Pathol 1967;24:89101. 8. Marosky JE, Patterson SS, Swartz M. Marginal leakage of temporary sealing materials used between endodontic appointments and assessed by calcium 45--an in vitro study. J Endodon 1977;3:110-3. 9. Allison DA, Weber CR, Walton E. The influence of the method of canal preparation on the quality of apical and coronal obturation. J Endodon 1979;5:298-304. 10. Wasserman F, Blayney JR, Groetzinger G, DeWitt TG. Studies on the different pathways of exchange of minerals in teeth with the aid of radioactive phosphorous. J Dent Res 1941;20:389-98. 11. Matloff IR, Jensen JR, Singer L, Tabibi A. A comparison of methods used in root canal sealability studies. Oral Surg 1982;53:203-8. 12. Mortensen DW, Boucber NE Jr, Ryge G. A method of testing for marginal leakage of dental restorations with bacteria. J Dent Res 1965;44:5863. 13. Krakow AA, deStoppelaar JD, Gren P. In vivo study of temporary filling materials used in endodontics in anterior teeth. Oral Surg 1977;43:615-20. 14. Goldman LB, Goldman K, Kronman JH, Letourneau J M Adaptation and porosity of poly-HEMA in a model system using two microorganisms. J Endodon 1980;66:863-6. 15. Spradling R, Senia ES. The relative sealing ability of paste-type filling materials. J Endodon 1982;8:543-9. 16. Williams S, Goldman M. Penetrability of the smeared layer by a strain of Proteus vulgaris. J Endodon 1985; 11:385-7. 17. Skinner RL, Himel VT. The sealing ability of injection-molded thermoplasticized gutta-percha with and without the use of sealer. J Endodon 1987;13:315-7. 18. Kronman JH, Goldman M, Goldman LB, Coleman E, Kliment CK. Microbiologic evaluation of poly-HEMA root canal filling material. Oral Surg 1979 ;48:175-7.
Printed in U.S.A.
VOL. 16, NO. 12, DECEMBER1990
In Vitro Bacterial Penetration of Coronally Unsealed Endodontically Treated Teeth Mahmoud Torabinejad, DMD, MS, Borasmy Ung, DDS, and James D. Kettering, PhD
In addition to dyes, radioisotopes have been used to study microleakage in alloy, resins, temporary filling substances, and root canal filling materials (4-9). Although isotopes may be a good tool for comparing relative leakage, they cannot give a true picture of the leakage which occurs clinically. This is because the ions used are much smaller than dye molecules and they diffuse much more rapidly than other small molecules (4). Isotopes are indicators of ion exchange, diffusion, or metabolism within the tissues rather than indicators of true leakage (10, 11). Mortensen et al. (12) and Krakow et al. (13) have stated that microorganism penetration might be more appropriate than dye or isotope penetration for studying leakage in vivo. Goldman et al. (14) have pointed out that bacteria performed better than dye in testing for leakage of hydrophilic materials and that dyes could give a false positive reading if their molecules were small enough. Because air bubbles can prevent dye leakage, the results of dye studies have been questioned (15; Goldman et al., personal communication). Because of inherent inadequacies in dye and radioisotope studies, it appears that bacterial leakage studies can provide more accurate information in clinical situations. We have found no reports on the length of time that passes before the entire obturated root canal is invaded by bacteria in obturated root canals without coronal seals. The purpose of this experiment was to determine the length of time needed for bacteria to penetrate a standardized length of obturated root canals which were intentionally exposed to one of two species of microorganisms.
Forty-five root canals were cleaned, shaped, and then obturated with gutta-percha and root canal sealer, using a lateral condensation technique. The coronal portions of the root filling materials were placed in contact with Staphylococcus epidermidis and Proteus vulgaris. The number of days required for these bacteria to penetrate the entire root canals was determined. Over 50% of the root canals were completely contaminated after 19-day exposure to S. epidermidis. Fifty percent of the root canals were also totally contaminated when the coronal surfaces of their fillings were exposed to P. vulgaris for 42 days.
Sealed root canals can be recontaminated under several circumstances: (a) if the patient has had endodontic treatment but has delayed placement of permanent restorations; (b) if the seal of the temporary filling material has broken down; or (c) if filling materials and/or tooth structures have fractured or been lost. When these situations occur, the coronal portion of the root canal system is exposed to oral flora. The question is how quickly the entire root canal system becomes contaminated again, to the point that retreatment of the canal may be necessary. Swanson and Madison (1) evaluated the length of time that the obturation material could be exposed to artificial saliva before compromising the integrity of the seal. They exposed the coronal portion of obturated root canals to artificial saliva for various time periods, and then immersed them in Pelikan ink for 48 h. They found that the dye penetrated from 79 to 85% of the root length in all exposed specimens. There was no leakage in the control group which was not exposed to artificial saliva, but was placed in contact with ink for 48 h. The follow-up study by Madison et al. (2) showed that in teeth exposed to artificial saliva for 7 days, the ink penetrated between 33 and 80% of the root length, depending on the type of sealer used. The latest findings by Madison and Wilcox (3) evaluating in vivo microleakage did not, however, confirm their in vitro studies. They found that some of the positive controls (no sealer) did not show microleakage, while some of the negative controls (temporary not removed) showed dye penetration.
MATERIALS AND METHODS To test bacterial penetration, a set-up similar to the one used by Goldman et al. (14) and Williams and Goldman (16) was used in this experiment.
Apparatus Set-up By using a high-speed handpiece and a #2 round bur, a small circular opening (about l mm in diameter) was made through the cap of a 20-ml scintillation flask. A paper clip was threaded through the opening and an alligator clip was hung on the inside. The outside end was bent to stabilize the alligator clip against the cap. The outside opening of the cap
566
Vol. 16, No. 12, December 1990
was resealed with acrylic material. The flasks, with the alligator clips attached to their caps, were steam sterilized. After instrumentation and obturation of root canals, a 25m m length of latex tubing was placed over the coronal portion of each tooth and the edges were sealed with epoxy resin (Quick Gel, non-run super-glue; Loctite Corp., Cleveland,
OH). The tubes, with the teeth attached, were sterilized in 5.25% sodium hypochlorite for 15 rain (based on the results of a pilot study) and then were rinsed with approximately 300 ml of sterile water. The tubes were then fastened to the caps of the previously sterilized scintillation vials. Ten milliliters of sterile phenol red broth with 3% lactose was added to the bottom of a flask, and the length of the tubing was adjusted so that a m i n i m u m of 2 m m of the apical part of each tooth was immersed in the solution (Fig. 1).
In Vitro Bacterial Penetration
567
Monitoring of the Samples The results from a pilot study showed that viable P. vulgaris were present 3 wk after incubation, while the S. epidermidis organisms were viable for only 1 wk. Fresh overnight cultures of organisms and sterile artificial saliva were added to the tubes at 5-day intervals for S. epidermidis and at 5- to 10-day intervals for P. vulgaris. When the bacterial culture was replenished, the old culture was plated to confirm continued viability o f the microorganisms. The samples were monitored daily until the red indicator solution at the bottom of the flask turned yellow. After color change, a sample of the yellow medium was plated on blood agar to assure that it contained the same type o f bacteria as that placed in the tubing.
Preparation of Teeth Bacterial Preparations Two species of bacteria were used as contaminants for this experiment; Proteus vulgaris, which is highly motile, and Staphylococcus epidermidis, which is nonmotile. These were grown overnight in 30 ml of trypticase soy broth (approximately 4.7 x l08 per ml o f / ' . vulgaris and 7.5 x 1 0 6 per ml of S. epidermidis). Two milliliters of the bacterial suspension and 0.7 ml of sterile artificial saliva as specified by Swanson and Madison (1) (1 mM CaC12, 3 mM NAH2PO4, 20 mM NaHCO3) were placed into the tubing. Since both organisms are acid formers, we expected the phenol red indicator solution to change to a yellow color when the bacteria reached it (16, 17).
Forty-five maxillary incisors and cuspids with straight canals were used in this study. The teeth had been stored previously in 10% formalin and were kept moist at all times throughout the experiment. After initial radiographs, standard access cavities were prepared, and the coronal portions of the canals were enlarged with #2 to #4 Gates Glidden drills. In order to obtain a standardized diameter, the apical foramina of the teeth were enlarged and kept patent to a #40 file, using a step-back filing technique. Approximately 2 ml of 5.25% NaOC1 were used between each file size, to remove debris. The prepared teeth were divided into experimental and control groups.
Experimental Groups The root canals of 33 teeth were obturated with guttapercha and Roth's sealer (Roth Drug Co., Chicago, IL) using the lateral condensation technique. To obtain a standardized length of filling, the coronal portion of the gutta-percha was removed with hot pluggers until only l0 m m of the filling material remained in the canal. To prevent bacteria from penetrating the root surfaces, two layers of fingernail polish were applied to the outside of the root except for 1 m m at the apex. GROUP 1 The coronal portions of the root canals of 16 teeth in this group were placed in contact with 2 ml of P. vulgaris in trypticase soy broth and 0.7 ml of sterile artificial saliva as described above. The tubing was then suspended over the phenol broth so that approximately 2 m m of the apex of the tooth was immersed in it. GROUP 2
FiG 1. Diagram of a tooth attached to a tube and suspended in the flask by an alligator clip.
The coronal portions of the root canals of the rest of the teeth in the experimental group (17 teeth) were exposed to 2 ml ofS. epidermidis and 0.7 ml of artificial saliva. The apices of these teeth were also immersed in phenol red broth.
568
Torabinejad et al.
Joumal of Endodontics
Control Groups
TABLE 2. Rate of total recontamination of obturated root canals exposed to S. epidermidis
To test the reliability of our results, the rest of the prepared teeth were divided into two control groups. GROUP 3 The root canal in each of the eight teeth that were used as positive controls was filled with a single gutta-percha cone without sealer, simulating poorly obturated root canals. Each of the two species of bacteria were placed separately in the tubing to contaminate four root canals (per microorganism), as described in the experimental group. GROUP 4 To verify that no contamination developed during the experiments (negative controls), four instrumented root canals were filled with gutta-percha and sealer. After removing the coronal portion of the filling material and leaving 10 m m of it in the root canal, the coronal portions of the filling were exposed to sterile artificial saliva applied as described above.
No. of Samples
% of Total
Cumulative %
1 1 5 2 2 4 1 1
6 6 29 12 12 23 6 6
6 12 41 53 65 88 94 100
Total 17
100%
No. of Days 15 16 17 19 20 30 45 51
Average: 24.1 days 0.4 mm/day
t test of independent samples shows that the difference is statistically significant (t = 4.68), with p < 0.01. Except for one sample in the positive control group (group 3), the rest of the samples (seven of eight) caused a color change in the phenol red medium after 1 to 4 days. The culture medium did not change color in teeth whose coronal segments were in contact with only sterile saliva (group 4) throughout the experiment (over 90 days).
RESULTS DISCUSSION In group 1, contaminated with P. vulgaris, two of the samples became positive after 2 days. One of these may have been a sample from the positive control, whereas the other one was found to have leakage through the latex tubing. These two samples were discarded. Table 1 shows the time it took P. vulgaris to reach the apex through 10 m m of the filling material. It varied from 10 to 73 days. The average length of time for leakage was 48.6 days. The time periods required for S. epidermidis to reach the apex in group 2 are shown in Table 2. Compared with those obtained in group 1, the results were more consistent, i.e. most apical leakage occurred between 15 and 30 days, with a range between 15 and 51 days. The average length of time for total penetration was 24.1 days. A statistical analysis using a
TABLE 1. Rate of total recontamination of obturated root canals exposed to P, vulgaris
No. of Samples 2 discarded 1 1 1 2 2 2 1 1 1 1 1 Total 14
% of Total 7 7 7 14.3 14.3 14.3 7 7 7 7 7
99.9%
Cumulative % 7 14 21 36 50 64 71 79 86 93 100
No. of Days
10 29 31 39 42 57 63 64 66 68 73 Average: 48.6 days 0.2 mm/day
Most of the samples in the positive control group with poorly filled canals showed leakage by 1 to 4 days, except for one which did not show a change until the 22nd day. There are two possible technical errors which could have caused the latter; one is that this tooth may have been switched by mistake with one of the experimental samples, the other is that the space prepared may have been circular enough to provide a very tight seal. The results of the positive control group are a confirmation of studies by Marshall and Massler (4), Evans and Simon (5), and Skinner and Himel (17) who showed that sealers are needed to improve the apical seal. As none of the negative controls led to a color change in the phenol red medium, it appears that our set-up did provide a contamination-free chamber. Over 85% of the teeth inoculated with P. vulgaris became completely penetrated in 66 days, whereas most (88%) of those inoculated with S. epidermidis were totally infected in 30 days, suggesting that motility may not be a factor in rate of penetration to the apices. We found a significant variability in the time it took the bacteria to penetrate the entire root canal system. Similar results were reported by Swanson and Madison (1) and Madison et al. (2) when they studied dye penetration in obturated root canals. This might be due to the shape of the prepared canal, type of sealer used, or the nature of the solution to which the coronal portions of the root canal were exposed. Goldman et al. (14) studied bacterial penetration in root canals filled with poly-HEMA, a hydrophilic, plastic polymer. They found no bacterial penetration after 42 days. The main reasons for lack of bacterial penetration in their results could be that poly-HEMA does not support bacterial growth as reported by Kronman et al., (18) because the pores are substantially smaller than the bacteria, or that the polymers have
Vol. 16, No. 12, December 1990
the same adhesive properties as the acrylic nail polish which was used to cover the external surfaces of the teeth. Compared with clinical conditions, the model used in our study was static, its media for bacterial growth was not totally similar to saliva and for ease of management of experimental conditions and interpretations of the data, its bacterial contents were purposely limited to only two species. Because of these limitations and their possible effects on the results, in vitro models simulating clinical conditions are needed to investigate the rate of leakage in unsealed, obturated root canals.
We gratefully acknowledge the assistance of Dr. Junichi Ryu and William Keeler with the bacterial culture aspects of the experiment. Dr. Torabinejad is director, Postgraduate Endodontics, Dr. Ung is a former graduate student in endodontics, and Dr. Kettering is professor of microbiology, Loma Linda University School of Medicine, Loma Linda, CA.
References 1. Swanson KS, Madkson S. An evaluation of coronal microleakage in endodontically treated teeth. Part I. Time periods. J Endodon 1987;13:56-9. 2. Madison S, Swanson KL, Chile SA. An evaluation of coronal microleakage in endodontically treated teeth. Part I1. Sealer types. J Endodon 1987;13:109-12. 3. Madison S, Wilcox LR. An evaluation of coronal microleakage in endodontically treated teeth. Part III. In vivo study. J Endodon 1988;14:455-8. 4. Marshall FJ, Massler M. The sealing of pulpless teeth evaluated with
In Vitro Bacterial Penetration
569
radio-isotopes. J Dent Med 1961 ;16:172-84. 5. Evans JT, Simon JHS. Evaluation of the apical seal produced by injected thermoplasticized gutta-percha in the absence of smear layer and root canal sealer. J Endodon 1986;12:101-7. 6. Kapsimalis P, Evans R. Sealing properties of endodontic filling materials using radioactive polar and nonpolar isotopes. Oral Surg Oral Med Oral Pathol 1966;22:355-8. 7. Higginbotham TL. A comparative study of the physical properties of five commonly used root canal sealers. Oral Surg Oral Med Oral Pathol 1967;24:89101. 8. Marosky JE, Patterson SS, Swartz M. Marginal leakage of temporary sealing materials used between endodontic appointments and assessed by calcium 45--an in vitro study. J Endodon 1977;3:110-3. 9. Allison DA, Weber CR, Walton E. The influence of the method of canal preparation on the quality of apical and coronal obturation. J Endodon 1979;5:298-304. 10. Wasserman F, Blayney JR, Groetzinger G, DeWitt TG. Studies on the different pathways of exchange of minerals in teeth with the aid of radioactive phosphorous. J Dent Res 1941;20:389-98. 11. Matloff IR, Jensen JR, Singer L, Tabibi A. A comparison of methods used in root canal sealability studies. Oral Surg 1982;53:203-8. 12. Mortensen DW, Boucber NE Jr, Ryge G. A method of testing for marginal leakage of dental restorations with bacteria. J Dent Res 1965;44:5863. 13. Krakow AA, deStoppelaar JD, Gren P. In vivo study of temporary filling materials used in endodontics in anterior teeth. Oral Surg 1977;43:615-20. 14. Goldman LB, Goldman K, Kronman JH, Letourneau J M Adaptation and porosity of poly-HEMA in a model system using two microorganisms. J Endodon 1980;66:863-6. 15. Spradling R, Senia ES. The relative sealing ability of paste-type filling materials. J Endodon 1982;8:543-9. 16. Williams S, Goldman M. Penetrability of the smeared layer by a strain of Proteus vulgaris. J Endodon 1985; 11:385-7. 17. Skinner RL, Himel VT. The sealing ability of injection-molded thermoplasticized gutta-percha with and without the use of sealer. J Endodon 1987;13:315-7. 18. Kronman JH, Goldman M, Goldman LB, Coleman E, Kliment CK. Microbiologic evaluation of poly-HEMA root canal filling material. Oral Surg 1979 ;48:175-7.
