0099-2399/90/1605-0207/$02.00/0 JOURNAL OF ENDOOONTICS Copyright 9 1990 by The American Association of Endodontists
Printed in U.S.A. VOL. 16, NO. 5, MAY 1990
SCIENTIFIC ARTICLES Root Canal Dentinal Tubule Disinfection Kamran E. Safavi, DMD, MEd, Larz S. W. Sp~,ngberg, DDS, PhD, and Kaare Langeland, DDS, PhD
Dentinal tubules of the root canal walls of human teeth were infected in vitro with a known bacterial isolate. The roots were exposed to either calcium hydroxide or iodine potassium-iodide for various periods of time and the viability of microorganisms was determined by incubation of entire root samples in a culture medium. The effects of the two agents on microbial viability were evaluated and compared. lodine potassium-iodide disinfected dentin effectively. In contrast, bacteria remained viable in the dentin after relatively extended periods of calcium hydroxide treatment.
of microorganisms. Nevertheless, some microorganisms growing in the dentinal tubules may not be removed by instrumentation ( 11 ). C o m m o n root canal microorganisms are readily killed when brought in contact with intracanal antiseptic solutions in vitro (2). Little is known of the continued viability of microorganisms in the tubules after infected dentin is exposed to intracanal antiseptics. The purpose of this study was to evaluate and compare the antimicrobial effects of two intracanal agents, calcium hydroxide and iodine potassium-iodide, on infected dentinal tubules in vitro.
MATERIALS AND METHODS The fundamental role of microorganisms in development of periapical inflammatory disease has been clearly demonstrated in recent years (l). It has been shown that necrotic pulp by itself does not cause apical bone resorption, but rather when a necrotic pulp becomes infected, apical periodontitis ensues. In modern endodontic practice, cleaning of the root canal by instrumentation, ample irrigation, and suction has reduced reliance on deposit antimicrobial agents that are known to be toxic to the host tissue (2). Microorganisms are also implicated in endodontic treatment failures (3). Residual pulp space infection appears to be due to clinical difficulties in removing the microbial substrate from the root canals and thus, in such instances, use of antimicrobial agents alone may not be sufficient. Nevertheless, it has been shown that if no antimicrobial agents are used during the root canal treatment, any microorganisms remaining in the canal will grow in the period between the treatments and rapidly increase in number (4-6). A wide variety of medicaments have been used as intracanal antiseptics (2). However, regardless of type and amount of antimicrobial agent used, the occurrence of persistent root canal infection is a c o m m o n clinical problem in endodontics (7-9). One possible reason for persistent endodontic infection may be retention of microorganisms in the dentin tissue of the root canal walls. Dentinal tubules of the root canal walls have been shown to harbor microorganisms (10-13). Routine root canal preparation procedures, including filing and irrigation, are likely to significantly reduce the number
Roots of freshly extracted, intact human teeth were used in this study. The teeth were stored in 4"C tap water until used. By using a rotating diamond saw and water irrigation, each root was separated from the crown apical to the cementoenamel junction, perpendicular to the long axis of the root. The apical end of the root was also cut off, again perpendicular to the long axis of the tooth, so that from each root a 4- to 6m m long specimen was prepared for the study. The cementum on the buccal surface of the specimens was removed using a diamond saw and water irrigation. The root canals were then instrumented with consecutively larger size reamers to a reamer which was three sizes larger than the original canal lumen. Copious tap water irrigation was used during root canal instrumentation. Specimens were kept in tap water at 40C before they were transferred to a 100-ml screw-capped glass flask containing 50 ml of a 5.25% solution of sodium hypochlorite. After agitation for 5 min, the specimens were placed under running tap water for 3 h and were then transferred to a flask containing 50 ml of a 50% solution of citric acid. After l-min agitation, the specimens were washed under running tap water for 3 h. The specimens in the water bath were autoclaved at 250~ for 20 min. Under strict aseptic conditions, the water was aspirated out o f the flasks, and 50 ml o f sterile fluid thioglycolate medium (BBL Microbiology Systems, Becton Dickinson and Co., Cockeysville, MD) were dispensed into each flask. To ensure sterility of the specimens, the flasks were incubated at 37~ for 2 wk before they were used. The confirmed sterile specimens were then divided into the three groups:
207
208
Safavi e t al.
Journal of Endodontics
Group 1 Specimens in this group were incubated in streptococcal cultures in the following manner: A flask containing autoclaved specimens in fluid thioglycolate was inoculated with Streptococcusfaecium (ATCC 9790) using a sterile platinum loop. The flask was placed in an incubator at 37"C for 27 days. During the incubation period, every 72 h, the turbid medium was aspirated and 50 ml of fresh sterile medium were dispensed into the flask. This group, hereafter, is referred to as the "infected" group.
Group 2 Specimens of this group were exposed to Streptococcus faecium for a short period of time in the following manner: The sterile medium was aspirated out of a flask containing autoclaved specimens and a 50-ml suspension of Streptococcus faecium (3 x 103 per ml) in fluid thioglycolate was dispensed into the flask and shaken for a few seconds. The specimens remained in contact with the microorganisms for approximately 10 m in at room temperature before antimicrobial experiments were begun as described below. This group is referred to as the "contaminated" group.
Group 3 Autoclaved specimens were incubated in sterile fluid thioglycolate medium at 37"C and were used throughout the experiments as negative controls. This group is referred to as the "control" group.
Histological Studies Under strict aseptic conditions, specimens were randomly removed from each of the three groups and were prepared for histological evaluation in the following manner. Each specimen was placed in a 5-ml solution of 10% neutral buffered formalin. After fixation for 48 h, the specimen was washed under running tap water for 8 to 10 h, decalcified in a 5% solution of nitric acid, rinsed again under running tap water for 8 to 10 h, and processed for paraffin embedding. Each specimen was placed in a paraffin block oriented for histological section perpendicular to the long axis of the root. Approximately 50 histological serial sections, each 5-#m thick, were obtained from each specimen. During cutting of histological specimens with the microtomc, the paraffin block was slightly reoriented after approximately every 20 cuts. This was to ensure that sections parallel to the dentinal tubules were obtained in all specimens. Five to six sections were placed on each slide and every third slide was stained using the Brown and Brenn method. The slides were studied under a light microscope.
Antimicrobial Experiments Each group of specimens was used in the following manner. Specimens were removed from the flasks under strict aseptic conditions and each specimen was submerged in 2 ml of
sterile saline solution and agitated three times for 30 s (fresh sterile saline each time), and then submerged in 2 ml of one of the following using cluster wells of cell culture plates (Costar, Cambridge, MA): (a) 2% iodine potassium-iodide (IKI) (iodine, 2%; potassium-iodide, 4%; and distilled water, 94%); (b) Ca(OH)2 (calcium hydroxide USP) mixed with saline to a toothpaste consistency; or (c) V M G III transport medium (14). The specimens were agitated for 30 s and left in the wells. After various specimen-medicament exposure times (Table 1), the specimens were washed three times in 2 ml of sterile saline for 30 s (fresh sterile saline each time) and transferred to a test tube containing 8 ml of fluid thioglycolate medium and incubated at 37"C. The test tubes were read periodically for turbidity for 60 days. The number of specimens used in each group is given in Table 1. RESULTS Microorganisms were observed only in the dentinal tubules of the infected group. Only a small percentage of tubules showed evidence of any microbial penetration (Fig 1). In some histological sections, no microorganisms could be observed in the tubules. However, some tubule infection could be found in all specimens of the infected group. The tubule infection occurred on the pulpal side of the dentin, mainly in the areas where the corresponding cementum had been removed. Except in one specimen, no microbial penetration was observed in the peripheral exposed dentin. No microbial penetration was observed in the cementum. The frequency of microbial penetration did not seem to vary in different specimens. The penetration of microorganisms was generally between 50 to 100 um (Fig. 1). However, microbial penetration as deep as 300 um was observed in some sections. No microorganisms were observed in histological sections of the contaminated or control groups. After 24-h incubation of the specimens in the medium, turbidity was observed in some of the test tubes. The frequency of positive cultures increased thereafter up to 72 h. Cultures that were not turbid at the 72-h observation period remained negative to the end of the observation period of 60 days. The frequency of positive cultures in each group is given in Table 1. In iodine potassium-iodide-treated specimens, a short period of medicament-dentin contact was sufficient to prevent
TABLE 1. N u m b e r of specimens and the time they w e r e exposed to intracanal agents or the transport medium* Infected Dentin
C o n t a m i n a t e d Dentin
Time Ca(OH)2
IKI
V M G III
Ca(OH)2
IKI
V M G III
1 min
10(10)
10(10)
--
15(13)
13(0)
--
5 min 10min l h
10 (10) 10(10) 10(10)
8 10 10
--5(5)
16 (14) 10(10) 17 (9)
13(0) 22(0) 10(0)
--5(5)
10(0)
5(5)
---
5(5) 5(5)
2h 12h 24h
(8) (0) (0)
10(10)
--
5(5)
10 (2)
10 8
---
5(5) 5(5)
10 10
(5) (0)
(3) (0)
* Values in parentheses are number of pos=t=veyields obtained from cuMudr~:j the Sl:)ec~mensafter they were exposed to the intracanalagent or t ~ transport medium.
Dentinal Tubule Disinfection
Vol. 16, No. 5, May 1990
FIG 1. Streptococcus faecium penetration into the dentinal tubules
(Brown and Brenn; original magnification x800). microbial growth. In calcium hydroxide-treated specimens, on the other hand, positive cultures were frequently observed despite relatively long periods of dentin-medicament contact periods (Table 1). In both iodine potassium-iodide- and calcium hydroxidetreated specimens, the positive cultures were more frequently observed in the infected than in the contaminated group. All specimens of infected or contaminated groups that were placed in VMG III transport medium yielded positive growth. No growth was observed in the control (sterile) group. DISCUSSION The cementum was removed because it was shown in vitro that in the areas of the roots of bovine teeth, where the cementum was removed and the tubules were patent, the infection of corresponding tubules from the pulpal side was considerably enhanced (13). Results of our experiments were generally in agreement with these findings. The choice of incubation period was based on results of experiments ( 12,13) that showed that Streptococcusfaecalis did not invade the dentinal tubules until after 2 wk of incubation, after 3 wk of incubation a dense tubule infection occurred, and thereafter the depth of tubule infection increased only slowly by time. These findings were confirmed in our pilot studies. In a method to infect the dentinal tubules (13), published recently, the inoculated medium was confined to the root canal. Our pilot experiments, however, indicated that maintaining the sterility o f the media surrounding the cementum was unnecessary, as bacteria did not penetrate the cementum, and penetration into the exposed dentinal tubules from the outer root surface was very infrequent. Iodine potassium-iodide was used as a test material because it has been shown to be an effective antimicrobial agent and, compared with commonly used root canal antiseptics, has a low level of cytotoxicity (2). Calcium hydroxide is advocated as a routine intracanal dressing agent (9,15), and it has been shown that its application in instrumented and irrigated root canals eliminates microorganisms effectively (15).
209
The penetration of antiseptics into the dentinal tubules has been shown to vary greatly (I 7). Iodine potassium-iodide, by way of its vapors, has been shown to have long distance bacteriocidal effects in vitro. Its duration of antimicrobial efficiency, however, has been shown to be very short (18) and its ability to penetrate the tubules has not been investigated. Calcium hydroxide, on the contrary, has a rather poor solubility, and its bacteriocidal effects on dentinal tubule microorganisms are questioned in a recent report (13). The results of pilot experiments indicated that washing of specimens in saline, after they were exposed to intracanal agents, was sufficient to prevent transportation of the medicaments in the culture media and thus no chemical inactivation ofintracanal agents, such as sodium thiosulfate (for iodine potassiumiodide) or citric acid (for calcium hydroxide) (14), was deemed necessary. Frequency of positive cultures obtained in our experiments (Table 1) confirmed the pilot study results. Black-pigmented Bacteroides and other Gram-negative strict anaerobes, rather than streptococci, are now considered to be the predominant microflora of the root canals of teeth with periapical pathosis (19). Streptococcusfaecium, however, was selected as a test microorganism in the present study because it is one of the more resistant microorganisms of root canal flora (15) and in histological sections, a blue-staining Gram-positive microorganism is more distinguishable from tissue components than a red-staining Gram-negative bacteria
(2o). In an in vitro model, using infected bovine dentin, it was shown that incubation of dentin chips, scraped from the pulpal surface of dentin wall with the aid of sterile burs, yielded no growth, whereas samples taken in the same manner from deeper parts of dentin, in the same specimen, often yielded positive findings (13). No such samples were taken in our study because pilot histological observations revealed that in our materials the depth of bacterial penetration in the tubules varied in different specimens, and, thus, a negative culture obtained from a certain depth o f dentin could not be correlated with the bacteriocidal effects of the intracanal agent, as one cannot be sure whether microorganisms penetrated to that level ofdentin to begin with. In our experiments, after treatment of dentin with medicaments, in order to allow any viable microorganism to grow, the specimens were incubated in the medium for a long period of time. The effectiveness of calcium hydroxide treatment in eliminating microorganisms from the root canals has been clearly demonstrated in vivo (15). Results of our experiments are in agreement with previous reports (13) indicating that enterococci may remain viable after relatively long exposures to calcium hydroxide. The significance of enterococci in the development of periapical pathosis is not clear, but it serves as an appropriate target organism in this type of investigation. In clinical situations where infection of the root canals is suspected or a contamination of the root canals occurs during treatment, it seems to be prudent that an antiseptic be applied in the root canals. Based on results obtained in our experiments, a "flooding" of the root canal for a few minutes with iodine potassium-iodide during root canal procedures may be advocated in such cases.
This study was supported by the University of Connecticut Research Foundation.
210
Journal of Endodontics
Safari et al.
Drs. Safavi, Sp~mgberg, and Langeland are affiliated with the School of Dental Medicine, University of Connecticut Health Center, Farmington, CT.
References 1. Sundqvist G. Bacteriological studies of necrotic dental pulps. Ume& University Odontological Dissertations No. 7, 1976. 2. Sp&ngberg L. Endodontic medicaments. In: Smith DC, Williams DF, eds. Biocompatibility of dental materials. Boea Raton, FL: CRC Press, 1982;22357. 3. Engstr(~ B. Lundberg M. The correlation between positive culture and the prognosis of root canal therapy after pulpectomy. Odontol Revy 1965;16:193-203. 4. Bystr6m A, Sundqvist G. Bacteriologic evaluation of the efficacy of mechanical root canal instrumentation in endodontic therapy. Scand J Dent Res 1981 ;89:321-8. 5. Bystr~)m A, Sundqvist G. Bacteriologic evaluation of the effect of 0.5 percent sodium hypochlorite in endodontic therapy. Oral Surg 1983;55:30712. 6. Bystr6m A, Sundqvist G. The antimicrobial action of sodium hypochlorite and EDTA in 60 cases of endodontic therapy. Int Endod J 1985;18:35-40. 7. Engstr6m B. The significance of enterococci in root canal treatment. Odontol Revy 1964;15:87-106. 8. Myers JW, James Marshal F, Rosen S. The incidence and identity of microorganisms present in root canals at filling following culture reversals. Oral Surg 1969;28:889-96. 9. Safavi KE, Dowden WE, Langeland K, Introcaso JH. A comparison of
antimicrobial effects of calcium hydroxide and iodine potassium iodide. J Endodon 1985;11.'454-6. 10. Chirnside IM. The bacteriological status of dentine around infected pulp canals. New Zealand Dent J 1958;54:173-83. 11. Shovelton DS. The presence and distribution of microorganisms within non-vital teeth. Br Dent J 1964; 117:101-7. 12. Akpata ES, Blechman H. Bacterial invasion of pulpal dentin wall in vitro. J Dent Res 1982;61:435-8. 13. Haapasalo M, (Z)rstavik D. In vitro infection and disinfection of dentinal tubules. J Dent Res 1987;66:1375-9. 14. M611er/~JR, Microbiological examination of the root canals and periapical tissues of human teeth. Methodological studies. Odontol Tidskr 1966;74(suppl): 1-360. 15. Bystrom A, Claeseon R, Sundqvist G. The antibacterial effect of camphorated paramonochlorophenol, camphorated phenol and calcium hydroxide in treatment of infected root canals. Endod Dent Traumato11985;1:170-5. 16. Ellerbruch ES, Murphy RA. Antimicrobial activity of root canal medicament vapors. J Endodon 1977;3:189-93. 17. Avny WY, Heiman GR, Madonia JV, Wood NK, Smulson MH. Autoradiographic studies of the intracanal diffusion of aqueous and camphorated parachloraophenol in endodontics. Oral Surg 1973;36:80-9. 18. Engstrom B. On duration of the antibacterial efficiency of four antiseptics used in root canal treatment in situ. Svensk Tandlakare-Tidskrift 1958;51:1 6. 19. Sundqvist G, Johansson E, Sj~:jren U. Prevalence of black-pigmented Bacteroides species in root canal infections. J Eedodon 1989;15:13-9. 20. Wijnbergen M, Van Mullem PJ. Effect of histological decalcifying agents on number and stainability of Gram-positive bacteria. J Dent Res 1987; 66:1029-31.
Printed in U.S.A. VOL. 16, NO. 5, MAY 1990
SCIENTIFIC ARTICLES Root Canal Dentinal Tubule Disinfection Kamran E. Safavi, DMD, MEd, Larz S. W. Sp~,ngberg, DDS, PhD, and Kaare Langeland, DDS, PhD
Dentinal tubules of the root canal walls of human teeth were infected in vitro with a known bacterial isolate. The roots were exposed to either calcium hydroxide or iodine potassium-iodide for various periods of time and the viability of microorganisms was determined by incubation of entire root samples in a culture medium. The effects of the two agents on microbial viability were evaluated and compared. lodine potassium-iodide disinfected dentin effectively. In contrast, bacteria remained viable in the dentin after relatively extended periods of calcium hydroxide treatment.
of microorganisms. Nevertheless, some microorganisms growing in the dentinal tubules may not be removed by instrumentation ( 11 ). C o m m o n root canal microorganisms are readily killed when brought in contact with intracanal antiseptic solutions in vitro (2). Little is known of the continued viability of microorganisms in the tubules after infected dentin is exposed to intracanal antiseptics. The purpose of this study was to evaluate and compare the antimicrobial effects of two intracanal agents, calcium hydroxide and iodine potassium-iodide, on infected dentinal tubules in vitro.
MATERIALS AND METHODS The fundamental role of microorganisms in development of periapical inflammatory disease has been clearly demonstrated in recent years (l). It has been shown that necrotic pulp by itself does not cause apical bone resorption, but rather when a necrotic pulp becomes infected, apical periodontitis ensues. In modern endodontic practice, cleaning of the root canal by instrumentation, ample irrigation, and suction has reduced reliance on deposit antimicrobial agents that are known to be toxic to the host tissue (2). Microorganisms are also implicated in endodontic treatment failures (3). Residual pulp space infection appears to be due to clinical difficulties in removing the microbial substrate from the root canals and thus, in such instances, use of antimicrobial agents alone may not be sufficient. Nevertheless, it has been shown that if no antimicrobial agents are used during the root canal treatment, any microorganisms remaining in the canal will grow in the period between the treatments and rapidly increase in number (4-6). A wide variety of medicaments have been used as intracanal antiseptics (2). However, regardless of type and amount of antimicrobial agent used, the occurrence of persistent root canal infection is a c o m m o n clinical problem in endodontics (7-9). One possible reason for persistent endodontic infection may be retention of microorganisms in the dentin tissue of the root canal walls. Dentinal tubules of the root canal walls have been shown to harbor microorganisms (10-13). Routine root canal preparation procedures, including filing and irrigation, are likely to significantly reduce the number
Roots of freshly extracted, intact human teeth were used in this study. The teeth were stored in 4"C tap water until used. By using a rotating diamond saw and water irrigation, each root was separated from the crown apical to the cementoenamel junction, perpendicular to the long axis of the root. The apical end of the root was also cut off, again perpendicular to the long axis of the tooth, so that from each root a 4- to 6m m long specimen was prepared for the study. The cementum on the buccal surface of the specimens was removed using a diamond saw and water irrigation. The root canals were then instrumented with consecutively larger size reamers to a reamer which was three sizes larger than the original canal lumen. Copious tap water irrigation was used during root canal instrumentation. Specimens were kept in tap water at 40C before they were transferred to a 100-ml screw-capped glass flask containing 50 ml of a 5.25% solution of sodium hypochlorite. After agitation for 5 min, the specimens were placed under running tap water for 3 h and were then transferred to a flask containing 50 ml of a 50% solution of citric acid. After l-min agitation, the specimens were washed under running tap water for 3 h. The specimens in the water bath were autoclaved at 250~ for 20 min. Under strict aseptic conditions, the water was aspirated out o f the flasks, and 50 ml o f sterile fluid thioglycolate medium (BBL Microbiology Systems, Becton Dickinson and Co., Cockeysville, MD) were dispensed into each flask. To ensure sterility of the specimens, the flasks were incubated at 37~ for 2 wk before they were used. The confirmed sterile specimens were then divided into the three groups:
207
208
Safavi e t al.
Journal of Endodontics
Group 1 Specimens in this group were incubated in streptococcal cultures in the following manner: A flask containing autoclaved specimens in fluid thioglycolate was inoculated with Streptococcusfaecium (ATCC 9790) using a sterile platinum loop. The flask was placed in an incubator at 37"C for 27 days. During the incubation period, every 72 h, the turbid medium was aspirated and 50 ml of fresh sterile medium were dispensed into the flask. This group, hereafter, is referred to as the "infected" group.
Group 2 Specimens of this group were exposed to Streptococcus faecium for a short period of time in the following manner: The sterile medium was aspirated out of a flask containing autoclaved specimens and a 50-ml suspension of Streptococcus faecium (3 x 103 per ml) in fluid thioglycolate was dispensed into the flask and shaken for a few seconds. The specimens remained in contact with the microorganisms for approximately 10 m in at room temperature before antimicrobial experiments were begun as described below. This group is referred to as the "contaminated" group.
Group 3 Autoclaved specimens were incubated in sterile fluid thioglycolate medium at 37"C and were used throughout the experiments as negative controls. This group is referred to as the "control" group.
Histological Studies Under strict aseptic conditions, specimens were randomly removed from each of the three groups and were prepared for histological evaluation in the following manner. Each specimen was placed in a 5-ml solution of 10% neutral buffered formalin. After fixation for 48 h, the specimen was washed under running tap water for 8 to 10 h, decalcified in a 5% solution of nitric acid, rinsed again under running tap water for 8 to 10 h, and processed for paraffin embedding. Each specimen was placed in a paraffin block oriented for histological section perpendicular to the long axis of the root. Approximately 50 histological serial sections, each 5-#m thick, were obtained from each specimen. During cutting of histological specimens with the microtomc, the paraffin block was slightly reoriented after approximately every 20 cuts. This was to ensure that sections parallel to the dentinal tubules were obtained in all specimens. Five to six sections were placed on each slide and every third slide was stained using the Brown and Brenn method. The slides were studied under a light microscope.
Antimicrobial Experiments Each group of specimens was used in the following manner. Specimens were removed from the flasks under strict aseptic conditions and each specimen was submerged in 2 ml of
sterile saline solution and agitated three times for 30 s (fresh sterile saline each time), and then submerged in 2 ml of one of the following using cluster wells of cell culture plates (Costar, Cambridge, MA): (a) 2% iodine potassium-iodide (IKI) (iodine, 2%; potassium-iodide, 4%; and distilled water, 94%); (b) Ca(OH)2 (calcium hydroxide USP) mixed with saline to a toothpaste consistency; or (c) V M G III transport medium (14). The specimens were agitated for 30 s and left in the wells. After various specimen-medicament exposure times (Table 1), the specimens were washed three times in 2 ml of sterile saline for 30 s (fresh sterile saline each time) and transferred to a test tube containing 8 ml of fluid thioglycolate medium and incubated at 37"C. The test tubes were read periodically for turbidity for 60 days. The number of specimens used in each group is given in Table 1. RESULTS Microorganisms were observed only in the dentinal tubules of the infected group. Only a small percentage of tubules showed evidence of any microbial penetration (Fig 1). In some histological sections, no microorganisms could be observed in the tubules. However, some tubule infection could be found in all specimens of the infected group. The tubule infection occurred on the pulpal side of the dentin, mainly in the areas where the corresponding cementum had been removed. Except in one specimen, no microbial penetration was observed in the peripheral exposed dentin. No microbial penetration was observed in the cementum. The frequency of microbial penetration did not seem to vary in different specimens. The penetration of microorganisms was generally between 50 to 100 um (Fig. 1). However, microbial penetration as deep as 300 um was observed in some sections. No microorganisms were observed in histological sections of the contaminated or control groups. After 24-h incubation of the specimens in the medium, turbidity was observed in some of the test tubes. The frequency of positive cultures increased thereafter up to 72 h. Cultures that were not turbid at the 72-h observation period remained negative to the end of the observation period of 60 days. The frequency of positive cultures in each group is given in Table 1. In iodine potassium-iodide-treated specimens, a short period of medicament-dentin contact was sufficient to prevent
TABLE 1. N u m b e r of specimens and the time they w e r e exposed to intracanal agents or the transport medium* Infected Dentin
C o n t a m i n a t e d Dentin
Time Ca(OH)2
IKI
V M G III
Ca(OH)2
IKI
V M G III
1 min
10(10)
10(10)
--
15(13)
13(0)
--
5 min 10min l h
10 (10) 10(10) 10(10)
8 10 10
--5(5)
16 (14) 10(10) 17 (9)
13(0) 22(0) 10(0)
--5(5)
10(0)
5(5)
---
5(5) 5(5)
2h 12h 24h
(8) (0) (0)
10(10)
--
5(5)
10 (2)
10 8
---
5(5) 5(5)
10 10
(5) (0)
(3) (0)
* Values in parentheses are number of pos=t=veyields obtained from cuMudr~:j the Sl:)ec~mensafter they were exposed to the intracanalagent or t ~ transport medium.
Dentinal Tubule Disinfection
Vol. 16, No. 5, May 1990
FIG 1. Streptococcus faecium penetration into the dentinal tubules
(Brown and Brenn; original magnification x800). microbial growth. In calcium hydroxide-treated specimens, on the other hand, positive cultures were frequently observed despite relatively long periods of dentin-medicament contact periods (Table 1). In both iodine potassium-iodide- and calcium hydroxidetreated specimens, the positive cultures were more frequently observed in the infected than in the contaminated group. All specimens of infected or contaminated groups that were placed in VMG III transport medium yielded positive growth. No growth was observed in the control (sterile) group. DISCUSSION The cementum was removed because it was shown in vitro that in the areas of the roots of bovine teeth, where the cementum was removed and the tubules were patent, the infection of corresponding tubules from the pulpal side was considerably enhanced (13). Results of our experiments were generally in agreement with these findings. The choice of incubation period was based on results of experiments ( 12,13) that showed that Streptococcusfaecalis did not invade the dentinal tubules until after 2 wk of incubation, after 3 wk of incubation a dense tubule infection occurred, and thereafter the depth of tubule infection increased only slowly by time. These findings were confirmed in our pilot studies. In a method to infect the dentinal tubules (13), published recently, the inoculated medium was confined to the root canal. Our pilot experiments, however, indicated that maintaining the sterility o f the media surrounding the cementum was unnecessary, as bacteria did not penetrate the cementum, and penetration into the exposed dentinal tubules from the outer root surface was very infrequent. Iodine potassium-iodide was used as a test material because it has been shown to be an effective antimicrobial agent and, compared with commonly used root canal antiseptics, has a low level of cytotoxicity (2). Calcium hydroxide is advocated as a routine intracanal dressing agent (9,15), and it has been shown that its application in instrumented and irrigated root canals eliminates microorganisms effectively (15).
209
The penetration of antiseptics into the dentinal tubules has been shown to vary greatly (I 7). Iodine potassium-iodide, by way of its vapors, has been shown to have long distance bacteriocidal effects in vitro. Its duration of antimicrobial efficiency, however, has been shown to be very short (18) and its ability to penetrate the tubules has not been investigated. Calcium hydroxide, on the contrary, has a rather poor solubility, and its bacteriocidal effects on dentinal tubule microorganisms are questioned in a recent report (13). The results of pilot experiments indicated that washing of specimens in saline, after they were exposed to intracanal agents, was sufficient to prevent transportation of the medicaments in the culture media and thus no chemical inactivation ofintracanal agents, such as sodium thiosulfate (for iodine potassiumiodide) or citric acid (for calcium hydroxide) (14), was deemed necessary. Frequency of positive cultures obtained in our experiments (Table 1) confirmed the pilot study results. Black-pigmented Bacteroides and other Gram-negative strict anaerobes, rather than streptococci, are now considered to be the predominant microflora of the root canals of teeth with periapical pathosis (19). Streptococcusfaecium, however, was selected as a test microorganism in the present study because it is one of the more resistant microorganisms of root canal flora (15) and in histological sections, a blue-staining Gram-positive microorganism is more distinguishable from tissue components than a red-staining Gram-negative bacteria
(2o). In an in vitro model, using infected bovine dentin, it was shown that incubation of dentin chips, scraped from the pulpal surface of dentin wall with the aid of sterile burs, yielded no growth, whereas samples taken in the same manner from deeper parts of dentin, in the same specimen, often yielded positive findings (13). No such samples were taken in our study because pilot histological observations revealed that in our materials the depth of bacterial penetration in the tubules varied in different specimens, and, thus, a negative culture obtained from a certain depth o f dentin could not be correlated with the bacteriocidal effects of the intracanal agent, as one cannot be sure whether microorganisms penetrated to that level ofdentin to begin with. In our experiments, after treatment of dentin with medicaments, in order to allow any viable microorganism to grow, the specimens were incubated in the medium for a long period of time. The effectiveness of calcium hydroxide treatment in eliminating microorganisms from the root canals has been clearly demonstrated in vivo (15). Results of our experiments are in agreement with previous reports (13) indicating that enterococci may remain viable after relatively long exposures to calcium hydroxide. The significance of enterococci in the development of periapical pathosis is not clear, but it serves as an appropriate target organism in this type of investigation. In clinical situations where infection of the root canals is suspected or a contamination of the root canals occurs during treatment, it seems to be prudent that an antiseptic be applied in the root canals. Based on results obtained in our experiments, a "flooding" of the root canal for a few minutes with iodine potassium-iodide during root canal procedures may be advocated in such cases.
This study was supported by the University of Connecticut Research Foundation.
210
Journal of Endodontics
Safari et al.
Drs. Safavi, Sp~mgberg, and Langeland are affiliated with the School of Dental Medicine, University of Connecticut Health Center, Farmington, CT.
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