The comparative antimicrobial effect of calcium hydroxide Kathryn G. Stuart, DDS, MSD,a Chris H. Miller, PhD,b Cecil E. Brown Jr., DDS, IMS,~ and Carl W. Newton, DDS, MSD.d Indianapolis, Ind. INDIANA
UNIVERSITY
SCHOOL OF DENTISTRY
The antimicrobial effectiveness of calcium hydroxide, camphorated paramonochlorophenol, and formocresol in root canals of extracted human teeth was’ compared. Canals in single-rooted teeth were enlarged and inoculated with Streptococcus mutans, Actinomyces viscosus, and Bacteroides gingivalis or Bacteroides fragilis. After treatment with a test agent and sealing and incubation for 1 hour, the canal contents were analyzed for the number of viable test bacteria and compared with that of inoculated teeth not treated with test agents. All test agents exhibited antimicrobial activity against all bacteria, with percent reductions in viable bacteria ranging from 64.3% to 100%. The combined data for Pulpdent paste and calcium hydroxide showed significantly higher antimicrobial activity than the combined data for camphorated paramonochlorophenol and formocresol for S. mutans and B. gingivalis or B. fragitis but showed no difference for A. viscosus. (ORAL SURC ORAL MED ORAL PATHOL 1991;72:101-4)
uccessful endodontic treatment depends on many S factors. Among the most important of these is the reduction or elimination of bacteria from the pulpal spaceand the removal of the substrate on which they depend. Biomechanical instrumentation and irrigation are successful in cleaning the root canal system, and intracanal medications traditionally have been used to maintain cleanliness between treatments. Selection of these medications has been based on effectiveness, toxicity, inflammation potential, and diffusibility. Unfortunately, most medications used today are nonspecific in toxicity and diffusibility and can themselves cause inflammation and patient discomfort. Calcium hydroxide has been used for apexification and pulp capping procedures and now is being used as an intracanal medication in endodontic therapy.le5 Clinically, it appears to control infection and to reduce the incidence of symptoms between treatments more effectively than the traditional medications of camphorated paramonochlorophenol (CMCP) and formocresol. This study compares the antibacterial
aEndodontist in private practice, Indianapolis. bProfessor and Chairman of Oral Microbiology. CAssociate Professor of Endodontics. dProfessor and Chairman of Endodontics. 7/15/28824
effectivenessof Ca(OH)z, CMCP, and formocresol in root canals of extracted human teeth against organisms shown to be related to pulpal infections. MATERIAL AND METHODS
The canals of single-rooted extracted teeth were prepared with K-type endodontic files to a size 50, sterilized, inoculated with a mixture of three test bacteria, and either treated with one of four test agents or left untreated before being sealed with dental stopping. After 1 hour at 37” C in a humid environment, the canal contents were removed and analyzed to determine the number of viable bacteria. The percent reduction in the number of viable bacteria remaining in the treated canals was calculated by comparing with the number of viable bacteria in the untreated control teeth. Tooth preparation
Ninety extracted, single-rooted adult human teeth, consisting of maxillary incisors, maxillary and mandibular canines, and mandibular premolars, were collected without regard to pulpal diagnosis. Teeth with large single canals were selected to accommodate the test medicaments and inoculum. The teeth were extracted, immediately placed in sterile water, and stored until the time of use. Before preparation the teeth were autoclaved twice in 15-minute cycles at a temperature of 250” F. The 101
102
Stuart
et al.
outer surfaces of the teeth were then debrided and scaled. The canals were prepared to a size 50 by a circumferential filing technique with a K-type file. All teeth were irrigated with 5 ml of sterile water during canal preparation with a tuberculin syringe. The specimenswere autoclaved again for 15 minutes at a temperature of 250” F before inoculation. After final autoclaving an aseptic technique was maintained with the use of sterile surgical gloves, drapes, and sterilized instruments. Before inoculation the canals were dried with sterile absorbent paper points and the external root surfaces were blotted dry with sterile gauze. The teeth were then dipped apex first in melted paraffin to the cementoenameljunction to prevent apical leakage of the inoculum and/or the medicament. Preparation
of standardized
inoculum
Four different microorganisms were used: Streptococcus mutans 6715 (obtained from P. H. Keyes, National Institute for Dental Research, Bethesda, Md.); Actinomyces viscosus ATCC 15987 (American Type Culture Collection [ATCC], Rockville, Md.); Bacteroides gingivalis ATCC 33277; and Bacteroides fragilis IUOM AN 1 (Department of Oral Microbiology, Indiana University, Indianapolis). The test bacteria were cultured from frozen stock in brain-heart infusion broth for 24 hours (S. mutans and A. viscosus) or for 48 hours with a subculture at 24 hours (Bacteroides species)in an anaerobic atmosphere (85% nitrogen, 10% hydrogen, 5% carbon dioxide) within an anaerobic chamber (model 1024, Forma Scientific Inc., Marietta, Ohio). Measured amounts of S. mutans, A. viscosus, and either B. gingivalis or B. fragilis culture were mixed together and centrifuged at 10,OOOg for 10 minutes at 7” C to pellet the cells. The pellet was resuspendedin 2.0 ml of a reduced transport fluid (RTF).6 A standardized volume of 15 ml of the RTF suspensionof the three speciesof bacteria was placed in the prepared canal of each tooth. This inoculum volume was previously determined by estimating the average root canal volume of prepared teeth. Becauseseveral batches of test bacterial cultures were prepared throughout the study, attempts were made to obtain the same relative number of cells of each speciesof bacterium in the final 15 ml inoculum preparations. This was performed by measuring the optical density (675 nm) of the individual cultures before mixing and relating these previously measured viable cell counts at that optical density. The volumes of each individual culture added to the mixture were then adjusted accordingly with each batch of inoculum prepared.
Treatment
of inoculated
canals
A total of 20 prepared teeth were used with each of the four test agents. All 20 teeth were inoculated; 10 were treated with the test agent, and 10 were not treated. The test agents were CMCP (Sultan Chemists Inc., Englewood, N.J.), formocresol (Sultan Chemists), Pulpdent paste (Pulpdent Corp., Watertown, Mass.) and a sterile saline solution slurry of Ca(OH)z. Inoculated teeth were treated with CMCP or formocresol by placing a size 4 sterile cotton pledget medicated with 1.0 ~1 of the agent. This was the amount found to remain on the pledget after squeezing it dry. Pulpdent paste was used as supplied and was placed with a sterile tuberculin syringe and spiraled into the canal to the apex with a lentulo instrument. Fifty milligrams of Ca(OH)z powder was slurried with sterile saline solution, and the thick paste was applied with an amalgam carrier and condensedwith endodontic pluggers. The canal access of untreated teeth was blocked with a sterile size 4 cotton pledget. All teeth were sealed with dental stopping (Hygenic Corp., Akron, Ohio). Ten teeth with prepared canals were not inoculated or treated but were sealed.These served as a control for aseptic technique. All teeth were placed in separate 10 ml sterile beakers in a covered sterile 1000 ml beaker also containing a 100 ml sterile beaker full of sterile water. This system provided a humid environment during the lhour period of incubation at 37” C. Recovery
of test bacteria
After the l-hour incubation period, each tooth canal was aseptically opened with a sterile endodontic explorer over a sterile 30 ml beaker containing 1.Oml of sterile phosphate-buffered RTF (BRTF). BRFT was used to neutralize the alkalinity of the Pulpdent and Ca(OH)z materials that would normally prevent growth of live bacteria remaining in the canal during subsequent culturing. A fresh, sterile size 50 file was used to remove the superficial layer of dentin, medicament, and bacteria into the BRTF. The instrument canal was flushed with 1.Oml of sterile BRTF into the samebeaker with a sterile tuberculin syringe. The file was added to the BRTF suspension,and after ultrasonic dispersion for 10 seconds(Artek sonic dismembrater, 60% maximum setting with a microtip) the suspensionwas serially diluted in lo-fold increments to 1:10,000. One-tenth milliliter of the undiluted suspension and of each dilution was spread plated on MM10 blood agar7 for recovery of A. viscosus and Bacteroides speciesand on Mitis salivarius-bacitratin-sucrose (MSBS) agar8 for recovery of S. mutans.
Volume 72 Number I
After anaerobic incubation at 31" C for 3 days (MSBS agar) or 5 days (MMlO), the number of colony-forming units (CFU) that developed on the plates was determined with the aid of a stereoscopic microscope. Bacteroides species were identified as black-pigmented colonies containing gram-negative rods from the MM 10 agar. A. viscosus was identified as dome-shaped, cream-colored colonies containing gram-positive branching rods that were catalase positive from the MM10 agar. S. mutans was identified as heaped, irregular granular colonies, sometimes with a drop of liquid on top that contained grampositive cocci from the selective MSBS agar.
RESULTS
Analysis of the uninoculated untreated control teeth yielded no growth after culturing the canal filings, indicating that the aseptic procedures used to manipulate the teeth were adequate to eliminate contamination. The mean levels of bacterial challenge (CFU per untreated tooth) varied from 2.68 X lo3 to 50.0 X lo6 for S. mutans, 5.55 X lo5 to 100.0 X lo6 for A. viscosus, and 1.61 X lo6 to 100.0 X lo6 for B. gingivalis. The mean CFU per untreated tooth for B. fragilis, used only with Ca(OH)z-treated teeth, was 100.0 X 106.These levels of bacterial challenge were shown to be appropriate to detect antibacterial activity with all four test agents. The nonparametric simultaneous test procedure of Dwass9 was used to compare statistically the differences in recovery of CFU per tooth between the untreated and treated teeth for each test agent and eachspeciesof bacterium tested (Table I). The differences between each respective control and test group were significant at p levels of
UNIVERSITY
SCHOOL OF DENTISTRY
The antimicrobial effectiveness of calcium hydroxide, camphorated paramonochlorophenol, and formocresol in root canals of extracted human teeth was’ compared. Canals in single-rooted teeth were enlarged and inoculated with Streptococcus mutans, Actinomyces viscosus, and Bacteroides gingivalis or Bacteroides fragilis. After treatment with a test agent and sealing and incubation for 1 hour, the canal contents were analyzed for the number of viable test bacteria and compared with that of inoculated teeth not treated with test agents. All test agents exhibited antimicrobial activity against all bacteria, with percent reductions in viable bacteria ranging from 64.3% to 100%. The combined data for Pulpdent paste and calcium hydroxide showed significantly higher antimicrobial activity than the combined data for camphorated paramonochlorophenol and formocresol for S. mutans and B. gingivalis or B. fragitis but showed no difference for A. viscosus. (ORAL SURC ORAL MED ORAL PATHOL 1991;72:101-4)
uccessful endodontic treatment depends on many S factors. Among the most important of these is the reduction or elimination of bacteria from the pulpal spaceand the removal of the substrate on which they depend. Biomechanical instrumentation and irrigation are successful in cleaning the root canal system, and intracanal medications traditionally have been used to maintain cleanliness between treatments. Selection of these medications has been based on effectiveness, toxicity, inflammation potential, and diffusibility. Unfortunately, most medications used today are nonspecific in toxicity and diffusibility and can themselves cause inflammation and patient discomfort. Calcium hydroxide has been used for apexification and pulp capping procedures and now is being used as an intracanal medication in endodontic therapy.le5 Clinically, it appears to control infection and to reduce the incidence of symptoms between treatments more effectively than the traditional medications of camphorated paramonochlorophenol (CMCP) and formocresol. This study compares the antibacterial
aEndodontist in private practice, Indianapolis. bProfessor and Chairman of Oral Microbiology. CAssociate Professor of Endodontics. dProfessor and Chairman of Endodontics. 7/15/28824
effectivenessof Ca(OH)z, CMCP, and formocresol in root canals of extracted human teeth against organisms shown to be related to pulpal infections. MATERIAL AND METHODS
The canals of single-rooted extracted teeth were prepared with K-type endodontic files to a size 50, sterilized, inoculated with a mixture of three test bacteria, and either treated with one of four test agents or left untreated before being sealed with dental stopping. After 1 hour at 37” C in a humid environment, the canal contents were removed and analyzed to determine the number of viable bacteria. The percent reduction in the number of viable bacteria remaining in the treated canals was calculated by comparing with the number of viable bacteria in the untreated control teeth. Tooth preparation
Ninety extracted, single-rooted adult human teeth, consisting of maxillary incisors, maxillary and mandibular canines, and mandibular premolars, were collected without regard to pulpal diagnosis. Teeth with large single canals were selected to accommodate the test medicaments and inoculum. The teeth were extracted, immediately placed in sterile water, and stored until the time of use. Before preparation the teeth were autoclaved twice in 15-minute cycles at a temperature of 250” F. The 101
102
Stuart
et al.
outer surfaces of the teeth were then debrided and scaled. The canals were prepared to a size 50 by a circumferential filing technique with a K-type file. All teeth were irrigated with 5 ml of sterile water during canal preparation with a tuberculin syringe. The specimenswere autoclaved again for 15 minutes at a temperature of 250” F before inoculation. After final autoclaving an aseptic technique was maintained with the use of sterile surgical gloves, drapes, and sterilized instruments. Before inoculation the canals were dried with sterile absorbent paper points and the external root surfaces were blotted dry with sterile gauze. The teeth were then dipped apex first in melted paraffin to the cementoenameljunction to prevent apical leakage of the inoculum and/or the medicament. Preparation
of standardized
inoculum
Four different microorganisms were used: Streptococcus mutans 6715 (obtained from P. H. Keyes, National Institute for Dental Research, Bethesda, Md.); Actinomyces viscosus ATCC 15987 (American Type Culture Collection [ATCC], Rockville, Md.); Bacteroides gingivalis ATCC 33277; and Bacteroides fragilis IUOM AN 1 (Department of Oral Microbiology, Indiana University, Indianapolis). The test bacteria were cultured from frozen stock in brain-heart infusion broth for 24 hours (S. mutans and A. viscosus) or for 48 hours with a subculture at 24 hours (Bacteroides species)in an anaerobic atmosphere (85% nitrogen, 10% hydrogen, 5% carbon dioxide) within an anaerobic chamber (model 1024, Forma Scientific Inc., Marietta, Ohio). Measured amounts of S. mutans, A. viscosus, and either B. gingivalis or B. fragilis culture were mixed together and centrifuged at 10,OOOg for 10 minutes at 7” C to pellet the cells. The pellet was resuspendedin 2.0 ml of a reduced transport fluid (RTF).6 A standardized volume of 15 ml of the RTF suspensionof the three speciesof bacteria was placed in the prepared canal of each tooth. This inoculum volume was previously determined by estimating the average root canal volume of prepared teeth. Becauseseveral batches of test bacterial cultures were prepared throughout the study, attempts were made to obtain the same relative number of cells of each speciesof bacterium in the final 15 ml inoculum preparations. This was performed by measuring the optical density (675 nm) of the individual cultures before mixing and relating these previously measured viable cell counts at that optical density. The volumes of each individual culture added to the mixture were then adjusted accordingly with each batch of inoculum prepared.
Treatment
of inoculated
canals
A total of 20 prepared teeth were used with each of the four test agents. All 20 teeth were inoculated; 10 were treated with the test agent, and 10 were not treated. The test agents were CMCP (Sultan Chemists Inc., Englewood, N.J.), formocresol (Sultan Chemists), Pulpdent paste (Pulpdent Corp., Watertown, Mass.) and a sterile saline solution slurry of Ca(OH)z. Inoculated teeth were treated with CMCP or formocresol by placing a size 4 sterile cotton pledget medicated with 1.0 ~1 of the agent. This was the amount found to remain on the pledget after squeezing it dry. Pulpdent paste was used as supplied and was placed with a sterile tuberculin syringe and spiraled into the canal to the apex with a lentulo instrument. Fifty milligrams of Ca(OH)z powder was slurried with sterile saline solution, and the thick paste was applied with an amalgam carrier and condensedwith endodontic pluggers. The canal access of untreated teeth was blocked with a sterile size 4 cotton pledget. All teeth were sealed with dental stopping (Hygenic Corp., Akron, Ohio). Ten teeth with prepared canals were not inoculated or treated but were sealed.These served as a control for aseptic technique. All teeth were placed in separate 10 ml sterile beakers in a covered sterile 1000 ml beaker also containing a 100 ml sterile beaker full of sterile water. This system provided a humid environment during the lhour period of incubation at 37” C. Recovery
of test bacteria
After the l-hour incubation period, each tooth canal was aseptically opened with a sterile endodontic explorer over a sterile 30 ml beaker containing 1.Oml of sterile phosphate-buffered RTF (BRTF). BRFT was used to neutralize the alkalinity of the Pulpdent and Ca(OH)z materials that would normally prevent growth of live bacteria remaining in the canal during subsequent culturing. A fresh, sterile size 50 file was used to remove the superficial layer of dentin, medicament, and bacteria into the BRTF. The instrument canal was flushed with 1.Oml of sterile BRTF into the samebeaker with a sterile tuberculin syringe. The file was added to the BRTF suspension,and after ultrasonic dispersion for 10 seconds(Artek sonic dismembrater, 60% maximum setting with a microtip) the suspensionwas serially diluted in lo-fold increments to 1:10,000. One-tenth milliliter of the undiluted suspension and of each dilution was spread plated on MM10 blood agar7 for recovery of A. viscosus and Bacteroides speciesand on Mitis salivarius-bacitratin-sucrose (MSBS) agar8 for recovery of S. mutans.
Volume 72 Number I
After anaerobic incubation at 31" C for 3 days (MSBS agar) or 5 days (MMlO), the number of colony-forming units (CFU) that developed on the plates was determined with the aid of a stereoscopic microscope. Bacteroides species were identified as black-pigmented colonies containing gram-negative rods from the MM 10 agar. A. viscosus was identified as dome-shaped, cream-colored colonies containing gram-positive branching rods that were catalase positive from the MM10 agar. S. mutans was identified as heaped, irregular granular colonies, sometimes with a drop of liquid on top that contained grampositive cocci from the selective MSBS agar.
RESULTS
Analysis of the uninoculated untreated control teeth yielded no growth after culturing the canal filings, indicating that the aseptic procedures used to manipulate the teeth were adequate to eliminate contamination. The mean levels of bacterial challenge (CFU per untreated tooth) varied from 2.68 X lo3 to 50.0 X lo6 for S. mutans, 5.55 X lo5 to 100.0 X lo6 for A. viscosus, and 1.61 X lo6 to 100.0 X lo6 for B. gingivalis. The mean CFU per untreated tooth for B. fragilis, used only with Ca(OH)z-treated teeth, was 100.0 X 106.These levels of bacterial challenge were shown to be appropriate to detect antibacterial activity with all four test agents. The nonparametric simultaneous test procedure of Dwass9 was used to compare statistically the differences in recovery of CFU per tooth between the untreated and treated teeth for each test agent and eachspeciesof bacterium tested (Table I). The differences between each respective control and test group were significant at p levels of
