tnternational Endodontic journal {1992) 2 5,15-19
Microbiological evaluation ofthe efficacy of chlorhexidine in a sustained-release device for dentine sterilization L HELING, M. SOMMER, D. STEINBERG*, M. FRIEDMAN* & M. N. SELAt Department of Endodontics, The Hebrew University-Hadassah School of Dental Medicine, the *Department of Pharmacy, School of Pharmacy, Hebrew University, and the f Department of Oral Biology, The Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel
Summary The aim of this in-vitro study was to evaluate the effect of chlorhexidine in solution and in a sustained-release device as an intracanal medication. Hollow cylindrical dentine specimens prepared from bovine incisors were incubated with Streptococcus faecalis for a period of 3 weeks. The intracanal medicaments tested were 0.2% chlorhexidine gluconate solution (CH), chlorhexidine in a sustained-release device (1.2 mg) (SRD), camphorated paramonochlorophenol (CMCP), and a control (no medication). Each medicament was introduced into the lumen of one dentine specimen and Incubated for 5 min. 24 h, 48 h or 7 days at 37°C. The bacteriological samples were taken by shaving the dentine inside the lumen with dental burs ranging in size from ISO 023-03 3. The dentine powder was collected in test-tubes containing growth medium and incubated for 24 h. The optical density ofthe medium was recorded by meiins of a spectrophotometer at a wavelength of 540 nm. There was a statistically significant difference between the control group and all the medicaments tested. Keywords: calcium hydroxide, camphorated monochlorophenol, chlorhexidine, root canal medication. Introduction The role of biomechanical preparation ofthe root canal system in successful endodontic treatment has been extensively studied. Although it has an important cleaning effect, biomechanical preparation alone cannot eliminate, in the majority of teeth treated, all the bacteria from the dentinal tubules and the smear layer formed during instrumentation. The remaining bacteria may multiply during the period between appointments, often reaching the same level as at the start of the previous Correspondeticer Dr liana Heling. Department of Endodontics, The Hebrew University-Hadassah Schoo! of Dental Medicine. POB 1172. Jerusaletn. 91010 Israel.
session (Seltzer et al. 1963, Engstrom & Lundberg 1966, Bystrom & Sundqvist 1981). These observations call for the use of an effective intracanal medication that will assist sterilization of the root canal system. Such a medicament should be effective throughout its period of application, and penetrate the dentinal tubules, eliminating any bacteria that may be present. Various formulations of chlorine, phenol and camphor have been used for root canal sterilization. However, because of their toxicity, small amounts have been used, and they have been employed only in the pulp chamber. The assumption has been that the vapours would kill bacteria throughout the root canal system (Spangberg 1982). Spangberg et al. (1979) demonstrated that the vapours of camphorated paramonochlorophenol (CMCP) could not kill bacteria. Furthermore, Messer & Chen (1984) showed that almost 95% ofthe CMCP from a cotton pellet placed inside the pulp chamber was lost within 24 h. The duration ofthe intracanal medication is short, due to a rapid inacUvation process which occurs a few hours after its exposure to tissue or fluids. Calcitmi hydroxide was shown by Bystrom et al. (1985) to be the intracanal medication of choice, being superior to CMCP in obtaining sterility ofthe root canal system. However, in a study performed by Haapasalo and Orstavik (1987), CMCP was more effective than calcium hydroxide in killing bacteria inside the dentinal tubules. According to this study it appeared that the antibacterial properties of CMCP were dose dependent (HaapasaioS 0rstavik 1987). A new concept of pharmaceutical formulation of sustained-release devices (SRD) for dental use in prevention of plaque and caries, and treatment of periodontal diseases, has been reviewed by Steinberg Emd Friedman (1988). The rationale for using SRD for intracanal medication is that it is necessary to sterilize the root canal system and to maintain its sterility throughout the treatment. It follows, therefore, that the medication should remain active at a constant concentration for a 15
16
I. Heling et al.
designated time period. In addition, if the antibacterial medicament has an affinity for dentine and will be slowly released from it, this in turn will prolong the pedod of antimicrobial activity. The degradable slow-release device was similar to that described by Steinberg et al. (1990). using glutaraidehyde as the crosslinking agent and 20% chiorhexidine gluconate as the active agent. The rate of degradation and the consequent release of chiorhexidine could be controlled for periods ranging from several hours to several days (Steinberg et al. 1990). The SRD is in the form of solid strips which can be cut to the desired size and shape. Chlorhexidine was chosen because it is active against a wide range of Gram-positive and Gram-negative organisms, as well as yeast, fungi, facultative anaerobes and aerobes (Fardal & TumbuU 1986). The aim of the present in-vttro study was to evaluate the suitability of chiorhexidine in solution and in a sustained-release device for intracanal medication.
Materials and methods The method of Haapasalo and 0rstavik (1987). with certain modifications, was used in this study. Intact freshly extracted bovine incisors were placed in 0.5% NaOCI overnight for surface disinfection. After removal of the apical 5 mm and the crown with a rotating diamond saw at 195 rpm with water coolant (Isomet, Buehler. Illinois, USA), the root cementum was removed at low speed with a water-cooled bur. The remaining piece of dentine was then sliced into 4-mm-thick sections with the rotating diamond saw; the total number of sections was 128. Each section had an external diameter of 5-6 mm. An ISO size 023 round bur was thea used to standardize the internal diameter of the blocks. Removal of the smear layer was performed in an ultrasonic bath (Bransonic 220) in 17% EDTA pH 7.8 (4 min) and 5.2 5% NaOCl (4 min). At this stage, sterilization ofthe blocks in test-tubes was carried out by autoclaving three times in tryptone soya broth (TSB) (Oxoid, Basingstoke, UK) for 30 min at 121 °C. After each sterilizing session the blocks were incubated for 24 h at 37°C to check their sterility. Following the third incubation, the blocks were immersed La TSB and placed in an ultrasonic bath for 10 min to enhance the penetration of the broth into the dentinal tubules. An overnight suspension of Streptticoccus faecalis was introduced into the test-tubes to infect the blocks. A fresh ovem^ht inoculum was added each day, and in addition the broth was changed daily. These procedures were carried out for 3 weeks.
Following 3 weeks of treatment, the blocks were removed from the test-tubes and the outer surface was coated with nail varnish. The blocks were then individually placed on separate sections of sterile aluminium foil which were moulded around the base and sides. The medicament to be tested was subsequently placed in the lumen and the foil was closed over the top of the block. There were 8 blocks in each experimental group. The blocks were then incubated at 3 7°C for periods of 5 min, 24 h, 48 h or 7 days. The medications used were as follows: (i) 0.1 ml of 0.2% chlorhexidine gluconate solution containing 0.2 mg chlorhexidine in the volume used: (ii) the sustained-release device with chlorhexidine (SRD) (containing 1.2 mg of 20% chlorhexidine gluconate solution in the pharmaceutical preparation) supplemented with 0.1 ml saline introduced in the lumen: (iU) 0.1ml chlorophenol camphor (CMCP) (60% camphor, 30% paramonochlorophenol) (Deiro 7070 Schwab, Gmund. Austria): (iv) a control. 0.1 ml sterile saline (no medication). At the end of each test period, sterile round burs (ISO sizes 023, 025. 027, 029, 031 and 033) were used sequentially to remove dentine powder from the inner surface of the blocks, which were held in sterile forceps (Figs 1 and 2). The powder was then placed in sterile TSB and incubated for 24 h, after which the optical density of the broth was measured by means of a spectrophotometer (Uvikon, Tegimenta, Switzerland), using a wave-length of 540 nm. The optical density of the broth was proportional to the number of bacteria present. The data collected were analysed using Student's t-test.
Results All control specimens, at each period tested, showed a constant level of infection at all levels of penetration examined. The penetration levels ranged from ISO bur size 02 3 to 033, i.e. from a depth of 0-0.5 mm. At the 5-nun application period inconsistent results were obtaLaed. which may be attributed to the fact that the m ^ c a m e n t in a number of specimens was applied for periods of more than 5 min. In this group the results showed that at the shallow penetration depth the SRD was more effective than the other medicaments, but the difference was not statistically significant (P
Microbiological evaluation ofthe efficacy of chlorhexidine in a sustained-release device for dentine sterilization L HELING, M. SOMMER, D. STEINBERG*, M. FRIEDMAN* & M. N. SELAt Department of Endodontics, The Hebrew University-Hadassah School of Dental Medicine, the *Department of Pharmacy, School of Pharmacy, Hebrew University, and the f Department of Oral Biology, The Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel
Summary The aim of this in-vitro study was to evaluate the effect of chlorhexidine in solution and in a sustained-release device as an intracanal medication. Hollow cylindrical dentine specimens prepared from bovine incisors were incubated with Streptococcus faecalis for a period of 3 weeks. The intracanal medicaments tested were 0.2% chlorhexidine gluconate solution (CH), chlorhexidine in a sustained-release device (1.2 mg) (SRD), camphorated paramonochlorophenol (CMCP), and a control (no medication). Each medicament was introduced into the lumen of one dentine specimen and Incubated for 5 min. 24 h, 48 h or 7 days at 37°C. The bacteriological samples were taken by shaving the dentine inside the lumen with dental burs ranging in size from ISO 023-03 3. The dentine powder was collected in test-tubes containing growth medium and incubated for 24 h. The optical density ofthe medium was recorded by meiins of a spectrophotometer at a wavelength of 540 nm. There was a statistically significant difference between the control group and all the medicaments tested. Keywords: calcium hydroxide, camphorated monochlorophenol, chlorhexidine, root canal medication. Introduction The role of biomechanical preparation ofthe root canal system in successful endodontic treatment has been extensively studied. Although it has an important cleaning effect, biomechanical preparation alone cannot eliminate, in the majority of teeth treated, all the bacteria from the dentinal tubules and the smear layer formed during instrumentation. The remaining bacteria may multiply during the period between appointments, often reaching the same level as at the start of the previous Correspondeticer Dr liana Heling. Department of Endodontics, The Hebrew University-Hadassah Schoo! of Dental Medicine. POB 1172. Jerusaletn. 91010 Israel.
session (Seltzer et al. 1963, Engstrom & Lundberg 1966, Bystrom & Sundqvist 1981). These observations call for the use of an effective intracanal medication that will assist sterilization of the root canal system. Such a medicament should be effective throughout its period of application, and penetrate the dentinal tubules, eliminating any bacteria that may be present. Various formulations of chlorine, phenol and camphor have been used for root canal sterilization. However, because of their toxicity, small amounts have been used, and they have been employed only in the pulp chamber. The assumption has been that the vapours would kill bacteria throughout the root canal system (Spangberg 1982). Spangberg et al. (1979) demonstrated that the vapours of camphorated paramonochlorophenol (CMCP) could not kill bacteria. Furthermore, Messer & Chen (1984) showed that almost 95% ofthe CMCP from a cotton pellet placed inside the pulp chamber was lost within 24 h. The duration ofthe intracanal medication is short, due to a rapid inacUvation process which occurs a few hours after its exposure to tissue or fluids. Calcitmi hydroxide was shown by Bystrom et al. (1985) to be the intracanal medication of choice, being superior to CMCP in obtaining sterility ofthe root canal system. However, in a study performed by Haapasalo and Orstavik (1987), CMCP was more effective than calcium hydroxide in killing bacteria inside the dentinal tubules. According to this study it appeared that the antibacterial properties of CMCP were dose dependent (HaapasaioS 0rstavik 1987). A new concept of pharmaceutical formulation of sustained-release devices (SRD) for dental use in prevention of plaque and caries, and treatment of periodontal diseases, has been reviewed by Steinberg Emd Friedman (1988). The rationale for using SRD for intracanal medication is that it is necessary to sterilize the root canal system and to maintain its sterility throughout the treatment. It follows, therefore, that the medication should remain active at a constant concentration for a 15
16
I. Heling et al.
designated time period. In addition, if the antibacterial medicament has an affinity for dentine and will be slowly released from it, this in turn will prolong the pedod of antimicrobial activity. The degradable slow-release device was similar to that described by Steinberg et al. (1990). using glutaraidehyde as the crosslinking agent and 20% chiorhexidine gluconate as the active agent. The rate of degradation and the consequent release of chiorhexidine could be controlled for periods ranging from several hours to several days (Steinberg et al. 1990). The SRD is in the form of solid strips which can be cut to the desired size and shape. Chlorhexidine was chosen because it is active against a wide range of Gram-positive and Gram-negative organisms, as well as yeast, fungi, facultative anaerobes and aerobes (Fardal & TumbuU 1986). The aim of the present in-vttro study was to evaluate the suitability of chiorhexidine in solution and in a sustained-release device for intracanal medication.
Materials and methods The method of Haapasalo and 0rstavik (1987). with certain modifications, was used in this study. Intact freshly extracted bovine incisors were placed in 0.5% NaOCI overnight for surface disinfection. After removal of the apical 5 mm and the crown with a rotating diamond saw at 195 rpm with water coolant (Isomet, Buehler. Illinois, USA), the root cementum was removed at low speed with a water-cooled bur. The remaining piece of dentine was then sliced into 4-mm-thick sections with the rotating diamond saw; the total number of sections was 128. Each section had an external diameter of 5-6 mm. An ISO size 023 round bur was thea used to standardize the internal diameter of the blocks. Removal of the smear layer was performed in an ultrasonic bath (Bransonic 220) in 17% EDTA pH 7.8 (4 min) and 5.2 5% NaOCl (4 min). At this stage, sterilization ofthe blocks in test-tubes was carried out by autoclaving three times in tryptone soya broth (TSB) (Oxoid, Basingstoke, UK) for 30 min at 121 °C. After each sterilizing session the blocks were incubated for 24 h at 37°C to check their sterility. Following the third incubation, the blocks were immersed La TSB and placed in an ultrasonic bath for 10 min to enhance the penetration of the broth into the dentinal tubules. An overnight suspension of Streptticoccus faecalis was introduced into the test-tubes to infect the blocks. A fresh ovem^ht inoculum was added each day, and in addition the broth was changed daily. These procedures were carried out for 3 weeks.
Following 3 weeks of treatment, the blocks were removed from the test-tubes and the outer surface was coated with nail varnish. The blocks were then individually placed on separate sections of sterile aluminium foil which were moulded around the base and sides. The medicament to be tested was subsequently placed in the lumen and the foil was closed over the top of the block. There were 8 blocks in each experimental group. The blocks were then incubated at 3 7°C for periods of 5 min, 24 h, 48 h or 7 days. The medications used were as follows: (i) 0.1 ml of 0.2% chlorhexidine gluconate solution containing 0.2 mg chlorhexidine in the volume used: (ii) the sustained-release device with chlorhexidine (SRD) (containing 1.2 mg of 20% chlorhexidine gluconate solution in the pharmaceutical preparation) supplemented with 0.1 ml saline introduced in the lumen: (iU) 0.1ml chlorophenol camphor (CMCP) (60% camphor, 30% paramonochlorophenol) (Deiro 7070 Schwab, Gmund. Austria): (iv) a control. 0.1 ml sterile saline (no medication). At the end of each test period, sterile round burs (ISO sizes 023, 025. 027, 029, 031 and 033) were used sequentially to remove dentine powder from the inner surface of the blocks, which were held in sterile forceps (Figs 1 and 2). The powder was then placed in sterile TSB and incubated for 24 h, after which the optical density of the broth was measured by means of a spectrophotometer (Uvikon, Tegimenta, Switzerland), using a wave-length of 540 nm. The optical density of the broth was proportional to the number of bacteria present. The data collected were analysed using Student's t-test.
Results All control specimens, at each period tested, showed a constant level of infection at all levels of penetration examined. The penetration levels ranged from ISO bur size 02 3 to 033, i.e. from a depth of 0-0.5 mm. At the 5-nun application period inconsistent results were obtaLaed. which may be attributed to the fact that the m ^ c a m e n t in a number of specimens was applied for periods of more than 5 min. In this group the results showed that at the shallow penetration depth the SRD was more effective than the other medicaments, but the difference was not statistically significant (P
