Z. Fuss, A.Mizrahi, S. Lin, O. Cherniak & E. I.Weiss
Department of Endodontology and Department of Restorative Dentistry, The Maurice and Gabriela Goldschleger School of Dental Medicine, Tel Aviv University, Tel Aviv, Israel.
Department of Prosthodontics, Faculty of Dental Medicine, Hebrew University, Jerusalem, Israel.

Introduction.
Success of root-canal treatment is dependent on the removal of bacteria from the root-canal space (Kakehashi et al.1965, Sundqvist1976). It is widely accepted that the bacteria invading dentinal tubules, root-canal ramifications, isthmuses and apical deltas are responsible for persistent endodontic infections (Akpata & Blechman 1982). Mechanical instrumentation and irrigation are the main methods of removing bacteria from the root canal space (Kettering&Torabinejad1998). In particular, irrigation with antibacterial agents and placement of intracanal medicaments are essential to kill bacteria in infected dentinal tubules and root-canal ramifications which cannot be eliminated mechanically.
A variety of intracanal medicaments can be used to disinfect the root-canal dentinal walls. In vitro test shave shown that the bacteria are killed when the yare in direct contact with low concentrations of some medicaments (Spangberg 1982). However, intracanal medicaments, such as calcium hydroxide (CH) or Ledermix1 (Laderle, Wolfratshausen, Germany) have a limited ability to penetrate and the disinfect dentinal tubules (Haapasalo & Grstavik1987, Siqueira & Uzeda1996).
Antibacterial agents, e.g. iodine-potassium iodide (IKI) are more effective than pure CH in the dentinal tubules but no complete disinfection has been established (Safavi et al. 1990). Knappwost (1993) has described a method to disinfect root canals using CH with copper. The paste is introduced into the coronal third of the root-canal system and an electrophoresis system is used to mobilize the ions.
The purpose of the present study was to evaluate the antibacterial effect of CH with additives of copper or IKI to disinfect dentinal tubules.

Materials and methods.
The in vitro model for dentinal tubule infection of root canals originally described by Haapasalo & Grstavik (1987) and modified by Assouline et al. (2001) was used in the present study. Extracted, intact bovine incisors were maintained in 0.5% NaOCl (Lever, Lovenkleen, Kiryat-Ata, Israel) overnight for surface disinfection. The apical 5 mm and two-thirds of the crown were removed with a rotarydiamondsawat1000 rpm (Isomet plus precision saw, Buehler, Cave Blu!, IL,USA)underwater cooling. The root canal of the remaining tooth was enlarged to a diameter of 2.3 mm with an ISO size 023 round bur using a slow speed dental handpiece. Cementum was removed using a polishing paper (Ecomet 3, variable speed grinder-polisher, Buehler) under water cooling, resulting in a root specimen with an external diameter of 6 mm. The root was then cut into 4-mm-thick sections with a diamond saw. All teeth and dentine slices were maintained in vials containing tap water during all procedures to avoid dehydration. Organic and inorganic debris, including smear layer was removed by placing the specimens in an ultrasonic bath with 17% EDTA (Sigma, Holon, Israel) (pH 7.8) followed by 0.5% NaOCl, for 5 min. Sterilization was conducted by autoclaving the specimens in the vials for 30 min at1218C. Six specimens serving as negative controls were incubated in growth media at 378C for 48 h to con￠rm sterility.
A total of 36 sterile specimens were aseptically transferred to separate test tubes containing 1.5 mL brain- heart infusion broth (BHI, Difco, Detroit, MI, USA) inoculated with the test microorganism. The medium was changed every 2 days for 21 days.
Enterococcus faecalis, a common isolate from infected root canals (Sundqvist1992) has been used in numerous studies because of its relative resistance (Haapasalo & Grstavik 1987). In the present study, a clinical isolate of E. faecalis T2 (Tel Aviv University stock, Israel) which is resistant to 2 mg mL_1 streptomycin (Weiss et al. 1996, Fuss et al.1997, Shalhav et al.1997) was used. Streptomycin sulphate (Sigma, Holon, Israel), at a concentration of 0.5 mg mL was included in all growth mediato overcome possible contamination in the experimental setup (Weiss et al.1996).
The 36 infected specimens were divided into four equal groups. CH in aqueous paste with different additives was introduced into the root-canal space of the experimental specimens using Hawe Composite-Gun tip no. 1914 (Hawe Neos Dental, Bioggio, Switzerland) as follows:

• Group1: commercial preparation of CH with copper in a creamy consistency (Humanchemie, Bremen, Germany).
• Group 2: CH powder (4 mg) with powder of iodine-potassium iodide (IKI) (1 mg, Medent Laboratory, Beer Sheba, Israel) mixed with distilled water to a creamy consistency.
• Group 3: CH powder (4 mg) mixed with distilled water to a creamy consistency.
• Group 4: served as a positive control with no medicament.

In group1, ions derived from the CH/copper paste were mobilized via a low current electric ￠field of 5 mA for 5 min. This was created by using an electrode inserted into the root-canal space whilst another electrode was located outside the specimen according to a method originally described by Knappwost (1993).To simulate clinical conditions, all specimens were incubated under humid condition for 1 week at 378C. At the end of the experimental period, the intracanal medicaments were removed by using sterile round burs ISO size 023,which has the same diameter of the root canal to ensure complete removal of the medicaments from the inner surface of the canal. The coronal and the apical surfaces of each specimen were gently curetted to prevent biased results due to possible remnants of bacteria and medicaments. Dentinal samples were then taken from each test specimen with sterile round burs mounted in a handpiece at low speed. Specimens were maintained in place with sterile forceps during sampling, and the burs were used in sequence in the following ISO sizes: 025, 027, 029, 031 and 033. Each bur removed a dentinal layer from the inner surface of the canal in the shape of a hollow cylinder100 mm thick with increasing radius as the burs were changed. The powder dentine samples obtained with each bur were immediately collected in separate test tubes containing1mL of BHI broth and streptomycin at a concentration of 0.5 mg mL.Tubes were vigorously mixed by vortex for 20 s before and after incubation for1 h at 378C. The content of each test tube was serially diluted, 100 mL in eppendorf tube with 100 mL of saline (￠ve times). Triplicate samples of 0.01mL were spread on BHI agar plates from each dilution, which were incubated for24 hat378C.The growing colonies were counted and recorded as colony forming units (cfu). For each dentinal layer at least triplicate samples from two agar plates were counted. Data were analyzed using a nova with repeated measures to indicate di!erences between treatment groups. One-way a nova was carried out on logarithmic transformation (Tukey’s method) to indicate di!erences within each layer.

The cfu represent a close estimate of viable bacteria penetrating into dentinal tubules at different layer depths. The numbers of cfu obtained from ￠ve consecutive dentinal layers are presented in Table 1 and Fig.1. In the positive control specimens, heavy bacterial infection was observed at the layer close to the lumen. This decreased from layer to layer up to the deepest layer tested (400-500 mm) which contained several 100 cfu. CH without additives significantly (P < 0.001) reduced the amount of viable bacteria in the ￠rst to fourth layers compared to the positive control group, but did not eliminate them completely. Application of low electric current to the group containing CH and copper completely eliminated bacterial survival in the dentinal tubules at least up to 500 mm from the root-canal lumen and in the second to ￠fth layers was statistically (P < 0.001) more effective than the experimental group and control. CH with IKI significantly (P < 0.001) reduced the amount of viable bacteria in all layers compared to the positive control group and was statistically (P < 0.001) more effective than CH without additives in the fourth and ￠fth layers. The negative control group showed no growth of bacteria, which indicate sterilization of the process and prevention of biased results.

Table 1. Logarithmic transformation of the number of colony forming units (cfu +1) at different dentine layer depths.


* One-way anova was performedonlog transformationof cfu for eachlayer. Horizontal lines connects data which does not show significant differences (Tukey's method).

Figure 1. Colony forming units (cfu) of bacteria in dentinal tubules facing the root-canal space following treatment with calciumhydroxide with no additives (CH), calciumhydroxide with IKI (CH þ IKI), and calciumhydroxide with electrophoretic activated copper (CH +CU + E). Each column represents the log transformation (cfu +1) obtained from nine dentinal specimens.

Discussion.
Microorganisms in dentinal tubules may constitute a reservoir from which root canal and surrounding tissue infection and reinfection may occur. Treatment strategies designed to eliminate this reservoir should include agents that can penetrate the dentinal tubules and destroy these microorganisms, since they are located beyond the host defense mechanism, and out of reach of systemically administered antimicrobial agents. The present study evaluated the ability of intracanal medicaments to disinfect dentinal tubules leading from the root-canal space. For this purpose, cylindrical root specimens prepared from bovine teeth were used in anin vitro model of dentinal tubule infection (Haapasalo & Grstavik 1987). We modified this experimental model slightly to obtain additional quantitative information.
Calcium hydroxide (CH) has a lasting antibacterial activity in the root-canal space due to its high pH (Fuss et al.1989,1996). However, CH has poor solubility and its bactericidal effect on penetration into the dentinal tubules is unsatis factory (Haapasalo & Grstavik 1987).
Safavi et al. (1990) have shown that IKI penetrates into the dentinal tubules from a distance and kills bacteria in vitro. However, the duration of its antimicrobial efficiency is short (Engstrom1958); it is expected to remain active from 1 to 3 days depending on the rate of fluid exchange through the apical foramen (Moller 1966). In the present study, the use of IKI combined with CH proved to be more effective in dentinal tubules than pure CH. The use of this combination is especially important in root canals with complex anatomy where packing CH is complicated or impossible.
Molander et al. (1999) tested the activity of CH in the root canal following pretreatment with 5% IKI for 3-7 days; CH had no increased antimicrobial effect following such pretreatment. They suggested that the smear layer should be removed and the IKI mixed with a vehicle. This could enhance its antibacterial effect in the dentinal tubules and increase its duration of activity. In the present study, CH was used as a vehicle for the IKI and the smear layer was removed. This may explain the significantly better effect in the dentinal tubules compared to the former study. In a pilot test, there were no significant changes in pH values of CH paste following the addition of iodine (pH >12.5).
In this in vitro model, the best results were obtained with the addition of copper to CH and with the use of an electric current, according to the method described by Knappwost (1993). The depth of complete disinfection was at least 500 mm, which is superior to other medicaments. It is generally accepted that the choice of an intracanal medicament should balance antibacterial potency with tissue toxicity, a balance that is often difficult to achieve because medicaments that are bactericidal, usually exhibit some toxicity to mammalian cells (Spangberg1990). Coppermay be toxic to the periradicular tissues, therefore, its clinical use should be questioned and limited. In addition, electrodes which do not penetrate to the root-end (apex) as described by Knappwost (1993), are limited in their effectiveness at the most important site, the apical delta.

References.

American Associationof Endodontists (1998) Glossary. Contemporary Terminology for Endodontics, 6th edn.
Bechelli C, Zecchi Orlandini S, Colafranceschi M (1999) Scanning electron microscope study on the efficacy of root canal wall debridement of hand versus Light speed instrumentation. International Endodontic Journal 32, 484-93.
Bolanos OR, Jensen JR (1980) Scanning electron microscope comparisons of the efficacy of various methods of root canal preparation. Journal of Endodontics 6, 815-22.
European Society of Endodontology (1994) Consensus report of the European Society of Endodontology on quality guidelines for endodontic treatment. International Endodontic Journal 27,115-24.
Gambarini G (1999) Shaping and cleaning the root canal system: a scanning electron microscopic evaluation of a new instrumentation and irrigationtechnique. Journal of Endodontics 25,800-3.
Haikel Y, Allemann C (1988) Effectiveness of four methods for preparing root canals: a scanning electron microscopic evaluation. Journal of Endodontics14, 340-5.
H lsmann M (2000) Entwicklung Einer Methodik Zur Standardisierten Uberprufung Verschiedener Aufbereitungsparameter und Vergleichende in-Vitro-Untersuchung Unterschiedlicher Systeme Zur Maschinellen Wurzelkanalaufbereitung (Habilitationsschrift). Gottingen, Germany: University of Gottingen. Hulsmann M, Rummelin C, Schafers F (1997) Root canal cleanliness after preparation with different endodontic handpieces and hand instruments: a comparative SEM investigation. Journal of Endodontics 23,301-6.
HulsmannM, Versumer J, SchadeM(2000) Acomparative study of Lightspeed, ProFile 0.04, Quantec and Hero 642. International Endodontic Journal 33,150 (abstract).
Kum K-Y, SpOngberg L, Cha BY, Il-Young J, Seung-Jong L, Chan- Young L (2000) Shaping ability of three ProFile rotary instrumentation techniques in simulated resin root canals. Journal of Endodontics 26,719-23. Mizrahi SJ, Tucker JW, Seltzer S (1975) A scanning electron microscopic study of the efficacy of various endodontic instruments. Journal of Endodontics1,324-33.
Nair PNR, Sjogren U, Krey G, Kahnberg KE, Sundqvist G (1990) Intraradicular bacteria and fungi in root-filled, asymptomatic human teeth with therapy-resistant periapical lesions: a long-term light and electron microscopic follow-up study. Journal of Endodontics16,580-8.
PetscheltA, Stumpf B, RaabW(1987) Tightness of root canal sealers with and without smear layer. Deutsche Zahnarztliche Zeitschrift 42,743-6.
Schafer E, Diez C, Hoppe W, Tepel J (in press) Roentgenographic investigation of frequency and degree of canal curvatures in human permanent teeth. Journal of Endodontics in press.
Schafer E, Lohmann D (2002) Efficiency of rotary nickeltitanium FlexMaster instruments compared with stainless steel hand K-Flexofile. Part 1. Shaping ability in simulated curved canals. International Endodontic Journal 35, 514-21.
Schafer E, Zapke K (2000) A comparative scanning electron microscopic investigation of the efficacy of manual and automated instrumentation of root canals. Journal of Endodontics 26, 660-4.
Schwarze T, GeurtsenW (1996) Comparative qualitative SEM study of automated vs. hand instrumentation of root canals. Deutsche Zahnarztliche Zeitschrift 51, 227-30.
SpOngberg L, Engstrom B, Langeland K (1973) Biological effects of dental materials. Part 3. Toxicity and antimicrobial effects on endodontic antiseptics in vitro. Oral Surgery 36, 856-71.
Thompson SA, Dummer PMH (1997) Shaping ability of ProFile .04 taper Series 29 rotary nickel-titanium instruments in simulated canals. Part 1 and 2. International Endodontic Journal 30,1-15.
Turkun M, Cengiz T (1997) The effects of sodium hypochlorite and calcium hydroxide on tissue dissolution and root canal cleanliness. International Endodontic Journal 30, 335-42.
West JD, Roane JB, Goerig AC (1994) Cleaning and shaping the root canal system. In: Cohen S, Burns RC, eds. Pathways of the Pulps,6th edn. St. Louis, MO, USA: Mosby.179-218.
Wu MK,WesselinkPR (1995) Efficacy of three techniques in cleaning the apical portion of curved root canals. OralSurgery 79, 492-6.
Yamada RS, Armas A, Goldman M, Lin PS (1983) A scanning electron microscopic comparison of high volume final flush with several irrigating solutions. Part 3. Journal of Endodontics 9, 137-42.