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 »  Home  »  Endodontic Articles 6  »  A laboratory study of the effect of calcium hydroxide mixed with iodine or electrophoretically activated copper on bacterial viability in dentinal tubules
A laboratory study of the effect of calcium hydroxide mixed with iodine or electrophoretically activated copper on bacterial viability in dentinal tubules
Introduction - Materials and methods.



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.