An In Vitro Test Model for Investigation of Disinfection of Dentinal Tubules Infected with Enterococcus faecalis

1Department of Conservative Dentistry and Endodontics, Selçuk University,
Faculty of Dentistry, Konya, Türkiye
2Department of Microbiology, Selçuk University, Faculty of Veterinary Medicine,
Konya, Türkiye

Braz Dent J (1997) 8(2): 67-72 ISSN 0103-6440

| Introduction | Material and Methods | Results | Discussion | References |

The aim of the present study was to develop an in vitro test model from human teeth to comparatively examine antibacterial effectiveness of calcium hydroxide, parachlorophenol (PCP) and camphorated parachlorophenol (CPCP) against Enterococcus faecalis in infected root canals. Cylindrical dentin specimens were prepared from freshly extracted human maxillary anterior teeth. The specimens were inoculated with  E. faecalis and then medicated with either CPCP, PCP or Ca(OH)2. The disinfecting efficacy of these agents was tested by collecting dentin chips from the inner (“canal”) walls of the specimens and counting viable  E. faecalis. The dentin chips were diluted and a classical bacterial count technique was used for recovery of  E. faecalis strains on 5% sheep blood agar. The effectiveness of CPCP and PCP at one day was superior to the effectiveness of Ca(OH)2. In the three-day group, CPCP was the most effective, followed by Ca(OH)2. The experimental model used in this study may be useful for investigation of the effect of intracanal medicaments on microorganisms lodged in the root dentinal tubules.

Key Words: calcium hydroxide, endodontic treatment, parachlorophenol.


Improvements in sampling and bacteriological culture techniques have made it possible to precisely determine the composition of the root canal microbial population, revealing the predominance of anaerobic microorganisms to be Gram-positive cocci and Gram-negative bacilli (Perez et al., 1993).

The population of microorganisms in an infected root canal may be reduced significantly by reaming, filing and irrigating. However, some microorganisms lodged in the root dentinal tubules may not be removed by instrumentation and irrigation (Akpata and Blechman, 1982; Perez et al., 1993). The need for an intracanal medicament to destroy or reduce the number of microorganisms seems evident. To date, many different types of medicaments have been used as root canal disinfectants and they may be grouped arbitrarily as essential oils, phenolic compounds, Ca(OH)2 and antibiotics.

In recent years, Ca(OH)2 has emerged as a popular choice of intracanal medicament. The effectiveness of Ca(OH)2 is due to its late antimicrobial effect, anti-inflammatory property and osteogenic potential. It also enhances the tissue dissolving effect of NaOCl (Foreman and Barnes, 1990; Chong and Pitt Ford, 1992). It has very low toxic effects on periapical tissues and the osteogenic repair potential of Ca(OH)2 has been shown in soft tissue and osseous wounds (Fisher, 1972).

It has been shown that dentinal tubules of infected roots might harbor bacteria to some extent (Ortstavik and Haapasalo, 1990; Sen et al., 1995), which may be a reason for persistent infection. It is questionable whether sufficient amounts of the commonly used medicaments can penetrate into dentinal tubules to achieve disinfection. A few attempts have been made to establish in vitro test models of root canal infections and disinfection (Akpata and Blechman, 1982; Haapasalo and Ortstavik, 1987). A simple method for studying the elimination of tubule infection by several medicaments is lacking.

The purpose of this study was to develop a simple in vitro test model for the infection of dentinal tubules and to evaluate the antibacterial efficacy of three root canal medicaments by using this method.

Material and Methods

Forty recently extracted human maxillary anterior teeth were obtained from the Department of Surgery, Faculty of Dentistry, Selçuk University, Turkey and stored in physiologic saline solution. The crowns were removed at the cemento-enamel junction by means of high-speed fissure burs. The canals of the teeth were first prepared with K-type endodontic files to size 70. The apical one-third of the root was removed with a rotating diamond saw under water cooling. With peeso reamers (number 2, 3, 4 and 5) (Largo, Maillefer, Switzerland), canals were widened to 2 mm in diameter, 9 mm in length. In the coronal and apical sections of the root specimens, small cavities were prepared to 2.5 mm in diameter and 1.5 mm in length in order to leave some space for composite and temporary filling. The apical section of the specimens were sealed with a composite resin (Degufill, Degussa AG, Frankfurt, Germany) (Figure 1). The prepared canals were irrigated with 5 ml 3% H2O2, 5 ml 5.25% NaOCl and 1 ml 17% EDTA which was left in place for 5 min to remove the smear layer. The canals were then given a final flush with 5 ml of 5.25% NaOCl.

Figure 1 - Prepared dentin test specimen in diameters (mm).

After sterilization by steam autoclave for 15 min at 121oC, each tooth was transferred to brain heart infusion (BHI) broth (Oxoid, England), and incubated for 24 h at 37°C as a test for sterility. These teeth were transferred to 2 ml sterile physiologic saline (SPS) in individual tubes for washing out BHI and to avoid dehydration and contamination; they were then incubated for 24 h at 37°C. Following incubation in SPS, each tooth was removed from SPS and root canals were carefully dried with sterile paper points under aseptic conditions. The root canals were then infected with a standard volume of 10 µl (1-3 x 108 cfu) of E. faecalis suspension (RSHI 10541 strain; Refik Saydam Health Institute, Ankara, Turkey) in reduced transport fluid (RTF). After this stage, the specimens were divided in two groups, one-day group and three-day group, for evaluation of the antibacterial efficacy of each root canal medicament as time dependent.

One-day group: Twenty teeth were immediately transferred to 2 ml SPS and incubated for infection of dentinal tubules for 48 h at 37°C. After incubation, 6 teeth were medicated with PCP (Cresophene, Septodont, France) and 6 with CPCP (Acide Phenic, Güler Chemical, Turkey) by placing a tiny cotton pledget in the orifices of the canals. The cotton pledgets were moistened with solution and excess liquid pressed onto cotton rolls. Another 6 teeth were filled with Ca(OH)2 paste (Superlux calcium hydroxide liner, DMG Dental Materials, Hamburg, Germany) by injecting the material with a syringe. The medicaments were left in the canals as dressings for 1 day by sealing the orifices of the canals with a temporary filling material. The 2 unmedicated teeth were kept as control. The medicated and unmedicated teeth were stored in sterile SPS for 24 h at 37°C. After medication the cotton pledgets and Ca(OH)2 were removed from the root canals. A sterile file was used for removal of Ca(OH)2. After drying the canals by using paper points, the dentin chips obtained with a peeso reamer (number 6) from the canal walls were immediately collected onto separate Petri dishes and weighed 10 mg.

Three-day group: Twenty teeth infected with 10 µl (1-3 x 108 cfu) of E. faecalis (RSHI 10541) were incubated for 48 h at 37°C and then 18 teeth were medicated under the same conditions. The medicated teeth and 2 incubated but unmedicated control teeth were stored in SPS for 72 h at 37°C. Dentin chips were then taken and weighed 10 mg.

Assessment of antibacterial efficacy: In order to count cfu of E. faecalis in dentin tubules, dentin chips of the teeth were diluted in 2 ml SPS and then serial dilutions as 10-1, 10-2 and 10-3 were obtained. The classical bacterial count technique was used for recovery of E. faecalis on blood agar (Difco, Detroit, MI, USA), containing 5% sheep blood.


The median numbers of bacterial cells recovered from medicated teeth of the two groups are presented in Table 1. In the teeth medicated for one day, there were fewer microorganisms than in the teeth of the three-day group. The control specimens had the highest number of microorganisms in both groups.

The one-day testing showed that PCP and CPCP were significantly more effective than Ca(OH)2 against E. faecalis. On the other hand, in the three-day group, CPCP was the most effective, followed by Ca(OH)2.


The persistence of bacteria in the root canal system often leads to the failure of treatment. Microorganisms can colonize spaces that are not accessible to instruments and irrigation. Several studies have attempted to demonstrate the possible ways in which bacteria invade the tubules (Akpata and Blechman, 1982; Perez et al., 1993). However, there are few reports on the efficacy of root canal medicaments against infected tubules under controlled conditions. Using an in vitro model with infected dentin specimens, it has been previously shown that sequential removal of dentin for bacterial analysis is possible (Haapasalo and Orstavik, 1990).

E. faecalis was chosen as a test organism in this study because it is among the few facultative organisms associated with persistent apical periodontitis (Haapasalo et al., 1983; Haapasalo and Orstavik, 1990) and it may be difficult to eliminate from root canals (Weine, 1989).

Many drugs, such as phenol, iodine solutions, formocresol and antibiotics, have been used for disinfection of the root canal. It is questionable whether sufficient amounts of commonly used medicaments can penetrate into tubules to achieve disinfection. The effectiveness of intracanal medicaments based on phenol has been shown to decrease rapidly. Camphorated parachlorophenol has been found to be effective in inhibiting the growth of microorganisms in the root canal (Wantulok, 1972; Stevens and Grossman, 1983). This preparation releases chlorine slowly and is more germicidal. The camphor serves as a vehicle and diluent and reduces the irritating effect of pure parachlorophenol. It also prolongs the antimicrobial effect (Grossman, 1988).

For Ca(OH)2 to act effectively as an intracanal medicament, the hydroxyl ion must be able to diffuse through the dentin. The results of a recent study show that hydroxyl ions derived from a Ca(OH)2 intracanal medicament diffuse in hours into the inner dentin but require 1 to 7 days to reach peak levels in the outer root dentin (Nerwich et al., 1993).

There is some uncertainty as to the efficacy of Ca(OH)2 compared with other medicaments when used as an intracanal disinfectant. However, some studies have shown that Ca(OH)2 has more favorable antibacterial effects than camphorated paramonochlorophenol (CMCP) (Georgopoulou et al., 1993; Stuart et al., 1991). In another study, when compared with 2% iodine-potassium iodide solution, Ca(OH)2-dressed root canals yielded fewer culture reversals (Safavi et al., 1985).

Sjögren et al. (1991) reported that Ca(OH)2 efficiently eliminates bacteria which may survive biomechanical instrumentation.

The findings of our study show similarity with results of some investigators (Haapasalo and Orstavik, 1987; Safavi et al., 1985). The inability of Ca(OH)2 to eliminate microorganisms during a short period of time (1 or 3 days) is not surprising in light of previous reports by other investigators. In the one-day group, CPCP and PCP showed the same effectiveness on microorganisms. In the three-day group, the effectiveness of CPCP was superior to PCP. This may be due to a prolonged effect of CPCP which is enhanced by camphor. Ca(OH)2 also elicited increased effectiveness in the three-day group. It may be assumed that more hydroxyl ions diffused into the dentinal tubules, and higher effectiveness of Ca(OH)2 against microorganisms in the tubules will be obtained with time. Our control specimens were uniformly infected and yielded the highest number of microorganisms in the dentinal tubules. In this study, after three days of medication, the cultures yielded more microorganisms than after one day of medication. This may be expected because microorganisms can easily grow in tooth tissue and even in SPS solution with time. Large numbers of microorganisms in control groups also confirm this situation.

We conclude that the method used in this study is suitable for in vitro testing of root canal medicaments. However, long-term in vivo investigations are needed for reliable testing of Ca(OH)2. It is believed that bacterial invasion from the exposed dentinal surfaces into the dentinal tubules is less extensive in vital teeth than in nonvital teeth, because vital teeth have living odontoblastic processes and supporting collagen fibers within the tubules that direct the flow of the dentinal fluid from the pulp at constant pressure (Nagaoka et al., 1995).


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Correspondence: Dr. Füsun Tanriverdi, Selçuk Üniversitesi, Dishekimligi Fakültesi, Dis Hastaliklan ve Tedavisi A. D. 42079, Kampüs, Konya, Türkiye.

Accepted  April 10, 1997
Electronic publication: March, 1998