Evaluation of the Antimicrobial Activity
of Three Irrigating Solutions
in Teeth with Pulpal Necrosis

Cláudio Maniglia FERREIRA1
Kleber Cortes BONIFÁCIO1
Izabel Cristina FRÖNER1
Izabel Yoko ITO2

1Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil
2Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil


Braz Dent J (1999) 10(1): 1-60 ISSN 0103-6440

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


The antimicrobial activity of 0.4% papaine gel (FCF-USP), an antibacterial product derived from 3.3% castor oil (IQSC-USP), and 0.5% sodium hypochlorite (FORP-USP) was evaluated in teeth with radiographically visible pulpal necrosis and periapical lesion in vivo. After cavity access, under aseptic conditions, a first harvesting was performed. The 3 irrigating solutions were used for biomechanical preparation. After 72 hours, a second harvesting was performed, also under aseptic conditions. The number of colony forming units (cfu) was counted with a stereomicroscope under reflected light. Castor oil and 0.5% sodium hypochlorite presented similar antimicrobial activities for the reduction of the anaerobe number, S. mutans and streptococci; however, the papaine gel showed lower activity. We conclude that both castor oil and sodium hypochlorite are effective as antimicrobial agents and can be used in the treatment of root canals with pulpal necrosis.


Key Words:  endodontics, anaerobic bacteria, dental pulp diseases, irrigating solution, antimicrobial solution.


Introduction

    The aim of endodontic treatment is the complete removal of pulpal tissues and/or microorganisms that remain in the root canal system providing an appropriate root canal filling and, consequently, the repair of the periapical tissues (Briseño et al., 1992). Infiltrated microorganisms are found in the root canal which are reached only by chemical agents used in biomechanical preparation or by temporary root canal fillings (Grossman and Meinmamn, 1941). Irrigating solutions used in endodontic treatment not only present antimicrobial action, but they also clean the pulp chamber (Grossman and Meinmamn, 1941; Senia et al., 1971; Hand et al., 1978; Leonardo and Leal, 1991).The irrigating solution must combine maximum antimicrobial action with minimum toxicity, physical and chemical properties associated with a feasible cost to the professional.
    Sodium hypochlorite has been used for more than 70 years, because its antimicrobial action is associated with low toxicity and the ability of tissue dissolution. Sodium hypochlorite toxicity is directly proportional to its concentration (Heggers et al., 1991; Alaçam et al., 1993; Yesilsoy et al., 1995). Lower concentrations of sodium hypochlorite do not present efficient antimicrobial action (Byström and Sundqvist, 1983), being ineffective for species of Staphyloccocus aureus present in the root canal of teeth with pulpal necrosis (Yesilsoy et al., 1995).
    For the dissolution of the pulpal tissue, Harlan (1900) used Carica papayna (papaine) successfully. This action of papaine was further supported by Hession (1977). Lund and Roger (1969) claimed that papaine is a proteolytic enzyme, comprised of 17 different amino acids, with a molecular weight of 20,900. Characterized for promoting, in small doses, proteolysis, it has been used in the food industry, in bacteriological and biochemical laboratories, in the rubber industry and pharmacopaea (Arnon, 1970). Flindt (1978) reported that papaine is a proteolytic enzyme which acts only on necrotic tissues. When associated to other pharmaceutical products or in gel form, papaine has shown to be effective in the healing process of skin lesions (Modolin and Bevilacqua, 1985; Velasco, 1993).
    Ricinus communis (tropical castor bean) is a typical plant of tropical climates, with great chemical-oil potential, which can guarantee the supply of polyol and prepolymers from greasy acids on a  large scale. The oil of Ricinus communis or castor oil is of vegetal origin which presents 81 to 96% castor oil in its composition. A polymer and a 33% solution were developed from castor oil. The biocompatibility of the castor oil polymer was demonstrated in implants placed in rat alveoli, in histologic and histometric evaluation showing to be compatible and progressively osteointegrated during tissue repair (Carvalho et al., 1997a,b).
    Papaine and a liquid derived from castor oil present excellent biological properties; however, there is no report in scientific dental literature about their use in endodontics.
    The purpose of this investigation is to compare in vivo the antimicrobial activity on anaerobes, streptococci and S. mutans of the 0.4% papaine gel, an antibacterial product derived from 3.3% castor oil, and 0.5% sodium hypochlorite in teeth with pulpal necrosis and radiographically visible apical lesion.


Material and Methods

    Sixty single-rooted teeth, radiographically and clinically diagnosed as having pulpal necrosis with periapical lesion, were selected from 49 patients of both sexes, age ranging from 10 to 52 years treated at the Ribeirão Preto Dental School-USP (Universidade de São Paulo). Three groups of 20 root canals each were used with one of 3 irrigating solutions: 0.5% sodium hypochlorite (FORP-USP), 3.3% castor oil (IQSC-USP), and 0.4% papaine gel (FCF-USP).
    Radiographs were carried out with the positioner (JON, São Paulo, Brazil) and millimetered screen (Flow-dental-X-Ray-adult grid-film, New York, USA). Complete isolation and antisepsis of the operating field was made with 1% PVPI solution of active iodine.
    A high-speed hand piece and sterilized round bur were used to remove the carious tissue and after access surgery to the root canal, new antisepsis of the operating field was carried out. Under aseptic conditions, the first harvesting of the root canal contents was made with sterile paper cones with metal wings compatible to the root canal diameter. These root canals had not had previous endodontic intervention. The material collected was placed in test tubes with reduced transport fluid (RTF) for transportation to the laboratory for microbiological processing (FCFRP-USP). Biomechanical preparation was then performed with K type files (Maillefer, Switzerland) and Gates-Glidden burs with classical neutralization and step-back technique with programmed backing, using the 3 irrigating solutions under evaluation. The root canals were enlarged three files above the initial one, obeying the working length, dried with sterile absorbent paper cones (Tanari, Manacapuru, AM, Brazil) and temporarily sealed with sterile cotton, gutta-percha and Cimpat (Septodont, Saint-Maur, France).
    Glass beads and metal wings sterilized in a laminar flow chamber were aseptically added to the test tubes with the collected material and subjected to mechanical motion for two min (Mixtron-Toptronix, São Paulo, Brazil). From this suspension, decimal dilutions were carried out up to 10-4 in PBS (phosphate buffer, pH 7.0, 1/15M), in a laminar flow chamber. Aliquots of 0.05 ml were deposited in blood-agar slabs (As), agar-mitis salivaris (Mas) and agar-sucrose Bacitracine (SB20), where hatching was performed by the technique of Westergreen and Krasse (1979). The slabs were placed in anaerobic jars and incubated at 37oC for 48 to 72 h (Mas and SB20) and 5 to 7 days (As). Following the incubation period, the counting of the colony forming units (cfu) of the anaerobic microorganisms, streptococci and S. mutans was carried out with the aid of a stereomicroscope (Nikon) with reflected light.
    After 72 h, under the same aseptic conditions described above, the temporary sealing was removed for the second material harvest and for microbiological processing which was carried out in the Laboratory of Microbiology and Immunology (FCFRP-USP).
    The data were analyzed statistically using software developed by Professor Dr. Geraldo Maia Campos from the Stomatology Department (FORP-USP).


Results

    Initial statistical analysis showed that the sampling distribution was not normal which led to the utilization of non-parametric statistical tests.
    Table 1 compares the total means of each microorganism present in the first and second harvesting.
    Table 2 shows the antimicrobial action of the 3 irrigating solutions on anaerobic microorganisms, streptococci and S. mutans, at the first and second harvestings.
    The Wilcoxon test was used for comparison between the first and second harvestings, evaluating the three irrigating solutions.
    There was a statistically significant difference (P<0.01) in the second harvesting compared to the first harvesting, suggesting that the irrigating solutions reduced the number of root canal microorganisms.
    Analyzing the action of each solution on the anaerobes separately comparing the 1st and 2nd harvestings (Table 2), the Wilcoxon test showed a statistically significant difference (P<0.01) with sodium hypochlorite and castor oil. However, there was no statistically significant difference for the papaine gel. At the first harvesting, the Kruskal-Wallis test showed there was no statistically significant difference between the solutions evaluated. At the second harvesting there was a statistically significant difference (P<0.05). There was no statistically significant difference between sodium hypochlorite and castor oil, and papaine did not reduce the number of anaerobic cfu.
    Analyzing each irrigating solution separately concerning streptococci, the Wilcoxon test presented a statistically significant difference at P<0.05 related to the first and second harvestings when sodium hypochlorite was used. However, castor oil and papaine did not present statistically significant results between the harvestings. Using the Kruskal-Wallis test to compare the solutions, there was no significant difference between the first and the second harvestings. However, at the second harvesting when comparing the means, sodium hypochlorite and castor oil behaved similarly and were statistically different from papaine (P<0.05) which did not reduce the number of streptococci.
    When comparing the 1st and 2nd harvestings, the Wilcoxon test showed a statistically significant reduction in S. mutans (P<0.01) at the second harvesting for the three solutions. The Kruskal-Wallis test confirmed the behavior of the evaluated solutions in reducing S. mutans between the harvestings, showing a statistically non-significant result between them.
    Figure 1 presents the effectiveness of the antimicrobial action of the irrigating solutions: 0.5% sodium hypochlorite, 3.3% castor oil, and 0.4% papaine gel on the number of microorganisms at the 1st and 2nd harvestings.


Discussion

    There is a large number of microorganisms in root canal infections (Moorer and Wesselink, 1982), and auxiliary substances may be necessary to aid in the removal of the microbiota in areas where instrumentation has no access (Grossman and Meinmamn, 1941).
    Table 1 shows the presence of microorganisms at the first harvesting. There was a larger number of anaerobes, followed by streptococci and a lower number of S. mutans. After the healing of the root canal, there was a significant reduction in the number of microorganisms, which supports the fact that endodontic instrumentation promotes an alteration in the microflora of root canals (Byström and Sundqvist, 1983).
    Sodium hypochlorite, at different concentrations, has been used in endodontics for 70 years due to its tissue dissolution ability and antimicrobial action (Senia et al., 1971; Hand et al., 1978; Bystöm and Sundqvist, 1983; Heggers, 1991; Leonardo and Leal, 1991; Yesilsoy et al., 1995). The results of the present study supported the antimicrobial action of 0.5% sodium hypochlorite on the evaluated microbiota.
    The castor oil plant polymer presents good biocompatibility (Carvalho et al., 1997a,b); however, the liquid derived from the castor oil plant is still in the initial evaluation stage in all levels of biological and microbiological tests.
    Papaine is a substance which has been tested in endodontic research (Harlan, 1900; Hession, 1977), and its action on the connective tissue of skin lesions showed biocompatibility and efficiency in the healing process (Modolin and Bevilacqua, 1985); however, there is no record to prove that this substance presents antimicrobial properties.
    With the purpose of evaluating the antimicrobial action of the solution derived from the castor oil plant and the papaine gel on microorganisms present in root canals, this study compared them to 0.5% sodium hypochlorite which is an irrigating solution
whose biological and microbiological effects are scientifically well known.
    The means of cfu presented in Table 2 allow the conclusion that the irrigating solutions evaluated present a significant capacity of reducing the number of microorganisms between the first and the second harvestings.
    When evaluating the antimicrobial action of the irrigating solutions on each microorganism, the statistical results showed a higher efficiency of 0.5% sodium hypochlorite and 3.3% castor oil in reducing the anaerobic microorganisms and streptococci compared to papaine.
    Comparing the antimicrobial action of the irrigating solutions at the first and second harvestings it was clear that the action of sodium hypochlorite and castor oil had a significant action on the anaerobes. The same did not occur with papaine gel. When the results between the two harvestings were compared, it could be seen that the antimicrobial action of sodium hypochlorite and castor oil was significant for streptococci. Papaine did not show a significant result. There was a reduction in S. mutans (Table 1) between the harvestings; however, there was no difference in antimicrobial action of the solutions related to this microorganism (Figure 1, Table 1).
    In Figure 1 we can note, in a general way, the antimicrobial action of the evaluated solutions highlighting the larger number of colony forming units (cfu) of the microflora present at the first harvesting and the reduction observed at the second harvesting. A similar antimicrobial action of 0.5% sodium hypochlorite and 3.3% castor oil and the weaker action of 0.4% papaine gel between the harvestings can be noted.
    Papaine gel promoted a significant reduction of S. mutans only and a slight increase of cfu of anaerobes and streptococci between the harvesting (Figure 1).
    Our results allow us to conclude that 3.3% castor oil presented an antimicrobial action similar to 0.5% sodium hypochlorite on the assessed microflora. Due to its antimicrobial action, the castor oil plant solution can be a substitute for sodium hypochlorite as an irrigating solution for root canals.
    Papaine gel has not been assessed as an antimicrobial agent particularly in endodontic treatment. Our results lead to the conclusion that the action of papaine gel did not show efficiency similar to the other solutions evaluated. Further studies are necessary on the proteolytic action of papaine gel on necrotic pulpal tissue.


Conclusions

    1. All three irrigating solutions showed antimicrobial action.
    2. Papaine gel (0.4%) presented less antimicrobial action on all the microorganisms evaluated.
    3. The 3.3% castor oil solution and 0.5% sodium hypochlorite showed similar antimicrobial action.
    4. At the first harvesting, the anaerobic microorganisms were present in a greater number followed by the streptococci and S. mutans.
    5. There was a decrease in the number of colony forming units (cfu) of all microorganisms evaluated using the 3 irrigating solutions.


Acknowledgments

    We would like to thank Dr. Gilberto Orivaldo Chierice (Institute of Chemistry of São Carlos - USP, Brazil) for supplying the castor oil, and Professor Maria Valéria Velasco (FCF-USP, Brazil) for supplying the 0.4% papaine gel.


References

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Correspondence:   Izabel Cristina Fröner, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Departamento de Odontologia Restauradora, Av. do Café, s/n, 14040-904 Ribeirão Preto, SP, Brasil.


Accepted September 28, 1998
Electronic publication: September, 1999


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