International Journal of Clinical Dentistry ISSN: 1939-5833 Volume 6, Number 3 Nova Science Publishers, Inc. BUFFERING EFFECT OF HYDROXYAPATITE AND DENTIN POWDER ON THE ANTIBACTERIAL ACTIVITY OF SODIUM HYPOCHLORITE Zahed Mohammadi', Luciano Giardino, Flavio Palazzi and Sousan Shalavi Department of Endodontics, Hamedan University of Medical Sciences, Hamedan, Iran, Iranian Center for Endodontic Research (ICER), Tehran, Iran Department of Periodontology, Endodontology, Phannacology and Microbiology, Dental, School, University of Brescia, Italy Department of Odontostomatological and Maxillofacial Sciences, Federico II University of Naples, Italy Hamedan University of Medical Sciences, Hamedan, Iran ABSTRACT The purpose of this study was to compare the inhibitory effect of dentin powder and hydroxyapatite (HA) on the antibacterial activity of sodium hypochlorite against Staphylococcus aureous and Streptococcus mutans. All materials were prepared according to the manufacturer's directions immediately before testing. The antibacterial effect of each group was determined by measuring the diameter of zone of inhibition in millimeters after incubation at 37 C for 24 hours in a humid atmosphere. Each test was repeated three times. Findings showed that the antibacterial activity of sodium hypochlorite group was significantly greater than two other groups against both tested bacteria (p<0.05). In conclusion, both dentin powder and HA reduced the antibacterial activity of sodium hypochlorite significantly. Keywords: Dentin powder, hydroxyapatite, inactivation, sodium hypochlorite INTRODUCTION The major causative role of microorganisms in the pathogenesis of pulp and periapical diseases has clearly been demonstrated [1-3]. The elimination of microorganisms from Corresponding address: Zahed Mohammadi, Dspartment of Endodontics, Hamedan University of Medical Sciences, Hamedan, Iran, Email: zahed_mohammadl @yahoo.com
238 Zabed Mohammadi, Luciano Giardino, Flavio Palazzi et al. infected root canal systems is a complicated task involving tbe use of various instrumentation tecbniques, irrigation regimens and intra-canal medicaments [4, 5]. Ex vivo and clinical evidence bas shown tbat mecbanical instrumentation leaves significant portions of tbe root canal walls untouched [6] and complete elimination of bacteria by instrumentation alone is unlikely to occur [6]. Tbis bas been attributed to tbe complex anatomy of tbe root canal system [4, 5]. Tberefore, some form of irrigation and disinfection is necessary to remove residual tissue and to kill microorganisms. Root canal milieu is a complex environment witb a massive organic, inorganic and microbial load. Microorganisms are readily killed in vitro by root canal irrigants and medicaments. However, in tbe clinical conditions, a small proportion of tbe same microorganisms survive even after prolonged exposure to tbese agents. There are several reasons for poorer in vivo performance of root canal irrigants and medicaments compared to in vitro results sucb as low concentration, poor penetration, sbort exposure time, small volume, and inactivation by tbe root canal contents [7]. Studies have demonstrated tbe inbibitory effect of dentin powder, bovine serum albumin (BSA), and bydroxyapatite (HA), and beat-killed microbial wbole cells on tbe antibacterial activity of endodontic irrigants and medicaments [7, 8]. Tbere are few studies on tbe inbibitory effect of root canal contents on tbe antibacterial activity of NaOCl [7, 8]. Tberefore, tbe purpose of tbis study was to assess tbe effect of dentin powder and HA on tbe antibacterial activity of 5.25% NaOCl using agar diffusion test (ADT). MATERIALS AND METHODS Tbe materials used in tbe present study were 5.25% sodium bypocblorite (Ogna Laboratori Farmaceutici, Muggio, Italy), HA (Bio-Rad Laboratories, Ricbmond, CA, USA), and dentin powder. Botb inbibitors (dentin powder and HA) were used in powder form. Dentin powder was prepared as follows: Human tbird molars were extracted and kept in 0.5% sodium bypocblorite to remove soft tissue and prevent bacterial growth. Before furtber preparation tbe teetb were rinsed and autoclaved (12rc, 15 min) in an excess of distilled water to remove sodium bypocblorite from tbe root canal system. Tbe crowns of tbe teetb were removed witb a diamond saw and tbe roots were crusbed between two clean metal blocks. Tbe crusbed dentin was tben ground witb a sbaking apparatus of a marble ball and bowl to obtain bomogenous dentin powder. Of eacb powder, 28 mg was suspended in 50 ^L water. Fifty nl of tbe different inbibitor suspensions were tborougbly mixed and incubated witb 50 \il NaOCl in sealed test tubes at 37 C for 1 bour before adding 50 pl of tbe bacterial suspension, giving a total volume of 150 (il. One control group consisted of 50 \il sterile water instead of dentin powder or HA and tbe otber control group consisted of 50 ^ll sterile water instead of NaOCl. Tbe suspensions were careftdly mixed and incubated at 37 C in air. Tbe microorganisms used in tbis study included Stapbylococcus aureous and Streptococcus mutans. Overnight cultures of tbe microorganisms were used. Tbe bacteria were grown in tryptic - soy brotb and were adjusted to tbe turbidity of a 0.5 Me Farland BaSo4 standard (~ 1.5x 10^ colony forming unit (CFU) ml''. Tbirty Petri disbes containing tryptic - soy agar enricbed witb 5% deftbrinated sbeep blood and supplemented witb bemin and vitamin K were seeded witb bacteria.
Buffering Effect of Hydroxyapatite and Dentin Powder on the Antibacterial... 239 Seeding was done using sterile cotton - tipped applicators that were bmshed across the agar surface. Three wells of 5-mm depth and 6-mm diameter were punched in each agar plate and filled with freshly mixed materials. Fifteen wells were used for each group. The plates were then maintained at room temperature for two hours for pre-difrision of the material. Later, the antimicrobial effect of each material was determined by measuring the diameter of zone of inhibition in millimeters after incubation at 37 c for 24 hours in a humid atmosphere. Each test repeated three times. Data were analyzed using ANOVA and Tukey's test. Differences at the 5% level (p < 0.05) were considered statistically significant. RESULTS All materials tested demonstrated some antibacterial activity. Both dentin powder and HA decreased the efficacy of NaOCl against both bacteria significantly (p<0.05). The means of the diameters of the zone of microbial inhibition for each group against each bacterium was shown in Table 1. Table 1. Means of the diameters of the zones of bacterial growth inhibition provided by tested materials (in mm) Bacterial Species Sodium hypochlorite Mean SD Sodium hypochlorite +Dentin powder Mean SD Sodium hypochlorite +HA Mean SD S. aureous S. mutans 19.10 17.22 1.30 1.59 11.84 10.65 0.54 1.20 12.25 n.2i L27 0.97 DISCUSSION Microorganisms are considered to be the primary etiologic agents in endodontic diseases [1-3]. The agar diffusion test is extensively used to assess the antimicrobial activity of endodontic materials [9-11]. Several factors that are relevant for the diffusion capacity of materials in agar must be considered, such as the contact between the experimental material and agar, molecular weight, size and shape of the antimicrobial agent, load and concentration of test material, agar gel viscosity, and ionic concentration in relation to the medium. In addition, control and standardization of inoculation density, evaluation of results, selection of agar medium, selection of microorganisms, depth of agar medium, incubation temperature of plates, and reading point of inhibition haloes are also restricting factors affecting the dynamics and variability of diffusion tests in an agar medium [10]. Nevertheless, if most of these variables are carefully controlled, then consistent and reproducible results may be obtained [9]. As a result of the obvious limitations of in vitro studies, clinical inferences should be drawn with strict caution. Although aerobic and facultative bacteria are usually minor constituents of primary infections, they have been found in cases in which the treatment had been protracted, in flare-
240 Zahed Mohammadi, Luciano Giardino, Flavio Palazzi et al. ups, and associated with endodontic failures. Therefore, in addition to anaerobic bacteria, it also seems important to evaluate the antimicrobial activity of endodontic materials against them [12]. Furthermore, test microorganisms used in the present study were utilized in several studies to evaluate the antimicrobial effects of root canal sealers [11, 13]. Findings of the present study showed that both dentin powder and HA reduced the antibacterial activity of NaOCl against both tested bacteria (S. aureus and S. mutans). HA is the major inorganic component of dentin [7]. Wang and Hume [14] showed that dentin was a strong buffer against acids. Inorganic apatite seems to be mainly responsible for the buffering effect of dentin [7] Portenier et al. [8] demonstrated that HA completely inhibited the antibacterial activity of calcium hydroxide; however, it revealed little to no inhibitory effect on chlorhexidine and iodine compound. Although HA is the main component of dentin, approximately 20% of the biomass of dentin consists of organic compounds, mainly type I collagen [15]. Furthermore, contents of the root canal system including remnants of the pulpal tissue (necrotic or vital), inflammatory exúdate, as well as killed microbial cells in the root canal system contribute considerably to the overall amount of organic material present [7]. Another important issue is the relationship between the concentration of inhibitory materials and the amount of reducing the antibacterial activity of root canal irrigants and medicaments. Portenier et al. [8] found that reducing the amount of dentin to one-tenth did not reduce its inhibitory effect on the antibacterial activity of calcium hydroxide. This was in contrary to chlorhexidine, in which the inhibitory effect of dentin reduced significantly by decreasing the concentration of dentin powder to one-tenth. In conclusion, within the limitations of the present study, both dentin powder and HA reduced the antibacterial activity of NaOCl significantly. REFERENCES [1] Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposure of dental pulps in germ-fi-ee and conventional laboratory rats. Oral. Surg. 1965; 18: 340-8. [2] Möller AJ, Fabricius L, Dahlén G, Öhman AE, Heyden G. Influence on periapical tissues of indigenous oral bacteria and necrotic pulp tissue in monkeys. Scand. J. Dent. Res 1981; 89: 475-84. [3] Sundqvist G. Ecology of the root canal flora. J. Endod. 1992; 18: 427-30. [4] Hess, W. Anatomy of root canals in the teeth of the permanent dentition. New York, 1925, William Wood & Co. [5] Peters OA, Laib A, Gohring TN, Barbakow F. Changes in root canal geometry after preparation assessed by high resolution computed tomography. J. Endod. 2001; 27: 1-6. [6] Mohammadi Z. Chemomechanical strategies to manage endodontic infections. Dent. Today 2010; [7] Haapasalo M, Qian W, Portenier I, Waltimo TM. Effects of dentin on the antimicrobial properties of endodontic medicaments. 7. Endod. 2007; 33: 917-25. [8] Portenier I, Haapasalo M, Rye A, Waltimo TM, Orstavik D, Haapasalo M. Inactivation of root canal medicaments by dentin, hydroxyapatite, and bovine serum albumin. Int. Endod. J. 2001; 34: 184-8.
Buffering Effect of Hydroxyapatite and Dentin Powder on the Antibacterial... 241 [9] Spangberg L, Engstrom B, Langland K. Biologic effects of dental materials. 3. Toxicity and antimicrobial effect of endodontic antiseptics in vitro. Oral Surg. Oral Med. Oral Pathol. 1973; 36: 856-71. [10] Al- Khatib ZZ, Baum RH, Morse DR, Yesilsoy C, Bhambhani S, Fürst ML: The antimicrobial effect of various endodontic sealers. Oral Surg. Oral Med. Oral Pathol. 1990; 70: 784-90. [11] Cobankara FK, Altinoz HC, Erganis O, Kav K, Belli S: In vitro antibacterial activities of root - canal sealers by using two different methods. J. Endod. 2004; 30: 57-60, 2004. [12] Tobias RS: Antibacterial properties of dental restorative materials: a review. Int. Endod. J. 1988; 21: 155-60. [13] Lai C-C, Huang F-M, Yang H-W, Chen Y, Huang M-S, Chang Y-C : Antimicrobial activity of four root canal sealers against endodontic pathogens. Clin Oral. Investig. 2001; 5: 236-9. [14] Wang JD, Hume WR. Diffusion of hydrogen ion and hydroxyl ion from various sources into dentin. Int. Endod. J. 1988; 21: 17-26. [15] Nanci A. Dentin-pulp complex. In: Nanci A, ed. Ten Cate oral histology. St. Louis: Mosby, 2003:193. [16] Scardina, G.A. Editorial: Critical reading of a scientific article: Scientific validity. Int. J. Clin. Denti. 2009; 2 (4):205-206.
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