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Cytotoxic Effects and Antibacterial Efficacy of a 3-Antibiotic Combination: An In Vitro Study
Basic Research—Biology
Cytotoxic Effects and Antibacterial Efficacy of a 3-Antibiotic Combination: An In Vitro Study Sorapong Chuensombat, DDS,* Saengusa Khemaleelakul, DDS, Dip Clin Dent, PhD,* Siriporn Chattipakorn, DDS, PhD,† and Tanida Srisuwan, DDS, Dip Clin Dent, PhD* Abstract Introduction: A 3-antibiotic combination (3Mix) is widely used in endodontics for root canal disinfection, particularly in pulp revascularization procedures. However, the cytotoxicity of 3Mix has not been evaluated. The purpose of this study was to determine the cytotoxicity and antibacterial efficacy of 3Mix and each single antibiotic component of 3Mix. Methods: For the cytotoxicity test, human dental pulp cells and apical papilla cells were exposed to either 3Mix or to each single antibiotic component of 3Mix using concentrations of 0.024, 0.097, 0.39, 1.56, 6.25, and 25.00 mg/mL for 1, 3, 5, and 7 days. Cell viability was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide (MTT) assay. For the antibacterial test, 25.00 mg/mL and 0.39 mg/mL 3Mix or single antibiotic were tested on bacteria isolated from necrotic teeth by measuring bacterial recovery on blood agar. Results: The 0.024-mg/mL concentration of all experimental groups generated the highest dental pulp cell or apical pulp cell viability at all time periods. On day 7, 0.39 mg/mL 3Mix produced more than 90% cell viability; 25.00 mg/mL 3Mix completely eliminated isolated bacteria, whereas 0.39 mg/mL was unable to eradicate all bacteria. However, the overall bacterial reduction was significantly different compared with the control group (P < .01). Conclusions: All drugs except metronidazole induced cytotoxicity on cultured cells. 3Mix generated higher cytotoxicity compared with a single drug. The cytotoxicity increased in a concentration- and time-dependent manner; 0.39 mg/mL 3Mix had less cytotoxicity and was able to significantly reduce bacteria isolated from necrotic teeth. (J Endod 2013;39:813–819)
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ental pulp plays an important role in tooth homeostasis (1). Various types of cells can be found inside the pulp (eg, odontoblasts, fibroblasts, stem cells, immune cells, and endothelial cells). During root formation, apical papilla tissue is found. This tissue has a high collateral circulation and consists of various cells types (eg, fibroblasts, immune cells, endothelial cells, and stem cells). These cells play a part in tooth development and pulpal infection control including the self-repairing process (1–3). Microorganisms, especially bacteria, are the main cause of pulpal and periradicular disease. The invasion of bacteria and their toxins into the pulp is able to induce pulpal inflammation and lead to pulp death (4, 5). When pulp necrosis occurs during tooth development, the formation of the root is limited, leaving the tooth with a thin root structure and wide apical closure, risking root fracture (6). The treatment for incomplete tooth development is difficult and challenging (7, 8). Recently, pulp revascularization has become a new treatment modality to manage immature necrotic tooth using the ‘‘lesion sterilization and tissue repair (LSTR)’’ concept (9, 10). In brief, the infection is removed, creating a sterile environment that promotes tissue regeneration and revitalization (11, 12) and permitting the tooth to continue the development (6, 13). The cell sources that play a role in this situation are still unknown. However, remaining vital tissue inside the tooth and surviving cells around the apical part of the tooth, which have differentiation potential, are suspected (14–17). A 3-antibiotic combination (3Mix), a mixture of minocycline, ciprofloxacin, and metronidazole, has been introduced by Hoshino et al (18) along with the LSTR concept. Various studies reported promising outcomes when 3Mix was used in necrotic teeth with incomplete root formation (16, 19). However, some recent histologic studies have shown that regenerated tissue found in the treated tooth was not exactly pulp tissue (20, 21). Still, the cause for these consequences is unknown. An interesting issue is the concentration of 3Mix used because the clinical use of 3Mix has been empirical. Local application of 3Mix using an excessive concentration might affect the host tissue, causing cell death, which limits tissue regeneration. Therefore, the purposes of this study were to determine the cytotoxic effects of 3Mix and each single antibiotic component of 3Mix on cultured human dental pulp cells (DPCs) and apical papilla cells (APCs) and to evaluate the antibacterial efficacy of the noncytotoxic concentration of 3Mix and each single antibiotic component of 3Mix against bacteria isolated from necrotic teeth.
Key Words 3-antibiotic combination, antibacterial efficacy, apical papilla cells, cytotoxicity, dental pulp cells
Materials and Methods Primary Cell Culture DPCs and APCs were extracted from human dental pulp tissue or apical papilla tissue obtained from nonpathologic mature or immature third molars. All procedures
From the Departments of *Restorative Dentistry and Periodontology and †Oral Biology and Diagnostic Sciences, Faculty of Dentistry, Chiang Mai University, Chiang Mai, Thailand. Address requests for reprints to Dr Sorapong Chuensombat, Department of Restorative Dentistry and Periodontology, Faculty of Dentistry, Chiang Mai University, Chiang Mai, Thailand. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.11.041
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Figure 1. DPCs treated with 3Mix at different concentrations for 7 days. (A) Control (cells in culture medium) and (B–G) 0.024-, 0.097-, 0.39-, 1.56-, 6.25-, and 25.00-mg/mL concentrations (40 magnification). DPCs treated with 0.024, 0.097, 0.39, and 1.56 mg/mL started to form a multilayer when reaching confluence, whereas DPCs treated with 6.25 and 25.00 mg/mL showed a mixed population of both spindle-shaped cells and polygonal-like cells. 3Mix at a concentration of 25.00 mg/mL produced fewer DPCs when compared with lower concentrations and the control.
were approved by the Human Experimentation Committee of the Faculty of Dentistry, Chiang Mai University, Chiang Mai, Thailand. Dental pulp tissues or apical papilla tissues were minced and digested with a mixture of 3.00 mg/mL collagenase (Gibco/Invitrogen, Gaithersburg, MD) and 4.00 mg/mL dispase (Sigma-Aldrich, St Louis, MO) at 37 C for an hour in order to extract DPCs or APCs. The extracted cells were cultured in the alpha modification of Eagle medium (a-MEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (Gibco/Invitrogen), 100.00 U/mL penicillin, 100.00 mg/mL streptomycin (SigmaAldrich), and 100.00 mmol/L L-ascorbic acid (Sigma-Aldrich). The cells were kept at 37 C in a humidified atmosphere with 95% air and 5% CO2. For the experiment, cells at the third through fifth passages were harvested and used.
Drug Preparation A stock solution of 3Mix was prepared by mixing the powder from minocycline (Qualimed, Samut Prakan, Thailand), ciprofloxacin (Khandelwal, Mumbai, India), and metronidazole (Piramal Healthcare,
Gujarat, India) at 50.00 mg/mL in distilled water. Then, a 4-time serial dilution was performed producing 25.00-, 6.25-, 1.56-, 0.39-, 0.097-, and 0.024-mg/mL concentrations using a-MEM. For the single-drug solution, minocycline, ciprofloxacin, and metronidazole were separately prepared in the same concentrations as previously described. All samples were prepared at room temperature and sterilized by a double-filtering technique using 2.00-mm pore size filter paper (Whatman, Maidstone, England) and 0.2-mm pore size microfilters (Corning, Oneonta, NY).
Cytotoxicity Test DPCs or APCs were seeded at 103 cells/well onto 96-well plates containing 150.00 mL a-MEM. After 24 hours, cells were stimulated with media containing 25.00, 6.25, 1.56, 0.39, 0.097, and 0.024 mg/mL either 3Mix or each single drug. The cells were monitored for 1, 3, 5, and 7 days under a light microscope (DP12; Olympus, Melville, NY). An 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide (MTT) assay was performed using
Figure 2. APCs treated with 3Mix at different concentrations for 7 days. (A) Control (cells in culture medium) and (B–G) 0.024-, 0.097-, 0.39-, 1.56-, 6.25-, and 25.00-mg/mL concentrations (40 magnification). 3Mix affected on APCs similar to DPCs. APCs treated with 0.024 mg/mL 3Mix showed densely cell number, a descending of cell number of APCs was demonstrated when the cells was exposed to higher concentrations of 3Mix. APCs treated with 6.25 and 25.00 mg/mL showed a mixed population of both spindle-shaped cells and polygonal-like cells.
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Basic Research—Biology 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide solution (Sigma-Aldrich) at 5.00 mg/mL in phosphate-buffered saline solution. The optical density was investigated at l570 nm using an enzyme-linked immunosorbent assay spectrophotometer (Sunrise; Tecan, Mannerdrof, Switzerland). Percentages of cell viability were calculated and grouped according to the criteria of Dalh et al (22) as follows: 1. 2. 3. 4.
<30% cell viability = severe cytotoxicity 30%–60% cell viability = moderate cytotoxicity 60%–90% cell viability = slight cytotoxicity >90% cell viability = noncytotoxicity
The results were analyzed using the Kruskal-Wallis and MannWhitney U tests.
Bacterial Collection Bacterial samples were collected from 20 necrotic teeth during routine endodontic treatment following the protocol of Sassone et al (23). Briefly, under sterile conditions, an access opening was made
using diamond burs with sterile normal saline solution irrigation. The operating field and pulp chamber were then swabbed with 2.50% NaOCl followed by sterile 5.00% sodium thiosulfate solution. Sterile normal saline solution was introduced into the canal, and a no. 15 K-type file was inserted 1.00–2.00 mm short of the root apex. After filing the canals, 2 sterile paper points were introduced into the canal and left for 1 minute. The paper points were transferred into cryotubes (Corning) containing 500.00 mL reduced transport fluid. The bacterial samples were immediately transferred to the laboratory, and 10 serial dilutions were performed under an anaerobic condition.
Bacterial Recovery Aliquots of 100.00 mL of the diluted bacterial samples were dropped on and gently spread over the surface of tryptic soy blood agar (Criterion, Santa Maria, CA) containing either 25.00 or 0.39 mg/mL 3Mix or of each single drug. The plates were incubated in an anaerobic glove box at 37 C for 7 days. The colonies were counted and recorded. The data were analyzed using the paired t test.
Figure 3. DPC and APC viability after being treated with 3Mix at different concentrations and times. (A) DPCs and (B) APCs. For both cell types, 0.024 mg/mL 3Mix produced the highest cell viability, whereas 25.00 mg/mL 3Mix generated the lowest cell survival at all time periods (P < .001). On day 7, DPC and APC viability was significantly reduced into less than 90% cell viability when exposed to 25.00, 6.25, and 1.56 mg/mL 3Mix (P < .001). Data are mean standard deviation. *A significant difference from the control group (P < .001).
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Basic Research—Biology Results Morphology of DPCs and APCs Monitoring DPCs and APCs under a light microscope showed that DPCs or APCs in the control group were a mixed population containing both spindle-shaped cells and polygonal-like cells. In the experimental groups that contained antibiotics, a reduced cell number was shown only for DPCs or APCs stimulated by 25.00 mg/mL 3Mix. Polygonal and round-shaped cells were observed in a monolayer (Figs. 1 and 2). Cytotoxicity of 3Mix and Each Single Antibiotic The 0.024-mg/mL concentration of 3Mix produced the highest cell viability, whereas 25.00 mg/mL 3Mix generated the lowest cell survival at all time periods (P < .001). On day 7, DPC and APC survival was reduced to less than 90% cell viability when stimulated by 25.00, 6.25, and 1.56 mg/mL 3Mix (Fig. 3). All experiments on day 1 produced the highest cell viability, whereas day 7 generated the lowest cell viability when 3Mix, minocycline, and ciprofloxacin were tested (P < .001, Fig. 4). When comparing all the drugs, 3Mix had greater cytotoxicity than the other single antibiotics. Minocycline and ciprofloxacin at 25.00, 6.25, and 1.56 mg/mL generated less than 90% cell viability, whereas metronidazole at all concentrations had little effect on cell viability (Fig. 5). Antibacterial Efficacy of Antibiotics 3Mix at 25.00 mg/mL completely eradicated all isolated bacteria with a significant reduction of bacterial recovery (P < .01).
In the presence of 0.39 mg/mL 3Mix or a single drug at the concentration of 25.00 mg/mL, the bacterial number was reduced, but bacteria were not completely eliminated. However, bacterial recovery also significantly decreased compared with the control group (P < .01, Fig. 6).
Discussion This study showed that all drugs except metronidazole were cytotoxic to cultured human DPCs and APCs in a dose- and time-dependent manner. When comparing all drugs at the same concentration, 3Mix had greater cytotoxicity than the other single drugs; 25.00 mg/mL 3Mix produced moderate cytotoxicity with the highest bacteria reduction, whereas 0.39 mg/mL 3Mix had a less cytotoxic effect on cultured cells but showed less bacterial elimination. However, this concentration was able to significantly reduce the number of bacteria when compared with the control group. A 3-antibiotic combination has been used in treating immature teeth with necrotic pulp using the LSTR concept (10, 18). Many studies have reported the successful outcomes by which immature teeth showed continued root formation (13, 24). However, histologic studies regarding the regenerated tissue inside the necrotic human tooth are lacking. Recent histologic studies (20, 21) of dogs’ teeth treated with the LSTR technique revealed that the newly generated tissue was not pulp tissue. Periodontal tissue, including bone, was generally observed inside the treated tooth. Therefore, the question should be raised whether this treatment option is valid and how to prevent these consequences.
Figure 4. DPC and APC viability after being treated with 3Mix at 25.00, 6.25, and 1.56 mg/mL for 1, 3, 5, and 7 days. (A) DPCs and (B) APCs. 3Mix generated the highest percentage of cell viability on day 1, whereas the lowest percentage of cell viability was observed on day 7. DPC and APC viability was significantly reduced when the exposure time increased (P < .001). Data are mean standard deviation. A significant difference from *day 1, **day 3, and ***day 5.
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Basic Research—Biology
Figure 5. Cell viability after being treated with antibiotics at different concentrations for 7 days. (A) DPCs and (B) APCs. For both cell types, 3Mix produced more cytotoxicity compared with each single drug at all concentrations. Minocycline and ciprofloxacin at 25.00, 6.25, and 1.56 mg/mL generated less than 90% cell viability, whereas metronidazole at all concentrations had a small effect on cell viability. Data are mean standard deviation. *A significant difference from the control group (P < .001).
One interesting issue is whether excessive concentrations of 3Mix are used in the treatment. Hoshino et al (10) reported that 25.00 mg/mL 3Mix was able to eradicate all isolated bacteria. However, the exact concentration used in the clinic, especially in the pulp revascularization technique, has never been measured. It seems that excessive concentrations are currently being used, which may damage the remaining cells and impair tissue healing and regeneration. It is possible that the remaining vital pulp tissue and periradicular area, both containing stem cells (14–17), are damaged by the high concentration of 3Mix. Therefore, an expected differentiation pattern (eg, odontoblast differentiation and dentin formation) might not occur. In this study, DPCs or APCs were exposed to different concentrations of 3Mix. DPCs were selected for this study because these cells may survive in the infected tooth. Many published case reports revealed the observation of vital tissue in the tooth diagnosed as necrotic (14–17). Moreover, APCs were also tested because stem cells may still remain in the apical region, which may have some roles in pulp regeneration. From our study, we discovered that 25.00 mg/mL 3Mix produced moderate cytotoxicity. However, in a pilot study, concentrations JOE — Volume 39, Number 6, June 2013
higher than 25.00 mg/mL produced even greater toxicity, leading to total cell death (data not shown). Therefore, these results ensure that the concentration of 3Mix in general clinical usage is too strong, causing cytotoxicity on the remaining vital tissues. On the other hand, 3Mix at 0.39 mg/mL had a less cytotoxic effect on DPC and APC viability. This concentration is suggested to be the best candidate for pulpal regeneration along the concentrations of drugs tested in this experiment. Interestingly, recently published data concerning the cytotoxicity of 3Mix reported that 0.01–0.10 mg/mL did not have an effect on cultured stem cells from apical papilla (25). This result was different from our study, which showed that 0.39 mg/mL 3Mix was a safe dose. Also, some possible reasons might be the different source of antibiotics used in the experiment, the different techniques for 3Mix solution preparation, and the different method for cell viability measurement that may affect the results. It is important for clinicians to be aware that diverse sources of antibiotics have varying levels of toxicity; these antibiotics also have varying levels of effectiveness, which is an additional concern. More information and research into this area are needed.
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Figure 6. The antibacterial efficacy of 3Mix and each single antibiotic against bacteria isolated form necrotic teeth. 3Mix at 25.00 mg/mL could completely eradicate all isolated bacteria with a significant reduction of bacterial recovery (P < .01). In the presence of 0.39 mg/mL 3Mix or a single drug at the concentration of 25.00 mg/mL, the bacteria number was reduced but not completely eliminated. Data are mean standard deviation. *A significant difference from the control group (P < .01).
The cytotoxicity of 3Mix was investigated regarding its combination because 3 antibiotics were mixed. Therefore, each drug was also tested at the same concentration as 3Mix using an MTT assay. The result indicated that all drugs except metronidazole generated cytotoxicity. The cytotoxic effect was dose and time dependent. 3Mix had greater cytotoxicity than other single drugs. The greater cytotoxicity of 3Mix might result from a combination of the toxicities of each drug. However, the study was conducted in an in vitro environment, which may not represent the real clinical situation. No evidence has been published regarding the depth of 3Mix penetration in immature tooth whether it reaches the apical and surrounding tissue. Therefore, the clinical implication of these results should be further tested. One reason for its cytotoxicity might be the low pH (pH = 4.0–4.6) of 3Mix. Minocycline hydrochloride and ciprofloxacin hydrochloride are generally used for 3Mix preparation. The release of hydrogen ions from HCl groups resulted in an acidic condition that was an unfavorable condition for culturing cells (26). In addition, a low pH allowed more agents to remain soluble and available for uptake into the cells; thus, the retained drug was able to develop more cytotoxicity (27). Conversely, in this study, metronidazole did not inversely affect DPCs and APCs even at the 25.00-mg/mL concentration. It is possible that metronidazole solution has a neutral pH (pH = 6.84); thus, cytotoxicity did not occur. In contrast, this result differed from that of a previous study by Ferreira et al (28), who reported that only 5.00 mg/mL metronidazole resulted in 74% human gingival fibroblast cell line viability. However, many studies indicated that there is a variation in the degree of drug-induced cytotoxicity in different cell types (29, 30). Therefore, the sensitivity of DPCs or APCs and the gingival fibroblast cell line on drugs might be different. From this point of view, the cytotoxic effect of 3Mix from its acidic condition should be a concern. Other antibiotics that have a neutral pH should be selected as a substitute for minocycline and ciprofloxacin. However, those alternative antibiotics should intensely offer a potent antibacterial efficacy. The antibacterial efficacy of antibiotics used in vital pulp treatment, especially the LSTR concept, is important because bacteria remnant might affect the treatment outcome. Interestingly, 0.39 mg/mL 3Mix, which had a less cytotoxic effect on cultured cells, significantly reduced isolated bacteria compared with the control group. However, this effective concentration differed from Hoshino et al (10), who reported much higher concentrations (100–200 mg/mL) with antibacterial effects. The reasons might be the differences in the source of antibiotics used and the bacteria isolation techniques. When single antibiotics were tested at the 818
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same concentrations as 3Mix, it was found that even a 25.00-mg/mL concentration of a single drug alone was unable to eradicate all isolated bacteria. This might be because each drug is not active against all bacterial types. Minocycline and ciprofloxacin, which are broad-spectrum antibiotics, have an efficient action against some gram-positive and gram-negative bacteria (31, 32), whereas metronidazole is able to kill only anaerobic bacteria (33). Thus, the combination of 3 drugs provides a broader antibacterial spectrum than a single drug alone. However, balancing between the cytotoxic effect and antibacterial efficacy is important and still needs further study. However, there is concern whether the types of bacteria presented in necrotic immature teeth are the same as in necrotic mature teeth. In this study, only bacteria isolated from necrotic mature teeth were applied using a protocol adapted from Sassone et al (23). The dentin was scratched by means of a K-file, assuming that bacteria presented in dentin might be dispersed and collected after absorbing with a paper point. After incubation, collected bacteria samples were grossly clarified using gram staining. Main types of bacteria observed were similar to previously published data representing bacteria found in necrotic tooth (34). However, these bacterial samples may or may not completely represent the bacteria observed in immature necrotic teeth. Further studies are suggested because current evidence is lacking. The use of 3Mix has been popular for a decade. It offers many benefits in treating patients with infected teeth. However, the results from this study should stimulate a concern with 3Mix usage, especially in pulp revascularization, because using high doses of 3Mix produced cytotoxicity. The reduction of the 3Mix concentration into an appropriate dose may improve the outcome of pulp revascularization treatment because it causes less cytotoxicity and still has a potent antibacterial efficacy.
Conclusions 3Mix, minocycline, and ciprofloxacin were cytotoxic to cultured DPCs and APCs. 3Mix induced a higher toxicity than minocycline, ciprofloxacin, and metronidazole. The cytotoxicity of each antibiotic except metronidazole increased in a concentration- and time-dependent manner. The 0.39-mg/mL concentration of 3Mix had no cytotoxicity to DPCs and APCs, yet it was able to significantly reduce bacteria isolated from necrotic teeth.
Acknowledgments The authors deny any conflicts of interest related to this study. JOE — Volume 39, Number 6, June 2013
Basic Research—Biology References 1. Pashley DH, Liewehr FR. Structure and functions of the dentin-pulp complex. In: Cohen S, Hargreaves KM, eds. Pathways of the Pulp. 9th ed. St Louis, MO: Mosby; 2006:460–513. 2. Casagrande L, Cordeiro MM, Nor SA, et al. Dental pulp stem cells in regenerative dentistry. Odontology 2011;99:1–7. 3. Rosa V, Botero TM, Nor JE. Regenerative endodontics in light of the stem cell paradigm. Int Dent J 2011;61(suppl 1):23–8. 4. Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Path 1965;20:340–9. 5. Keyes PH. The infectious and transmissible nature of experimental caries. Findings and implications. Arch Oral Biol 1960;1:304–20. 6. Banchs F, Trope M. Revascularization of immature permanent teeth with apical periodontitis: new treatment protocol? J Endod 2004;30:196–200. 7. Lovelace TW, Henry MA, Hargreaves KM, et al. Evaluation of the delivery of mesenchymal stem cells into the root canal space of necrotic immature teeth after clinical regenerative endodontic procedure. J Endod 2011;37:133–8. 8. Petrino JA, Boda KK, Shambarger S, et al. Challenges in regenerative endodontics: a case series. J Endod 2010;36:536–41. 9. Alam T, Nakazawa F, Nakajo K, et al. Susceptibility of Enterococcus faecalis to a combination of antibacterial drugs (3Mix) in vitro. J Oral Biosci 2005;47: 315–20. 10. Hoshino E, Kurihara-Ando N, Sato I, et al. In-vitro antibacterial susceptibility of bacteria taken from infected root dentine to a mixture of ciprofloxacin, metronidazole and minocycline. Int Endod J 1996;29:125–30. 11. Murray PE, Garcia-Godoy F, Hargreaves KM. Regenerative endodontics: a review of current status and a call for action. J Endod 2007;33:377–90. 12. Torabinejad M, Corr R, Buhrley M, et al. An animal model to study regenerative endodontics. J Endod 2011;37:197–202. 13. Jung IY, Lee SJ, Hargreaves KM. Biologically based treatment of immature permanent teeth with pulpal necrosis: a case series. J Endod 2008;34:876–87. 14. Chueh LH, Ho YC, Kuo TC, et al. Regenerative endodontic treatment for necrotic immature permanent teeth. J Endod 2009;35:160–4. 15. Cotti E, Mereu M, Lusso D. Regenerative treatment of an immature, traumatized tooth with apical periodontitis: report of a case. J Endod 2008;34: 611–6. 16. Ding RY, Cheung GS, Chen J, et al. Pulp revascularization of immature teeth with apical periodontitis: a clinical study. J Endod 2009;35:745–9. 17. Shah N, Logani A, Bhaskar U, et al. Efficacy of revascularization to induce apexification/apexogensis in infected, nonvital, immature teeth: a pilot clinical study. J Endod 2008;34:919–25.
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18. Hoshino E. 3MIX-MP method-better and efficient clinical procedure of lesion sterilization and tissue repair (LSTR) therapy. Dent Rev 1998;666:57–106. 19. Chen MY, Chen KL, Chen CA, et al. Responses of immature permanent teeth with infected necrotic pulp tissue and apical periodontitis/abscess to revascularization procedures. Int Endod J 2012;45:294–305. 20. Wang X, Thibodeau B, Trope M, et al. Histologic characterization of regenerated tissues in canal space after the revitalization/revascularization procedure of immature dog teeth with apical periodontitis. J Endod 2010;36:56–63. 21. Yamauchi N, Nagaoka H, Yamauchi S, et al. Immunohistological characterization of newly formed tissues after regenerative procedure in immature dog teeth. J Endod 2011;37:1636–41. 22. Dahl JE, Frangou-Polyzois MJ, Polyzois GL. In vitro biocompatibility of denture relining materials. Gerodontology 2006;23:17–22. 23. Sassone LM, Fidel RA, Faveri M, et al. A microbiological profile of symptomatic teeth with primary endodontic infections. J Endod 2008;34:541–5. 24. Reynolds K, Johnson JD, Cohenca N. Pulp revascularization of necrotic bilateral bicuspids using a modified novel technique to eliminate potential coronal discolouration: a case report. Int Endod J 2009;42:84–92. 25. Ruparel NB, Teixeira FB, Ferraz CCR, et al. Direct effect of intracanal medicaments on survival of stem cells of the apical papilla. J Endod 2012;38:1372–5. 26. Kobayashi M, Kagawa T, Takano R, et al. Effect of medium pH on the cytotoxicity of hydrophilic statins. J Pharm Pharm Sci 2007;10:332–9. 27. Seay TM, Peretsman SJ, Dixon PS. Inhibition of human transitional cell carcinoma in vitro proliferation by fluoroquinolone antibiotics. J Urol 1996;155:757–62. 28. Ferreira MB, Myiagi S, Nogales CG, et al. Time- and concentration-dependent cytotoxicity of antibiotics used in endodontic therapy. J Appl Oral Sci 2010;18: 259–63. 29. Arenholt-Bindslev D, Bleeg H. Characterization of two types of human oral fibroblast with a potential application to cellular toxicity studies: tooth pulp fibroblasts and buccal mucosa fibroblasts. Int Endod J 1990;23:84–91. 30. Pissiotis E, Spangberg LS. Toxicity of Pulpispad using four different cell types. Int Endod J 1991;24:249–57. 31. Almazin SM, Dziak R, Andreana S, et al. The effect of doxycycline hyclate, chlorhexidinegluconate, and minocycline hydrochloride on osteoblastic proliferation and differentiation in vitro. J Periodontol 2009;80:999–1005. 32. Mohammadi Z. Systemic, prophylactic and local applications of antimicrobials in endondontic: and update review. Int Dent J 2009;59:178–86. 33. Soares GM, Figueiredo LC, Favevi M, et al. Mechanisms of action of systemic antibiotics used in periodontal treatment and mechanisms of bacterial resistance to these drugs. J Appl Oral Sci 2012;20:295–309. 34. Love RM, Jenkinson HF. Invasion of dentinal tubules by oral bacteria. Crit Rev Oral Biol Med 2002;13:171–83.
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Cytotoxic Effects and Antibacterial Efficacy of a 3-Antibiotic Combination: An In Vitro Study Sorapong Chuensombat, DDS,* Saengusa Khemaleelakul, DDS, Dip Clin Dent, PhD,* Siriporn Chattipakorn, DDS, PhD,† and Tanida Srisuwan, DDS, Dip Clin Dent, PhD* Abstract Introduction: A 3-antibiotic combination (3Mix) is widely used in endodontics for root canal disinfection, particularly in pulp revascularization procedures. However, the cytotoxicity of 3Mix has not been evaluated. The purpose of this study was to determine the cytotoxicity and antibacterial efficacy of 3Mix and each single antibiotic component of 3Mix. Methods: For the cytotoxicity test, human dental pulp cells and apical papilla cells were exposed to either 3Mix or to each single antibiotic component of 3Mix using concentrations of 0.024, 0.097, 0.39, 1.56, 6.25, and 25.00 mg/mL for 1, 3, 5, and 7 days. Cell viability was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide (MTT) assay. For the antibacterial test, 25.00 mg/mL and 0.39 mg/mL 3Mix or single antibiotic were tested on bacteria isolated from necrotic teeth by measuring bacterial recovery on blood agar. Results: The 0.024-mg/mL concentration of all experimental groups generated the highest dental pulp cell or apical pulp cell viability at all time periods. On day 7, 0.39 mg/mL 3Mix produced more than 90% cell viability; 25.00 mg/mL 3Mix completely eliminated isolated bacteria, whereas 0.39 mg/mL was unable to eradicate all bacteria. However, the overall bacterial reduction was significantly different compared with the control group (P < .01). Conclusions: All drugs except metronidazole induced cytotoxicity on cultured cells. 3Mix generated higher cytotoxicity compared with a single drug. The cytotoxicity increased in a concentration- and time-dependent manner; 0.39 mg/mL 3Mix had less cytotoxicity and was able to significantly reduce bacteria isolated from necrotic teeth. (J Endod 2013;39:813–819)
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ental pulp plays an important role in tooth homeostasis (1). Various types of cells can be found inside the pulp (eg, odontoblasts, fibroblasts, stem cells, immune cells, and endothelial cells). During root formation, apical papilla tissue is found. This tissue has a high collateral circulation and consists of various cells types (eg, fibroblasts, immune cells, endothelial cells, and stem cells). These cells play a part in tooth development and pulpal infection control including the self-repairing process (1–3). Microorganisms, especially bacteria, are the main cause of pulpal and periradicular disease. The invasion of bacteria and their toxins into the pulp is able to induce pulpal inflammation and lead to pulp death (4, 5). When pulp necrosis occurs during tooth development, the formation of the root is limited, leaving the tooth with a thin root structure and wide apical closure, risking root fracture (6). The treatment for incomplete tooth development is difficult and challenging (7, 8). Recently, pulp revascularization has become a new treatment modality to manage immature necrotic tooth using the ‘‘lesion sterilization and tissue repair (LSTR)’’ concept (9, 10). In brief, the infection is removed, creating a sterile environment that promotes tissue regeneration and revitalization (11, 12) and permitting the tooth to continue the development (6, 13). The cell sources that play a role in this situation are still unknown. However, remaining vital tissue inside the tooth and surviving cells around the apical part of the tooth, which have differentiation potential, are suspected (14–17). A 3-antibiotic combination (3Mix), a mixture of minocycline, ciprofloxacin, and metronidazole, has been introduced by Hoshino et al (18) along with the LSTR concept. Various studies reported promising outcomes when 3Mix was used in necrotic teeth with incomplete root formation (16, 19). However, some recent histologic studies have shown that regenerated tissue found in the treated tooth was not exactly pulp tissue (20, 21). Still, the cause for these consequences is unknown. An interesting issue is the concentration of 3Mix used because the clinical use of 3Mix has been empirical. Local application of 3Mix using an excessive concentration might affect the host tissue, causing cell death, which limits tissue regeneration. Therefore, the purposes of this study were to determine the cytotoxic effects of 3Mix and each single antibiotic component of 3Mix on cultured human dental pulp cells (DPCs) and apical papilla cells (APCs) and to evaluate the antibacterial efficacy of the noncytotoxic concentration of 3Mix and each single antibiotic component of 3Mix against bacteria isolated from necrotic teeth.
Key Words 3-antibiotic combination, antibacterial efficacy, apical papilla cells, cytotoxicity, dental pulp cells
Materials and Methods Primary Cell Culture DPCs and APCs were extracted from human dental pulp tissue or apical papilla tissue obtained from nonpathologic mature or immature third molars. All procedures
From the Departments of *Restorative Dentistry and Periodontology and †Oral Biology and Diagnostic Sciences, Faculty of Dentistry, Chiang Mai University, Chiang Mai, Thailand. Address requests for reprints to Dr Sorapong Chuensombat, Department of Restorative Dentistry and Periodontology, Faculty of Dentistry, Chiang Mai University, Chiang Mai, Thailand. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.11.041
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Figure 1. DPCs treated with 3Mix at different concentrations for 7 days. (A) Control (cells in culture medium) and (B–G) 0.024-, 0.097-, 0.39-, 1.56-, 6.25-, and 25.00-mg/mL concentrations (40 magnification). DPCs treated with 0.024, 0.097, 0.39, and 1.56 mg/mL started to form a multilayer when reaching confluence, whereas DPCs treated with 6.25 and 25.00 mg/mL showed a mixed population of both spindle-shaped cells and polygonal-like cells. 3Mix at a concentration of 25.00 mg/mL produced fewer DPCs when compared with lower concentrations and the control.
were approved by the Human Experimentation Committee of the Faculty of Dentistry, Chiang Mai University, Chiang Mai, Thailand. Dental pulp tissues or apical papilla tissues were minced and digested with a mixture of 3.00 mg/mL collagenase (Gibco/Invitrogen, Gaithersburg, MD) and 4.00 mg/mL dispase (Sigma-Aldrich, St Louis, MO) at 37 C for an hour in order to extract DPCs or APCs. The extracted cells were cultured in the alpha modification of Eagle medium (a-MEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (Gibco/Invitrogen), 100.00 U/mL penicillin, 100.00 mg/mL streptomycin (SigmaAldrich), and 100.00 mmol/L L-ascorbic acid (Sigma-Aldrich). The cells were kept at 37 C in a humidified atmosphere with 95% air and 5% CO2. For the experiment, cells at the third through fifth passages were harvested and used.
Drug Preparation A stock solution of 3Mix was prepared by mixing the powder from minocycline (Qualimed, Samut Prakan, Thailand), ciprofloxacin (Khandelwal, Mumbai, India), and metronidazole (Piramal Healthcare,
Gujarat, India) at 50.00 mg/mL in distilled water. Then, a 4-time serial dilution was performed producing 25.00-, 6.25-, 1.56-, 0.39-, 0.097-, and 0.024-mg/mL concentrations using a-MEM. For the single-drug solution, minocycline, ciprofloxacin, and metronidazole were separately prepared in the same concentrations as previously described. All samples were prepared at room temperature and sterilized by a double-filtering technique using 2.00-mm pore size filter paper (Whatman, Maidstone, England) and 0.2-mm pore size microfilters (Corning, Oneonta, NY).
Cytotoxicity Test DPCs or APCs were seeded at 103 cells/well onto 96-well plates containing 150.00 mL a-MEM. After 24 hours, cells were stimulated with media containing 25.00, 6.25, 1.56, 0.39, 0.097, and 0.024 mg/mL either 3Mix or each single drug. The cells were monitored for 1, 3, 5, and 7 days under a light microscope (DP12; Olympus, Melville, NY). An 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide (MTT) assay was performed using
Figure 2. APCs treated with 3Mix at different concentrations for 7 days. (A) Control (cells in culture medium) and (B–G) 0.024-, 0.097-, 0.39-, 1.56-, 6.25-, and 25.00-mg/mL concentrations (40 magnification). 3Mix affected on APCs similar to DPCs. APCs treated with 0.024 mg/mL 3Mix showed densely cell number, a descending of cell number of APCs was demonstrated when the cells was exposed to higher concentrations of 3Mix. APCs treated with 6.25 and 25.00 mg/mL showed a mixed population of both spindle-shaped cells and polygonal-like cells.
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Basic Research—Biology 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide solution (Sigma-Aldrich) at 5.00 mg/mL in phosphate-buffered saline solution. The optical density was investigated at l570 nm using an enzyme-linked immunosorbent assay spectrophotometer (Sunrise; Tecan, Mannerdrof, Switzerland). Percentages of cell viability were calculated and grouped according to the criteria of Dalh et al (22) as follows: 1. 2. 3. 4.
<30% cell viability = severe cytotoxicity 30%–60% cell viability = moderate cytotoxicity 60%–90% cell viability = slight cytotoxicity >90% cell viability = noncytotoxicity
The results were analyzed using the Kruskal-Wallis and MannWhitney U tests.
Bacterial Collection Bacterial samples were collected from 20 necrotic teeth during routine endodontic treatment following the protocol of Sassone et al (23). Briefly, under sterile conditions, an access opening was made
using diamond burs with sterile normal saline solution irrigation. The operating field and pulp chamber were then swabbed with 2.50% NaOCl followed by sterile 5.00% sodium thiosulfate solution. Sterile normal saline solution was introduced into the canal, and a no. 15 K-type file was inserted 1.00–2.00 mm short of the root apex. After filing the canals, 2 sterile paper points were introduced into the canal and left for 1 minute. The paper points were transferred into cryotubes (Corning) containing 500.00 mL reduced transport fluid. The bacterial samples were immediately transferred to the laboratory, and 10 serial dilutions were performed under an anaerobic condition.
Bacterial Recovery Aliquots of 100.00 mL of the diluted bacterial samples were dropped on and gently spread over the surface of tryptic soy blood agar (Criterion, Santa Maria, CA) containing either 25.00 or 0.39 mg/mL 3Mix or of each single drug. The plates were incubated in an anaerobic glove box at 37 C for 7 days. The colonies were counted and recorded. The data were analyzed using the paired t test.
Figure 3. DPC and APC viability after being treated with 3Mix at different concentrations and times. (A) DPCs and (B) APCs. For both cell types, 0.024 mg/mL 3Mix produced the highest cell viability, whereas 25.00 mg/mL 3Mix generated the lowest cell survival at all time periods (P < .001). On day 7, DPC and APC viability was significantly reduced into less than 90% cell viability when exposed to 25.00, 6.25, and 1.56 mg/mL 3Mix (P < .001). Data are mean standard deviation. *A significant difference from the control group (P < .001).
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Basic Research—Biology Results Morphology of DPCs and APCs Monitoring DPCs and APCs under a light microscope showed that DPCs or APCs in the control group were a mixed population containing both spindle-shaped cells and polygonal-like cells. In the experimental groups that contained antibiotics, a reduced cell number was shown only for DPCs or APCs stimulated by 25.00 mg/mL 3Mix. Polygonal and round-shaped cells were observed in a monolayer (Figs. 1 and 2). Cytotoxicity of 3Mix and Each Single Antibiotic The 0.024-mg/mL concentration of 3Mix produced the highest cell viability, whereas 25.00 mg/mL 3Mix generated the lowest cell survival at all time periods (P < .001). On day 7, DPC and APC survival was reduced to less than 90% cell viability when stimulated by 25.00, 6.25, and 1.56 mg/mL 3Mix (Fig. 3). All experiments on day 1 produced the highest cell viability, whereas day 7 generated the lowest cell viability when 3Mix, minocycline, and ciprofloxacin were tested (P < .001, Fig. 4). When comparing all the drugs, 3Mix had greater cytotoxicity than the other single antibiotics. Minocycline and ciprofloxacin at 25.00, 6.25, and 1.56 mg/mL generated less than 90% cell viability, whereas metronidazole at all concentrations had little effect on cell viability (Fig. 5). Antibacterial Efficacy of Antibiotics 3Mix at 25.00 mg/mL completely eradicated all isolated bacteria with a significant reduction of bacterial recovery (P < .01).
In the presence of 0.39 mg/mL 3Mix or a single drug at the concentration of 25.00 mg/mL, the bacterial number was reduced, but bacteria were not completely eliminated. However, bacterial recovery also significantly decreased compared with the control group (P < .01, Fig. 6).
Discussion This study showed that all drugs except metronidazole were cytotoxic to cultured human DPCs and APCs in a dose- and time-dependent manner. When comparing all drugs at the same concentration, 3Mix had greater cytotoxicity than the other single drugs; 25.00 mg/mL 3Mix produced moderate cytotoxicity with the highest bacteria reduction, whereas 0.39 mg/mL 3Mix had a less cytotoxic effect on cultured cells but showed less bacterial elimination. However, this concentration was able to significantly reduce the number of bacteria when compared with the control group. A 3-antibiotic combination has been used in treating immature teeth with necrotic pulp using the LSTR concept (10, 18). Many studies have reported the successful outcomes by which immature teeth showed continued root formation (13, 24). However, histologic studies regarding the regenerated tissue inside the necrotic human tooth are lacking. Recent histologic studies (20, 21) of dogs’ teeth treated with the LSTR technique revealed that the newly generated tissue was not pulp tissue. Periodontal tissue, including bone, was generally observed inside the treated tooth. Therefore, the question should be raised whether this treatment option is valid and how to prevent these consequences.
Figure 4. DPC and APC viability after being treated with 3Mix at 25.00, 6.25, and 1.56 mg/mL for 1, 3, 5, and 7 days. (A) DPCs and (B) APCs. 3Mix generated the highest percentage of cell viability on day 1, whereas the lowest percentage of cell viability was observed on day 7. DPC and APC viability was significantly reduced when the exposure time increased (P < .001). Data are mean standard deviation. A significant difference from *day 1, **day 3, and ***day 5.
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Figure 5. Cell viability after being treated with antibiotics at different concentrations for 7 days. (A) DPCs and (B) APCs. For both cell types, 3Mix produced more cytotoxicity compared with each single drug at all concentrations. Minocycline and ciprofloxacin at 25.00, 6.25, and 1.56 mg/mL generated less than 90% cell viability, whereas metronidazole at all concentrations had a small effect on cell viability. Data are mean standard deviation. *A significant difference from the control group (P < .001).
One interesting issue is whether excessive concentrations of 3Mix are used in the treatment. Hoshino et al (10) reported that 25.00 mg/mL 3Mix was able to eradicate all isolated bacteria. However, the exact concentration used in the clinic, especially in the pulp revascularization technique, has never been measured. It seems that excessive concentrations are currently being used, which may damage the remaining cells and impair tissue healing and regeneration. It is possible that the remaining vital pulp tissue and periradicular area, both containing stem cells (14–17), are damaged by the high concentration of 3Mix. Therefore, an expected differentiation pattern (eg, odontoblast differentiation and dentin formation) might not occur. In this study, DPCs or APCs were exposed to different concentrations of 3Mix. DPCs were selected for this study because these cells may survive in the infected tooth. Many published case reports revealed the observation of vital tissue in the tooth diagnosed as necrotic (14–17). Moreover, APCs were also tested because stem cells may still remain in the apical region, which may have some roles in pulp regeneration. From our study, we discovered that 25.00 mg/mL 3Mix produced moderate cytotoxicity. However, in a pilot study, concentrations JOE — Volume 39, Number 6, June 2013
higher than 25.00 mg/mL produced even greater toxicity, leading to total cell death (data not shown). Therefore, these results ensure that the concentration of 3Mix in general clinical usage is too strong, causing cytotoxicity on the remaining vital tissues. On the other hand, 3Mix at 0.39 mg/mL had a less cytotoxic effect on DPC and APC viability. This concentration is suggested to be the best candidate for pulpal regeneration along the concentrations of drugs tested in this experiment. Interestingly, recently published data concerning the cytotoxicity of 3Mix reported that 0.01–0.10 mg/mL did not have an effect on cultured stem cells from apical papilla (25). This result was different from our study, which showed that 0.39 mg/mL 3Mix was a safe dose. Also, some possible reasons might be the different source of antibiotics used in the experiment, the different techniques for 3Mix solution preparation, and the different method for cell viability measurement that may affect the results. It is important for clinicians to be aware that diverse sources of antibiotics have varying levels of toxicity; these antibiotics also have varying levels of effectiveness, which is an additional concern. More information and research into this area are needed.
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Figure 6. The antibacterial efficacy of 3Mix and each single antibiotic against bacteria isolated form necrotic teeth. 3Mix at 25.00 mg/mL could completely eradicate all isolated bacteria with a significant reduction of bacterial recovery (P < .01). In the presence of 0.39 mg/mL 3Mix or a single drug at the concentration of 25.00 mg/mL, the bacteria number was reduced but not completely eliminated. Data are mean standard deviation. *A significant difference from the control group (P < .01).
The cytotoxicity of 3Mix was investigated regarding its combination because 3 antibiotics were mixed. Therefore, each drug was also tested at the same concentration as 3Mix using an MTT assay. The result indicated that all drugs except metronidazole generated cytotoxicity. The cytotoxic effect was dose and time dependent. 3Mix had greater cytotoxicity than other single drugs. The greater cytotoxicity of 3Mix might result from a combination of the toxicities of each drug. However, the study was conducted in an in vitro environment, which may not represent the real clinical situation. No evidence has been published regarding the depth of 3Mix penetration in immature tooth whether it reaches the apical and surrounding tissue. Therefore, the clinical implication of these results should be further tested. One reason for its cytotoxicity might be the low pH (pH = 4.0–4.6) of 3Mix. Minocycline hydrochloride and ciprofloxacin hydrochloride are generally used for 3Mix preparation. The release of hydrogen ions from HCl groups resulted in an acidic condition that was an unfavorable condition for culturing cells (26). In addition, a low pH allowed more agents to remain soluble and available for uptake into the cells; thus, the retained drug was able to develop more cytotoxicity (27). Conversely, in this study, metronidazole did not inversely affect DPCs and APCs even at the 25.00-mg/mL concentration. It is possible that metronidazole solution has a neutral pH (pH = 6.84); thus, cytotoxicity did not occur. In contrast, this result differed from that of a previous study by Ferreira et al (28), who reported that only 5.00 mg/mL metronidazole resulted in 74% human gingival fibroblast cell line viability. However, many studies indicated that there is a variation in the degree of drug-induced cytotoxicity in different cell types (29, 30). Therefore, the sensitivity of DPCs or APCs and the gingival fibroblast cell line on drugs might be different. From this point of view, the cytotoxic effect of 3Mix from its acidic condition should be a concern. Other antibiotics that have a neutral pH should be selected as a substitute for minocycline and ciprofloxacin. However, those alternative antibiotics should intensely offer a potent antibacterial efficacy. The antibacterial efficacy of antibiotics used in vital pulp treatment, especially the LSTR concept, is important because bacteria remnant might affect the treatment outcome. Interestingly, 0.39 mg/mL 3Mix, which had a less cytotoxic effect on cultured cells, significantly reduced isolated bacteria compared with the control group. However, this effective concentration differed from Hoshino et al (10), who reported much higher concentrations (100–200 mg/mL) with antibacterial effects. The reasons might be the differences in the source of antibiotics used and the bacteria isolation techniques. When single antibiotics were tested at the 818
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same concentrations as 3Mix, it was found that even a 25.00-mg/mL concentration of a single drug alone was unable to eradicate all isolated bacteria. This might be because each drug is not active against all bacterial types. Minocycline and ciprofloxacin, which are broad-spectrum antibiotics, have an efficient action against some gram-positive and gram-negative bacteria (31, 32), whereas metronidazole is able to kill only anaerobic bacteria (33). Thus, the combination of 3 drugs provides a broader antibacterial spectrum than a single drug alone. However, balancing between the cytotoxic effect and antibacterial efficacy is important and still needs further study. However, there is concern whether the types of bacteria presented in necrotic immature teeth are the same as in necrotic mature teeth. In this study, only bacteria isolated from necrotic mature teeth were applied using a protocol adapted from Sassone et al (23). The dentin was scratched by means of a K-file, assuming that bacteria presented in dentin might be dispersed and collected after absorbing with a paper point. After incubation, collected bacteria samples were grossly clarified using gram staining. Main types of bacteria observed were similar to previously published data representing bacteria found in necrotic tooth (34). However, these bacterial samples may or may not completely represent the bacteria observed in immature necrotic teeth. Further studies are suggested because current evidence is lacking. The use of 3Mix has been popular for a decade. It offers many benefits in treating patients with infected teeth. However, the results from this study should stimulate a concern with 3Mix usage, especially in pulp revascularization, because using high doses of 3Mix produced cytotoxicity. The reduction of the 3Mix concentration into an appropriate dose may improve the outcome of pulp revascularization treatment because it causes less cytotoxicity and still has a potent antibacterial efficacy.
Conclusions 3Mix, minocycline, and ciprofloxacin were cytotoxic to cultured DPCs and APCs. 3Mix induced a higher toxicity than minocycline, ciprofloxacin, and metronidazole. The cytotoxicity of each antibiotic except metronidazole increased in a concentration- and time-dependent manner. The 0.39-mg/mL concentration of 3Mix had no cytotoxicity to DPCs and APCs, yet it was able to significantly reduce bacteria isolated from necrotic teeth.
Acknowledgments The authors deny any conflicts of interest related to this study. JOE — Volume 39, Number 6, June 2013
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18. Hoshino E. 3MIX-MP method-better and efficient clinical procedure of lesion sterilization and tissue repair (LSTR) therapy. Dent Rev 1998;666:57–106. 19. Chen MY, Chen KL, Chen CA, et al. Responses of immature permanent teeth with infected necrotic pulp tissue and apical periodontitis/abscess to revascularization procedures. Int Endod J 2012;45:294–305. 20. Wang X, Thibodeau B, Trope M, et al. Histologic characterization of regenerated tissues in canal space after the revitalization/revascularization procedure of immature dog teeth with apical periodontitis. J Endod 2010;36:56–63. 21. Yamauchi N, Nagaoka H, Yamauchi S, et al. Immunohistological characterization of newly formed tissues after regenerative procedure in immature dog teeth. J Endod 2011;37:1636–41. 22. Dahl JE, Frangou-Polyzois MJ, Polyzois GL. In vitro biocompatibility of denture relining materials. Gerodontology 2006;23:17–22. 23. Sassone LM, Fidel RA, Faveri M, et al. A microbiological profile of symptomatic teeth with primary endodontic infections. J Endod 2008;34:541–5. 24. Reynolds K, Johnson JD, Cohenca N. Pulp revascularization of necrotic bilateral bicuspids using a modified novel technique to eliminate potential coronal discolouration: a case report. Int Endod J 2009;42:84–92. 25. Ruparel NB, Teixeira FB, Ferraz CCR, et al. Direct effect of intracanal medicaments on survival of stem cells of the apical papilla. J Endod 2012;38:1372–5. 26. Kobayashi M, Kagawa T, Takano R, et al. Effect of medium pH on the cytotoxicity of hydrophilic statins. J Pharm Pharm Sci 2007;10:332–9. 27. Seay TM, Peretsman SJ, Dixon PS. Inhibition of human transitional cell carcinoma in vitro proliferation by fluoroquinolone antibiotics. J Urol 1996;155:757–62. 28. Ferreira MB, Myiagi S, Nogales CG, et al. Time- and concentration-dependent cytotoxicity of antibiotics used in endodontic therapy. J Appl Oral Sci 2010;18: 259–63. 29. Arenholt-Bindslev D, Bleeg H. Characterization of two types of human oral fibroblast with a potential application to cellular toxicity studies: tooth pulp fibroblasts and buccal mucosa fibroblasts. Int Endod J 1990;23:84–91. 30. Pissiotis E, Spangberg LS. Toxicity of Pulpispad using four different cell types. Int Endod J 1991;24:249–57. 31. Almazin SM, Dziak R, Andreana S, et al. The effect of doxycycline hyclate, chlorhexidinegluconate, and minocycline hydrochloride on osteoblastic proliferation and differentiation in vitro. J Periodontol 2009;80:999–1005. 32. Mohammadi Z. Systemic, prophylactic and local applications of antimicrobials in endondontic: and update review. Int Dent J 2009;59:178–86. 33. Soares GM, Figueiredo LC, Favevi M, et al. Mechanisms of action of systemic antibiotics used in periodontal treatment and mechanisms of bacterial resistance to these drugs. J Appl Oral Sci 2012;20:295–309. 34. Love RM, Jenkinson HF. Invasion of dentinal tubules by oral bacteria. Crit Rev Oral Biol Med 2002;13:171–83.
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