Stability of Doxycycline Absorbed on Root Canal Dentin After Obturation with Gutta-Percha/AH26 and Resilon/RealSeal at Different Time Intervals

Headnote

Abstract

Objective: Eradication of microorganisms present in the root canal system is paramount for the successful outcome of root canal therapy. The purpose of this study was to compare the of doxycycline absorbed from MTAD into root canal dentin after obturation with gutta-percha/AH26 and Resilon/RealSeal at different time intervals.

Materials and Methods: Fifty-one extracted human teeth were instrumented. Thirty samples were obturated with either gutta-percha/AH26 or Resilon/self-etch RealSeal after final irriga-tion with MTAD. Fifteen samples were kept unobturated (positive control); six samples were obturated with either gutta-percha/AH26 or Resilon/self-etch RealSeal without MTAD irriga-tion (negative control).

After aging for 1, 3 or 6 weeks, dentin debri were collected, the Doxycycline compound was extracted and its amount was quantified using high performance liquid chromatography. The statistical significance of the change in Doxycycline concentrations was tested with two-way ANOVA.

Results: The mean concentration of Doxycycline in dentin for one, three and six-week gutta-percha/AH26 samples was 1.8±0.36, 1.22±0.22 and 0.67±0.11 respectively, whereas these con-centrations in Resilon/RealSeal samples were 1.60±0.26, 0.80±0.14 and 0.59±0.01 respective-ly. Regarding the positive control group, these concentrations were 2.09±0.11, 1.54±0.12 and 0.72±0.07 respectively for 1, 3 and 6-week intervals. No Doxycycline was detected in negative control groups. The Doxycycline concentrations showed a significant difference forobturating materials (p=0.008). These concentrations were higher in the gutta-percha/AH26 samples than Resilon/RealSeal samples in each time interval.

Conclusion: The remaining amount of Doxycycline bonded to dentin was higher when root canals were obturated with gutta-percha/AH26 compared to Resilon/RealSeal. The stability of Doxycycline showed a time dependent decrease.

Key Words: MTAD; Substantivity; Real Seal; AH26 Sealer; Resilon Sealer

Journal of Dentistry, Tehran University of Medical Sciences, Tehran, Iran (2013; Vol. 10, No.4)

INTRODUCTION

The outcome of endodontic therapy is influ-enced by the presence of bacteria in the root canal system at the time of obturation [1]. One of the strategies for preventing bacterial reco-lonization or eliminating the remaining bacteria after endodontic treatment is to apply a fi-nal irrigating solution with substantivity [2].

MTAD is an aqueous solution consisting of 3% doxycycline, 4.25% citric acid, and 0.5% polysorbate 80 detergents [3]. It is effective in removing endodontic smear layers [3] with less erosion [4], it eliminates microbes that are resistant to common endodontic irrigants and dressings [5] such as E. faecalis [6], it pro-vides residual antimicrobial activity through the affinity of doxycycline towards dental hard tissues [7], and it subsequently releases with-out losing its antibacterial activity [8]. One study revealed that MTAD does not adversely affect the sealing ability of Resilon/Epiphany [9]. Its half-life in unobturated root canals has been reported to be 3.0 weeks (10). Some stu-dies have shown that doxycycline has a signif-icantly greater zone of inhibition than 6% NaOCl for P. micros, P. intermedia, and S. sanguis [11]. Resilon and RealSeal have been recently developed as an alternative root-filling material. RealSeal's second generation is an acidic self-adhesive sealer [12, 13]. Self-adhesives have the advantage of reducing er-rors that might occur during bonding steps be-cause they are less technique sensitive and re-quire fewer steps and less time [13]. No stu-dies have shown the changes in MTAD's sta-bility overtime. There is also no information about probable effects of obturation material on MTAD's stability. The present study was aimed to compare the stability of doxycycline absorbed into root canal dentin after obtura-tion with gutta-percha/AH26 or Resi-lon/Realseal after 1, 3 and 6-week time inter-vals.

MATHERIALS AND METHODS

Sample preparation

Fifty-one extracted human single rooted, sin-gle canal mandibular premolars with no ca-rious lesion that were extracted due to peri-odontal problems were collected. Straight and angulated radiographs were taken to confirm the root canal anatomy. Teeth with more than one canal or with calcified root canals were excluded. After disinfection and access cavity preparation, the root canals were instrumented with Protaper rotary instruments (Dentsply Maillefer, Ballaigues,Switzerland) in the fol-lowing sequence: S1, S2, F1 and F2. The root portion and apical foramen were covered with nail polish. Forty-five samples were selected and irrigated with MTAD (Dentsply Tulsa, Tulsa, OK) according to the manufacturer's instructions: a 2-minute rinse with 3 ml of 1.3% NaOCl, a brief rinse with 1 ml of MTAD leftfor 5 minutes, and finally a 1-minute flush with 4 ml of MTAD. After drying all canals with paper points (Ariadent, Iran), 30 of the samples were randomly divided into two groups: A, obturation with gutta-percha (Ga-padent Co, Ltd, Korea)/AH26 (Dentsply, De-Trey, Germany); B: obturation with Resi-lon/self-etch RealSeal (SybronEndo, USA). Fiftenn samples were kept unobturated (posi-tive control); six samples were obturated with either gutta-percha/AH26 or Resilon/self-etch RealSeal (three samples for each group) with-out MTAD irrigation (negative control). After obturation, all teeth were restored with Colto-sol (AriaDent, Iran). The samples were coded and placed in 37 degrees centigrade and 100% humidity. Then experimental samples in all groups were divided into three subgroups ac-cording to the tested time intervals: 1 week, 3 and 6 weeks. After aging, samples were split in half by a high speed diamond bur, obtura-tion material was taken out with a spatula and dentin debris were collected in coded eppen-dorf tubes from 2 mm below the CEJ from both sides of the root canal by using a low speed round #7 bur to a width and depth equivalent to the bur's radius and a length of 3 mm (Fig 1).

Recovery of active compound

0.02 gr of dentinal debris from each sample was measured by a digital Analytic Balance (Sartorius, Germany) and transferred to anoth-er coded eppendorf tube.

The recovery solution for the samples was 5% pH 7.5 EDTA (Merck, Germany), after adding 0.9 mL of the solution, the debris were vigo-rously shaken with Vortex mixer (Labnet in-ternational,USA) then 0.3 ml of 0.4% per-chloric acid (Merck,Germany) in acetonitrile (Merck, Germany) was added to each sample. All samples were syringe-filtered with 13 mm 0.22 mm PTFE filters (MS®, USA) before HPLC analysis.

Quantification of doxycycline

Doxycycline was quantitatively analyzed ac-cording to the method described by Rasimick et al. [10] with some modifications. The sepa-ration was performed by High Performance Liquid Chromatography (HPLC) system (Wa-ters Chromatography Division, Milford, MA, USA) with a C-8 column (Lichrospher 100RP8EC 5µm 250.46, Teknokroma, Spain). The protocol consisted of isocratic system with mobile phase including 49.5:49.5:0.5 (volume) water: acetonitrile: THF, respective-ly that was neutralized with NaOH to pH=2.5.

The flow rate was 1 ml/min for 20 min and injection volume was 20 µL. Detection was carried out at a wavelength of 350 nm.

Linearity in the range of 0-60 µg/mL doxycyc-line was confirmed by using known dilutions of standard doxycycline hyclate (Merck, Ger-many) in mobile phase.

The limit of detection (LOD), S/N =3.3, and the limit of quantitation (LOQ), S/N=10, were 1.49 and 4.52 µg/ml, respectively.

The statistical significance of the change in doxycycline concentrations was tested with two-way ANOVA. (The interaction effect of the obturation material and time was insignifi-cant; p=0.25). Post hoc analysis for pairwise comparison between time intervals was done by Tukey test. Correlation between time inter-vals and docycycline concentrations was cal-culated by Spearman correlation test. P-values less than 0.05 were considered as significant.

RESULTS

The comparative chromatograms of the ex-tracted doxycycline in samples are illustrated in figure 2. The peak at the retention time of 3.8 min is contributed to oxytetracycline com-pared to the one in the standard chromato-gram. The concentration of doxycycline in liquid is the concentration calculated based on the area under the curve from the recovered samples obtained from 0.02gr of dentinal debris.

The concentration of doxycycline in dentin is its concentration in 1 gr dentin that was calcu-lated by mathematical proportion from the concentration in liquid.

The mean concentration of doxycycline in dentin for one, three and six-week gutta per-cha/AH26 samples was 1.8±0.36, 1.22±0.22 and 0.67±0.11, respectively, whereas these concentrations in Resilon/RealSeal samples were 1.60±0.26, 0.80±0.14 and 0.59±0.01, re-spectively.

These concentrations were higher in the gutta-percha/AH26 samples than Resilon/RealSeal samples in each time interval.

A significant difference was seen between doxycycline concentrations in different time intervals regardless of the type of obturating material (p<0.001).

Regarding the positive control group, these concentrations were 2.09±0.11, 1.54±0.12 and 0.72±0.07, respectively for 1, 3 and 6-week intervals.No doxycycline was detected in the negative control groups.

The statistical analysis showed there was a significant difference for doxycycline concen-trations between different obturating materials (p=0.008). No significant difference was seen between the doxycycline concentrations in the positive control group and gutta-percha/AH26 samples in each time interval (p>0.05), but a significant difference was seen between this concentration in the positive control and Resi-lon/RealSeal samples in each time interval (p<0.05). All groups showed a time dependent decrease in doxycycline concentrations (Spearman's correlation coefficient=0.91, p<0.001).

The highest concentration of doxycycline was found in the one-week positive control sam-ples and then in the one-week gutta per-cha/AH26 samples and the lowest concentra-tion was found in six-week Resilon/RealSeal samples.

The mean concentration of doxycycline in dentin for one, three and six-week gutta per-cha/AH26 samples was 1.8±0.36, 1.22±0.22 and 0.67±0.11, respectively, whereas these concentration in Resilon/RealSeal samples were 1.60±0.26, 0.80±0.14 and 0.59±0.01, re-spectively.

Regarding the positive control group, these concentrations were 2.09±0.11, 1.54±0.12 and 0.72±0.07, respectively for 1, 3 and 6-week intervals.

No doxycycline was detected in the negative control groups.

The statistical analysis showed there was a significant difference for doxycycline concen-trations between different obturating materials (p=0.008).

These concentrations were higher in the gutta-percha/AH26 samples than Resilon/RealSeal samples in each time interval.

A significant difference was seen between doxycycline concentrations in different time intervals regardless of the type of obturating material (p<0.001). No significant difference was seen between the doxycycline concentra-tions in the positive control group and gutta-percha/AH26 samples in each time interval (p>0.05), but a significant difference was seen between this concentration in the positive con-trol and Resilon/RealSeal samples in each time interval (p<0.05). All groups showed a time dependent decrease in doxycycline con-centrations (Spearman's correlation coeffi-cient=0.91, p<0.001). The highest concentra-tion of doxycycline was found in the one-week positive control samples and then in the one-week gutta percha/AH26 samples and the lowest concentration was found in six-week Resilon/RealSeal samples.

The mean concentrations of doxycycline found in our experimental groups are shown in Table 1.

DISCUSSION

Various methods for determination of dox-ycycline in-vitro and in-vivo have been re-ported such as microbiology, fluorimetry, TLC-fluorescence scanning densitometry, spectrophotometry, sequential injection chro-matography (SIC) and HPLC [14].

High-performance liquid chromatography (HPLC) is a chromatographic technique used to separate a mixture of compounds with the purpose of identifying and quantifying the in-dividual components of the mixture [15, 16]. Hplc separation is accomplished by means of sample mixture interacts with solid particles within the hplc column [15-17].

This technique offers several advantages over other techniques, including minimal sample manipulation before chromatography, rapid analysis and the simultaneous analysis of mul-tiple compounds with good specificity, preci-sion and accuracy [14].

Portenier et al [18] reported that the minimum concentration of doxycycline that provided a bactericidal effect against E. faecalis in the presence of dentin within 24 hours of contact, was 0.3% (3mg/ml). The measured concentra-tion of doxycycline in the dentin in both groups of our study was lower. On the other hand, Newberry et al [19] reported that MTAD killed most strains of E. faecalis when diluted to 1:512. Upon dilution of MTAD to 1:512 the concentration of doxycycline would be 0.05mg; therefore, according to the results of the present study, the concentration of dox-ycycline in root canal dentin within 6 weeks might be able to kill most strains of E. faecalis which deserves further studies.

The decomposition and diffusion rate of the antimicrobial compounds is likely a function of pH [10]. It has been reported that acidic so-lutions and environments result in the dehy-dration of tetracycline [20, 21]. Acidic sealers like Epiphany SE might substantially affect the local pH of dentin and hence, the stability of doxycycline [10]. This might be one of the reasons for the lower concentration of dox-ycycline found in Resilon/RealSeal groups in the present study.

The bond strength of AH26 to dentin is related to the formation of covalent bonds between epoxide rings and exposed amino groups in the collagen network [22]. Doxycycline also has an amino group in its molecular structure [23]; Therefore, AH26 might be able to form covalent bonds with doxycycline too.

Although there is no study that documented the aforementioned reaction, in theory, the higher concentration of doxycycline in gutta-percha/AH26 samples might be due to this bonding. Further studies in this regard are rec-ommended.

Rasimick et al. [10] analyzed the doxycycline concentration in teeth that were not obturated and showed that its concentration decreased over a 9-week period of time, which was con-sistent with our results.

The primary and most important factor in de-termining the long term success of an endo-dontic treatment is the presence and/or persis-tence of microorganisms [24]. There is strong evidence that all microorganisms cannot be removed after chemo-mechanical preparation of the root canal system [25]. However, it is possible to minimize the amount of bacteria present in the root canal [26]. Maintaining a good coronal seal after completion of obtura-tion is also important to minimize bacterial colonization [27]. Microorganisms may pene-trate into the root canal system via temporary restorations and any defects in the coronal tooth structure. Furthermore, reinfection of the root canal system can occur because of re-growth of residual microorganisms that have survived the endodontic treatment procedures [28]. A disinfection protocol that provides substantivity might improve the outcome of endodontic treatment [29] and optimize single visit disinfection [30]. The long term antibac-terial efficacy of absorbed doxycycline in den-tinal walls and its effects on success/failure of the treatment is not determined yet.

The possibility and mechanisms of leakage in root canals obturated with gutta-percha/AH26 [31-33] and Resilon/RealSeal [13, 31, 33] has been well documented. Because of the poten-tial of leakage after obturation with either of these materials it might be beneficial to use irrigants with antimicrobial substantivity. Ac-cording to outcomes of the present study, after using MTAD as the final irrigant, root canal obturation with non-acidic sealers like AH26 might be better in terms of stability of dox-ycycline.

Although the present study showed that the stability of doxycycline following MTAD irri-gation decreased more in the presence of Resi-lon/RealSeal in the root canal space, the clini-cal relevance of the issue is still unclear. Be-sides, the stability of doxycycline showed a time dependent decrease that raises questions about the long-term antibacterial effectiveness of MTAD. Therefore, randomized clinical tri-als on the effects of MTAD on success/failure of the endodontic treatments are recommend-ed.

CONCLUSION

The outcomes of this study showed that the stability of doxycycline following irrigation with MTAD was lower when the root canals were obturated with Resilon/RealSeal com-pared to gutta-percha/AH26. The stability of doxycycline showed a time dependent de-crease.

ACKNOWLEDGMENTS

This study was supported by a grant from Te-hran University of Medical Sciences and Health Services (grant no. 15658).

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