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Tetracycline HCl Solution as a Root Canal Irrigant
Printed in U.S.A. VOL. 27, No. 12, DECEMBER 2001
JOURNAL OF ENDOWNTICS Copyright 0 2001 by The American Association of Endodontists
Tetracycline HCI Solution as a Root Canal lrrigant Faruk Haznedaroglu DDS, PhD, and Handan Ersev, DDS, PhD
access cavities were prepared, and pulp tissues were removed by means of barbed broaches. A size 10 K-type file (Thomas, Bourges, France) was penetrated through the root canal until it could be seen from the apical foramen, and 1 mm short of this length was accepted as the working length. Root canals were mechanically prepared with step-back technique using K- type files and Gates-Glidden burs to size 45 at the working length. During instrumentation 2 ml of an irrigant was used after each file, delivered by a 30-gauge needle placed as far apically as possible into the canal without binding. Five teeth were irrigated with bidistilled water as group 1 (negative control group), and the remaining 15 teeth were irrigated with a solution of 2.5% sodium hypochlorite (NaOCl), as groups 2 to 4. Throughout the instrumentation procedure the canals were kept flooded with the irrigant. On completion of instrumentation apical and coronal thirds of the roots were removed with diamond discs under continuous water coolant. The rest of the study was conducted on the middle thirds of the roots. Longitudinal grooves were made on the buccal and palatal surfaces of the root segments without penetrating the canal. The roots were then split into two halves with a hammer and a chisel. Ten root halves were obtained for each group. In all groups the split root halves were placed in separate 10-ml vials containing the corresponding irrigant and treated under constant agitation by shaking on a rocking plate. The specimens were treated with bidistilled water in group 1 and with 2.5% NaOCl in group 2 for 2 min, and the solutions were changed after the first minute. Groups 3 and 4 were first treated with 50% citric acid or tetracycline HCl, respectively, for 1 min and then with 2.5% NaOCl for another minute. Finally the specimens in all groups were rinsed with bidistilled water for 1 min. After treatment all samples were washed, fixed, and dehydrated in an ascending series of alcohol. Then they were mounted on aluminium stubs, vacuum-dried, coated with palladium-gold, and examined under SEM (JFiOL JSM T-330, JEOL Co., Tokyo, Japan) at 20 kV. Photomicrographs of representative areas were taken at magnifications of X350, X1000, X 1500, and X5000, coded, and qualitatively evaluated by two investigators who rated the degree of cleanliness on a scale of 0 to 2 (score 0 = no smear layer, dentinal tubules are open and free of debris; score 1 = partial removal of smear layer, some dentinal tubules are open; score 2 = intact smear layer, all of the dentinal tubules are obliterated or single tubules are open) (10). Data were statistically analyzed using the nonparametric Mann-Whitney U test.
The effect of tetracycline hydrochloride as an endodontic irrigant on smear layer removal was examined by scanning electron microscopy and compared with bidistilled water, 2.5% sodium hypochlorite (NaOCI), and 50% citric acid in 20 extracted teeth. Five teeth, as a control, were irrigated with bidistilled water only in group 1. All the other groups were irrigated with NaOCl during instrumentation. The final rinses were NaOCl in group 2, citric acid in group 3, and 1% tetracycline hydrochloride in group 4. Scanning electron microscopic results and statistical analysis revealedthat bidistilled water and NaOCl were ineffective in removing the smear layer, whereas citric acid and tetracycline hydrochloridewere significantly more effective (p c 0.001). No statistically significant difference was detected between tetracycline hydrochloride and citric acid (p > 0.05). However tetracycline hydrochloride demineralized less peritubular dentin.
In 1975 McComb and Smith (1) were the first to describe the smear layer in instrumented root canals by scanning electron microscopy (SEM). It has been shown that complete removal of the smear layer requires both organic and inorganic solvents used alternately (2,3). Surface demineralization of radicular dentin removes the smear layer (4),formed as a result of scaling and root planing procedures (4,5) and enhances regeneration of the lost periodontal attachment during periodontal therapy (6). Tetracycline was one of the agents proposed for the demineralization procedure (7-9). Tetracycline hydrochloride (HCl), an acidic solution with bacteriostatic properties, has been considered to be suitable for endodontic irrigation. Therefore the aim of the present investigation was to evaluate the effect of tetracycline HC1 as a root canal irrigant on removal of debris and smear layer from instrumented root canals.
MATERIALS AND METHODS A total of 20 recently extracted human maxillary canine teeth with fully developed apices were included in this study. Standard 738
Vol. 27, No. 12, December 2001
FIG. 1. Specimen treated with 2.5% NaOCI. At high magnification dentinal tubules are covered with the smear layer. (Original magnification x5000.)
FIG. 2. Specimen treated with 50% citric acid. High magnification shows that peritubular dentin is totally removed and dentinal tubules are wide open. (Original magnification ~5000.)
Tetracycline HCI as Endodontic lrrigant
739
FIG. 3. Specimentreated with 1% tetracycline HCI. High-power view of the surface shows that the smear layer is completely removed, and the dentinal tubule apertures are slightly enlarged. (Original magnification x5000.)
Group 4,treated with tetracycline HCl, presented an appearance similar to that seen in group 3. No smear layer was present, and the surface was found to be free of debris. The apertures of the dentinal tubules were enlarged. However, in contrast to the results obtained from the 50% citric acid treatment, the tubule apertures were not widened extensively and peritubular dentin was not destroyed as much in the tetracycline HC1-treated group (Fig. 3). In groups 3 and 4 the score obtained for all of the samples was 0, except one sample in group 3 and two samples in group 4 with a score of 1. Statistical analysis revealed that bidistilled water (negative control) and 2.5% NaOCl were not effective in removing the smear layer, and the difference between these two imgants was not statistically significant (p > 0.05). Citric acid (50%) and tetracycline HCl(l%) completely removed the smear layer from dentinal walls, and no statistically significant difference was detected between them (p > 0.05). Citric acid (50%) and tetracycline HCI (1%) were found to be significantly more effective on smear layer removal than bidistilled water and 2.5% NaOCl (p < 0.001).
DISCUSSION
RESULTS In group 1, in which bidistilled water was used as the imgant, dentinal walls were thoroughly covered by smear layer and residual pulpal tissue was observed. Even at high power no dentinal tubules could be detected. Typical appearance of the smear layer could be seen on surface of these specimens. In group 2, treated with 2.5% NaOCl, there was practically no superficial debris present on the dentinal walls, but the smear layer was still present. Dentinal tubules seemed to be plugged with the layer, and no patent dentinal tubule could be seen (Fig. 1). In groups 1 and 2 the score obtained for all of the samples was 2. In group 3, the citric acid group, both low- and high-power magnification revealed that the smear layer was completely removed by the 50% citric acid treatment, and dentinal tubule apertures were widened. Moreover peritubular dentin around enlarged tubular apertures was totally removed by the treatment (Fig. 2). and some calcium citrate crystal formations on the dentinal walls were noted.
This study was conducted on split root halves rather than root canals treated with traditional final irrigation to determine the maximum cleaning effect of the imgants and to eliminate morphological variations that can reduce the efficacy of the irrigation solutions. Furthermore, to enhance the overall cleaning effect, the jet effect produced by delivery through a needle in clinical conditions was simulated by agitating the imgating solution in the vials. Bidistilled water and 2.5% NaOCl, when used alone during and after instrumentation were found to be ineffective on smear layer removal, in agreement with the results of many other investigators (2, 3, 11-13). GutiCrrez et al. (14) have demonstrated that EDTA used during the mechanical preparation of the root canals favors the diffusion of microorganisms within the dentinal tubules and have noted that complete removal of smear layer with EDTA increases the risk of rapid decay formation, if the coronal dentin of the endodontically treated tooth is accidentally exposed to the oral fluid (15). In addition, according to $en et a1.(16), once the smear layer is
740
Journal of Endodontics
Haznedaroglu and Ersev
removed, there is always a risk of dentinal tubule reinfection, if the seal fails. SEM studies have revealed that chelating agents or organic acids not only eliminate this layer, but also remove the peritubular dentin (3, 13) subsequent to a marked demineralization of the dentin surface (1 1, 13) and soften the root dentin (13). Citric acid (50%),an organic acid, has been shown to cause an extensive demineralization of dentin ( 11, 17), and to open ( 11, 17) and widen the tubular apertures by removing almost the entire peritubular dentin (17). Therefore in our study the use of 50% citric acid was considered to be appropriate to compare the extent of demineralization caused by tetracycline HCI. These solutions were applied to the split root halves of the same tooth, respectively, to better determine their relative efficacy and also to minimize the structural variations of the sample teeth. In specimens treated with 50% citric acid the smear layer was completely removed. This was essentially in agreement with published evidence (2, 11, 17). The finding of widened apertures of dentinal tubules due to the use of citric acid was also confirmed in our investigation (8, 17). Furthermore, as described by Yamada et a1.(2), calcium citrate crystal formation resulting from citric acid treatment was observed in some cases. SEM evaluation revealed that application of tetracycline HCl resulted in complete removal of the smear layer, and the effect of tetracycline on smear layer and root dentin was found to be comparable with that observed using 50% citric acid. Labahn et al. (8) have examined the effects of tetracycline HCI and citric acid on root dentin surface during periodontal treatment and found that both removed the smear layer. However citric acid caused more extensive peritubular dentin demineralization and greater degrees of morphological alteration in root dentin than tetracycline HCl. In the present study tetracycline HCI was also found to demineralize less peritubular dentin than citric acid. Tetracyclines, broad-spectrum antimicrobials (S), are reported to bind directly to demineralized dentinal surfaces (9) and maintain their antimicrobial activity by being subsequently released (7). However it is also well known that the tetracycline group of drugs can chelate with calcium (8, 18) and, as one side effect, cause staining of teeth (18). Therefore how using this drug group with the aforementioned properties in endodontic therapy relates to clinical situations remains to be investigated. We thank Professor Dr. Adnan Tekin and Huseyin Sezer, Istanbul Technical University, for providing technical support; Dr. Bilge Hakan $en, Ege University, for kindly reviewing the article; and Dr. Pembe Cagatay, Istanbul University, for her help with statistical analysis.
Dr. Haznedaroglu is an associate professor and Dr. Ersev is a research assistant, Department of Endodontics, Faculty of Dentistry, Istanbul University, Istanbul, Turkey. Address requests for reprints to Dr. Haznedaroglu, Department of Endodontics, Faculty of Dentistry, Istanbul University, Gapa, 34390 Istanbul, Turkey.
References 1. McComb D, Smith DC. A preliminary scanning electron microscopic study of root canals after endodontic procedures. J Endodon 1975;1:238-42. 2. Yamada RS, Armas A, Goldman M, Lin PS. A scanning electron microscopic comparison of a high volume final flush with several irrigating solutions: part 111. J Endodon 1983;9:137-42. 3. Karagoz-Kupukay I, Bayirli G. An apical leakage study in the presence and absence of the smear layer. Int Endod J 1994;27:87-93. 4. Hanes PJ, Polson AM, Frederick GT. Root and pulpal dentin after surface demineralization. Endod Dent Traumatol 1986;2:190-5. 5. Polson AM, Frederick GT, Ladenheim S, Hanes PJ. The production of a root surface smear layer by instrumentation and its removal by citric acid. J Periodontol 1984;55:443-6. 6. Register AA. Bone and cementum induction by dentin, demineralized in situ. J Periodontol 1973;44:49-54. 7. Wikesjo UME, Baker PJ, Christersson LA, Genco RJ, Lyall RM, Hic S, DiFlorio RM, Terranova VP. A biochemical approach to periodontal regeneration: tetracycline treatment conditions dentin surfaces. J Periodontol Res 1986;21:322-9. 8. Labahn R, Fahrenbach WH, Clark SM, Lie T, Adams DF. Root dentin morphology after different modes of citric acid and tetracycline hydrochloride conditioning. J Periodontol 1992;63:303-9. 9. Bjortvan K, Skaug N, Selvig KA. Tetracycline-impregnated enamel and dentin: duration of anti-microbial capacity. Scand J Dent Res 1985; 931192-7. 10. Abbott P, Heijkoop PS, Cardaci SC, Hume WR, Heithersay GS. A SEM study of the effects of different irrigation sequences and ultrasonic. Int Endod J 1991;24:308-16. 11. Wayman BE, Kopp VM, Pinero GJ, Lazzari EP. Citric and lactic acids as root canal irrigants in vitro. J Endodon 19793258-65. 12. Goldman LB, Goldman M, Kronman JH, Lin PS. The efficacy of several irrigating solutions for endodontics: a scanning electron microscopic study. Oral Surg Oral Med Oral Pathol 1981;52:197-204. 13. Garberoglio R, Becce C. Smear layer removal by root canal irrigants: a comparative scanning electron microscopic study. Oral Surg Oral Med Oral Pathol 1994;78:359-67. 14. Gutierrez JH, Villena F, Jofre A, Amin M. Bacterial infiltration of dentin as influenced by proprietary chelating agents. J Endodon 1982;8: 448-54. 15. Gutierrez JH, Herrera VR, Berg EH, Villena F, Jofre A. The risk of intentionaldissolutionof the smear layer after mechanical preparation of root canals. Oral Surg Oral Med Oral Pathol 1990;70:96-108. 16. $en BH, Wesselink PR, Turkun M. The smear layer: a phenomenon in root canal therapy. Int Endod J 1995;28:141-8. 17. Brannstrom M, Johnson G. Effects of various conditioners and cleaning agents on prepared dentin surfaces: a scanning electron microscopic evaluation. J Prosthet Dent 1974;31:422-30. 18. Bridges JB, Owens PDA, Stewart DJ. Tetracyclines and teeth: an experimental investigation into five types in the rat. Br Dent J 1969;1:30611.
JOURNAL OF ENDOWNTICS Copyright 0 2001 by The American Association of Endodontists
Tetracycline HCI Solution as a Root Canal lrrigant Faruk Haznedaroglu DDS, PhD, and Handan Ersev, DDS, PhD
access cavities were prepared, and pulp tissues were removed by means of barbed broaches. A size 10 K-type file (Thomas, Bourges, France) was penetrated through the root canal until it could be seen from the apical foramen, and 1 mm short of this length was accepted as the working length. Root canals were mechanically prepared with step-back technique using K- type files and Gates-Glidden burs to size 45 at the working length. During instrumentation 2 ml of an irrigant was used after each file, delivered by a 30-gauge needle placed as far apically as possible into the canal without binding. Five teeth were irrigated with bidistilled water as group 1 (negative control group), and the remaining 15 teeth were irrigated with a solution of 2.5% sodium hypochlorite (NaOCl), as groups 2 to 4. Throughout the instrumentation procedure the canals were kept flooded with the irrigant. On completion of instrumentation apical and coronal thirds of the roots were removed with diamond discs under continuous water coolant. The rest of the study was conducted on the middle thirds of the roots. Longitudinal grooves were made on the buccal and palatal surfaces of the root segments without penetrating the canal. The roots were then split into two halves with a hammer and a chisel. Ten root halves were obtained for each group. In all groups the split root halves were placed in separate 10-ml vials containing the corresponding irrigant and treated under constant agitation by shaking on a rocking plate. The specimens were treated with bidistilled water in group 1 and with 2.5% NaOCl in group 2 for 2 min, and the solutions were changed after the first minute. Groups 3 and 4 were first treated with 50% citric acid or tetracycline HCl, respectively, for 1 min and then with 2.5% NaOCl for another minute. Finally the specimens in all groups were rinsed with bidistilled water for 1 min. After treatment all samples were washed, fixed, and dehydrated in an ascending series of alcohol. Then they were mounted on aluminium stubs, vacuum-dried, coated with palladium-gold, and examined under SEM (JFiOL JSM T-330, JEOL Co., Tokyo, Japan) at 20 kV. Photomicrographs of representative areas were taken at magnifications of X350, X1000, X 1500, and X5000, coded, and qualitatively evaluated by two investigators who rated the degree of cleanliness on a scale of 0 to 2 (score 0 = no smear layer, dentinal tubules are open and free of debris; score 1 = partial removal of smear layer, some dentinal tubules are open; score 2 = intact smear layer, all of the dentinal tubules are obliterated or single tubules are open) (10). Data were statistically analyzed using the nonparametric Mann-Whitney U test.
The effect of tetracycline hydrochloride as an endodontic irrigant on smear layer removal was examined by scanning electron microscopy and compared with bidistilled water, 2.5% sodium hypochlorite (NaOCI), and 50% citric acid in 20 extracted teeth. Five teeth, as a control, were irrigated with bidistilled water only in group 1. All the other groups were irrigated with NaOCl during instrumentation. The final rinses were NaOCl in group 2, citric acid in group 3, and 1% tetracycline hydrochloride in group 4. Scanning electron microscopic results and statistical analysis revealedthat bidistilled water and NaOCl were ineffective in removing the smear layer, whereas citric acid and tetracycline hydrochloridewere significantly more effective (p c 0.001). No statistically significant difference was detected between tetracycline hydrochloride and citric acid (p > 0.05). However tetracycline hydrochloride demineralized less peritubular dentin.
In 1975 McComb and Smith (1) were the first to describe the smear layer in instrumented root canals by scanning electron microscopy (SEM). It has been shown that complete removal of the smear layer requires both organic and inorganic solvents used alternately (2,3). Surface demineralization of radicular dentin removes the smear layer (4),formed as a result of scaling and root planing procedures (4,5) and enhances regeneration of the lost periodontal attachment during periodontal therapy (6). Tetracycline was one of the agents proposed for the demineralization procedure (7-9). Tetracycline hydrochloride (HCl), an acidic solution with bacteriostatic properties, has been considered to be suitable for endodontic irrigation. Therefore the aim of the present investigation was to evaluate the effect of tetracycline HC1 as a root canal irrigant on removal of debris and smear layer from instrumented root canals.
MATERIALS AND METHODS A total of 20 recently extracted human maxillary canine teeth with fully developed apices were included in this study. Standard 738
Vol. 27, No. 12, December 2001
FIG. 1. Specimen treated with 2.5% NaOCI. At high magnification dentinal tubules are covered with the smear layer. (Original magnification x5000.)
FIG. 2. Specimen treated with 50% citric acid. High magnification shows that peritubular dentin is totally removed and dentinal tubules are wide open. (Original magnification ~5000.)
Tetracycline HCI as Endodontic lrrigant
739
FIG. 3. Specimentreated with 1% tetracycline HCI. High-power view of the surface shows that the smear layer is completely removed, and the dentinal tubule apertures are slightly enlarged. (Original magnification x5000.)
Group 4,treated with tetracycline HCl, presented an appearance similar to that seen in group 3. No smear layer was present, and the surface was found to be free of debris. The apertures of the dentinal tubules were enlarged. However, in contrast to the results obtained from the 50% citric acid treatment, the tubule apertures were not widened extensively and peritubular dentin was not destroyed as much in the tetracycline HC1-treated group (Fig. 3). In groups 3 and 4 the score obtained for all of the samples was 0, except one sample in group 3 and two samples in group 4 with a score of 1. Statistical analysis revealed that bidistilled water (negative control) and 2.5% NaOCl were not effective in removing the smear layer, and the difference between these two imgants was not statistically significant (p > 0.05). Citric acid (50%) and tetracycline HCl(l%) completely removed the smear layer from dentinal walls, and no statistically significant difference was detected between them (p > 0.05). Citric acid (50%) and tetracycline HCI (1%) were found to be significantly more effective on smear layer removal than bidistilled water and 2.5% NaOCl (p < 0.001).
DISCUSSION
RESULTS In group 1, in which bidistilled water was used as the imgant, dentinal walls were thoroughly covered by smear layer and residual pulpal tissue was observed. Even at high power no dentinal tubules could be detected. Typical appearance of the smear layer could be seen on surface of these specimens. In group 2, treated with 2.5% NaOCl, there was practically no superficial debris present on the dentinal walls, but the smear layer was still present. Dentinal tubules seemed to be plugged with the layer, and no patent dentinal tubule could be seen (Fig. 1). In groups 1 and 2 the score obtained for all of the samples was 2. In group 3, the citric acid group, both low- and high-power magnification revealed that the smear layer was completely removed by the 50% citric acid treatment, and dentinal tubule apertures were widened. Moreover peritubular dentin around enlarged tubular apertures was totally removed by the treatment (Fig. 2). and some calcium citrate crystal formations on the dentinal walls were noted.
This study was conducted on split root halves rather than root canals treated with traditional final irrigation to determine the maximum cleaning effect of the imgants and to eliminate morphological variations that can reduce the efficacy of the irrigation solutions. Furthermore, to enhance the overall cleaning effect, the jet effect produced by delivery through a needle in clinical conditions was simulated by agitating the imgating solution in the vials. Bidistilled water and 2.5% NaOCl, when used alone during and after instrumentation were found to be ineffective on smear layer removal, in agreement with the results of many other investigators (2, 3, 11-13). GutiCrrez et al. (14) have demonstrated that EDTA used during the mechanical preparation of the root canals favors the diffusion of microorganisms within the dentinal tubules and have noted that complete removal of smear layer with EDTA increases the risk of rapid decay formation, if the coronal dentin of the endodontically treated tooth is accidentally exposed to the oral fluid (15). In addition, according to $en et a1.(16), once the smear layer is
740
Journal of Endodontics
Haznedaroglu and Ersev
removed, there is always a risk of dentinal tubule reinfection, if the seal fails. SEM studies have revealed that chelating agents or organic acids not only eliminate this layer, but also remove the peritubular dentin (3, 13) subsequent to a marked demineralization of the dentin surface (1 1, 13) and soften the root dentin (13). Citric acid (50%),an organic acid, has been shown to cause an extensive demineralization of dentin ( 11, 17), and to open ( 11, 17) and widen the tubular apertures by removing almost the entire peritubular dentin (17). Therefore in our study the use of 50% citric acid was considered to be appropriate to compare the extent of demineralization caused by tetracycline HCI. These solutions were applied to the split root halves of the same tooth, respectively, to better determine their relative efficacy and also to minimize the structural variations of the sample teeth. In specimens treated with 50% citric acid the smear layer was completely removed. This was essentially in agreement with published evidence (2, 11, 17). The finding of widened apertures of dentinal tubules due to the use of citric acid was also confirmed in our investigation (8, 17). Furthermore, as described by Yamada et a1.(2), calcium citrate crystal formation resulting from citric acid treatment was observed in some cases. SEM evaluation revealed that application of tetracycline HCl resulted in complete removal of the smear layer, and the effect of tetracycline on smear layer and root dentin was found to be comparable with that observed using 50% citric acid. Labahn et al. (8) have examined the effects of tetracycline HCI and citric acid on root dentin surface during periodontal treatment and found that both removed the smear layer. However citric acid caused more extensive peritubular dentin demineralization and greater degrees of morphological alteration in root dentin than tetracycline HCl. In the present study tetracycline HCI was also found to demineralize less peritubular dentin than citric acid. Tetracyclines, broad-spectrum antimicrobials (S), are reported to bind directly to demineralized dentinal surfaces (9) and maintain their antimicrobial activity by being subsequently released (7). However it is also well known that the tetracycline group of drugs can chelate with calcium (8, 18) and, as one side effect, cause staining of teeth (18). Therefore how using this drug group with the aforementioned properties in endodontic therapy relates to clinical situations remains to be investigated. We thank Professor Dr. Adnan Tekin and Huseyin Sezer, Istanbul Technical University, for providing technical support; Dr. Bilge Hakan $en, Ege University, for kindly reviewing the article; and Dr. Pembe Cagatay, Istanbul University, for her help with statistical analysis.
Dr. Haznedaroglu is an associate professor and Dr. Ersev is a research assistant, Department of Endodontics, Faculty of Dentistry, Istanbul University, Istanbul, Turkey. Address requests for reprints to Dr. Haznedaroglu, Department of Endodontics, Faculty of Dentistry, Istanbul University, Gapa, 34390 Istanbul, Turkey.
References 1. McComb D, Smith DC. A preliminary scanning electron microscopic study of root canals after endodontic procedures. J Endodon 1975;1:238-42. 2. Yamada RS, Armas A, Goldman M, Lin PS. A scanning electron microscopic comparison of a high volume final flush with several irrigating solutions: part 111. J Endodon 1983;9:137-42. 3. Karagoz-Kupukay I, Bayirli G. An apical leakage study in the presence and absence of the smear layer. Int Endod J 1994;27:87-93. 4. Hanes PJ, Polson AM, Frederick GT. Root and pulpal dentin after surface demineralization. Endod Dent Traumatol 1986;2:190-5. 5. Polson AM, Frederick GT, Ladenheim S, Hanes PJ. The production of a root surface smear layer by instrumentation and its removal by citric acid. J Periodontol 1984;55:443-6. 6. Register AA. Bone and cementum induction by dentin, demineralized in situ. J Periodontol 1973;44:49-54. 7. Wikesjo UME, Baker PJ, Christersson LA, Genco RJ, Lyall RM, Hic S, DiFlorio RM, Terranova VP. A biochemical approach to periodontal regeneration: tetracycline treatment conditions dentin surfaces. J Periodontol Res 1986;21:322-9. 8. Labahn R, Fahrenbach WH, Clark SM, Lie T, Adams DF. Root dentin morphology after different modes of citric acid and tetracycline hydrochloride conditioning. J Periodontol 1992;63:303-9. 9. Bjortvan K, Skaug N, Selvig KA. Tetracycline-impregnated enamel and dentin: duration of anti-microbial capacity. Scand J Dent Res 1985; 931192-7. 10. Abbott P, Heijkoop PS, Cardaci SC, Hume WR, Heithersay GS. A SEM study of the effects of different irrigation sequences and ultrasonic. Int Endod J 1991;24:308-16. 11. Wayman BE, Kopp VM, Pinero GJ, Lazzari EP. Citric and lactic acids as root canal irrigants in vitro. J Endodon 19793258-65. 12. Goldman LB, Goldman M, Kronman JH, Lin PS. The efficacy of several irrigating solutions for endodontics: a scanning electron microscopic study. Oral Surg Oral Med Oral Pathol 1981;52:197-204. 13. Garberoglio R, Becce C. Smear layer removal by root canal irrigants: a comparative scanning electron microscopic study. Oral Surg Oral Med Oral Pathol 1994;78:359-67. 14. Gutierrez JH, Villena F, Jofre A, Amin M. Bacterial infiltration of dentin as influenced by proprietary chelating agents. J Endodon 1982;8: 448-54. 15. Gutierrez JH, Herrera VR, Berg EH, Villena F, Jofre A. The risk of intentionaldissolutionof the smear layer after mechanical preparation of root canals. Oral Surg Oral Med Oral Pathol 1990;70:96-108. 16. $en BH, Wesselink PR, Turkun M. The smear layer: a phenomenon in root canal therapy. Int Endod J 1995;28:141-8. 17. Brannstrom M, Johnson G. Effects of various conditioners and cleaning agents on prepared dentin surfaces: a scanning electron microscopic evaluation. J Prosthet Dent 1974;31:422-30. 18. Bridges JB, Owens PDA, Stewart DJ. Tetracyclines and teeth: an experimental investigation into five types in the rat. Br Dent J 1969;1:30611.