The Effect of Irrigation with Doxycycline or Citric Acid on Leakage and Osseous Wound Healing

Transcription

1 JOURNAL OF ENDODONTICS Printed in U.S.A. Copyright 2002 by The American Association of Endodontists VOL. 29, NO. 1, JANUARY 2003 The Effect of Irrigation with Doxycycline or Citric Acid on Leakage and Osseous Wound Healing Joanna L. Davis, DDS, MS, Billie G. Jeansonne, DDS, PhD, William D. Davenport, PhD, and Diana Gardiner, PhD This study investigated the effect of doxycycline irrigation on wound healing and the apical seal of three filling materials. Part 1: 220 extracted teeth received root canal therapy followed by root resection (2 mm) and ultrasonic root end preparations (3 mm). Groups of 20 were irrigated with saline, citric acid, or doxycycline and filled with amalgam, Super EBA, or MTA. Leakage was measured (mm) after decalcification and clearing. Part 2: two defects were made on each side of the mandible of 10 New Zealand rabbits. On each side, one defect was irrigated with saline and one with either citric acid or doxycycline. The animals were killed in groups of five at 9 and 18 days. Sections of each defect were stained with hematoxylin and eosin for evaluation of healing and bone fill. Super EBA and MTA leaked significantly less than amalgam, regardless of irrigant. Leakage after irrigation with doxycycline compared to citric acid or saline was not significantly different for Super EBA or MTA but was lower for amalgam. There was no significant difference in healing or bone fill among irrigants at 9 or 18 days. responsive to fibroblast adhesion and growth (4). Terranova et al. (5) showed in a 1986 study that tetracycline-treated dentin slabs showed three times more fibroblast attachment to their surface than did citric acid-treated dentin. Tetracycline has been shown to inhibit mammalian collagenases that are released during inflammatory diseases, such as periodontitis. This anticollagenase activity can lead to enhanced formation of collagen and bone (6). Doxycycline, which is a hydroxy derivative of tetracycline, is the most potent anticollagenase of the available tetracyclines (7). Irrigation with doxycycline to remove the smear layer from dentinal surfaces has been shown to improve the apical seal of root end filling materials, such as intermediate restorative material (IRM) and amalgam (8). Although there are leakage studies of several materials used for apical fillings (9, 10), no study comparing the effect of smear layer removal on the apical seal of Super EBA and MTA has been completed. Also to date, no study has been performed to evaluate the effects of direct irrigation with acidic solutions like doxycycline, for smear layer removal and demineralization, on the healing of trabecular and cortical bone. Because successful periapical surgery involves wound healing and effectively sealing the root end, the purpose of this study was 2-fold: (a) to compare the effect on the apical seal of Super EBA, MTA, and amalgam when irrigation with saline, citric acid, or doxycycline HCl is used to remove the smear layer; and (b) to evaluate the effects of those same irrigation materials on the healing and osseous repair ability of bone. Root resection during periapical surgery creates a smear layer. The smear layer has both organic and inorganic components, such as dentin, remnants of odontoblasts, tissue debris, pulp, and bacteria (1). The smear layer has been shown to act as a barrier to new attachment of connective tissue with supracrestal cementum or dentin after mucoperiosteal flap surgery in periodontal therapy (2). The smear layer present on the resected root end may act as a functional barrier to cementogenesis possibly delaying or preventing periradicular wound healing. Removal of the smear layer by demineralizing the root surface with citric acid has been shown to increase cementogenesis and promote periradicular wound healing by exposing the collagen matrix, which stimulates fibroblast attachment and growth (3). Tetracycline has also been shown to remove the smear layer and widen the orifice of the dentinal tubules making the surface more MATERIALS AND METHODS Part 1: Apical Leakage In this study 220 human, single-rooted, extracted teeth were used. The crowns were removed to the CEJ, and the canals were instrumented to within 1 mm of the apex using the 0.04 taper Profile Series 29 files (Tulsa Dental Products, Tulsa, OK), (#2 8 in a step-back technique) with 2.5% NaOCl irrigation between each file. The canals were obturated with gutta-percha and Roth s 811 sealer (Roth International, Chicago IL) using a warm vertical technique. The coronal opening for each was sealed with Cavit (ESPE, Seefeld, Germany). All teeth were stored in physiological saline for a minimum of 1 week to allow for complete setting of the 31

2 32 Davis et al. Journal of Endodontics sealer. All teeth were then painted with two coats of fingernail polish. Forty teeth served as leakage controls. Ten were left completely coated with nail polish (negative controls), resection and root end filling preparations were completed in the remaining 30 teeth. Groups of 10 teeth were irrigated with each irrigant for 1 min, but no filling materials were placed (positive controls). The apical 2 mm of the remaining 180 teeth was resected with a #557 bur in a water-cooled, high-speed handpiece. Root end preparations were made to a depth of 3 mm with a P-5 Booster Suprasson P-MAX ultrasonic unit (Satelec, Cherry Hill, NJ) at a setting of 6 to 7. All visible gutta-percha was removed along the cavity walls. Sixty root end preparations were irrigated with 10% citric acid solution (ph 3.1) for 1 min, 60 were irrigated with saline for 1 min, and 60 were irrigated with doxycycline (100 mg/ml solution, ph 3.3) for 1 min. The final irrigation for each group was saline for 1 min. Each group was then subdivided into subgroups of 20 teeth. Cavity preparations in each subgroup were filled with amalgam (Tytin, Kerr Dental Corporation, Romulus, MI), Super EBA (Bosworth Co., Skokie, IL), or MTA (Mineral Trioxide Aggregate, Pro Root Dentsply, Tulsa Dental), each mixed and placed according to manufacturer s instructions. All teeth received a final 1-min rinse with saline. The teeth were wrapped in moist gauze and stored in a sealed container at 37 C for a minimum of 1 week before submerging in India ink under vacuum pressure (605 mmhg) for 30 min. They were stored in India ink, in a closed container, for 1 week. All teeth were rinsed of excess India ink and the nail polish was removed by using a #15 blade. The teeth were cleared by decalcification in 5% nitric acid, dehydrated in alcohol, and stored in methyl salicylate for 48 h until rendered transparent. Dye penetration was measured in three dimensions to the nearest 0.1 mm by using a stereoscopic microscope. The largest measurement of apical to coronal leakage was recorded for each tooth. The leakage results were evaluated using a two-way ANOVA to determine if there was an overall significant difference in dye leakage based on type of irrigant or filling material. The Scheffe test was used to determine significant differences among the irrigants and materials separately. The one-way ANOVA with Scheffe follow-up test was used to determine significant differences among the nine groups considering irrigant and filling materials together. Citric acid and doxycycline groups were compared to saline by using a t test. Significance testing was performed based on a probability level of p Part 2: Effect on Wound Healing Ten New Zealand White rabbits ( kg) were used to evaluate osseous healing. The rabbits were anesthetized for all procedures with a combination of xylazine (7 mg/kg) and ketamine (30 mg/kg) via an intramuscular injection. In addition, 16 mg of 2% lidocaine with 1/100,000 epinephrine was infiltrated into the surgical area. On both sides of the mandible, an incision was made along the alveolar crest in the naturally edentulous space between the incisor and premolar teeth. A full-thickness mucoperiosteal flap was reflected to expose the alveolar cortical bone. Two osseous defects, the size of a #8 round bur (approximately 2 mm in diameter), were made on each side of the mandible. Defects were placed approximately 10 mm apart using the position of the incisors and the mandibular ridge as identifiable landmarks for each animal. On each side, one defect was irrigated with saline and the other defect with either 10% citric acid solution or 100 mg/ml of doxycycline solution for 1 min. Each defect received a final 1-min rinse with saline. Surgical flaps were reapproximated with resorbable sutures. A combination of penicillin (100,000 units of procaine G) and streptomycin (base equivalent of 125 mg) was given by intramuscular injections for infection control after surgery. The rabbits were given three injections for pain control (Buprenorphine 0.5 mg/kg q 12 h) and fed a soft diet of crushed rabbit pellets to minimize trauma to the surgical sites. The rabbits were killed in groups of five at 9 days and 18 days postoperatively by using an overdose of ketamine. Specimens were sectioned to include a defect and adjacent bone at each surgical site. Each specimen was decalcified in a 10% sodium formateformic acid solution and processed for routine paraffin embedding for histological examination. Seven-micrometer sections were cut in a coronal plane through the approximate center of the defect and stained with hematoxylin and eosin. Selected samples for each specimen were submitted to two independent examiners for evaluation of inflammation, evidence of healing, and amount of bone fill, using the following scales. Healing Scores 4 Complete healing in the surgical site with healthy cancellous bone; 3 Fibrosis with dense collagen with or without early bone formation; 2 Granulation tissue filling site with or without chronic inflammation; 1 Acute inflammation with or without granulation tissue; 0 Abscess formation. Bone Fill Scores 4 Complete bone fill with cortical plate intact; 3 Incomplete cortical plate with > 50% lesion center filled with bone; 2 Incomplete cortical plate with > 50% peripheral bone fill; 1 Incomplete cortical plate with < 50% periphery with evidence of early bone formation; 0 Connective tissue with minimal or no evidence of early bone formation. If the scores between the examiners differed, they conferred and came to an agreement on the final score. The final scores for each site were analyzed for 9- and 18-day specimens using Wilcoxon sign rank, nonparametric analysis to determine any significant difference comparing irrigants on each side of the mandible within the same animal, and comparing irrigants from one side to the other within the same animal. Then using a Friedman analysis, all irrigants for each time period were compared by using a significant difference tested at p RESULTS Part 1: Leakage Study The leakage found in the amalgam groups, regardless of irrigant, was significantly greater than the leakage of the Super EBA and the MTA groups. Among the amalgam groups, leakage was significantly less with doxycycline than with the other two irrigants.

3 Vol. 29, No. 1, January 2003 Doxycycline or Citric Acid Irrigation 33 There was no significant difference in the leakage of Super EBA or MTA with any irrigant (Fig. 1). A follow-up comparison for the nine groups ranks the groups from least amount of leakage to the most. The best combinations were Super EBA with doxycycline or MTA with saline, both with very comparable results. The worst combination was amalgam with citric acid (Fig. 2). The pattern of leakage between amalgam and the other two materials was different. Both MTA and Super EBA had an initial washing out of the materials before the final set. The leakage for both materials seemed to be confined to a circumferential pattern when this occurred. This was observed more frequently in the MTA samples than in the Super EBA samples. Amalgam samples leaked mainly in the apical to coronal direction. In most samples, the leakage extended the entire length of the root end preparation along side the filling material. In some samples the leakage also extended into the dentinal tubules. When comparing the amount of apical to coronal leakage of samples irrigated with citric acid or doxycycline to those irrigated with saline, there was no significant difference with any material. When a similar comparison of leakage into the dentinal tubules FIG. 1. Overall leakage results. Mean for each group displayed two-way ANOVA. was performed, there was a significant difference only with the MTA samples. The MTA samples irrigated with doxycycline or citric acid leaked significantly more laterally especially in the washed out area, than samples irrigated with saline. Part 2: Animal Study Healing and bone fill were measured in the rabbits at 9 days and at 18 days. No significant differences in healing were found when comparing defects on the same side or when comparing right side to left side within the same animal. Also no significant differences were found when comparing all irrigants to each other. At 9 days (Fig. 3), there was very little difference in healing between the doxycycline and saline defects, which both had slightly higher scores than the citric acid defects. At 9 days, the patterns of bone fill scores were very similar to that of the healing scores. At 18 days, 18/20 (90%) of the defects showed complete healing while 12/20 (60%) of the defects showed complete bone fill. There were no significant differences for any comparisons for both healing and bone fill in the 18-day specimens (Fig. 4). A typical histological section of a doxycycline irrigated site at 9 days is shown in Figs. 5 and 6. The healing score of 2 reflects large amounts of granulation tissue in the center of the defect with minimal peripheral bone formation. The higher magnification in Fig. 6 shows connective tissue cells, the inflammatory cells of the granulation tissue, collagen fiber formation, and new bone formation along the edge of the defect. A typical histological section of a doxycycline irrigated site at 18 days is shown in Figs. 7 and 8. Complete healing occurred with healthy cancellous bone filling the defect and the cortical plate is intact. Higher magnification (Fig. 8) shows a reversal line and the first stages of remodeling of the new bone of the lesion and the older bone of the wound edge. In terms of both healing and bone fill, saline irrigated samples had the higher average scores at 9 days and the lowest scores at 18 days. FIG. 2. Rank order of leakage for all groups. Scheffe for one-way ANOVA. FIG. 3. Average healing and bone fill scores at 9 days.

4 34 Davis et al. Journal of Endodontics FIG. 4. Average healing and bone fill scores at 18 days. FIG. 6. Higher magnification ( 125) of Fig. 5. B new bone formation; C collagen formation; G granulation tissue. FIG. 5. Specimen irrigated with doxycycline at 9 days: healing 2; bone fill 1. Granulation tissue fills the site. There is an incomplete cortical plate and less than 50% peripheral bone formation (magnification 50;H&Estain). FIG. 7. Specimen irrigated with doxycycline at 18 days: healing 4; bone fill 4. Complete healing with healthy cancellous bone and an intact cortical plate (magnification 50;H&Estain). DISCUSSION Super EBA and MTA showed less leakage with all irrigants compared with amalgam. In almost every sample of amalgam, leakage could be measured through the entire depth of the preparation. Most leakage studies testing amalgam as a root end filling material have suggested that amalgam fails to provide an adequate seal when compared with other materials (11, 12). One long-term clinical study showed a clinical success rate of 60% after 10 yr (13), and another 10-yr study showed a 75% success rate (14) for amalgam. This suggests that there may be factors other than leakage that can affect the clinical success. The results of this study agree with other in vitro studies comparing the sealing ability of Super EBA and MTA (15, 16). This study showed that irrigating with citric acid or doxycycline (removal of the smear layer) did not significantly improve the apical seal of Super EBA or MTA. One observation from this study was the difference in the pattern of leakage for each material. Amalgam leaked primarily in an apical to coronal pattern often extending the entire length of the root end filling. MTA had distinctive washing out of the material FIG. 8. Higher magnification ( 125) of upper left portion of Fig. 7. B interface of new bone formation to wound edge before complete remodeling. before the final set. This was most obvious in the citric acid samples. Leakage was usually confined to this circumferential area. This may be due to the extended setting time of MTA.

5 Vol. 29, No. 1, January 2003 Doxycycline or Citric Acid Irrigation 35 Irrigation, with acidic solutions such as citric acid, to remove the smear layer may delay the setting of MTA, allowing the washing out of some of the material before complete setting. Most samples had no significant leakage beyond the point of the washout. The amount of remaining MTA and Super EBA, which ranged from 1.0 to 2.0 mm, prevented apical to coronal leakage beyond the surface of the filling material. In part 2 of this study, the inflammatory response and ultimate healing observed in the rabbits was the result of intentional wounding when defects were created in the mandible. At 9 days, only 3 out of 10 sites irrigated with citric acid or doxycycline showed greater than 50% peripheral bone fill, whereas 6 of 10 sites irrigated with saline exhibited complete peripheral bone fill and greater than 50% of the center of the lesion with new bone; however, the difference was not significant. By 18 days, most of the defects irrigated with doxycycline or citric acid had completely healed and had complete bone fill, including a new cortical plate, whereas only 50% of the sites irrigated with saline showed complete bone fill with an intact cortical plate; but this difference was also not significant. Demineralization of resected root ends has been shown to improve associated alveolar healing as well as enhance cementogenesis and dentoalveolar healing (17). Irrigation with doxycycline may provide a similar beneficial effect in dentoalveolar healing by removal of the smear layer and exposing the collagen matrix for increased fibroblast attachment. The lower ph of doxycycline and citric acid may initiate a more intense inflammatory response initially when compared with saline. This may inhibit the healing process as measured by new bone formation. As healing progresses, the potential benefits of the anticollagenase activity may allow for more rapid collagen formation and ultimately allow more rapid new bone formation. Doxycycline has been shown to be a potent anticollagenase (18). Inhibiting the degradation of collagen may be beneficial in the healing of inflammatory lesions and the formation of new bone. This may be especially significant in areas where chronic lesions have resulted in periradicular tissue destruction. Dr. Davis is an endodontic postgraduate student; Dr. Jeansonne is associate professor and coordinator of postgraduate endodontics; Dr. Davenport is associate professor, Department of Oral Pathology; and Dr. Gardiner is director, Instructional Services, Accountability and Planning, Louisiana State University Health Science Center, School of Dentistry, New Orleans, Louisiana. Dr. Davis is currently in private practice in Austin, Texas. Address requests for reprints to Dr. Billie G. Jeansonne, LSUSD Endodontics, Box 135, 1100 Florida Avenue, New Orleans, LA References 1. Mader CL, Baumgartner JC, Peters DD. Scanning electron microscopic investigation of the smeared layer on root canal walls. J Endodon 1984;10: Slagle JL. Regeneration of connective tissue attachment after citric demineralization in periodontal flap surgery: a review of the literature. VA Dentistry J 1984;61: Craig KR Harrison JW. Wound healing following demineralization of resected root ends in periradicular surgery. J Endodon 1993;19: Wikesjo UM, Baker PJ, Christersson LA, et al. A biochemical approach to periodontal regeneration: tetracycline treatment conditions dentin surfaces. J Periodontal Res 1986;21: Terranova VP, Franzetti LC, Hic S. A biochemical approach to periodontal regeneration: tetracycline treatment of dentin promotes fibroblast adhesion and growth. J Periodontal Res 1986;21: Rifkin BR, Vernillo AT, Golub LM. Blocking periodontal disease progression by inhibiting tissue destructive enzymes: a potential therapeutic role for tetracyclines and their chemically modified analogs. J Periodontol 1993; 64(Suppl): Burns FR, Stack MS, Gray RD, Paterson CA. Institution of purified collagenase from alkali-burned rabbit corneas. Invest Ophthalmol Vis Sci 1989;30: Barkhordar RA, Russel T. Effect of doxycycline on the apical seal of retrograde filling materials. J Calif Dent Assoc 1998;26: Wu M, Kontakiotis E, Wesselink P. Long-term seal provided by some root-end filling materials. J Endodon 1998;24: Torabinejad M, Pitt Ford TR. Root-end filling materials. a review. Endod Dent Traumatol 1996;12: Thirawat J, Edmunds DH. The sealing ability of materials used as retrograde fillings in endodontic surgery. Int Endod J 1989;22: Trope M, Lost C, Schmitz HJ, Friedman S. Healing of apical periodontitis in dogs after apicoectomy and retrofilling with various filling materials. Oral Surg Oral Med Oral Pathol 1996;81: Frank A, Glick D, Patterson S, Weine F. Long-term evaluation of surgically placed amalgam fillings. J Endodon 1992;18: Dorn S, Gartner A. Retrograde filling materials: a retrospective success-failure study of amalgam, EBA, and IRM. J Endodon 1990;16: Torabinejad M, Pitt Ford TR. Root end filling materials: a review. Endod Dent Traumatol 1996;12: Bates CF, Carnes DL, del Rio CE. Longitudinal sealing ability of mineral trioxide aggregate as a root end filling material. J Endodon 1996;22: Craig KR, Harrison JW. Wound healing following demineralization of resected root ends in periradicular surgery. J Endodon 1993;19: Burns FR, Stack MS, Gray RD, Paterson CA. Institution of purified collagenase from alkali-burned rabbit corneas. Invest Ophthalmol Vis Sci 1989;30: