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

Joanna L. Davis, DDS, MS, Billie G. Jeansonne, DDS, PhD, William D. Davenport, PhD, andDiana Gardiner, PhD

This study investigated the effect of doxycyclineirrigation on wound healing and the apical seal ofthree filling materials. Part 1: 220 extracted teethreceived root canal therapy followed by root resec-tion (2 mm) and ultrasonic root end preparations (3mm). Groups of 20 were irrigated with saline, citricacid, or doxycycline and filled with amalgam, SuperEBA, or MTA. Leakage was measured (mm) afterdecalcification and clearing. Part 2: two defectswere made on each side of the mandible of 10 NewZealand rabbits. On each side, one defect was ir-rigated with saline and one with either citric acid ordoxycycline. The animals were killed in groups offive at 9 and 18 days. Sections of each defect werestained with hematoxylin and eosin for evaluationof healing and bone fill. Super EBA and MTA leakedsignificantly less than amalgam, regardless of irri-gant. Leakage after irrigation with doxycyclinecompared to citric acid or saline was not signifi-cantly different for Super EBA or MTA but waslower for amalgam. There was no significant dif-ference in healing or bone fill among irrigants at 9or 18 days.

Root resection during periapical surgery creates a smear layer. Thesmear layer has both organic and inorganic components, such asdentin, remnants of odontoblasts, tissue debris, pulp, and bacteria(1). The smear layer has been shown to act as a barrier to newattachment of connective tissue with supracrestal cementum ordentin after mucoperiosteal flap surgery in periodontal therapy (2).The smear layer present on the resected root end may act as afunctional barrier to cementogenesis possibly delaying or prevent-ing periradicular wound healing.

Removal of the smear layer by demineralizing the root surfacewith citric acid has been shown to increase cementogenesis andpromote periradicular wound healing by exposing the collagenmatrix, which stimulates fibroblast attachment and growth (3).Tetracycline has also been shown to remove the smear layer andwiden the orifice of the dentinal tubules making the surface more

responsive to fibroblast adhesion and growth (4). Terranova et al.(5) showed in a 1986 study that tetracycline-treated dentin slabsshowed three times more fibroblast attachment to their surface thandid citric acid-treated dentin. Tetracycline has been shown toinhibit mammalian collagenases that are released during inflam-matory diseases, such as periodontitis. This anticollagenase activ-ity can lead to enhanced formation of collagen and bone (6).

Doxycycline, which is a hydroxy derivative of tetracycline, isthe most potent anticollagenase of the available tetracyclines (7).Irrigation with doxycycline to remove the smear layer from den-tinal surfaces has been shown to improve the apical seal of root endfilling materials, such as intermediate restorative material (IRM)and amalgam (8).

Although there are leakage studies of several materials used forapical fillings (9, 10), no study comparing the effect of smear layerremoval on the apical seal of Super EBA and MTA has beencompleted. Also to date, no study has been performed to evaluatethe effects of direct irrigation with acidic solutions like doxycy-cline, for smear layer removal and demineralization, on the healingof trabecular and cortical bone.

Because successful periapical surgery involves wound healingand effectively sealing the root end, the purpose of this study was2-fold: (a) to compare the effect on the apical seal of Super EBA,MTA, and amalgam when irrigation with saline, citric acid, ordoxycycline HCl is used to remove the smear layer; and (b) toevaluate the effects of those same irrigation materials on thehealing and osseous repair ability of bone.

MATERIALS AND METHODS

Part 1: Apical Leakage

In this study 220 human, single-rooted, extracted teeth wereused. The crowns were removed to the CEJ, and the canals wereinstrumented to within 1 mm of the apex using the 0.04 taperProfile Series 29 files (Tulsa Dental Products, Tulsa, OK), (#2–8in a step-back technique) with 2.5% NaOCl irrigation betweeneach file. The canals were obturated with gutta-percha and Roth’s811 sealer (Roth International, Chicago IL) using a warm verticaltechnique. The coronal opening for each was sealed with Cavit(ESPE, Seefeld, Germany). All teeth were stored in physiologicalsaline for a minimum of 1 week to allow for complete setting of the

JOURNAL OF ENDODONTICS Printed in U.S.A.Copyright © 2002 by The American Association of Endodontists VOL. 29, NO. 1, JANUARY 2003

31

sealer. All teeth were then painted with two coats of fingernailpolish.

Forty teeth served as leakage controls. Ten were left com-pletely coated with nail polish (negative controls), resection androot end filling preparations were completed in the remaining30 teeth. Groups of 10 teeth were irrigated with each irrigant for1 min, but no filling materials were placed (positive controls).The apical 2 mm of the remaining 180 teeth was resected witha #557 bur in a water-cooled, high-speed handpiece. Root endpreparations were made to a depth of 3 mm with a P-5 BoosterSuprasson P-MAX ultrasonic unit (Satelec, Cherry Hill, NJ) ata setting of 6 to 7. All visible gutta-percha was removed alongthe cavity walls. Sixty root end preparations were irrigated with10% citric acid solution (pH 3.1) for 1 min, 60 were irrigatedwith saline for 1 min, and 60 were irrigated with doxycycline(100 mg/ml solution, pH 3.3) for 1 min. The final irrigation foreach 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 (Bos-worth Co., Skokie, IL), or MTA (Mineral Trioxide Aggregate,“Pro Root” Dentsply, Tulsa Dental), each mixed and placed ac-cording to manufacturer’s instructions. All teeth received a final1-min rinse with saline.

The teeth were wrapped in moist gauze and stored in a sealedcontainer at 37°C for a minimum of 1 week before submerging inIndia ink under vacuum pressure (605 mmHg) for 30 min. Theywere stored in India ink, in a closed container, for 1 week. All teethwere rinsed of excess India ink and the nail polish was removed byusing a #15 blade. The teeth were cleared by decalcification in 5%nitric acid, dehydrated in alcohol, and stored in methyl salicylatefor 48 h until rendered transparent. Dye penetration was measuredin three dimensions to the nearest 0.1 mm by using a stereoscopicmicroscope. The largest measurement of apical to coronal leakagewas recorded for each tooth.

The leakage results were evaluated using a two-way ANOVA todetermine if there was an overall significant difference in dyeleakage based on type of irrigant or filling material. The Scheffetest was used to determine significant differences among the irri-gants and materials separately. The one-way ANOVA with Scheffefollow-up test was used to determine significant differences amongthe nine groups considering irrigant and filling materials together.Citric acid and doxycycline groups were compared to saline byusing a t test. Significance testing was performed based on aprobability level of p � 0.05.

Part 2: Effect on Wound Healing

Ten New Zealand White rabbits (2.8–3.9 kg) were used toevaluate osseous healing. The rabbits were anesthetized for allprocedures with a combination of xylazine (7 mg/kg) and ketamine(30 mg/kg) via an intramuscular injection. In addition, 16 mg of2% lidocaine with 1/100,000 epinephrine was infiltrated into thesurgical area. On both sides of the mandible, an incision was madealong the alveolar crest in the naturally edentulous space betweenthe incisor and premolar teeth. A full-thickness mucoperiostealflap was reflected to expose the alveolar cortical bone. Two osse-ous defects, the size of a #8 round bur (approximately 2 mm indiameter), were made on each side of the mandible. Defects wereplaced approximately 10 mm apart using the position of the inci-sors and the mandibular ridge as identifiable landmarks for each

animal. On each side, one defect was irrigated with saline and theother defect with either 10% citric acid solution or 100 mg/ml ofdoxycycline solution for 1 min. Each defect received a final 1-minrinse with saline. Surgical flaps were reapproximated with resorb-able sutures. A combination of penicillin (100,000 units of pro-caine G) and streptomycin (base equivalent of 125 mg) was givenby intramuscular injections for infection control after surgery. Therabbits were given three injections for pain control (Buprenorphine0.5 mg/kg q 12 h) and fed a soft diet of crushed rabbit pellets tominimize trauma to the surgical sites.

The rabbits were killed in groups of five at 9 days and 18 dayspostoperatively by using an overdose of ketamine. Specimens weresectioned to include a defect and adjacent bone at each surgicalsite. Each specimen was decalcified in a 10% sodium formate-formic acid solution and processed for routine paraffin embeddingfor histological examination. Seven-micrometer sections were cutin a coronal plane through the approximate center of the defect andstained with hematoxylin and eosin. Selected samples for eachspecimen were submitted to two independent examiners for eval-uation of inflammation, evidence of healing, and amount of bonefill, using the following scales.

Healing Scores

4 � Complete healing in the surgical site with healthy cancel-lous bone;

3 � Fibrosis with dense collagen with or without early boneformation;

2 � Granulation tissue filling site—with or without chronicinflammation;

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 ev-

idence 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 andcame to an agreement on the final score. The final scores for eachsite were analyzed for 9- and 18-day specimens using Wilcoxonsign rank, nonparametric analysis to determine any significantdifference comparing irrigants on each side of the mandible withinthe same animal, and comparing irrigants from one side to the otherwithin the same animal. Then using a Friedman analysis, allirrigants for each time period were compared by using a significantdifference tested at p � 0.05.

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 andthe MTA groups. Among the amalgam groups, leakage was sig-nificantly less with doxycycline than with the other two irrigants.

32 Davis et al. Journal of Endodontics

There was no significant difference in the leakage of Super EBAor MTA with any irrigant (Fig. 1).

A follow-up comparison for the nine groups ranks the groupsfrom least amount of leakage to the most. The best combinationswere Super EBA with doxycycline or MTA with saline, both withvery comparable results. The worst combination was amalgamwith citric acid (Fig. 2).

The pattern of leakage between amalgam and the other twomaterials was different. Both MTA and Super EBA had an initial“washing out” of the materials before the final set. The leakage forboth materials seemed to be confined to a circumferential patternwhen this occurred. This was observed more frequently in theMTA samples than in the Super EBA samples. Amalgam samplesleaked mainly in the apical to coronal direction. In most samples,the leakage extended the entire length of the root end preparationalong side the filling material. In some samples the leakage alsoextended into the dentinal tubules.

When comparing the amount of apical to coronal leakage ofsamples irrigated with citric acid or doxycycline to those irrigatedwith saline, there was no significant difference with any material.When a similar comparison of leakage into the dentinal tubules

was performed, there was a significant difference only with theMTA samples. The MTA samples irrigated with doxycycline orcitric acid leaked significantly more laterally especially in thewashed out area, than samples irrigated with saline.

Part 2: Animal Study

Healing and bone fill were measured in the rabbits at 9 days andat 18 days. No significant differences in healing were found whencomparing defects on the same side or when comparing right sideto left side within the same animal. Also no significant differenceswere found when comparing all irrigants to each other.

At 9 days (Fig. 3), there was very little difference in healingbetween the doxycycline and saline defects, which both hadslightly higher scores than the citric acid defects. At 9 days, thepatterns of bone fill scores were very similar to that of the healingscores.

At 18 days, 18/20 (90%) of the defects showed complete healingwhile 12/20 (60%) of the defects showed complete bone fill. Therewere no significant differences for any comparisons for both heal-ing and bone fill in the 18-day specimens (Fig. 4).

A typical histological section of a doxycycline irrigated site at9 days is shown in Figs. 5 and 6. The healing score of 2 reflectslarge amounts of granulation tissue in the center of the defect withminimal peripheral bone formation. The higher magnification inFig. 6 shows connective tissue cells, the inflammatory cells of thegranulation tissue, collagen fiber formation, and new bone forma-tion along the edge of the defect.

A typical histological section of a doxycycline irrigated site at18 days is shown in Figs. 7 and 8. Complete healing occurred withhealthy cancellous bone filling the defect and the cortical plate isintact. Higher magnification (Fig. 8) shows a reversal line and thefirst stages of remodeling of the new bone of the lesion and theolder bone of the wound edge.

In terms of both healing and bone fill, saline irrigated sampleshad the higher average scores at 9 days and the lowest scores at 18days.FIG. 1. Overall leakage results. Mean for each group displayed

two-way ANOVA.

FIG. 2. Rank order of leakage for all groups. Scheffe for one-wayANOVA. FIG. 3. Average healing and bone fill scores at 9 days.

Vol. 29, No. 1, January 2003 Doxycycline or Citric Acid Irrigation 33

DISCUSSION

Super EBA and MTA showed less leakage with all irrigantscompared with amalgam. In almost every sample of amalgam,leakage could be measured through the entire depth of the prepa-ration. Most leakage studies testing amalgam as a root end fillingmaterial have suggested that amalgam fails to provide an adequateseal when compared with other materials (11, 12). One long-termclinical study showed a clinical success rate of 60% after 10 yr(13), and another 10-yr study showed a 75% success rate (14) foramalgam. This suggests that there may be factors other thanleakage that can affect the clinical success.

The results of this study agree with other in vitro studiescomparing 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 theapical seal of Super EBA or MTA.

One observation from this study was the difference in thepattern of leakage for each material. Amalgam leaked primarily inan apical to coronal pattern often extending the entire length of theroot end filling. MTA had distinctive washing out of the material

before the final set. This was most obvious in the citric acidsamples. Leakage was usually confined to this circumferentialarea. This may be due to the extended setting time of MTA.

FIG. 4. Average healing and bone fill scores at 18 days.

FIG. 5. Specimen irrigated with doxycycline at 9 days: healing � 2;bone fill � 1. Granulation tissue fills the site. There is an incompletecortical plate and less than 50% peripheral bone formation (magni-fication �50; H & E stain).

FIG. 6. Higher magnification (�125) of Fig. 5. B � new bone forma-tion; C � collagen formation; G � granulation tissue.

FIG. 7. Specimen irrigated with doxycycline at 18 days: healing � 4;bone fill � 4. Complete healing with healthy cancellous bone and anintact cortical plate (magnification �50; H & E stain).

FIG. 8. Higher magnification (�125) of upper left portion of Fig. 7. B� interface of new bone formation to wound edge before completeremodeling.

34 Davis et al. Journal of Endodontics

Irrigation, with acidic solutions such as citric acid, to remove thesmear layer may delay the setting of MTA, allowing the washingout of some of the material before complete setting. Most sampleshad no significant leakage beyond the point of the washout. Theamount of remaining MTA and Super EBA, which ranged from 1.0to 2.0 mm, prevented apical to coronal leakage beyond the surfaceof the filling material.

In part 2 of this study, the inflammatory response and ulti-mate healing observed in the rabbits was the result of intentionalwounding when defects were created in the mandible. At 9 days,only 3 out of 10 sites irrigated with citric acid or doxycyclineshowed greater than 50% peripheral bone fill, whereas 6 of 10sites irrigated with saline exhibited complete peripheral bonefill and greater than 50% of the center of the lesion with newbone; however, the difference was not significant. By 18 days,most of the defects irrigated with doxycycline or citric acid hadcompletely healed and had complete bone fill, including a newcortical plate, whereas only 50% of the sites irrigated withsaline 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 im-prove associated alveolar healing as well as enhance cementogen-esis and dentoalveolar healing (17). Irrigation with doxycyclinemay provide a similar beneficial effect in dentoalveolar healing byremoval of the smear layer and exposing the collagen matrix forincreased fibroblast attachment.

The lower pH of doxycycline and citric acid may initiate a moreintense inflammatory response initially when compared with sa-line. This may inhibit the healing process as measured by new boneformation. As healing progresses, the potential benefits of theanticollagenase activity may allow for more rapid collagen forma-tion 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 inthe healing of inflammatory lesions and the formation of new bone.This may be especially significant in areas where chronic lesionshave resulted in periradicular tissue destruction.

Dr. Davis is an endodontic postgraduate student; Dr. Jeansonne is asso-ciate professor and coordinator of postgraduate endodontics; Dr. Davenportis associate professor, Department of Oral Pathology; and Dr. Gardiner isdirector, 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. Addressrequests for reprints to Dr. Billie G. Jeansonne, LSUSD Endodontics, Box 135,1100 Florida Avenue, New Orleans, LA 70119.

References

1. Mader CL, Baumgartner JC, Peters DD. Scanning electron microscopicinvestigation of the smeared layer on root canal walls. J Endodon 1984;10:477–83.

2. Slagle JL. Regeneration of connective tissue attachment after citricdemineralization in periodontal flap surgery: a review of the literature. VADentistry J 1984;61:32–7.

3. Craig KR Harrison JW. Wound healing following demineralization ofresected root ends in periradicular surgery. J Endodon 1993;19:339–47.

4. Wikesjo UM, Baker PJ, Christersson LA, et al. A biochemical approachto periodontal regeneration: tetracycline treatment conditions dentin surfaces.J Periodontal Res 1986;21:322–9.

5. Terranova VP, Franzetti LC, Hic S. A biochemical approach to peri-odontal regeneration: tetracycline treatment of dentin promotes fibroblastadhesion and growth. J Periodontal Res 1986;21:330–7.

6. Rifkin BR, Vernillo AT, Golub LM. Blocking periodontal disease pro-gression by inhibiting tissue destructive enzymes: a potential therapeutic rolefor tetracyclines and their chemically modified analogs. J Periodontol 1993;64(Suppl):819–26.

7. Burns FR, Stack MS, Gray RD, Paterson CA. Institution of purifiedcollagenase from alkali-burned rabbit corneas. Invest Ophthalmol Vis Sci1989;30:1569–75.

8. Barkhordar RA, Russel T. Effect of doxycycline on the apical seal ofretrograde filling materials. J Calif Dent Assoc 1998;26:842–5.

9. Wu M, Kontakiotis E, Wesselink P. Long-term seal provided by someroot-end filling materials. J Endodon 1998;24:557–60.

10. Torabinejad M, Pitt Ford TR. Root-end filling materials. a review.Endod Dent Traumatol 1996;12:161–78.

11. Thirawat J, Edmunds DH. The sealing ability of materials used asretrograde fillings in endodontic surgery. Int Endod J 1989;22:295–8.

12. Trope M, Lost C, Schmitz HJ, Friedman S. Healing of apical periodon-titis in dogs after apicoectomy and retrofilling with various filling materials.Oral Surg Oral Med Oral Pathol 1996;81:221–7.

13. Frank A, Glick D, Patterson S, Weine F. Long-term evaluation ofsurgically placed amalgam fillings. J Endodon 1992;18:391–8.

14. Dorn S, Gartner A. Retrograde filling materials: a retrospective suc-cess-failure study of amalgam, EBA, and IRM. J Endodon 1990;16:391–3.

15. Torabinejad M, Pitt Ford TR. Root end filling materials: a review. EndodDent Traumatol 1996;12:161–78.

16. Bates CF, Carnes DL, del Rio CE. Longitudinal sealing ability of mineraltrioxide aggregate as a root end filling material. J Endodon 1996;22:575–8.

17. Craig KR, Harrison JW. Wound healing following demineralization ofresected root ends in periradicular surgery. J Endodon 1993;19:339–47.

18. Burns FR, Stack MS, Gray RD, Paterson CA. Institution of purifiedcollagenase from alkali-burned rabbit corneas. Invest Ophthalmol Vis Sci1989;30:1569–75.

Vol. 29, No. 1, January 2003 Doxycycline or Citric Acid Irrigation 35