Printed in U.S.A. VOL. 26, No. 3, MARCH 2000
JOURNAL OF ENDOCONTICS Copyright 0 2000 by The American Association of Endodontists
Apical Microleakage of Radiolabeled Lysozyme Over Time in Three Techniques of Root Canal Obturation Youssef Haikel, DCD, DSO, PhD, Michel Freymann, DCD, DU, Virginie Fanti, DCD, Anne Claisse, DCD, DSO, FranGoise Poumier, DCD, DU, and Morag Watson, BDS
techniques include scanning electron microscopy, bacterial penetration studies (7), pressurized fluid measurements (8), electrochemical methods (9), radioactive isotopes detected by autoradiography (6), scintillation spectrometry (lo), and gamma counters (1 1). These procedures have given varying and often contradictory results. Lateral condensation has often been used as a reference for comparison with Thermafd (4) or with McSpadden techniques (6, 12), but to date few experiments have compared all three of the aforementioned techniques to each other. The objective of this study was to compare the Thermafil and McSpadden techniques with each other and with the more traditional lateral condensation technique. Apical leakage was analyzed quantitatively using radiolabeled tracers that were detected and measured by a gamma counter using a new method described in a previous study (13). For each technique, samples were immersed for varying periods of time in the radioactive solution so that changes in the quality of the seal over time could be analyzed.
The three methods of obturation assessed in this study were lateral condensation (80 teeth), Thermafil(40teeth), and McSpadden(40 teeth). All teeth were prepared to the master apical file 30 and widened coronally by Gates Glidden burs before being randomly assigned to experimental groups to be filled by each technique; they were then sealed with nail polish, except for the apical 1 mm. Quantitative evaluation of apical microleakage for each technique was obtained after periods of 1 day, 7 days, 14 days, and 28 days of immersion in a lysozyme solution labeled with radioactive iodine by preparing horizontal sections of the teeth and measuring the level of radioactivity in each section using a gamma counter. Initial (1-day) leakage was least in the Thermafil group and was significantly different from the other techniques. Leakage was greatest in the laterally condensed samples. By the end of the study (28th day) values for lateral condensation were lowest, but were significantly different only for the McSpadden group. For all techniques leakage was most significant in the first 3 mm from the apex and was very low below this level so that all methods can be considered as giving a hermetic seal below 3 mm.
MATERIALS AND METHODS The study involved 192 single-rooted human teeth without marked apical curvature that had been stored in a 10% formalin solution.
Endodontic Preparation Teeth were prepared and obturated by a single operator. Access cavities were cut with a diamond bur to gain entry to the root canals. Apical patency was confirmed by inserting a #15 file (Maillefer, Ballaigues, Switzerland) until it was just visible at the apical foramen and the working length was taken as 1 mm short of this. The canal was instrumented with frequent sodium hypochlorite irrigation, alternating between K-files and H-files (Maillefer) until a master apical file of 30 was reached and the coronal third flared using Gates Glidden burs (Maillefer).
Obturation is the final stage of endodontic treatment and has an important role in preventing recontamination of the root canal and encouraging healing of the penapical tissues (1). Classically canals have been filled by cold lateral condensation, but limitations in this technique have been identified (2). In an attempt to overcome these problems and make obturation easier and less time-consuming (3), other methods have been introduced, such as Thermafil (4, 5) and the McSpadden technique (2, 6). As techniques have been introduced, they have been assessed using varying experimental designs. Most commonly dye penetration ( 2 ) has been used to evaluate the apical seal, but other
OBTURATION OF CANALS The teeth were randomly divided into 1 group of 80 teeth and 2 groups of 40 teeth according to the method of obturation to be used. To reduce variables between the groups, the same sealer AH 148
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plus (Dentsply De Trey, Konstanz, Germany), which had been shown to be the most effective in a previous study (13), was used for each technique. The group of 80 teeth were filled by lateral condensation; a medium-sized, nonstandardized master gutta-percha point (Hygienic, Akron, OH) was inserted and adjusted until apical tugback was achieved at the working length of 1 mm short of the apex, then the canal walls were coated with sealer and the master cone placed and condensed and accessory points added until the canal was full. One of the groups of 40 teeth was obturated by Thermafil (Thermafil Endodontic Obturators). The size was chosen to correspond to the size of the verifier that best fit the working length of the canal; sealer was placed in the entrance of the canal and the Thermafil device heated in the oven supplied (Thermaprep) for 4 min at 145°C and inserted to the working length of the canal in one firm movement. The handle was removed by cutting with a tapered diamond bur, and excess gutta-percha was plugged round the carrier. The final group was filled by the McSpadden technique: after coating the walls of the root canal with sealer, a gutta-percha point was placed to the working length and the compactor introduced running at 10 to 12,000 rpm for 1 min to compress the gutta-percha. The samples were then stored for 48 h at 37°C in a humid environment to allow the sealer to set.
PREVENTION OF LATERAL LEAKAGE Two coats of nail polish (L'Oreal, Clichy, France) were applied to the surface of each root leaving 1 mm free at the apex so that tracer could only penetrate the canal via the apical region.
CONTROL GROUPS Two other groups of 16 teeth each were prepared as positive and negative controls. The positive control group was obturated by lateral condensation, but without sealer and the test group was coated in nail polish except for the apical 1 mm. The negative control group was left unobturated and was totally coated in nail polish, including the apical region.
IMMERSION OF THE SAMPLES The experiment was undertaken in a modified syringe (Fig. 1) from which the end had been removed and the chamber was enclosed in lead foil to limit ionization from the radioactive solution, as described in a previous study (13). Teeth were mounted by their coronal aspects in transparent tubes attached to the pistons of' the syringes. This equipment allowed each root to be immersed up to 2 mm from the apex in radioactive solution. The three experimental groups were each further subdivided into four equal groups, depending on the duration of immersion-1 day, 7 days, 14 days, or 28 days-and were stored in the solution for the allotted time at a temperature of 23°C to limit evaporation.
PREPARATION OF SECTIONS On removing the teeth from the solution, the root surfaces were wiped with cellulose impregnated with a decontaminant (DCcon 90 diluted to 5%) until they no longer gave a reading when exposed to a Geiger-Muller tube (Nardeux), indicating that all the radioactive solution had been removed from the surface of the root. Each root was then cut into six transverse sections using a fine diamond wire (ESCIL diamented thread saw, Well 3032-4, Chassieu, France) with lubrication to prevent overheating. The sections had a thickness of 0.8 mm each, beginning 1 mm from the apical foramen. The first section from each tooth was discarded, because the filling did not begin until 1 mm from the apex. Each subsequent section was placed in a series of Pyrex tubes for measurement of the radioactivity present by the gamma counter (Minaxi Auto Gamma 5000 Series, United Technologies Packard, Downers Grove, IL). This allowed the amount of apical infiltration and thus the quality of seal of the filling to be evaluated.
STATISTICAL ANALYSIS Results were analyzed using the Kruskal-Wallis one-way analysis of variance on ranks and the all painvise multiple comparison procedure (Dunn) to determine the significance of the values attained.
RADIOACTIVE SOLUTION The radioactive tracer used was Iz5I combined with lysozyme at a concentration of 20 mg/100 ml. This solution contains molecules small enough to penetrate an inadequate apical seal due to the small size (40 nm) and low molecular weight (14,600 Da) of the lysozyme, while not reacting with the crystalline phase of the tooth substance. The protein was made radioactive using the McFarlane technique (14, 15) in which '*'I attaches to the lysozyme molecule at its tyrosyl and histidyl sites by an iodination reaction. The labeled protein solution was then divided into 2 ml aliquots and frozen. The concentration of this solution was determined using the values of protein absorbance by spectrophotometry and radioactivity using a gamma counter, allowing the samples to be thawed and diluted with phosphate-buffered saline to a concentration of 20 mg/100 ml for the experiment. The iodine is a gamma emitter with a half-life of 60 days. The lysozyme used was extracted from egg white and had a purity in excess of 96%.
FIG1. Diagram of apparatus for immersion of samples. System used to mount the samples during the study. 7, piston;2, sliding chamber; 3, lead foil; 4 , syringe tip; 5, PVC tube; 6, sample; 7, sample support; 8, sterile tube; and 9,radioactive solution.
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RESULTS The positive control showed high levels of tracer penetration present from day 1, but increasing as immersion time increased. This verified that penetration of the tracer through the apical foramen was possible when sealer was absent (Fig. 2). The negative control gave very low readings that did not increase with duration of immersion. This indicated that very little penetration had occurred and confirmed that nail polish was effective in sealing the external root surface (Fig. 3).
1 Day (Fig. 4)
Readings at this stage were fairly low; they were greatest in the first (1 to 1.8 mm) section, especially with lateral condensation. In the second (2 to 2.18 mm) section, levels of radioactivity seemed to settle at a lower level. There was a significant difference between Thermafil and lateral condensation and between Thermafil and McSpadden at the level of the first section. Comparison between all the techniques showed that, after 1 day, Thermafil performed best with no significant differences between the two others.
2 3 4 CrOOS root section from the apex
FIG4. Radioactiveintensity signal (cpm) as a function of the distance (mm) from the apex: 1-day period of immersion. Cond. Lat., lateral condensation.
7 Days (Fig. 5) After 1 wk, radioactivity had not increased very much, but the order of the leakage values had changed. Again readings were highest in the first section and reduced in subsequent sections. Statistical analysis of sections at the first level and overall illustrated no significant differences between the techniques.
Cross root sections from the apex
FIG5. Radioactiveintensity signal (cpm) as a function of the distance (mm) from the apex: 7-day period of immersion. Cond. Lat., lateral condensation.
Cross root section from the apex
FIG2. Positive control group.
Cross root sections from the apex
FIG 6. Radioactiveintensity signal (cprn) as a function of the distance (mm) from the apex: 14-day period of immersion. Cond. Lat., lateral condensation.
14 Days (Fig. 6) 200
Cross root sections from the apex
FIG3. Negative control group.
After 2 wk higher levels of infiltration were evident. Values were again higher at the level of the first section, reducing toward the second section, and leveling out, with all techniques showing similar low levels of infiltration from the third section. Statistical
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analysis of results at the first level showed no significant difference between the techniques, as was the case when all sections were considered together.
-Cond. -Mc 6000
28 Days (Fig. 7) After 1 month, highest readings were attained in the first section, with infiltration being much lower in the teeth filled by lateral condensation than the other techniques. After the second level, however, readings from the Thermafil and McSpadden groups had greatly reduced to levels similar to those of lateral Condensation. Statistical analysis of the results shows a significant difference, with lateral condensation performing better than McSpadden at the level of the first section and also when all sections are taken into account.
Throughout the experiment, infiltration was mainly important in the first 2 to 3 mm (i.e. sections 1 and 2 ) , where the infiltration increased over the time of the experiment and varied between the techniques. More coronal sections showed very little infiltration, and levels of leakage did not appear to increase with time. It would therefore appear that all techniques give a hermetic obturation from the level of the third section; so all methods studied can be considered to result in effective sealing of the root canals. Comparison of the total infiltration of lysozyme at each time period allowed the changes in permeability over time to be investigated. Initially, Thermafil seems the most effective technique; but, over time, infiltration increased (Fig. 8). Lateral condensation and McSpadden show similar levels of leakage at 1 day and 7 days, and leakage began to rise after this time.
DISCUSSION One of the major problems with studies of apical microleakage is the lack of standardization of samples and techniques (5, 16). In this study all canals were prepared to the same size and a single operator conducted the preparation and obturation to try to overcome variations. An attempt was made to make the samples of
1500 IOOa 500 0
FIG 8. Total radioactive intensity signal (cprn) as a function of the immersion period in days. Cond. Lat., lateral condensation.
Cross root section from the apex FIG7. Radioactive intensity signal (cpm) as a function of the distance (mm) from the apex: 28-day period of immersion. Cond. Lat., lateral condensation.
teeth comparable by using teeth with a single straight root, to avoid the problems of multiple canals or differences between roots with different curvatures. The teeth were also all of similar lengths so that they would have the same volume available for infiltration. Despite these attempts, natural anatomical variation among the teeth was still a factor, but it was hoped that randomly assigning the teeth to experimental groups and examining each group as a whole rather than looking at individual teeth would give a fair comparison between the groups. The different obturation techniques also brought the potential for variation. For example, Beatty et al. ( 2 ) noted that leakage was reduced if the finger spreader tip was inserted to within 1 mm of the working length. Uniformity of the depth reached by the finger spreader is, however, difficult to standardize. In the same study variations in the Thermafil technique were noted as a result of nonuniform heating of the gutta-percha in a flame. This problem was reduced by a newer Thermafil technique in which the carrier is heated in an oven at a standard temperature for a uniform time. Variations had also been shown with the McSpadden technique by Harris et al. (6). who found that when used, sealer may become incorporated in the gutta-percha giving the same effect as if no sealer had been used, but this does not happen in every case. Again the use of a single operator and analyzing the samples in groups rather than as individual teeth attempts to reduce these problems. Although it is important to minimize such variables, they will not detract significantly from the results obtained because, clinically, such anatomical and operator variations are also evident. Subjective measurements in the previous studies have also increased the margin of error (9, 13). In this study the use of the Iz5I radioactive tracer detected by a gamma counter allows for quantitive measurements to be made at each root level, without affecting the filling. The measurements were not subject to observer variation and could be easily analyzed and compared. The gamma counter gave accurate values for radioactivity in each sample while taking into account the background radiation by measuring it before beginning each sample. Various isotopes have been used to study leakage, because they are smaller than dye molecules and diffuse more rapidly than other small molecules (7). The '2sI-labeled lysozyme used in this case was chosen because of its low molecular weight. Although penetration of bacteria into the root canal may be minimal due to their large particle size and the fact that they are absorbed specifically
Haikel et al.
to the root surface by the action of immunoglobulins, penetration of much smaller nutrients into the root canal system may encourage multiplication of bacteria that have not been removed during canal preparation (8),and some antigenic substances released from bacteria in the periapical region may easily enter the canal because they may have a molecular weight as low as 1,000 D (10). Another reason for choosing lysozyme as the tracer was that it does not interact with tooth substance, whereas 45Ca that has been used in the past undergoes ionic exchange with the apatite crystals and so interferes with the true pattern of leakage (9). To prevent isotope penetration apart from in the apical region, nail polish was used to seal the external root surface. This technique is frequently used, but a study of surface sealing materials (17) found high leakage levels with nail polish and concluded that nail polish was not an adequate sealant for short-term microleakage studies. Despite these findings, the negative control group showed very low levels of isotope penetration, suggesting that nail polish would seal the roots sufficiently for this study. The high levels of leakage seen in the positive control group indicated that lack of sealant cement will result in greatly increased levels of apical microleakage and so confirmed the necessity for its use in root canal obturation. Another potential problem with the experimental technique that had occurred in previous studies (12) was loss of tooth substance on preparing the horizontal sections. The use of a fine diamond wire (0.17 mm diameter), which cut slowly and without pressure under lubrication, enabled the amount of tooth substance lost to be minimized. One of the main findings of this study was that levels of apical penetration varied over time. The results obtained support the opinion of Delvanis and Chapman (9), who suggested that 2 days (the period used in many apical leakage studies) is not long enough for maximal leakage to be detected. Rhome et al. (1 8) undertook a 6-month study of leakage that demonstrated a rise and fall of leakage values over the time for all methods of condensation (lateral and vertical gutta-percha condensation and Hydron). They found leakage to be greatest in the initial weeks (which is the time period of this study). Changes in permeability of the apical seal may be due to a change in the properties of the filling over time. These changes may be due to the material used, for example, the sealer cement is likely to resorb over time resulting in increased leakage (19). The obturation techniques involving heating of gutta-percha may also cause an increase in permeability in the initial stages when shrinkage occurs as the gutta-percha cools (2).It should, however, not be forgotten that, as the samples are immersed for a longer time period, readings will increase simply because infiltration is occurring with the extended time. Throughout the experiment, the variations in permeability meant that different methods of obturation appeared effective at different stages, so that no one method could be said to be overall better than any other. In general the levels of infiltration observed in all samples were low coronal to the third section so that any of the techniques can be considered effective.
Journal of Endodontics The authors thank B. Senger for his statistical assistance. Drs. Haikel, Freymann, Fanti, and Poumier are affiliated with the Universite Louis Pasteur, Faculte de Chirurgie Dentaire, Strasbourg, France. Drs. Hai’kel and Poumier are affiliated with the Centre de Recherches Odontologiques. National Institute of Health and Medical Research, Strasbourg, France. Dr. Watson is affiliated with the ERASMUS Exchange Program, University of Glasgow, Glasgow, Scotland. Dr. Claisse is affiliated with the Faculte de Chirurgie Dentaire, Place de Verdun, Lille, France. Address requests for reprints to Dr. Y. Haikel, Universite Louis Pasteur, lnserm U-424, Faulte de Chirurgie Dentaire, 1 Place de I’Hbpital, 67000 Strasbourg, France.
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