Leakage of bovine serum albumin (bsa) in root canals obturated with super-eba and irm abstract

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The aim was to determine the leakage of SuperEBA and IRM in root canal samples with or without orthograde filling, by evaluating Bovine Serum Albumin microleakage using spectrophotometry.

Thirty five single-rooted teeth were divided into five groups, instrumented, and had apices resected. Root-end cavities in Groups I and II were filled with SuperEBA and IRM. The samples from the Groups III, IV and V were filled with gutta-percha and sealer. In Groups IV and V, root-end cavities were filled with SuperEBA and IRM.

The greatest microleakage of BSA after 60 days was observed in Group II (4.1±0.0011 ng), then in Groups III (3.4±0.0064 ng) and I (2.6±0.0019 ng). Samples from Groups IV and V leaked the least (0.7±0.0014 ng).

Significantly less leakage (p<0.05) occurred in samples filled with orthograde and root-end fillings than did in samples filled only with an orthograde approach and the samples with IRM root-end fillings.

Key words: SuperEBA, IRM, spectrophotometry, BSA
Root-end resection and placement of a root-end filling is a widely applied procedure in surgical endodontics. This is indicated in the cases where orthograde therapy of the root canal does not ensure restitution of the periapical tissues and healing (1). An ideal root-end filling material should: produce a complete apical seal, have antibacterial activity, be non-toxic, be biocompatible, be non-absorbent, be dimensionally stable, be easy to manipulate, be unaffected by moisture and radiopaque (2). Several different materials have been proposed as root-end fillings, included are: amalgam, thermoplasticized gutta-percha, zinc-oxide-eugenol (ZOE) cements, composite resins, polycarboxylate and glass-ionomer cements, mineral trioxide aggregate (MTA) (3). Amalgam is still a widely used material, regardless of its disadvantages including initial leakage, secondary corrosion, galvanic interaction, a suggestion of mercury and tin into the body, retentive undercut in the cavity preparation and tissue discoloration (4). Materials based on ZOE have been proposed as root-end filling materials quite some time ago, and are still used and studied (5,6). A potential advantage of these materials is their antibacterial effect and good sealing ability though their solubility has been questioned (7,8,9). Modified forms of ZOE cements have been developed in order to increase strength and decrease solubility. Intermediate Restorative Material (IRM), a zinc-oxide and eugenol material reinforced with polymethylmethacrylate, has been recommended due to its biocompatibility, marginal integrity and acceptable progression of wound healing (10). Super EBA cements were developed in the 1960's as a less irritant substitute for zinc phosphate cements. Its liquid contains 68% o-ethoxy benzoic acid (EBA) as well as 32% eugenol (6). It has been proposed as a root-end filling as a result of its good sealing properties as stated in in vitro dye leakage and fluid filtration studies (11) as well as the good tissue response to Super EBA cement (12). Dorn and Gartner (4) reported good clinical results when using ZOE cements reinforced with ethoxybenzoic acid as root-end fillings. Recent studies report mineral trioxide aggregate (MTA) to seal off all the pathways of communication between the root canal system and the external surface of the tooth, which together with the studies concerned with its biocompatibility and regeneration of periradicular tissues, makes MTA prefarable to SuperEBA (13, 14, 15, 16, 17). However, some comparative studies involving SuperEBA and MTA reported that there were no significant differences in microleakage between the two materials (18, 19).

Several research models have been applied to evaluate the quality of root-end filling materials and to estimate apical microlekeage including the use of dyes, radioisotopes, bacteria and endotoxins, electrical current, SEM microscopy, fluid filtration, albumine protein lekeage (20). Each technique has been questioned and has its limitations (20, 21, 22).

By determining the quantity of bovine serum albumin (BSA) leaked using spectrophotometry, Bradford's (23) protein essay enables the estimation of root-end filling microleakage in all planes. We considered this method to be suitable for testing the sealing of SuperEBA and IRM sincs no signs of desintegration of the two cements in buffered phosphate and bovine serum were noticed after 6 months (16).

The purpose of this study was to quantitatively determine the leakage of two root-end filling materials: SuperEBA and IRM, with or without orthograde gutta-percha and Diaket sealer filling, by measuring BSA microleakage using Bradford (23) method of protein quantitation.

Teeth preparation

Forty six single-rooted teeth with straigth canals were collected for the experiment. The teeth had been extracted for periodontal and orthodontic reasons and were stored in phosphate-buffered saline solution until use.

The teeth were randomly devided into five experimental groups wherein each group with seven teeth. Seven teeth served as negative control and four teeth as positive control. The roots of the teeth were scaled and planed using Gracey curets. Standard access was made with a round diamond bur under water cooling. The root canals were prepared with the conventional step-back technique using K-files and Hedstrom files (Maillefer, Ballaigues, Swiss). The master apical file was #30. After each instrument the root canal was irrigared by 5 ml of 2.5% NaOCl solution. Gates-Glidden burs #'s 2 and 3 were used in widening the acces opening.

The teeth from Groups III, IV and V were filled conventionally with gutta-percha points (Maillefer, Ballaigues, Swiss) and Diaket sealer (ESPE, Seefeld, Germany) using a cold lateral condensation technique. The teeth were stored in a saline solution for 7 days to allow full setting of the orthograde filling materials. The apices of all teeth (Groups I-V) were resected using a diamond bur under a continous water spray at an angle of 90° 3 mm from the apex. In groups I, II, IV and V, root-end cavities 3 mm deep were prepared using a round carbide bur. Root-end cavities and root canals were dried with paper points #30 (Johnson & Johnson, Slough, GB). Root-end cavities in Groups I and IV were filled with Super-EBA (Staident International, Staines, Great Britain) and in Groups II and V with IRM (DeTrey, Dentsply, Germany). In the teeth that had not recieved orthograde filling (Groups I and II), the root-end filling was placed against a finger spreader in the root canal to avoid the material spreading into the canal. The teeth from Group III did not receive a root-end cavity preparation and filling. The samples were finally filled as follows: I. SuperEBA, II. IRM, III. orthograde filling, IV. orthograde filling and SuperEBA, V. orthograde filling and IRM. All teeth were stored in saline solution for another 2 weeks at room temperature in order for the root-end filling material to set. A day prior to setting the apparatus for protein lekeage, two coats of nail polish were applied to the whole surface of the total length of each root except the resected root-end.

The teeth used as neagtive control were instrumented, obturated with gutta-percha and sealer and their entire root surfaces were covered with two coats of nail polish. The teeth used as positive control were instrumented, had apices resected, root-end cavity prepared and recieved no root-end filling. In the positive control group, the glass vile was filled with 8,5 ml of redistilled water to allow full leakage.
Lekeage of Bovine Serum Albumin protein

The experimental model was similar as in Valois CRA et al. (20) study (Figure 1.). Using a heated hand instrument (diameter 1 mm), a hole in the rubber stopper of a 10 ml glass vial was made wherein a tooth was inserted through it so that the resected root-end was pointed upwards and the crown downwards, in the vial. Rappid-setting cyanoacrylate was used to seal up the gaps between the tooth and stopper, and the plastic cylinder made of syringe and the stopper. The glass vial was filled with 9.5 ml of redistilled water and closed with stopper to which the tooth and plastic cylinder were attached as described. The cylinder was filled with 1 ml of 22% BSA (A-7906, Sigma Chemical Co, St Louis,Mo) solution. Parafilm was used to fasten the stopper to the vial. The upper side of the plastic cylinder was covered with aluminium foil.

The apparatus was set for all 35 teeth, the positive and negative control. The experiment was conducted in humidor at 37 o C during 60 day period. The protein solution was replaced every day by fresh solution stored at 4 o C.
Quantitation of root-end filling lekeage through absorbance (A) measurement

Two sets of measurements were conducted; a seven day and a 60 day period. The protein reagent and the calibration curve were made before each measurement (23). Bradford protein reagent is an aqueous solution of Coomassie Brilliant Blue G (B5133; 100mg; Sigma Chemical Co, St Louis, Mo), ethanole and phosphoric acid. The reaction is based on the observation that the maximum absorbance of Coomassie Brilliant Blue G is shifted from 465 nm to 596 nm when in complex with albumin (23). The test solution was the solution from the glass vial. In the first set of measurements, the stopper was gently removed together with tooth and plastic cylinder, and only 100µl of the test solution was pipetted into an Eppendorf tube. One ml of freshly filtered Bradford protein reagent was added to the test solution, vortexed, transferred into disposable civettes and the absorbance was recorded at 596 nm. Glass vial was refilled with 100 µl of water and the apparatus was reset.

After 60 days, the test solution was transferred into a test tube and vortexed in order for the solution to be homogenised. 100 μl of the homogenised test solution was pippeted into an Eppendorf tube and 1 ml of the protein reagent was added. The solution was vortexed, left for 5 minutes, transferred into disposable civettes, and the absorbance for each specimen was measured. Redistilled water served as blind probe (A=0).

The mass values (ng) of the BSA protein that leaked besides the filling, were calculated using absorbance values and calibration curve coefficient. Statistical analysis was carried out using ANOVA test and Tuckey's method at the significance level of 5% (p<0.05).

The positive control samples showed complete leakage of BSA during first 24 hours. In the negative control group the absorbance was 0 after 60 days, i.e. BSA protein leakage did not take place.

After 7 days, the microleakage was observed in only two specimens: in Group II (0.64 ng of BSA) and in Group III (8489.5 ng). Due to an improperly set apparatus, the specimen in Group III showed a very large leakage and was excluded from further measurements.

After 60 days, the highest mickroleakage was observed in Group II (4.1±0.0011 ng), then in Group III (3.4±0.0064 ng) and Group I (2.6±0.0019 ng). Samples from Groups IV and V leaked the least and at equal rate (0.7±0.0014 ng) (Figure 2.). ANOVA analysis showed that there was significant difference between the groups (F=7,054; p=0,000428) and the Tuckey's analysis showed that the statistically significant difference (p<0,05) existed between Groups II and IV, Groups II and V, Groups III and IV and Groups III and V (Table 1).

Our results show significantly less BSA leakage in the samples filled with orthograde and root-end fillings than in the samples filled only with orthograde fillings and the samples with IRM root-end fillings (Table 1.). The samples with SuperEBA root-end fillings did not leak significantly more than the samples with orthograde and root-end fillings. This would suggest that SuperEBA is good apical plug which is in concordance with the results of some microleakage studies where fluid filtration and dye leakage methods were used (11, 14, 24, 25).

The leakage in the samples filled only with orthograde approach with gutta-percha and Diaket sealer, and only retrograde, either with SuperEBA or IRM, was not significantly different. These results agree with the results obtained by Sullivan et al. (11) and Fogel et al. (14) who had used fluid filtration model and reported no statistical differences between the microleakage with SuperEBA and IRM. Nevertheless, Theodosopoulou & Niederman (26) systematically reviewed studies concerned with retrograde obturation materials evaluated in vitro by dye/ink penetration method. They reported that SuperEBA cement provided a less effective sealing than IRM and orthgrade gutta-percha and sealer after 10 days, based on the linear regression analysis (26). However, in the studies included in the review IRM was examined over a period ranging from 0 to 7 days while SuperEBA and orthograde condensed gutta-percha with sealer were examined over periods of time ranging from 0-84 days and 0.1-180 days, respectively (26). This could bring into question the conclusion about SuperEBA providing less effective sealing than IRM and orthograde gutta-percha and sealer. Whitworth & Baco (27) reported that gutta-percha with epoxide amide and addition curing silicon sealers, known to expand on setting, did not provide significantly better coronal seal than gutta-percha alone (p>0.01), but, the sealers alone performed significantly better than gutta-percha alone and gutta-percha with either sealer (p<0.001). This interesting finding may lead to testing root-end sealing in vitro where apices were resected after the canals had been filled with well adapted resin based and silicon sealers without root-end cavity preparation and any other root-end material placement.

Martell & Chandler (28) studied the sealing properties of three root-end filling materials; included were IRM and SuperEBA which were compared using electrochemical and dye leakage methods. The study did not show any significant difference between SuperEBA and IRM using dye leakage method. However, the samples filled with SuperEBA leaked significantly less then the samples filled with IRM in the electrochemical test. Fischer et al. (13), used bacterial leakage to estimate the sealing properties of four root-end materials and reported a significantly better seal with Super EBA than with IRM. This could be for the reason that bacteria need 24-48 hours to grow after contamination which could lead to different results then with a dye leakage and fluid filtration method (20). Moreover, bacteria-sized particles are larger then albumin protein molecule which has relatively low molecular weight compared to large protein molecules such as globulins and fibrinogens (29) . This is supported by the Kersten and Moorer (22) study in which they reported that while the leakage of bacteria-sized particles and large size protein molecules could be prevented with some of the obturation techniques, microleakage of the small molecules could not be prevented. The protein molecules involved in this experiment are larger then dye molecules and smaller then bacteria. Although the method enables the measurement of total leakage, it is important to remember that the dynamic interaction between the root canal and the periradicular tissue is not represented by the static model used in this study.


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Figure 1. Apparatus used in the study to determine bovine serum albumin (BSA) protein microleakage.
Figure 2. Box & Whiskers diagram showing the microleakage of bovine serum albumin (BSA) expressed in nanograms (ng). Group I- SuperEBA; Group II- IRM; Group III- Orthograde gutta-percha and sealer; Group IV- Orthograde gutta-percha and sealer + SuperEBA; Group V- Orthograde gutta-percha and sealer +IRM.


Table 1. Thuckey's analysis of the leakage of bovine serum albumine (BSA) in 5 experimental groups (p<0,05).

Group I- SuperEBA; Group II- IRM; Group III- Orthograde gutta-percha and sealer; Group IV- Orthograde gutta-percha and sealer + SuperEBA; Group V- Orthograde gutta-percha and sealer +IRM.
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