The study was performed on 25 fresh cadaveric porcine mandibles of male and female pigs that were obtained from the slaughterhouse in Kiel- Wellsee- Germany. Animals were between seven and nine months old at the time of slaughter. The heads were stored frozen after decapitation at –21°C and then thawed in the refrigerator at 4°C for 48 hours before the root resection and root canal filling was performed.
Root-end filling materials
Five different root filling materials have been studied; Diaket™, AH-Plus™, Super-EBA™ regular,
Super-EBA™ fast und ProRoot® MTA. (Table 1).
Apicoectomy and root-end filling procedure
Apical resection was performed on 100 canals of 50 maxillary premolars. 48 hours before the operation, frozen pork jaw halves were thawed in the refrigerator at 4°C. They were disinfected with Betaisodona® solution (Mundipharma GmbH, D–65549 Limburg). An incision was made with a no. 11 Scalpel and a full thickness mucoperiosteal flap was reflected. Osteotomy was performed with a ball mill (0.5 mm) under irrigation with isotonic saline solution exposing the apical third of the experimental tooth roots. The root tips were removed 2 - 3 mm away from the apex with a Lindemann cutter in vestibulo-oral direction with an angle of less than 45 °. Retrograde cavities were prepared with dental hand piece (D–88400 Biberach / Riss INTRAmatic, KaVo Dental GmbH,) and washed with 3% H2O2 and 70% alcohol and then dried.
The root-end of 24 canals were retrogradely filled with Diaket ™, 37 root canals with Super-EBA ™– (18 with Super-EBA ™ Regular and 19 with Super-EBA ™ fast), 20 canals with ProRoot® MTA and 19 canals with AH-Plus ™. (Table 2) All materials were prepared and applied according to the manufacturers guidelines.
Preparation of the samples for SEM
After completion of the root–end fillings, teeth were osteotomised with hammer and chisel en bloque from the bone segment (Figure 1) and stored in 4% formalin. The teeth were cut in half in vestibulo-oral direction and were placed in in phosphate buffered solution (pH 7.4 to Sörensen KH2PO4 / Na2HPO4) (Walter GmbH & Co. KG, D–24116 Kiel) for 20 minutes. The procedure was repeated three-times. The samples were trimmed with a wet grinder (Jean Wirtz, D-Düsseldorf Germany) and Siliconkarbitpaper with a grain of 400 in the transverse or longitudinal axis at a predetermined cutting plane, before the surface could be polished to a high gloss with a grain of 1200 - 4000.
Before taking an impression of the roots with Silagum® AV light, an addition-cross linked silicone (DMG Chemical Pharmaceutical Factory GmbH, D–22547 Hamburg Germany), with 30% isopropyl alcohol was used to clean the surfaces. The impressions were prepared with epoxy resin (Stycast (TE-adhesive technology, D–30165 Hannover Germany) and left at room temperature for 24 hours.
The replica models were placed on the alluminum specimen stubs (Agar Scientific Ltd., Essex CM24 8
DA, UK) of the SEM. Electronic conductivity has been reached by vapor deposition (sputtering) of the samples with a 20 micron thin gold alloy at a pressure of 0.1 bar and at a voltage of 15 mA for 90 seconds under a vacuum.
To evaluate the samples obtained from the apical portion of the root tips, 60, 190, 320 and 600 fold magnification options were required on the screen of the SEM. To determine the marginal gap value, two points were determined in the area of greatest expansion and the distance was measured in microns.
Preparation of the samples for Microradiography
After completion of the root–end fillings, teeth were osteotomised with hammer and chisel en bloque from the bone segment and stored in 4% formalin. With a band saw (Metabo, D–78822 Nürtingen Germany), the teeth were initially cut in half in vestibulo-oral direction, so that two root canals could be obtained seperately. The preparations were then placed over a period of two days in an embedder (PSI, CH–5232 Villingen) containing an alcohol series (20%, 40%, 60%, 80%, 90%, 2 times 100% for each 45 - 60 minutes and finally 100% for 6 - dehydrated 8 hours) and then were soaked in methacrylate. The samples were placed in a methacrylate solution for another two weeks before they were placed in glasses capped with methacrylate and polymerized with fresh methacrylate solution in water bath at 38°C. The composition of the embedding medium is given in Table 3.
The polymerized samples were first cut with the band saw and the surfaces of the blocks were polished to 4000 with a Siliconcarbitpaper with a grain size of 400, before the cutting plane for the actual recovery preparations was determined. Each sample was cut along its sectional plane with the band saw again, surface was polished, glued to a microscope slide and reduced with a precision hole saw (Leica GmbH, D- 64625 Bensheim) to 100–200 micron thickness.
Via a wet grinding machine (Struers A / S, DK–2610 Rodovre) the samples were refined up to 70 - 110 micron and polished with a Silicon carbitpaper with a grain size of 800 to 4000. The samples were taken from the slide and put on high-resolution Microradiography plates in which the resolution is 2000 lines per millimeter ( High Resolution plates ( Kodak, Rochester, NY 14650, USA). Subsequently, the exposure was performed in a Mikroradiographiekammer Faxitron 53855A (Hewlett -Packard, McMinnville, OR 97128, USA) at a focus distance of 16 cm. The voltage was set to 25 kV at a power of 3 mAs, exposure time of 6 minutes and 30 seconds for 70 micron samples and up to 10 minutes and 30 seconds for 110 microns film thickness. The exposure time was increased by increments of one minute per 10 microns thickness of the specimen.
The plates were embedded for five minutes in Kodak® HRP developer at 20 ° C under constant movement swung (HRP developers /distilled water: 1/3). In a 1% acetic acid bath, the development was stopped after one minute. For fixation, Kodak® fixer 300A was used for 10 minutes with agitation ( Kodak® fixer 3000A / distilled water:1/3). This was followed for 15 minutes by washing with water and a final rinse with Agepon® ( 400 mL of distilled water and 2 ml Agepon®) for one minute.The plates were air dried and then covered with cover glasses ( 4 x 4.5 cm) using 1 - 2 drops of n - butyl acetate ( xylene substitute) and a drop of Eukitt-air for 24 hours. After a drying period of seven days under an air extractor, the preparations were digitally photographed under a light microscope at a magnification of 1:18. The evaluation was performed using an image editing program (Adobe Photoshop 7.0 for Windows)
The statistical analysis was performed by using SPSS for Windows, version 14.0 (SPSS Inc., USA). The continuous variables were analyzed using the Kolmogorov-Smirnov test with respect to their normal distribution. In analysis of the abnormal distribution of the tested variables (Kolmogorov-Smirnov test: p <0.05), non-parametric tests were used.
Since the comparisons template more than two independent, not normally distributed random samples, the H-test was used by Kruskal and Wallis. A p-value <0.05 was taken as statistically significant for all statistical tests. In the graphs, which were also created using SPSS, error bars were used to illustrate the mean values.