Materials
TheraCal LC (Bisco Inc, Schaumburg, IL, USA)
Light-curing, resin-modified calcium silicate in single paste [Composition: Calcium silicates (Portland cement type III), Bis-GMA (Bisphenol A diglycidyl methacrylate), PEGDMA (Polyethylene glycol dimethacrylate) and Barium zirconate].
Mineral trioxide aggregate (Angelus, Londrina, PR, Brazil)
[Composition: Powder: Tricalcium silicate, Dicalcium silicate, Tricalcium aluminate, Tetracalcium aluminoferrite and Bismuth oxide. Liquid: Distilled water].
Biodentine (Septodont, Saint-Maurdes- Fosses, France)
[Composition: Powder: Tricalcium silicate, Dicalcium silicate, Calcium carbonate, Iron oxide and Zirconium oxide. Liquid: Water, Calcium chloride and Modified polycarboxylate].
Adaptability evaluation
Sample selection and preparation
Three freshly human extracted single rooted teeth were obtained from patients who received orthodontic treatment at the outpatient clinic, Faculty of Dentistry, Ain Shams University, Egypt. The institutional ethical committee approved the work on the extracted teeth without consents. The teeth were cleaned by removing the hard deposits using curette and the soft tissues by soaking in 5.25% NaOCl for 10 min. Sample preparation was performed by removing the crown part and apical segment of each tooth to leave the coronal and middle third measuring 10 mm in length using a high speed hand piece under water spray. The roots were placed in acrylic blocks and three horizontal sections (2 ± 0.1 mm thick) were made from root segments of each tooth using a diamond disc under continuous water irrigation and the final thickness of each slice was measured with a digital caliper having an accuracy of 0.001mm (CenTech 4, Harbor Freight Tools, Calabasas, CA, USA). Low speed round bur size 1.2mm diameter (ISO size 012, Komet Dental, Gebr, Brasseler GmbH &Co. KG, Germany) was used to drill three perforations like cavities parallel to the root canal in each root slice. A minimum distance of 1 mm was maintained between the cavities, the external cementum and the root canal wall. Afterwards, all root slices were immersed in a 2.5% sodium hypochlorite (NaOCl) solution for 15 min and then immersed in distilled water to neutralize the NaOCl solution. The smear layer was removed using 17% Ethylenediaminetetraacetic acid (EDTA) solution for 3 min, distilled water for 1 min, 2.5% NaOCl for 1 min and a final flush with distilled water for 1 min [10]. The cavities were then dried with absorbent paper and the three cavities of each root slice were randomly filled with one of the selected experimental materials. The cavities filled with TheraCal LC were light cured for 20s while Biodentine, MTA-Angelus were mixed according to the manufacture's instructions as described before and delivered to the cavities with the special MTA carrier. Nine root slices with twenty seven cavities were produced then divided into 3 groups (9 cavities each); group I: filled with MTA-Angelus, group II: filled with Biodentine and group III: filled with TheraCal. Each slice was marked with an indelible marker. The filled root slices were incubated in contact with gauze moistened with saline solution at 37°C for 24h for a complete setting of the materials.
Samples evaluation
All samples were examined by a scanning electron microscope (SEM Model Quanta 250 FEG (Field Emission Gun) attached with EDX Unit, NTS Gmbh, Germany) at low vacuum. The perimeter of each cavity was divided into 4 quarters and the presence of any gap between the dentin surface and filing material in each quadrant was analyzed at X1000 magnification in scanning electron microscope. The marginal adaptation was classified according to Aggarwal et al. [11] into; continuous non gapped margin (continuous interface between the filling material and dentin with less than 1um gap) and gapped margin (interface between the filling material and dentin with gaps more than 1um wide).
Solubility evaluation
Classification and preparation of samples
Split Teflon ring Molds (2mm thick and 8mm internal diameter) were used to prepare 30 identical specimens. These specimens were divided into three groups (10 specimens each) according to the material used; group I: MTA-Angelus, group II: Biodentine and group III: TheraCal. Each group was subjected to solubility and disintegration testing according to the International Standards Organization (ISO) 6876 method [12] and with American Dental Association (ADA) specification No.30 [13].
Solubility test
The Split Teflon ring Molds were placed on a glass plate covered with cellophane paper while the waxed thread was placed inside the mold that was filled to slight excess with tested materials. After filling the mold, another glass plate covered with cellophane paper was placed on the top of mold exerting light pressure to remove any excess material. The molds were stored in an incubator (Heraeus incubator, West Germany) at 37oC and 95% humidity then the materials were left to set. The specimens were removed from the mold and the net weight of each specimen was recorded (W0) by high precision electrical weighting balance (Precision electrical weighting balance, Percisa 120A,West Germany) adjusted to give 0.0001-gram accuracy.
Each specimen was suspended vertically by the waxed thread in a clean glass beaker filled with distilled water at 37oC in a special incubator for 1 week. At the end of the period, the specimens were removed from their glass beakers and rinsed with a little distilled water and the surplus water was removed from the specimens by gentle blotting with paper tissues. The specimens were stored in desiccators containing thoroughly dry anhydrous calcium sulfate (CaSO4) for 24h and reweighed to nearest 0.001gram (W1). Solubility percent for each sample was calculated as follows: % Wight loss = W0 - W1 / W0 x 100
Biocompatibility evaluation
Animal model
The present study was approved by the Institutional Animal Care and Use Committee at Faculty of Dentistry, Ain Shams University, Egypt. The authors followed up all institutional regulations and The Animal Research: Reporting in Vivo Experiments guidelines (ARRIVE).
Eight adults clinically free mongrel male dogs were obtained commercially from Al-Fahad Trading Company of Animals (Abu-Rawash, Giza, Egypt). The average weight of these dogs was15 to 20 kg and the average age was 2 to 3 years. The animals were quarantined in separate cages at Department of Surgery, Anesthesiology and Radiology, Faculty of Veterinary Medicine, Cairo University, Egypt. The dogs were fed, examined and kept under observation of an expert veterinarian for two weeks before they were used as experimental animals in this study.
Classification of samples
In each dog 10 bi-rooted premolars were used with total number. The animals were divided according to the evaluation period into 2 main groups (4 dogs each); group I: after 1 month and group II: after 3 months. Each group then subdivided into 4 subgroups (10 teeth each) according to the material used for perforation repair as follows; subgroup (A): MTA-Angelus, subgroup (B): Biodentine, subgroup (C): TheraCal and subgroup (D): Positive control (perforation without sealing). All subgroups were represented in each dog.
Surgical procedure
Each dog was anesthetized with general anesthesia after fasting for 12h using Atropine sulphate (Atropine sulphate®, ADWLA Co., Egypt) at a dose of 0.05 mg/kg body weight injected subcutaneous, Xylazine HCl (Xylaject®, ADWIA Co., Egypt) at a dose of 1mg/kg body weight injected intramuscular and Ketamine HCl (Keiran®, EIMC Pharmaceuticals Co., Egypt) injected intravenous through a cannula in the cephalic vein at a dose of 5mg/kg body weight. The anesthesia was then maintained by using a 2.5 % solution of Thiopental sodium (Thiopental sodium®, EIPICO, Egypt) at a dose of 25 mg/kg body weight injected intravenous (dose to effect). Preoperative periapical radiograph was taken for each tooth to confirm the absence of periodontal defect and bone loss.
Access cavities were made through the occlusal surface of the bi-rooted premolars using tapered diamond stone with a conventional speed hand piece mounted on the electric motor. The chamber was irrigated with Naocl 2.5% and the pulp tissue was removed using suitable excavator. Size 15 K file was carried into the canals till the apex and the working lengths were confirmed by apex locator. The canals were cleaned and shaped up to the working length using StSt hand K files size 35 and 40. The canals were irrigated with Naocl 2.5% and saline solution, then dried with paper points. The canals were obturated with gutta-percha cones and resin sealer using the lateral compaction technique.
A perforation was made in the center of the floor of pulp chamber using a sterile, low speed round bur 1.2mm diameter (Komet Dental, Gebr, Brasseler GmbH &Co. KG, Germany) (ISO size 012) [14] to penetrate the furcation area into the periodontal tissue. The penetration depth was estimated 2mm into the alveolar bone using a rubber stopper as a marker on the shank of the bur. The observed bleeding was controlled with paper points and the perforations sites were irrigated with saline solution.
The perforations were sealed immediately by one of the three tested materials according to the subgroup. The perforation area was left open without repair in the positive control subgroup. The materials were prepared and mixed according to manufacturer, s instruction as follows:
MTA-Angelus
One part of water was added to 3 parts of cement, then gradually incorporated the liquid into the cement using a plastic mixing stick for about 1 min. The paste was carried out into the perforation by the special MTA carrier and compacted with a suitable size plunger.
Biodentine
The capsule was gently tapped on a hard surface to loosen the powder, then opened and placed on the white capsule holder. Then 5 drops of the liquid were poured into the capsule. The capsule was closed and placed on a mixing device (amalgamator) at a speed of 4000 rotations/min for 30s. Then the material was collected and carried out to the perforation site by a special MTA carrier and compacted with a suitable size plugger.
TheraCal LC
Ready to use paste in a single syringe (1g) was injected directly into the perforation site and then cured for 20s with light cure (MINIS curing light/Woodpecker medical instrument).
The coronal access cavities of all teeth were filled with chemical cured Glass ionomer cement as a permanent restoration (Riva Light Cure LC/Southern Dental Industries SDI).
Radiographic evaluation
Two periapical radiographs were taken for each tooth using conventional size 2 dental films (Kodak Ultra-Speed Dental Film®). Postoperative periapical radiograph was taken for each tooth immediately after perforation repair and follow-up periapical radiograph was taken for each tooth after the evaluation period of each group. The radiographs were processed using an automatic film processor (Durr, Periomat plus) and transferred to a PC computer with transparency scanner (HP Scanjet G4050 Hewlett-Packard Development Company, L.P.) then evaluated. According to a previous study by Vanni et al. [17], the evaluation was performed by an experienced operator unaware of different experimental groups with respect to the presence or absence of radiolucency in the furcation region.
Histopathological evaluation
According to the group, all dogs were euthanized by anesthetic overdose using 20mL of 5% Thiopental sodium injected rapidly through the cephalic vein. Both jaws were dissected and sectioned into halves at the midline using a saw. Block sections including the experimental teeth with its surrounding bone were obtained. The blocks were fixed at 10% buffered formalin solution with a ratio of 1:50. After two weeks of fixation, blocks were decalcified by 17% EDTA solution for 120 days. After decalcification, the specimens were prepared as usual and stained with hematoxylin and eosin. The stained sections were blindly examined by a pathologist using a light microscope (OLYMPUS BX60 Microscope, Olympus Inc, Japan) and photographs were taken using a digital camera fitted to the light microscope (Canon EOS 750 D, Canon Inc, Japan). The inflammatory cell count was assessed. For each slide, three microscopic fields were captured at magnification 400X. The inflammation was categorized by counting visible inflammatory cells on each field, according to previous studies [15,16] as follows:
Score 0: represented non or few inflammatory cells
Score 1: represented less than 25 cells
Score 2: represented 25-125 cells
Score 3: represented more than 125 cells
Statistical Analysis
Quantitative data were presented as mean, standard deviation (SD), range (Minimum – Maximum) for numerical values. Data were explored for normality by checking the data distribution and using Kolmogorov-Smirnov and Shapiro-Wilk tests. ANOVA was used and followed by Tukey’s post-hoc test for pairwise comparisons when ANOVA test was significant. Chi square test was used for categorical data. The significance level was set at P = 0.05 and 95% Confidence interval. Statistical analysis was performed using Graph Pad Instat (Graph Pad, Inc.) software for windows.