Background
Perforations represent pathologic or iatrogenic connections between the root canal space and the external tooth surface. Furcation perforation of the pulpal floor in multi-rooted teeth results in periodontitis and irreversible attachment loss [
1].
Several materials have been applied for repairing furcation perforation such as; reinforced zinc oxide eugenol, amalgam, super EBA, calcium hydroxide, composite resins, glass ionomer, MTA, bioaggregate, biodentine, platelet rich plasma (PRP), platelet rich fibrin (PRF) and others, but none of them has the characteristics of an ideal repair material [
1‐
3]. An ideal perforation repair material should be inducing osteogenesis, cementogenesis and good sealing, nontoxic, noncarcinogenic, biocompatible, dimensionally stable and insoluble in the body's fluids [
2,
3].
The MTA is one of the most commonly used root repair materials due to its good biocompatibility, marginal adaptation, bacterial leakage, and low cytotoxicity, but it has some drawbacks such as long setting time (3 h), difficult applicability and expensiveness [
1‐
3]. The MTA-Angelus (Angelus, Londrina, PR, Brazil) is another formulation of ProRoot MTA that has short sitting time (10 min) [
4].
Biodentine (Septodont, Saint-Maurdes-Fosses, France) is an alternative to MTA due to its similar properties when compared with the MTA, but with better handling properties and faster setting time [
3]. A modified powder constituents, addition of setting accelerators and softeners, and a new predosed capsule formulation are responsible for the improvement of the physical properties of biodentine [
5].
In recent years, innovations are still carried out to introduce recent endodontic repair materials that overcome the shortcomings of the available materials. TheraCal LC (Bisco Inc, Schaumburg, IL, USA) is a new light-curing Resin Modified Calcium Silicates (RMCS) material that has been recorded to enhance apatite formation and secondary dentin. Due to its high physical properties and low solubility, TheraCal is applied mainly as a barrier and for protection of dental pulp complex [
6]. Therefore, evaluation of the physical and biological properties of this newly introduced resin modified calcium silicate-based material (TheraCal LC) is thought to be valuable.
The marginal adaptation, solubility and biocompatibility are the main tested properties of the furcation perforation repair materials [
7‐
9]. The hypothesis of this study was that TheraCal LC will alternate the MTA and Biodentine as a furcation perforation repair material. Therefore, this study compared the repair potential of TheraCal LC Resin Modified Calcium Silicate material against the MTA-Angelus and Biodentine as a furcation perforation repair material, in terms of marginal adaptation, solubility and biocompatibility.
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 (Protocol No. 349-Endo). 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 weight of these dogs was15 to 20 kg and the 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. The animals were randomly 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 was randomly 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 LC 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 12 h using Atropine sulphate (Atropine sulphate®, ADWLA Co., Egypt) at a dose of 0.05 mg/kg injected subcutaneous, Xylazine HCl (Xylaject®, ADWIA Co., Egypt) at a dose of 1 mg/kg injected intramuscular and Ketamine HCl (Keiran®, EIMC Pharmaceuticals Co., Egypt) injected intravenous through a cannula in the cephalic vein at a dose of 5 mg/kg. 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 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 a 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 a 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 stainless steel hand K files size 35 and 40. The canals were irrigated with NaOCl 2.5% and saline solutions, 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 1.2 mm 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 2 mm 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 30 s. Then the material was collected and carried out to the perforation site by the special MTA carrier and compacted with a suitable size plugger.
TheraCal LC
Ready to use paste in a single syringe (1 g) was injected directly into the perforation site and then cured for 20 s 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).
For pain and infection control, all dogs were given intra-muscular injections of Cefotaxime sodium at a dose of 10 mg/kg (Cefotax 250 mg vial®, T3A Co., Egypt) and Diclofenac sodium at a dose of 1.1 mg/ kg (Voltaren 75 amp®, Novartis Co., Egypt) once daily for five postoperative days [
15]
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. [
16], 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 an anesthetic overdose using 20 mL 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 surrounding bone were obtained. The blocks were fixed in 10% buffered formalin solution with a ratio of 1:50. After two weeks of fixation, the 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 a previous study [
17] 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) and 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.
Discussion
Furcation perforation has usually occurred at the furcal site of the posterior teeth, leading to a bad effect on the prognosis of the affected teeth [
18]. In order to decrease the inflammation and enhance the periodontal ligament (PDL) attachment, an ideal furcal perforation repair material should be used. Calcium silicate cements are the materials of choice for treatment of the furcation perforation due to their good biocompatibility and ability to induce calcium-phosphate precipitation at the interface to the periodontal tissue with high quality of the material-dentin interface [
19]. Therefore, the present study compared the TheraCal LC with the MTA-Angelus and Biodentine, in terms of marginal adaptation, solubility and biocompatibility. The results of this study recorded significant differences between the tested materials regarding the evaluated properties with superiority of the MTA-Angelus that was followed by Biodentine then TheraCal LC. Therefore, the hypothesis of this study is rejected.
The three-dimensional hermetic seal is one of the most important requirements during furcation perforation repair. This seal is a complex outcome of marginal adaptation, adhesion, solubility, and volume changes of the cement used. Therefore, the gap size between the dentin and repair material and the fluid leakage constitute the quantitative manifestation of the material sealing ability [
20]. The evaluation of marginal adaptation of furcation perforation repair materials by SEM can give information about their sealing ability [
21]. The results of the present study showed that TheraCal LC exhibited a lower sealing ability than that of the Biodentine and MTA-Angelus and Biodentine exhibited a better sealing ability than the MTA-Angelus. These results agree with that of a previous study [
22]. The good adaptation property of Biodentine may be attributed to the small size of Biodentine particles which may enhance the adaptation at the cavity surface and filling interface. On the other hand, the marginal adaptability and microlakage of the MTA were better than that of the Biodentine [
20]. The difference between our results and other controversial results may be related to the method of sample preparation and method of evaluation.
As regards the TheraCal LC, it exhibited the highest frequency distribution of gap compared to MTA-Angelus and Biodentine. This poor adaptability might be due to the presence of resin matrix in the TheraCal LC that may maximize its polymerization shrinkage and consequently reflects on the material adaptation. In contrast, our result is not in agreement with the study done by Makkar et al
. [
22] who compared the sealing ability of TheraCal LC, Biodentine and MTA by using the dye penetration method and Confocal Laser Scanning Microscope for evaluation. They found that TheraCal LC exhibits less microleakage than other materials. This difference in the results could be related to the difference of sample preparation and the evaluation method. Therefore, it is worth to mention that in comparing the results of studies on marginal adaptation of root repair materials, several factors such as the design of studies, plane of root sectioning and methods of gap measurement should be considered in order to obtain a better comparison [
23].
The second property tested in this study was the solubility. Lack of solubility is a favorable property of the root repair material for obtaining a long term seal without microleakage [
24]. Solubility is evaluated by the ISO standards after a period of 24 h, but longer analysis periods may be used [
25]. The most widely used time interval is 7 days [
9,
25].
In this study, the Biodentine exhibited the highest solubility after 7 days followed by that of the MTA-Angelus then the TheraCal LC. Similar findings were recorded in several previous studies that reported a higher solubility of the Biodentine than MTA [
26,
27]. The high solubility of Biodentine could be attributed to the presence of Calcium carbonate and water soluble polycarboxylate in its composition for lowering the water-powder ratio and obtaining good workable cement. The lower solubility of MTA-Angelus than Biodentine could be explained by the absence of Calcium carbonate and presence of setting accelerator in its composition that result in shortening of the setting time [
28]. In other studies, the Biodentine exhibited the lowest solubility among the Calcium silicate materials after one day, including the ProRoot MTA [
29,
30]. These different results could be related to the differences in the evaluation period as well as conditions of mixing and curing such as water to powder ratio, temperature, environmental humidity and pH, entrapped air and water, the rate of packing and the condensation pressure applied [
31].
Regarding the TheraCal LC, it exhibited the lowest solubility compared to the MTA and Biodentine probably due to its immediate setting which is related to the presence of a light-curable resin.
On the other hand, it must bear in mind that the repair materials such as Calcium silicate materials producing Calcium hydroxide or Calcium oxide during setting should present a certain degree of solubility to improve the mineralization process in contact with vital tissue [
31].
In the present study, dogs were used as an animal model due to their large, well-developed dental roots that allow better accessibility and visibility. However, in dogs, the furcation lies more superficial within the alveolus (1–2 mm from the cemento-enamel junction) than that of human [
32].
Biocompatibility may be evaluated by either cell adhesion assay, cell proliferation, cytotoxicity or inflammatory cell response. In the present study, the inflammatory cell response and radiographic presence or absence of radiolucency adjacent to the perforation site were used to evaluate the biocompatibility after one month and three months. The highest biocompatibility was recorded in the MTA-Angelus subgroup that was followed by that of the Biodentine and TheraCal LC subgroups. In this regard, the MTA-Angelus and Biodentine were less cytotoxic on human dental pulp stem cells (hDPSCs) than TheraCal LC in a previous in vitro study [
33]. Moreover, similar findings of the MTA and Biodentine were reported after repairing furcation perforation in dog’s primary teeth [
7]. These results could be attributed to the chemical composition of each material. Unlike MTA, the Biodentine has no aluminate phase that does not form ettringite on hydration. Presence of aluminate phase in certain amounts improves the biocompatibility of Portland cement systems [
34]. Also, unlike MTA, the Biodentine contains Zirconium oxide that decreases the cell viability [
35]. On the other hand, another study reported that addition of Zirconium oxide to the Calcium silicate cement provides a good biocompatibility [
36]. Therefore, the exact effect of Zirconium oxide on biocompatibility needs further investigations. In contrast to our findings, Jung et al
. [
37] found that Biodentine is more biocompatible than the MTA on the cells of human periodontal ligament (PDL) after 20 days. This difference might be attributed to the difference of evaluation periods, the type of MTA used and nature of the study (in vitro).
TheraCal LC subgroup exhibited mild to moderate inflammation after one month and moderate to severe inflammation after 3 months of evaluation. These findings mean that TheraCal LC material is less biocompatible than the MTA-Angelus and Biodentine. This may be due to the presence of resin in its composition. These findings are in agreement with other studies [
38,
39]. Also the depth of cure should be considered [
39].
It is worth to mention that no furcation in any tested subgroup was free of inflammatory cells during both evaluation periods. This could be attributed to the somewhat shorter periods of evaluation. In this regard, various degrees of tissue reactions in contact with the MTA were reported in dogs after 30 days while after 180 days the periodontium was almost free of inflammation [
40]. This allows the speculation that a better result might be observed over a longer post-operative period than those of this study.
The radiographic evaluation demonstrates the effectiveness of the tested material on sealing of the perforation and preventing the formation of bony defect adjacent to the perforation site. The results of radiographic evaluation agree with those of the histological findings. The frequency distribution of radiolucency presented in the TheraCal LC subgroup was higher than that of the MTA-Angelus and Biodentine subgroups of both groups with non-significant difference between TheraCal LC and control subgroups. This could be attributed to the lower biocompatibility of the TheraCal LC than that of the MTA-Angelus and Biodentine.
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