| Abstract|| |
Aim: The purpose of the study is to evaluate and compare the biological and chemical-physical properties of four different root-end filling materials.
Materials and Methods: Cytotoxicity towards murine odontoblasts cells (MDPC-23) was evaluated using the Transwell insert methodology by Alamar blue test. Streptococcus salivarius, S. sanguis, and S. mutans strains were selected to evaluate the antimicrobial activity by agar disc diffusion test. Solubility was determined after 24 h and 2 months. pH values were measured after 3 and 24 h. To evaluate radiopacity, all materials were scanned on a GE Healthcare Lunar Prodigy.
Results: Excellent percentage of vitality were obtained by mineral trioxide aggregate (MTA)-based materials and Biodentine. MTA-Angelus, ProRoot MTA, and Intermediate Restorative Material (IRM) showed the highest values for the inhibition zones when tested for S. mutans, while Biodentine showed the largest inhibition zone when tested for S. sanguis. All the materials fulfilled the requirements of the International Standard 6876, demonstrating low solubility with a weight loss of less than 3%. No significant reduction in pH value was demonstrated after 24 h. ProRoot MTA and MTA-Angelus showed the highest values of radiographic density.
Conclusions: The differences showed by the root-end filling materials tested do not cover completely the ideal clinical requests.
Keywords: Antibacterial activity; cytotoxicity; pH; root-end filling materials; solubility
|How to cite this article:|
Ceci M, Beltrami R, Chiesa M, Colombo M, Poggio C. Biological and chemical-physical properties of root-end filling materials: A comparative study. J Conserv Dent 2015;18:94-9
|How to cite this URL:|
Ceci M, Beltrami R, Chiesa M, Colombo M, Poggio C. Biological and chemical-physical properties of root-end filling materials: A comparative study. J Conserv Dent [serial online] 2015 [cited 2020 May 31];18:94-9. Available from: http://www.jcd.org.in/text.asp?2015/18/2/94/153058
| Introduction|| |
Root-end filling materials are in contact with periradicular tissues, so they should have good sealing ability and be biocompatible to promote healing. Because many of these materials are not able to ensure a perfect seal, a microscopic space is likely to create at the root-end cavity/filling interface,  through which micro organisms and their products can penetrate. Thus, besides good sealing ability and biocompatibility, root-end filling materials should ideally have some antibacterial activity.  Low solubility played a fundamental role on the success rate of the surgical procedure. More over, these materials should have low solubility to avoid that components leaching from the endodontic space might exercise undesirable biologic effects on the surrounding tissues.  Finally, radiopacity is a very important property for all root-end filling materials because the material has to be detected radiographically, and thus distinguished from surrounding anatomic structures. 
Intermediate restorative material (IRM) has addressed some drawbacks including moisture sensitivity, irritation to vital tissues and difficulty in handling. 
MTA was developed by Parirokh and Torabinejad  to address shortcomings of commonly used root-end filling materials. Several studies have recognized MTA as a bioactive material,  that is, hard tissue conductive, hard tissue inductive, and biocompatible.  The sealing ability of MTA in root-end fillings was found to be superior to that of amalgam, IRM, and super-ethoxy benzoic acid (EBA) with both dye and bacterial leakage methods.  However, its handling characteristics are less than ideal as a result of long setting time and difficulty in maintaining consistency of mixture. 
Several new calcium silicate-based materials have recently been developed; , aiming to improve some MTA drawbacks such as its difficult handling property and long setting time.  Biodentine is amongst these materials and is claimed to be used as a dentine restorative material in addition to endodontic indications similar to those of MTA. 
The purpose of the present study is to evaluate and compare the biological (cytotoxicity and antibacterial efficacy) and chemical-physical (solubility, pH, and radiopacity) properties of four different root-end filling materials: Biodentine (Septodont, Saint-Maur-des-Fosses, France), MTA-Angelus (Angelus, Londrina, PR, Brazil), ProRoot MTA (Dentsply, Johnson City, TN, USA), and IRM (Dentsply, Johnson City, TN, USA).
| Materials and methods|| |
Mouse odontoblast cell line (MDPC-23) was used for the cytotoxicity tests. Transwell insert methodology (Sigma-Aldrich, St Louis, MO, USA) was performed. Cytotoxicity was assessed with MDPC-23 cells grown in the lower chamber of a 24-mm diameter Transwell plate with a 0.3 mm pore size polycarbonate membrane (Sigma-Aldrich, St. Louis, MO, USA). All the materials tested were prepared and mixed under sterile hood. The Transwell membrane of the inner chamber, filled with the paper disks, were placed into the lower chamber of the 24-well culture plate containing at the bottom 5 × 10 4 cells/well and incubated at 37°C in 5% CO 2 atmosphere for 24, 48, and 72 h, respectively. Some wells were incubated with only tissue culture media (negative control) whereas others with a 10% dilution of 30% H 2 O 2 (positive control). At the end of incubation time the cell viability was performed with Alamar blue test. The results were presented as percentage of cell viability referred at cells incubated in absence of materials set at 100%. The vitality test to Alamar blue reagent acts as an indicator of cell health, determining the reducing power in order to measure quantitatively the proliferative capacity. The reagent was added in a ratio of 1:10 to the cell culture and then the cells were kept in the incubator for 3-4 h at 37°C. The degree of fluorescence and the relative values of absorbance were then acquired by using a spectrophotometer at a wavelength of 595 nm. For a further control, the percentage of vitality of murine odontoblasts, at 72 h, was also assessed with the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, St Louis, MO, USA). The MTT test is a standard colorimetric assay for measuring the activity of enzymes that reduce the MTT to formazan (a salt blue) in the mitochondria, giving the substance a blue/purple color. This reaction was assessed and measured by the spectrophotometric reading of the sample, at a wavelength of 570 nm by a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
The Streptococcal strains used in this study were provided from the Culture Collection of University of Goteborg (CCUG): Streptococcus mutans (CCUG 35176), S. salivarius (CCUG 11878), and S. sanguis (CCUG 17826). The cultures were grown and maintained in a Brain Heart Infusion (BHI; Difco Lab., Detroit, MI, USA). S. mutans culture medium was supplemented with 10% (v/v) heat-inactivated horse blood serum (Oxoid S.p.A., Rodano, MI, Italy) to improve its growth. The culture of all bacterial strains was statically incubated for 16 h at 37°C under aerobic conditions. This overnight culture, used as source for the experiments, was reduced at a final density of 1 × 10 10 cells/ml as determined by comparing the optical density at 600 nm (OD 600 ) of the sample with a standard curve relating OD 600 to cell number.
Agar disc diffusion test was performed. Sterile paper discs (diameter: 6 mm, thickness: 1 mm) (Watman International Ltd. Maidstone, UK) were impregnated with 10 μl of each root-endfilling material. Then, BHI-agar plates were incubated with 1 × 10 7 cells/ml of an overnight culture of each Streptococcal strain at 37°C for 20 min. The excess of bacterial suspension was removed from the plates and incubated with the paper disks impregnated with the root-end filling materials at 37°C for 24 h. The diameter of the halo formed around the paper disc (inhibition zone) was measured by the same operator in two perpendicular locations with a millimeter ruler with accuracy of 0.5 mm, after 24 and 48 h. The size of the inhibition zone was calculated as follows:
Size of inhibition zone = (diameter of halo - diameter of specimen) × ½.
All the assays were conducted in triplicate and the results were recorded in terms of the average diameter of inhibition zone.
Stainless steel ring molds with an internal diameter of 20 ± 0.1 mm and a height of 1.5 ± 0.1 mm were used for sample preparation. All molds were weighed three times prior to use (accuracy ± 0.0001 g) on a Mettler AE-163 balance (Mettler-Toledo S.p.A., Novate Milanese, MI, Italy), which was used throughout the experiment. After filling the molds, a glass plate covered with a Mylar strip was placed on top of the molds, exerting a light pressure in order to remove any excess. All samples were left to set for 24 h on a grating in a cabinet at 37°C and 100% relative humidity. The samples in their molds were then exposed to air for 15 min, weighed three times, and the average reading was recorded to three decimal places. The specimens of each material were individually placed in tarred bottles, containing 5 ml of distilled water. The bottles were then transferred to an oven at 37°C where they remained for 24 h. They were removed from the oven and rinsed with distilled water, which was then collected in the same bottle. The water was evaporated at a temperature slightly below boiling point. Bottles and residues were dried in an oven at 105°C, cooled down in desiccators, and weighed. The differences found between this weight and the original bottle weight were divided by the initial dry weight of the specimens and multiplied by 100. The result was recorded as solubility. , The solubility test was performed again at 2 months using the same method. The solubility of the set root-end filling materials should not exceed 3% mass fraction (ISO 6876 clause 4.3.6). 
Each specimen was mixed and placed onto cylindrical Teflon molds 2-mmheight and 10-mmdiameter. Each sample was placed into a separate vial, containing 10 mL distilled water. The samples were stored at 37°C, and pH measurement was performed after incubation of 3 and 24 h. The pH value was measured by a digital HI 2210 pH meter (Hanna Instruments US, Woonsocket, RI, USA), previously calibrated.
Thirty standardized specimens of each material were prepared. Biodentine, MTA-Angelus, ProRoot MTA, and IRM were mixed and placed into silicon molds measuring 1 mm in thickness and 4 mm in internal diameter. The specimens were covered with glass plates on each side to allow for the removal of excessive material. The specimens were kept in a chamber at 37°C and 95% relative humidity for 24 h. Ten arrays were set by collecting three samples for each material in order to scan them with Prodigy DXA System (GE Healthcare, Madison, WI, USA).
All materials were scanned on a GE Healthcare Lunar Prodigy and iDXA in routine clinical manner per manufacturer recommendations. For the Prodigy, Encore software (GE Healthcare, Madison, WI, USA) version 9.2 was used for acquisition and 11.4 for analysis; with iDXA, Encore software version 9.3 was used to acquire scans with version 11.0 used for analyses. The precision assessments were performed in routine clinical manner according to International Society for Clinical Densitometry (ISCD) recommendations.
Data collected were analyzed with Stata 12 (StataCorp. 2011. Stata Statistical Software: Release 12. College Station, TX: StataCorp LP.). To assess the normality of the data obtained from the cytotoxicity assays, the antibacterial tests, the solubility evaluations, and from the radiographic densities; the Shapiro-Wilk test was applied (P < 0.05). The number of vital cells was analyzed with Tukey's test. The effects of the paper disc method were analyzed by Mann-Whitney test; the inhibition zones values after 48 h were used for the statistical analysis.  Data obtained from solubility test were analyzed with Mann-Whitney test. To determine whether time influenced the solubility of the pulp capping materials, an analysis of longitudinal data was performed using t-test for paired data (P < 0.05). t-test for paired data test was applied to determine whether significant differences existed in pH values after 3 and 24 h of incubation (P < 0.05). Tukey's test was then applied to evaluate the differences in pH values among the materials ( P < 0.05). Radiographic densities obtained from dual-energy X-ray absorptiometry (DXA) measurements were analyzed using Tukey's test. Significance for all statistical tests was predetermined at P < 0.05.
| Results|| |
Descriptive statistics analysis for biocompatibility investigation is reported in [Table 1]. As regards Alamar blue test [Table 1], after 24 h there were no differences in the number of vital cells among ProRoot MTA, MTA-Angelus, Biodentine, and the negative control (P > 0.05). IRM showed a significant reduction in the number of vital cells (P < 0.05). These results changed uniformly after 48 h and maintained no significant differences among the materials, except for IRM. After 72 h all three materials varied from the negative control, in particular ProRoot MTA and MTA-Angelus gained a similar and lower number of vital cells than the control, while Biodentine showed no decrease, as confirmed by the analysis for paired data. IRM maintained lower values than the other materials. When the MTT test [Table 1] was applied, no significant differences were recorded among ProRoot MTA, MTA-Angelus, Biodentine, and the negative control (P > 0.05), while IRM maintained lower percentages of vitality (P < 0.05).
|Table 1: Mean ± standard deviation of the number of vital cells for each material tested with Alamar blue at different times|
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[Table 2] shows the mean diameter of the inhibition zones after 48 h and the standard deviation. Except for Biodentine when tested for S. mutans, the root-end filling materials caused zones of inhibition. However; MTA-Angelus, ProRoot MTA, and IRM showed the highest values for the inhibition zones when tested for S. mutans (P < 0.05). No significant differences between MTA-Angelus, ProRoot MTA, and IRM (P > 0.05) were found when materials were tested for S. salivarius. Biodentine showed the largest inhibition zone when tested for S. sanguis (P < 0.05), while MTA-Angelus and ProRoot MTA produced similar smaller inhibition zones (P > 0.05).
|Table 2: Mean diameter and standard deviation of inhibition zones (mm) formed by the root-endfilling materials after 48 h by the paper disc method|
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The results of solubility test (after 24 h and 2 months) are listed in [Table 3]. All the materials fulfilled the requirements of the International Standard 6876, demonstrating a weight loss of less than 3%. There was no statistical significance in solubility among the materials tested after 24 h (P < 0.05). Similar results were obtained after 2 months, except for IRM for which solubility significantly raised (P < 0.05). For remnant materials the weight loss after 2 months was not significantly different from the weight loss after 24 h: The materials tested provided a low solubility in time. All the materials tested provided a very alkaline pH after 3 h [Table 3]; ProRoot MTA showed the highest value among the materials tested. Biodentine and IRM provided similar pH values after 3 h (P > 0.05). For all materials tested, a nonsignificant reduction in pH value was recorded after 24 h (P > 0.05). For each root-end filling material descriptive statistics for radiographic densities and the results obtained with the analyses of multiple comparisons were calculated and reported in [Table 4]. ProRoot MTA and MTA-Angelus showed the highest values of density when compared with the other materials (P < 0.05). No significant differences are reported between Biodentine and IRM (P > 0.05).
|Table 3: Mean percentage values of solubility and standard deviation (SD) for each material and for each immersion period|
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|Table 4: Radiographic densities: Descriptive statistics for each material tested and multiple comparisons calculated with Tukey' test (P = 0.05)|
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| Discussion|| |
When comparing the cytotoxicity of the root-end materials tested in this study, IRM demonstrated the lower rates of vitality and a strong cytotoxic capability. IRM has shown the lowest mean number of cells in the colorimetric assay performed with Alamar blue, with assessments at 24, 48, and 72 h, and in the MTT assay at 72 h. These results are in agreement with previous studies.  It has been demonstrated that cell response to IRM was characterized by marked rounding of the cells and depletion of cell numbers, indicating that IRM was not biocompatible.  The toxic component of IRM is eugenol. Very different results were obtained by MTA-based materials. At 72 h, with Alamar blue test and MTT assay, they have reported excellent percentage of vitality. Similar results have been reported in various studies. , Parirokh and Torabinejad  found that ProRoot MTA was less toxic than amalgam, Super EBA, and IRM. However, Biodentine proved to be the more biocompatible material tested. Biodentine, in measurements made at 24, 48, and 72 h; reported percentage of vitality above the negative control. Biodentine is new bioactive cement based on calcium silicate, whose high biocompatibility has been shown in recent studies. ,
The agar diffusion test has been widely used to evaluate the antibacterial activity of dental materials.  In our study, except for Biodentine against S. mutans, all the materials demonstrate antibacterial activity, causing zones of inhibition against the three Streptococcal strains tested small differences in the antibacterial effect on the different Streptococcal strains were recorded. This is not in accordance with a study by Eldeniz et al.,  in which ProRoot MTA and IRM proved to be strong inhibitors. While we have little data about the antibacterial capability of Biodentine; various authors evaluated the effect of MTA on microorganisms, showing conflicting results. , It has been shown that in aerobic conditions, MTA could generate reactive oxygen species with antimicrobial activity. Parirokh and Torabinejad  found that MTA had no antibacterial effect against any of the strictly anaerobic bacteria. However, as showed by our results, it is possible that MTA's highly alkaline pH affords its antimicrobial activity even when in anaerobic condition.
The solubility test followed the procedures laid out in ISO 6876. The test was repeated after 2 months, because Schafer and Zandbiglari  stated that the 24-h period of the specification test is not sufficient. In our study all the materials tested showed minimal solubility, fulfilling the requirements of the ISO 6876 and demonstrating a weight loss of less than 3%. These conclusions are in accordance with our previous study about solubility of ProRoot MTA, IRM, and Super-EBA. 
A very alkaline pH was recorded after 3 h; with the highest value registered for ProRoot MTA. No significant reduction in pH value was recorded after 24 h. ProRoot MTA showed pH values greater than Biodentine. Similar outcomes were reported by other researchers.  This may be probably caused by the higher presence of metallic oxides in MTA that could promote ion dissociation.
Finally, as concerned for radiopacity, in our study MTA-based materials demonstrated higher values of density compared to IRM and Biodentine. Radiopacity of Biodentine has been recently evaluated. Comparing the density of Biodentine and IRM, Grech et al.,  found higher values for IRM; while the relatively lower radiopacity of Biodentine, compared to MTA, has been recently shown in a study by Tanalp et al. 
| Conclusions|| |
Within the limitation of this in vitro study, the differences shown by the materials do not cover completely the ideal clinical requests for root-end filling. IRM could not be considered as the ideal material due to the high cytotoxicity; while MTA-based cement and Biodentine proved to be biocompatible. However, Biodentine, comparing to MTA, did not cause zone of inhibition for the entire microorganism tested and it showed lower values of radiopacity. Therefore, MTA-Angelus and ProRoot MTA can be considered as the best material for root-end filling among those tested due to the fact that they presented at once biocompatibility, antibacterial properties, radiopacity, and low solubility.
| Acknowledgements|| |
We would like to thank Dr Jacques Eduardo (Department of Cardiology, Restorative Sciences, Endodontics; University of Michigan School of Dentistry) for providing us the mouse odontoblast cell line (MDPC-23).
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Department of Clinical, Surgical, Diagnostic and Pediatric Sciences - Section of Dentistry, Policlinico "San Matteo", Piazzale Golgi 3, 27100 Pavia
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4]