Basic Research—Technology
Cytotoxicity Comparison of Mineral Trioxide Aggregates and EndoSequence Bioceramic Root Repair Materials Beth Ann Damas, DDS, MS, Michelle A. Wheater, PhD, Josef S. Bringas, DMD, DDS, MS, and Michael M. Hoen, DDS Abstract Introduction: The purpose of this bench top evidence level 5 in vitro study was to compare the cytotoxic effect of 2 brands of white mineral trioxide aggregate cement (ProRoot MTA and MTA-Angelus), Brasseler EndoSequence Root Repair Material, and Brasseler EndoSequence Root Repair Putty by using human dermal fibroblasts. Methods: The cells were cultured in recommended culture conditions and exposed to the tested materials. The cytotoxic effects were recorded at an observation period of 24 hours by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)–based colorimetric assay. Results were analyzed by using oneway analysis of variance with significance of p < .05. Results: All materials tested demonstrated cell viability $91.8%. Overall, there was no statistically significant difference in cell viability of ProRoot MTA, MTA-Angelus, and Brasseler EndoSequence Root Repair Material. However, there was a statistically significant difference negatively associated with the cell viability of human dermal fibroblasts in association with the Brasseler EndoSequence Root Repair Putty. Conclusions: The Brasseler EndoSequence Root Repair Materials were shown to have similar cytotoxicity levels to those of ProRoot MTA and MTA-Angelus. (J Endod 2011;37:372–375)
Key Words Biocompatibility, brasseler root repair material, MTA, MTA-angelus, MTT assay, root end filling materials
From the Department of Endodontics, University of Detroit Mercy, Detroit, Michigan. Address requests for reprints to Dr Michael M. Hoen/ Dr Beth Ann Damas, University of Detroit Mercy, Department of Endodontics, 2700 Martin Luther King Jr Blvd, Detroit, MI 48208-2576. E-mail address:
[email protected] 0099-2399/$ - see front matter Copyright ª 2011 American Association of Endodontists. doi:10.1016/j.joen.2010.11.027
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n annual estimation of endodontic procedures suggests that approximately 5.5% of all treatments performed involve apical root-end surgery and root perforation repair (1). The aim of the surgical procedures is to correct problems and successfully eliminate inflammatory processes that would not otherwise be successfully treated with nonsurgical root canal treatment. Iatrogenic complications that can occur during the nonsurgical root canal treatment might also be treated by surgical means (1, 2). Surgical procedures usually involve sealing the root canal in a retrograde rather than orthograde manner. Whether the aim is to place a retrofill material or repair an iatrogenic procedural accident, the material used should demonstrate the ability to form a seal with the dentin and maintain biocompatibility with the periapical tissues (3). Ideal characteristics of a root-end filling or root repair material would include the ability to adhere to dentin, maintain a sufficient seal, be insoluble in tissue fluids, be dimensionally stable, nonresorbable over time, radiopaque, easily manipulated, adequate compressibility, adequate working time, quick setting time, and be biocompatible with human tissue (3). Since the implementation of surgical procedures into endodontic practices, many different materials have been used. These materials included amalgam, composites, zinc oxide–eugenol cements, and glass ionomer cements. None of these materials have been able to meet all of the characteristics of an ideal material (3–5). Certain endodontic clinical procedures involve placement of a dental material directly over the pulp tissues or into direct contact with the periapical oral tissues. In both cases, the ultimate goal is to promote healing and function of the tooth. Several factors influence the success of the procedure; one is that the dental material either stimulates repair or is neutral. It is important to avoid dental materials that are toxic to the pulpal and periapical tissues that might counteract this purpose (6, 7). Cytotoxicity can be determined by using the MTT-based colorimetric assay. MTT is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and exists as a yellow tetrazolium salt. MTT is reduced in metabolically active cells by a mitochondrial enzyme to form insoluble purple formazan crystals, which are solubilized by the addition of a detergent. The resulting purple color can then be quantified by measuring the absorbance. There is a linear relationship between cell number and absorbance, so the amount of MTT formed in cell cultures is correlated to the absorbance, with less absorbance correlating to fewer cells, and increased levels of cytotoxicity. MTT assays are a standard method to determine the cytotoxicity of dental materials in cultured cells (8, 9). Although mineral trioxide aggregate (MTA) as a root-end filling material and root repair material has gained widespread use since its introduction into the endodontic market, it is not an easy material to handle (10). A major drawback of MTA is the inability to obtain consistent results when mixed according to the manufacturer’s directions. This results in difficulty while placing the product during treatment (2). The second most quoted disadvantage of MTA is the long setting time (11). Attempts have been made to overcome the handicaps associated with MTA. Variations on the formulation have been produced and subsequently introduced into the dental market. MTA-Angelus (AMTA) (Angelus, Londrina, Brazil) is one of these products (12). AMTA is composed of 80% Portland cement (PC) and 20% bismuth oxide. In comparison, white MTA is composed of 75% PC, 5% calcium, and 20% bismuth oxide. AMTA’s PC makeup has been shown to contain a greater amount of calcium carbonate,
JOE — Volume 37, Number 3, March 2011
Basic Research—Technology TABLE 1. Computation of Percentage of Viable Cells of the Samples and Chlorhexidine in Comparison to the Control Consisting of Culture Medium Only, Arbitrarily Set to 100% Material tested
Mean OD 570 nm
Standard error of mean
% Viable cells compared with control
Standard error of the mean
Control, culture medium only Chlorhexidine control MTA AMTA RRM RRP
0.200 0.072 0.198 0.193 0.197 0.184
0.001 0.002 0.003 0.003 0.003 0.004
100.0 36.0 99.2 96.7 94.0 91.8
0.02 0.01 0.01 0.04 0.02
AMTA, MTA-Angelus; MTA, mineral trioxide aggregate; OD, optical density; RRM, root repair material; RRP, EndoSequence Root Repair Putty.
calcium silicate, and barium zinc phosphate. These elements contribute to the improved setting time and workability of AMTA (13–15). Brasseler USA (Savannah, GA) has recently introduced the EndoSequence Root Repair Material (RRM) and EndoSequence Root Repair Putty (RRP), which use bioceramic technology to address some of the inconsistencies associated with conventional MTA. Bioceramics refers to the combination of calcium silicate and calcium phosphate that is applicable for biomedical or dental use (16–18). These new materials are produced as a premixed product to provide the clinician with a homogeneous and consistent material. The bioceramic material is produced with nanosphere (1 10–3 mm in its greatest diameter) particles that allow the material to enter into the dentinal tubules and interact with the moisture present in the dentin (19). This creates a mechanical bond on setting. The technology eliminates the potential for shrinkage of the root-end filling material, rendering the material with exceptional dimensional stability (19, 20). According to the manufacturer, the bioceramic materials are bright white in color, rendering a highly radiopaque material. This property makes it easy to place during treatment and to identify in radiographs. A high alkaline pH is partially responsible for its antibacterial nature. The bioceramic materials are said to reach a pH of 12.8 during the time of placement. During a 7-day period, the pH steadily decreases. This property of bioceramic root repair material is implied to provide superior biocompatibility characteristics (18–20). The purpose of this bench top evidence level 5 in vitro study was to compare the cytotoxicity of 2 brands of white MTA cement (ProRoot MTA [DENTSPLY Tulsa Dental, Johnson City, TN] and MTA-Angelus), Brasseler EndoSequence Root Repair Material, and Brasseler EndoSequence Root Repair Putty by using human dermal fibroblasts.
Materials and Methods This study used 4 different root repair materials. The materials included white ProRoot MTA, white MTA-Angelus (AMTA), Brasseler EndoSequence Root Repair Material (RRM), and Brasseler EndoSequence Root Repair Putty (RRP). Ten samples of each material were tested. Both MTA materials were prepared with a powder-to-liquid ratio of 3:1 and mixed on a glass slab for 1 minute, according to the manufacturer’s instructions (21, 22). The Brasseler USA products are packaged premixed by the manufacturer and do not require preparation before use (19). The sample cements were placed into polyvinyl siloxane molds with an inner diameter of 4 mm and a thickness of 5 mm. The 2 types of MTA samples were carried to the molds with an amalgam plugger and hand condensed with a standard amalgam condenser. The Brasseler RRP was carried to the molds with the beaver tail end of a Glick (Vista Dental Products, Racine, WI) hand instrument and hand condensed JOE — Volume 37, Number 3, March 2011
with an amalgam condenser. The Brasseler RRM was dispensed into the molds via the plastic microcannula tips provided by the manufacturer. Each individual sample was covered with a sopping wet cotton pellet before placement in the incubator. The samples were stored at 37 C in a chamber of 100% relative humidity for 1 week.
Cytotoxicity Assay The 4 5 mm disks of white ProRoot MTA, white AMTA, Brasseler RRM, and Brasseler RRP were sterilized by ultraviolet radiation. Ten cylinders of each material were placed in a 15-mL culture tube, and 8 mL of culture medium (dermal fibroblast culture medium as formulated by the manufacturer; Zen-Bio, Inc, Research Triangle Park, NC) was added. The cylinders were incubated in the culture medium for 24 hours at 37 C to allow the soluble materials to leach from the samples into the medium. The medium was filtered through 0.2-mm syringe filters to remove particulate matter before use. Adult human dermal fibroblasts, pre-plated in wells of a 48-well tissue culture plate, were purchased from Zen-Bio. The medium conditioned by each of the 4 materials was diluted with fresh culture medium as follows: no dilution, 1:2 dilution, and 1:5 dilution. One milliliter of each dilution was added to each of 3 wells in the tissue culture plate. As a negative control, cells were cultured in medium only, and as a positive control, cells were cultured in medium containing 0.010% chlorhexidine, which has been shown to be cytotoxic to cells (23). Fibroblasts were cultured for 24 hours at 37 C in 5% CO2. At 24 hours in culture, 100 mL MTT solution (Cell Growth Determination Kit MTT Based; Sigma-Aldrich, St Louis, MO) was added to each well, and cells were incubated for an additional 3 hours. The resulting formazan crystals were dissolved by removing the culture medium and adding 1 mL MTT solvent to each well. The plate was shaken at room temperature for 10 minutes to dissolve the crystals. The 200-mL samples were transferred to wells of a 96-well microtiter plate, and the absorbance at 570 nm (A570nm) was determined by using a microtiter plate reader (6, 8). The experiment was repeated twice with similar results. The mean and standard error of the mean for each treatment were calculated from triplicate samples. Statistical analysis of the data was performed by using one-way analysis of variance and Tukey multiple comparison post test, with significance of p
