L 929 Cell Response to Root Perforation Repair Cements : an In Vitro Cytotoxicity Assay

Root perforations allow the root canal system to communicate with the periradicular tissues and may cause the loss of the tooth due to secondary periodontal involvement. The ideal repair material must show good sealing ability in addition to physical, chemical and biological properties. The properties of an ideal repair material have been demonstrated by several studies since Lee et al. (1) presented mineral trioxide aggregate (MTA). Currently, MTA is considered the material of choice for the repair of root perforations. MTA is composed by tricalcium silicate, tricalcium oxide, silicate oxide and other mineral oxides that are responsible for its physical and chemical properties. An alkaline pH and calcium release probably stimulate tissue mineralization (2). The major difference between gray and white MTA (WMTA) is the concentration of iron oxide (FeO). The amount of FeO is extremely reduced in the WMTA (3), while the biological properties of both types of cement are comparable (4-5). A new experimental epoxy resin and calcium hydroxide based cement (MBPc) has been developed at the Department of Endodontics of Bauru Dental School, University of São Paulo, Brazil as another option to repair root perforations. The ability of the epoxy resin to seal and the biocompatibility of calcium hydroxide have been proven in vitro (6-8) and have shown good results in vivo (9). Generally, cytotoxicity is the first test used to investigate biocompatibility. The evaluation of new materials using in vitro methods must first be done to avoid unnecessary animal sacrifice. The agar overlay method with neutral red dye allows great sensitivity using the material as it is used clinically, while protecting the cell monolayer against physical damage (10). The aim of this study was to evaluate the toxicity of gray MTA, white MTA and the experimental MBPc by comparing their effect on L929 cells, using the agar overlay method with neutral red dye.


INTRODUCTION
Root perforations allow the root canal system to communicate with the periradicular tissues and may cause the loss of the tooth due to secondary periodontal involvement.The ideal repair material must show good sealing ability in addition to physical, chemical and biological properties.The properties of an ideal repair material have been demonstrated by several studies since Lee et al. (1) presented mineral trioxide aggregate (MTA).Currently, MTA is considered the material of choice for the repair of root perforations.
MTA is composed by tricalcium silicate, tricalcium oxide, silicate oxide and other mineral oxides that are responsible for its physical and chemical properties.An alkaline pH and calcium release probably stimulate tissue mineralization (2).The major difference between gray and white MTA (WMTA) is the concentration of iron oxide (FeO).The amount of FeO is extremely reduced in the WMTA (3), while the biological properties of both types of cement are comparable (4)(5).
A new experimental epoxy resin and calcium hydroxide based cement (MBPc) has been developed at the Department of Endodontics of Bauru Dental School, University of São Paulo, Brazil as another option to repair root perforations.The ability of the epoxy resin to seal and the biocompatibility of calcium hydroxide have been proven in vitro (6)(7)(8) and have shown good results in vivo (9).
Generally, cytotoxicity is the first test used to investigate biocompatibility.The evaluation of new materials using in vitro methods must first be done to avoid unnecessary animal sacrifice.The agar overlay method with neutral red dye allows great sensitivity using the material as it is used clinically, while protecting the cell monolayer against physical damage (10).
The aim of this study was to evaluate the toxicity of gray MTA, white MTA and the experimental MBPc by comparing their effect on L929 cells, using the agar overlay method with neutral red dye.

Test Materials
Gray ProRoot MTA (Dentsply/Tulsa Dental, Tulsa, OK, USA) and Angelus WMTA (Angelus Soluções Odontológicas, Londrina, PR, Brazil) were mixed in a 1:1 ratio of powder: liquid, and the MBPc experimental cement was mixed in a 3:1 ratio of base paste (0.2 g): catalyze paste (0.6 g), according to the manufacturers' instructions.The mixtures were then immediately placed into a silicone mold to prepare uniform pellets (6 x 6 x 1 mm 3 ).The cements were allowed to set for 48 h.

Agar Overlay Method
The study was performed according to the Standard Operating Procedures (SOP) of the Oswaldo Cruz Foundation (FIOCRUZ, Rio de Janeiro, RJ, Brazil) (11).Cells were seeded in 6-well plates (Costar) at a density of 2 x10 5 cells/mL.Each well, which had an inner diameter of 35 mm, received 4 mL of the prepared suspension.The plates were incubated for 48 h at 37ºC in an atmosphere of 5% CO 2 in air and 100% humidity, to allow adhesion and cell monolayer confluence.The medium was removed from the wells and replaced with agar 1.8% (Bacto; Difco Laboratories Inc., Detroit, MI, USA) added neutral red dye 0.01%, staining the viable cells while allowing diffusion of leachable chemicals from cements.Samples in triplicate, positive (natural rubber) and negative (Teflon) controls on the agar overlay featured the 6-well plates.One well in each plate received the agar only to serve as a cell control.
After an incubation period of 24 h at 37ºC in a humidified atmosphere of 5% CO 2 in air, discolored areas could be seen around specimens related to their toxicity.A milimetric paper was placed underneath the plates before pictures were taken with a digital camera (Nikon Coolpix 4300; Kinon, Tokyo, Japan).Then, the areas of nonviable cell were measured per quadrant using Image Tool image-analysis software (The University of Texas Health Science Center at San Antonio, Texas, USA) adjusting the image magnification tool that allowed improved view of focused areas.The milimetric paper allowed calibrating the digital measurement tool.The method evaluated the biological reactions quantitatively, comparing the extension (in mm) of nonviable cell areas.
The results were classified according to a 5-point cytotoxicity grading system related to the agar overlay method, according to which grade 2 or lower means satisfactory samples: 0 = absent cytotoxicity (absence of a zone of lysis underneath the sample; 1= slight (zones of cell lysis underneath the sample only); 2= mild (zones of cell lysis  ≤5 mm from the sample); 3=Moderate (zones of cell lysis >5 mm and ≤10 mm from the sample); 4= severe (zones of cell lysis >10 mm, but not involving the entire well).In addition, the wells were occasionally examined with an inverted microscope (Nikon Eclipse TS 100; Nikon) to observe cell changes.

Statistical Analysis
Parametric analyses of data were performed by one-way ANOVA and Tukey's test at 5% significance level using GraphPad Prism statistical software package (GraphPad Software Inc., San Diego, CA, USA).

RESULTS
There were statistically significant differences (p<0.05)among the mean zones of cell lysis comparing the cements to each other and comparing the cements to the controls from the respective plates (Fig. 1).
Classified according to the 5-point cytotoxicity grading system, ProRoot MTA and the Angelus WMTA received grade 1 (slight cytotoxicity) and the MBPc received grade 2 (mild cytotoxicity).Negative controls were graded 0 (absent cytotoxicity), while positive controls were graded 3 (moderate cytotoxicity).Cell controls showed viable cells during the entire experimental period.
When examining morphological changes in the cells with the inverted microscope, ProRoot MTA showed viable cells around the pellets.Dead cells could be observed underneath the material after removal from the agar layer.
A ×100 magnification of an Angelus WMTA well clearly showed 4 different moments in the cell morphology around the cement: cell lysis, rounded cells loosened from the substrate, some morphological alterations, and viable cells with homogeneous cytoplasm in a monolayer (Fig. 2).MBPc revealed the largest zones of cell lysis and morphological cell changes among the 3 cements.Because of this only one section of all wells examined in the study exhibited the different zones within the same filed of view (Fig. 3).

DISCUSSION
Studies with established cell lines are used because of the reproducibility of the results, besides they multiply rapidly with an unlimited life span (12).It is interesting to evaluate the reaction of fibroblasts to the materials because those cells are involved with the healing process.The established L929 cell line used in this study is commonly employed in biocompatibility studies (10,(13)(14)(15).
Using the agar overlay method with vital red dye, Torabinejad et al. ( 13) recorded a zone of lysis around the samples of fresh and set gray MTA, without any difference between them.Saidon et al. ( 14) and Haglung et al. (15) found denatured proteins and dead cells underneath fresh MTA samples, morphological changes around them and viable cells in most of the area of the well.The present study obtained similar results for the 48-h set MTA, in agreement with Koulaouzidou et al. ( 16) who observed little cytotoxicity of 48-h set gray MTA.
Although gray and white MTA cements are slightly cytotoxic and have exhibited similar effects on the cells in the present study, it was interesting to note that during the microscopic analysis the cell behavior in the presence of ProRoot MTA was the most favorable.
Accordingly, Pérez et al. (17) and Camilleri et al. (18) showed less cell growth in the presence of white MTA, but they confirmed its biocompatibility.
Epoxy resin has a favorable property of adhesion to dental structure and calcium hydroxide is known to be biocompatible (19).Cohen et al. (10) demonstrated severe cytotoxicity of resin-based cements in the first 48 h after preparation, period in that the most toxic substances are released from resin-based cement, according to the same authors.The present investigation revealed grade 2 (mild) for 48-h set MBPc, probably because the cement had been left to set for 48 h before it has been used, which reduced its toxicity in a considerable way (20).Even though our morphological findings showed that fibroblasts appeared rounded, unstained and probably in the process of apoptosis in the greatest part of the discolored area as an effect of MBPc, only close to the cement the cells presented vacuolated cytoplasms or lysis, differently from positive controls that determined the entire discolored area with dead cells.
ProRoot MTA and Angelus WMTA were slightly cytotoxic.Although the experimental MBPc was the most cytotoxic cement among the 3 tested, the sample was considered satisfactory.The changes in cell morphology occurred in the following sequence of intensity: gray ProRoot MTA < Angelus WMTA < MBPc, considering that the changes associated with the positive controls were significantly more intense than those observed these materials.
A further biochemical evaluation would be interesting to count viable cells after contact with the tested materials.The experimental MBPc cement must be subjected to other tests to allow an appropriate clinical indication of its biocompatibility.This material may become a good alternative to repair root perforations.

Figure 1 .
Figure 1.Comparison of the mean zones of cell lysis among cements and among the cements and their respective controls.

Figure 2 .
Figure 2. Well 2 of Angelus WMTA: a) cell lysis; b) rounded cells loosened from the substrate; c) some morphological cell changes; d) viable cells exhibiting a homogeneous cytoplasm and arranged in a monolayer (×100 magnification).