Root end filling materials designed to stimulate hard and soft tissue repair in periradicular tissues are highly recommended.1 The characteristics of an ideal apical root end filling material include adherence to the dentinal walls of the retrograde preparation, periradicular tissue tolerance, and bioactive promotion of healing.2 Many materials have been used for root end fillings in endodontic surgery. These materials include amalgam, gutta-percha, zinc oxide eugenol, calcium hydroxide, composite resin, gold foil, glass ionomers, mineral trioxide aggregate (MTA), and others.
Amalgam has historically been the most widely used root end filling material, but studies showed disadvantages such as mercury toxicity.3 Prevention of irritants leaking from the root end filling material into the periradicular tissues is the key to treatment success. Recently, MTA was shown to be biocompatible with periradicular tissues and had good physical and chemical properties.4, 5
Cytotoxicity testing is one of the most commonly used in vitro measures of biocompatibility. The method is a simple, rapid, and cost-effective biocompatibility screening test. It gives valuable indications as to which materials should be discarded and which should be subjected to further testing.6 In toxicity studies of root end filling materials, there were correlations between the extracts of freshly mixed or set root end filling materials and cytotoxicity. Using the agar overlay method of cytotoxicity testing, freshly mixed root end filling materials were cytotoxic to L929 cells.7 Bruce et al. suggested increased cytotoxicity over time for amalgam samples.8 They reported that this might be due to the possible accumulation of corrosive products on the amalgam surface. The root end filling material extraction duration and the extract exposure time to cells influences the toxicity.8
The aim of the present study was to evaluate the biocompatibility of three types of root end filling materials under different extraction and exposure times: 1-day and 1-week extraction times of freshly mixed materials and exposure times of 24 and 48 h.
MATERIALS AND METHODS
Test Materials and Extraction Preparations
Three different root end filling materials were studied. They were calcium hydroxide based cement (CHBC) (Life, Kerr Co., Romulus, MI), eugenol based cement (EBC) (Super EBA, Bosworth Co., Durham, England), and mineral trioxide aggregate (MTA) (Proroot, Tulsa Dental, Tulsa, OK) (Table I).
The biocompatibility methods were as reported previously by Keiser et al.9 The root end filling materials were mixed according to the manufacturer's recommendations and placed into the bottom of 48 well tissue culture plates to achieve a thickness of about 5 mm. The surface area of the test material exposed was 64 mm2.
Fresh extracts of the test materials were made as follows: after the test materials were mixed, the 1-day extractions were made by covering each exposed test material with 20 μL of complete McCoy's medium (Sigma Co., St. Louis, MO) and incubating the plates at 37°C with 100% relative humidity for 24 h. The 1-week extraction time extracts were made using the same procedure, but were incubated for 1 week. The medium was then withdrawn and sterile filtered through a 0.22-μm filter. To observe a dose-response relationship, the extracts were serially diluted with complete McCoy's medium to concentrations of 100, 1, and 0.01% (vol/vol). The extracts were then used in the following assays.
The biocompatibility testing was done according to the methods of Schweikl and Schmalz10 and Wataha et al.11 Cell suspensions of the U2OS human osteogenic sarcoma cell line (BCRC no. 60187, Food Industry Research and Development Institute, Taiwan) were seeded into 96-well flat-bottomed plates at 5 × 103 cells/well, as determined using a hemocytometer, in complete McCoy's medium, and incubated in a humidified atmosphere with 5% CO2 at 37°C for 24 h. The culture medium was then replaced with 200-μL aliquots of the test extracts or control media (DMSO 5% was prepared as the positive control and complete culture medium served as the negative control), and the cells were then incubated for 24 h at 37°C in humidified air with 5% CO2. The experimental samples were then divided into two groups. In the first group, cells were exposed to the test extracts for 24 h, whereas in the second group, the cells were exposed for 48 h. Each extract concentration was tested in triplicate.
After the exposure, cell viability was determined by the ability of the cells to cleave the tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) (MTT) to a formazan dye. The medium was removed with a sterile pipette and 200 μL of phosphate buffered saline was added to each well, swirled gently for 1 min, and then replaced with 100 μL of complete medium and 10 μL of a 5-mg/L solution of MTT. The cells were incubated in the MTT/medium solution for 4 h at 37°C in an atmosphere of 5% CO2. Then, 100 μL of a 6.25% (vol/vol) 0.1 mol/L NaOH in DMSO solution was added to each well, and the plates were incubated overnight to solubilize any formazan crystals that had formed. Plates were shaken for 60 min at room temperature on a plate shaker to achieve uniform color. Optical densities were then measured at 550 nm in a multiwell spectrophotometer. The survival rates were calculated as experimental group optical density divided by negative control optical density and presented as mean ± standard deviation (%, Mean ± SD). The results were compared using one-way analysis of variance (ANOVA). Differences in treatment means were analyzed using the Student Newman-Keul test and were considered to be significant at probabilities of less than 0.05.
The morphologies of the U2OS added with the 100% concentrations of root end filling materials are shown in Figure 1. The MTA group showed good survival of the U2OS cells but not the EBC and CHBC groups.
The survival rates of U2OS cells upon incubation with CHBC for 24 h using the 1-day extract solution at 1% (97.77 ± 2.24) and 0.01% (91.01 ± 3.56) concentrations were similar (p = 0.05). Incubation with CHBC for 24 h using the 1-week extract solution resulted in higher survival rates at the 0.01% (88.29 ± 10.0) concentrations (p < 0.05) [Figure 2(A)]. The survival rate of one week group resulted in a dose-response increase effect (F = 89.17, p = 0.00).
The cell survival rates upon incubation with CHBC for 48 h, regardless of the extraction times (1 day or 1 week), were similar for the 1% (104.88 ± 7.20 in 1-day and 97.64 ± 7.20 in 1-week) and 0.01% (105.27 ± 4.43 in 1- day and 106.54 ± 4.84 in 1-week) concentrations (p > 0.05) [Figure 2(B)]. The survival rate of 48-h exposure was higher than that of 24-h exposure in the CHBC group (p < 0.05).
The cell survival rates were decreased upon incubation with EBC for 24 h using the 1-day extract concentrations of 1% (107.25 ± 12.23) and 0.01% (82.81 ± 8.23) (p < 0.05). Incubation of EBC for 24 h using the 1-week extract at 1% (82.25 ± 16.4) and 0.01% (71.65 ± 5.72) concentrations survival rate did not differ (p = 0.773) [Figure 3(A)].
The survival rates of cells incubated with EBC for 48 h using 1-day extract solution increased from the 1% (84.27 ± 3.09) to the 0.01% (114.43 ± 1.65) concentration (p < 0.05). Incubation of EBC for 48 h using the 1-week extract at 1% (101.30 ± 7.27) and 0.01%(106.71 ± 4.14) concentrations did not differ (p > 0.05). Both 1-day (10.57 ± 0.76) and 1-week (13.13 ± 0.46) extraction groups at 100% concentration showed the lowest survival rate (p < 0.05) [Figure 3(B)].
The survival rates of U2OS cells upon incubation with MTA for 24 h using the 1-day extract solution showed a decline with decreasing MTA concentrations (F=26.72, p = 0.001). Incubation for 24 h using the 1-week extract resulted in survival rates similar to the 1-day extract rates (F=5.57, p = 0.043) [Figure 4(A)].
The survival rates of cells upon incubation with MTA for 48 h using the 1-day extract showed a dose-response effect at the 100% (103.74 ± 8.03), 1% (89.19 ± 7.47), and 0.01% (87.07 ± 2.05) concentrations (F = 5.75, p = 0.04). Incubation of MTA for 48 h using the 1-week extract at 100% (85.04 ± 1.56), 1% (83.87 ± 1.15), and 0.01% (82.76 ± 5.33) concentrations showed no dose-response effect (F = 0.36, p = 0.709) [Figure 4(B)].
Using the extracts of the root end filling materials is useful for toxicity screening in vitro. It offers the advantages of being easily sterilized by filtration and the ability to examine the effect of materials on cells that are both distant to and in contact with them.9 The in vitro extract testing simulates the immediate postsurgical periradicular environment in which toxic elements of the root end filling materials might leach into the surrounding fluids in the bony crypt because the root end filling material is in contact with the osseous tissue. Thus, we employed a human osteosarcoma U2OS cell culture system in our study. These cells closely resemble human osteoblasts in their ability to express high levels of bone markers.
We tested two different exposure times, 24 and 48 h, to observe their effects on survival. The 24- and 48-h exposure groups showed similar results among the three different materials studied. CHBC and EBC resulted in decreased cell survival rates (<20%), while MTA resulted in the highest cell survival rates (>100%) at concentrations of 100% (Figures 2–4). One possible reason for the CHBC low survival results might be that the pH of the extracts was too high. For EBC, eugenol is the main component, which might be damaging to cells. When freshly mixed zinc oxide eugenol cements contact fluid, an immediate and initially high release of eugenol occurs.12 Eugenol is toxic to Chinese hamster lung fibroblast V79 cells and can also cause chromosome damage.13 Thus, when the original extracts of root end filling materials were added to the cultures, most of the cells died. If the original extracts were diluted to 1 and 0.01% concentrations, the survival rates increased significantly. The MTA survival rate was higher than that of the CHBC and EBC survival rates. Calcium hydroxide was the main compound released by MTA in water.14 The calcium hydroxide is biocompatible to tissue. The present study results are similar results for MTA exposure to MG63 osteosarcoma cells and human periodontal ligament cells.4, 9 The present study showed the cell survival rate was independent of exposure time or fresh mixed extraction time.
In clinical treatment, the root end filling material is placed immediately on the root end area. The material is set freshly in the tissue. There might be many unknown substances degraded or leached into the periradicular tissue. To simulate this clinical condition, we evaluated extracts from freshly set materials immersed for 1 day and for 1 week in culture medium. The CHBC sample extracted after incubation for 1 week and exposed for 24 h showed lower survival rates at higher concentrations (100 and 1%) than did the 1-day extraction group, while the survival rates were similar for both 1-week and 1-day groups at the lowest concentration (0.01%) [Figure 2(A)]. For CHBC samples with 48-h exposure intervals, the survival rates did not differ significantly (p > 0.05) between the 1-week extract group and 1-day extract group [Figure 2(B)].
For the EBC samples with 24-h exposure, the survival rates for the 1-day extract were higher than for the 1-week extract at concentrations of 1 and 0.01% (p < 0.05) [Figure 3(A)]. In EBC samples with 48-h exposure, the survival rates for the 1-day extract was lower at the 1% concentration, but higher at the 0.01% concentration (p < 0.05) [Figure 3(B)]. The study showed that U2OS cell survival on short (24 h) exposure to EBC was longer when the extraction time was shorter. EBC contains ortho-ethoxybenzoic acid and eugenol. With subcutaneous implantation of EBC, there is a moderate inflammatory response.15 This suggests that ortho-ethoxybenzoic acid is released from freshly mixed material.16
For MTA samples with 24-h exposure, the 1-day extract survival rate was higher than was the 1-week survival rate at concentrations of 100% (p < 0.05), but there was no difference between the two extraction times at 1 and 0.01% concentrations [Figure 4(A)]. In MTA samples with 48-h exposure, except at concentrations of 100%, the survival rate did not statistically differ between 1-day extraction and 1-week extract [Figure 4(B)].
The most significant finding for MTA samples was that when the MTA original extract solutions were added to the cell cultures, they did not decrease U2OS survival rates. On the contrary, it actually increased the cell growth. This is because in order to stimulate mineralization, a root end material should have an alkaline pH level and release calcium.17–19 MTA has higher values for pH and calcium ion release,20 which might explain the present findings.
When comparing the original extract concentrations of the three root end materials for survival rate, MTA resulted in the highest survival rate. This result is similar to that of Torabinejad et al.7 They found that freshly mixed and set MTA was less toxic than amalgam, EBC, and intermediate restorative material when the radiochromium method was used.7 In the present study, the toxicity assay was used to measure mitochondrial dehydrogenase activity, as shown by the cleavage of MMT to a formazan dye. The reaction only occurs in living, metabolically active cells. The decision to use the MTT assay was made because MTA is a hydrophilic substance likely to release ionic components; it is more apt to interfere with intracellular enzyme activities than influence membrane permeabilities.
The present study used U2OS cells for evaluation of toxicity. Established cell lines have the advantage of enhanced reproducibility of results and are recommended by the ISO standard for preliminary cytotoxicity screening. The present study indicated that the MTA was less toxic to U2OS cells than the other materials tested. Similar results were reported by Osorio et al.21 and Keiser et al.,9 who showed that MTA was less toxic to human periodontal ligament cells than was EBC at all concentrations in both the freshly mixed and 24-h set states.
This study supports the view that regardless of the extraction time (1 day or 1 week), the original extracts of EBC and CHBC presented the highest toxicity to U2OS cells compared to MTA. Conversely, MTA extracts were more biocompatible with U2OS cells than were EBC and CHBC.