Cytotoxicity in culture of cells (MTT assay)

The cytotoxicity of NRRFMs in both fresh state and set state was investigated. After being mixed, the materials which were placed in a circular mold (5 mm diameter × 2 mm) and set for 10 min at 37°C in 100% humidity under aseptic conditions were designated as fresh specimens, and those that were incubated for 24 hours were designated as set specimens [25, 26]. Then the disks were removed from the molds and immediately immersed in extraction medium (RPMI 1640, Gibco Laboratories, Grand Island, USA) by using the ratio 1.25 cm²/mL between the surface of the sample and the volume of medium for 24 hours at 37°C according to ISO 10993–12 (2007) [27]. The medium stored under the same conditions was used as a negative control group and the medium with 0.7% acrylamide was used as a positive control group. Subsequently, the tested extracts and the control groups were sterile filtered by using Millex-GS sterile filter. Each of the various tested extracts and the controls was supplemented with 10% fetal calf serum (FCS, Gibco Laboratories, Grand Island, USA).

L-929 mouse fibroblasts (Institute of Cell Biology, Chinese Academy of Sciences, Shanghai, PRC) were cultured in RPMI 1640, supplemented with 10% FCS, 0.07% sodium bicarbonate, 20 mM HEPES, penicillin (100 U/mL) and streptomycin (100 mg/mL), incubated at 37°C in 5% CO₂. For the cell cytotoxicity assay, cells were seeded in 96-well plates (n = 5) at an initial density of approximately 6×10³ cells per well in 200 μL of culture medium and allowed to adhere overnight. Then the cell culture medium was removed, and 200 μL of the tested and control extracts (supplemented with 10% FCS) from different elute groups were placed into the culture wells. After incubation in a humidified atmosphere for 24 hours or 48 hours, cell morphology of the test specimens was observed by using an Axiovert-25 CFL inverted microscope (Carl Zeiss Inc., Oberkochen, Germany). According to ISO 10993–5 (2009) [28], the cell viability was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, MO, USA) assays. The optical density (OD) was measured by a microplate reader (Multiskan MK3, Thermo Labsystems Co., Shanghai, PRC) at a wavelength of 492 nm. The cell viability was expressed as percentage using the following formula (2):

where OD_{test material} is the mean value of the measured optical density of the test sample, and OD_{negative control} is the mean value of the measured optical density of the negative control group. The value of cell viability was used to evaluate the cytotoxicity grade of each group according to the 6-level (0–5) definition (see the notation under Table 1) [29, 30]. To ensure the reproducibility, the experiments were repeated three times.

Different letters indicate significant differences between the materials at the same times (one-way ANOVA followed by Fisher’s LSD test, P<0.05).

Toxicity grade was defined as follows: 0 (cell viability≥100%), 1 (75%-99%), 2 (50%-74%), 3 (25%-49%), 4 (1%-24%) and 5 (0%).