4.5 ml tube

For each replicate, ten material disks were placed in a 4.5 ml tube (Nunc, Roskilde, Denmark) containing 2 ml D-MEM (serum free) and then incubated at 37°C for 24 h (day 1). After the incubation period, the disks were removed using sterile forceps. Following initial elution of the materials for 24 h, the disks were removed and then re-eluted in fresh serum-free media for another 24 h (day 2). After the second elution of the materials, the disks were removed again and re-eluted in fresh serum-free media for an additional 24 h (day 3). Finally, the resulting day-1, day-2, and day-3 elutes were subjected to analysis for calcium ions using ion chromatography (Dionex ICS-1000, USA).
Since the elution volume for each elution period was 2 ml, two-fifths that required for ion chromatography, only two samples from each elution period were analyzed for this work by adding 1 ml of each elute into 4 ml of diluting ultradistilled water (Advantec, Tokyo, Japan) to a final volume of 5 ml and a 1:5 dilution factor.

For each material, four replicates were used, each replicate was incubated in DMEM for a different time interval, namely 1 day; 2 weeks; 4 weeks; and 6 weeks. For each replicate, ten material disks were placed in a 4.5 ml tube (Nunc, Roskilde, Denmark) containing 2 ml D-MEM, and then incubated at 37°C. After the incubation period, the disks were removed using sterile forceps. For the control, four tubes of 2 ml D-MEM each were incubated for the same time intervals at 37°C. At the end of the incubation period, the contents of each vial of the elutes and the control were refrigerated until use.
The effect of the material elutes on cell viability was evaluated by means of the MTT assay. Four sets of experiments were performed, which represent the effects of 1-day, 2-week, 4-week, and 6-week elutes on cell viability. In each experiment, a 96-well culture plate was used. Each well received 100 μl of cell suspension (5 × 10⁵ cells/ml) and 10 μl of the material elute or the medium control. Ten replicates were made for each group. Cells were incubated with the elutes for 24 h at 37°C and 5% CO₂.