Elx800uv

The specimens were placed in a multi-well plate, and 1 mL of artificial saliva was dispensed into each well and stirred at room temperature for 2 h to coat specimens. The artificial saliva was aspirated, and the specimens were allowed to dry naturally for 15 min. The liquid culture medium (1.5 × 10⁷ CFU/mL) was dispensed onto each specimen and incubated for 48 h under anaerobic conditions. The specimens on which the bacteria were cultured were treated with NTP for 60 s and 120 s (Figure 2). Immediately after the plasma treatment, osteoblasts (MCT3-E1; 4 × 10⁴ cells/mL) were dispensed onto the specimens and incubated in a CO₂ incubator set at 5% CO₂ and 37 °C. After incubation for 48, 72, and 96 h, osteoblast activation was assessed using a water-soluble tetrazolium salt (WST-8) assay. WST-8 reagent (100 μL; EZ-Cytox, Itsbio, Inc., Seoul, Korea) was dispensed into each well, and the specimens were cultured in an incubator set at 37 °C and 5% CO₂. When the color orange developed into 100 μL of the solution was transferred from each well into a 96-well plate and absorbance was measured at 450 nm with an absorbance meter (ELISA reader: ELx 800UV, Bio-Tek Instrument., Winooski, VT, USA). The contrasting wavelength was set to 630 nm. All specimens were sterilized, and each group was divided into 30 specimens, and 10 specimens were used at each time.

The ability of the scaffolds to support cell metabolism was evaluated through cell adhesion and proliferation studies.
The scaffolds were sterilized with 70% ethanol and washed with PBS. Then, the materials for the cell culture and material controls were placed in 24-well plates and fixed with silicone O-rings.
Saos-2 cells were seeded at a concentration of 30 k cells/cm² directly over the sample’s surface and at the bottom of the wells for the cell controls. The cells were maintained in McCoy’s 5A medium and incubated for 24 h at 37 °C in a controlled 5% CO₂ atmosphere.
The cell adhesion rate was determined by the reduction in resazurin, as in the cytotoxicity tests. For this process, the medium was substituted by a 1:1 solution of resazurin/McCoy’s medium and incubated for 3 h. Then, the incubated media was transferred to a 96-well plate, and the absorbance at 570 nm and 600 nm was read in a microplate reader (Biotek ELx 800 UV) [24 ]. The resazurin assay was repeated at 3 and 7 days to evaluate the cell proliferation in each of the materials.
The data of the three independent biological assays were statistically analyzed using an ANOVA, comparing all samples in each time point and the same samples throughout the time. The statistical analysis was performed using OriginPro software with 95%, 99% and 99.5% levels of significance.

To evaluate the mitochondrial activity of the seeded cells, that is, cell viability on the scaffolds during culture, the samples and controls were washed 2-3 times with McCoy' 5A medium without serum and then incubated with 200 μL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT—Sigma-Aldrich) in McCoy's medium (0.5 mg mL⁻¹). The plates were incubated for 4 h in a cell incubator at 37°C, after which the MTT solution was replaced with 200 μL of dimethylsulphoxide (DMSO—Sigma-Aldrich). After 10 min, 100 μL aliquots were transferred to empty wells and the absorbances were measured in a microplate reader (Elx-800-UV, Bio-Tek Instruments) at 570 nm and 650 nm.

Assessment of cell viability was done utilizing the Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Kumamoto, Japan). In brief, 100 μL of PC12 cells, Nrg-1 transfected PC12 cells, and shNrg-1 transfected PC12 cells (1 × 105/mL) underwent seeding in a 96-well plate for 12 h and subsequently treated using 0.5 percent DMSO, 300 μM CORT and/or 100 nM AsVI for 24 h. Afterward, the removal of the medium was done, and each of the wells was grown with 10 μL of CCK-8 solution and 90 μL of culture medium at a temperature of 37 °C for 2 h. Finally, the optical density at 450 nm was assessed using a microplate reader (ELX 800 UV, BIO-TEK, USA).