2.5.1. Live/Dead Assay

The live/dead examinations were performed after 3, 7, and 10 days. Three samples per scaffold (DD, Curasan) per time period were placed in cell culture plates. Subsequently, 50,000 cells each, which were in 200 μL of medium, were placed directly onto the samples and incubated for 2 h at 37 ◦C, with a CO2 saturation of 5% in the incubator so that the cells could adhere to the surface of the samples. After two hours, 2.5 mL of a DMEM-F12 (Art.

No. BE12-719F, Lonza, Basel, Switzerland) complete medium was added to each well and incubated in the incubator for a defined time (3, 7, and 10 days). After this, the samples were prepared for staining. The staining solution was first prepared by adding 2 mL of DPBS (Art. No. 14190-094, Gibco, Grand Island, NE, USA) to a Falcon and 4 μL of Ethidium Homodimer III (Eth D-III) solution. The solution was then mixed. Then, 1 μL of calcein dye was added and mixed again. Finally, the prepared solution was covered with aluminum foil due to the sensitive fluorescent dye. For staining after the first cultivation, the medium was removed, and the cells were washed to eliminate serum esterase activity. Subsequently, the cells were stained according to the protocol [19]. After incubation, the cells were inspected under a fluorescence microscope. For evaluation, images were taken with an Olympus fluorescence microscope (BX51, Olympus, Tokyo, Japan) from five different positions, with 5× and 10× magnification on the scaffolds. Then, the ceramics were cut horizontally and viewed at the same three positions with the known magnifications. Living cells fluoresced green under blue light, and dead cells fluoresced red.

#### 2.5.2. Cell Proliferation Assay

Three samples of each of the differently sized scaffolds were examined after 3, 7 and 10 days using the WST-1 test. A NuncTM ThermanoxTM Coverslip (Thermo Fisher Scientific, Waltham, MA, USA) membrane served as the positive control. All samples and controls were equally covered with 50,000 cells in 200 μL. The cells were incubated for 2 h at 37 ◦C, with a CO2 saturation of 5% in the incubator so that they could adhere to the surface of the sample. At the end of this period, 2.5 mL of the DMEM-F12 complete medium was added to each sample and incubated. A medium change with the DMEM-F12 with the 10% FBS and 1% P/S additives was performed for days 7 and 10. The plate from day 3 was prepared for the WST evaluation. The medium was aspirated, and the wells were washed three times with PBS. The samples and the Thermanox coverslips were then transferred to a new well, and then 2.5 mL of the DMEM-F12 phenol red free (Art. No. 11039-021, Gibco, Grand Island, NE, USA) with the 1% P/S and 1% FBS additives were added to the wells with the sample (TCP + R). A total of 400 microliters of the medium was added to the previously used empty sample wells (TCP), positive control (C + R), empty control well (C+) and the blank. The blank contained only the DMEM medium without phenol red and was measured to account for background absorption. A 10% WST reagent (Art. No. 05015944001, Roche, Basel, Switzerland) was added to the corresponding volume of medium. Thus, 250 μL WST was added to the wells with sample (TCP + R), and 40 μL was added to the old wells (TCP and C+), the blank wells and the positive control (C+). This was incubated in an incubator at 37 ◦C for 2 h. After this time, the liquids were transferred into a 96-well plate. Three times in a row, 100 μL of each solution was added to the wells. The absorption was then measured at 450 nm using a Spectrostar Nano microplate reader (BMG Labtech, Ortenberg, Germany). The experiment was performed at least three times for each time point (3, 7, and 10 days).
