*2.5. Elisa*

In order to analyze protein secretion, a human Osteoprotegerin Instant ELISA (a ffymetrix, San Diego, CA, USA) and a MicroVue ® Bone CICP EIA Elisa (Quidel, San Diego, CA, USA) were used according to the manufacturers' instructions. The Elisas were analyzed using a Tecan Infinite M200 Pro (Tecan, Männedorf, Switzerland) for readout and the software iControlTM V. 1.2 or MagellanTM V. 7.2 for, all from Tecan, Männedorf, Switzerland).

#### *2.6. MTT Assay*

Cell proliferation was tested according to DIN ISO 10993-5 by using tetrazolium salt MTT (3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid) as described elsewhere. Staining was conducted 24 h, 3 days, and 7 days after cell seeding on the specimen, which was cultivated under standard conditions [15]. At the indicated time points, the discs were placed into fresh wells of a 24-well plate in order to only include cells seeded on the titanium surface. The wells were filled with 1 mL MTT solution and incubated for 3 h. Then, the MTT solution was replaced by 200 μL of a 0.04 M HCl/Isopropanol mixture. The discs were reversed using forceps and sonicated for 3 min. The staining was analyzed with a Tecan Infinite M200 Pro (570 nm/650 nm), as described above (Section 2.5).

#### *2.7. Alizarin Red S Staining*

To prove the matrix calcification of the seeded specimens, Alizarin red S staining was performed. Therefore, 20,000 cells per disc were cultivated for 14 days in basal medium with the additional supplements as indicated above (Section 2.4). Then, the discs were placed into a new well and washed three times with 1 mL phosphate bu ffered saline (Biochrome, Berlin, Germany). Subsequently, 1 mL of 70% ethanol was added and incubated for 1 h. Thereafter, 1 mL of a 40 nM Alizarin staining solution was added and incubated on a horizontal shaker for 10 min. After several washing steps with pure water, an incubation for 15 min with 10% cetylpyrimidinchloride was performed. 100 μL was used to analyze the concentration of Alizarin red S using a Tecan Infinite M200 Pro plate reader (absorbance 652 nm), as described above (Section 2.5).

#### *2.8. Statistical Testing*

Statistical analysis was carried out using GraphPad Prism version 8.4 for Macintosh (GraphPad Software, La Jolla, San Diego, CA, USA, (www.graphpad.com)). Depending on the experimental setup, a one-way-Analysis of Variance (ANOVA) or two-way-ANNOVA was used. A Tukey post hoc test was performed for one-factorial analysis of variance; a Bonferroni post hoc test was used for two-factorial analysis. The level of significance was set to *p* < 0.05. The data are presented by a Scatter-dot plot with Median and interquartile range.
