## 2 Materials and Methods
### Methods
#### 2.2.4 Biocompatibility Tests  
In this work, the biocompatibility tests were performed with three different cell types (MG-63, MLO-Y4 and MSC). Each of these cell types required individual cultivation. All three cell types were cultivated in 75 cm\({}^{2}\) cell culture flasks from CELISTAR in an incubator (37 \({}^{\circ}\)C, CO2 saturation 5%). The cell culture flasks for MLO-Y4 were coated with Rat Tail Collagen type I (0.15 mg/mL) in advance. The culture solutions for the cell types are listed below:  
* MG-63: DMEM-F12 medium with L-glutamine and 15 mM HEPES (2-(4-(2-hydroxyethyl)) -1-piperazinyl)-ethanesulfonic acid) and the additions of 1% penicillin/streptomycin (P/S) and 10% fetal bovine serum (FBS). * MLO-Y4: Alpha-Mem medium with L-glutamine and deoxyribonucleosides and the additions 1% P/S, 2.5% FBS inactivated and 2.5% Newborn Calf Serum. * MSC: bmMSC expansion medium (GMP+); DMEM medium (500 mL), 1 M HEPES (12.5 mL), L-glutamine (5.0 mL), P/S (5.0 mL), heparin (1 mL), Human Plasma centrifuged, filtered (25 mL), Human Platelet Lysate (25 mL). Live/Dead Assay  
On each scaffold 100 uL of cell specific medium were pipetted with the following cell concentrations: MG-63: 25,000 cells/100 uL; MLO-Y4: 50,000 cells/100 uL; MSC: 50,000 cells/100 uL. The well plates were then incubated for 2 h at 37 \({}^{\circ}\)C and a CO\({}_{2}\) saturation of 5% in an incubator. After two hours, 1 mL of the specific cell medium described previously was added to each well before incubating the well plates in the incubator for 3, 7 and 10 days. The staining solution was prepared by adding 2 mL DPBS (art. no. 14190-094, Gibco, Grand Island, NE, USA) to a Falcon tube (Greiner Bio-One International GmbH, Kremsmunster, Austria) and 4 uL ethidium homodimer III (Eth D-III) solution (together with calcein part of the Live/Dead Cell Staining Kit II (PromoCell, Heidelberg, Germany)) according to the manufacturer's protocol (PromoCell). An amount of 1 uL of calcein dye was added after mixing the staining solution. All steps were performed in the dark to avoid photobleaching of staining solution and samples. To eliminate serum esterase activity, all samples at a time point had the medium removed and the cells washed. Staining was then performed according to a previously published protocol [19]. Evaluation was performed using an Olympus fluorescence microscope (BX51, Olympus, Osaka, Japan) at five different positions on the samples at 5\(\times\) and 10\(\times\) magnification. LDH Assay  
LDH measurements were performed after 24, 48 and 72 h. In addition to the scaffolds, positive control (Triton X, 100% toxicity) and negative controls (cells only, 0% toxicity) were used for the measurements at the different times. Both coated and uncoated scaffolds were also used. Cells were seeded onto the scaffolds and membranes in 100 uL of their medium (MG-63: 25,000 cells/100 uL, MLO-Y4: 50,000 cells/100 uL, MSC: 50,000 cells/100 uL). These were then incubated for 2 h in an incubator at 37 \({}^{\circ}\)C and 5% CO\({}_{2}\) saturation. Following this, 1 mL of DMEM-F12 medium without phenol red was added to all wells with the additions of 1% P/S and 1% FBS. Since FBS in higher concentrations may induce background absorption, only 1% FBS was used. In the positive controls (C+), an additional 1% Triton X 100 was added to kill 100% of the cells. After 24 h incubation in the incubator, 100 uL from each well was transferred to three new wells of a 96-well plate. Thus, from one well, three wells of 100 uL each were obtained. To ensure that the \"blank\" had the same concentration of phenol red, 100 uL of DMEM-F12 medium containing phenol red was added to this well, prior to transfer. To evaluate the cytotoxicity, the Cytotoxicity Detection Kit solution was prepared. For this, 111.1 uL of catalyst solution was mixed with 5 mL of staining solution. Of this, 100 uL was pipetted into each well before incubating the well plate in darkness for 30 min. At the end of the 30 min, the absorbance at 490 nm could be measured using a spectrometer.