Cell scraper

To investigate the effect of transfection on osteoclast differentiation, a coculture of PDLF and human macrophages (THP‑1; TIB-202; ATCC; Manassas, VA, USA) was established. THP‑1 monocytes were cultured in RPMI (61870-010, Gibco™, Carlsbad, CA, USA) supplemented with 20% FBS (P30-3302; PAN-Biontech, Aidenbach, Germany) and 1% antibiotics/antimycotic (A5955, Sigma-Aldrich, St. Louis, MO, USA). First, suspension THP‑1 monocytes were differentiated to adherent macrophages by addition of 25 ng/ml phorbol 12-myristate 13-acetate (PMA; 19-144, Sigma-Aldrich, St. Louis, MO, USA) for 3 days. PDLFs were seeded in 24-well plates (662 160; Greiner Bio-One, Frickenhausen, Germany) according to the experimental setup for transfection and compressed with 6 g/cm² for 6 h (supplemental Fig. 1c). After the compression period, we checked, whether the suspension THP‑1 monocytes were differentiated into adherent macrophages under the microscope. The macrophages were scraped off using a cell scraper (83.3951, Sarstedt, Nümbrecht, Germany) and 50,000 macrophages per well of the 24-well plate were added to the previously compressed PDLFs and incubated at 37 °C for an additional 3 days. Finally, TRAP staining was performed.

MDA-MB468 and LNCaP cells were cultured for 72 h at normoxia or hypoxia. About 5x10^7 cells were washed with PBS (PAA) supplemented with protease inhibitor and harvested from culture flasks with a cell scraper (Sarstedt). After 10 min centrifugation at 500xg at 4°C, subcellular fractionation was conducted with the Qproteome Cell Compartment kit (Qiagen). Protein samples were precipitated in four volumes of ice-cold acetone and solved in PBS. Protein concentration was determined by bicinchoninic acid assays (BCA), using the BCA Protein Assay reagent (Pierce) with BSA standard dilutions for quantification. Samples were electrophoresed on 12% SDS polyacrylamide gels and blotted to a nitrocellulose membrane. Membranes were incubated in 5% non-fat dry milk for 1 h and washed in 0.1% PBS-T for 5 min. Primary antibodies were applied (Table 3) overnight at 4°C. Secondary antibodies (Table 3) were incubated for 1 h at room temperature. ECL solution was applied for time frames varying from 45 sec to 5 min to detect light emission via photo film (Kodak) exposure. Membrane stripping was conducted with Mild Stripping buffer (Abcam).

BS were generated as previously described^{18 (link)}. In order to incorporate glioblastoma cells into the BS, the protocol was slightly modified as follows: NPCs were grown (as above) in 175 mm² poly-l-ornithine and laminin-coated flasks. When NPCs were at 90% confluency, 7 × 10⁵ glioblastoma cells were plated on top of the NPCs. After 24 hours the cells were detached mechanically using a cell scraper (Sarstedt). The mixture of cells was pipetted repeatedly to disaggregate cell clumps. A density of 2 × 10⁶ cells per well were plated on a non-coated 6 plate-well. Cells were grown in differentiation medium (Neurobasal^® electro Medium (Gibco) supplemented with 5% B-27^® Electrophysiology (Gibco), 1% Glutamax (Gibco), 0.01 μg/ml human recombinant GDNF (Gemini), 0.01 μg/ml human recombinant BDNF (Gemini). Cultures were kept at 37 °C in an atmosphere of 5% CO₂ under constant gyratory shaking (88 rpm, 19 mm orbit) for up to 7 weeks, Fig. 1 Supplementary Data.

Spheroids (500 for hanging drop and 750 for spheroid plate culture) were snap-frozen after 7 days (each at least n = 3–6) and stored at −80 °C until homogenized in RLT-buffer containing 1% mercaptoethanol (Qiagen GmbH, Hilden, Germany). Monolayers (0.6 × 10⁶ ligamentocytes) cultured for 24 h in T25 flasks were incubated in RLT-buffer containing 1% mercaptoethanol and scraped off using a cell scraper (Sarstedt AG & Co. KG, Nürnbrecht, Germany). RNA was isolated using the RNeasy Mini kit according to the manufacturer’s instructions (Qiagen GmbH), with on-column DNA elimination. The purity and quantity of the RNA samples were analyzed using the Nanodrop ND-1000 spectrophotometer (Peqlab, Biotechnologie GmbH, Erlangen, Germany) applying the 260/280 absorbance ratio.

The effect of the material extracts, as well as the suspension of material particles, on the metabolic activity of RAW 264.7 macrophages in the presence and absence of low concentration of an inflammatory agent, was tested as well. The cells were detached using a cell scraper (Sarstedt, Nümbrecht, Germany), centrifuged, washed, counted, and seeded in 96-well culture plates. For this assay, 2 × 10⁴ cells per well were seeded, and after 24 h of cultivation, the material extracts (concentrations 100, 25, and 5%) and the suspension of the material particles (concentrations 100 and 20 µg/mL) with and without the addition of 1 ng/mL of lipopolysaccharide (LPS, 0111: B4 Escherichia coli, Sigma-Aldrich, St. Louis, MO, USA) were added to the cells. As controls, complete DMEM (unstimulated macrophages), and LPS (LPS-stimulated macrophages) were used. The cells were incubated with material extracts and suspension, as well as control media, for the next 24 h, under standard cell culture conditions. After the incubation period ended, an MTT test was performed.

Control and test cells (2 × 10⁶ each) were grown over a period of 5, 10, 15, and 20 days in dish culture, rinsed twice with PBS and scraped off from the dishes using a cell scraper (Sarstedt, Nümbrecht, Germany). After centrifugation the cells were re-suspended and dissolved in 1 N HCl. Calcium content was measured using the Abcam Calcium Detection Assay Kit ab102505 (Abcam, Berlin). This colorimetric endpoint assay measures the amount of calcium–cresolphthalein complexone formed when cresolphthalein complexone binds to free calcium in alkaline solution within the physiological range of 0.1 mM–3.0 mM. Absorbance was measured at a wavelength of 575 nm with background subtraction of 490 nm using a microplate reader (Dynatech MR5000, Denkendorf, Germany).

The RNA was extracted with an RNeasy mini kit (#74104; Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Briefly, after a 24-h incubation with the drugs, the cell culture media were removed from the wells (four replicates) and replaced with 350 µL of RLT buffer containing 1% of mercaptoethanol (#M6250, Sigma). The cells were scraped into the RLT buffer with a cell scraper (#83.1832, Sarstedt, Newton, NC, USA) and transferred to a 1.5-Ml centrifuge tube. The cells were homogenized by vortexing for 1 min. Then, 350 µL of 70% ethanol was added to the lysate. The lysate was then transferred to a column, centrifuged, and washed with RW1 buffer. For the DNA removal, DNAse I (#79256, Qiagen) and an RDD buffer mixture were added to the column and the mixture was incubated at room temperature for 15 min. The column was washed with RW1 and an RPE buffer according to the manufacturer’s protocol. The remaining RNA was eluted to 50 µL of RNase-free water, aliquoted, and stored at −70 °C for quality control and RNA-seq.