Nhost

Commercially available human mesenchymal stromal cells (hMSCs; Lonza, Walkersville, MD, USA) were cultured in Mesenchymal Stem Cell Growth Medium (MSCGM™; Bullet Kit, Lonza, Walkersville, MD, USA) in a humidified atmosphere of 5% of CO₂ at 37 °C. To obtain osteogenic differentiation and consequent pre-osteoblast cells, hMSCs were treated with hMSC Mesenchymal Stem Cell Osteogenic Differentiation Medium (OM) (hMSC; Osteogenic Differentiation BulletKit™, Lonza). Commercially available normal human osteoblast cells (Nh-Ost; Lonza) were cultured in Osteoblast Growth Medium (OGM; Osteoblasts BulletKit™, Lonza). Culture medium was changed every three days, and cells were split at 70–80% of confluence using StemProAccutase (Gibco by Life Technologies Italia, Monza, Italy). Cells were used at an early passage for all experiments.

The human osteosarcoma cell lines, U-2OS (origin from a female 15y old osteosarcoma patient), SaOS, MNNG/HOS, and MG63 were purchased from the American Type Culture Collection (Rockville, MD) in 2014. The human osteosarcoma KHOS (origin from a female 13y old osteosarcoma patient) cell line was provided by Dr. Efstathios Gonos (Institute of Biological Research & Biotechnology, Athens, Greece) in 2008. KHOS and U-2OS are two commonly used cell lines in osteosarcoma research. The human osteoblast cell lines NHOst and HOB-c were purchased from LonzaWalkersville (Walkersville, MD) and PromoCell GmbH (Heidelberg, Germany) respectively in 2013. The osteosarcoma cell lines were cultured in RPMI-1640 (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin (Life Technologies, Carlsbad, CA). The osteoblast cell lines were cultured in osteoblast growth medium (PomoCell) with 10% fetal bovine serum. All cells were maintained in a humidified atmosphere containing 5% CO₂ at 37 °C. Bicalutamide (Sigma-Aldrich, St. Louis, MO) is a synthetic, non-steroidal AR antagonist and a pure antiandrogen used in the treatment of prostate cancer, hirsutism, and other androgen-dependent conditions. Doxorubicin was purchased from APP pharmaceuticals, LLC, (Lake Zurich, Illinois).

Primary human osteoblasts (NHOst, Lot Number: 0000288136; LONZA, Basel, Switzerland) and fibroblasts (NHDF, Lot Number: 0061502; PromoCell, Heidelberg, Germany) were cultivated on titanium and PEEK samples (both: diameter: 12 mm, density: 2.5 mm; MEDICON, Tuttlingen, Germany). After shipping, the material probes were cleaned in 70% ethanol overnight to remove residue of the production by the companies PromoCell/LONZA. The next day, the samples were dried under a laminar floor and autoclaved (121 °C, 1 bar, 20 min.). The probes were stored at room temperature and, before use in the cell culture, all materials were sterilized by UV light for 15 min.
The cells were cultured following slight modifications of the protocols provided by the companies PromoCell and LONZA, respectively. The seeding density of the fibroblasts NHDF were 5000 cells/cm² and for the NHOst, 11,000 osteoblasts/cm². Instead of trypsin, Accutase was used for detaching the cells from the culture flasks. For the 24 h LPS incubation, LPS from the bacterium Escherichia coli (Sigma-Aldrich, Taufkirchen, Germany) was used at a concentration of 10 µg/mL, as provided by Tilakaratne et al. [49 (link)]. E. coli is able to bind to TLR4 and to trigger an inflammatory response. The handling of all human samples strictly adhered to the “Declaration of Helsinki”.

Normal human osteoblasts (NHOst, Lonza, USA) were cultured in OGM culture medium, supplemented with 10 % FBS, ascorbic acid and 5 % solution of gentamicin and amphotericin-B (OGM BulletKit, Lonza, USA) in an atmosphere of 5 % CO₂ at 37 °C. The tests were conducted on cells from passages 5 to 6. The cell suspension was obtained by addition of 5 % trypsin with EDTA (Lonza, USA). After flushing and centrifugation, the cells were concentrated to 2 × 10⁴ cells/mL in OGM medium supplemented with Differentiation SingleQuots (Lonza, USA). Next, cell were seeded on sterilized discs—test samples. Bottom of the well plate (TCPS) was used as a positive control. Cells were cultivated up to 21 days; cell viability/cytotoxicity tests were conducted at day 3 and 7, while cell mineralization was assessed at days: 7, 14 and 21.

The human osteoblast cell line NHOST was acquired from Lonza Walkersville Inc. (Walkersville, MD, USA) and cultured in osteoblast growth medium (PromoCell, Heidelberg, Germany). The human osteosarcoma cell line KHOS was generously provided by Dr. Efstathios Gonos (Institute of Biological Research & Biotechnology, Athens, Greece) [27 ]. Other human osteosarcoma cell lines MG63, MNNGHOS, U2OS, and 143B were purchased from the American Type Culture Collection (ATCC) (Rockville, MD, USA). The recurrent human osteosarcoma cell line OSA1777 was established in our lab and previously authenticated by the ATCC database [28 (link)]. These cell lines were cultured in RPMI 1640 (GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Sigma‐Aldrich, St. Louis, MO, USA) and 2% penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA). All cell lines were cultured in a humidified 5% CO₂ atmosphere at 37 °C.