Human osteoblast like mg63 cells

For cell culture experiments, samples were sterilized under UV light, inserted into 12-well polystyrene cell culture plates (TPP, Switzerland; internal well diameter 21.4 mm) and seeded with: i) human osteoblast-like MG-63 cells (European Collection of Cell Cultures, Salisbury, UK; Cat. No. 86051601) suspended in Dulbecco's modified Eagle's Minimum Essential Medium (DMEM; Sigma, USA, Cat. No. D5648), ii) Saos-2 cells (European Collection of Cell Cultures, Salisbury, UK, Cat. No. 89050205) suspended in McCoy medium (Sigma, Cat. No. M4892), or iii) murine macrophage-like RAW 264.7 cells (ATCC, TIB-71, USA) suspended in RPMI-1640 medium (Sigma, Cat. No. R8758). All culture media were supplemented with 10% fetal bovine serum (Sebak GmbH, Aidenbach), and gentamicin (40 µg/mL, LEK). Each well contained i) 36,000 MG-63 cells (i.e., approximately 10,000 cells/cm²), ii) 20,000 Saos-2 cells (5,600 cells/cm²) or iii) 50,000 RAW 264.7 cells (14,000 cells/cm²), and 2 mL of the medium. The cells were cultured for 1 and 3 days (to evaluate cell numbers) or for 7 days (for PCR detection of markers of osteoblast differentiation, and to measure the potential immune activation of RAW cells) at 37°C in a humidified air atmosphere containing 5% CO₂. Two samples were used for each experimental group and time interval.

Adipose-derived human mesenchymal stem cells (ADSCs) were purchased from PromoCell. The culture medium consisted of Mesenchymal Stem Cell Growth Medium® and 10% Supplement Mix® (PromoCell GmbH, Heidelberg, Germany), with 100 IU/mL of penicillin and 100 µg/mL streptomycin (Sigma-Aldrich, Darmstadt, Germany), according to the manufacturer’s protocol. The cells were passaged using 0.04% trypsin-EDTA solution (Sigma-Aldrich) when reaching 70%–80% of confluency.
Human osteoblast-like MG 63 cells (European Collection of Cell Cultures, Salisbury, UK, cat. no. 86051601) were cultured in Eagle’s Minimum Essential Medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, MEM non-essential amino acid, 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin). All components were provided from Sigma Aldrich (Darmstadt, Germany).
L929 murine fibroblast cells (American Type Culture Collection) were cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% fetal bovine serum and 1% antibiotics (penicillin/streptomycin). All cell lines were cultured at 37 °C in humidified atmosphere of 5% CO₂.

In the process of obtaining bioglasses and composites, the following components were used: tetraethoxysilane; zinc nitrate (V) hexahydrate; calcium nitrate (V) tetrahydrate (Avantor Performance Materials Poland S.A., Gliwice, Poland); strontium nitrate (V) (Sigma-Aldrich, St. Louis, MO, USA); triethyl phosphate (V) (Fluka, Chemie GmbH, Buchs, Switzerland); chitosan of a 75% deacetylation degree and 500 mPas viscosity (HMC+—Heppe Medical Chitosan GmbH company, Halle, Germany); and 80% pure acetic acid, ethanol 96% p.a. grade.
Human osteoblast-like MG63 cells (European Collection of Cell Cultures, Salisbury, UK) were used for cytocompatibility studies of the chitosan/bioglass composites.