C 12974

The current study used human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) as in vitro models to analyze MSC differentiation into adipocytes and osteoblast. Cells were purchased from PromoCell (C-12974, Heidelberg, Germany). For the experiments, cells at passage numbers between 3 and 6 were cultured in 6-well plates (1 × 10⁶ cells/well) unless otherwise noted. Prior to differentiation, cells were grown to confluency during which they were fed mesenchymal stem cell growth medium (C-28009, PromoCell). Following confluency, hBM-MSCs were induced to differentiate into adipocytes or osteoblasts by swapping the culture medium with mesenchymal stem cell adipogenic differentiation medium 2 (C-28016, PromoCell) or mesenchymal stem cell osteogenic differentiation medium (C-28013, PromoCell), respectively. Cells introduced to differentiation media were incubated for 10 days, swapping the differentiation medium with fresh media every third day. Treatment with M3G was carried out along with initial differentiation inducement only. Incubation of hBM-MSCs before and during the differentiation was in controlled incubators set to 37 °C temperature and 5% CO₂ level.

Murine osteoblast-like MC3T3-E1 cells obtained from ATCC (CRL-2593^™) and huBM-MSCs obtained from PromoCell (C-12974) were cultured in 6-well plates unless otherwise noted. MC3T3-E1 cells were fed with α-Modified minimal essential medium (αMEM) containing 10% fetal bovine serum (heat-inactivated, v/v), 1 mM sodium pyruvate, 100 units/L penicillin and 100 mg/L streptomycin in an atmosphere of 5% CO₂ at 37 °C. Following the confluence, osteoblast differentiation was induced with a differentiation cocktail of 50 μg/mL ascorbic acid and 10 mM β-glycerophosphate in cell culture medium. PFF-A was introduced to the cells with differentiation medium and included in every medium change (every second day).
huBM-MSCs were fed with mesenchymal stem cell growth medium (C-28009, PromoCell) and incubated in an atmosphere of 5% CO₂ at 37 °C. Following 100% confluence cells were induced by MSC osteogenic differentiation medium (C-28013, PromoCell) and incubated until the time of analysis. PFF-A was introduced with differentiation medium and included in all medium changes (every third day).

Human bone-marrow mesenchymal stem cells (bMSC) were purchased from PromoCell (C-12974) and cultivated in low glucose Dulbecco’s modified Eagle medium (DMEM; Sigma-Aldrich, Milan, Italy) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, Milan, Italy) and 1% antibiotics at 37 °C, 5% CO₂ atmosphere. Cells were cultivated until 80–90% confluence, detached by a trypsin EDTA solution (0.25% in PBS), harvested and used for experiments.

Cells representative for the bone healing process after composite implantation were tested. Accordingly, human mesenchymal stem cells (hMSCs), human primary osteoblasts progenitors, and human endothelial cells were directly cultivated in contact with control 100% BG and test composites.
Human mesenchymal stem cells (hMSC) were purchased from Merck (C-12974 from PromoCell GmbH, Heidelberg, Germany, distributed by Merck, Milan, Italy) and cultivated in low-glucose Dulbecco’s modified Eagle Medium (DMEM, Merck) supplemented with 15% fetal bovine serum (FBS, Merck) and 1% antibiotics (penicillin/streptomycin, Merck) at 37 °C, 5% CO₂ atmosphere. Human primary osteoblasts progenitors (hFOB 1.19 CRL-11372) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultivated in MEM/F12 mix medium (50:50, from Sigma, Darmstadt, Germany) 10% fetal bovine serum (FBS, from Sigma) 1% antibiotics, and 3 mg/mL neomycin (G418 salt, Sigma) at 34 °C, 5% CO₂ atmosphere. Human endothelial cells (EA.hy926, CRL-2922) were purchased from ATCC and cultivated in high-glucose Dulbecco’s modified Eagle Medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% antibiotics (penicillin/streptomycin) at 37 °C, 5% CO₂ atmosphere. Cells were cultivated until 80–90% confluence, detached by trypsin-EDTA solution, harvested, and used for experiments.