Analysis of Intracellular Magnesium and Mineral Depositions during Osteogenic Commitment of 3D Cultured Saos2 Cells

Abstract

In this study, we explore the behaviour of intracellular magnesium during bone phenotype modulation in a 3D cell model built to mimic osteogenesis. In addition, we measured the amount of magnesium in the mineral depositions generated during osteogenic induction. A two-fold increase of intracellular magnesium content was found, both at three and seven days from the induction of differentiation. By X-ray microscopy, we characterized the morphology and chemical composition of the mineral depositions secreted by 3D cultured differentiated cells finding a marked co-localization of Mg with P at seven days of differentiation. This is the first experimental evidence on the presence of Mg in the mineral depositions generated during biomineralization, suggesting that Mg incorporation occurs during the bone forming process. In conclusion, this study on the one hand attests to an evident involvement of Mg in the process of cell differentiation, and, on the other hand, indicates that its multifaceted role needs further investigation.

Keywords: biomineralization; osteoblastic differentiation; osteogenesis; osteosarcoma.

Figure 1

Assessment of 3D cell culture conditions. (a) cell growth curve; (b) cell cycle analysis. The figure depicts the results obtained in one experiment representative of three.

Figure 1

Assessment of 3D cell culture conditions. (a) cell growth curve; (b) cell cycle analysis. The figure depicts the results obtained in one experiment representative of three.

Figure 2

Analysis of SaOS2 cells cultured in 3D collagen scaffolds (3 and 7 days) treated with the osteogenic cocktail (20 nM 1α,25-Dihydroxyvitamin D3, 50 µM L-Ascorbic acid 2-phosphate, 10 mM β-Glycerophosphate). qPCR analysis of osteogenic markers (RUNX2, COL1A1, BGLAP, and SPP1) was performed using GAPDH and HPRT1 as reference genes (2-ΔΔCT method). Fold changes from control untreated cells at day 3 were calculated. Data are reported as mean ± SEM of three biological replicates. * p < 0.05; ** p < 0.01.

Figure 3

H/E ((a), left panel) or Alizarin Red ((a), right panel) staining of paraffin-embedded sections of 10 μm thickness (n = 3). (b) Fluorimetric assay of total intracellular magnesium content by DCHQ5 probe (panel b) [19]; panel (c) quantification of Alizarin-stained Ca depositions. Data are reported as means ± standard error of the mean, values deriving from a triplicate experiment. * p < 0.05.

Figure 4

X-ray analysis of depositions released by SaOS2 cells. Panels (a) and (b) depict the 3D distribution of mineral depositions in the whole control and treated scaffolds imaged by microfocus X-ray µCT at the TomoLab station of Elettra (isotropic voxel size = 5 µm for the control and 3.3 µm for the treated sample). The dimensions of the scaffolds are approximately 80 mm³. Panel (c) and (d) show two selected VOIs reporting the depositions distribution in CTRL (6000 × 4300 × 700 µm³) and treated scaffolds (7300 × 1200 × 800 µm³) respectively. Table and graph show the descriptive statistical analysis of the depositions in the two representative VOIs showed in panel c (CTRL) and d (treated), respectively. The graph classified the particles dimension (µm³) in relation to the number of particles within the class. Panel (e) describes the Mg and P co-localization in a deposition released by a differentiating SaOS2 cell at the TwinMic beamline of Elettra.

Figure 5

Synchrotron-based X-ray µCT analysis of one treated scaffold imaged at the SYRMEP beamline of Elettra. Panel (a) represents a reconstructed axial slice of the scaffold (isotropic voxel size = 0.9 µm) highlighting the presence of mineral depositions (white dots) along brighter filaments. Panel (b) shows a zoom of the green ROI indicated in panel (a). Panel (c) depicts a 3D rendering of 50 adjacent slices (400 × 400 × 50 µm³) showing the 3D distribution of the mineral depositions. Scale bars = 100 µm.