Calcein am staining

We used microfluidic
chambers (channel width, 3.8 mm; channel length, 17 mm; channel height,
0.4 mm; μ-slide VI 0.4) purchased from Ibidi (Martinsried, Germany).
We chose either polymer culture-treated coverslips or glass coverslips,
depending on the type of subsequent microscopy used. Regardless of
the used coverslip’s material, the channels were coated with
Fibronectin (75 μg mL^–1) to enhance the attachment
of CHO cells. After preparing the cell suspension at a density of
2 × 10⁶ cells mL^–1, we injected
it into the microchannel from the inlet and cultured it at 37 °C
to promote cell adhesion. After at least 1 h, we added culture media
to the inlet and outlet of the microchannel, preventing evaporation
and change in salt concentrations affecting cell viability. Cells
were then kept in culture for at least 24 h before using them for
subsequent experiments. Cell viability in the chambers was routinely
verified with calcein AM staining (Sigma-Aldrich).

Osteoblasts were seeded at 3 × 10³ cells/cm² within a 96‐well plate with basal medium and incubated for 24 h. Media was replaced with fresh basal medium supplemented with/without TSA (Sigma‐Aldrich, UK) (5, 10, 20, 50, 100 nM) and incubated for 1, 3 and 7 days. At each time point, AlamarBlue reagent (Thermo Scientific, UK) was added and incubated for 4 h at 37°C. Fluorescence readings was acquired using a SPARK spectrophotometer (TECAN, CH) at an excitation/emission wavelength of 540/590 nm, respectively. Employing the same protocol, the osteoblast morphology via calcein‐AM staining (Sigma‐Aldrich, UK) after 3 days of culture, observed under an EVOS fluorescent inverted microscope (Thermo Scientific, UK).

hBMSCs were seeded at 3 × 10³ cells/cm² within a 96-well plate with a basal medium and incubated for 24 h. The medium was replaced with a fresh basal medium supplemented with/without AZT (Sigma-Aldrich, Gillingham, UK) (5, 10, 20, 50 µM) or DFO (Sigma-Aldrich, Gillingham, UK) (5, 10, 20, 50 µM), and incubated for 1, 3 and 7 days. At each time point, AlamarBlue reagent (Thermo Scientific, Paisley, UK) was added and incubated for 4 h at 37 °C. Fluorescence readings were acquired using a SPARK spectrophotometer (TECAN, Männedorf, Switzerland) at an excitation/emission wavelength of 540/590 nm, respectively. Employing the same protocol, the cell morphology was assessed via calcein-AM staining (Sigma-Aldrich, Gillingham, UK) after 3 days of culture, and observed under an EVOS fluorescent inverted microscope (Thermo Scientific, Paisley, UK).