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To analyze the presence of calcium ECM in hydrogels, Alizarin Red S staining was applied. Briefly, fixed hydrogels were washed in distilled water, incubated for 10 min in Alizarin Red S working solution, washed three times in water and observed with inverted microscope as a whole mount. Additionally, fixed hydrogels were embedded in paraffin, cut for 5 µm sections and stained for 5 min. Documentation was made with Canon PowerShot digital camera.

After osteogenic differentiation, the cells were fixed with 4% PFA (paraformaldehyde) for 15 min at room temperature. To visualise the calcium deposits, the specimens were stained with Alizarin Red for 20 min at room temperature as described previously [20 (link), 28 (link)]. Then, the specimens were washed three times with distilled water and observed under Axio Observer A1 inverted microscope (3832000970, Zeiss, Oberkochen, Germany). The photographs were taken by Canon PowerShot digital camera (Woodhatch, UK) under 100 × magnification. The resolution of obtained images was: 3648 × 2736 pixels. The differences in staining intensity between the specimens were based on the number of colour pixels. The number of colour pixels was determined in three technical repetitions and using three different thresholds in Pixel Counter plugin (ImageJ Software version 1.52n, Wayne Rasband, National Institutes of Health, USA).

To visualize the accumulation of lipid droplets within the cells after PA treatment, they were stained with Oil Red O dye. Prior to staining, cells were washed with phosphate buffer saline (PBS) and fixed with 4% paraformaldehyde (PFA). An additional wash was performed to remove the fixation solution, and cells were treated with 60% isopropanol for 5 min. After the removal of alcohol and washing, cells were incubated with Oil Red O solution for 15 min at room temperature (RT). Specimens were observed under an inverted microscope (Leica), and pictures were acquired using a Canon PowerShot digital camera. Accumulation of dye was quantified using spectrophotometrical measurement. Briefly, the accumulated dye was washed out from the cells using 100% isopropanol and subjected to 96-well plates for the measurement of absorbance at 490 nm/570 nm wavelengths using a plate reader (Epoch, Biotek, Bad Friedrichshall, Germany).
Alternatively, neutral lipid accumulation was observed in cells using Lipid Tox™ Green Neutral Lipid Stain (ThermoFisher). Fixed samples were incubated with the dye solution (1:200) for 30 min at RT. After washing, the dye, the specimens were mounted on glass slides using ProLong™ Diamond Antifade Mountant with DAPI (ThermoFisher) and observed in a confocal microscope (Leica TCS SPE).

The intracellular accumulation of neutral lipids within the differentiated and undifferentiated ASCs was evaluated by Oil Red O staining according to supplier’s instructions. Briefly, the cells were fixed in 4% paraformaldehyde for 40 min at room temperature, and additionally for 5 min with 60% isopropanol. The fixed cells were subsequently stained with 0.5 g/mL Oil Red prepared in 60% aqueous isopropanol for 15 min at room temperature, and then washed with 60% aqueous isopropanol and PBS. The nuclei were counterstained with hematoxylin solution for 1 min. All stained cells were monitored under an inverted microscope (Axio Observer A1, Zeiss), and micrographs were acquired using a Canon PowerShot digital camera.

The protocol of extracellular matrix staining was described previously.²¹, ²⁹ Briefly, differentiated cultures of BMSCs were fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature and stained with specific dyes. Safranin‐O was used for proteoglycans detection and Alizarin Red for calcium deposits detection. Obtained specimens were analysed using Axio Observer A1‐inverted microscope (Zeiss, Oberkochen, Germany) and documented with Canon PowerShot digital camera (Woodhatch, UK). The photographs of visualized proteoglycan and calcium deposits were taken under 100× magnification. In order to visualize the neutral lipid droplets, HCS LipidTOX Green Neutral Lipid Stain was used according to manufacturer's protocol (H34475, Sigma‐Aldrich) and observed under a confocal microscope (Leica TCS SPE, Leica Microsystems, KAWA.SKA Sp z o.o., Zalesie Górne, Poland). The photographs of visualized neutral lipid droplets were taken under 630× magnification. Obtained signals were measured using Fiji (ImageJ) and Pixel Counter plugin (version 1.52n, Wayne Rasband, National Institutes of Health, USA) as described previously.³⁰ The differences between the amount of colour pixels were determined in three technical repetitions and using three different thresholds (osteogenesis/chondrogenesis—239, 240 and 241; adipogenesis—48, 50 and 51) within ImageJ Software.