4.2. Mineralization Assay

For mineralization assay, cell nanofragments were incubated with α-MEM supplemented with 10 mM β-glycerophosphate (β-GP, Sigma-Aldrich), unless otherwise mentioned, in 48-well or 96-well tissue culture plates for different time intervals (1 to 7 days), at 37 °C, 5% CO₂, and 99% humidity. After incubation, the samples were then fixed with 4% paraformaldehyde (PFA) without PBS overnight, and gently washed with distilled water (DW) before staining. Mineralization was confirmed by 1% Alizarin Red S (ARS) staining (Sigma-Aldrich), as reported [40]. Briefly, after fixation and washing, the samples were incubated with 1% ARS solution for 10 min, and thoroughly washed (at least 3 times) to remove stain excess.

Alkaline phosphatase (ALP) staining was performed by incubating the cell nanofragments with 5-bromo-4-chloro-3-indolyl-phosphate and nitro blue tetrazolium (BCIP/NBT) substrate solution (Roche, Basel, Switzerland) for at least 20 min. The samples were then washed gently 3 times to remove the staining substrate. Photographs were taken, and the intensity of the staining was calculated using ImageJ.