*3.6. Immunofluorescence Staining*

At the proliferation assay timepoints (24 and 72 h), two replicates of each sample were fixed with 4% formaldehyde (Panreac AppliChem, Barcelona, Spain) and subjected to immunofluorescence staining, to analyze their behavior in the di fferent culture patterns. Cell's cytoskeleton was stained with 1 μg.mL−<sup>1</sup> of phalloidin tetramethylrhodamine (TRITC, Sigma Aldrich, Sintra, Portugal) solution for 45 min at room temperature, and cells' nucleus with 1 μg.mL−<sup>1</sup> of a 4,6-diamidino-2-phenylindole (DAPI, Sigma Aldrich, Sintra, Portugal) solution for 5 min. Samples were washed with PBS 1× before, after, and during the steps. Finally, the samples were visualized with fluorescence microscopy (Olympus BX51 Microscope, Lisboa, Portugal).

#### *3.7. Quantification of DNA and Alkaline Phosphatase Activity*

Cells' DNA content wasmeasured using a CyQUANT ® Cell Proliferation Assay Kit (Life Technologies, Porto, Portugal) and the osteogenic capacity was determined through an ALP (Sigma Aldrich, Sintra, Portugal), both of which are described in [26]. Briefly, after 7 days of cell culture on both GM and DM, cells were lysed with Triton 0.1% buffer and frozen at 70 ◦C. After thawing, 50 μL *p*-nitrophenyl phosphate and 50 μL 2-amino-2-methyl-1-propanol were added, according to manufacturer's protocol. Using a microplate reader, the amount of the produced *p*-NP (*p*-nitrophenol) was measured, reading the absorbance at 405 nm. To proceed to the ALP activity normalization, cells' DNA content was quantified from the cell lysate using the CyQUANT ® Cell Proliferation Assay Kit, according to the manufacturer's protocol, measuring its fluorescence by exciting the sample at 480 nm and measuring the emission at 520 nm, using the same microplate reader. The results will be presented as mean ± SEM of quadriplicated samples.
