## 2 Materials and Methods
### Evaluation of Alkaline Phosphatase Activity and Alizarin Red S Staining in Osteogenic Media  
These spheroids were grown in osteogenic media contained 82% of minimal essential medium alpha modification, with L-glutamine, ribo- and deoxyribonucleosides (Cat. No. SH30265.01; HyClone(r), GE Healthcare Life Sciences, Thermo Fisher Scientific, Inc., Logan, UT, USA), 15% of fetal bovine serum (Cat. No. SH30084.03; HyClone(r)), 1% of L-glutamine (Cat. No. G7513; Sigma-Aldrich), 1% of penicillin-streptomycin solution (Cat. No. SV30010; HyClone(r)) and 1% of Dulbecco's phosphate buffered saline modified without calcium, magnesium (Cat. No. SH30028.02; HyClone(r)) with 0.019 g of dexamethasone (Cat. No. D4902; Sigma-Aldrich, St Louis), 1 g of \(\beta\)-glycerophosphate disodium salt hydrate (Cat. No. G9422) and 0.0144 g of ascorbic acid (Cat. No. A4544; Sigma-Aldrich). An alkaline phosphatase (ALP) activity assay (Cat. No. AS-72146; SensoLyt(r) p-nitropenyl palmitate [pNPP] colorimetric alkaline phosphatase assay kit, AnaSpec, EGT Group, Fremont, CA, USA) and Alizarin Red S staining (Cat. No. 0223; ScienCell Research Laboratories, Carlsbad, CA, USA) were used for osteogenic differentiation. Figure 1: Study flow diagram illustrating the overview. The ALP assay kit was used to detect ALP in biological samples by providing a convenient colorimetric assay. A mixture of 50% component A (pNPP, a colorimetric alkaline phosphatase substrate), 5% component B (10\(\times\) assay buffer), and 45% distilled water was added to the sample. The sample was incubated at room temperature for one hour, and the spectrophotometric absorbance was evaluated at 405 nm on Days 1, 7, and 14. An anthraquinone dye was used to stain calcium deposits on Days 2, 8, 14, and 22. The stem cell spheroids were washed with phosphate buffered saline (pH 7.4 \(\pm\) 0.1; Cat. No. LB204-02, fresh media, Welgene, Gyeongsan-si, Gyeongsangbuk-do, Korea) and fixed with 4% paraformaldehyde (Cat. No.P2031, Biosesang, Seongnam-si, Gyeonggi-do, Korea) for 30 min at RT. The spheroids were washed with dH2O. Ten percent cetylpyridinium chloride (Cat. No. C0732; Sigma-Aldrich, St. Louis, MO, USA) was applied for 15 min at RT to solubilize the bound dye on Days 2, 8, 15, and 22. The spectrophotometric absorbance at 560 nm was measured to quantify the bound dyes. Total RNA Extraction and Quantification of Runt-Related Transcription Factor 2 (RUNX2), Bone Sialoprotein (BSP), Osteocalcin (OCN) and COLA1 mRNA by Real-Time Quantitative Polymerase Chain Reaction (qPCR)  
Total RNA extraction was performed using a commercially available kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's instructions [17]. The quantity of RNA was evaluated with a bioanalyzer (Agilent 2100) using a kit (RNA 6000 Nano Chip; Agilent Technologies), and RNA quality was evaluated with the ratio of absorbance at 260 nm and 280 nm using a spectrophotometer (ND-2000, Thermo Fisher Scientific, Inc.). RNA was used as reverse transcription template applying reverse transcriptase (SuperScript II; Invitrogen, Carlsbad, CA, USA). mRNA expression was detected by qPCR on Days 1, 7, and 14. We used GenBank to design the sense and antisense primers for PCR. The primer sequences were as follows: RUNX2 (accession No. : NM_001015051.3; forward: 5\({}^{\prime}\)-CAGTTCCCAAGCATTTCATCC-3\({}^{\prime}\), reverse: 5\({}^{\prime}\)-AGGTGCTGATGGCATT-3\({}^{\prime}\)); BSP (accession No. : NM_004967.4; forward: 5\({}^{\prime}\)-CCTCCTCCCAAATGGTGGTTT-3\({}^{\prime}\), reverse: 5\({}^{\prime}\)-ATTCAACGGTGGTTTTTC-3\({}^{\prime}\)); OCN (accession No. : NM_199173.6; forward 5\({}^{\prime}\)-GGTCAGAGTCCAGCAAAGG-3\({}^{\prime}\), reverse: 5\({}^{\prime}\)-GCGCCTGGGCTCTTTC-3\({}^{\prime}\)); COL1A1 (accession No. : NM_000088.4; forward: 5\({}^{\prime}\)-TACCACCACCAGCTG-3\({}^{\prime}\), reverse: 5\({}^{\prime}\)-CCGAACCAGACATGCCTTT-3\({}^{\prime}\)); and \(\beta\)-actin (accession No.