T 25 cell culture flasks

To study melanogenesis in vitro, a murine melanoma B16F10 cell line was employed. The cell line was purchased from the Korea Cell Line Bank (Seoul, Republic of Korea). Cells were cultured in T-25 cell culture flasks (#70025, SPL Life Sciences, Pocheon, Republic of Korea) and fed with Dulbecco’s modified Eagle’s medium (DMEM, Welgene, Republic of Korea) containing 10% fetal bovine serum (FBS, Welgene) and 1% antibiotic solution (Welgene). For the assays, cells were transferred onto 6- or 96-well plates. Melanogenesis in B16F10 cells was induced by stimulating the cells with the addition of 400 nM α-MSH for 24 h. A separate group was only fed DMEM without α-MSH for comparison. During experiments, cells were kept in 37 °C incubators with controlled atmosphere containing 5% CO₂.

Alizarin Red S staining assay was performed to investigate the in vitro osteogenic capability of the samples. For this purpose, osteosarcoma cells (MG-63 cell line) in a concentration of 6 × 10⁴ cells were plated in T25 cell culture flasks (SPL Life Sciences, Gyeonggi-do, Korea) and incubated in a 5% CO₂ humidified atmosphere at 37 °C overnight. Then, the RPMI-1640 media were replaced with the conditioned media (containing 4 mg/mL of crushed scaffold) and followed by a further incubation for 14 days. The media were exchanged each 3–4 days once. After finishing the incubation time, all the conditioned media were aspirated, and the cells were carefully washed with PBS (three times) and fixed with a sufficient amount of 10% neutral buffered formalin (NBF) for 30 min. Afterwards, the NBF was removed, and the cells were washed with distilled water. Finally, the cells were stained with Alizarin Red S (Sigma-Aldrich, USA) for 45 min at room temperature in the dark. The amount of generated calcium deposits was detected by optical microscopy observations.