Osteodiff medium

For adipogenic differentiation, cells were cultured in AdipoDiff medium (Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with 1% penicillin/streptomycin (Lonza, Basel, Switzerland). Thereafter, cells were fixed with 4% paraformaldehyde (PFA) for 15 min and stained with Oil red O solution (Sigma-Aldrich, St. Quentin Fallavier Cedex, France) for 10 min, followed by two washes with distilled water. Cells with red lipid vacuoles were counted under a light microscope.
For osteogenic differentiation, cells were cultured in an OsteoDiff medium (Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with 1% penicillin/streptomycin (Lonza, Basel, Switzerland) and replaced every three days. Alizarin red staining (Sigma-Aldrich, St. Quentin Fallavier Cedex, France) was performed to analyse calcium deposits after cell fixation with 4% PFA for 15 min. Cells were washed with distilled water and stained with Alizarin red solution (Sigma-Aldrich, St. Quentin Fallavier Cedex, France) 40 mM, pH 4.2 for 10 min.
The calcium deposits were assessed under optical microscopy, and staining intensity was graded as follows: 0 = absent, 1 = 20%, 2 = 40%, 3 = 60%, 4 = 80%, 5 = 100%. Each condition was assayed in triplicate.

For osteogenic differentiation, cells were cultured in a 6-well plate with different undiluted extract materials. On the next day, the medium was replaced with a differentiation medium (OsteoDiff medium; Miltenyi Biotec, San Diego, CA, USA) composed of MEM-Alpha, 10% FBS, ascorbic acid-2-phosphate 2.5 mg/L (Sigma Aldrich, Steinheim, Germany), dexamethasone 0.1 μM (Sigma), and beta-glycerophosphate 10 mM (Merck, Darmstadt, Germany). The differentiation was performed for 14 days. The negative control group consisted of cells cultured in a normal medium, and the positive control group consisted of cells cultured in the OsteoDiff medium. Both the osteogenic induction medium and normal medium were changed twice per week.

For osteogenic differentiation, the hMSC were cultured with specific differentiation medium NH OsteoDiff Medium (Miltenyi Biotec, Germany). The hMSC culture was changed every 3 days during 10 days [25 (link)]. Afterwards, the monolayer was washed with PBS (phosphate-buffered saline), cooled 70% ethanol solution fixed for 10 min at room temperature, and then incubated for 30 min with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP-NBT, Sigma, B5655). For a better contrast, an incubation in 1 ml of hematoxylin for 2 min was done. Then, the monolayer was washed with distilled water and observed under an optical inverted microscope (Olympus BX41).
For adipogenic differentiation, the hMSCs were cultured with differentation medium NH AdipoDiff Medium (Miltenyi Biotec, Germany). The hMSC culture was changed every 3 days for 21 days. Afterwards, the monolayer was washed with PBS, 10% formalin fixed for 2 min at room temperature, and then incubated for 1 hour with 1 ml Oil Red O solution (Merk, Darmstadt, Germany) at room temperature.