Stemmacs osteodiff media

Four aliquots of 45 × 10³ cells were grown in 35 mm Petri dishes: two plates were set up with differentiating medium StemMACS OsteoDiff Media (Miltenyi, Germany), and the other two were used as control. To verify cell differentiation, after 15 days, differentiated BM-MSCs were fixed with acetone/methanol (1:1), stained with Van Kossa and observed morphologically using an optical microscope. The presence of calcium crystals and mineral deposits was evaluated.

For differentiation experiments, E-MSCs were cultured at the third passage in osteogenic, adipogenic, and chondrogenic mediums, according to the manufacturer’s instructions. Briefly, for osteogenic induction, 45,000 cells were plated in each well of six well plates and cultivated in StemMACS OsteoDiff Media (Miltenyi, Bergisch Gladbach, Germany). After 21 days, osteogenic differentiation was demonstrated by the accumulation of calcium (crystalline hydroxyapatite detection by Von Kossa staining). For adipogenic differentiation, 75,000 cells were cultured in StemMACS AdipoDiff Media differentiation medium (Miltenyi, Bergisch Gladbach, Germany) for 21 days, after which the presence of intracellular lipid vesicles was assessed after fixation with paraformaldehyde vapors and Oil Red O staining. For chondrogenic differentiation, an aliquot of 250,000 cells were cultured in StemMACS ChondroDiff Media differentiation medium (Miltenyi, Bergisch Gladbach, Germany) for 21 days in 15 mL polypropylene culture tubes. During chondrogenic differentiation, cellular growth occurred as cellular aggregates floated freely in suspension. The pellet was included in paraffin and stained with Alcian Blue to identify the presence of hyaluronic acid and sialomucin.

The differentiation capacity was assessed following the manufacturer's instructions for commercial differentiation kits. For rADSCs, cells were cultured in a differentiation medium (StemXVivo Osteogenic/Adipogenic Media-R&D Systems, Minneapolis, MI, USA) with adipogenic or osteogenic supplements contained in the kit. Adipocytes were detected by Oil Red O (Sigma-Aldrich, Saint Louis, MO, USA) staining and osteoblasts by NBT/BCIP (Sigma-Aldrich, Saint Louis, MO, USA) staining. To differentiate into chondrocytes, 2.5 × 10⁵ cells were cultured in DMEM/F-12 media (Sigma-Aldrich, Saint Louis, MO, USA) plus chondrogenic supplements (StemXVivo Chondrogenic Supplement-R&D Systems) in the bottom of a 15 ml conical tube. The cell pellet formed a rounded ball approximately 1-2 mm in diameter on the 3^rd day. On the 14-28^th days, the pellet was fixed in 4% paraformaldehyde and cut into 10 μm sections. They were treated with chondrocyte-specific primary antibody anti-aggrecan (R&D Systems, Minneapolis, MI, USA) and anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific, Waltham, MS, USA) as a secondary antibody. For hADSCs, cells were cultured in StemMACS AdipoDiff media and StemMACS OsteoDiff media (Miltenyi Biotec, Bergisch Gladbach, Germany). The procedure of detection was similar to that of rADSCs.

Multipotent differentiation was assessed evaluating the ability of UC-MSCs to differentiate into adipogenic, osteogenic and chondrogenic lineages under adapted media conditions. UC-MSCs were plated at 2 × 10⁴cells/well in 24-multiwell culture plate and grown in StemPro^® Adipogenesis Differentiation Kit (Gibco, ThermoFisher Scientific) for 3 weeks, replacing medium every 3 days. Adipogenesis was assessed using Oil Red O (Sigma-Aldrich) staining to detect intracellular lipid vacuoles. For osteogenic differentiation UC-MSCs were plated at 10⁴ cells/well in 24-multiwell culture plate and grown in StemMACS OsteoDiff Media (Miltenyi Biotec GmbH, Germany) for 10 days, replacing medium every 3 days. Osteogenesis was assessed by alizarin red S (Sigma-Aldrich) staining to detect the deposition of intracellular calcium. Chondrogenic differentiation was assessed by plating 4 × 10⁴ cells/well in NH ChondroDiff Medium (Miltenyi Biotec GmbH, Germany) for 3 weeks, replacing medium every 3 days. Chondrogenesis was confirmed using Alcian Blue staining (Sigma-Aldrich).

The genetic expression of human alkaline phosphatase (ALP), collagen type 1 (Col1A1), dentin sialophosphoprotein (DSPP), osteonectin (ON), runt-related transcription factor 2 (RUNX2), and bone sialoprotein progenitor (BSP) was determined by quantitative polymerase chain reaction (qPCR) from cells treated with discs of the different material eluates after 14 days (n = 3) as described previously [16 (link)]. Briefly, cells were harvested and total RNA was isolated (Purelink RNA Mini Kit, Invitrogen, Thermo Fisher Scientific). Following that, cDNA synthesis was performed (iScript RT Supermix, Bio-Rad, Hercules, CA, USA). The 2^‑ΔΔCT method was used to calculate the relative gene expression values obtained by qPCR analysis for each gene compared to human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression. Cells cultured with basal growth media were used as negative control, and cells treated with a commercial differentiation media (StemMACS OsteoDiff Media, Miltenyi Biotec, Bergisch Gladbach, Germany) acted as the positive control. Each experimental condition was carried out in triplicate for each sample and analyzed in three independent experiments.