Stempro osteogenesis differentiation kit

To quantify the cell viability, hAECs (3.2 × 10³ cells/well) and hDPSCs (3.2 × 10³ cells/well) were seeded in 96-well plates to reach 80% confluency. The hAECs were exposed to the following media: (i) DMEM 10% FBS; (ii) DMEM 10% FBS and 500 pg/mL eDMPs; and (iii) osteogenic differentiation medium with 10 ng/mL EGF (StemPro Osteogenesis Differentiation Kit; Thermo Fisher Scientific, Waltham, MA, USA). Likewise, hDPSCs were cultured in (i) α-MEM with 10% FBS, (ii) α-MEM with 10% FBS and 500 pg/mL eDMPs and (iii) osteogenic differentiation medium (StemPro Osteogenesis Differentiation Kit; Thermo Fisher Scientific, Waltham, MA, USA). MTT assays were performed after 2, 4 and 8 days. The cells were incubated with 100 µL/well of a 0.5 mg/mL MTT solution (Thiazolyl Blue Tetrazolium Bromide; Sigma-Aldrich, Saint Louis, MO, USA) for 60 min at 37 °C and 5% CO₂. Subsequently, the dye was dissolved in 200 µL/well of dimethyl sulfoxide (DMSO; Merck Millipore, Billerica, MA, USA) on a shaker (540 rpm) at room temperature for 10 min. Optical density readings were performed on a microplate reader at λ = 540 nm (Infinite 200; Tecan, Männedorf, Switzerland) and the results were summarized as median values with 25–75% percentiles (n = 8).

The multilinear differentiation potential was evaluated by testing the ability of the product obtained after treatment with Rigenera (60 s) and the enzymatic method to differentiate into adipocytes, chondrocytes, and osteocytes. Briefly, adipocyte differentiation was achieved after 16 days culture of MSCs with adipogenic medium, containing 10⁻⁶ M dexamethasone, 10 μg/mL insulin, and 100 μg/mL 3-isobutyl-1-methylxantine (Sigma). Chondrocyte differentiation was achieved after 14 days culture with the StemPro osteogenesis differentiation kit (GIBCO Life Technology, Italy). Osteoblast differentiation was achieved after 21 days culture with the StemPro osteogenesis differentiation kit (GIBCO Life Technology, Italy). Oil Red O, Alcian blue, and Alizarin Red Stain were employed to identify adipocytes, chondrocytes, and osteocytes, respectively.

The differentiation potential of MSCs was assessed using the StemPro® Adipogenesis Differentiation Kit and StemPro® Osteogenesis Differentiation Kit according to the manufacturer’s instructions (Life Technologies). To detect adipogenesis, MSCs were fixed in 10 % neutral buffered formalin after two weeks in culture and lipid vacuoles were stained with Oil red O (Sigma-Aldrich, Sydney, Australia) in 60 % (v/v) isopropanol. Cells were then counterstained with haematoxylin. To detect osteogenesis, MSCs were fixed in 10 % neutral buffered formalin after three weeks in culture and calcium deposits were stained with 2 % (w/v) Alizarin red S (Sigma-Aldrich) in distilled water.