Stempro osteogenesis

Nano-engineered MSCs were seeded at a density of ~4 × 10⁴ cells/well in a 24-well plate, and then incubated with adipogenic or osteogenic differentiation media (StemPro Osteogenesis or Adipogenesis Differentiation Kits, Life Technologies, Carlsbad, CA, USA) for 3 weeks. The medium was replaced every 3–4 days. The untreated MSCs incubated with differentiation media and those incubated with regular culture media were used as positive and negative controls, respectively. After a 3-week incubation period, cells were fixed with 4% formalin and stained with alizarin red or oil red O to visualize osteogenic and adipogenic differentiation, respectively. Images were captured using a light microscope at 20× magnification.

BMCs were plated in triplicate in 4-well plate (SPL Life Sciences, Pocheon-si, Korea) for chondrogenic a pellet cultured incubated at 37 °C and 5% CO₂. After one day, the culture medium was removed, and then replaced with StemPro chondrogenesis (Thermo Fisher Scientific, Waltham, MA, USA) medium. Osteogenic and Adipogenic differentiation were incubated at 37 °C and 5% CO₂. After reaching 80% confluence, the culture medium was removed, and StemPro osteogenesis and StemPro adipogenesis (Thermo Fisher Scientific, Waltham, MA, USA) were added to the cultures. All differentiation medium was renewed every three days, and their differentiation potential was examined after 15 days of differentiation. Chondrogenic differentiation was examined by staining with Alcian Blue staining kit (Lifeline Cell Technology, Frederick, MD, USA) to identify sulfated proteoglycans deposits. Osteogenic differentiation was examined by staining with 2% Alizarin Red staining kit (Lifeline Cell Technology, Frederick, MD, USA) to identify calcium deposits. Adipogenic differentiation was examined by staining with Oil Red O staining (Sigma-Aldrich, St. Louis, MO, USA) to identify lipid droplets.

Differentiation assay was conducted to confirm the MSC plasticity. At the fifth passage, cultured cells were harvested and transferred to specific inducing media for chondrogenic, osteogenic, and adipogenic differentiations. The cells were cultured in complete medium to induce spontaneous chondrogenic differentiation. Osteogenic and adipogenic potencies were evaluated by culturing the cells in StemPro Osteogenesis and Adipocyte Differentiation Kits (ThermoFisher Scientific, USA), respectively. All cultures were incubated at 37 °C under a normoxia condition. The induced cells were analyzed after 7, 14, and 21 days of culturing.
For the differentiation assays, the cells were stained with 1% of alcian blue, 2% of alizarin red, and 1.4% oil red O for evaluating their capacity to undergo chondrogenesis, osteogenesis, and adipogenesis, respectively. Percentage of staining area was observed in five fields of view with 100-fold magnification. Differentiation potential was measured as percentage of stained area by ImageJ 1.50i software [17 (link), 18 (link)].

During the third passage (P3), BM-MSC, AT-MSC and UC-MSC samples were placed in triplicate in six-well plates (Sarstedt, USA) for osteogenic and adipogenic differentiation and were incubated at 37°C in a humidified atmosphere containing 95% air and 5% CO₂. After reaching 80% confluence, the culture medium was removed and the differentiation media StemPro adipogenesis and StemPro osteogenesis (Invitrogen, USA) were added to the cultures. The media were changed every three days, and adipogenic differentiation was confirmed by the deposition of lipid droplets in the cytoplasm using 0.5% Oil Red O staining (Sigma-Aldrich Corp., USA). The osteogenic differentiation was confirmed by positive staining of the extracellular calcium matrix using 2% Alizarin Red S staining (Sigma-Aldrich Corp., USA).
For chondrogenic differentiation, a pellet of MSCs was cultured in a Falcon tube and incubated at 37°C in a humidified atmosphere containing 95% air and 5% CO₂. After two days, the culture medium was removed and the differentiation medium StemPro chondrogenesis (Invitrogen, USA) was added and changed every three days. To confirm chondrogenic differentiation, pellets were stained with Alcian Blue (pH = 2.5) and toluidine blue (pH = 1) to identify proteoglycans.