Osteogenic medium

To evaluate the osteogenic differentiation potential in vitro, 1×10⁵ MSCs (three and six passages) from five healthy male subjects were seeded on 6 well plates (BD Biosciences). Subcon?uent cultures were incubated in the osteogenic medium (Invitrogen) for 2 weeks. The medium was changed every 2 days. Then cells were fixed with 70% ethanol, and Alizarin Red staining was used to determine the potential of osteogenic differentiation of MSCs. The mineralization was measured by using the Image-Pro Plus 6.0 program (Media Cybernetics, Rockville, MD, USA).

The multipotent capacity of rabbit MSCs was proven after in vitro culturing with specific supplements by inducing differentiation into osteogenic, chondrogenic, and adipogenic. To induce osteogenic differentiation, confluent passaged-2 cells were cultured in the osteogenic medium (Invitrogen, Gibco, USA). After 21 days, Alizarin Red staining was used to observe the matrix mineralization. STEMPRO Chondrogenesis Differentiation Kit (Gibco, Invitrogen) was used to induce chondrogenic differentiation of rabbit MSCs in a micromass pellet culture system. Feeding of chondrogenic medium was carried out over a period of 21 days and the pellet was then processed for histology staining using Safranin O/fast green solution. For adipogenesis, adipogenic medium (Invitrogen, Gibco, USA) was used to induce the differentiation in the confluent culture of passaged-2 cells. Fourteen days after culture initiation, the cells were fixed with methanol at room temperature for 10 minutes, rinsed by 60% isopropanol, and stained by using freshly prepared Oil Red O solution in 99% isopropanol for 15 minutes. The images at different magnifications were captured using a camera attached to the light microscope (Nikon Eclipse E200, Nikon, Japan).