2.3. In vitro Tri-lineage differentiation and histological analysis
Passage 1 SPFP-MSCs samples from ten knee osteoarthritis patients were subjected to directed differentiation to induce osteogenesis, adipogenesis, and chondrogenesis (Table 1). All directed-differentiation media were freshly prepared in house.
Osteogenesis: SPFP-MSCs were seeded at a cell density of 5,000 cells/cm2 in osteogenic medium, prepared freshly in house and containing 10 nM dexamethasone, 10 mMβ -glycerophosphate and 50 μg/ml ascorbate-2 phosphate (all Sigma-aldrich, MO, USA) and with 10% FBS. SPFP-MSC cultures were maintained into AG containing media at various concentrations (1, 5, and 10 μM) for 21 days. Medium was changed every 3 days. The extracellular calcium deposits were determined from 100% ethanol-fixed cell cultures by incubating with 0.2% w/v Alizarin Red S solution for 40 minutes at room temperature. Excess dye was removed by several washing steps using water and observed under an inverted light microscope.
Adipogenesis: SPFP-MSCs were seeded at a cell density of 5,000 cells/cm2 and when the SPFP-MSCs were >90% confluent, the growth medium was substituted with in-house, freshly prepared differentiation medium containing 10 µg/mL insulin, 100 nM dexamethasone, 0.45 mM IBMX (3-isobutyl-methyl-xantine), and 50 µg/mL indomethacin (all from Sigma-Aldrich, MO, USA). The cells were then incubated in AG-containing media at varying concentrations (1, 5, and 10 μM) for 21 days. The adipogenic differentiation was evaluated from formalin-fixed cell cultures by incubating with 0.3% (w/v) Oil Red O (Sigma) solution for 15 minutes at room temperature. Then, the excess staining was washed and removed with water. The stained lipid vacuoles were observed under inverted light microscope.
Chondrogenesis: The SPFP-MSCs were cultured to form a cell pellet in a 15 mL polypropylene tube (Wuxi NEST Biotechnology, Jiangsu, China). Approximately 2 × 105 cells were cultured in chondrogenic medium with in-house ,freshly prepared medium with the presence of 10 nM dexamethasone (Sigma-Aldrich, MO, USA), 10 ng/ml transforming growth factor-β3 (TGF-β3) (ProSpec, Rehovot, Israel) and 6.25 μ g/ml insulin-transferrin-selenium (ITS supplement) (MP Biomedicals, California, USA) were expanded. The cells were then incubated with andrographolide-containing media at different concentrations (1, 5, and 10 μM) for 21 days with medium changes every 3 days. Alcian blue, Toluidine blue, and Haematoxylin & Eosin (H&E) were used to detect the presence of enrichment of glycosaminoglycans in cartilage, polysaccharides and morphology, respectively. Before staining, the cell pellet cultures were fixed in formalin then embedded in paraffin and sectioned into 4-5 μ m and stained with H&E, Alcian blue and Toluidine blue. The sections were observed under an inverted light microscope.