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.