Figure 2

Calcification potential of human synovial and bone marrow MSCs cultured with autologous human serum or FBS. Human serum with heat-inactivation and FBS without heat-inactivation were used for further analyses. Synovial and bone marrow MSCs precultured with autologous human or FBS at passage 0 were replated at 100 cells in 150-cm² dishes. These cells were cultured in the medium containing the same serum used at passage 0 for 14 days to form cell colonies. Then, the medium was switched into calcification medium containing the same serum used at passage 0 and cultured for an additional 21 days. The dishes were stained with Alizarin Red for calcification. (A) Microscopic features of Alizarin Red-positive calcium nodule. Bar 100 μm. (B) Calcified cell colonies stained with Alizarin Red. Bar 5 cm. (C) Total cell colonies. The same dishes shown in (B) were stained with Crystal Violet. (D) Alizarin Red-positive colony forming efficiency of synovial MSCs (%). The ratio of Alizarin Red positive colony number to the total colony number was calculated (n = 3). Average value with standard deviation from 4 donors is shown respectively. (E) Alkaline phosphatase activity. Synovial or bone marrow MSCs at passage 0 were replated at 5000 cells/cm² and cultured in the absence or presence of calcification medium with autologous human serum or FBS for 10 days. Average value with standard deviation is shown (n = 3). (*P < 0.01 vs. control, **P < 0.05 vs. FBS) (F) Reverse-transcription-PCR analysis for osteocalcin. Synovial or bone marrow MSCs at passage 1 were replated at 5000 cells/cm² and cultured in the absence or presence of calcification medium with autologous human serum or FBS for 21 days.