Fig 1.
(A) Two cell-contained culture systems: (I) MSC-chondrocyte indirect system for evaluating the paracrine effect of MSCs. (II) MSC-chondrocyte direct contact system to evaluate the combined MSC paracrine and direct cell-to-cell contact effects, and (B) one cytokine-only culture system: conditioned medium system to examine the effect of MSC-produced soluble mediators on chondrocytes were evaluated in this study.
Table 1.
Cell number and MSC-chondrocyte ratios in the indirect co-culture system.
Fig 2.
Effects of lipopolysaccharide (LPS) on inflammatory gene expressions on chondrocytes.
Chondrocytes were incubated for 4 h in medium alone (control) or medium containing 2, 20, or 200 μg/mL of LPS, then levels of mRNAs for TNF-α, IL-6, iNOS, and IL-1β were determined. The data are expressed as the mean ± standard deviation (n = 4). * p < 0.05, significant difference compared to the control group.
Fig 3.
Effects of LPS on chondrocyte morphology and cell numbers.
Chondrocytes were incubated with medium alone (control) or medium containing 2, 20, or 200 μg/mL of LPS for 24 h (A and C) or 72 h (B and C), when morphological changes were assessed (A and B) and cell numbers quantified (C). The experiments in A and B were repeated 5 times with similar results. In (C), the data are expressed as the mean ± standard deviation (n = 5). * p < 0.05, significant difference compared to the control group at the same time point.
Fig 4.
Gene expression in MSC-chondrocyte indirect system in chondrocytes with LPS-induced inflammation.
In the control group, chondrocytes were incubated with medium alone. In all of the other groups, the chondrocytes were incubated with 2 μg/mL of LPS group for 4 h, then were incubated for a further 24 h (A, C, E) or 72 h (B, D, F) either in the presence of LPS alone (LPS group) or LPS plus MSCs at different MSC:chondrocyte ratios (M1C1, M1C3, and M1C5 groups). (A, B) are the inflammation-related genes results; (C, D) are anti-inflammation and free-radical-related genes results; (E, F) are ECM synthesis-related genes results. * p < 0.05, significant difference compared to the control group. # p < 0.05, significant difference compared to the LPS group. § p < 0.05, significant difference between the indicated groups.
Fig 5.
Gene expression in MSC-chondrocyte indirect and direct system in chondrocytes with LPS-induced inflammation.
The setup was essentially as in Fig 4 and compared the results between indirect (M1C3-IND) and direct (M1C3-D) in a single MSCs-chondrocyte ratio of 1:3, with mRNA levels measured at 24 h (A, C, E) and 72 h (B, D, F). (A, B) are the inflammation-related genes results; (C, D) are anti-inflammation and free-radical-related genes results; (E, F) are ECM synthesis-related genes results. * p < 0.05, significant difference compared to the control group. # p < 0.05, significant difference compared to the LPS group. § p < 0.05, significant difference between the indicated groups.
Fig 6.
Gene expression MSC-conditioned medium in chondrocytes with LPS-induced inflammation.
In the control group, chondrocytes were incubated with medium alone. In all other groups, the chondrocytes were treated with medium containing LPS (2 μg/mL) for 4 h, then the LPS-containing medium was removed and replaced with fresh medium (LPS group) or PT5X (pass-through from the 5-fold concentration of MSC-CM), CM1X (MSC-CM without concentration), CM5X (5-fold MSC-concentrated CM), or CM10X (10-fold concentrated MSC-CM) for a further 24 h (A, C, E) or 72 h (B, D, F). * p≤ 0.05, significant difference compared to the Control group. # p≤ 0.05, significant difference compared to the LPS group. § p≤ 0.05, significant difference between the indicated groups.
Fig 7.
Cell viability for chondrocytes incubated with medium or LPS with or without subsequent treatment with different MSC-CM.
Cells were treated as in Fig 6, then cell viability was estimated after 24 h or 72 h. * p ≤ 0.05, significant difference compared to the Control group. # p ≤ 0.05, significant difference compared to the LPS group. § p ≤ 0.05, significant difference between the indicated groups.