Modulation of Hyaluronan Synthesis by the Interaction between Mesenchymal Stem Cells and Osteoarthritic Chondrocytes

Abstract

Bone marrow mesenchymal stem cells (BM-MSCs) are considered a good source for cellular therapy in cartilage repair. But, their potential to repair the extracellular matrix, in an osteoarthritic environment, is still controversial. In osteoarthritis (OA), anti-inflammatory action and extracellular matrix production are important steps for cartilage healing. This study examined the interaction of BM-MSC and OA-chondrocyte on the production of hyaluronan and inflammatory cytokines in a Transwell system. We compared cocultured BM-MSCs and OA-chondrocytes with the individually cultured controls (monocultures). There was a decrease in BM-MSCs cell count in coculture with OA-chondrocytes when compared to BM-MSCs alone. In monoculture, BM-MSCs produced higher amounts of hyaluronan than OA-chondrocytes and coculture of BM-MSCs with OA-chondrocytes increased hyaluronan production per cell. Hyaluronan synthase-1 mRNA expression was upregulated in BM-MSCs after coculture with OA-chondrocytes, whereas hyaluronidase-1 was downregulated. After coculture, lower IL-6 levels were detected in BM-MSCs compared with OA-chondrocytes. These results indicate that, in response to coculture with OA-chondrocytes, BM-MSCs change their behavior by increasing production of hyaluronan and decreasing inflammatory cytokines. Our results indicate that BM-MSCs per se could be a potential tool for OA regenerative therapy, exerting short-term effects on the local microenvironment even when cell:cell contact is not occurring.

Figure 1

BM-MSCs decrease cell number after coculture with OA-chondrocytes. Number of cells cultivated for 3 and 6 days in monoculture (n = 6; bone marrow mesenchymal stem cells (BM-MSCs) and chondrocytes) or in coculture (n = 6; BM-MSC_Coc and Chondro_Coc). Error bars represent the SEM for the mean value. Statistical significance (two-way ANOVA) is set according to the number of asterisks, as follows: ^∗ P ≤ 0.05, ^∗∗∗ P ≤ 0.0001.

Figure 2

Coculture upregulates hyaluronan production. Hyaluronan concentration (pg/mL/cell) after 3 and 6 days in monoculture (BM-MSCs and chondrocytes) or in coculture. Bars represent the mean and SEM; bars with gray line (expected coculture) show “expected hyaluronan production in coculture,” based on production of monoculture and cell number counted. Table shows the cell number in each group. The asterisk (∗) indicates a significant (P ≤ 0.05) difference between cell culture groups based on a two-way ANOVA followed by Tukey's posttest.

Figure 3

mRNA expression of hyaluronan enzymes by BM-MSCs and OA-chondrocytes. Relative mRNA expression of hyaluronan-related enzymes after 3 days (a) or 6 days (b) in monoculture. Bars represent the mean ± SEM of hyaluronan synthase- (HAS-) 1, HAS-2, and HAS-3 and hyaluronidase- (HYAL-) 2 and HYAL-3 mRNA expression. All fold changes were calculated relative to a calibrator sample. Statistical significance based on unpaired t-test was set according to the number of asterisks, as follows: ^∗ P ≤ 0.05, ^∗∗ P ≤ 0.001.

Figure 4

Hyaluronan enzyme mRNA expression in BM-MSC after coculture. mRNA expression of hyaluronan synthase- (HAS-) 1, HAS-2, and HAS-3 (a-b) and hyaluronidase- (HYAL-) 1, HYAL-2, and HYAL-3 (c-d) after coculture relative to time-matched and cell-matched controls. Expression of HAS in cocultivated BM-MSC relative to BM-MSC monoculture after 3 days (a) and 6 days (n = 5) (b); expression of hyaluronidases in cocultivated BM-MSC relative to BM-MSC monoculture after 3 days (c) and 6 days (d) (n = 5). Bars represent the mean fold change value ± SEM relative to BM-MSC in monoculture of hyaluronan enzymes. Statistical significance based on unpaired t-test was set according to the number of asterisks, as follows: ^∗ P ≤ 0.05, ^∗∗ P ≤ 0.001.

Figure 5

Hyaluronan enzyme mRNA expression in OA-chondrocytes after coculture. mRNA expression of hyaluronan synthase- (HAS-) 1, HAS-2, and HAS-3 (a-b) and hyaluronidase- (HYAL-) 1, HYAL-2, and HYAL-3 (c-d) after coculture relative to time-matched and cell-matched controls. Expression of HAS in cocultivated chondrocytes (Chon) relative to Chon monoculture after 3 days (a) and 6 days (b) (n = 5); expression of hyaluronidases in cocultivated chondrocyte relative to Chon monoculture after 3 days (c) and 6 days (d) (n = 5). Bars represent the mean fold change value ± SEM relative to chondrocyte in monoculture of hyaluronan enzymes. Statistical significance based on unpaired t-test was set according to the number of asterisks, as follows: ^∗ P ≤ 0.05, ^∗∗ P ≤ 0.001.

Figure 6

OA-chondrocytes produce higher IL-6 and IL-8 levels than BM-MSCs and cocultured cells and decrease these cytokines. Interleukin (IL) production for 3 and 6 days in monoculture (n = 5; BM-MSCs and chondrocytes) or in coculture (n = 5). IL-6 concentration (ng/mL/cell) (a). IL-8 concentration (ng/mL/cell) (b). Overall, bars represent the mean value with the SEM; bars with gray line (expected coculture) show expected ILs synthesis in coculture, based on production of monoculture and cell number counted. Table shows cell number in each group. Statistical significance based on a two-way ANOVA with Tukey's posttest was set according to the number of asterisks, as follows: ^∗ P ≤ 0.05, ^∗∗ P ≤ 0.001, and ^∗∗∗ P ≤ 0.0001. Tendency of statistical differences between cell culture groups was identified by #.