PPAR-δ agonist affects adipo-chondrogenic differentiation of human mesenchymal stem cells through the expression of PPAR-γ

Abstract

Introduction: Peroxisome proliferator-activated receptor (PPAR) subfamily play an important role in chondrogenesis. Previous study has reported that mixture of GW0742 (PPAR-δ agonist), hyaluronic acid (HA) and mesenchymal stem cells (MSCs) enhance chondrogenesis. The purpose of this study is to compare with efficacies of commercially available HA and demonstrate correlation of PPAR-γ and PPAR-δ.

Methods: In this experimental study, MSCs were cultured with chondrogenic media and clinical HA gels (Euflexxa®, Synvisc®, Orthovisc® and Supartz®) using micormass culture method. Expression of type Ⅰ, Ⅱ collagen and matrix metalloprotease-13 (MMP-13) was measured by immunoblotting. MSCs were cultured with chondrogenic media and/or HA and/or GW0742 and/or rosiglitazone (PPAR-γ agonist) and/or human osteoarthritis synovial fluid. Immunoblotting was used to measure expression of type Ⅱ collagen and PPAR-γ. To identify the effective dose for chondrogenesis and adipogenesis, either 0.1, 1, 5 or 10 μM of rosiglitazone was added to MSCs in chondrogenic media or adipogenic media.

Results: Clinical HA gels inhibited expression of type Ⅰ collagen and enhanced the expression of MMP-13. Type Ⅱ collagen expression was significantly elevated in all treatment groups except Supartz®. GW0742 decreased the expression of PPAR-γ with/without inflammation condition. Rosiglitazone enhanced adipogenesis in a dose-dependent manner and enhanced the expression of type Ⅱ collagen under inflammation condition. Otherwise, the expression of type Ⅱ collagen and formation of chondrocyte spheroids showed a dose-dependent manner with a peak at 1 μM of rosiglitazone.

Conclusions: PPAR-γ has a considerable anti-inflammatory effect and a strong pro-adipogenic effect, which inhibits the chondrogenic effect. PPAR-γ is related with PPAR-δ and shows a chondrogenic effect at lower concentrations. And clinical HA gels shows various efficacy of chondrogenesis. This study suggested that PPAR-γ and PPAR-δ are key regulatory factors of chondrogenesis.

Keywords: Adipogenesis; Chondrogenesis; DJD, degenerative joint disease; ECM, extracellular matrix; FBS, fetal bovine serum; GAG, glycosaminoglycans; HA, hyaluronic acid; MMP, matrix metalloprotease; MSC, mesenchymal stem cells; Mesenchymal stem cells; OA, osteoarthritis; PBS, phosphate-buffered saline; PPAR, Peroxisome proliferator–activated receptor; PPAR-γ; PPAR-δ; TGF, Transforming growth factor; Type Ⅱ collagen; α-MEM, α-minimum essential medium.

Graphical abstract

Fig. 1

Comparison of chondrogenesis efficacy between various HA gels. Chondrocytes differentiated 80,000 cells of hMSCs were seeded either on matrigel-coated wells on 60 μL of hyaluronan gel (Euflexxa®, Orthovisc®, Supartz® and Synvisc®) or without HA (control) in the wells of 96-well plates and incubated in chondrogenic medium for 15 days. (a) The expression of Type Ⅰ collagen, Type Ⅱ collagen and MMP-13 was measured by immunoblot analysis. (b) The expression of Type Ⅰ collagen was only shown in MSC group. (c) Type Ⅱ collagen expression was significantly elevated in all treatment groups except Supartz®. There was no statistical difference among Euflexxa®, Orthovisc® and Supartz®, (α, β, γ P > 0.05) (d) MMP-13 expression was highly elevated in all HA treatment groups.

Fig. 2

Treatment with PPAR-δ agonist inhibits the expression of PPAR-γ. (a) MSCs were cultured with chondrogenic media and PPAR-δ agonist (GW0742) was administered every other day for 14 days. GW0742 increased the expression of type Ⅱ collagen, while inhibiting the expression of PPAR-γ. ∗P < 0.05 versus control. ∗∗P < 0.05 versus control (b) MSCs were cultured plus/minus 1 μM GW0742 to chondrogenic medium with 50% human OA synovial fluid for 14 days. The complete combination of hBM-MSCs, GW0742, and HA gel (CHAMP) significantly increased the protein expression of type Ⅱ collagen and inhibited the expression of PPAR-γ within human OA synovial fluid. αP < 0.05 versus control. βP < 0.05 versus control. †P < 0.001 versus control. ‡P < 0.05 versus control.

Fig. 3

Synergistic effect of PPAR-γagonist (rosiglitazone) and HA. MSCs were cultured plus/minus 70 μL Euflexxa® for 2 h, plus/minus 1 μM GW0742, plus/minus 1 μM rosiglitazone to chondrogenic medium with 50% human OA synovial fluid for 14 days. The expression of type Ⅱ collagen were significantly enhanced in treatment of PPAR-ã agonist (Rosiglitazone) and HA. ∗P < 0.001 versus control. †P < 0.05 versus control. ‡P < 0.05 versus control. ∗∗P < 0.05 versus control. ∗∗∗P < 0.05 versus control.

Fig. 4

Chondrogenic effect according to concentration of rosiglitazone. hBM-MSCs in HA gel were cultured with 0, 0.1, 1, 5, or 10 μM of rosiglitazone in chondrogenic media for 14 days. The protein level of TGF-β and type Ⅱ collagen also increased in a dose-dependent manner with a peak at 1 μM. ∗P < 0.05 versus control. †P < 0.05 versus control.

Fig. 5

The production of chondrocyte according to concentration of rosiglitazone. The production of chondrocyte was quantified by number of spheroids stained by Alcian blue. hBM-MSCs on 96 well plate in chondrogenic medium were treated with 0, 0.1, 1, 5, or 10 μM of rosiglitazone for 14 days. hBM-MSCs with 0.1 μM rosiglitazone generated significantly more chondrocyte spheroids than the control group (without rosiglitazone). More than 1 μM of rosiglitazone was reduced the production of chondrocyte spheroids, compared to the results of the control group. ‡P < 0.05 versus control. ∗P < 0.05 versus control.

Fig. 6

Adipogenic effect of PPAR-δ. hBM-MSCs in adipogenic medium were treated with 0, 0.1, 1, 5, or 10 μM of rosiglitazone for 14 days. The lipid droplets of adipocytes increased in a dose-dependent manner with a peak at 10 μM. ∗P < 0.05 versus control. †P < 0.001 versus control. ‡P < 0.001 versus control. ∗∗P < 0.05 versus control.

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