Conditioned medium of IGF1-induced synovial membrane mesenchymal stem cells increases chondrogenic and chondroprotective markers in chondrocyte inflammation

Abstract

Recently, mesenchymal stem cells (MSCs) have been the most explored cells for cell therapy for osteoarthritis (OA) that can be obtained from various sources. Synovial membrane MSCs (SMMSCs) provide best potential for OA therapy, however they are not widely explored. Conditioned medium of SMMSCs (SMMSCs-CM) rich in growth factors and cytokines can inhibit apoptosis and increase chondrocytes cell proliferation. The aim of the present study was to determine growth factors content in SMMSCs-CM as well as the chondrogenic and chondroprotective markers expression in OA model after insulin-like growth factor (IGF)1-induced and non-induced SMMSCs-CM treatments. Chondrocyte cell line (CHON002) was induced by IL1β as OA model (CHON002 with IL1β (IL1β-CHON002)) and treated with SMMSCs-CM with or without IGF1 induction to determine its effectiveness in repairing OA cells model. ELISA was used to assay BMP2, fibroblast growth factor 18 (FGF18) and transforming growth factor (TGF) β1 (TGFβ1) levels in SMMSCs-CM, matrix metalloproteinase (MMP) 13 (MMP13) and a disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS4) levels in OA cells model treated with SMMSCs-CM. RT-qPCR analyses were used to investigate the gene expression of SOX9, COL2, and COL10. CM from SMMSCs cultured and induced by IGF1 150 ng/mL was the most effective concentration for increasing the content of growth factor markers of SMMSCs-CM, which had successfully increased negative cartilage hypertrophy markers (SOX9 and COL2) and reduced hypertrophy markers (COL10, MMP13, and ADAMTS4). Preconditioning with IGF1 has better and very significant results in lowering MMP13 and ADAMTS4 levels. The present study supports IGF1 pre-conditioned SMMSCs-CM to develop a new therapeutic approach in OA improvement through its chondrogenic and chondroprotective roles.

Keywords: Chondrogenesis; Col2; IGF1; MMP13; Mesenchymal Stem Cells.

Figure 1. Effects of various concentrations of IGF1 towards BMP2, FGF18, and TGFβ1 levels of SMMSCs-CM

(A) Concentration of BMP2 (pg/mL) in IGF1-induced SMMSCs-CM. (B) Concentration of FGF18 (pg/mL) in IGF1-induced SMMSCs-CM. (C) Concentration of TGFβ1 (pg/mL) in IGF1-induced SMMSCs-CM. *The data were presented as a histogram of mean ± standard deviation. Different letters (a, ab, b) marked significant difference among treatments towards BMP2 level (A). Different letters (a, ab, b) marked significant difference among treatments towards FGF18 level (B). Different letters (a, b) marked significant difference among treatments towards TGFβ1 level (C). The data were analyzed using ANOVA followed by Tukey’s HSD post hoc test (P<0.05).

Figure 2. Effects of IGF1-induced SMMSCs-CM toward SOX9, COL2, and COL10 gene expressions on OA model

(A) Relative SOX9 gene expression in IGF1-induced SMMSCs-CM. (B) Relative COL2 gene expression in IGF1-induced SMMSCs-CM. (C) Relative COL10 gene expression in IGF1-induced SMMSCs-CM. (I) CHON002 (healthy cells as negative control), (II) IL1β-CHON002 (OA model), (III) OA model+SMMSCs-CM 15% (IV), OA model+SMMSCs-CM 30% (V) OA model+IGF1-SMMSCs-CM 15% (VI) OA model+IGF1-SMMSC-CM 30%. *The figure above shows gene expressions relative to CHON002 (negative control). Data are presented as mean ± standard deviation. Double star sign (**) marks statistical difference compared with negative control group at 0.01 significance level, double hashtag (##) marks statistical difference compared with OA model at 0.01 significance level.

Figure 3. Effects of IGF1-induced SMMSCs-CM toward MMP13 and ADAMTS4 levels on OA cells model

(A) MMP13 level (pg/mL) on OA cells model. (B) ADAMTS4 level (pg/mL) on OA cells model. (I) CHON002 (healthy cells as negative control), (II) IL1β-CHON002 (OA model), (III) OA model+SMMSCs-CM 15% (IV), OA model+SMMSCs-CM 30% (V) OA model+IGF1-SMMSCs-CM 15% (VI) OA model+IGF1-SMMSC-CM 30%. *Data are presented as mean ± standard deviation. Double star sign (**) marks statistical difference compared with negative control group at 0.01 significance level, double hashtag (##) marks statistical difference compared with OA model at 0.01 significance level.

Figure 4. Proposed mechanism on cartilage repair and chondroprotective effects of IGF1-induced SMMSC-CM therapy

SMMSCs secrete many growth factors, such as TGFβ1, FGF18, and BMP2 whose concentrations increase with the IGF1 induction. These factors are contained in the SMMSCs-CM. IL1β induces chondrocyte inflammation and triggers OA involving two different pathways: the NFκB and P38-MAPK. During inflammation, the NFκB protein plays a role in decreasing in SOX9 gene expression. SMMSCs treatment up-regulate SOX9 expression mainly through TGFβ1 action via SMAD2/SMAD3 pathway. SOX9 acts as an activator of COL2 gene, and an inhibitor of COL10. During IL1β-induced inflammation of chondrocytes in which MMP13 and ADAMTS4 are up-regulated through P38/MAPK pathway. SMMSCs treatments act in alleviating the OA symptoms through: (1) BMP2 down-regulates COL10; (2) FGFs, mainly through FGF18 that acts by inhibiting MMP13 and ADAMTS4 synthesis through P38/MAPK pathway. COL2 deposition results in healthy and elastic joint. Meanwhile, COL10 causes stiffness in the joint, which is one characteristic of OA.