Umbilical cord mesenchymal stem cells relieve osteoarthritis in rats through immunoregulation and inhibition of chondrocyte apoptosis

Abstract

This study aims to investigate the effectiveness of umbilical cord mesenchymal stem cells (UCMSCs) in treating osteoarthritis (OA). Sprague-Dawley rats were used in in vivo experiments and divided into four groups: normal, OA model, saline, and UCMSC-treated groups (n = 6). An OA model was established by injecting iodoacetic acid into the joint cavity. The results indicate that UCMSC transplantation significantly reduced joint surface and articular cartilage damage, and the levels of IL-1β, TNF-α, and MMP13 in the joint fluid were significantly reduced after UCMSC treatment. In vitro experiments showed that co-culturing UCMSCs and chondrocytes promoted the expression of aggrecan, COL2, SOX9, and BCL-2; downregulated the expression of BAX and BAD in chondrocytes; and promoted the expression of IL-10 and TGF-β1 in UCMSCs. Additionally, the supernatant of UCMSCs inhibited the expression of IL-1β and TNF-α in the articular cavity and promoted the expression of COL2 and aggrecan in vivo. These effects were impaired when IL-10 and TGF-β1 were removed. Collectively, UCMSC transplantation appears to improve joint pathology, reduce inflammatory factors, and decrease chondrocyte apoptosis, likely through the involvement of IL-10 and TGF-β1, thus providing a potential therapeutic option for patients with OA.

Figure 1

UCMSCs transplantation improved the knee joint structure of rats with OA. One week after the third UCMSC transplantation, X-rays were used to examine the structure of the rats’ knee joints. The OA model group had irregular articular surfaces, deformities, cartilage defects, tangential fissures, and obvious osteophytes. The articular surface of the UCMSC group was relatively flat. (A) Joint X-ray imaging. (B) Kellgren Lawrence score of joint X-ray imaging. Normal, normal group; Model, OA model group; Vehicle, OA model with saline injection; UCMSC, OA model with UCMSC transplantation, n = 6. *P < 0.05 analyzed by unpaired two-tailed t-test after normality and equal variance test using Shapiro–Wilk test and ANOVA plus Brown-Forsythe test in the GraphPad software.

Figure 2

UCMSCs alleviated the pathological process of OA. (A) Safranin O staining of joint tissues. There was an increased number of cartilage cells that survived and thickened the cartilage surface in the UCMSC group compared with the OA model group. Scale bars = 100 µm. (B) OARSI classification of Safranin O staining. OARSI classification was used to assess the degree of pathological damage to articular cartilage. Normal, normal group; Model, OA model group; Vehicle, OA model with saline injection; UCMSC, OA model with UCMSC transplantation, n = 6. **P < 0.01 analyzed by unpaired two-tailed t-test after normality and equal variance test using Shapiro–Wilk test and ANOVA plus Brown-Forsythe test in the GraphPad software.

Figure 3

Inflammation inhibition and chondrocyte protection by UCMSCs. (A) Detection of inflammatory factors in synovial fluid. ELISAs were performed to detect IL-1β, TNF-α, and MMP13 in synovial fluid. The levels of these three factors decreased significantly in the UCMSC group compared with the model group, indicating that UCMSC transplantation inhibited the expression of key inflammatory factors in OA. Normal, normal group; Model, OA model group; Vehicle, OA model with saline injection; UCMSC, OA model with UCMSC transplantation. n = 6. (B) The effect of UCMSCs on the gene expression levels of aggrecan, Col2, and Sox9 in chondrocytes induced by IL-β1. Chondrocytes co-cultured with UCMSCs and IL-β1 were collected, and RT-PCR was used to detect gene expression levels in chondrocytes. Treatment of chondrocytes with high (1 × 10⁶ cells) and low (0.5 × 10⁶ cells) doses of UCMSCs promoted the expression of aggrecan, Col2, and Sox9 genes. Control, normal cultured rat chondrocytes; Model, rat chondrocytes pre-treated by 10 ng/mL IL-1β; UCMSC-L, rat chondrocytes pre-treated by 10 ng/mL IL-1β were co-cultured with 0.5 × 10⁶ UCMSCs; UCMSC-H, rat chondrocytes pre-treated by10ng/mL IL-1β were co-cultured with 1 × 10⁶ UCMSCs, n = 3. *P < 0.05, **P < 0.01 analyzed by unpaired two-tailed t-test after normality and equal variance test using Shapiro–Wilk test and ANOVA plus Brown–Forsythe test in the GraphPad software.

Figure 4

The effect of UCMSCs on the apoptosis of chondrocytes induced by IL-β1. Chondrocytes co-cultured with UCMSCs and IL-β1 were collected to test the rate of chondrocyte apoptosis by flow cytometry. Both high (1 × 10⁶ cells) and low (0.5 × 10⁶ cells) doses of UCMSCs co-cultured with chondrocytes for 24 and 48 h significantly reduced the apoptosis rate of chondrocytes. Model, chondrocytes pre-treated with IL-1β; UCMSC-L, chondrocytes pre-treated with IL-1β co-cultured with a low dose of UCMSCs; UCMSC-H, chondrocytes pre-treated with IL-1β co-cultured with a high dose of UCMSCs, n = 3. *P < 0.05, **P < 0.01 analyzed by unpaired two-tailed t-test after normality and equal variance test using Shapiro–Wilk test and ANOVA plus Brown-Forsythe test in the GraphPad software.

Figure 5

The effect of UCMSCs on apoptosis-related proteins in chondrocytes induced by IL-β1. After co-culture, chondrocytes were collected and used to extract total protein. Cropped gels and western blots show that, compared with the model group, treatment with different doses of UCMSCs significantly upregulated the protein expression levels of Bcl-2. In contrast, they inhibited the expression of Bax and Bad. Full-length blots and gels are presented in Supplementary Fig. 3. Normal, normal group; Model, OA model group; UCMSC-L, Low dose UCMSCs treatment group; UCMSC-H, High dose UCMSCs treatment group, n = 3. *P < 0.05, **P < 0.01 analyzed by unpaired two-tailed t-test after normality and equal variance test using Shapiro–Wilk test and ANOVA plus Brown-Forsythe test in the GraphPad software.

Figure 6

UCMSCs inhibited the inflammatory process of OA by secreting IL-10 and TGF-β1. (A) After co-culture with chondrocyte, the expression levels of IL-10 and TGF-β1 in UCMSCs increased significantly. Normal, normal cultured UCMSCs; Induced, chondrocyte co-cultured with UCMSCs. (B) Injection of the supernatant from co-cultured UCMSCs into OA model rats reduced the expression levels of IL-1β and TNF-α in the joint cavity fluid of rats. (C) Transplantation of UCMSC supernatant into OA model rats increased the expression levels of aggrecan and Col2a1 genes in chondrocytes. Normal, normal group; Model, OA model group; S-UCMSC, supernatant of co-cultured UCMSCs without removing IL10 or TGF-β1 transplantation group; − IL10, supernatant of co-cultured UCMSCs with IL-10 removed; − TGF-β1, supernatant of co-cultured UCMSCs with TGF-β1 removed; − IL10–TGF-β1, supernatant of co-cultured UCMSCs with IL-10 and TGF-β1 removed. n = 6. **P < 0.01, ***P < 0.001 analyzed by One-way ANOVA with Bonferroni test after normality and equal variance test using Shapiro–Wilk test and ANOVA plus Brown–Forsythe test in the GraphPad software.