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Mesenchymal stem cells improve osteoarthritis by secreting superoxide dismutase to regulate oxidative stress response

BMC Musculoskeletal Disorders volume 26, Article number: 409 (2025) Cite this article

Abstract

Background

To investigate the therapeutic effect of intraarticular injection of mesenchymal stem cells (MSC) in a rabbit osteoarthritis (OA) model. And to suppose whether MSC play a pivotal role in OA therapy by improving oxidative stress through secreting superoxide dismutase (SOD).

Methods

MSC and chondrocytes were isolated and cultured in vitro. SOD gene of MSC was silenced by siRNA technology to prepare the SOD-siRNA-MSC for in-vivo study. Twenty healthy adult New Zealand white rabbits underwent papain injection to induce OA and then received intra-articular injection with MSC, siRNA-MSC, or normal saline. The rabbits were divided into 4 groups (n = 5), such as the control group, the model group, the MSC group, the siRNA-MSC group. Cytokines determination was performed 2 and 4 weeks after treatment. Magnetic resonance imaging (MRI) and histopathology and immunohistochemistry determination were performed 4 weeks after treatment. Normal chondrocytes, OA chondrocytes, OA chondrocytes + MSC group, and OA chondrocytes + siRNA-MSC were incubated for 24 h. Then β-galactosidase staining and reactive oxygen species (ROS) level was detected to establish the senescence of the cells.

Results

COMP, TNF-α, MMP-2 and MMP-13 in the MSC group were significantly decreased compared to those in model group (P < 0.05). However, MMP2 and MMP13 in the siRNA-MSC group were not significantly decreased compared to the model group (P < 0.05). Magnetic resonance results revealed a significant improvement in cartilage and synovial membrane 4 weeks after MSC injection. Histopathology determination showed that cartilage structure was also significantly improved in MSC group. Immunohistochemical analysis revealed amelioration in the expression levels of proteoglycan, COL-2, P21 and P53 in MSC group. On the other hand, MRI, histopathologic and immunohistochemical analysis also indicated a decreased therapeutic effect with SOD-siRNA -MSC. The positive rate of β-galactosidase staining and ROS level of OA chondrocytes were significantly higher than those in normal chondrocytes, which was decreased in OA chondrocytes + MSC (P < 0.05). In addition, it was increased in OA chondrocytes + siRNA-MSC (P < 0.05).

Conclusion

Our study demonstrated for the first time that MSC might be a promising therapy in OA through anti-apoptosis and regeneration in chondrocyte by secreting SOD and improving oxidative stress.

Key points

1. It is another proof that MSC may be a promising therapy in OA.

2. MSC might play a pivotal role in the treatment of OA through anti-apoptosis and regeneration in chondrocyte by secreting SOD and improving oxidative stress.

Peer Review reports

Introduction

Osteoarthritis (OA) is a highly prevalent degenerative condition of the joints, most commonly the knee and hip, which affects more than 300 million people in the world according to a review in 2019 [1]. Patients with OA suffer from mobility problems and chronic pain, resulting in a lifelong disability and reduced quality of life quality [2]. In addition, chronic OA management imposes a heavy burden on the family and medical resources. The pathogenesis of OA is complex and is not fully understood, but it is characterized by skeletal inflammation, cartilage degeneration, subchondrical bone sclerosis, fibrosis and synovial hyperplasia, subchondral bone remodeling, menisci degeneration and infrapatellar fat pad role [3]. In addition to physical therapy and weight loss program, paracetamol or non-steroidal anti-inflammatory drugs (NSAIDs), and intraarticular injections of corticosteroids or hyaluronic acid may be the main current non-surgical treatments for OA [4]. Patients with no response to the treatment mentioned above, would consider other pain relief treatment such as tramadol or amitriptyline. The final option for end-stage knee OA is joint replacement. However, this surgery is not suitable for all patients for the risk of complications [5]. Furthermore, all these treatments have limited effects on the healing potential of damaged cartilage.

Although several types of cells are involved in pathogeny of OA, chondrocytes are primarily considered to play an important role in OA induction by cellular senescence [6]. Normal chondrocytes usually show moderate metabolic and proliferative activity under normal conditions. However, some senescence chondrocytes lose their differentiated phenotype under inflammation conditions. Recent studies have identified that OA pathology is obviously related to oxidative stress and reactive oxygen species (ROS), which would regulate chondrocyte apoptosis and senescence, matrix metalloproteinase (MMP) production, and extracellular matrix synthesis and degradation [7].

MSC play an important role in repairing cartilage by tissue regeneration, producing factors that promote resident chondrocytes in the joint to form articular cartilage, and inhibiting inflammation [8]. A summary paper reviewed MSC therapy for OA in preclinical and clinical studies in the last 5 years, which certified the benefits and safety of intra-articular injections of MSC [9]. Nevertheless, the effects and mechanisms of intra-articular injections of MSC in OA are not well defined due to the low-quality evidence. Furthermore, in vivo MSC differentiation is relatively rare, indicating that MSC differentiation in chondrocytes could not explain the therapeutic effect [10]. Klein D et al. verified that the antioxidant enzyme superoxide dismutase 1 (SOD1) could be an MSC secreted factor, which could mediate ROS [11].

The aim of our study was to observe whether intra-articular injection of MSC could change knee joint morphology using magnetic resonance imaging (MRI). Furthermore, inflammation determination, histopathological examination and immunohistochemical analysis were also carried out to establish cartilage injury and senescence improvement of intra-articular MSC injection. In addition, SOD gene was silenced to suppose whether MSC play a pivotal role in OA treatment through oxidative stress pathway.

Materials and methods

Culture and characterization of UCB-MSC and chondrocytes

hUCB-MSC (passage 4–6, Jiangsu Keygen Biotech Corp, China) were cultured in RPMI-1640 medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Gaithersburg, MD, USA) at 37 °C with 5% CO2. Characterization of hUCB-MSC were performed according to published procedures through a flow cytometer [12]. Complete RPMI-1640 media was carefully aspirated and replaced with PBS. hUCB-MSC were digested with 0.25% trypsin (Gibco, Gaithersburg, MD, USA) and were obtained by centrifugation at 300 × g for 5 min. After making with anti-human CD29 (BioLegend, USA) and CD90 (BioLegend, USA), flow cytometer was performed (Ex = 488 nm; Em = 575 nm and Ex = 488 nm; Em = 620 nm) to identify MSC.

A total of 6 patients with OA who were hospitalized in the Department of Orthopedics of Nanjing Drum Tower Hospital Affiliated to the Medical School of Nanjing University from January 2018 to December 2020 were selected. The diagnostic criteria for OA refer to the diagnostic criteria for knee osteoarthritis of the American College of Rheumatology. Another 6 patients with joint trauma matching age and sex were selected as control. The patients who received glucocorticoids or nonsteroidal anti-inflammatory drugs were excluded. Cartilage tissue was obtained from joint replacement arthroscopic surgery (This study had passed clinical medical ethics review and informed consent). After removing from the operating room, the joint cartilage specimen was immediately immersed in sterile RPMI-1640 (Gibco, Gaithersburg, MD, USA) and preserved at 4℃. The tissue was cut into 1-2mm3 fragments, and was continuously stirred and digested with 0.125% pancrease (Gibco, Gaithersburg, MD, USA) at 37℃ for 1 h, and then digested with 0.075% collagenase II solution (Abcam, UK) at 37℃ overnight. After filtering and collecting, the chondrocytes were centrifuged at1500r/min for 5 min. Then the chondrocytes were rinsed with PBS solution twice. Finally, chondrocytes were inoculated at a density of 1.0 × 106/ml. Chondrocytes were identified with toluidine blue solution staining solution (Best-reagent, China) and immunohistochemistry (chondrocyte type II collagen expression), which were observed under an optical microscope, and 3 areas were taken for photo preservation.

The identified MSC and chondrocytes at P2-3 generation were used for the next experiment.

SOD SiRNA construction and transfection

The siRNA primer sequence of SOD1 was designed following GENE BANK. There were three sequences listed as below.

Gene

Sequence

SOD1siRNA1

forward 5’-GGG CAA AGG UGG AAA UGA ATT-3’

 

Reverse 5’-UUC AUU UCC ACC UUU GCC CTT-3’

SOD1 siRNA2

Forward 5’-GUU CAU GAG UUU GGA GAU ATT-3’

 

Reverse 5’-UAU CUC CAA ACU CAU GAA CTT-3’

SOD1 siRNA3

Forward 5’-CAA UGU GAC UGC UGA CAA ATT-3’

 

Reverse 5’-UUU GUC AGC AGU CAC AUU GTT-3’

One day before transfection, an appropriate number of MSC cells were inoculated into 6-well cell culture plates, so that the cell confluency at transfection could reach 80–90%. SiRNA was diluted to 20 μm with 125µL serum-free Opti-MEM medium (Gibco, Gaithersburg, MD, USA), mixed gently, and incubated at room temperature for 5 min. Five µL lipo3000 (Invitrogen, thermos, USA) was also diluted with 125µL serum-free Opti-MEM medium, mixed gently, and incubated at room temperature for 5 min. Then siRNA and lipo3000 was gently mixed and incubated for 20 min at room temperature. The mixture was added to the cell in 750µL complete medium and cultured in CO2 incubator at 37℃ for 48 h. QPCR was used for identification the best silencing sequence, which decreased the expression of SOD mostly. And the follow-up experiment was conducted with the best silenced SOD-siRNA-MSC. In addition, cell viability of MSC and siRNA-MSC was both verified through Trypan blue (Jiangsu Keygen Biotech Corp, China) dye exclusion test after transfection for 24 h. IL-1β(Sigma, Aldrich, USA) at the concentration of 10 µg/L was added to the siRNA-MSC, whose cell viability was also detected.

Experimental animals and groups

This experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of Drum Tower Hospital affiliated with the Nanjing University School of Medicine and was in accordance with ARRIVE guidelines. Twenty healthy adult New Zealand white rabbits with body weight between 2.5 and 3.0 kg (provided by the Experimental Animal Centre of Drum Tower Hospital affiliated to Nanjing University School of Medicine) were selected and divided into 4 groups (n = 5), such as control group, model group, MSC group and SOD-siRNA-MSC group. The environment of the animal laboratory was controlled at a temperature of 23 ± 2 °C, a relative humidity of 55 ± 10%, a ventilation frequency of 12 times/h, and a lighting cycle of 12 h. And a normal diet and sterile distilled water was provided without restriction.

Establishment of osteoarthritis model and experimental procedures

Papain (Sigma, USA) was dissolved in PBS to 4% concentration, while 0.5mL was locally articular injected into the posterior knee cavity of the model group, MSC group and siRNA-MSC group on day 1, 4 and 7 for three times in total. The control group was injected with normal saline instead. Two weeks later, the experimental joints were observed by magnetic resonance imaging (MRI) to verify whether the OA model had been established successfully. And serum was collected through the marginal ear vein for determination of cytokines. Subsequently, the MSC group was injected once with 5 × 105MSC, and the siRNA-MSC group was injected once with 5 × 105 siRNA-MSC. Both the MSC and siRNA-MSC concentration is 1 × 106/mL. Cell viability was confirmed to be > 95% by trypan blue exclusion immediately before injection. The model group and control group were injected with 0.5 ml normal saline as a negative comparison. Aseptic technique was maintained throughout using laminar flow hood and sterile surgical procedures.

After 2 weeks of treatment, the serum of the 4 groups was collected through the marginal ear vein. After 4 weeks of treatment, the morphology of the joints was observed by MRI. After observation, the experimental animals were sacrificed and serum, synovial membrane and articular cartilage were collected for further study.

MRI evaluation

MRI was performed 4 weeks after treatment. In this study, the Philips 3.0T magnetic resonance imaging system (Ingenia, Philips, The Netherlands) was used. After anesthesia through propofol (5 mg/kg) injection, rabbits were fixed in the supine position on the experimental plate. Coronal and sagittal scans were performed with 8-channel orthogonal magnetic resonance coils of the head to obtain PDW-SPAIR sequence images. The following sequences were utilized: TR3000 MS, TE30 MS, turn angle 90°, field of vision 10 cm, layer thickness 1.5 mm, acquisition time 2 min 30 s. Soft tissue swelling and joint effusion were observed through MRI to assess synovitis and osteophytes. The grade criteria of synovitis and osteophytes was as follows: none (0), mild (1), moderate (2) and severe (3).

Detection of cytokines in serum

Cytokine determination was performed 2 and 4 weeks after treatment. Rabbit serum was collected through marginal ear vein. Serum levels of IL-1β, L-6, TNFα, MMP2, 13 and COMP were determined by enzyme-linked immunosorbent assay (ELISA lab, Hubei, China) according to each instruction, respectively.

Histopathologic examination and immunohistochemistry

Rabbits were sacrificed after MRI evaluation at week 4. The supracondylar portion of the femur was collected, including the arthritis-induced site. The joint tissue specimens were fixed with 10% formalin solution, embedded in paraffin, sectioned into 4 μm sections and stained with HE. The articular cartilage was fixed in 10% formalin solution, decalcified with EDTA, rinsed with water, and then treated with conventional embedding, sectioning and HE staining. All tissues were observed under a light microscope. Expression of P21, P53, COL-2 and proteoglycan in articular cartilage tissues was detected by Streptomyces antibiotin protein-peroxidase immunohistochemical method (Abcam, UK). The percentage of microscopically positive cells and staining intensity were evaluated by semiquantitative interpretation. Percentages of positive staining cell scores were calculated as follows: 0 for positive cells < 5%, 1 for 5-25%, 2 for 26-50%, 3 for 51-75%, and 4 for 76-100%. Positive stain intensity scores were calculated as follows: 0 for colorless, 1 for light yellow, and 2 for brownish yellow, 3 for tan. The multiplication of 2 scores can be considered a positive rating: 0 for negative (-), 1–4 for weakly positive (+), 5–8 for positive (+ +), 9–12 for strong positive (+ + +). Two independent assessors performed the scoring blinded to each other’s evaluations.

β-galactosidase detection of chondrocytes

Normal chondrocytes, OA chondrocytes, OA chondrocytes + MSC and OA chondrocytes + siRNA-MSC were incubated for 24 h in six-well plates with Transwell system. Then β-galactosidase staining was detected to performed the senescence of the cells. The chondrocytes culture medium in different treatment group was removed, washed once with PBS and added with 1 ml β-galactosidase solution (Invitrogen, Thermo Fisher, USA) for 15 min at room temperature. The cell fixative was removed and the chondrocytes were washed with PBS 3 times for 3 min each time. One ml of dye working solution was added into the chondrocytes. Then the staining was observed under an optical microscope, and 3 areas were taken for photo preservation.

ROS detection of chondrocytes

Normal chondrocytes, OA chondrocytes, OA chondrocytes + MSC and OA chondrocytes + siRNA-MSC were incubated for 24 h in six-well plates with Transwell system. Chondrocytes in different treatment groups were collected and adjusted to concentration at 1 × 106/ml. DCFH-DA was diluted with serum-free culture solution at 1:1000, so that the final concentration was 10 µM. After collection, the cells were suspended in the diluted DCFH-DA and incubated at 37℃ for 20 min. The cells were washed with serum-free cell culture solution 3 times to fully remove the DCFH-DA. Flow cytometry (Ex = 488 nm; Em = 530 nm) was performed to detect intracellular reactive oxygen species.

Statistical analysis

Multiple comparison tests were conducted to compare different groups. The homogeneity of variance was satisfied among the groups. If data were normally distributed, one-way analysis of variance (ANOVA) and T test were used to analyze the data. If data were skewed distributed, Wilcoxon signed rank tests and Mann-Whitney U tests were used to analyze statistical differences. SPSS 22.0 statistical software was used for analysis.

Results

MSC and chondrocytes identification

hUCB-MSC were usually spindle-shaped, with uniform cell distribution and regular morphology under a microscope. Flow cytometry was used to monitor the proportion of CD29 and CD90 expression cells. It was indicated that the number of CD29 positive expression cells was 99.76%, and the number of CD90 positive expression cells was 94.87%, both of which were more than 90%. The number of CD29 positive cells was 0.13% and CD90 positive cells was 0.28% in the homotype control group (Fig. 1).

Toluidine blue staining was used to identify chondrocyte. As shown in Fig. 2A, the primary chondrocytes were fusiform and polygonal, which was dyed blue by fuel Toluidine blue staining. The expression of type II collagen was detected by IHC, and the brown color indicated type II collagen (Fig. 2B), which was indicated that chondrocytes were isolated and cultured successfully.

Fig. 1
figure 1

Identification of MSC through flow cytometry. A and C present homo-type control cells. B and D present MSC. The cells were identified CD29 and CD90 positive expression, which was consistent with the performance of MSC

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Fig. 2
figure 2

A Identification of chondrocyte. A represents the result of toluidine blue staining of chondrocytes. B represents the expression of type II collagen detected by IHC method (×200)

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SOD1 SiRNA sequence design and the best choice

The siRNA primer sequence of SOD1 was designed by GENE BANK, and the synthesized primer sequence was transfected into MSC by liposome. qPCR was used to verify whether the sequence could block SOD1 expression in MSC. The results suggested that the expression of SOD1 in MSC was significantly decreased, either transfected with sequence 1, 2 or 3 (P < 0.05). MSC transfected with sequence 3 had a stronger inhibition effect on SOD1 expression than those transfected with sequence 1 or 2, but there was no statistical difference (P > 0.05) (Table 1). Sequence 3 was applied as the SOD-siRNA sequence in subsequent experiments.

Table 1 The relative expression level of SOD1 after MSC transfection
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Morphology of SOD siRNA-MSC was also spindle-shaped or fusiform, with uniform cell distribution and regular morphology under a microscope which was similar with MSC. Flow cytometry was also used to identify siRNA-MSC, which result was similar with MSC (data not shown). In addition, the cell viability assay indicated that more than 95% cell survivor after transfection (Table 2), which was suitable for intraarticular injection. Moreover, there was no significant difference in cell viability between SOD siRNA-MSC and MSC. The cells remained active in an inflammatory environment.

Table 2 Cell viability of MSC and siRNA-MSC after transferring for 24 h
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Cytokines outcomes

The results of serum cytokines and cartilage damage markers are shown in Fig. 3. COMP, IL-1β, IL-6, TNF-α, MMP-2 and MMP-13 levels in model group were significantly higher than those in control group (P < 0.05), which indicated OA model was established successfully. Two weeks after intra-articular injection, the levels of COMP, IL-1β, IL-6, TNF-α, MMP-2 and MMP-13 in MSC group were significantly decreased compared with those in model group (P ≤ 0.05). Moreover, the levels of COMP, TNF-α, MMP-2 and MMP-13 in the MSC group were still lower than those in model group at 4 weeks after intra-articular injection (P ≤ 0.05).

Two weeks after intra-articular injection, IL-1β, MMP-2, MMP-13 and TNF-α in the SOD- siRNA-MSC group was decreased compared with model group (P < 0.05), while COMP, and IL-6 did not significantly change (P > 0.05). Moreover, IL-1β, IL-6 and MMP2 in SOD-siRNA-MSC group were elevated compared with MSC group (P < 0.05). Four weeks after intraarticular injection, IL-1β, IL-6 and MMP-13 in the siRNA-MSC group did not significantly decrease compared to the model group (P < 0.05).

Fig. 3
figure 3

Inflammatory index results of each group after 2w and 4w treatment. Different bars represent control, model, MSC and siRNA-MSC for 2weeks (A) and 4weeks (B). The horizontal line shows the comparison between the two groups. * P < 0.05, significant difference between groups

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MRI outcomes

All rabbits were performed MRI (Fig. 4A-D). Images showed that the cartilage surface of the femoral condyle joint was smooth, no abnormal signal was found in the subchondral bone, the synovial signal was normal, and a small amount of synovial fluid was found in the joint cavity on T2WI fat saturation sequence in control group. However, rough cartilage surface of femoral condylar article, poor meniscus polish, slightly increased signal of anterior cruciate ligament, slight thickening of synovium, slightly increased signal of subchondral bone, and moderate effusion in the joint cavity were showed in model group. The MRI images on T2WI fat saturation sequence of the joint after 4w treatment in the siRNA-MSC group showed that the cartilage surface of the femoral condyle joint was rough, the meniscus was not polished, the anterior cruciate ligament signal was increased, the synovium was slightly thickened, the subchondral bone signal was slightly increased, and there was fluid in the joint cavity, which did not change much compared with the model group. In MSC group, MRI findings showed that the cartilage signal of femoral condyle joint and tibial plateau was uneven, the meniscus was normal and intact, the anterior cruciate ligament was normal, the synovial signal was slightly increased, which was improved compared with the model group. In addition, the subchondral bone signal was normal, and the joint effusion was significantly reduced compared with the model group. The synovitis and osteophyte score of groups were displayed in Fig. 4E-F. Significant differences of synovitis scores between control and other groups (p<0.05). However, only the synovitis score in MSC group was significantly decreased compared with model group (p<0.05). Significant differences of osteophyte score between control and model group were observed (p<0.05), which was similar between control and siRNA-MSC group. However, there was no significant difference between MSC group and control group (p>0.05).

Fig. 4
figure 4

Magnetic resonance of knee joint of rabbits in each group after 4w treatment. A represents control group, B represents OA group, C represents MSC group. D represents siRNA-MSC group. 1 and 2 represent sagittal and coronal magnetic resonance images in each group. The arrows pointe to the fluid in the joint cavity in each group. E and F represent the synovitis and osteophyte score of different groups. * P < 0.05, significant difference between groups

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Histopathologic outcomes

HE staining was performed on articular cartilage and synovial membrane of rabbits in all experimental groups (Fig. 5). The results suggested that the posterior articular cartilage of rabbits in the control group was continuous and smooth. There was no obvious infiltration of cartilage, inflammatory cells, infiltration of synovial tissue inflammatory cells, or thickening of the synovial membrane. In the model group, the articular surface of the posterior knee cartilage was not smooth, the cartilage became thin, the synovial membrane was infiltrated by a large number of inflammatory cells and the synovial membrane was hyperplasia and edema. Compared with the model group, the infiltration of inflammatory cells in MSC group was obviously reduced, indicating that MSC treatment of OA could improve the inflammation of articular cartilage and have a protective effect on articular cartilage. Four weeks after intra-articular injection with siRNA-MSC, the articular surface of the posterior knee cartilage was not smooth, the articular cartilage was fractured, the synovial membrane was infiltrated by a large number of inflammatory cells, and the synovial membrane was hyperplastic and edema, and the inflammatory infiltration was worse than that of the model group, suggesting that siRNA-MSC had no protective effect on the joint, and the inflammatory injury was aggravated.

Fig. 5
figure 5

Histological findings of each group. A represents control group, B represents OA group, C represents MSC group. D represents siRNA-MSC group. 1 and 2 represent synovial membrane and cartilage, respectively (HE staining ×100). The arrows in the left column pointed to lymphocyte, while the arrows in the right column pointed to the cartilage surface

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Immunohistochemistry outcomes

Immunohistochemical results of expression of P21, P53, proteoglycan and COL-2 in articular cartilage of experimental rabbit joints are shown in Fig. 6. Semiquantitative results showed that proteoglycan expression was 3+, col-2 expression was 2+, P21 expression was +, P53 expression was + in the control group. Additionally, in the model group, proteoglycan expression was +, col-2 expression was 2+, P21 expression was 3 + and P53 expression was 3+. Moreover, in MSC group, proteoglycan expression was 2+, col-2 expression was 2+, P21 expression was + and P53 expression was +. However, in the siRNA group, proteoglycan expression was 2+, col-2 expression was 2+, P21 expression was 3 + and P53 expression was 2 + (Table 3).

Fig. 6
figure 6

Immunohistochemical results of each group. A represents control group, B represents OA group, C represents MSC group. D represents siRNA-MSC group. 1 to 4 represent proteoglycan, COL-2, P21 and P53 in each group, respectively (×200)

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Table 3 Intensity scores of intensity scores of 4 proteins in articular cartilage
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MSC inhibit the senescence of OA chondrocytes

The results showed that the positive rate of β-galactosidase staining in OA chondrocytes was significantly higher than that in the control chondrocytes, indicating obvious senescence (Fig. 7). The positive rate of β-galactosidase staining in MSC + OA chondrocytes was lower than that in the OA chondrocytes. The positive rate of β-galactosidase staining in siRNA-MSC + OA chondrocytes was higher than that in OA chondrocytes, suggesting that MSC could not ameliorate chondrocyte senescence after blocking SOD secretion.

Fig. 7
figure 7

Changes of β-galactosidase staining of chondrocytes A-D corresponds to β-galactosidase staining results of control chondrocytes, OA chondrocytes, MSC + OA chondrocytes and siRNA-MSC + OA chondrocytes, respectively (×200)

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MSC decrease the ROS level of OA chondrocytes

As shown in Fig. 8, ROS level in OA chondrocytes was significantly higher than that in control chondrocytes (P < 0.05). The ROS level of MSC + OA chondrocytes was significantly lower than that in OA chondrocytes (P < 0.05). However, the ROS level of siRNA-MSC + OA chondrocytes was obviously higher than that in MSC + OA chondrocytes and OA chondrocytes (P < 0.05). In other words, SOD secretion and regulation of ROS level was blocked through siRNA, resulting in similar ROS level in siRNA-MSC + OA chondrocytes and OA chondrocytes. The increased level of ROS in siRNA-MSC + OA chondrocytes might be related to the increased senescence of MSC after SOD silencing.

Fig. 8
figure 8

Changes of ROS level of chondrocytes. A-D represents the control chondrocytes, OA chondrocytes, MSC + OA chondrocytes, and siRNA-MSC + OA chondrocytes respectively. E represents the statistical result graph. * indicates P ≤ 0.05. NS indicates no statistical difference

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Discussion

In this study, we confirmed that intra-articular injection with MSC could improve OA and the mechanism could be anti-chondrocyte senescence by secreting SOD. To our knowledge, several papers have demonstrated the therapeutic effects of MSC in either rabbit, beagles or sheep [13]. Some studies have shown that the MSC would protect or repair damaged cartilage through downregulating the expression of catabolic enzymes such as matrix metalloproteases 3 and 13. Some studies have indicated that MSCs would not reduce degenerative changes in OA despite improvements in pain outcomes. The mechanism of MSC in treating OA is still under debate. The ability of MSC to differentiate along a mesodermal lineage, such as chondrocytes, has suggested its intrinsic potential for tissue repair and regeneration [14,15,16]. However, it is likely that MSC manipulate the OA local environment via paracrine signaling, rather than direct differentiation, which leads to the disease modification [17]. The production of immunomodulatory and growth factors by MSC seemed to regulates the tissue immunological microenvironment and cartilage regeneration in OA, which provided a new approach to cell-free therapy [18]. MSC transferred by siRNA SOD was applied to the model rabbit to verify the mechanism of MSC through SOD secretion. Although protein-level validation of SOD knockdown was not performed in this study, the well-established correlation between mRNA interference and protein suppression in prior literature [19], coupled with our observed consistent antioxidant effects following siRNA treatment, suggests novel molecular mechanisms underlying osteoarthritis pathogenesis.Furthermore, the therapy effect was also contrasted between two different treatment courses. Thus, we believe that our study was valuable for assessing the effect and mechanism of MSC as an OA treatment strategy.

Due to large and thick joints for cartilage, there are rabbit, beagle, and sheep suitable for cartilage repair experiments. Rabbits offer advantages as they are cost effective, easy to handle, and to house. Rabbits have been widely applied to in vivo OA research in previous studies [20]. Papain is a proteolytic enzyme which could induce OA through releasing chondroitin sulfate from a protein-polysaccharide complex of the articular cartilage matrix and producing inflammatory cytokines (such as TNF-α and IL-1β) [21, 22]. Along with inflammatory cytokine secretion, MMPs levels and free radical products increase, which could promote the pathology of OA [23]. Therefore, rabbits were selected as our model animals in this study and induced OA via papain injection.

It is common sense that defect articular cartilage in OA limits self-healing capability. Originally, destructive processes limited the supply of nutrients and oxygen. Then, it will reduce the synthesis of extracellular matrix (ECM) components and increase the synthesis of tissue-destructive proteinases. Ultimately, there will be general apoptosis of chondrocytes and synovial inflammation [24]. In addition, apoptosis chondrocytes have little self-repairing abilities, resulting in further progressive degradation of articular cartilage, which is a major challenge for OA therapy [25]. As a result, OA therapy should aim to repair cartilage that recapitulates the native properties of healthy cartilage. MSC might be a potential cell-based therapies in OA for its excellent tissue repair ability [26].

According to our study, intra-articular injection of MSC could reduce the secretion of cytokine factors associated with OA, even after 4 weeks. The MRI and HE results showed that MSC therapeutic effect might due to anti-chondrocyte senescence of chondrocyte. Recent studies had concluded that intra-articular MSC injection was effective in OA through inhibiting cartilage degradation [27,28,29]. Furthermore, Gupta et al. [30] indicated that MSC were a potential treatment strategy of OA and contributed cartilage regeneration via 3 distinct pathways: differentiating to chondrocytes, suppressing inflammatory and secreting trophic factors that modulate ECM synthesis. The injection of MSC weekly was found to provide a more significant chondroprotective effects compared to a single injection [31]. In our study, the amelioration effect of MSC was relatively decreased at 4 weeks in both anti-inflammation and anti-cartilage degradation. Our findings were consistent with this hypothesis.

In previous studies, it was demonstrated that oxidative stress and reactive oxygen species (ROS) was critical in pathogenesis of OA through regulating chondrocyte apoptosis and senescence, MMPs production and extracellular matrix degradation [7]. In this study, β-galactosidase staining was used to establish senescence of chondrocytes. Moreover, ROS level detection was related to β-galactosidase staining result, which was accordance with previous study. Among multiple types of endogenous and exogenous antioxidants, SOD is a major catalytic antioxidant in articular cartilage [32]. However, both preclinical and clinical studies confirmed that direct use of free SOD only provided a slight protective effect against oxidative damage due to its inadequate retention and rapid inactivation at the site of the disease [33]. Gui T’s work demonstrated the therapeutic potential of SOD-loaded porous polymersome nanoparticles (SOD-NP) and inferred that targeting SOD could be a great promise for OA therapy [34]. In addition, MSC secreted SOD would be a cellular based antioxidant therapy in OA [35]. In our study, SOD-siRNA MSC was prepared and injected to another group of rabbits. The results showed that SOD inhibition in MSC would decrease the therapeutic effect of MSC in cytokines and tissue structure. Furthermore, the p53/p21 protein was determined through immunohistochemistry assay. In addition to tumor suppressant function, p53 is also involved in a serious number of pathologies associated with ageing [36]. P53/P21 signaling is associated with pathogenesis of OA. And p53 expression is obviously increased in OA patients than in healthy volunteers [37]. Loss of Sirt1 in cartilage led to accelerate OA pathogenesis through aberrant activation of p53/p21, which mediated senescence associated secretory phenotype, hypertrophy and apoptosis [38]. The results in current study showed that the expression of p53 and p21 in articular cartilage were decreased by MSC treatment, which was reversed by SOD-siRNA MSC. Watanabe K et al. also verified that SOD loss decreased the mitochondrial membrane potential of chondrocytes and led to p53 activation, which contributed to growth arrest and apoptosis of chondrocyte [39]. And the in-vitro study demonstrated that MSC could not ameliorate chondrocyte senescence after blocking SOD secretion. Despite being characteristically intracellular, SOD appears to exert paracrine effects on ROS homeostasis [40]. Therefore, we have reasons to believe that MSC might be a promising therapy in OA through anti-apoptosis and promoting regeneration in chondrocyte by secreting SOD and improving oxidative stress. However, there was still some residue improvement in MMP2 and TNF-α levels even if SOD expression is blocked in MSC either in 2 weeks or 4 weeks. We assumed that MSC can still secrete anti-inflammatory factors in addition to SOD silencing.

Despite the findings, this study still had some limitations. Firstly, the further invitro study was lacking to verify the SOD mechanism in MSC treating OA. This study was an observational study in vivo to find the relationship between SOD and MSC therapy function in OA. While we evaluated chondrocyte ROS levels, mitochondrial membrane potential, and β-galactosidase-positive cell counts, further studies are needed to directly attribute these effects to MSC-secreted SOD in cartilage aging. We had no idea that how long does the SOD remain biologically active in the joint cavity. Secondly, we did not track the fate and distribution of the injected MSC. However, the role of mesenchymal stem cell secretion factor was investigated in this study, which seems to be independent of cell distribution.

Conclusion

In conclusion, our study demonstrated that intra-articular MSC injection inhibited the progression of OA through cartilage regeneration. Furthermore, we first discussed that the mechanism might be that SOD secreted by MSC played an important role in improving oxidative stress and anti-apoptosis chondrocyte. To adequately evaluate the effectiveness and mechanism of MSC therapy in preclinical studies, a reliable animal model with appropriate doses and injection frequency should be considered in future.

Data availability

The datasets used during the current study are available from the corresponding author on reasonable request.

Abbreviations

ECM:

Extracellular Matrix

MRI:

Magnetic Resonance Imaging

MMP:

Matrix Metalloproteinase

MSC:

Mesenchymal Stem Cells

NSAIDs:

Non-Steroidal Anti-Inflammatory Drugs

OA:

Osteoarthritis

ROS:

Reactive Oxygen Species

SOD:

Superoxide Dismutase

References

  1. Kloppenburg M, Berenbaum F. Osteoarthritis year in review 2019: epidemiology and therapy. Osteoarthritis Cartilage. 2020;28(3):242–8.

    Article  CAS  PubMed  Google Scholar 

  2. Lohan C, Coates G, Clewes P, Stevenson H, Wood R, Tritton T, Massey L, Knaggs R, Dickson AJ, Walsh D. Estimating the cost and epidemiology of mild to severe chronic pain associated with osteoarthritis in England: a retrospective analysis of linked primary and secondary care data. BMJ Open. 2023;13(11):e073096.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Loeser RF, Collins JA, Diekman BO. Ageing and the pathogenesis of osteoarthritis. Nat Rev Rheumatol. 2016;12(7):412–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Gibbs AJ, Gray B, Wallis JA, Taylor NF, Kemp JL, Hunter DJ, Barton CJ. Recommendations for the management of hip and knee osteoarthritis: A systematic review of clinical practice guidelines. OSTEOARTHR Cartil. 2023;31(10):1280–92.

    Article  Google Scholar 

  5. Jarecki J, Waśko MK, Widuchowski W, Tomczyk-Warunek A, Wójciak M, Sowa I, Blicharski T. Knee cartilage lesion Management-Current trends in clinical practice. J Clin Med. 2023;12(20):6434.

    Article  PubMed  PubMed Central  Google Scholar 

  6. McCulloch K, Litherland GJ, Rai TS. Cellular senescence in osteoarthritis pathology. Aging Cell. 2017;16:210–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Lepetsos P, Papavassiliou AG. ROS/oxidative stress signaling in osteoarthritis. Biochim Biophys Acta. 2016;1862(4):576–91.

    Article  CAS  PubMed  Google Scholar 

  8. Sekiya I, Ojima M, Suzuki S, Yamaga M, Horie M, Koga H, Tsuji K, Miyaguchi K, Ogishima S, Tanaka H, Muneta T. Human mesenchymal stem cells in synovial fluid increase in the knee with degenerated cartilage and osteoarthritis. J Orthop Res. 2012;30(6):943–9.

    Article  PubMed  Google Scholar 

  9. Guishan W, Dan X, Wei L, Yuanyuan Z, Haifeng L, Lei Y, Kenan F, Peidong, Baofeng Y, Jiao Jiao L, Bin W. Preclinical studies and clinical trials on mesenchymal stem cell therapy for knee osteoarthritis: A systematic review on models and cell doses. Int J Rheum Dis. 2022;00:1–31.

    Google Scholar 

  10. Katsha AM, Ohkouchi S, Xin H, Kanehira M, Sun R, Nukiwa T, Saijo Y. Paracrine factors of multipotent stromal cells ameliorate lung injury in an elastase-induced emphysema model. Mol Ther. 2011;19(1):196–203.

    Article  CAS  PubMed  Google Scholar 

  11. Klein D, Steens J, Wiesemann A, Schulz F, Kaschani F, Röck K, Yamaguchi M, Wirsdörfer F, Kaiser M, Fischer JW, Stuschke M, Jendrossek V. Mesenchymal stem cell therapy protects lungs from Radiation-Induced endothelial cell loss by restoring superoxide dismutase 1 expression. ANTIOXID REDOX SIGN. 2016;26(11):563–82.

    Article  Google Scholar 

  12. Seo Y, Yang SR, Jee MK, Joo EK, Roh KH, Seo MS, Han TH, Lee SY, Ryu PD, Jung JW, Seo KW, Kang SK, Kang KS. Human umbilical cord blood-derived mesenchymal stem cells protect against neuronal cell death and ameliorate motor deficits in Niemann pick type C1 mice. Cell Transplant. 2011;20(7):1033–47.

    Article  PubMed  Google Scholar 

  13. Wang G, Xing D, Liu W, Zhu YY, Liu HF, Yan L, Fan K, Liu PD, Yu BF, Li JJ, Wang B. Preclinical studies and clinical trials on mesenchymal stem cell therapy for knee osteoarthritis: A systematic review on models and cell doses. INT J RHEUM DIS. 2022;25(5):532–62.

    Article  CAS  PubMed  Google Scholar 

  14. Arinzeh TL. Mesenchymal stem cells for bone repair: preclinical studies and potential orthopaedic applications. Foot Ankle Clin. 2015;10(4):651–65.

    Article  Google Scholar 

  15. Wright A, Arthaud-Day ML, Weiss ML. Therapeutic use of mesenchymal stromal cells: the need for inclusive characterization guidelines to accommodate all tissue sources and species. Front Cell Dev Biol. 2021;9:632717.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Xie RH, Gong SG, Song J, Wu PP, Hu WL. Effect of mesenchymal stromal cells transplantation on the outcomes of patients with knee osteoarthritis: A systematic review and meta-analysis. J ORTHOPAED RES. 2023; 2023: 1–16.

  17. Caplan A. What are MSC therapeutic? New data: new insight. J Pathol. 2009;217:318–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Yuan F, Qi Q, Wang L, Pan JH, Shi XY, Zhang Y. Deciphering the Immunomodulatory pathways of mesenchymal stem. Cells CURR STEM CELL RES T; 2024. p. 122542.

  19. Li X, Chen Y, Zhao J et al. The Specific Inhibition of SOD1 Selectively Promotes Apoptosis of Cancer Cells via Regulation of the ROS Signaling Network. OXID MED CELL LONGEV, 2019, 2019: 9706792.

  20. Moran CJ, Ramesh A, Brama PA. The benefits and limitations of animal models for translational research in cartilage repair. J Exp Orthop. 2016;3(1):1.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Lampropoulou-Adamidou K, Lelovas P, Karadimas EV, Liakou C, Triantafillopoulos IK, Dontas I, Papaioannou NA. Useful animal models for the research of osteoarthritis. Eur J Orthop Surg Traumatol. 2014;24(3):263–71.

    Article  PubMed  Google Scholar 

  22. Rasheed MS, Ansari SF, Shahzadi I. Formulation, characterization of glucosamine loaded transfersomes and in vivo evaluation using Papain induced arthritis model. Sci Rep. 2022;12(1):19813.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Horváth E, Sólyom Á, Székely J, Nagy EE, Popoviciu H. Inflammatory and metabolic signaling interfaces of the hypertrophic and senescent chondrocyte phenotypes associated with osteoarthritis. Int J Mol Sci. 2023;24(22):16468.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Bijlsma JW, Berenbaum F, Lafeber FP. Osteoarthritis: an update with relevance for clinical practice. Lancet. 2011;377(9783):2115–26.

    Article  PubMed  Google Scholar 

  25. Diekman BO, Guilak F. Stem cell-based therapies for osteoarthritis: challenges and opportunities. Curr Opin Rheumatol. 2013;25(1):119–26.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Harrell CR, Markovic BS, Fellabaum C, Arsenijevic A, Volarevic V. Mesenchymal stem cell-based therapy of osteoarthritis: current knowledge and future perspectives. Biomed Pharmacother. 2019;109:2318–26.

    Article  CAS  PubMed  Google Scholar 

  27. Saulnier N, Viguier E, Perrier-Groult E, Chenu C, Pillet E, Roger T, Maddens S, Boulocher C. Intra-articular administration of xenogeneic neonatal mesenchymal stromal cells early after meniscal injury down-regulates metalloproteinase gene expression in synovium and prevents cartilage degradation in a rabbit model of osteoarthritis. Osteoarthritis Cartilage. 2015;23(1):122–33.

    Article  CAS  PubMed  Google Scholar 

  28. Sato M, Uchida K, Nakajima H, Miyazaki T, Guerrero AR, Watanabe S, Roberts S, Baba H. Direct transplantation of mesenchymal stem cells into the knee joints of Hartley strain guinea pigs with spontaneous osteoarthritis. Arthritis Res Ther. 2012;14(1):R31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Li M, Luo X, Lv X, Liu V, Zhao G, Zhang X, Cao W, Wang R, Wang W. In vivo human adipose-derived mesenchymal stem cell tracking after intra-articular delivery in a rat osteoarthritis model. Stem Cell Res Ther. 2016;7(1):160.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Gupta PK, Das AK, Chullikana A, Majumdar AS. Mesenchymal stem cells for cartilage repair in osteoarthritis. Stem Cell Res Ther. 2012;3(4):25.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Ozeki N, Muneta T, Koga H, Nakagawa Y, Mizuno M, Tsuji K, Mabuchi Y, Akazawa C, Kobayashi E, Matsumoto K, Futamura K, Saito T, Sekiya I. Not single but periodic injections of synovial mesenchymal stem cells maintain viable cells in knees and inhibit osteoarthritis progression in rats. Osteoarthritis Cartilage. 2016;24(6):1061–70.

    Article  CAS  PubMed  Google Scholar 

  32. Koike M, Nojiri H, Kanazawa H, Yamaguchi H, Miyagawa K, Nagura N, Banno S, Iwase Y, Kurosawa H, Kaneko K. Superoxide dismutase activity is significantly lower in end-stage Osteoarthritic cartilage than non-osteoarthritic cartilage. PLoS ONE. 2018;13(9):e0203944.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Gao X, Ma YQ, Zhang GJ, Tang FY, Zhang JJ, Cao JC, Liu CH. Targeted elimination of intracellular reactive oxygen species using nanoparticle-like chitosan- superoxide dismutase conjugate for treatment of monoiodoacetateinduced osteoarthritis. Int J Pharm. 2020;590:119947.

    Article  CAS  PubMed  Google Scholar 

  34. Gui T, Luo L, Chhay B, Zhong LL, Wei YL, Yao LT, Yu W, Li J, Nelson CL, Tsourkas A, Qin L, Cheng ZL. Superoxide dismutase-loaded porous polymersomes as highly efficient antioxidant nanoparticles targeting synovium for osteoarthritis therapy. Biomaterials. 2022;283:121437.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Nightingale H, Kemp K, Gray E, Hares K, Mallam E, Scolding N, Wilkins A. Changes in expression of the antioxidant enzyme SOD3 occur upon differentiation of human bone marrow-derived mesenchymal stem cells in vitro. STEM CELLS DEV. 2012;21(11):2026–35.

    Article  CAS  PubMed  Google Scholar 

  36. Vigneron A, Vousden KH. P53, ROS and senescence in the control of aging. Aging. 2010;2(8):471–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Zhu XB, Yang SW, Lin WJ, Wang L, Ying JW, Ding YW, Chen X. Roles of cell Cyle regulators Cyclin D1, CDK4, and p53 in knee osteoarthritis. Genet Test Mol Biomarkers. 2016;20(9):529–34.

    Article  CAS  PubMed  Google Scholar 

  38. Xu M, Feng M, Peng H, Qian Z, Zhao LT, Wu SF. Epigenetic regulation of chondrocyte hypertrophy and apoptosis through Sirt1/P53/P21 pathway in surgery-induced osteoarthritis. BIOCHEM BIOPH RES CO. 2020;528(1):179–85.

    Article  CAS  Google Scholar 

  39. Watanabe K, Shibuya S, Koyama H, Ozawa Y, Toda T, Yokote K, Shimizu T. Sod1 loss induces intrinsic superoxide accumulation leading to p53-mediated growth arrest and apoptosis. Int J Mol Sci. 2013;14(6):10998–1010.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Khanh VC, Yamashita T, Ohneda K, et al. Rejuvenation of mesenchymal stem cells by extracellular vesicles inhibits the elevation of reactive oxygen species. Sci Rep. 2020;10(1):17315.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

Not applicable.

Funding

This work was supported by Nanjing Health Bureau Medical Science and technology development project (No. ZKX17020 and No. YKK15067).

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Authors and Affiliations

  1. Department of Pharmacy, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China

    Yao Yao

  2. Department of Geriatrics, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China

    Juan Cao & Congzhu Ding

Authors
  1. Yao Yao
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  2. Juan Cao
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  3. Congzhu Ding
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Contributions

Congzhu Ding was responsible for the overall research work arrangement. Yao Yao was responsible for animal research. Juan Cao was responsible for Statistic work and. Yao Yao wrote the first draft of the manuscript. Yao Yao and Juan Cao revised the manuscript. All authors reviewed the manuscript.

Corresponding authors

Correspondence to Juan Cao or Congzhu Ding.

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This experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of Drum Tower Hospital affiliated with the Nanjing University School of Medicine.

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Yao, Y., Cao, J. & Ding, C. Mesenchymal stem cells improve osteoarthritis by secreting superoxide dismutase to regulate oxidative stress response. BMC Musculoskelet Disord 26, 409 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12891-025-08670-4

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12891-025-08670-4

Keywords

BMC Musculoskeletal Disorders

ISSN: 1471-2474

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