Abstract
Background
Knee osteoarthritis causes pain and disability in many people worldwide, for which no definitive treatment has yet been proposed. In this study, we investigated the safety and efficacy of placental mesenchymal stromal cells-derived extracellular vesicles in patients with knee osteoarthritis.
Methods
This triple-blind, randomized clinical trial included patients suffering from bilateral knee osteoarthritis with grade 2 or 3. The knees of each patient were randomized to intervention and control. For the intervention knee, 5 cc of placental mesenchymal stromal cells-derived extracellular vesicles were injected, and for the control knee, 5 cc of normal saline was injected. The patients’ symptoms were evaluated before the intervention and 2 and 6 months after the intervention using VAS, WOMAC questionnaire, and Lequesne index. MRI was performed before the intervention and 6 months after the intervention to evaluate retropatellar and tibiofemoral cartilage volume, medial and lateral meniscal disintegrity, ACL injury, and effusion-synovitis.
Results
62 knees (31 patients) were enrolled in this study. There were 31 knees as intervention and 31 knees as control. Finally, the data of 58 knees (29 patients) were analyzed, including 28 women and 1 man. The mean age of the patients was 55.38 ± 6.07 years. No statistically significant difference was detected between the two groups in clinical outcomes (including VAS, WOMAC, and Lequesne scores) before treatment and 2 and 6 months after treatment. Also, no statistically significant difference was detected between the two groups in MRI findings before treatment and 6 months after treatment. No systemic complications or severe local reactions occurred in the patients.
Conclusion
A single intra-articular injection of placental mesenchymal stromal cells-derived extracellular vesicles (5 cc, 7 × 109 particles/cc) is safe, but does not improve clinical symptoms or MRI findings in knee osteoarthritis beyond placebo. The protocol of this study was approved on 11 May 2022 with registration number IRCT20210423051054N1.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12891-024-07979-w.
Keywords: Extracellular vesicles, Knee osteoarthritis, MRI
Introduction
Knee osteoarthritis (OA) is a degenerative disease that affects the tibiofemoral and patellofemoral joints and the soft tissues surrounding them. This disease causes pain and disability in patients and thus limits their quality of life. Another important issue is the financial burden that knee OA places on the patient and the healthcare system [1]. Since the middle of the 20th century, the prevalence of knee OA has doubled [2]. The global prevalence of symptomatic knee OA is 3.8% and by 2050, approximately 642 million people will suffer from knee OA [3, 4].
Despite the high prevalence of knee OA, there is no clear non-surgical treatment for it. Intra-articular injections of corticosteroids, hyaluronic acid (HA), ozone, botulinum toxin A, platelet rich plasma (PRP), stem cells, and oral or topical drug treatments in combination with physical modalities and exercise have been proposed [5, 6]. Exercise is a common non-surgical treatment for knee OA, prescribed alone or in combination with other treatments. Also, exercises can be combined with electromyographic biofeedback [7]. A new modality that has recently received attention is extracellular vesicles (EVs), which are derived from Mesenchymal stromal cells (MSCs).
MSCs are multipotent stem cells which are commonly explored in cell therapy research. MSCs can move to the site of injury, increase peripheral tolerance, decrease pro-inflammatory substances release, reduce activation of monocytes, improve tissue repair, and promote the survival of damaged cells. Previous clinical trials show that intra-articular injection of MSCs improves knee OA [8]. MSCs can be obtained from bone marrow, adipose tissue, and placenta or umbilical cord [9]. The regenerative potential of placental MSCs (PMSCs) is comparable to MSCs derived from bone marrow or adipose tissue. PMSCs are more accessible than other types of MSCs and their preparation method is non-invasive [10]. Therefore, PMSCs are a more accessible and non-invasive source of EVs.
EVs are nano-sized vesicles released by cells into the extracellular space. EVs are categorised into three major subtypes: (1) Exosomes, (2) Microvesicles, and (3) Large EVs (including apoptotic bodies and oncosomes). All three types of EVs have different sizes, contents and formation mechanisms. Exosomes and microvesicles are released physiologically by normal cells. Exosomes have a diameter of 30–100 nm and are produced by the adhesion of endosomal compartments to the plasma membrane. The diameter of microvesicles is 100–1000 nm and they are produced by cell membrane budding [11].
EVs play a substantial role in cell-to-cell communication, in the regulation of tissue homeostasis, and in biological processes. EVs are derived from MSCs and have the valuable properties of donor MSCs. However, compared to stromal cells, EVs elicit fewer immunological responses and are more stable for storage and in vivo use. The components of EVs include miRNAs, lipids and proteins, and they promote tissue regeneration and modulate immunity through these components. Reports suggest that mesenchymal stromal cells-derived EVs (MSCs-EVs) improve the phenotype of chondrocytes, attenuate cartilage degradation in vitro and improve the progression of OA in vivo [12].
The results of various clinical studies on the treatment of knee OA with intra-articular injection of MSCs have confirmed safety and, in some cases, efficacy. Although the details of the mechanism of osteoarthritis treatment by stromal cells are not yet clear, many studies have shown that this therapeutic effect is caused by the paracrine function of stromal cells, including the secretion of EVs [13]. So far, EVs derived from different types of stromal cells have been hypothesised to regulate cartilage regeneration and reduce OA progression in preclinical studies. Reviews by D’Arrigo et al. and Kim et al. include these preclinical studies [14, 15].
Numerous studies on stromal cells have already been published, reporting on their safety and various therapeutic effects in knee OA. Our study is one of the first articles on the use of MSCs-EVs for the treatment of knee OA in humans. This clinical trial was designed to evaluate the safety of a single intra-articular injection of placental mesenchymal stromal cells-derived EVs (PMSCs-EVs) and to achieve preliminary data about its therapeutic effects in patients with knee OA.
Methods
Study design and patient selection
The proposal of this study was approved by the ethics committee of Shahid Beheshti University of Medical Sciences on 8 March 2022 with ethics code IR.SBMU.MSP.REC.1400.773. Subsequently, we registered the study protocol on the IRCT website (www.irct.ir) and it was approved on 11 May 2022 with registration number IRCT20210423051054N1.
The sample size was calculated based on previous studies to achieve an effect size of 0.7 with an alpha level of 0.05 and a power of 80% [16]. This calculation was done through the mean difference between two groups and using G-Power 3.1.9.7. Based on this calculation, we required 52 subjects (26 subjects per group) for a 1:1 allocation ratio.
Participants were selected from patients with knee OA attending the physical medicine and rehabilitation (PM&R) clinics of four medical training centers based on the following criteria.
Inclusion criteria: (1) Bilateral knee pain in the last 6 months, related to the degenerative process of the knee, requiring pain medication and lasting longer than one month; (2) Radiologic confirmation of grade 2 or 3 bilateral knee osteoarthritis based on the Kellgren-Lawrence classification; (3) Visual analog scale (VAS) ≥ 4.
Exclusion criteria: (1) Age under 45 or over 65 years; (2) History of rheumatoid arthritis; (3) History of malignant disease; (4) Body mass index over 35 kg/m2; (5) History of coagulation disorders or use of anticoagulants; (6) Intra-articular knee injections in the last 3 months (including corticosteroids, HA, dextrose, PRP, etc.); (7) History of severe knee trauma in the last 1 month; (8) Septic arthritis of the knee or active ulcer of the knee joint; (9) Genovarum or genovalgum of more than 20 degrees; (10) Knee joint replacement; 11) Pacemaker; 12) Cochlear implant; 13) Shrapnel or metallic foreign body in the knee or around the vessels, nerves or brain.
After the patients were selected, a resident of physical medicine and rehabilitation explained to them the aims, methods and available evidence of the study, as well as the benefits and possible complications of the procedure. Only patients who signed the informed consent form could participate in the study. First, personal data including age, sex, weight, height, and OA grade (based on Kellgren-Lawrence classification in X-ray image) were recorded. Subsequently, a VAS for pain assessment, a WOMAC questionnaire (Western Ontario and McMaster Universities) and a Lequesne index for functional assessment were completed for each patient by a physician who was blinded to treatment allocation.
Blinding of the study
This study is a randomized, triple-blind clinical trial. The patient, physician, interviewer, and both radiologists did not know which treatment was assigned to the patient. Data analysis was performed in such a way that the analyst was blinded to treatment allocation.
Clinical evaluation
Clinical evaluation was performed using VAS, WOMAC questionnaire, and Lequesne index. WOMAC and Lequesne are specific to knee assessment, but VAS is a general scale for pain assessment. The VAS is a valid and reliable scale for pain. On this scale, 0 indicates no pain and 10 indicates unbearable pain [17]. The WOMAC questionnaire has 24 items (5 items for pain, 2 items for stiffness and 17 items for function) and each item is scored from 0 to 4, with a lower score indicating fewer symptoms and better function. The total WOMAC score is 0 to 96. The Lequesne index consists of 11 items, including 5 items for pain or discomfort, 2 items for maximum distance walked (MDW) and 4 items for activities of daily living (ADL), with a lower score indicating fewer symptoms and better function. The total Lequesne score is 0 to 24 [18]. VAS, WOMAC, and Lequesne have been used in most studies on knee osteoarthritis for clinical evaluation of patients. For example, we can refer to the studies of Raeissadat et al. and Bahrami et al. [3, 6]. VAS is a public domain for pain assessment, which has been published in previous articles including the article by Bao et al. [17]. Also, the English and Arabic versions of WOMAC have been published in the article by Guermazi et al. [19].
Radiological imaging
The MR images of the patients’ knees were taken before (3–4 days before injection) and 6 months after treatment. All images were taken with a Siemens AVANTO (1.5 Tesla) MR system. Also, a circumferential knee coil was used for all patients. Sequences included transverse 3D-TRUFISP, coronal fat-saturated proton density (PD) and sagittal fat-saturated PD. Each MR image was evaluated and reported by two radiologists separately. If the two reports were different, the image was rechecked by a third radiologist. All three radiologists were uniformly trained to evaluate MRI findings. They did not know whether the images were in the intervention or control group and whether they were taken before or after treatment. Also, all images were evaluated and reported in unpaired mode. Five features were evaluated in the knee joint as follows.
Retropatellar cartilage volume: The retropatellar cartilage was evaluated in the transverse 3D-TRUFISP sequence using 3D-Slicer 5.0.3 (a free software for image analysis) and manually segmented to obtain the cartilage area in all axial images. Then the software calculated the cartilage volume using the obtained area and the thickness of the slide [5].
Tibiofemoral cartilage volume: All the above steps were performed for the tibiofemoral cartilage in the sagittal images.
Medial and lateral meniscal disintegrity: In both meniscus, three segments including anterior horn, posterior horn and body segment were evaluated separately in sagittal and coronal images and each segment was scored 0–3; 0: intact, 1: spherical signal within the meniscus that did not extend to the articular surface, 2: linear signal within the meniscus that did not extend to the articular surface, 3: abnormal signal within the meniscus extended to the articular surface [5].
ACL injury: The anterior cruciate ligament (ACL) was evaluated in the sagittal images and given a score of 0–2; 0: intact, 1: partial tear, 2: complete tear [20].
Effusion-synovitis: High intensity in the joint cavity indicates a combination of effusion and thickening of the synovium, graded on a scale of 0–3; 0: there is a normal amount of fluid; 1: small – there is continuous fluid in the retropatellar space; 2: medium – there is a mild convexity in the suprapatellar bursa; 3: large – there is evidence of capsular expansion [21].
Administered dose
Sariboyaci et al. investigate MSCs-derived exosomes in degenerative meniscal injuries [22], where the dose of exosomes is 1 × 106 particles/kg. Also, Espinoza et al. investigate MSCs-derived exosomes in knee osteoarthritis [23], where the dose of exosomes is 3–5 × 1011 particles/dose. Based on these studies, we chose the PMSCs-EVs dose of 35 × 109 particles/dose (5 cc, 7 × 109 particles/cc), which is between the mentioned doses.
Randomization
We used Random Allocation Software v2.0 to ensure that the allocation process was random and unbiased. Using this software, the knees of each patient were randomized to intervention and control.
Intervention
In each patient, one knee was the intervention and the contralateral knee was the control. After sterilization of the joint, 5 cc of PMSCs-EVs (7 × 109 particles/cc) were injected into the intervention knee and 5 cc of normal saline was injected into the control knee. The injection was performed by the anterolateral approach in the flexed knee position with a 22-gauge needle. No anesthetic was used in any of the patients. All injections were performed by a specialist physician who had no knowledge of the treatment allocation and no role in the follow-up of the patients. The drug and placebo were prepared by a nurse in two identical syringes in a completely sterile manner. All patients were advised to relative rest for 48 h after the injection. If they were in pain, they could take 500 mg of paracetamol every 6 h and apply a cold compress to the knee for 20 min. One week after the injection, all patients began isometric strengthening of the quadriceps, hip adductors and abductors (10 times, 10 s each) and stretching of the hamstrings (3 times, 30 s each), twice daily (morning and evening). These exercises were continued until the end of the 6 months. All exercises were taught to the patients by the PM&R resident and followed up by phone calls.
Follow up
All patients were followed up 2 and 6 months after treatment and questionnaires (including VAS, WOMAC, and Lequesne) were completed again for them. Also, another MRI was performed at month 6.
Preparation of PMSCs-EVs
Passage 3 PMSCs were used in this study. PMSCs were obtained from placenta tissue of healthy women with informed consent [24]. The cells were cultured in serum-free DMEM/MEM/GlutamaX/NEAA medium (Gibco, USA) and incubated in 5% CO2 at 37 ° C for 72 h. Then the conditioned cell culture medium was collected and centrifuged at different speeds [300-g (10 min), 2000-g (10 min) and 10000-g (30 min)] to separate the cells and remove dead cells, cell debris and large particles. The supernatant was ultracentrifuged [110000-g (70 min)] using a W32Ti rotor (L-80XP, Beckman Coulter, USA) to isolate the vesicles. The vesicles were eluted with phosphate buffered saline (PBS) (Sigma-Aldrich, USA) and centrifuged again to remove contaminating proteins. In the last step, the vesicles were suspended in 100 cc of normal saline (Sigma-Aldrich, USA). All steps were performed in a good manufacturing practice (GMP) certified clean room according to current GMP guidelines.
Morphological analysis of EVs
Morphological evaluation of vesicles was done using scanning electron microscope (SEM) (XL30 SEM, Philips, Germany). For this purpose, the vesicles were imaged at an accelerating voltage of 20.0 kV and then the mean diameter was calculated using Clemex Vision software by recording at least 100 vesicles.
Dynamic light scattering analysis of EVs
The size distribution was determined by dynamic light scattering (DLS) technique using a SZ-100 Nanopartica series instrument (HORIBA, Japan) operating in a dynamic range from 0.3 nm to 8 μm with a scattering angle of 90°. For this purpose, the vesicles were diluted in PBS (1:1000 v/v) and the sample was measured with the standard setting (count rate = 29 kcps, viscosity of the dispersion medium = 0.892 mPa.s, temperature of the holder = 25.2 °C).
Flow cytometric analysis of EVs
A flow cytometric analysis was performed to examine the surface markers of the vesicles. In brief, the vesicle suspension was added to each vial containing anti-CD81 (1:1000) and anti-CD63 (1:500) antibodies (Abcam, Cambridge, UK) and then incubated for 60 min at 4 °C. Finally, the vesicles were fixed in 100 µl paraformaldehyde (1%) (Sigma-Aldrich, USA) and statistical recording was analyzed using a Becton Dickinson instrument (BD Biosciences, USA) and Flowing Software 2.5.1 (BD Company, USA). Data were obtained from three measurements and reported as mean ± standard deviation (n = 3).
Western blot assay of EVs
To perform the Western blot assay, PMSCs-EVs were lysed with RIPA buffer (pH = 8.0) containing a protease inhibitor cocktail, sodium deoxycholate (0.5%), sodium chloride (150 mM), Tris-HCl (50 mM), Igepal (1.0%) and sodium dodecyl sulfate (0.1%) (all from Sigma-Aldrich, USA). The concentration of total protein in the vesicles was determined using the bicinchoninic acid assay (Thermo Scientific, Waltham, USA). The vesicles were then re-suspended and boiled in the loading buffer for 5 min at 100 °C. The same amount of protein from the sample was added to the reducing Laemmli buffer and then loaded onto a Tris-glycine-sodium dodecyl sulfate-polyacrylamide gel and electrophoresed. The proteins were transported to polyvinylidene fluoride to block the membrane in 5% non-fat milk. The sample was then incubated with primary antibodies including anti-CD9 (1:50) (Abcam, Cambridge, UK) and anti-CD81 (1:100) for 15 h at 4 °C. Finally, the signals were observed with an enhanced chemiluminescence kit (Bio-Rad, USA) using Chemidoc Touch (Bio-Rad, USA) [25].
Statistical analysis
The obtained data were statistically analyzed using SPSS v26 for Windows (IBM Inc, NY) by a statistician who was blinded to treatment allocation. Frequency index and percentage were used to indicate qualitative variables and descriptive statistics such as mean ± standard deviation were used for quantitative variables. The Kolmogorov-Smirnov test (K-S test) was used to check the normal distribution of variables. Independent T-test, Paired T-test, and ANOVA were used to analyze variables with normal distribution. Variables that did not follow normal distribution were analyzed using Mann-Whitney, Wilcoxon Signed Rank, and Kruskal-Wallis tests. Pearson correlation coefficient and Spearman correlation coefficient were used to check the correlation between variables. The significance level for P-value was considered less than 0.05.
Results
Characteristics of PMSCs-EVs
We evaluated the morphology, diameter and size distribution of vesicles by examining SEM images and analyzing DLS data. The round shape of the vesicles with an average diameter of 28.23 nm confirms their structure as EVs (Fig. 1A). The DLS results also show that the Z-average of the vesicles is 92.7 nm and the polydispersity index (PDI) is 0.471 (Fig. 1B). Flow cytometric analysis revealed strong signals for CD63 and CD81 as markers on EVs, indicating over 73% expression for CD63 and over 88% expression for CD81 (Fig. 1C). Western blot assay also confirmed the surface markers of EVs including CD9 and CD81 (Fig. 1D).
Fig. 1.
Characteristics of PMSCs-EVs. (A) SEM images; (B) DLS analysis; (C) Flow cytometric analysis; (D) Western blot assay
Patients
62 knees (from 31 patients with bilateral knee OA) were enrolled in this study, 31 knees for intervention and 31 knees for control. 2 knees from each group were eliminated and leaving 29 knees in each group after 6 months (Fig. 2). 28 patients were female and 1 patient was male, with an average age of 55.38 ± 6.07 years and an average BMI of 30.19 ± 2.53 kg/m2 (Table 1). There was no significant difference in the basic characteristics of the patients between the two groups (Table 2).
Fig. 2.
CONSORT flow diagram
Table 1.
Individual characteristics of the study population
| PMSCs-EVs | Control | ||
|---|---|---|---|
| Sex (%) | Female | 28 (96.5%) | 28 (96.5%) |
| Male | 1 (3.5%) | 1 (3.5%) | |
| Age (Mean ± SD) | 55.38 ± 6.07 | 55.38 ± 6.07 | |
| BMI (Mean ± SD) | 30.19 ± 2.53 | 30.19 ± 2.53 | |
| Grade (%) | Grade 2 | 17 (58.6%) | 17 (58.6%) |
| Grade 3 | 12 (41.4%) | 12 (41.4%) | |
Table 2.
Basic characteristics of the study population
| PMSCs-EVs | Control | P-value | ||
|---|---|---|---|---|
| VAS (Mean ± SD) | 6.55 ± 2.33 | 6.10 ± 1.89 | 0.42 | |
| WOMAC (Mean ± SD) | Pain | 9.86 ± 3.61 | 9.45 ± 4.30 | 0.69 |
| Stiffness | 2.07 ± 2.34 | 1.90 ± 2.12 | 0.77 | |
| Function | 32.79 ± 12.19 | 30.93 ± 11.40 | 0.55 | |
| Total | 44.72 ± 16.14 | 42.28 ± 15.76 | 0.56 | |
| Lequesne (Mean ± SD) | Pain or Discomfort | 5.07 ± 1.71 | 4.79 ± 1.84 | 0.62 |
| MDW | 2.24 ± 1.50 | 2.41 ± 1.74 | 0.81 | |
| ADL | 5.06 ± 1.70 | 4.86 ± 1.82 | 0.62 | |
| Total | 12.37 ± 3.16 | 12.06 ± 4.46 | 0.64 | |
| MRI | Retropatellar Cartilage Volume (Mean ± SD) | 1249.93 ± 695.46 | 1098.60 ± 648.51 | 0.39 |
| Tibiofemoral Cartilage Volume (Mean ± SD) | 686.26 ± 329.73 | 610.38 ± 341.44 | 0.39 | |
| Meniscal Disintegrity (Mean ± SD) | 5.07 ± 3.64 | 5.38 ± 3.21 | 0.73 | |
|
ACL Injury (Frequency) Intact Partial tear Complete tear |
8 20 1 |
5 24 0 |
0.35 | |
|
Effusion-Synovitis (Frequency) Grade 0 Grade 1 Grade 2 Grade 3 |
9 8 6 6 |
8 14 2 5 |
0.28 | |
Safety
No systemic complications or severe local reactions occurred in the patients after the injection. Moderate pain with mild warmness occurred in 4 patients (13%), which improved within 48 h with cold compresses every 6 h for 20 min and paracetamol 500 mg every 6 h. Other patients (n = 27, 87%) either had no local symptoms or reported mild pain (without warmness, erythema or swelling) after the injection, which improved within 24 h without the need for treatment.
Efficacy
Before treatment and in months 2 and 6, no significant difference was detected between the clinical results of the two groups (Tables 3, 4 and 5). Figure 3 shows the changes in VAS, WOMAC, and Lequesne scores during 6 months in two groups. Also, no significant difference was detected in MRI findings between the two groups before and after treatment (Tables 6 and 7). Figures 4 and 5 show MRI before and after treatment.
Table 3.
Mean ± SD of VAS before treatment and 2 and 6 months after treatment in both groups
| PMSCs-EVs | Control | Mean Difference 95% CI | P-value | ||
|---|---|---|---|---|---|
| Mean ± SD | Mean ± SD | ||||
| VAS | T0 | 6.55 ± 2.33 | 6.10 ± 1.89 | 0.44 95% [-0.67, 1.56] | 0.42 |
| T1 | 4.86 ± 2.69 | 4.76 ± 2.55 | 0.10 95% [-1.27, 1.48] | 0.88 | |
| T2 | 5.10 ± 2.45 | 4.62 ± 2.47 | 0.48 95% [-0.81, 1.77] | 0.45 | |
Table 4.
Mean ± SD of WOMAC before treatment and 2 and 6 months after treatment in both groups
| WOMAC Parameters | PMSCs-EVs | Control | Mean Difference 95% CI | P-value | |
|---|---|---|---|---|---|
| Mean ± SD | Mean ± SD | ||||
| Pain | T0 | 9.86 ± 3.61 | 9.45 ± 4.30 | 0.41 95% [-1.67, 2.50] | 0.69 |
| T1 | 7.31 ± 3.68 | 6.93 ± 4.24 | 0.37 95% [-1.71, 2.47] | 0.71 | |
| T2 | 6.66 ± 4.74 | 6.76 ± 4.93 | -0.10 95% [-2.65, 2.44] | 0.93 | |
| Stiffness | T0 | 2.07 ± 2.34 | 1.90 ± 2.12 | 0.17 95% [-1.0, 1.35] | 0.77 |
| T1 | 1.69 ± 1.92 | 1.97 ± 2.19 | -0.27 95% [-1.36, 0.81] | 0.61 | |
| T2 | 1.34 ± 1.93 | 1.0 ± 1.60 | 0.34 95% [-0.58, 1.27] | 0.46 | |
| Function | T0 | 32.79 ± 12.19 | 30.93 ± 11.40 | 1.86 95% [-4.35, 8.07] | 0.55 |
| T1 | 22.14 ± 12.33 | 21.83 ± 13.72 | 0.31 95% [-6.55, 7.17] | 0.92 | |
| T2 | 21.0 ± 12.30 | 20.48 ± 12.70 | 0.51 95% [-6.32, 7.35] | 0.88 | |
| Total | T0 | 44.72 ± 16.14 | 42.28 ± 15.76 | 2.44 95% [-5.94, 10.84] | 0.56 |
| T1 | 31.14 ± 17.19 | 30.72 ± 19.47 | 0.41 95% [-9.24, 10.07] | 0.93 | |
| T2 | 29.0 ± 18.88 | 28.24 ± 18.24 | 0.75 95% [-9.01, 10.52] | 0.87 | |
Table 5.
Mean ± SD of Lequesne before treatment and 2 and 6 months after treatment in both groups
| Lequesne Parameters | PMSCs-EVs | Control | Mean Difference 95% CI | P-value | |
|---|---|---|---|---|---|
| Mean ± SD | Mean ± SD | ||||
| Pain or Discomfort | T0 | 5.07 ± 1.71 | 4.79 ± 1.84 | 0.27 95% [-0.65, 1.21] | 0.62 |
| T1 | 3.90 ± 1.81 | 3.90 ± 1.71 | 0.0 95% [-0.93, 0.93] | 0.95 | |
| T2 | 4.24 ± 1.97 | 3.93 ± 1.87 | 0.31 95% [-0.70, 1.32] | 0.62 | |
| MDW | T0 | 2.24 ± 1.50 | 2.41 ± 1.74 | -0.17 95% [-1.02, 0.68] | 0.81 |
| T1 | 2.34 ± 1.42 | 2.31 ± 1.51 | 0.03 95% [-0.74, 0.81] | 0.76 | |
| T2 | 1.93 ± 1.36 | 1.79 ± 1.20 | 0.13 95% [-0.53, 0.81] | 0.69 | |
| ADL | T0 | 5.06 ± 1.70 | 4.86 ± 1.82 | 0.20 95% [-0.72, 1.13] | 0.62 |
| T1 | 4.25 ± 2.22 | 4.05 ± 2.05 | 0.20 95% [-0.91, 1.33] | 0.60 | |
| T2 | 4.34 ± 2.27 | 3.96 ± 1.85 | 0.37 95% [-0.71, 1.47] | 0.47 | |
| Total | T0 | 12.37 ± 3.16 | 12.06 ± 4.46 | 0.31 95% [-1.72, 2.34] | 0.64 |
| T1 | 10.50 ± 4.54 | 10.25 ± 4.44 | 0.24 95% [-2.12, 2.60] | 0.73 | |
| T2 | 10.51 ± 5.15 | 9.69 ± 4.41 | 0.82 95% [-1.69, 3.35] | 0.64 | |
Fig. 3.
Changes in scores during 6 months in two groups. (A) VAS scores; (B) Total WOMAC scores; (C) Total Lequesne scores
Table 6.
Mean ± SD of MRI findings before treatment and 6 months after treatment in both groups
| PMSCs-EVs | Control | Mean Difference 95% CI | P-value | ||
|---|---|---|---|---|---|
| Mean ± SD | Mean ± SD | ||||
| Meniscal disintegrity | T0 | 5.07 ± 3.64 | 5.38 ± 3.21 | -0.31 95% [-2.11, 1.49] | 0.73 |
| T2 | 5.52 ± 3.92 | 5.66 ± 3.68 | -0.13 95% [-2.14, 1.86] | 0.89 | |
| Retropatellar cartilage volume | T0 | 1249.93 ± 695.46 | 1098.60 ± 648.51 |
151.32 95% [-202.40, 505.61] |
0.39 |
| T2 | 1273.84 ± 620.33 | 1316.94 ± 604.72 | -43.09 95% [-365.36, 279.16] | 0.79 | |
| Tibiofemoral cartilage volume | T0 | 686.26 ± 329.73 | 610.38 ± 341.44 | 75.88 95% [-100.68, 252.45] | 0.39 |
| T2 | 546.79 ± 279.58 | 549.11 ± 312.09 | -2.32 95% [-158.19, 153.54] | 0.97 | |
Table 7.
Frequency of MRI findings before treatment and 6 months after treatment in both groups
| Study Group | Total | P-value | |||
|---|---|---|---|---|---|
| PMSCs-EVs | Control | ||||
| ACL – T0 | Intact | 8 | 5 | 13 | 0.35 |
| Partial tear | 20 | 24 | 44 | ||
| Complete tear | 1 | 0 | 1 | ||
| Total | 29 | 29 | 58 | ||
| ACL – T2 | Intact | 10 | 5 | 15 | 0.14 |
| Partial tear | 16 | 23 | 39 | ||
| Complete tear | 3 | 1 | 4 | ||
| Total | 29 | 29 | 58 | ||
| Effusion - Synovitis – T0 | Grade 0 | 9 | 8 | 17 | 0.28 |
| Grade 1 | 8 | 14 | 22 | ||
| Grade 2 | 6 | 2 | 8 | ||
| Grade 3 | 6 | 5 | 11 | ||
| Total | 29 | 29 | 58 | ||
| Effusion - Synovitis – T2 | Grade 0 | 4 | 2 | 6 | 0.79 |
| Grade 1 | 13 | 16 | 29 | ||
| Grade 2 | 9 | 8 | 17 | ||
| Grade 3 | 3 | 3 | 6 | ||
| Total | 29 | 29 | 58 | ||
Fig. 4.
Retropatellar cartilage was evaluated using 3D-Slicer software. Left image before PMSCs-EVs injection (1056 mm3) and right image after PMSCs-EVs injection (1115 mm3)
Fig. 5.
Effusion-synovitis was evaluated in axial images. Left image before PMSCs-EVs injection (Grade 1) and right image after PMSCs-EVs injection (Grade 1)
Discussion
The use of MSCs-EVs is a very new method for the treatment of diseases that has recently received a lot of attention. For this reason, published studies in this field are very limited, and this limitation is especially true for musculoskeletal disorders. As there are no comparable studies, it is difficult to discuss the results of this study and draw conclusions. There are more studies on MSCs as a treatment for knee OA. Therefore, comparing this study with studies on MSCs is the only solution to this problem.
In 2019, D’Arrigo et al. reviewed 20 preclinical studies and concluded that EVs exert positive effects on cartilage, subchondral bone, and synovium in OA [14]. The difference between the results of these studies and our study can be summarized in 3 reasons: First, in most of these studies, the source of EVs was bone marrow or adipose tissue, not the placenta. Second, most of these studies used pure exosome, not EVs. Third, in most of the invivo studies, 2 to 4 injections have been performed, not a single injection.
In 2019, Lee et al. investigated adipose tissue-derived MSCs in knee OA [26]. The results in both parts (clinical and MRI) are in contrast to the results obtained in our study.
In 2019, Lu et al. investigated adipose tissue-derived MSCs in knee OA [27]. The results in both parts (clinical and MRI) are in contrast to the results obtained in our study, which could be due to the double injection in Lu’s study.
In 2018, Emadedin et al. investigated bone marrow-derived MSCs in knee OA [16]. Their results were in contrast to the results obtained in our study.
In 2019, Matas et al. investigated umbilical cord-derived MSCs in knee OA [28]. The results of the Matas study were very similar to the results of our study, especially for the MRI findings.
In 2018, Khalifeh et al. investigated placenta-derived MSCs in knee OA [10]. The results at month 6 were completely similar to our study except for the cartilage defect.
In 2021, a systematic review and meta-analysis was published by Dai et al. [29]. Their results were similar to our study. They concluded that intra-articular injection of MSCs does not improve pain and function in knee OA more than placebo. However, they noted that more clinical trials with different types of cells, doses, and number of injections are needed to draw conclusions about efficacy.
None of the studies reported systemic or severe local complications with treatment, which was similar to our study.
A comparison between the studies shows that the therapeutic effect of stromal cells derived from different tissues is significantly different. This fact is also true for EVs. For this reason, the results of our study are consistent with studies that used stromal cells from the placenta or umbilical cord, but inconsistent with other studies. Therefore, the lack of efficacy in our study could be related to the source of the EVs.
One of the challenges with intra-articular treatments is the dose and frequency of injections, and this challenge is even greater in the field of cell therapy, especially in preliminary studies. EVs cannot self-replicate and have a short half-life in vivo. Therefore, repeated injections may be required to achieve a therapeutic effect. In contrast, stromal cells can self-replicate and survive in the body for a longer time. Therefore, the low dose of the drug or the low frequency of injections could be the reason for the lack of efficacy in our study.
The results of the study are also related to the follow-up period. Perhaps an extension of the follow-up period will show the positive effects of this treatment in the long term. The number of participants is also effective in the results of the study. Perhaps the increase in the number of participants will cause a significant difference between the two groups.
As discussed, different factors including the source of EVs, dose, frequency of injections, follow-up period, and number of participants can be considered as the reason for the negative results. But considering that most of the cell therapy studies that reported positive results included two or more injections, probably the single injection was the main reason for the lack of effectiveness in our study.
Finally, it should be noted that our study is a preliminary study including middle-aged patients with moderate osteoarthritis and the results may not be generalizable to other age groups or patients with mild or severe osteoarthritis.
Limitations
In each patient, one knee was the intervention and the contralateral knee was the control. The main reason for choosing this allocation method is to reduce the effect of individual and environmental factors on the evaluated outcomes. For example, the volume of knee cartilage is dependent on gender of the patient and grade of osteoarthritis, which this allocation method reduces these differences. Thus, bias is reduced and data analysis becomes easier. As you can see in Table 1, all these factors (including age, sex, BMI, and grade) are the same in the two groups. It is very difficult to compare the function, ADL, and MDW between the two knees. For this reason, most patients have reported the same function, ADL, and MDW for both knees. Therefore, these outcomes were not significantly different between the two groups. This limitation may have affected the total score in WOMAC and Lequesne. For this reason, clinical trials with different allocation methods are recommended. Although the follow-up period is 6 months in most knee OA studies, but this may not be sufficient to determine long-term results. Therefore, clinical trials with longer follow-up are recommended. Also, the relatively small number of participants can be a factor for the negative results. Therefore, clinical trials with more participants are also recommended.
Conclusion
A single intra-articular injection of placental mesenchymal stromal cells-derived extracellular vesicles (5 cc, 7 × 109 particles/cc) is safe, but does not improve clinical symptoms or MRI findings in knee osteoarthritis beyond placebo. Nevertheless, a longer study with more participants as well as studies with different doses and number of injections or different source of EVs are recommended to evaluate the efficacy.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Acknowledgements
We thank Mr. Hatam Chaakhorzadeh for taking care of the PMSCs-EVs vials and providing an appropriate environment for injections.
Abbreviations
- OA
Osteoarthritis
- HA
Hyaluronic Acid
- PRP
Platelet Rich Plasma
- EVs
Extracellular Vesicles
- MSCs
Mesenchymal Stromal Cells
- PMSCs
Placental Mesenchymal Stromal Cells
- MSCs-EVs
Mesenchymal Stromal Cells-derived Extracellular Vesicles
- PMSCs-EVs
Placental Mesenchymal Stromal Cells-derived Extracellular Vesicles
- PM&R
Physical Medicine and Rehabilitation; VAS: Visual Analog Scale
- WOMAC
Western Ontario and McMaster Universities
- MDW
Maximum Distance Walked
- ADL
Activities of Daily Living
- PD
Proton Density
- ACL
Anterior Cruciate Ligament
- PBS
Phosphate Buffered Saline
- GMP
Good Manufacturing Practice
- SEM
Scanning Electron Microscope
- DLS
Dynamic Light Scattering
- K-S test
Kolmogorov-Smirnov test
- PDI
Polydispersity Index
- BMI
Body Mass Index
Author contributions
NSB, SAR, SMR, and MB designed the clinical trial. RSO selected and followed up the patients and prepared the first draft and final version of the article. SHK and MS prepared the PMSCs-EVs. Knee injections were done by MZ. The review and report of MR images was done by HH and AZ. ZBR followed up the patients using questionnaires. Data analysis was done by KA. The final revision was done by NSB, SAR, SMR, MB, and FS. All authors contributed to interpretation of the results and preparation of the article. All authors approved the final version of the article to be published.
Funding
No external funding was involved in this study.
Data availability
We can share the Excel file of unidentified data at the request of the editorial board through the corresponding author’s email.
Declarations
Ethics approval and consent to participate
The proposal of this study was approved by the ethics committee of Shahid Beheshti University of Medical Sciences on 8 March 2022 with ethics code IR.SBMU.MSP.REC.1400.773. Subsequently, we registered the study protocol on the IRCT website (www.irct.ir) and it was approved on 11 May 2022 with registration number IRCT20210423051054N1. Verbal and written informed consent was obtained from the participants and they could withdraw from the study at any time.
Consent for publication
Not applicable.
Consort guidelines
We reported this study following CONSORT guidelines.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- 1.Felson DT. The epidemiology of knee osteoarthritis: results from the Framingham Osteoarthritis Study. Semin Arthritis Rheum. 1990;20:42–50. [DOI] [PubMed] [Google Scholar]
- 2.Wallace IJ, Worthington S, Felson DT, Jurmain RD, Wren KT, Maijanen H, et al. Knee osteoarthritis has doubled in prevalence since the mid-20th century. PNAS. 2017;114(35):9332–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Raeissadat SA, Ghazi Hosseini P, Bahrami MH, Salman Roghani R, Fathi M, Gharooee Ahangar A, et al. The comparison effects of intra-articular injection of platelet Rich plasma (PRP), plasma Rich in Growth factor (PRGF), Hyaluronic Acid (HA), and ozone in knee osteoarthritis; a one year randomized clinical trial. BMC Musculoskelet Disord. 2021;22(1):134. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Steinmetz JD, Culbreth GT, Haile LM, Rafferty Q, Lo J, Fukutaki KG, et al. Global, regional, and national burden of osteoarthritis, 1990–2020 and projections to 2050: a systematic analysis for the global burden of Disease Study 2021. Lancet Rheumatol. 2023;5(9):e508–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Raeissadat SA, Ghorbani E, Sanei Taheri M, Soleimani R, Rayegani SM, Babaee M, et al. MRI changes after platelet Rich plasma injection in knee osteoarthritis (Randomized Clinical Trial). J Pain Res. 2020;13:65–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Bahrami MH, Raeissadat SA, Cheraghi M, Rahimi Dehgolan S, Ebrahimpour A. Efficacy of single high-molecular-weight versus triple low-molecular-weight hyaluronic acid intra-articular injection among knee osteoarthritis patients. BMC Musculoskelet Disord. 2020;21(1):550. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Raeissadat SA, Rayegani SM, Sedighipour L, Bossaghzade Z, Abdollahzadeh MH, Nikray R, et al. The efficacy of electromyographic biofeedback on pain, function, and maximal thickness of vastus medialis oblique muscle in patients with knee osteoarthritis: a randomized clinical trial. J Pain Res. 2018;11:2781–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Song Y, Jorgensen C. Mesenchymal stromal cells in Osteoarthritis: evidence for Structural Benefit and Cartilage Repair. Biomedicines. 2022;10(6):1278. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Hass R, Kasper C, Böhm S, Jacobs R. Different populations and sources of human mesenchymal stem cells (MSC): a comparison of adult and neonatal tissue-derived MSC. Cell Communication Signal. 2011;9:12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Khalifeh Soltani S, Forogh B, Ahmadbeigi N, Hadizadeh Kharazi H, Fallahzadeh K, Kashani L, et al. Safety and efficacy of allogenic placental mesenchymal stem cells for treating knee osteoarthritis: a pilot study. Cytotherapy. 2019;21(1):54–63. [DOI] [PubMed] [Google Scholar]
- 11.Yuan S, Li G, Zhang J, Chen X, Su J, Zhou F. Mesenchymal stromal cells-derived extracellular vesicles as potential treatments for Osteoarthritis. Pharmaceutics. 2023;15(7):1814. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Zhuang Y, Jiang S, Yuan C, Lin K. The potential therapeutic role of extracellular vesicles in osteoarthritis. Front Bioeng Biotechnol. 2022;10:1022368. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Ni Z, Zhou S, Li S, Kuang L, Chen H, Luo X, et al. Exosomes: roles and therapeutic potential in osteoarthritis. Bone Res. 2020;8:25. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.D’Arrigo D, Roffi A, Cucchiarini M, Moretti M, Candrian C, Filardo G. Secretome and Extracellular vesicles as New Biological therapies for knee osteoarthritis: a systematic review. J Clin Med. 2019;8(11):1867. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Kim KH, Jo JH, Cho HJ, Park TS, Kim TM. Therapeutic potential of stem cell-derived extracellular vesicles in osteoarthritis: preclinical study findings. Lab Anim Res. 2020;36:10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Emadedin M, Labibzadeh N, Ghorbani Liastani M, Karimi A, Jaroughi N, Bolurieh T, et al. Intra-articular implantation of autologous bone marrow-derived mesenchymal stromal cells to treat knee osteoarthritis: a randomized, triple-blind, placebo-controlled phase 1/2 clinical trial. Cytotherapy. 2018;20(10):1238–46. [DOI] [PubMed] [Google Scholar]
- 17.Bao X, Tan JW, Flyzik M, Ma XC, Liu H, Liu HY. Effect of therapeutic exercise on knee osteoarthritis after intra-articular injection of botulinum toxin type A, hyaluronate or saline: a randomized controlled trial. J Rehabil Med. 2018;50(6):534–41. [DOI] [PubMed] [Google Scholar]
- 18.Raeissadat SA, Rayegani SM, Gharooee Ahangar A, Hassan Abadi P, Mojgani P, Gharooi Ahangar O. Efficacy of Intra-articular Injection of a Newly Developed Plasma Rich in Growth Factor (PRGF) Versus Hyaluronic Acid on Pain and Function of Patients with Knee Osteoarthritis: A Single-Blinded Randomized Clinical Trial. Clinical Medicine Insights: Arthritis and Musculoskeletal Disorders. 2017; 10:1179544117733452. [DOI] [PMC free article] [PubMed]
- 19.Guermazi M, Poiraudeau S, Yahia M, Mezganni M, Fermanian J, Habib Elleuch M, et al. Translation, adaptation and validation of the Western Ontario and McMaster universities osteoarthritis index (WOMAC) for an arab population: the Sfax modified WOMAC. Osteoarthr Cartil. 2004;12(6):459–68. [DOI] [PubMed] [Google Scholar]
- 20.Peterfy CG, Guermazi A, Zaim S, Tirman PF, Miaux Y, White D, et al. Whole-organ magnetic resonance imaging score (WORMS) of the knee in osteoarthritis. Osteoarthr Cartil. 2004;12(3):177–90. [DOI] [PubMed] [Google Scholar]
- 21.Hunter DJ, Guermazi A, Lo GH, Grainger AJ, Conaghan PG, Boudreau RM, et al. Evolution of semi-quantitative whole joint assessment of knee OA: MOAKS (MRI osteoarthritis knee score). Osteoarthr Cartil. 2011;19(8):990–1002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Sariboyaci AE. A phase II trial to investigate clinical efficacy of autologous synovial fluid mesenchymal stem cell-derived exosome application in patients with degenerative Meniscal Injury. 2022; [Available from: ClinicalTrials.gov].
- 23.Espinoza FA, Phase I. Study Aiming to Assess Safety and Efficacy of a Single Intra-articular Injection of MSC-derived Exosomes (CelliStem®OA-sEV) in Patients With Moderate Knee Osteoarthritis. 2021; [Available from: ClinicalTrials.gov].
- 24.Hashemian SR, Aliannejad R, Zarrabi M, Soleimani M, Vosough M, Hosseini SE, et al. Mesenchymal stem cells derived from perinatal tissues for treatment of critically ill COVID-19 induced ARDS patients: a case series. Stem Cell Res Ther. 2021;12(1):91. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Jahedi Zargar M, Heidari Keshel S, Kaviani S, Zahraei M, Izadyari Aghmiuni A, Savadkoohi AA, et al. Safety and efficacy of placental mesenchymal stem cell-derived extracellular vesicle in severe COVID-19 patients: phase I & II clinical trials. Anat Sci. 2022;19(2):79–88. [Google Scholar]
- 26.Lee WS, Kim HJ, Kim KI, Kim GB, Jin W. Intra-articular injection of autologous adipose tissue-derived mesenchymal stem cells for the treatment of knee osteoarthritis: a phase IIb, Randomized, Placebo-Controlled Clinical Trial. Stem Cells Translational Med. 2019;8(6):504–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Lu L, Dai C, Zhang Z, Du H, Li S, Ye P, et al. Treatment of knee osteoarthritis with intra-articular injection of autologous adipose-derived mesenchymal progenitor cells: a prospective, randomized, double-blind, active-controlled, phase IIb clinical trial. Stem Cell Res Ther. 2019;10(1):143. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Matas J, Orrego M, Amenabar D, Infante C, Tapia-Limonchi R, Cadiz MI, et al. Umbilical cord-derived mesenchymal stromal cells (MSCs) for knee osteoarthritis: repeated MSC Dosing is Superior to a single MSC dose and to Hyaluronic Acid in a controlled Randomized Phase I/II Trial. Stem Cells Translational Med. 2019;8(3):215–24. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Dai W, Leng X, Wang J, Shi Z, Cheng J, Hu X, Ao Y. Intra-articular mesenchymal stromal cell injections are no different from placebo in the treatment of knee osteoarthritis: a systematic review and Meta-analysis of Randomized controlled trials. Arthroscopy. 2021;37(1):340–58. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
We can share the Excel file of unidentified data at the request of the editorial board through the corresponding author’s email.