Regulatory Role of Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Loaded with miRNA-122 in the Progression of Osteoarthritis

doi:10.21203/rs.3.rs-4681217/v1

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Research Article

Regulatory Role of Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Loaded with miRNA-122 in the Progression of Osteoarthritis

https://doi.org/10.21203/rs.3.rs-4681217/v1

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Objective The aim was to design and construct engineered exosomes carrying miR-122 and miR-122 inhibitors, and to observe the effects of intra-articular injection on articular cartilage and the progression of osteoarthritis (OA) in rats, with the goal of providing new insights and options for cartilage repair, regeneration, and early treatment of OA.

Methods Forty-eight SPF male rats aged 6-8 weeks were selected to establish an OA model in the rat knee joints. Rats were randomly divided into four groups: blank control group (n=12) received injection of physiological saline, normal exosome group (n=12) received injection of normal exosome suspension, miR-122 engineered exosome group (n=12) received injection of miR-122 engineered exosome suspension, and miR-122 inhibitors engineered exosome group (n=12) received injection of miR-122 inhibitors engineered exosome suspension. Bone marrow mesenchymal stem cells were cultured, identified, and passaged. Exosomes were extracted and identified using transmission electron microscopy, nanoparticle size analysis, and western blotting. Engineered exosomes loaded with miR-122 and miR-122 inhibitors were prepared using magnetic bead method, and identified by nanoparticle size analysis and real-time fluorescence quantitative PCR. The experiment was conducted according to the experimental design. General behavioral observation analysis, imaging evaluation, gross histological observation evaluation, histological detection, and immunohistochemical detection were performed on the four groups to compare and evaluate the progression of arthritis.

Results (1) In terms of general behavioral observation, rats in the miR-122 engineered exosome group exhibited poorer performance compared to the blank control group in indicators such as pain, gait, joint mobility, and swelling; the miR-122 inhibitors engineered exosome group outperformed both the blank control group and normal exosome group in all indicators, with the most significant difference observed at 12 weeks. (2) Micro-CT imaging results showed that the progression of arthritis was most rapid in the miR-122 engineered exosome group, followed by the blank control group, while the normal exosome group exhibited mild arthritis, and no obvious signs of arthritis were observed in the miR-122 inhibitors engineered exosome group. Gross scoring evaluation revealed that the progression of arthritis was slowest in the miR-122 inhibitors engineered exosome group, while it was most rapid in the miR-122 engineered exosome group. (3) Histological staining results showed that at week 4, the articular surfaces of the blank control group, miR-122 engineered exosome group, and normal exosome group exhibited mild damage and thinning of cartilage; the normal exosome group and miR-122 inhibitors engineered exosome group showed relatively thicker cartilage layers. At week 8, the articular surfaces of the blank control group and miR-122 engineered exosome group were uneven, with decreased and disorganized chondrocytes, while those of the normal exosome group and miR-122 inhibitors engineered exosome group were relatively smooth, with more chondrocytes observed in the miR-122 inhibitors engineered exosome group. At week 12, a significant reduction in chondrocytes was observed in the blank control group, almost no chondrocytes were present in the miR-122 engineered exosome group, and severe cartilage damage was observed; the articular surfaces were relatively smooth in the normal exosome group, with thickening of the cartilage layer, while the miR-122 inhibitors engineered exosome group showed the best performance. (4) Immunohistochemical detection of type II collagen and proteoglycan revealed that the extracellular matrix of chondrocytes in the miR-122 inhibitors engineered exosome group and normal exosome group was better than that in the blank control group, with the miR-122 inhibitors engineered exosome group showing superiority over the normal exosome group, while the extracellular matrix of the miR-122 engineered exosome group was inferior to that of the blank control group.

Conclusion MiR-122 engineered exosomes disrupt the stability of the extracellular matrix, accelerating the progression of OA, while miR-122 inhibitors engineered exosomes delay this process, maintaining the stability of the extracellular matrix and protecting articular cartilage. This may provide new insights and options for the treatment of early OA and the repair and regeneration of cartilage.

miR-122

osteoarthritis

engineered exosomes

intra-articular injection

miR-122 inhibitors

Osteoarthritis (OA) is one of the most common chronic joint diseases, and its prevalence has been gradually increasing in recent years, posing significant challenges to both individuals^[1] and society due to substantial economic burdens^[2]. The etiology of OA remains unclear, although factors such as age, hormones, obesity, and joint injuries are involved in its pathogenesis^[3–5]. Pathological changes in OA primarily include cartilage degradation, osteophyte formation, subchondral sclerosis, and synovial tissue hyperplasia^[6–8], leading to pain and functional impairment as major clinical manifestations. Knee osteoarthritis (KOA) is among the most prevalent types of OA, with a global incidence increase of 48% from 1990 to 2019^[9]. KOA alone accounts for 85% of the global burden caused by OA^[10], with a symptomatic KOA prevalence of 8.1% in China, making it one of the most common disabling diseases^[11]. Thus, prevention and treatment of KOA are crucial to effectively mitigate this global health issue.

Currently, treatment options for OA are limited and mainly include non-pharmacological treatments, pharmacological therapies, and surgery, focusing primarily on symptom relief and improving joint function^[12]. Studies indicate that engaging in exercise, maintaining a healthy lifestyle, adopting joint protection measures, and weight management play positive roles in maintaining joint health^[13–15]. Additionally, commonly used non-pharmacological treatments such as laser therapy^[15], transcutaneous electrical nerve stimulation^{[16, 17]}, ultrasound^[18], electrotherapy^[19], and acupuncture^[20] have shown limited efficacy due to the complex etiology of OA. In terms of pharmacological treatment, current options mainly involve analgesics, hyaluronic acid joint supplements, and intra-articular corticosteroid injections aimed at alleviating pain, restoring synovial fluid viscosity, and physiological homeostasis to improve symptoms. However, these treatments provide only limited assistance and do not address the root cause of OA^[21]; for example, non-steroidal anti-inflammatory drugs (NSAIDs) can relieve pain associated with hip and knee arthritis but do not delay OA progression. Moreover, clinicians recognize that OA is typically definitively diagnosed only in the later stages, impacting treatment outcomes^[22]. For advanced OA patients, joint replacement surgery is a preferred treatment method to alleviate pain and restore daily functioning; however, complications such as prosthesis revision greatly limit its application^[23]. Therefore, early intervention in the development of OA is critical. Minimally invasive treatments at outpatient levels are currently the preferred methods for early OA treatment, with disease-modifying osteoarthritis drugs (DMOADs) administered via intra-articular injection attracting widespread attention^[24].

With advances in genomics in recent years, numerous microRNAs (miRNAs) have been found to exhibit tissue-specific or developmental stage-specific expression and are associated with orthopedic diseases^[25]. Research indicates that many miRNAs play crucial roles in regulating the occurrence and development of OA^[26], affecting the synthesis and metabolism of bone and cartilage matrices^{[27, 28]}. miR-122 is involved in regulating cellular proliferation, programmed cell death, invasion, and metastasis. In the study by Kong et al.^[29], elevated expression of miR-122 in the plasma of OA patients was observed, suggesting a new research direction for OA treatment. Bai et al.^[30] found that miR-122 can regulate ECM degradation through the miR-122/SIRT1 axis in OA. However, research on the role of miR-122 in OA disease is currently limited^{[30, 31]}.

Bone marrow mesenchymal stem cells (BMSCs) have the potential for multilineage differentiation and self-renewal, capable of differentiating into various cell types such as bone, cartilage, and adipose tissues^[32–34]. Extracellular vesicles (Exos) produced by BMSCs represent one approach to achieve therapeutic effects^[23]. Exos are natural cell-derived vesicles with lipid bilayer structures, ranging in size from 30–120 nm, secreted by most cells into body fluids^[35]. They carry proteins, lipids, RNAs, growth factors, and cytokines, exhibiting superior biocompatibility, biological barrier penetration, targeted delivery, low immunogenicity, and high reparability^[36]. Once entering target cells, Exos can regulate cellular functions and signal transduction, playing crucial roles in intercellular communication^[37].In the treatment of bone-related diseases, Exos are considered a promising therapeutic approach and have gained attention in recent years. Research has shown that BMSCs-Exos can partially delay OA progression^[38].However, with advancements in biomedical technology and the clinical application of Exos, it has been found that relying solely on the natural targeting of Exos cannot accurately deliver loaded drugs to specific locations. Therefore, researchers have engineered and modified Exos to enhance their targeting specificity, thereby optimizing drug delivery precision^[39].In recent years, research focus has shifted towards targeted studies on Exos, and with improved experimental techniques and conditions, more studies have demonstrated the immense potential of Exos in clinical therapy. Chen et al.^[40] found that Exos enriched with has-miR-23a-3p can enhance cell proliferation, reduce apoptosis, and promote cartilage regeneration. Liang et al.^[41] injected CAP-Exos loaded with miR-140 into the knee joints of rats, inhibiting cartilage-degrading enzymes and alleviating OA progression. Liu et al.^[42] indicated that BMSCs-Exos can enhance chondrocyte proliferation and inhibit apoptosis through the lncRNA-KLF3-AS1/miR-206/GIT1 signaling pathway. These studies underscore the potential of Exos in bone tissue engineering and treating OA, providing an experimental basis for future research and clinical applications.

In our study, we prepared two engineered types of Exos, BMSCs-miR-122-Exos (BME) and BMSCs-miR-122 Inhibitors-Exos (BMIE). After establishing a rat model of KOA, Exos suspensions were administered via intra-articular injection according to experimental groups and time points. We comprehensively compared and analyzed the regulatory effects of these two engineered Exos on KOA progression in rats using behavioral, imaging, histological, and other assessments, aiming to provide a new approach for future clinical treatment of KOA.

Cells and animals

BMSCs were purchased from Zhejiang Meisen Cell Technology Co., Ltd.

48 male SD rats aged 6 weeks were purchased from Liaoning Changsheng Biotechnology Co., Ltd. They were housed in SPF conditions at the Animal Experimental Center, with standard light/dark cycles. The rats were provided with SPF feed ad libitum and had access to water freely. All animals received humane care, and the experimental protocols involving animals were approved by the Medical Ethics Committee. The experimental procedures strictly followed the Guide for the Care and Use of Laboratory Animals.

The revival and cultivation of BMSCs

Prepare a water bath by preheating and adjusting the temperature to 40°C. Retrieve the cryopreserved cells from the liquid nitrogen tank and quickly place them into the preheated water bath. Shake the cryovial vigorously to facilitate rapid thawing of the cells. Transfer the thawed cells into a centrifuge tube containing 4 ml of cell culture medium. Mix well and centrifuge at 650 rpm for 10 minutes to remove the supernatant. Add 1 ml of cell culture medium to the centrifuge tube, mix thoroughly, and transfer to a culture dish containing 10 ml of cell culture medium (specifically serum-free medium for mesenchymal stem cells). Mix again and place the dish in a cell culture incubator for cultivation.

Extraction and Identification of BE

Extraction and of BE:The supernatant was centrifuged at 10,000 × g, 4°C for 10 minutes, and then the pellet was transferred to a new centrifuge tube. Magnetic beads in the vortex were oscillated for 30 seconds, and 350 μL was transferred to the centrifuge tube. After centrifugation at 3,000 × g, 4°C for 2 minutes, the supernatant was removed. Subsequently, after adding 10 mL PBS pre-cooled to 4°C and filtered through a 0.22 μm filter, the mixture was vortexed for 30 seconds, followed by centrifugation at 3,000 × g, 4°C for 5 minutes, and removal of the supernatant. Next, 4 mL Buffer EXA, 1 mL Buffer EXB, and 14.65 mL supernatant were added to a rotating mixer and mixed at 4°C for 40 minutes. The centrifuge tube was placed on a magnetic rack and left to stand at 4°C for 10 minutes. After magnetic bead aggregation, the supernatant was discarded following centrifugation at 3,000 × g, 4°C for 1 minute. The residue was discarded, and 0.5 mL Buffer EXE was added and mixed. After centrifugation at 7,000 × g, 4°C for 2 minutes, the supernatant was transferred to an EP tube. Exos solution was filtered through EXE rinsed syringe filters A and B for purification, obtaining purified Exos solution. Purified Exos can be immediately used for testing or stored at -80°C.

Identification of BE:(1) Transmission Electron Microscopy (TEM)

A volume of 10 µL of Exos solution was applied onto a copper grid and incubated at room temperature for 10 minutes. Subsequently, the grid was washed with sterile distilled water and excess liquid was blotted dry with absorbent paper. Then, 10 µL of 2% uranyl acetate was added onto the copper grid for negative staining treatment for 1 minute, followed by blotting with filter paper and air-drying at room temperature for 2 minutes. The copper grid was observed under transmission electron microscope at 80 kV for imaging.

(2) Concentration and Size Distribution of BE (NTA method)

A laser light source was used to irradiate the suspension of nanospheres, and their concentration was determined by measuring the scattered light of nanospheres. Particle matrix ZetaView PMX 110 was used to measure the concentration of isolated Exos under 405 nm emission light. To ensure accuracy, Exos were diluted in PBS to concentrations ranging from 1x10^7 particles/ml to 1x10^9 particles/ml and their size and mass were measured. Simultaneously, particle trajectories of Exos were analyzed.

(3) Protein Immunoblotting Detection of BE Surface Markers (Western Blot, WB)

The steps for protein concentration determination were as follows, following the instructions of the BCA method protein concentration determination kit. Firstly, the A and B solutions in the kit were mixed in a 50:1 ratio to prepare the working solution. Then, the protein standard was diluted to 0.5 mg/ml using sterile ddH2O, followed by serial dilution with ddH2O. Diluted standards and samples were added to a 96-well plate, and 200 μL of prepared working solution was added to each well for incubation at 37°C for 30 minutes. Finally, absorbance was measured at 562 nm using an enzyme-linked immunosorbent assay reader to establish a standard curve and calculate the protein concentration in the samples.

The steps for gel electrophoresis and blotting were as follows: a polyacrylamide gel set was used, and SDS-PAGE gel concentrations were adjusted according to the molecular weight of the target antibody. Based on the protein concentration of the sample, the required sample volume was calculated and added to the polyacrylamide gel wells. The samples were then loaded into the electrophoresis chamber and electrophoresed under a constant voltage of 80 V until the bromophenol blue indicator entered the separating gel, followed by switching to 120 V until the indicator reached the bottom of the gel. After electrophoresis, the polyacrylamide gel was removed, and the concentrating gel was excised, leaving only the separating gel. Subsequently, the separating gel was placed on a PVDF membrane, sandwiched between filter paper and sponge to create a sandwich structure. The membrane was placed in an electrophoresis tank, filled with transfer buffer, and electrophoresed at a constant current of 200 mA for 2 hours. The PVDF membrane was then removed, washed three times with TBST for 5 minutes each time, and blocked with 5% skim milk (in TBST) at room temperature for 1 hour.

Antibody processing: For primary antibody treatment, the PVDF membrane was removed from the blocking solution, washed three times with TBST for 5 minutes each time, and then incubated in the prepared primary antibody solution overnight at 4°C on a rocking platform. For secondary antibody treatment, the membrane was washed three times with TBST for 5 minutes each time, followed by transfer to the prepared secondary antibody solution. The secondary antibody dilution solution contained 5% skim milk (prepared in TBST). Incubation was carried out at room temperature for 1.5 hours.

Visualization and imaging: After completion of secondary antibody incubation, the PVDF membrane was washed three times with TBST for 5 minutes each time. Excess wash solution was removed from the PVDF membrane. The membrane was laid flat on a developer sheet, ECL reaction solution (previously prepared) was added dropwise, and then placed into a chemiluminescence imaging system for photography.

Production and of BME

(1) Cell Transfection: Cells were collected and counted after digestion with trypsin, then resuspended in T25 flasks. Upon reaching approximately 60% confluency after 24 hours of culture, miRNA-122 dry powder was dissolved in RNase-free ddH2O to a final concentration of 20 μM. Specifically, 125 μL of RNase-free ddH2O was used to dissolve 1 OD of siRNA dry powder, ensuring complete dissolution through vortexing. Two sterile EP tubes were prepared, each containing 250 μL of OPTI-MEM medium. In one tube, 6.8 μL of Omifection-R was added and allowed to stand at room temperature for 5 minutes. In the other tube, 13.75 μL of 20 μM miRNA-122 solution was added and mixed thoroughly. Omifection-R mixture was then added dropwise to the miRNA-122 mixture, thoroughly mixed, and left to stand at room temperature for 30 minutes to prepare the transfection complex. The prepared transfection complex was added dropwise to the cell culture medium, gently mixed, and returned to the CO2 incubator. The medium was replaced with fresh culture medium within the next 6 hours. Cell supernatants were collected after 36 hours of transfection.

(2) Extraction of BME: Supernatant was centrifuged at 10,000 × g for 10 minutes at 4°C and transferred to a new centrifuge tube. Magnetic beads were prepared by vortexing bottle-contained beads for 30 seconds, taking 350 μL and adding it to the centrifuge tube, which was then centrifuged at 3,000 × g for 2 minutes at 4°C, and the supernatant was discarded. Washing of the magnetic beads involved adding 10 mL of 4°C pre-cooled, 0.22 μm filtered PBS to the centrifuge tube, vortexing for 30 seconds, centrifuging at 3,000 × g for 5 minutes at 4°C, and discarding the supernatant. Exosome adsorption was achieved by adding 4 mL of Buffer EXA, 1 mL of Buffer EXB, and 14.65 mL of supernatant into the rotation mixer, rotating at 4°C for 40 minutes. The centrifuge tube was placed on a magnetic rack, left to stand for 10 minutes at 4°C until magnetic bead aggregation, the supernatant was discarded after centrifugation at 3,000 × g for 1 minute at 4°C, and the residue was discarded. Elution of exosomes involved adding 0.5 mL of Buffer EXE, mixing thoroughly, centrifuging at 7,000 × g for 2 minutes at 4°C, and transferring the supernatant to an EP tube. After passing through EXE-rinsed syringe filters A and B respectively, the Exos solution was filtered to obtain purified Exos solution. The purified Exos were immediately used for testing or stored in a -80°C freezer.

Identification of BME

(1) Nanoparticle Tracking Analysis (NTA): BME was diluted in sterile PBS to a concentration ranging from 1x10^7 particles/mL to 1x10^9 particles/mL, and its size and quality were determined. Particle movement trajectories of exosomes were also analyzed.

(2) qRT-PCR for microRNA-122 Extraction: 1 mL of Exos solution was mixed with 3 mL of lysis buffer (miRNA-specific Lysis Buffer), and incubated at room temperature for 3-5 minutes. The mixture was then transferred into a DNA removal column, centrifuged at 12,000 g for 2 minutes at 4°C, retaining the filtrate and discarding the DNA removal column. An equal volume of isopropanol was added to the filtrate, mixed by inversion. Subsequently, 50 μL of magnetic beads were added, and the mixture was incubated at 4°C for 30 minutes. The centrifuge tube was placed on a magnetic rack to aggregate the beads, and the supernatant was discarded. 1 mL of Wash Buffer C (proteinase K solution) was added to the beads, vortexed briefly (1,000 rpm for 30 seconds) to wash the beads. The beads were then precipitated by centrifugation or magnetic separation, and the supernatant was discarded. This process was repeated with 1 mL of Wash Buffer D, followed by centrifugation or magnetic separation to pellet the beads, and discarding the supernatant. The beads were centrifuged at 3,000 × g for 2 minutes at 4°C to pellet them, and the supernatant was removed using a pipette. 30 μL of RNase-free ddH2O was added to elute RNA, vortexed at 2,000 rpm for 30 seconds, and placed on a magnetic rack to aggregate and pellet the beads. The supernatant was transferred to a new EP tube, which contained the microRNA-122 solution. The concentration and purity of microRNA-122 were measured for subsequent reverse transcription.

Based on the sample concentration (ng/μL), the required amount of reverse transcription template was calculated as 550 ng per sample volume, followed by immediate centrifugation. The specific calculation formula is as follows: V1 (RNA sample) = 550 ng / concentration of the sample (ng/μL), V2 (water volume) = 11 μL - V1. The reverse transcription reaction mixture was prepared as follows: each tube contained 11 µL of quantified total RNA, followed by the addition of a total of 9 µL of the following mixed reagents, forming a total reaction system of 20 μL per tube. After thorough mixing and immediate centrifugation, the tubes were placed in a PCR machine for cDNA synthesis. The reaction conditions were: 42°C for 60 minutes; 70°C for 5 minutes.

Primer Synthesis and Sequencing

The gene-specific primers sequence:RT- Primer (5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGA

CCAAACA-3’),miR-122-5p Forward Primer(5’-CGCGTAGCACCATCTGAAAT-3’),miR-122-5p Reverse Primer(5’-AGTGCAGGGTCCGAGGTATT-3’).

Production and Identification of BMIE

Production and Identification of BMIE using methods similar to BME.

Subjects and Groups

48 SD rats were divided into four groups as shown in Table 1.

Surgical Model of Knee Osteoarthritis (KOA) in Rats

The surgical induction of left knee osteoarthritis was performed using anterior cruciate ligament transection (ACLT) combined with destabilization of the medial meniscus (DMM). Rats were anesthetized with intraperitoneal injection of 40 mg/kg pentobarbital sodium (UK). The surgical site around the knee joint was prepared by shaving with an electric razor and disinfecting with povidone-iodine solution. Rats were positioned supine on the operating table, and the surgical area was draped and sterilized.A longitudinal incision was made along the left patellar ligament to expose the joint capsule after sequentially incising the skin and subcutaneous layers. The patellar ligament was longitudinally opened, and the joint capsule was dissected to fully flex the knee, exposing the anterior cruciate ligament for transection. Complete transection was confirmed using anterior drawer test, followed by partial resection of the medial meniscus. After hemostasis and irrigation, the joint cavity was closed and layered sutures were performed up to the skin. The surgical date was marked, and rats were randomly assigned to experimental groups.

All surgeries were conducted under aseptic conditions. Postoperatively, rats were allowed free movement, access to food, and water ad libitum. In the pre-experimental phase, one rat was randomly selected at 4 weeks post-establishment of left knee OA model for MicroCT examination and three-dimensional reconstruction to assess the OA condition.

Anesthesia and Intra-articular Injection Procedure in Rats

Rats were anesthetized with pentobarbital sodium and carefully monitored for anesthesia depth. The knee joint spaces of rats were inspected and marked. Following standard disinfection procedures, a 1 mL syringe was used to inject 100 μL of solution into the joint cavity on the medial side of the patellar ligament, according to group assignment. The NC group received saline injection, the BE group received BE suspension (10^9/mL), the BME group received BME suspension (10^9/mL), and the BMIE group received BMIE suspension (10^9/mL). Care was taken to ensure blood-free aspiration before slow injection. The syringe was stabilized during injection, and rats' knee joints were gently moved post-injection to facilitate even distribution of the solution and prevent leakage.

Macroscopic observation of articular cartilage

The articular surface of the left knee joints of rats was exposed for visual comparison among groups, and photographic documentation was conducted. The joints were graded according to the Pelletier macroscopic scoring system^[43].

Histological staining procedures

Hematoxylin-Eosin (HE) staining was conducted as follows: staining with hematoxylin (1.5 minutes) - distilled water rinse (5 minutes) - differentiation in differentiation solution (3 minutes) - tap water rinse (2 minutes) - staining with eosin (1.5 minutes) - distilled water rinse (5 minutes) - dehydration in graded ethanol (75%, 85%, 95%, each step for 3 seconds) - dehydration in absolute ethanol II (1 minute) - clearing in xylene (2 times, each for 3 minutes) - mounting with neutral resin - observation under a microscope.

Detailed steps for Orange G-Alcian blue staining are as follows: Firstly, use Weigert's iron hematoxylin stain solution (A:B=1:1) for fresh Weigert treatment for 5 minutes. Subsequently, rinse with water for 5 minutes. Next, treat with acid differentiation solution for 15 seconds followed by thorough rinsing with distilled water. Then, stain with Alcian blue solution for 5 minutes. Rinse with acidic solution for 15 seconds after staining. After completing the process, air dry the samples. Then, apply Orange G stain solution for 5 minutes. Proceed with dehydration in graded ethanol (95% and absolute ethanol, each step for 3 seconds/step), followed by immersion in absolute ethanol for 10 minutes. After dehydration, clear in xylene for 10 seconds. Finally, mount with neutral resin for microscopy and photography.

Immunohistochemistry staining

PBS rinse 3 times (5 minutes each) - circumscribe tissue with immunohistochemistry pen - block with 3% hydrogen peroxide (18 minutes) - PBS rinse 3 times again (5 minutes each) - block with goat serum (15 minutes). For primary antibody incubation, dilute the primary antibody and apply it onto the tissue ensuring complete coverage, then overnight incubation in a humidified chamber (4°C). In the secondary antibody labeling step, equilibrate at room temperature for 5 minutes, followed by PBS rinse 3 times (5 minutes each), then apply the secondary antibody (room temperature, 15 minutes), and finally PBS rinse 3 times again (5 minutes each). For the chromogenic reaction, use DAB solution under dark conditions, apply onto the tissue ensuring complete coverage, then incubate in a humidified chamber at room temperature for 5 minutes, monitoring color development, and stop the reaction with distilled water at the appropriate time. Counterstaining with hematoxylin involves applying hematoxylin staining (30 seconds) followed by three rinses with distilled water (5 minutes each). The final steps of dehydration, clearing, mounting, and observation include dehydration and clearing with 75%, 85%, 95% ethanol, followed by xylene I and xylene II (each step for 3 minutes), mounting with neutral resin, and finally observing under a microscope.

Statistical analysis

Statistical analysis was performed using SPSS 23.0 software. Student's t-test was employed for comparisons between two groups, while one-way analysis of variance (ANOVA) was used for comparisons among multiple groups. A p-value less than 0.05 was considered statistically significant, p < 0.01 indicated significant differences, and p < 0.001 indicated highly significant differences. A p-value greater than 0.05 indicated no statistically significant difference.

Extraction and identification of BE

As shown in Figure 1.Figure A shows: Transmission electron microscopy (TEM) images of Exos reveal a near-spherical shape with typical cup-shaped characteristics. Figure B demonstrates: Western blot analysis of Exos surface markers indicating expression of HSP70, TSG101, and CD9. Figure C illustrates: The average diameter of Exos is 129.0 nm, with a main peak diameter of 93.2 nm, comprising 86.4% of particles by number, and a final concentration of 6.8 × 10^9 particles/mL. Figure D displays: BMSCs exhibit uniform spindle-shaped or stellate morphology. These results collectively confirm the identity of BE.

Identification of BME

After preparation of BME using magnetic bead method, the resulting samples were diluted 1.2-fold with PBS and subjected to nanoparticle tracking analysis (NTA). The detection results showed that the average size of BME was 139.7 nm, with a narrow size distribution. Compared to BE, BME exhibited a significantly increased average particle size (Figure 2).The qPCR results indicated that miR-122 expression was significantly upregulated in the BME group compared to the BE group (p < 0.01)(Figure 3).

Identification of BMIE

The NTA results showed that the average diameter of BMIE was 135.4 nm, with a main peak diameter of 91.1 nm, accounting for 61.3% of particles by number, and a final concentration of 1.6 × 10¹⁰ particles/mL(Figure 4).The qRT-PCR results in Figure X show that the secretion of Exos transfected with miR-122 inhibitors exhibited reduced miR-122 content, confirming successful preparation of BMIE(Figure 5).

Analysis of the effects of BME and BMIE knee injections on OA progression

Forty-eight rats in four groups all recovered well postoperatively with good wound healing and no deaths or occurrences of other diseases. According to the Lequesne MG^[44] grading system(table 2), we compared pain, gait, knee joint range of motion, and swelling at three time points.At the pre-experimental stage, four weeks after establishing the rat knee osteoarthritis (OA) model, one randomly selected SD rat underwent MicroCT examination and the results were reconstructed in 3D. As shown in Figure 6, significant cartilage erosion, irregular surface loss, decreased smoothness, and osteophyte formation were observed on the joint surface, indicating successful establishment of the rat knee OA model.

During the experimental phase, according to the results shown in Figure 7

(1) 4 weeks: In the NC group, there was cartilage surface damage and subchondral bone fracture. The BME group exhibited more severe damage compared to the NC group, while the BMIE group had relatively smoother joint surfaces compared to the other two groups.

(2) 8 weeks: The NC group showed joint surface damage with irregular areas of uneven surface. The BME group had defects on the joint surface and subchondral bone fractures, worse than the NC group. The BE group had mild joint surface damage, while the BMIE group showed relatively smooth joint surfaces.

(3) 12 weeks: The NC group exhibited severe joint surface damage, subchondral bone fractures, and decreased bone density around the joint. The BME group showed more severe joint surface damage compared to the NC group, with fractures in the subchondral bone and decreased bone density. The BE group had increased density and sclerosis on the joint surface with preserved joint space, while the BMIE group showed relatively smooth joint surfaces and visible sclerotic bands forming in the joint space.

(4)The Pelletier macroscopic scoring results showed significant differences among groups: at week 4, the BME group scored higher than the NC group, the NC group scored higher than the BE group, and there was no significant difference between the BE and BMIE groups; at week 8, the BME group scored higher than the NC group, the NC group scored higher than the BE group, and the BE group scored higher than the BMIE group; at week 12, the BME group scored higher than the NC group, the NC group scored higher than the BE group, and the BE group scored higher than the BMIE group. These results were statistically significant (p < 0.05)(Figure 8).

Macroscopic observations of rat knee joints(Figure 9)

(1) 4 weeks: In the NC group, roughening of the articular cartilage surface was evident. The BME group showed synovial hyperplasia on the articular cartilage surface, while no significant OA changes were observed in the BE and BMIE groups.

(2) 8 weeks: Thinning of the articular cartilage layer was observed in the NC group. The BME group exhibited rough and irregular articular cartilage surfaces with obvious pits and granular damage. The BE group showed small depressions on the articular cartilage surface but relatively smooth overall, while the BMIE group had relatively smooth articular cartilage surfaces without significant defects.

(3) 12 weeks: The NC group displayed significant wear and loss of glossiness on the articular cartilage surface. The BME group exhibited severe destruction of the articular cartilage surface, with fibrosis and decreased elasticity of the cartilage tissue. The BE group showed thinning of the articular cartilage surface but relatively smooth appearance, whereas the BMIE group had glossy articular cartilage surfaces with slightly thinner cartilage layers and no apparent OA changes.

Histological staining analysis

HE staining(Figure 10): At 4 weeks, the NC group, BME group, and BE group showed mild damage to the joint surface and thinning of articular cartilage. The cartilage layer in the BE group was thicker than that in the NC and BME groups, while the BMIE group exhibited the thickest cartilage layer and a relatively smooth joint surface. At 8 weeks, the NC group and BME group displayed irregular joint surfaces with significantly reduced and disordered arrangement of chondrocytes. The BMIE and BE groups showed relatively smooth joint surfaces with more organized arrangement of chondrocytes, and the BMIE group had more chondrocytes compared to the BE group. By 12 weeks, the NC group exhibited a more pronounced decrease in chondrocytes, the BME group showed almost no visible chondrocytes with severe joint surface damage, and the BE group had a relatively smooth joint surface with a thicker cartilage layer compared to the NC and BME groups. The BMIE group had the smoothest joint surface among the four groups and the thickest cartilage layer.
Safranin O-fast green staining(Figure 11): At 4 weeks, the NC group exhibited uneven cartilage thickness with areas of calcified cartilage formation on the surface. The BME group showed reduced cartilage staining intensity with a relatively smooth joint surface, while the BMIE and BE groups had thicker and more uniformly stained cartilage layers, with BMIE showing better organization than BE. By 8 weeks, the NC group showed significantly thinner cartilage layers, the BME group exhibited reduced cartilage staining and uneven joint surface, and the BMIE group had thicker cartilage layers compared to BE with more even distribution of chondrocytes. By 12 weeks, the BME group had thinner cartilage layers compared to BE, while both BMIE and BE groups had thicker cartilage layers compared to the other two groups. The BE group showed cracks on the cartilage surface, whereas the BMIE group still maintained a relatively smooth joint surface.
The modified Mankin's scoring(Figure 12)results showed significant differences among groups: at 4 weeks, there was no significant difference between the NC and BME groups, with the NC group scoring higher than the BE group, and the BE group scoring higher than the BMIE group; at 8 weeks, the BME group scored higher than the NC group, the NC group scored higher than the BE group, and the BE group scored higher than the BMIE group; at 12 weeks, the BME group scored higher than the NC group, the NC group scored higher than the BE group, and the BE group scored higher than the BMIE group (p < 0.05).

Immunohistochemical staining analysis

Expression of type II collagen(Figure 13): At 4 weeks, the NC group showed relatively low positive expression of type II collagen in chondrocyte cytoplasm, with light staining. The BME group exhibited poorer positive expression compared to the NC group, while the BE group was better than the NC group, and the BMIE group showed the best expression. By 8 weeks, the NC group displayed severe destruction of the articular cartilage surface, with few chondrocytes showing positive expression of type II collagen. The BME group exhibited uneven areas on the joint surface with cracks in the cartilage and severe damage, and the expression of type II collagen in chondrocytes was worse than that in the NC group. In contrast, the BMIE and BE groups had relatively smooth joint surfaces with better expression of type II collagen in chondrocytes compared to the other two groups. By 12 weeks, the NC group showed severe destruction of the joint surface, reduced numbers of chondrocytes, and decreased expression of type II collagen. The BME group exhibited cartilage ulceration and pit formation, with decreased expression of type II collagen. The BMIE and BE groups had relatively smoother joint surfaces, with better expression of type II collagen in the BMIE group compared to the BE group.
Proteoglycan expression(Figure 14): At 4 weeks, both the NC and BME groups showed minimal proteoglycan expression, with only a few cells exhibiting weak yellowish positive staining. The BMIE and BE groups exhibited significantly better proteoglycan positive expression compared to the former two groups, with relatively smoother joint surfaces. By 8 weeks, the NC and BME groups showed joint surface damage with uneven areas and no apparent positive expression. The BMIE and BE groups showed more proteoglycan positive expression and relatively smoother joint surfaces. By 12 weeks, the NC and BME groups exhibited limited positive expression, with severe joint surface damage. The BE group showed mild joint surface damage with clusters of chondrocytes and more positive expression of proteoglycans. The BME group showed relatively intact joint surfaces with abundant chondrocytes exhibiting deep yellowish positive staining, indicating the most pronounced expression intensity compared to the other three groups.

Osteoarthritis (OA) is a severe joint disease characterized by degenerative changes in cartilage and surrounding tissues^[45]. It is also one of the most common chronic diseases and has become a significant societal burden from both medical and economic perspectives^[46,47]. The pathogenesis of OA involves cartilage degeneration, compounded by the limited proliferative capacity of chondrocytes within avascular and aneural cartilage tissue surrounded by abundant extracellular matrix^[48]. Unfortunately, despite extensive research efforts over the past decade aimed at finding new treatment methods, significant progress has not been achieved^[21,49,50]. Current treatments still primarily focus on symptomatic relief. Therefore, there is an urgent need to develop more innovative and effective therapeutic strategies for OA to reduce healthcare costs, alleviate societal burdens, improve patient quality of life, and advance scientific and medical knowledge.

Knee osteoarthritis (KOA) is one of the leading causes of disability and chronic pain worldwide^[51]. Therefore, in this study, we chose to establish a KOA model. After determining the required number of experimental animals, we compared the costs, breeding periods, infection resistance, and cartilage conditions of commonly used experimental animals. Ultimately, we selected SD rats to establish the KOA model. There are various methods to establish KOA^[52]. After reviewing the literature and analyzing the compatibility of various methods with the objectives of this study, we focused on investigating the regulatory effects of intra-articular injections of BME and BMIE on OA progression. We paid particular attention to the development process of KOA, comparing the timeframes required for various modeling methods. Considering the study design, we found that the anterior cruciate ligament transection combined with partial medial meniscectomy method aligned well with our research objectives. Therefore, we chose to establish the KOA model in rats using the anterior cruciate ligament transection combined with partial medial meniscectomy method^[53,54]. In the pilot phase, after modeling for 4 weeks, we randomly selected one SD rat for Micro-CT examination and three-dimensional reconstruction. According to the reconstruction results, we observed significant cartilage degradation, decreased joint surface smoothness, and osteophyte formation in the knee joints of rats. Macroscopic observations revealed slight swelling in the left knee joint area of the rats, with pain responses upon palpation and restricted joint movement. During the experimental phase, after establishing the model, rats in the NC group exhibited symptoms of knee joint pain and restricted movement, which worsened over time. Through Micro-CT scans, histological staining, and macroscopic observations, we observed significant cartilage damage in the NC group rats at 4 weeks post-modeling, severe joint cartilage destruction at 8 weeks, and exacerbated cartilage damage accompanied by osteophyte formation at 12 weeks post-modeling. These results indicate the successful establishment of the KOA model in rats.

In recent years, significant progress has been made in the use of BMSCs for treating osteoarthritis (OA), showing potential to delay OA progression and promote joint tissue repair and regeneration^[55-57]. However, the application of BMSCs raises ethical concerns, issues of transplant rejection, and potential risks of tumor formation^[58]. Consequently, researchers have shifted their focus to exosomes derived from BMSCs, which also exhibit anti-inflammatory and immunomodulatory properties. Exosomes can alleviate inflammation, improve OA pathology, and are rich in growth factors, cytokines, regulate ECM, and possess low immunogenicity. Building upon BMSCs, exosomes provide a new direction for OA therapy. However, researchers have noted that natural exosomes exhibit low targeting precision. With advancements in research techniques and experimental conditions, engineered exosomes are increasingly recognized for their enormous clinical potential in OA treatment.Inspired by Bai et al.’s^[30] in vitro cell experiments, which revealed SIRT1 as a direct target of miR-122, regulating ECM degradation in OA through the miR-122/SIRT1 axis, we conducted the current study. Using magnetic bead technology, we prepared BME, BMIE, and BE, designed animal experiments, established a rat KOA model, and investigated the effects of intra-articular injection of BME and BMIE on OA progression in the KOA rat model.

In general behavioral observations, rats in the BME group exhibited poorer performance compared to the NC group in indicators such as pain, gait, joint mobility, and swelling. Conversely, the BMIE group outperformed both the NC and BE groups across all metrics. Specifically, after 4 weeks of observation, the BMIE group showed the least pain, whereas by the 8th week, the BME group's pain and joint mobility significantly declined compared to other groups. By the 12th week, the gait of the BMIE group gradually normalized and surpassed that of the BE group, while the BME group showed significantly poorer gait and joint mobility compared to the NC group. Radiographic examination results indicated that joint arthritis progressed most rapidly in the BME group, followed by the NC group, with the BE group showing mild arthritis, and no significant arthritis observed in the BMIE group. According to gross scoring results, joint arthritis progression was slowest in the BMIE group, followed by the BE group, whereas the BME group showed the fastest OA progression.Histological staining results revealed that at 4 weeks, the NC group had uneven cartilage thickness with surface hardening, while the BME group exhibited reduced cartilage staining and relatively smooth joint surfaces. The BMIE and BE groups showed significantly thicker cartilage layers compared to the other two groups, with uniform staining and regular cell arrangement, with the BMIE group superior to the BE group. By the 8th week, the NC group had significantly thinner cartilage layers, while the BME group exhibited uneven surfaces despite reduced staining. The BMIE group showed significantly thicker cartilage layers compared to the BE group, with more uniform distribution of chondrocytes. By the 12th week, the BME group had thinner cartilage layers compared to the BE group, whereas both the BMIE and BE groups showed significantly thicker cartilage layers than the other two groups. At this point, fissures appeared on the cartilage surface in the BE group, while the joint cartilage surface remained relatively smooth in the BMIE group.

Overall, compared to the NC group, the joint cartilage condition was significantly better in the BMIE and BE groups, while the BME group exhibited the least favorable joint surface condition. Immunohistochemical analysis of type II collagen and proteoglycan indicated that ECM conditions were superior in the BMIE and BE groups compared to the NC group, while the BME group showed relatively poorer ECM conditions. These results are consistent with our initial hypothesis.

BME negatively impacts the stability of the extracellular matrix, accelerating OA progression, which in turn leads to joint cartilage damage and functional decline. BMIE, to a certain extent, slows down this process, maintaining the stability of the extracellular matrix and protecting joint cartilage. This offers new perspectives and options for OA treatment.

Osteoarthritis	OA
Knee osteoarthritis	KOA
non-steroidal anti-inflammatory drugs	NSAIDs
disease-modifying osteoarthritis drugs	DMOADs
microRNA	miRNA
Bone marrow mesenchymal stem cells	BMSCs
Extracellular vesicles	Exos
BMSCs-Exos	BME
BMSCs-miR-122 Inhibitors-Exos	BMIE
Nanoparticle Tracking Analysis	NTA
anterior cruciate ligament transection	ACLT
destabilization of the medial meniscus	DMM

Ethics approval and consent to participate: The study procedure conformed to the ethical guidelines of the Declaration of Helsinki, and the approval for the study was obtained from the Zhongshan Hospital Affiliated with Dalian University.

Consent for publication: Not applicable.

Competing interests: On behalf of all authors, the corresponding author states that there is no conflict of interest.

Funding: This work was supported by Dalian Medical Science Research Project (NO. 2111038), State Key R&D Program (2020JH2/10300093), Liaoning Provincial Department of Education Key Research Project(LJKZZ20220148),Liaoning Provincial Health Commission, "Xingliao Plan of Excellence" Medical Masters Program(YXMJ-QN-21)

Acknowledgements: Not applicable.

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Regulatory Role of Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Loaded with miRNA-122 in the Progression of Osteoarthritis

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