Derivation and characterization of induced MSCs
The local ethics committee approved using human iPSC in this study of the Shanghai Sixth People's Hospital affiliated with Shanghai Jiao Tong University. The generation of mesenchymal stem cells from human induced pluripotent stem cells as previously described (27). Flow cytometry was used to detect phenotypical markers of iMSCs. The cells were incubated with 1% (w/v) bovine serum albumin (BSA) (Gibco) to block the non-specific antigens. Then, 1 × 106 cells were stained with the following conjugated mouse monoclonal antibodies: CD24-PE (1:100, 560991, BD Biosciences), CD29-PE (1:100, 561795, BD Biosciences), CD44-FITC (1:100, 560977, BD Biosciences), CD146-PE (1:100, 561013, BD Biosciences), CD133-PE (1:100, 130080081, MACS), CD105-FITC (1:100, 560943, BD Biosciences), CD73-PE (1: 100, 561014, BD Biosciences), CD90-PE (1:100, 328109, Biolegend), CD34-APC (1: 100, 560940, BD Biosciences), CD45-FITC (1:100, 560976, BD Biosciences), and HLA-DR-PE (1:100, 560943, BD Biosciences). After being washed in 1% (w/v) BSA twice, the cells were resuspended in 1% BSA and analyzed by CytoFLEX flow cytometer (Beckman Coulter Life Science, USA).
Isolation of iMSC-sEV
The iMSC-sEV were isolated from the cell culture medium of iMSCs by differential ultracentrifugation protocols. Briefly, the obtained medium was centrifuged at 300g for 10 min and 2000g for 10 min. After centrifugation at 10,000g for 1 h, the supernatant was filtered through a 0.22-µm filter sterilize Steritop™ (Millipore) to remove cellular debris and microvesicles. The collected medium was further ultracentrifuged at 100,000g for 70 min twice. After removal of the supernatant, the pellet was resuspended in PBS.
Size distribution and particle concentration of iMSC-sEVs
The size and concentration of the iMSC-sEVs were assessed using nano-flow cytometer (N30 Nanoflow Analyzer, NanoFCM Inc., Xiamen, China) as previously described(28). Briefly, isolated iMSC-sEVs diluted with 100-fold PBS (for a nanoparticle concentration of approximately 108/mL) were loaded to the nano-flow to measure the side scatter intensity (SSI). The concentration of iMSC-sEVs was calculated according to the ratio of SSI to particle concentration in the standard polystyrene nanoparticles. The size distribution of iMSC-sEVs sample was calculated according to the standard cure generated by standard silica nanoparticles.
Western blot analysis
To identify sEV using western blot analysis, three positive markers of iMSC-sEV, including CD9, TSG101, and CD63, and one negative marker GM130, were evaluated. Cells or iMSC-sEV proteins were harvested using RIPA lysis buffer (Beyotime biotechnology, China, P0013C) supplemented with protease inhibitor cocktail (Beyotime biotechnology, China, ST505). Lysates were cleared by centrifugation at 12,000 g for 20 min. The supernatant fractions were used for western blot analysis. Protein extracts were resolved by 10% SDS-PAGE and probed with the indicated antibodies. The antibodies against the following proteins were used for western blot analysis: rabbit monoclonal anti-CD9 (1:1000, Cell Signaling Technology, USA, 13174 s), mouse monoclonal anti-TSG-101 (1: 1000, Abcam, UK, ab83), Rabbit monoclonal anti-CD63 (1:1000, Abcam, UK, ab134045), and mouse polyclonal anti-GM130 (1:500, Abcam, UK, ab169276). Anti-rabbit IgG or anti-mouse IgG, HRP-linked antibody (1: 2,000; Cell Signaling Technology) was used as the secondary antibody. Protein level was detected using the ECL detection system.
Animal model and experimental design
Animal care and experimental procedures were approved by the Animal Research Committee of the Shanghai Jiao Tong University Affiliated Sixth People's Hospital (approval code: DWLL2021-0910). Previous studies have established a rat tendinopathy model by carrageenan(29). Hence, we injected 100ul 4% (w/v) carrageenan into the peritendon space of the quadriceps tendon under ultrasound guidance to induce a rat tendinopathy model while sham rats received a PBS injection. One week later, injection of iMSC-sEVs was performed in the sEVs group and PBS in the control group once a week for 4 weeks. Pain-related behaviors were analyzed one week after the administration of iMSC-sEVs or PBS. Reversal (%) of pain-related behaviors was calculated as follows:
$$Reversal \left(\%\right)=100 \times \frac{(\text{p}\text{o}\text{s}\text{t}\text{t}\text{r}\text{e}\text{a}\text{t} \text{v}\text{a}\text{l}\text{u}\text{e}, \text{t}\text{e}\text{n}\text{d}\text{i}\text{n}\text{o}\text{p}\text{a}\text{t}\text{h}\text{y} \text{r}\text{a}\text{t}\text{s}) - (\text{p}\text{r}\text{e}\text{t}\text{r}\text{e}\text{a}\text{t} \text{v}\text{a}\text{l}\text{u}\text{e}, \text{t}\text{e}\text{n}\text{d}\text{i}\text{n}\text{o}\text{p}\text{a}\text{t}\text{h}\text{y} \text{r}\text{a}\text{t}\text{s})}{(\text{a}\text{v}\text{g}. \text{p}\text{r}\text{e}\text{t}\text{r}\text{e}\text{a}\text{t} \text{v}\text{a}\text{l}\text{u}\text{e} \text{s}\text{h}\text{a}\text{m} \text{r}\text{a}\text{t}\text{s}) - \left(\text{p}\text{r}\text{e}\text{t}\text{r}\text{e}\text{a}\text{t} \text{v}\text{a}\text{l}\text{u}\text{e} \text{i}\text{n} \text{t}\text{e}\text{n}\text{d}\text{i}\text{n}\text{o}\text{p}\text{a}\text{t}\text{h}\text{y} \text{r}\text{a}\text{t}\text{s}\right)}$$
where "value" represents the values for static weight-bearing or hind paw withdrawal threshold.
Pain assessment
Hind-Paw Withdrawal Threshold
Hind-paw withdrawal thresholds were measured as described previously(30). The electronic von Frey instrument (model BIO-EVF4; Bioseb, Vitrolles France) was used to vertically stimulate the center of the rat hind paw with increasing intensity. The probe tip was gently placed perpendicularly into the mid-plantar surface of the paw, and steadily increasing pressure was applied until the hind paw was first lifted. Until the withdrawal reaction was positive, and there were 3 positive withdrawal reactions within the 5 consecutive stimuli, the value was defined as PWT and was expressed in grams (g).
Static Weight Bearing
The static weight-bearing (SWB) distribution over the right and left knee was assessed by measuring the postural balance between the injected and non-injected leg (30). Briefly, a rat was placed in the chamber of a weight-bearing measuring device (model #BIO-SWB-TOUCH-M; Bioseb). The force applied through each hind limb to the paw resting on the floor of the chamber was measured in grams (g), and an SWB index was calculated as follows:
$$SWB index=\frac{\text{F}\text{o}\text{r}\text{c}\text{e} \text{a}\text{p}\text{p}\text{l}\text{i}\text{e}\text{d} \text{t}\text{o} \text{r}\text{i}\text{g}\text{h}\text{t} \text{l}\text{i}\text{m}\text{b}}{\text{F}\text{o}\text{r}\text{c}\text{e} \text{a}\text{p}\text{p}\text{l}\text{i}\text{e}\text{d} \text{t}\text{o} \text{r}\text{i}\text{g}\text{h}\text{t} \text{l}\text{i}\text{m}\text{b} +\text{F}\text{o}\text{r}\text{c}\text{e} \text{a}\text{p}\text{p}\text{l}\text{i}\text{e}\text{d} \text{t}\text{o} \text{l}\text{e}\text{f}\text{t} \text{l}\text{i}\text{m}\text{b}}$$
For each rat, the test was given at least three times at each assessment period.
Gait analysis
Dynamic pain-related behavior was measured by the gait of the rats(31). The Catwalk system objectively quantifies behavioral gait adaptation after daily use of a painful limb, automatically documenting paw placements on a surface and related parameters of inter-limb coordination(32). Briefly, rats were placed on a walkway apparatus (Shanghai Mobiledatum Information Technology, Shanghai, China). A camera below the walkway captures and digitally records footprint images. These paw print placements and gait parameters were collected and further analyzed by WalkAnalysator (Shanghai Mobiledatum Information Technology). The CatWalk gait test was administered at weeks 4 after treatment. Print area, swing speed, duty cycle and max contact mean intensity were recorded and analyzed as Right/Left.
Histology and Immunohistochemistry
For histological analysis, the rat tendon samples were fixed en bloc in 4% PFA for 24 hours, dehydrated with a graded ethanol series, embedded in paraffin, and sectioned (5 µm thick) parallel to the long axis of the tendon. The sections were prepared for hematoxylin and eosin (H&E) and immunohistochemical analysis.
To assess the proinflammatory cytokine distribution in rat tendon samples, we performed immunohistochemistry (IHC) staining on paraffin-embedded sections. The following antibodies were used: anti-interleukin-1β (IL-1β) (1:500, Abcam, UK, ab283818), anti-tumor necrosis factor-α (TNF-α) (1:500, Abcam, UK, ab217706), anti-interleukin-6 (IL-6) (1:500, Abcam, UK, ab ab9324), and anti-nerve growth factor (NGF) (1:500, Abcam, UK, ab52987). HRP-conjugated antibodies were used with DAB as the chromogen for visualization. In some cases, a hematoxylin counterstaining was done for nuclear counterstaining. Histological and immunohistochemical staining was evaluated and photo-documented digitally with the microscope (Leica, DM6B, Germany). Interpretation of the slides was performed by semi-quantitative grading scale of Movin score for tendon abnormalities(33).
Immunofluorescence staining
The rats were sacrificed, and cardiac perfusion was performed with ice-cold saline, followed by 4% (w/v)paraformaldehyde perfusion. Lumbar dorsal root ganglion (DRG) at levels L3–L5 and tendon tissues were dissected from the surrounding tissue, fixed in 4% formaldehyde overnight at 4°C, and dehydrated with gradient sucrose solutions (20%, 30%, and 35% (w/v)). After being embedded and frozen in an optimal cutting temperature compound (OCT), the tissues were sliced into 10-µm-thick coronal sections. The sections were then stained with specific markers, including calcitonin gene-related peptide (CGRP, 1:400, Cell Signaling Technology, 14959), iNOS (1:400, Cell Signaling Technology, 13120), Tryptase (1:100, Abcam, UK, ab2378) and PGP9.5(1:100, Abcam, UK, ab108986). Fluorescence images were acquired using a fluorescence microscope (Leica, DM6B, Germany). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was performed to quantify Tryptase+ cell number, staining area, and relative expression of CGRP and iNOS.
Uptake of iMSC-sEVs by mast cells in vitro
To determine the uptake of iMSC-sEVs into RBL-2H3 cells in vitro, we labeled iMSC-sEVs with Dil fluorochrome (Thermo Fisher, USA) under room temperature for 15 min, followed by ultracentrifugation at 100,000g in PBS to get rid of the unlabeled dye. Next, Dil-labeled sEV were incubated with RBL-2H3 cells for 12 hours. And then, the culture medium was discarded, and the cells were rinsed twice with PBS before image capture under the fluorescence microscope (Leica, DM6B, Germany).
SP-induced degranulation in RBL-2H3 cells
RBL-2H3 cells were kindly provided by Stem Cell Bank, Chinese Academy of Sciences. The cells were grown in Eagles Modified Essential Medium (EMEM) supplemented with glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 µg/ml), and 10% fetal bovine serum (FBS, Gibco), in a humidified 5% CO2 atmosphere at 37°C, plated on 6-well culture dishes at a cell density of 9 × 105 cells per well, at 37°C in 5% CO2 atmosphere. After 24 hours, RBL- 2H3 cells were stimulated with SP (10 µM) or vehicle (PBS) and incubated for 15 min at 37°C in 5% CO2 atmosphere.
β-Hexosaminidase release assay
The RBL-2H3 cells were conducted β-Hexosaminidase release assay as previously described with a small modification to determine the degranulation activity(34). Briefly, after being stimulated by SP, the RBL-2H3 cells were treated with iMSC-sEVs (109/ml) or vehicle for different time (6h/ 9h/ 12h/ 24h) at 37°C in 5% CO2 atmosphere. The supernatants (15 µl) were incubated with 60 µl of the substrate (1 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide in citrate 0.05 M, pH 4.5) for 1 h at 37°C. Furthermore, the cells were lysed with 0.1% Triton X-100 and incubated with the substrate to determine the degranulation activity in the supernatants to determine the total amount of released β-hexosaminidase. The reaction was stopped by 150 µl of 0.1 M sodium bicarbonate buffer (pH 10.0), and the reaction product was monitored by measuring the optical density (OD) at 405 nm by using a reader GENios Pro (Tecan). The results were calculated by using the following formula: % degranulation = [OD-supernatant/(OD-supernatant + OD-triton x−100)] × 100.
Real-time quantitative polymerase chain reaction (RT-qPCR) analysis
The expression of targeted genes was analyzed by RT-qPCR. Briefly, The total RNA of samples was extracted using EZ-press RNA Purification Kit (EZBioscience, USA). RNA quantity and purity were confirmed with a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE). A 4× Reverse Transcription Master Mix (EZBioscience, USA) was used for reverse transcription reaction. PCR reactions were run using the ABI Prism 7900HT Real-Time System (Applied Biosystems, Carlsbad, CA) with 2× SYBR Green qPCR Master Mix (EZBioscience, USA). The primer sequences used in this study are listed in Additional file 1: Table S1.
Additional file 1: Table S1. The primer sequences were used in this study.
Enzyme-linked Immunosorbent Assays (ELISA)
The supernatant collected at 18h after different treatment was evaluated for proinflammatory molecules and NGF by ELISA. IL-1β, TNF-α, IL-6, IL-10, and NGF concentrations were measured by using a rat ELISA kit (Shanghai Westang Bio-Tech Co., LTD., Shanghai, China) according to the manufacturer's instructions. The absorbance was measured by a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm.
Statistical analysis
Data were presented as mean ± SD. Student t-test was used to assess the difference between two groups, and the one-way ANOVA with the Bonferroni post hoc test was applied for comparisons among multiple groups. All experiments were independently performed at least three times. Statistical analysis was performed using GraphPad Prism software (version 8.0). The significant difference was considered to be P-value < 0. 05.