Derivation of iMSCs
The derivation of iMSCs was described in our previous studies [12, 13]. Briefly, one iPSC cell line, iPSCs-(C1P33), which was provided by the South China Institute for Stem Cell Biology and Regenerative Medicine Group of the Chinese Academy of Sciences in agreement with Professor Pei [23], was used to generate MSCs. After 5 days in culture, the medium was replaced by Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mM l-glutamine, 1% penicillin/streptomycin (P/S), and 0.1 mM nonessential amino acids (all supplements from Gibco, Grand Island, NY, USA). Cells were passaged upon reaching approximately 80% confluence. After cells developed a homogeneous fibroblastic morphology, they were frozen at –80 °C for downstream experiments.
Derivation of SMMSCs
The Ethics Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital approved the use of SMMSCs (Approval Number: YS-2016-063). Written informed consent was obtained from all donors. The SMMSC preparation method was described previously [24, 25]. In brief, synovium was harvested from three donors (two males/one female, age range 22–28 years) during anterior cruciate ligament (ACL) reconstruction surgery for acute ACL injuries. The harvested synovial membrane specimens were kept in high-glucose DMEM at 4 °C. Within 1 h, the specimen was rinsed with phosphate-buffered saline (PBS), finely minced, and digested with 0.2% collagenase I (Sigma–Aldrich, Saint Louis, MO, USA) in high-glucose DMEM containing 10% FBS and 1% P/S. After overnight incubation at 37 °C, the released cells were centrifuged, washed, resuspended in expansion medium (high-glucose DMEM supplemented with 10% FBS and 1% P/S), and plated in a T25 culture flask. The medium was changed after 4 days, and nonadherent cells were removed by thorough washing with PBS.
Characterization of iMSCs and SMMSCs
Surface antigens of iMSCs and SMMSCs were analyzed by flow cytometry. Cells were harvested and incubated for 30 min with 3% bovine serum albumin (Gibco) in PBS to block nonspecific antigen binding. The iMSCs were then incubated with monoclonal antibodies against CD29, CD34, CD44, CD45, CD73 CD90, or HLA-DR; SMMSCs were incubated with monoclonal antibodies against CD34, CD44, CD45, CD73, CD90, CD166, or HLA-DR (all antibodies from BD Biosciences, Sparks Glencoe, MD, USA). The cells were then washed to remove unbound antibody. Surface antigens were analyzed using the Guava easyCyte™ flow cytometer (Millipore, Billerica, MA, USA).
Isolation and identification of iMSC-Exos and SMMSC-Exos
iMSC-Exos and SMMSC-Exos were isolated and purified following our established protocol [13, 26]. After reaching 80% confluency, MSCs were washed with PBS and the culture medium was replaced with MesenGro hMSC medium (StemRD, San Francisco, CA, USA). The cells were then cultured for an additional 48 h at 37 °C in 5% CO2. The conditioned medium was collected and centrifuged at 300 × g for 10 min and then at 1500 × g for 10 min at 4 °C. After centrifugation, the supernatant was filtered using a 0.22-μm filter (Steritop™; Millipore) to remove the remaining cells and cellular debris. The supernatant was then transferred to an Ultra-clear tube (Millipore) and centrifuged at 4000 × g until the volume in the upper compartment was reduced to approximately 200 μl. The ultrafiltration liquid was resuspended in PBS and re-ultrafiltrated at 4000 × g to 200 μl. This step was then repeated once. Exosomes were stored in aliquots at –80 °C or used for other downstream experiments.
The concentration and size distribution of iMSC-Exos and SMMSC-Exos were measured using tunable resistive pulse sensing (TRPS) analysis by qNano (Izon Science, Cambridge, MA, USA). Aliquots of iMSC-Exos, SMMSC-Exos, or calibration particles (CPC100 particles; Izon Science) were placed in the Nanopore (NP150, A37355; Izon Science) at 47.0-mm stretch with a voltage of 0.6 V. Izon Control Suite software v2.2 (Izon Science) was used for data analysis. Exosome morphologies were observed using an FEI Tecnai G2 spirit transmission electron microscope (TEM; FEI, Eindhoven, the Netherlands). Antibodies against CD9 (1:1000; Abcam, Cambridge, UK), CD63 (1:1000; Abcam), and TSG101 (1:1000; Santa Cruz, Dallas, TX, USA) proteins were used to analyze the incorporation of each protein into exosomes in western blots.
Collagenase-induced OA model
All procedures were approved by the Animal Research Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital (Approval Number: SYXK2011-0128). Six-week-old female C57B/L10 mice were randomized into four groups: normal (n = 5), iMSC-Exos treatment (n = 10), SMMSC-Exos treatment (n = 10), and OA (n = 10). On day 0, collagenase was used to induce OA in all mice in the iMSC-Exos, SMMSC-Exos, and OA treatment groups. The collagenase-induced model of OA was described previously [27, 28]. Mice were anesthetized by intraperitoneal injection of 10 ml/kg 4% chloral hydrate. The knee joints of the mice were injected once intra-articularly through the patellar ligament with 12 U of collagenase VII (Clostridium histolyticum; Sigma–Aldrich) in 8 μl saline. In the normal group, 8 μl of saline without collagenase was injected into the knee joints in the same way. On days 7, 14, and 21, mice in the iMSC-Exos and SMMSC-Exos treatment groups were injected intra-articularly with 8 μl iMSC-Exos (1.0 × 1010/ml) or 8 μl SMMSC-Exos in PBS (1.0 × 1010/ml), respectively. Mice in the OA and normal groups were injected intra-articularly with 8 μl PBS at each time point. On day 28, mice were euthanatized for further analysis.
Macroscopic examination
After euthanasia, the surface of the proximal tibia was exposed. The surrounding soft tissue including joint capsule and meniscus was removed. The cartilage surface was then fully exposed and examined macroscopically. The evaluation was performed by two blinded investigators, and the score was based on the International Cartilage Research Society (ICRS) for cartilage repair [29].
Histology
Mice tibias were fixed in 10% paraformaldehyde for 24 h and were then decalcified in 10% EDTA for 7 days at 37 °C. After serial dehydration, the tibial bones were embedded in paraffin and sectioned coronally through the tibial plateau at 5 μm thickness, and then stained with hematoxylin and eosin (H&E) and safranin O/fast green. Each specimen was scored for the medial tibial plateau by two blinded observers using the Osteoarthritis Research Society International (OARSI) cartilage OA histopathology grading system to histologically grade the severity of cartilage destruction [30].
Immunohistochemistry analysis
Immunohistochemical (IHC) staining for type I and II collagens was performed. All sections were deparaffinized, washed with PBS, treated for antigen retrieval, and blocked with mouse IgG for 30 min. Sections were incubated with primary antibodies against mouse anti-collagen I (1:200; Abcam) and mouse anti-collagen II (1:200; Abcam) overnight at 4 °C. Biotinylated secondary antibody and streptavidin peroxidase solution were then used to visualize the sections.
Chondrocyte migration assay
Human cartilage was harvested after obtaining informed consent from donors. Chondrocyte preparation was described previously [25]. The scratch wound assay was used to analyze the effect of iMSC-Exos and SMMSC-Exos on migration of chondrocytes, as described previously [13]. Briefly, 1.5 × 104 cells were seeded into 12-well plates and maintained at 37 °C for 8 h. Next, the confluent monolayer of cells was scratched using the tip of a P200 pipet tip. The medium was removed and the cells were washed once with PBS. The medium was then replaced with fresh DMEM F-12 medium containing 108/ml iMSC-Exos, 108/ml SMMSC-Exos, or control medium. Wound closure was monitored by collecting digital images at 0, 24, and 48 h after the scratch using an inverted microscope (Leica, Wetzlar, Germany). The images were obtained at the same position before and after incubation. Scratched areas were measured using Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA).
Chondrocyte proliferation assay
The effect of iMSC-Exos and SMMSC-Exos on the proliferation of human chondrocytes was evaluated using the Cell Counting Kit-8 (CCK-8; Dojindo, Kyushu Island, Japan) as described previously [12, 13]. Chondrocytes were seeded into 96-well plates at 2 × 103 cells/well. After 8 h, different doses of iMSC-Exos or SMMSC-Exos were added to the wells. The medium was changed daily for 5 days, using fresh DMEM F-12 medium containing 10% FBS and the same exosome concentrations. Cell proliferation curves were constructed by measuring the amount of formazan dye generated by cellular dehydrogenase activity with a microplate reader at a wavelength of 450 nm.
Statistical analysis
The data were presented as means ± standard deviation. Comparisons of macroscopic and histological scores as well as scratch wound assay results were made using the Mann–Whitney U test. Comparisons of chondrocyte proliferation assays were performed using unpaired Student’s t test. P < 0.05 was considered statistically significant.