Culture of human and rat synovial MSCs
Appropriate approvals were obtained for this study from the Medical Research Ethics Committee and the Animal Committee of our facility for the human and rat synovium harvesting, respectively. Human synovium was harvested from the knees of six osteoarthritis (OA) patients who underwent total knee arthroplasty (age range 59–84 years; two males and four females). Rat synovium was harvested from the knees of 11-week-old Lewis rats. The harvested synovium was minced with a scalpel and digested in 3 mg/mL collagenase (Sigma, St. Louis, MO, USA) for 3 h at 37℃. The resulting nucleated cells were filtered through a 70 µm cell strainer (Greiner Bio-One, Kremsmunster, Austria), plated in 145 cm2 dishes at a density of 2000 cells/cm2, and expanded in growth medium consisting of α-minimum essential medium (α-MEM; Invitrogen, Carlsbad, CA, USA), 10% fetal bovine serum (FBS; Invitrogen), and 1% antibiotic-antimycotic (Invitrogen) at 37℃ in a 5% CO2 atmosphere. Confluent cells were detached with 0.25% trypsin and 1 mM EDTA (Invitrogen) and cryopreserved in growth medium supplemented with 5% dimethyl sulfoxide (Wako, Tokyo, Japan) for future use.
In vitro synovial MSC migration model
Synovial MSCs at passage 1 were serum-starved in α-MEM supplemented with 0.5% FBS for 24 h. The cells were suspended in a solution containing 2 mg/mL type 1 collagen (Corning, NY, USA) and 2 mg/mL growth factor–reduced Matrigel (Corning) at a density of 2×106 cells/mL, and then 60 µL was placed onto transwell inserts with 8.0 µm pores (Corning) in 24-well plates (Corning). The hydrogels were solidified for 30 min at 37℃ and then 140 µL 0.5% FBS was added to the upper chamber. An 800 µL volume of either 0.5% or 10% FBS was added to the lower chamber as a negative and positive control, respectively. The migration assays were performed in triplicate. After 12, 24, and 48 h incubation at 37℃ in 5% CO2, the inserts were fixed with 10% formalin in a neutral buffer solution (Wako) and stained with 0.5% crystal violet. The gels on the upper side of the inserts were then removed with a cotton swab. The lower side of the inserts viewed by light microscopy (BZ-X700, Keyence Co., Ltd., Osaka, Japan) and photographed, and the numbers of migrating cells were counted in five randomly selected fields (×200).
Pretreatment of inflammatory cytokines to synovial MSCs
Synovial MSCs were cultured in 0.5% FBS containing 10 ng/mL recombinant human interleukin-1β (IL-1β; R&D Systems, Minneapolis, MN, USA) and tumor necrosis factor-α (TNF-α; R&D Systems) for 24 h. The cells were then detached and subjected to migration assay for 48 h.
Exploration of cytokines for synovial MSC mobilization
We used recombinant human bone morphogenetic protein-2 (BMP-2; Medtronic, Minneapolis, MN, USA), hepatocyte growth factor (HGF; Peprotech, Rocky Hill, NJ, USA), insulin-like growth factor-1 (IGF-1; Peprotech), platelet-derived growth factor-AA (PDGF-AA; R&D Systems), PDGF-AB (R&D Systems), PDGF-BB (R&D Systems), PDGF-CC (R&D Systems), PDGF-DD (R&D Systems), stromal cell-derived factor-1α (SDF-1α; R&D Systems), transforming growth factor-β3 (TGF-β3; Miltenyi Biotec, Bergisch Gladbach, Germany). The medium containing 0.5% FBS in the lower chamber was supplemented with 100 ng/mL BMP-2, 100 ng/mL HGF, 100 ng/mL IGF-1, 10 ng/mL PDGF-BB, 100 ng/mL SDF-1α, or 10 ng/mL TGF-β3. After 48 h incubation, migrated cells were counted. The PDGF isoforms were compared by adding PDGF-AA, AB, BB, CC, or DD to the lower chamber at 100 ng/mL. Crenolanib (Selleck Chemicals, Houston, TX, USA), an inhibitor of PDGF receptor (PDGFR) α and β, was added to both the upper and lower chambers during the migration assay.
Transmission electron microscopy (TEM)
The hydrogels were fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer (PB) for 2 h, post-fixed in PB buffered 1% OsO4 for 2 h, dehydrated in a graded series of ethanol, and then embedded in Epon 812. Ultrathin sections (70 nm) were collected on copper grids and then double-stained with uranyl acetate and lead citrate . The sections were observed by transmission electron microscopy (JEM-1400Flash, JEOL, Japan).
PDGF-BB injection into rat knee joints
All animal care and experiments were conducted in accordance with the institutional guidelines of the Animal Committee of our facility. Fourteen male Lewis rats at 14 weeks old were used for this study. Mean weight was 350 ± 10 g (328 to 362 g). All animals were housed at 2 animals per cage, allowed food and water ad libitum under a 12 h light/dark cycle, and acclimatized for 1 week before the start of the study. The animals were placed under general anesthesia with isoflurane and 100 ng recombinant rat PDGF-BB (ProSpec-Tany TechnoGene, Rehovot, Israel) in 50 µL PBS was injected with a 30G needle into left or right knee joint, which was randomly selected. As a control, 50 µL PBS was injected into the other knee joint. Rats were euthanized 48 h after the intra-articular injection.
Culture of colony-forming cells in rat synovial fluid
Synovial fluid was collected from rat knee joints by pumping with 50 µL PBS three times. The fluid was centrifuged and the sediments were plated in 145-cm2 dish and cultured in growth medium at 37℃ in 5% CO2. After 1 week of cultivation, the cells from 8 rats were fixed and stained with 0.5% crystal violet for colony counting. Colonies less than 1 mm in diameter were ignored. Colony-forming cells from the other 6 rats were pooled and further passaged at the density of 500 cells/cm2 to assess their MSC properties.
Matrigel-collagen hydrogels containing human synovial MSCs were fixed with 10% formalin in neutral buffer for 24 h. Rat knee joints were fixed for 2 days and decalcified with 20% EDTA (Wako) for 2–3 weeks. The hydrogels and knee joints were embedded in paraffin and specimens were cut into 5 μm sections and stained with hematoxylin and eosin (HE). The severity of synovitis around anterior horn of medial meniscus was quantified using the score proposed by Krenn et al . Immunohistochemistry of type I collagen was performed by incubating sections of the hydrogels with 0.3% hydrogen peroxide/methanol for 30 min and then blocking with 10% normal goat serum (Abcam, Cambridge, UK). Rabbit anti-type I collagen antibody (Rockland Immunochemicals, Limerick, PA, USA) was applied at 4℃ overnight. The slides were then washed and incubated with horseradish peroxidase (HRP)-linked second antibody (Abcam) for 1 h and then with diaminobenzidine (DAB) substrate (Dako, Glostrup, Denmark). All slides were counterstained with hematoxylin.
Colony-forming cells derived from rat synovial fluid at passage 1 were plated in 6-well plates at a density of 500 cells/cm2. After 2, 4, and 6 days of cultivation, the number of cells in each well were counted. The effect of PDGF-BB on the proliferation ability of human and rat synovial MSCs was examined by culturing 5,000 cells in 96-well plates with or without PDGF-BB. After 24 and 48 h, Cell Counting Kit-8 solution (Dojindo, Tokyo, Japan) was added and the cells were incubated for a further 2 h. The optical density values were then measured at 450 nm with a micro-plate reader (Tecan, Männedorf, Switzerland). The cell proliferation rate was calculated based on a standard curve.
Colony formation assay
One hundred colony-forming cells derived from rat synovial fluid at passage 1 were plated in a 145-cm2 dish and cultured for 14 days. The cells were fixed with 10% formalin in neutral buffer solution and stained with 0.5% crystal violet for colony counting. Colonies less than 2 mm in diameter were ignored.
Human synovial MSCs at passage 1 and colony-forming cells in rat synovial fluid at passage 1 were detached with TrypLE (Thermo Fisher Scientific, MA, USA) for 10 min and analyzed for surface markers. Human synovial MSCs were tested with PDGFRα-BV421 (BD Biosciences, San Jose, CA, USA) and PDGFRβ-BB700 (BD Biosciences) antibodies. Colony-forming cells in rat synovial fluid were tested with CD44-PE (eBioscience, San Diego, CA, USA), CD90-PE-Cy7 (eBioscience), CD105-APC (Novus Biologicals, Littleton, CO, USA), CD34-PerCP-Cy5.5 (Novus Biologicals), and CD45-FITC (BD Pharmigen, San Jose, CA, USA) antibodies. Cells were incubated with conjugated antibodies at 4℃ for 1 h in the dark. Isotype controls were prepared as a negative control. The cell fluorescence and percentage of antigen-positive cells were evaluated by flow cytometry with a FACSVerse instrument (BD Biosciences).
The chondrogenic ability was assayed by suspending 2.5×104 cells in a chondrogenic induction medium consisting of high glucose Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific), 1% ITS+ Premix (BD Biosciences), 50 µg/mL ascorbate-2-phosphate, 40 µg/mL L-proline (Sigma), 100 nM dexamethasone, 100 µg/mL pyruvate (Sigma), 1% antibiotic-antimycotic, 10 ng/mL TGF-β3, and 500 ng/mL BMP-2. The cells were placed in a 15 mL polypropylene tube and centrifuged at 450 g for 10 min to form cell pellets. After 3 weeks of cultivation in a chondrogenic induction medium, the pellets were weighed and embedded in paraffin. Sections (5 µm thick) were stained with Safranin O to evaluate the production of glycosaminoglycan.
For adipogenic induction, colony-forming cells in rat synovial fluid at passage 1 were plated in 12-well plates and cultured in growth medium until confluent. The medium was then switched to an adipogenic induction medium consisting of growth medium supplemented with 0.5 mM isobutyl-methylxanthine (Sigma), 100 μM indomethacin (Sigma), 100 nM dexamethasone (Wako), 4.5 mg/mL D-(+)-glucose (Sigma), and 10 μg/mL insulin (Wako). The medium was changed twice a week. After 2 weeks, the cells were stained with Oil Red O (Sigma). The amount of intracellular lipid was determined by extracting the Oil Red O dye with 300 μL isopropanol (Wako) and determining the optical density at 510 nm with an Infinite F200 PRO spectrophotometer (Tecan Systems Inc., San Jose, CA, USA).
For osteogenic induction, 500 cells were plated in a 50 cm2 dish and cultured in growth medium for 7 days. The medium was then shifted to osteogenic induction medium consisting of growth medium supplemented with 50 µg/mL ascorbate-2-phosphate (Sigma), 20 mM β-glycerophosphate (Sigma), and 1 nM dexamethasone. After 3 weeks, the cells were stained with alizarin red (Sigma). Calcification was quantified by extracting the alizarin red dye with 10% cetylpyridinium chloride in 10 mM Na2PO4 (pH 7.0) and measuring the optical density at 560 nm.
All data were expressed as mean ± standard deviation. All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria) . The statistical analysis methods are described in each of the figure legends. Statistical significance was accepted at p < 0.05.