Isolation of human synovial MSCs

This study was approved by the Medical Research Ethics Committee of Tokyo Medical and Dental University, and informed consent was obtained from all study subjects. Human synovium was harvested from the knees of four donors during total knee arthroplasty operations performed to treat osteoarthritis. The synovium was minced and digested in a solution of 3 mg/mL collagenase (Sigma-Aldrich Japan, Tokyo, Japan) at 37 °C for 3 h, and the digested cells were filtered through a 70 μm cell strainer (Greiner Bio-One GmbH, Frick-enhausen, Germany). The obtained nucleated cells were cultured in a growth medium consisting of α-MEM (Thermo Fisher Scientific, Rockford, IL, USA), 1% antibiotic-antimycotic (Thermo Fisher Scientific), and 10% fetal bovine serum (FBS, Thermo Fisher Scientific) under 5% CO2 at 37 °C. After 14 days, the human synovial MSCs were detached with 0.25% trypsin and 1 mM EDTA, harvested, and cryopreserved at − 80 °C for future use at a concentration of 106 cells/mL in a freezing vessel (BICELL, Japan Freezer, Tokyo, Japan). The cryopreservation medium consisted of 95% growth medium plus 5% dimethyl sulfoxide (DMSO, Fujifilm Wako Pure Chemical, Osaka, Japan). For colony formation assays, 100 cells, at 1.67 cells/cm2 in a 60 cm2 dish (Nunc EasYDish, Thermo Fisher Scientific) were cultured for 14 days and then stained with crystal violet (Fujifilm Wako Pure Chemical).

Differentiation assays

Chondrogenesis was examined by suspending 2.5 × 105 human synovial MSCs in 0.5 mL chondrogenic induction medium consisting of DMEM (Thermo Fisher Scientific) supplemented with 10 ng/mL transforming growth factor-β3 (Miltenyi Biotec, Bergisch Gladbach, Germany), 500 ng/mL bone morphogenetic protein 2 (BMP-2, Medtronic, Minneapolis, MN, USA), 40 μg/mL proline, 100 nM dexamethasone (Fujifilm Wako Pure Chemical), 100 μg/mL pyruvate, 50 μg/mL ascorbate-2-phosphate (Fujifilm Wako Pure Chemical), and 1% ITS Premix (Becton Dickinson [BD], NJ, USA). The cells were pelleted by centrifugation at 500×g for 10 min and then cultured for 21 days. The pellets were sectioned and stained with safranin O (Fujifilm Wako Pure Chemical).

For adipogenesis, 100 synovial MSCs were cultured in a 60 cm2 dish for 14 days in growth medium to produce cell colonies. The adherent cells were cultured for a further 21 days in an adipogenic induction medium consisting of α-MEM supplemented with 100 nM dexamethasone, 0.5 mM isobutylmethylxanthine (Sigma-Aldrich), and 50 mM indomethacin (Fujifilm Wako Pure Chemical). The generated adipocytes were stained with oil red O (Muto Pure Chemicals, Tokyo, Japan).

For calcification, 100 synovial MSCs were cultured in a 60 cm2 dish for 14 days in growth medium to produce cell colonies. The adherent cells were cultured for a further 21 days in calcification medium consisting of growth medium supplemented with 50 μg/mL ascorbate-2-phosphate, 10 nM dexamethasone, and 10 mM β-glycerophosphate (Sigma-Aldrich). Calcification was assessed by alizarin red staining (Merck Millipore, Billerica, MA, USA).

Flow cytometry

Human synovial MSCs were detached with TrypLE (Thermo Fisher Scientific) and suspended in FACS buffer (0.2% FBS and 5 mM EDTA [Thermo Fisher Scientific] in phosphate-buffered saline [PBS]) at a density of 5 × 105 cells/mL. Thawed MSCs were used immediately after thawing, with no further culture. The cells were stained for 30 min with the following antibodies: CD44 (PE-Cy7), CD45 (APC-H7), CD73 (V450), CD90 (PE), and CD105 (APC) (all from BD). Cell fluorescence was evaluated using a FACS Verse instrument (BD). The data were analyzed using FlowJo software (Tree Star Software, CA, USA).

Preparation of cultured and thawed human synovial MSCs

For cultured MSCs, the cryopreserved cells were thawed (ThawSTAR, Astero Bio, Menlo Park CA, USA), cultured for 14 days, and suspended in PBS for use in transplantation. For thawed MSCs, the cells were similarly thawed and cultured for 14 days, but they were subsequently cryopreserved at − 80 °C in 95% FBS and 5% DMSO at a density of 107 cells/mL [9] for 3 days. The cells were then thawed and suspended in PBS for use in transplantation (Fig. 1). The effect of FBS concentration on microspikes was evaluated by cryopreserving the cells were in the following solutions: (i) α-MEM supplemented with 10% FBS and 5% DMSO, (ii) α-MEM supplemented with 50% FBS and 5% DMSO, (iii) 95% FBS and 5% DMSO.

Fig. 1

Schematic of the study design. For cultured MSCs, the thawed cells were cultured for 14 days, and then suspended in PBS. For thawed MSCs, the thawed cells were cultured for 14 days, cryopreserved in 95% FBS and 5% DMSO for 3 days, and then suspended in PBS without further culturing. The cultured and thawed MSCs were observed by SEM and TEM. The MSCs were placed on porcine menisci, and the proportion of adhering cells and the cell SEM morphology were analyzed immediately and 10 min after MSC placement

Adhesion of human synovial MSCs to procine menisci

The surface of the menisci excised from fresh porcine knees (Shibaura Zoki Co., Ltd., Tokyo, Japan) was abraded to reproduce a degenerative meniscus. Each meniscus was cut into a cylindrical shape 12 mm in diameter. A cell suspension containing 106 cultured or thawed MSCs in 100 μL PBS (107 cells/mL) was placed on the meniscus. Either immediately after placement or 10 min later, the meniscus was washed with 1 mL PBS. The proportion of MSCs adhering to the meniscus was then calculated by counting the numbers of non-adherent cells in the washes [8].

Scanning electron microscopy

Cells and menisci were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB) for 2 h and washed overnight in 0.1 M PB at 4 °C. The specimens were then post-fixed with 1% osmium tetroxide (OsO4) in 0.1 M PB for 2 h at 4 °C and dehydrated in graded ethanol solutions. After exchanging with 3-methyl butyl acetate and critical point drying, the specimens were coated with platinum, and the meniscus surface was observed by SEM (S-4500; Hitachi Ltd., Tokyo, Japan). For quantification of microspike-positive cells, 50 cells were randomly selected per examination. The selected cells were classified by the presence or absence of microspikes. Microspike-positive cells were defined as cells that contained at least three microspikes [8].

Transmission electron microscopy (TEM)

Cells were fixed in 2.5% glutaraldehyde in 0.1 M PB for 2 h, washed with 0.1 M PB, and post-fixed in 1% OsO4 in 0.1 M PB for 2 h. After washing with PB, cells were resuspended in 2% gelatin (Sigma-Aldrich) and pelleted again. Microcentrifuge tubes were plunged into ice-cold water to quickly solidify the gelatin with the cells. The tip of the tube was cut open, and the cell pellets were cut into 1 mm3 blocks, dehydrated in a graded series of ethanol, and embedded in Epon 812. Ultrathin sections 70 nm thick were collected on copper grids and double-stained with uranyl acetate and lead citrate. The sections were examined by TEM (JEM-1400Flash, JEOL, Tokyo, Japan).

Statistical analysis

The proportions of adhered cultured versus thawed MSCs were compared using Student’s paired t-test. The correlation between the proportion of cells with microspikes in the cell suspension and the proportion of adhered cells was statistically evaluated with Pearson’s product-moment correlation. The effect of FBS concentration on microspikes was evaluated using Tukey’s test. Data were expressed as the average ± standard deviation (SD). P values < 0.05 were considered statistically significant. All statistical analyses were performed using GraphPad Prism 6 (GraphPad Software, CA, USA).