Collection of Synovial Fluids
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.
The subjects were 7 patients who had complex degenerative tears of the medial meniscus and underwent meniscal repair and synovial MSC transplantation. Their ages ranged from 29 to 60 years old. The meniscus symptoms, which included feeling instable in addition to pain, developed gradually in all patients, and the duration of the meniscus symptoms ranged from 4 months to 4 years. OA patients who were diagnosed as Kellgren–Lawrence grades 2-4 were not included. According to arthroscopic observation, all patients had complex degenerative meniscal tears, including a horizontal tear and flap tear at the avascular area of the middle and posterior segment of the medial meniscus.
Synovial fluid (Pre) was aspirated from knees with degenerative meniscus injury before harvest of approximately 0.5 g synovium (consisting of approximately 20 pieces by pituitary rongeur) and meniscus repair (Fig. 1). After 2 weeks, synovial fluid (Post) was aspirated again before transplantation of synovial MSCs.
Cultures of Synovial Fluid MSCs
Synovial fluid was filtered through a 70-μm nylon filter (Greiner Bio-One, Kremsmünster, Austria) to remove debris, and a half volume of the synovial fluid was plated in 6 culture dishes of 60 cm2 (Thermo Fisher Scientific, Waltham, MA, USA) in complete culture medium: α-modified essential medium (α-MEM; Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS) (Thermo Fisher Scientific), 100-U/mL penicillin, 100-μg/mL streptomycin, and 250-ng/mL amphotericin B (Thermo Fisher Scientific). The dishes were incubated at 37°C with 5% humidified CO2. After 24 hours, the nonadherent cells were washed out with phosphate-buffered saline (PBS). Fourteen days after initial plating, 3 dishes were stained with 0.5% crystal violet (Wako, Osaka, Japan) in 4% paraformaldehyde for 10 minutes for colony formation analysis. Colonies less than 2 mm in diameter and faintly stained colonies were ignored. The other 3 dishes were harvested with 0.25% trypsin and 1-mM ethylenediaminetetraacetic acid (EDTA) (Invitrogen, CA, USA) (Passage 0), and the number of isolated cells were counted. Then, the cells were replated at 500 cells/cm2 in a 145-cm2 culture dish (Thermo Fisher Scientific) and cultured for 14 days for surface epitopes and differentiation analyses.
Surface Epitopes
For synovial fluid MSCs from 7 donors, surface markers were examined using a FACSVerse instrument (Becton, Dickinson and Company; BD, NJ, USA). The cells were suspended in FACS buffer (2%FBS, 5mM EDTA in PBS) using Hank’s balanced salt solution (HBSS) at a density of 5 × 105 cells/mL and stained for 30 minutes with the antibodies CD44 (APC-A), CD90 (PerCP-Cy5.5-A), CD73 (V450-A), CD105 (PE-A), CD31 (PE-Cy7-A), and CD45 (FITC-A) (all from BD) and Ghost Dye Violet 510 for dead cells (Tonbo Biosciences, CA, USA). BD FACSuite software (BD) was used for the analysis.
Differentiation Assay
Differentiation assay was conducted as previously described.(22) For chondrogenesis, 2.5 × 105 cells were transferred to a 15-mL tube (BD Falcon) and cultured in chondrogenic induction medium containing 10-ng/mL transforming growth factor-β3 (Miltenyi Biotec Japan, Tokyo, Japan) and 500-ng/mL bone morphogenetic protein 2 (BMP-2, Infuse; Medtronic, Minneapolis, MN); this medium was changed every 3-4 days. After 21 days, the cell pellets were embedded, sectioned, and stained with safranin‑o and fast green (Wako, Tokyo, Japan).
Calcification was studied by plating 100 cells in a 60-cm2 dish and culturing for 14 days in α-MEM with 10% FBS. Adherent cells were subsequently cultured in an osteogenic induction medium containing 50-μg/mL ascorbic acid 2‑phosphate (Wako), 10-nM dexamethasone (Wako), and 10-mM β‑glycerophosphate (Sigma-Aldrich, St Louis, MO, USA); this medium was changed every 3-4 days. After 14 days, calcification was assessed by alizarin red (Sigma-Aldrich) staining.
Adipogenesis was evaluated by plating 100 cells in a 60-cm2 dish and culturing for 14 days to allow colony formation. Adherent cells were cultured in an adipogenic induction medium supplemented with 100-nM dexamethasone, 0.5-mM isobutylmethylxanthine (Sigma-Aldrich), and 50-mM indomethacin (Sigma-Aldrich) for an additional 14 days; this medium was changed every 3-4 days. Adipocyte colonies were stained with oil red-o (Sigma-Aldrich).
Antibody Array Analyses
After filtration, the half volume of synovial fluid (Post) was cryopreserved at ‑80°C and used to assess the expression levels of 507 proteins using RayBiotech’s L‑Series human antibody array L-507 membrane kit (RayBiotech, Peachtree Corners, GA, USA) according to the manufacturer’s instructions. Briefly, all synovial fluid samples were diluted 100 times, followed by quantification and biotinylation. Overnight incubation of the membrane array with biological samples at 4°C resulted in the binding of proteins to corresponding antibodies. Signals were visualized using horseradish peroxidase (HRP)-conjugated streptavidin and were imaged by Chemi Doc XRS (Bio-Rad Laboratories, Hercules, CA, USA). The final spot intensities were measured as the original intensities, subtracting the background. The data were normalized to Angiopoietin-like 1 for the positive controls, as this did not change in amount among patients. The correlation coefficients between the signal intensity and the colony number per dish were measured.
Transwell Migration of Synovial Fluid MSCs
After being cultured for 5-7 days with α-MEM containing 10% FBS, the medium was changed to α-MEM containing 0.5% FBS and incubated for 24 hours for serum starvation. A total of 5 x 104 synovial fluid MSCs were seeded to the upper chambers with 8-μm Transwell microporous membranes (Cell Culture Insert; Falcon, Franklin Lakes, New Jersey, USA) in 24-well plates (Falcon, Franklin Lakes, New Jersey, USA) (Fig. 2). The cells in the upper chambers were allowed to migrate across the membrane at 37°C and 5% CO2. Calcitonin gene-related peptide (CGRP) (Sigma-Aldrich, St Louis, MO, USA) at a concentration of 10-8 M, 10-10 M, and 10-12 M or hepatocyte growth factor (HGF) (Peprotech, Rocky Hill, NJ, USA) at a concentration of 10 ng/mL, 1.0 ng/mL, and 0.1 ng/mL was added in the lower chamber containing α-MEM with 0.5% FBS. After 6 hours, the upper chambers were stained with 0.5% crystal violet in 4% paraformaldehyde for 10 minutes. MSCs that did not migrate across the pores were wiped out with a cotton swab, and the number of MSCs on the lower side of the membrane was counted by light microscopy (BZ-X700, Keyence Co., Ltd, Osaka, Japan).
Statistical Analysis
All data were statistically evaluated using GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA). All data were expressed in dot plots. Each statistical analysis method is described in the figure legends. P values less than 0.05 were considered statistically significant.