Reagents and antibodies
The mBMSC culture and differentiation medium was purchased from Cyagen (China). The chondrocyte activator IL-1β was purchased from Sigma-Aldrich (USA). The primary antibodies used in this study included mouse anti-CD9, CD63, CD81, and SOX9 (Santa Cruz Biotechnology, USA); rabbit anti-COL II (Santa Cruze Biotechnology); rabbit anti-PDGFBB, VEGF, SDF-1, COL X, anti-cleaved caspase-3, Bcl-2, Bax, and GAPDH (Abcam, USA); rabbit anti-TGFβ1, Smad2/3, p-Smad2/3, EKR1/2, p-EKR1/2, p38, and p-p38 (Cell Signaling Technology, USA); rabbit anti- COL X and Ki67 (Novus, USA); rabbit anti-Aggrecan, and MMP13 (Proteintech; China).The secondary antibodies used in this study included Alexa-488 conjugated-goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch, USA); horseradishperoxidase–conjugated-goat anti-rabbit IgG (H+L) and horseradish peroxidase–conjugated-goat anti-Mouse IgG (H+L) (Invitrogen, USA); Nuclei was stained with DAPI dihydrochloride (Thermo Fisher Scientific, USA). Flow cytometry anlysis was performed to identify the characterization of mBMSCs stained with FITC-conjugated or PE-conjugated anti-mouse CD44, CD45, CD90, and CD105 (BD, USA).
All mice in this study were acquired from Gempharmatech (China). 6-8w old male C57/B6 mice were used for STOA model. 3w-old mice were used for BMSCs isolation, and newborn pups at postnatal 1d were used for chondrocytes isolation. All experimental procedures were conducted in conformity with institutional guidelines for the care and use of laboratory animals and protocols, which were approved by the Animal Care and Use Committee of Affiliated Drum Tower Hospital, Medical School of Nanjing University (No. YKK1760).
Cell culture of mBMSCs
BMSCs were obtained from bone marrow in 3w-old C57/B6 male mice, and seeded inT25 cell culture flasks with 6ml BMSC culture medium at 37℃. The cells (P0) were passaged when they fused at 80%. These reseeded cells were considered to be the first generation (P1), and so on as the second (P2), third (P3), etc. The BMSC culture medium was refreshed every 3 days, and P3 cells were used in followup experiments.
Cell culture of chondrocytes
To extract chondrocytes, 1d-old pups were sacrificed for collection of cartilage from knees. First, cartilage was into small pieces after washing with PBS. Second, the samples were digested in 0.25% trypsin-EDTA (Gibco, USA) solution for 5min, and DMEM-F12 (Gibco) containing 10% collagenase type II (Sigma-Aldrich) for 6h at 37°C, successively. The released chondrocytes were seeded in T25 cell culture flasks. Cells were passaged at a ratio of 1:3 at 80% confluence. The culture medium was refreshed every 3 days.
Identification of BMSCs
The morphology of P0 and P3 mBMSCs was observed via microscopy (NikonTS2, Japan). Flow cytometry cytoflex (Beckman, USA) was used to detect the surface markers of mBMSCs (CD44, CD45, CD90, and CD105). The data was analyzed by Flowjo 7.6 (TreeStar, USA). Adipogenic, Osteogenic, and chondrogenic differention of mBMSCs were performed according to standard culture methods, and measured via Oil red O staining, Alizarin Red staining, and Alcian blue (AB) staining, respectively. These images were obtained through bright-field microscope (Nikon TS2, Japan).
Chondrogenic differentiation of mBMSCs
According to the instructions, induction medium (Cyagen, China; containing: TGFβ3 1mg per 100ml medium) of chondrogenic differentiation was replaced every 3 days. TGFβ1 neutralizing antibody (1μg/ml, BD) was administrated in chondrogenic differentiation process. AB staining and COL II-immunofluorescence were conducted after 2w of culture to evaluation of chondrogenic differentiation ability. These results were observed through microscope (Nikon TS2) and Cell imager (Bio-tek Cytation1, USA), respectively.
Based on the methods of Tao et al.  and Guo et al., whole blood samples were collected from healthy volunteers and placed with acid-citrate Dextrose solution A (ACD-A) in anticoagulant tubes (1ml ACD-A/9ml blood). After centrifugation at 160g for 10min, platelet-containing plasma was carefully absorbed and transferred to a new centrifuge tube (Beckman coulter, USA) and centrifuged again at 250g for 15min. The supernatant plasma was discarded, before the platelet pellet was resuspended in the residual plasma to obtain 4ml PRP.
Isolation of PRP-Exo
According to the methods of Torreggiani et al., PRP samples were centrifuged at 250g for 15min to obtain PRP microspheres and the platelet pellet was washed with PBS (Ca2+-free and Mg2+-free, Gibco). After activating 4ml PRP suspension with 1ml of 10% CaCl2 and 1, 000U thrombin (Hunan Yige Pharmaceutical, China), the suspension was centrifuged in series at low speeds (300g for 10min, 2, 000g for 10min) to discard cell debris. Then, the supernatant was filtered through a 0.22μm filter (Millipore, Germany), and the filtrate was transferred to a 15ml ultrafiltration centrifuge tube (Millipore) under 4, 000g centrifugation for 50min. The liquid was washed with PBS and ultrafiltered at 4, 000g again. To purify the Exo, the medium was added onto a 30% sucrose/D2O cushion in an Ultra-ClearTM tube (Beckman Coulter, USA) and ultra-centrifuged at 100, 000g for 70min. After washing by PBS, Exo suspension was ultracentrifuged again at the same high speed for 70min. The Exo were then carefully resuspended in sterile PBS and stored at -80°C for subsequent experiments.
Identification of PRP-Exo
Nanosight tracking analysis (NTA; Nanosight, UK) was used to measure the concentration and size distribution of Exo. Transmission electron microscopy (TEM; Tecnai 12, Philips, The Netherlands) was used to identify the morphology of Exo after incorporated in Gel. The biosignature proteins of PRP-Exo, consisting of CD81, CD9, CD63, Tgfβ1, PDGFBB, VEGF, and SDF-1 were determined by western blotting assays.
Gel and Exo-Gel preparation
The production methods of Gel have been described in our previous report, the total polymer concentration of blank Gel was set to 22.9% (w/w), of which the concentration of Poloxamer 407 (PEO101-PPO66-PEO101, Sigma-Aldrich) and 188 (PEO80-PPO27-PEO80, Aladdin, China) was fixed at 17.9% (w/w) and 5% (w/w) respectively. Poloxamer 407 and 188 were added into ddH2O according to the ratio to stir and mix evenly at 4ºC. Then, the Gel in liquid state was fully mixed with extracted Exo, and this Exo incorporated Gel (Exo-Gel) system was freeze-dried by lyophilizer (Christ Alpha1-2, Germany). After that, it was further detected by SEM (Philips). Liquid Gel was transferred to transwell inserts with 8µm pore size (Millipore) and solidifed by raising temperature to 37.0℃ in the cell incubator. The conditioned medium from Exo-Gel (200μg Exo incorporated in 100μl thermossensitive Gel) incubation for 1w was obtained by placing the transwell inserts in wells containing 1ml of medium, and used to coculture with mBMSCs and chondrocytes for multiple functional tests, such as proliferation, migration, differentiation, and apoptosis.
Thermoresponsive release profile of Exo-Gel
For release studies, the same amount of Exo-Gel was placed into transwell inserts as described above and incubated in medium at 25°C and 37°C. The supernatant was removed and fresh medium was added at different time-points. The number of Exo released into the medium was determined using the Bradford Protein Assay Kit (Beyotime, China) and the size distribution of Exo at 2d postincubation was detected by NTA (Nanosight)
Uptake of Exo by mBMSCs and chondrocytes
For the fluorescent labeling of Exo, DiR-solution (Eugene, USA) was added to PBS and incubated as manufacturer instructed, then centrifuged 100, 000g at 4°C for 1h to remove excessed dye. These DiR-labeled Exo incorporated Gel (200μg/100μl) placed in a transwell insert and and cocultured with mBMSCs and chondrocytes in the bottom well for 48h. Cells were washed with PBS thrice, fixed in 4% paraformaldehyde (PFA), and permeabilized with 0.05% Triton X-100 for 5min, followed by incubation with FITC Phalloidine (Yeasen, China). Nuclei was stained with DAPI dihydrochloride. Fluorescent images were photographed by Cell imager (Bio-tek Cytation1).
Cell Counting Kit-8 analysis
Viability of mBMSCs and chondrocytes was evaluated with a Cell Counting Kit-8 (CCK-8) assay (Dojindo, Japan). After 0, 12h, 1d, and 2d, the wells were washed with PBS thrice, and CCK8 solution (10μl; 1:10 dilution) in fresh culture medium was added to wells and incubated for 2h at 37°C. The absorbance was measured at 450nm with a microplate reader (ELx800, Bio-tek). The cell growth curve is drawn according to the measured OD value.
5-Ethynyl-2'-deoxyuridine (EDU) analysis
Cell proliferation was evaluated using an EDU kit (RiboBio, China) in accordance with the manufacturer’s protocol. Briefly, mBMSCs and chondrocytes were seeded into 24-well plates 20, 000 per well and incubated with EDU medium for 2h. After washing with PBS, the cells were fixed in 4% PFA and permeabilized with 0.05% Triton X-100 for 5min. At last, we added staining solution to each well followed by incubating at room temperature for 30min. Nuclei was stained by Hoechst 33342 and the images were taken under Cell imager (Bio-tek Cytation1).
Transwell assay was used to analyze the migration ability of BMSCs and chondrocytes under different treatments. Briefly, 20, 000 cells were seeded into the upper chamber of a 24-well transwell plate (Millipore; pore size: 8µm) after which 600µL/well medium treated differently were added to the lower chamber. Following incubation for 24h, cells that migrated to the lower surface of the filter membrane were stained with 0.5% Crystal Violet for 30min and observed by microscopy (Nikon TS2). Migratory activity was assessed by counting the number of cells.
A scratch wound assay was also performed to evaluate cell migration capability. In brief, cells were cultured in 6-well plates to 100% confluence. We used a sterile 200μl pipette tip to scrape the cell layer. After washing with PBS, the images of each processing groups were recorded at 0 and 24h after scratching by microscopy (Nikon TS2).
Real-time quantitative PCR
Total RNA was extracted from the mBMSCs using the RNA-Quick Purification Kit (Vazyme, China) and the cDNA was amplified using the HiScript II Q RT SuperMix for qPCR (Vazyme) according to the manufacturer’s instructions. The qPCR was performed with SYBR Green PCR Master Mix (Vazyme) on using ABI steponeplus real-time PCR system (Applied Biosystems, USA). The level of expression was standardized to GAPDH, and the relative expression level was evaluated using the 2−ΔΔCT approach. The primers used in this experiment were synthesized by GENEbay (China) with the following sequence: GAPDH (Forward: 5'-TCATGGGTGTGAACC ATGAGAA-3', Reverse: 5'-GGCATGGACTGT GGTCATG AG-3'), COL II (Forward: 5'-CACACTGGTAAGTGGGGCAAGACCG-3', Reverse: 5'-GGATTGTGTTGTTTC AGGGTTCGGG-3'), ACAN (Forward: 5'-CCTGCTACTTCATCGACCCC-3', Reverse: 5'-AGATGCTGTTGACTCGAACCT-3'), and SOX9 (Forward: 5’-GGTGC T CAAGGGCTACGACT-3’, Reverse: 5’-GGGTGGTCTTTCTTGTGCTG-3’).
Chondrocyte apoptosis by flow cytometry
Chondrocyte apoptosis were assessed using an Annexin V-FITC/PIapoptosis detection kit (Keygen Biotech, China). According to the manufacturer’s protocol, the chondrocytes were digested by 0.25% trypsin without EDTA, washed with PBS and centrifuged at 300g for 5min. The collected cells (500, 000/per tube) were resuspended in binding buffer (500μl) containing 5μl Annexin V-FITC and 5μl Propidium Iodide (PI) after 10 min incubation in dark and detected by flow cytometer (Beckman, USA).
Cell fluorescence analysis
Cells were fixed with 4% PFA for 20min, washed with PBS three times, permeabilized with 0.05% Triton X‐100 for 10min and blocked with 10% goat serum for 2h. Then, samples were incubated with primary antibodies overnight at 4°C, followed by Alexa-488 conjugated-goat secondary antibody (Jackson ImmunoResearch) for 2h at room temperature. After triple washing by PBS, nuclei were stained with DAPI and fluorescent images were acquired using fluorescence microscope (Leica DMI3000B, Germany) and Cell imager (Bio-tek Cytation1).
Western blot assay
Total protein was purified from cells by RIPA lysis (keygen Biotech, China). Protein concentration was quantified by Bradford Protein Assay Kit (Beyotime). Equal amounts of protein were separated by SDS-PAGE, and transferred to PVDF membranes (Millipore). Subsequently, PVDF membrane was blocked by 5% bovine serum albumin for 2h at room temperature prior to incubating overnight at 4 °C in respective primary antibodies. Membranes were then incubated for 2h at room temperature with the appropriate secondary antibodies. The bands were exposed by applying chemiluminescence kit (Beyotime) in an imaging system (Bio-Rad, USA). Quantification of band intensity was also performed by ImageJ (National Institutes of Health, USA).
C57 male mice aged 6-8w were used to performed chronic ankle instability model by transecting lateral ligaments (ATFL/CFL) as previously described[12, 13]. Under anesthetic conditions, CFL, connecting from the apex of the fibular malleolus to the lateral surface of the calcaneus, and ATFL, running from the distal anterior tip of the fibula to the lateral talar neck were both excised at its attachment sites. The lateral ankle capsule, which connects from anterior aspects of fibula to the lateral side of the talus, was incised after removal of CFL and ATFL. After surgery, 2μl PBS, 2μl blank Gel, 2μl PBS containing 2μg Exo, or 2μl Exo-Gel (4μg Exo/2μl Gel) were respectively injected into subtalar joint, and consequently depending on the composition of the injection, the mice were assigned to below groups: PBS, Gel, Exo, or Exo-Gel (n = 6/group). A sham operation was performed on the opposite ankle using the same skin incision without ligament/tendon resection. To identify sustained-release of Gel, DiR-labeled Exo (4μg/2μl PBS) and Exo-Gel (4μg/2μl) were locally injected into subtalar joints via precooled Hamilton precision syringe (Hamilton Co., Ltd, Switzerland), respectively, and the IVIS Spectrum imaging system (PerkinElmer, USA) was used to observe right subtalar joints on 3, 7, 14, and 28d after surgery. A sham operation was performed on the opposite ankle using the same skin incision without ligament/tendon resection. The entire operation was finished after the suture of the incision. Mice were allowed to move freely in the cages and had free access to food and water. The mice were sacrificed at 4 and 8w after the surgery.
Ankle samples at 8w postinjury were fixed in 4% PFA, decalcified in EDTA, and embedded in paraffin. Five-μm frontal sections were prepared and stained with safranin O and fast green, HE, toluidine blue (TB), and alcian blue (AB) respectively, according to the standard protocol, and the morphology of the tissue was observed by microscopy (Nikon TS2, Japan).
Ankle samples at 4w postinjury were prepared according to standard dewaxing procedure. The slices were soaked in citric acid buffer (10mM citric acid, pH 6.0) at 100°C for 10min to display antigens and 3% hydrogen peroxide was added for 10min to inactivate endogenous peroxidase. Then, immunohistochemical staining was performed using a previously reported protocol. After incubation with the primary antibodies at 4°C overnigh, the slides were subsequently stained with horseradish peroxidase-conjugated secondary antibody (Invitrogen). The presence of antigen in cartilage was determined by counting the number of positively stained chondrocytes by microscopy (Nikon TS2).
TUNEL (Roche, Switzerland) staining of cells and tissue slides was performed in dark for 30min at 37°C based on the manufacturer’s protocol. Nuclei was stained with DAPI. Images were captured by using Cell imager (Bio-tek Cytation1).
GraphPad 7.0 and SPSS 19.0 software were used to process and analyze the data. All independent experiments were performed in triplicate and all quantitative data were expressed means ± SEM. Data were analyzed using the Student’s T-test or one-way or two-way ANOVA to determine statistical differences. A value of p<0.05 was considered as statistically significant.