Induction of OA in rat
Male Wistar rats (180–250 g, 7 weeks of age) were purchased from Central Lab Animal Inc. (Seoul, South Korea). Animals were randomly assigned to the treatment or control group before the study began. After anesthetization with isoflurane, seven-week-old male Wistar rats (n = 5) were injected with 3 mg of monosodium iodoacetate (MIA; I2512, Sigma, St. Louis, MO), dissolved in a 50 μL of saline, using a 26.5 G needle inserted through the patellar ligament into the intra-articular space of the right knee. Mitochondria were administered to 10 μg/50 μL volume twice weekly in the right knee intra-articular space of monosodium iodoacetate (MIA)-induced rats, and rats in the control group were injected with the same volume of saline.
Isolation of mitochondria from L6 cells and human muscle
Mitochondria were isolated from L6 rat cells using a Mitochondrial Isolation Kit for Cultured Cells (89874, Thermo, MA). Briefly, 3 ´ 106 L6 cells were incubated with 400 μL of reagent A for 2 min on ice with vortexing every minute and incubated with 5 μL of reagent B for 5 min on ice. After adding 400 μL of reagent C, cells were centrifuged at 700 ´ g for 10 min at 4°C. The supernatant was transferred to a new tube and centrifuged at 12,000 ´ g for 15 min at 4°C. The pellet (mitochondria) was washed with 400 μL of reagent C and centrifuged at 12,000 ´ g for 5 min at 4°C. Mitochondria from muscle tissue of OA patients were isolated according to Boutagy et al. (46). Protein concentrations were quantified by bicinchoninic acid (BCA) assay (23235, Thermo).
Rat and human articular chondrocyte differentiation and culture
Chondrocytes were isolated from the cartilage of MIA-induced OA rats and patients. Cartilage was digested with 0.5 mg/mL hyaluronidase, 5 mg/mL protease type ⅩⅣ, and 2 mg/mL collagenase type V. Chondrocytes were incubated Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. Rat chondrocytes of passage 1 were cultured and analyzed. Human chondrocytes were cultured in the presence or absence of IL-1β (20 ng/mL) and 5 μg of isolated mitochondria for 24 h.
Assessment of pain behavior
Nociception in MIA-treated rats was tested using a dynamic plantar aesthesiometer (Ugo Basile, Gemonio, Italy). The device is an automated version of the von Frey hair assessment procedure and is used to assess mechanical sensitivity. When the instrument was activated, a fine plastic monofilament advanced at a constant speed and touched the paw in the proximal metatarsal region. The filament exerted a gradually increasing force on the plantar surface, starting below the threshold of detection and increasing until the stimulus became painful, as indicated by the rat’s withdrawal of its paw. The force required to elicit a paw-withdrawal reflex was recorded automatically and measured in g. A maximum force of 50 g and a ramp speed of 25 s were used for all aesthesiometer tests. Weight balance in MIA-treated rats was analyzed using an incapacitance meter (IITC Life Science, CA). The rats were allowed to acclimate for 5 min in an acrylic holder. After 5 min, both feet of the rat were fixed to the pad and the weight balance was measured for 5 s. Three measurements were repeated in the same manner. The weight of the unguided and guided legs was determined and substituted in the formula to calculate the percentage. The percentage value was calculating by comparing the legs with and without osteoarthritis.
Histopathological analysis
Knee joints were collected from each group 3 weeks after MIA induction. The tissues were fixed in 10% formalin after decalcification using Decalcifying Solution-Lite (Sigma, St. Louis, MO), and embedded in paraffin. Tissue was stained with hematoxylin and eosin (H&E) and safranin O and analyzed by the Osteoarthritis Research Society International (OARSI) and the Mankin scoring system (26).
Immunohistochemistry
Paraffin-embedded sections were incubated at 4°C with the following primary monoclonal antibodies: anti-IL-1β (ab9722, Abcam, Cambridge, UK), anti-TNF-α (ab6671, Abcam), anti-MMP13 (ab39012, Abcam), anti-MCP-1 (ab7202, Abcam) and anti-phospho-mixed lineage kinase domain-like protein (ab196436, Abcam). The samples were next incubated with the respective secondary biotinylated antibodies, followed by incubation for 30 min with streptavidin–peroxidase complex. The reaction product was developed using 3,3-diaminobenzidine chromogen (K3468, Dako, CA).
In vivo micro-computed tomography
Micro-computed tomography (CT) was performed using a bench-top cone-beam type in vivo animal scanner (mCT 35; SCANCO Medical, Wangen-Brüttisellen, Switzerland). The animals were imaged at settings of 70 kVp and 141 μA using a 0.5-mm-thick aluminum filter. The pixel size was 8.0 μm and the rotation step was 0.4°. Cross-sectional images were reconstructed using a filtered back-projection algorithm (NRecon software, Bruker micro CT, Belgium). For each scan, a stack of 286 cross-sections was reconstructed at 2,000 × 1,335 pixels. Bone volume and surface were analyzed at the femur.
Electron microscopy
Cells were fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer overnight at 4°C. The cells were washed in 0.1 M phosphate buffer, postfixed with 1% osmium tetroxide for 1 h at 4°C, dehydrated in graded ethyl alcohol solutions, exchanged in acetone, and embedded in Epon 812. Ultrathin sections (70–80 nm) were obtained on an ultramicrotome (Leica Ultracut, Leica, Vienna, Austria) and stained with uranyl acetate and lead citrate. Images were acquired at 60 kV using a transmission electron microscope (JEM 1010, JEOL, Tokyo, Japan).
CTX-Ⅱ enzyme-linked immunosorbent assay
CTX-II level from the serum of MIA-induced OA model was performed based on the method of the CTX-II (E-EL-R2554, Elabscience, TX) kit.
ATP assay
Isolated mitochondrial ATP content was analyzed using the ATP Lite Luminescence Assay System (6016943, Perkin Elmer, UK)
Coculture of osteoarthritic human chondrocytes with mitochondria
OA chondrocytes were seeded in 24-well plates at 5 × 104/well with isolated mitochondria (5 μg) in 10% DMEM. One day later, the cell culture medium was replaced with 0.1% insulin–transferrin–selenium-A (ITSA)–DMEM, and isolated mitochondria were layered onto the OA chondrocytes. Cells were incubated at 37°C for 24 h and harvested for further experiments.
Mitochondrial analysis by flow cytometry
Delivery of exogenous mitochondria to human chondrocytes was confirmed by Mitotracker deep red (M22426, Thermo) staining. The mitochondrial membrane potential was measured by JC-1 (T3168, Thermo) staining. The mitochondrial ROS level was measured by MitoSOX (M36008, Thermo) staining, and analyzed by flow cytometry on a FACS Calibur instrument (BD Biosciences).
Annexin V and propidium-iodide staining
Chondrocytes were harvested and stained with fluorescein isothiocyanate-conjugated Annexin V and propidium iodide (K101-100, BD Biosciences) for 5 min at room temperature. Flow cytometric analysis was performed on a FACS Calibur instrument (BD Biosciences).
Autophagy staining and confocal laser scanning microscopy
OA chondrocytes with isolated mitochondria were cocultured for 1 day. For confocal staining of LC3 and p62, LAMP1 cells were plated in four-well chamber slides, washed with PBS, fixed in 4% paraformaldehyde, washed with PBS, and blocked with 10% normal goat serum for 30 min. The cells were stained at 4°C overnight with anti-LC3 (ab48394, Abcam), anti-LAMP1 (sc-20011, Santa Cruz Biotechnology, CA), and anti-p62 (ab56416, Abcam) antibodies. The primary antibody was detected using a phycoerythrin-conjugated antirabbit IgG secondary antibody for 2 h at room temperature, and nuclei were stained with DAPI (D3571, Invitrogen). Stained cells were analyzed using a confocal microscope (LSM 510 Meta; Zeiss). The expression of LC3, p62, and LAMP1 was estimated by comparing the mean fluorescence intensity using LSM 510 Meta.
Western blotting
The protein levels of LAMP1 (sc-20011, Santa Cruz), p62 (ab56416, Abcam), LC3 (ab48394, Abcam), phosphor MLKL (ab184718, Abcam), and GAPDH (ab181602, Abcam) were measured by western blotting (SNAP i.d. Protein Detection System, Merck Millipore, Danvers, MD). Cells were incubated in six-well plates with IL-1β (20 ng/mL) in the presence or absence of mitochondria (5 μg) for 24 h, and mitochondrial and whole-cell lysates were prepared. Protein concentration was measured by BCA assay (23235, Thermo), and samples were separated on a 4–12% sodium dodecyl sulfate-polyacrylamide gel and transferred to a nitrocellulose membrane (Amersham Pharmacia, Uppsala, Sweden). Primary antibodies to LC3, p62, LAMP1, pMLKL, and GAPDH were diluted in 0.1% skim milk in Tris-buffered saline Tween-20 and incubated for 15 min at room temperature. The membrane was washed and incubated with horseradish peroxidase-conjugated secondary antibody for 10 min at room temperature. Band density was estimated by image-capture densitometry.
Reverse transcription-quantitative real-time PCR
mRNA was extracted using TRIzol (Molecular Research Center, Cincinnati, OH). cDNA was synthesized using the Superscript Reverse Transcription system (Takara, Shiga, Japan), and qPCR was performed using LightCycler FastStart DNA Master SYBR Green I (Takara) following the manufacturer’s instructions. Expression values were normalized to that of β-actin. Primer sequences are listed in Supplementary Table 1.
Statistical analysis
Results are means ± standard error of the mean (SEM). Data were analyzed by Student’s t-test or Mann–Whitney U-test using Prism 5 software (GraphPad, La Jolla, CA). P < 0.05 (two-tailed) was considered indicative of significance.