Animals, human cartilage, and recombinant Atsttrin
We performed all animal studies under institutional guidelines. All of the protocols were approval by the Institutional Animal Care and Use Committee, New York University, NY, USA. We generated, maintained, and genotyped the mice with the genetic background of C57BL/6 wildtype (WT), PGRN-deficient (PGRN–/–), TNFR1-deficient (TNFR1–/–), and TNFR2-deficient (TNFR2–/–) mice as reported previously . Sprague–Dawley rats were obtained from Charles River (Wilmington, MA, USA). Eight-week-old male mice and 14-week-old male rats were used for this experiment [19, 20]. For human primary chondrocyte culture, human cartilage samples were harvested from patients receiving total knee joint replacement surgery from New York University, Hospital for Joint Diseases (NY, USA). Acquisition and use of human tissue was conducted in accordance with an Institutional Review Board (IRB#12758) approved protocol. Recombinant Atsttrin was manufactured and provided by Atreaon, Inc.
Noninvasive anterior crucial ligament rupture rat model
The noninvasive OA rat model was established as described previously . Animals were anesthetized and maintained using isoflurane, and the noninvasive anterior crucial ligament rupture model was established using the indicated machine: Electroforce 3200 (Bose Corp., MN, USA), Solidworks (Dassault Systemes, MA, USA), or Mojo 3D printer (Stratasys, MN, USA). After the model was established, we intraarticularly injected PBS or recombinant Atsttrin once a week for 4 weeks in total. After 4 weeks of treatment, the rats were sacrificed for histological evaluation.
Surgically induced OA mouse models
For the surgically induced destabilization of medial meniscus (DMM) mouse model, we took advantage of 8-week-old male PGRN–/– mice and their age-matched WT control littermates. The medial meniscotibial ligament of the right knee joint was cut to generate a destabilized medial meniscus. Six mice were used in each group. After surgery, WT mice received local delivery of 6 μl PBS via intraarticular injection, while PGRN–/– mice received local delivery of 6 μl PBS or recombinant Atsttrin (1 μg/μl). Four weeks after model induction, mice were sacrificed and knee joints were collected. The tissues were then processed for histological analysis.
To investigate the preventative as well as the therapeutic effect of Atsttrin, we also established the anterior cruciate ligament transection (ACLT) mouse model. To determine which TNFR was predominantly responsible for Atsttrin’s effect, we established the ACLT mouse model in age-matched WT, TNFR1–/–, or TNFR2–/– male mice (n = 6, respectively). To address the preventative potential of Atsttrin in OA, we intraarticularly injected 6 μl Atsttrin or PBS once a week for 4 weeks beginning on the day of surgery, as based on our previous study . For examination of Atsttrin’s therapeutic effect, 6 μl Atsttrin or PBS were intraarticularly injected once a week for 4 weeks beginning 4 weeks postoperatively, as based on our previous study . Ambulatory behavior of mice was monitored and recorded throughout the study. After 4 weeks of treatment, mice were sacrificed for dorsal root ganglia (DRG) harvest and histological evaluation.
Sandwich ELISA for cartilage oligomeric matrix protein
The serum concentration of cartilage oligomeric matrix protein (COMP) was analyzed by our established sandwich ELISA . Protein A agarose (Invitrogen) purified rabbit anti-COMP pAb and anti-COMP type III mAb 2127F5B6 were used as capture and detection antibodies, respectively. Anti-COMP type III mAb 2127F5B6 was labeled with horseradish peroxidase (HRP) using the Lightning-Link™ Horseradish Peroxidase Labeling Kit (Innova) as per the manufacturer’s protocol. Results were interpreted based on the linear range of the standard curve. All of the samples were assayed in triplicate.
Primary cultures of chondrocytes
Human articular chondrocytes were harvested by enzymatic digestion in accordance with established methodology . Briefly, human cartilage slices were cut into small pieces and washed several times with PBS, pH 7.4. Minced tissues were incubated with agitation in digestion medium comprised of 0.25% collagenase II in DMEM medium with 5% FBS in a spinner flask for 16 hours at 37 °C with 5% CO2. After digestion, the suspended cells were collected and seeded into six-well plates for subsequent study. For mouse primary chondrocyte culture, knee joints were collected from 6-day-old WT, TNFR1–/–, or TNFR2–/– mice following sacrifice. Under magnification, the cartilage samples were isolated with special attention to avoid damaging the subchondral bone and tissues were rinsed completely three times in PBS. Primary cartilage samples were placed in a 10-cm dish containing the aforementioned digestion buffer and incubated for 16 hours at 37 °C with 5% CO2. After full digestion, suspended cells were collected and seeded in a six-well plate for use. All chondrocytes used for experiments are first-generation cells.
von Frey test
von Frey filaments (Stoelting, Wood Dale, IL, USA) were applied with ascending force intensities on the plantar surface of the hind paw to determine the tactile pain threshold as based on a previous publication . Rapid withdrawal of the hind paw was recorded as a positive response. Hind paws were subjected to 10 trials at a given intensity with a 30-second interval maintained between trials. The number of positive responses for each von Frey filament’s stimulus was recorded. Animals were considered to have reached tactile threshold when five in 10 trials generated a positive response. The examiner was blinded to the groups.
Dorsal root ganglia isolation
Eight weeks after ACLT surgery, mice were sacrificed and L3–L5 DRG were isolated based on a previous publication . Briefly, mice were anesthetized using isoflurane and fur was cleared from the dorsal surface. A longitudinal incision was made, the spinal column was exposed, and L3–L5 DRG were extracted and tissues flash-frozen using liquid nitrogen. These tissues were processed using the Qiagen RNeasy kit (Qiagen, Valencia, CA, USA) for RNA extraction.
Luciferase assay was performed as reported previously . Lipofectamine2000 DNA transfection reagent was used to cotransfect NF-κB and renilla plasmids in C28I2 cells according to the manufacturer’s protocol (Life Technologies). Eighteen hours after transfection, C28I2 cells were treated without or with 10 ng/ml TNFα in the absence or presence of 200 ng/ml recombinant Atsttrin. After 24-hour incubation, we measured luciferase activity using the Reporter Assay System of Dual-Luciferase® in accordance with the manufacturer's instructions (Promega).
Histological analysis and immunostaining
Histological analysis was conducted as described previously . Briefly, knee joints were fixed immediately after sacrifice in 4% PFA at room temperature for 48 hours. After washing three times in PBS, the tissues were decalcified at 4 °C in 10% w/v EDTA for 2 weeks. Tissues were measured using a vernier caliper before paraffin processing. Knee joints were dehydrated and embedded; the blocks were trimmed to the midpoint as calculated previously from caliper measurements and serial 5-μm sections were placed on slides for staining. H&E or Safranin O/fast green staining was performed following the established protocol. The extent of synovitis was determined using a graded scale based on H&E staining: grade 0, no signs of inflammation; grade 1, mild inflammation with hyperplasia of the synovial lining without cartilage destruction; and grades 2–4, increasing degrees of inflammatory cell infiltrate and cartilage/bone destruction. For immunohistochemistry staining, sections were pretreated with 0.1% trypsin for 30 minutes at 37 °C. Sections were washed with PBS three times, followed by treatment with 0.25 U/ml chondroitinase ABC (Sigma-Aldrich) for 1 hour and then 1 U/ml hyaluronidase (Sigma-Aldrich) for 1 hour at 37 °C. To reduce nonspecific staining, sections were blocked at room temperature with 20% normal horse serum diluted in 3% BSA for 1 hour. Without washing after blocking, Col X antibody (1:200 dilution; DSHB), MMP-13 antibody (ab3208, 1:200 dilution; Abcam), and affinity-purified monoclonal anti-COMP fragments (1:200 dilution) were diluted in 20% normal horse serum with 3% BSA at 4 °C overnight. Sections were prepared for detection using the Vectastain Elite ABC kit following the manufacturer’s guidelines at 25 °C for 1 hour. Immunorecativity was visualized using 0.5 mg/ml 3,3′-diaminobenzidine (DAB) in 50 mM Tris–HCl substrate, pH 7.8. Methyl green (1%) was used for counterstaining.
Histological analysis and score
The articular cartilage proteoglycan content was defined on the basis of Safranin O staining. In this study, we used the well-accepted Osteoarthritis Research Society International (OARSI) scoring system : 0 = normal cartilage without any damage; 0.5 = loss of Safranin O staining with no detectable structural change; 1 = small fibrillation; 2 = vertical damage of cartilage limited to superficial layer; 3 = vertical damage, no more than 25% of the cartilage surface; 4 = vertical damage, 25–50% of the cartilage surface; 5 = vertical damage, 50–75% of the cartilage surface; and 6 = vertical damage, more than 75% of the cartilage surface.
Total RNA were extracted from cultured chondrocytes using the RNeasy kit (Qiagen) and reverse transcribed into cDNA using the ImProm-II reverse transcription system (Promega). Data were normalized to the internal control, GAPDH. The primers for specific amplification of murine genes are as follows: 5′-AATGCTGGTACTCCAAACCC-3′ and 5′-CTGGATCGTTATCCAGCAAACAGC-3′ for Aggrecan; 5′-ACTAGTCATCCAGCAAACAGCCAGG-3′ and 5′-TTGGCTTTGGGAAGAGAC-3′ for Col II; 5′-AATCTCACAGCAGCACATCA-3′ and 5′-AAGGTGCTCATGTCCTCATC-3′ for IL-1β; 5′-ACAGGAGGGGTTAAAGCTGC-3′ and 5′-TTGTCTCCAAGGGACCAGG-3′ for NOS-2; 5′-GCATTGACGCATCCAAACCC-3′ and 5′-CGTGGTAGGTCCAGCAAACAGTTAC-3′ for ADAMTS-4; 5′-ACTTTGTTGCCAATTCCAGG-3′ and 5′-TTTGAGAACACGGGGAAGAC-3′ for MMP-13; 5′-CATAGCAGCCACCTTCATTCC-3′ and 5′-TCTCCTTGGCCACAATGGTC-3′ for MCP-1; 5′-AGAGAGCTGCAGCAAAAAGG-3′ and 5′-GGAAAGAGGCAGTTGCAAAG-3′ for CCR-2; and 5′-AGAACATCATCCCTGCATCC-3′ and 5′-AGTTGCTGTTGAAGTCGC-3′ for GAPDH. Melting curve analysis was used to verify the PCR product. Each experiment was repeated three times.
Western blot analysis
Proteins extracted from chondrocytes were processed on 8% SDS-polyacrylamide gel, followed by electrotransfer to nitrocellulose membrane. The membrane was blocked in 3% BSA in 10 mM Tris–HCl, pH 8.0, 150 mM NaCl, and 0.5% Tween 20. After washing three times, blots were incubated at 4 °C overnight with polyclonal anti-Erk1/2 (#4695, 1:1000 dilution; Cell Signaling Technology), anti-phosphorylated Erk1/2 (#4370S, 1:1000 dilution; Cell Signaling Technology), polyclonal anti-Akt (#9272, 1:1000 dilution; Cell Signaling Technology), anti-phosphorylated Akt (#4058S, 1:1000 dilution; Cell Signaling Technology), polyclonal anti-MMP-3 (ab52915, 1:1000 dilution; Abcam), polyclonal anti-MMP-13 (ab3208, 1:1000 dilution; Abcam), polyclonal anti-ADAMTS-4 (PA1-1750, 1:1000 dilution; Thermo Fisher Scientific), polyclonal anti-NOS-2 (SC651, 1:1000 dilution; Santa Cruz Biotechnology), polyclonal anti-GAPDH (SC25778, 1:1000 dilution; Santa Cruz Biotechnology), polyclonal anti-tubulin (#5346, 1:1000 dilution; Cell Signaling Technology), or diluted polyclonal anti-lamin B (SC-6217, 1:500 dilution; Santa Cruz Biotechnology). After washing three times, blots were incubated with an appropriate HRP-conjugated anti-rabbit/mouse immunoglobulin secondary antibody at 25 °C for 1 hour. The bound antibody was visualized using an enhanced chemiluminescence system (Amersham Life Science, Arlington Heights, IL, USA).
Cartilage explant cultures
Cartilage explants were cultured as described previously . Briefly, mouse femoral head cartilage was isolated and finely minced to 1 mm diameter and 1 mm thickness. The cartilage explants were then dispensed into serum-free DMEM containing 25 mM HEPES and 2 mM glutamine, in the absence or presence of recombinant Atsttrin (200 ng/ml).
Dimethylmethylene blue assay of GAG
The mouse cartilage culture medium was collected and GAG release was quantified by dimethylmethylene blue assay (DMMB) (Polysciences, Warrington, PA, USA). Hyaluronidase (0.5 unit/ml; Seikagaku, Tokyo, Japan) was incubated with collected medium for 3 hours at 37 °C to remove hyaluronan in order to reduce inhibition of the DMMB assay. The DMMB signal from digests was measured at 520 nm using a SpectraMax 384 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). The GAG content was calculated based on linear regression of readings from chondroitin-6-sulfate standards from Shark cartilage (Sigma Aldrich, St. Louis, MO, USA). Each sample was read in triplicate. The average values of the triplicates were normalized to the standard curve.
Results were expressed as mean ± SEM. Statistical analysis included Student’s t test performed by SPSS software (SPSS Inc., Chicago, IL, USA). p < 0.05 was considered statistically significant.