- Email: [email protected]
Attenuation of subchondral bone abnormal changes in osteoarthritis by inhibition of SDF-1 signaling
Accepted Manuscript Attenuation of subchondral bone abnormal changes inosteoarthritis by inhibition of SDF-1 signaling Yuanfeng Chen, Sien Lin, Yuxin Sun, Jia Guo, Yingfei Lu, Chun Wai Suen, Jinfang Zhang, Zhengang Zha, Ki Wai Ho, Xiaohua Pan, Gang Li PII:
S1063-4584(17)30034-1
DOI:
10.1016/j.joca.2017.01.008
Reference:
YJOCA 3940
To appear in:
Osteoarthritis and Cartilage
Received Date: 21 July 2016 Revised Date:
28 December 2016
Accepted Date: 17 January 2017
Please cite this article as: Chen Y, Lin S, Sun Y, Guo J, Lu Y, Suen CW, Zhang J, Zha Z, Ho KW, Pan X, Li G, Attenuation of subchondral bone abnormal changes inosteoarthritis by inhibition of SDF-1 signaling, Osteoarthritis and Cartilage (2017), doi: 10.1016/j.joca.2017.01.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Attenuation of subchondral bone abnormal changes inosteoarthritis by
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inhibition of SDF-1 signaling
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Chen Yuanfeng1,2,3, Lin Sien2,3, Sun Yuxin2,3, Guo Jia2, Lu Yingfei2, Suen Chun Wai2,3, Zhang
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* * * Jinfang2,3, Zha Zhengang1, Ho Ki Wai2 , Pan Xiaohua4 , Li Gang2,3,5
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1. Institute of Orthopedic Diseases and Department of Orthopedics, the First Affiliated Hospital,
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Jinan University, Guangzhou, China
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2. Department of Orthopaedics& Traumatology, Li Ka Shing Institute of Health Sciences and Lui
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Che Woo Institute of Innovative Medicine, Faculty of Medicine, The Chinese University of Hong
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Kong, Prince of Wales Hospital, Shatin, Hong Kong SAR, PR China.
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3. The CUHK-ACC Space Medicine Centre on Health Maintenance of Musculoskeletal System, The
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Chinese University of Hong Kong Shenzhen Research Institute, Shenzhen, PR China.
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4. Department of Orthopaedics and Traumatology, Bao-An District People’s Hospital, Shenzhen, PR
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China.
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5. Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences,
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Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, PR China.
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* Correspondence: [email protected]; [email protected] or [email protected]
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Abstract: Background: Current conservative treatments for osteoarthritis (OA) are largely
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symptoms control therapies. Further understanding on the pathological mechanisms of OA is crucial
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for new pharmacological intervention. Objective: In this study, we investigated the role of Stromal
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cell-derived factor-1(SDF-1) in regulating subchondral bone changes during the progression of
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osteoarthritis. Methods: Clinical samples of different stages of OA severity were analyzed by
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histology staining, micro-CT, enzyme-linked immunosorbent assay (ELISA) and western blotting, to
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compare SDF-1 level in subchondral bone. The effects of SDF-1 on human mesenchymal stem cells
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(MSCs) osteogenic differentiation were evaluated. In vivo assessment was performed in an anterior
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cruciate ligament transaction plus medial meniscus resection in the SD rats. The OA rats received
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continuous infusion of AMD3100 (SDF-1 receptor blocker) in osmotic mini-pump implanted
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subcutaneously for 6 weeks. These rats were then terminated and subjected to the same in vitro
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assessments as human OA samples. Results: SDF-1 level was significantly elevated in the
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subchondral bone of human OA samples. In the cell studies, the results showed SDF-1 plays an
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important role in osteogenic differentiation of MSCs. In the OA animal studies, there were less
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cartilage damage in the AMD3100-treated group; microCT results showed that the subchondral bone
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formation was significantly reduced and so did the number of positive Nestin or Osterix cells in the
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subchondral bone region. Conclusions: Higher level of SDF-1 may induce the subchondral bone
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abnormal changes in osteoarthritis and inhibition of SDF-1 signaling could be a potential therapeutic
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approach for OA.
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Keywords: Stromal cell-derived factor-1, osteoarthritis, subchondral bone.
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INTRODUCTION Osteoarthritis (OA) is one of the leading cause of physical disability affecting nearly 80% of the
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individuals aged 75 years or over[1]. Current pharmacologic therapies mainly target at symptoms
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controlling and their efficacy in altering the progression of OA are disappointing. Further
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understanding of the pathological mechanisms of OA development is crucial for the design of the
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new pharmacological intervention.
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Various inflammatory cytokines including interleukin-1β(IL-1β), tumor necrosis factor-α (TNF-
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α)and NF-κB signal, attribute to the development and progress of OA[2-8]. Articular cartilage
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degeneration is the main feature in OA and many factors such as matrix metalloproteinase 13
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(MMP13)[9], collagen fragments (C2C)[10] as well asnerve growth factor (NGF)[11] are involved.
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The homeostasis and integrity of articular cartilage rely on its biochemical and biomechanical
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interplays with the subchondral bone and the surrounding soft tissues[12]. Subchondral bone
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provides mechanical support for the articular cartilage and constantly undergoes bone
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remodeling[13]. Bone marrow lesions are closely associated the severity of cartilage damage and
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pain in OA [14]. However, the relationship between the abnormal formation of subchondral bone and
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progression of OA still remains largely unknown.
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The role of stromal derived factor-1 (SDF-1) in the pathogenesis of OA or rheumatoid arthritis
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(RA) has drawn increasing attention in recent years[4, 15-17]. A dramatic elevation of SDF-1 is
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found in the knee synovium from RA and OA patients[4]. SDF-1 has been shown that it could
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regulate chondrocyte catabolic activities by stimulating the release of MMP-3 and MMP-13[4].
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Synovectomy could significantly reduce the concentrations of SDF-1, MMP-9, and MMP-13 in
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blood serum[15]. In animal study, blockage of SDF-1 signaling pathway using AMD3100, a CXCR4
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antagonist of the SDF-1 receptor, attenuated the cartilage degeneration [18]. SDF-1 is a well-known
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osteogenic differentiation of MSCs[22, 23]. Furthermore, in 2006 Lisignoli G and colleagues
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demonstrated that SDF-1 could significantly induce proliferation and collagen type I expression in
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osteoblasts from OA patients[17]. This study implies that SDF-1 may play a role in the abnormal
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changes in subchondral bone with OA. SDF-1 influences cartilage degeneration through stimulating
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the release of MMPs from chondrocytes, however, whether SDF-1 contributes to subchondral bone
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changes in OA is still not fully understood. Therefore, the objective of this study is to investigate the
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role of SDF-1 in subchondral bone changes during OA development.
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MATERIALS AND METHODS
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Clinical sample collection
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This study was approved by the Joint Chinese University of Hong Kong-New Territories East
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Cluster Clinical Research Ethics Committee and informed consent was obtained from each donor. All
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experiments were carried out in accordance with the research guidelines of the Chinese University of
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Hong Kong. The clinical specimens were obtained from patients with OA at the time of total knee
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arthroplasty surgery (n = 12; 8 women and 4 men; age 65.8±6.5 years, range 49–76 years). Various
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regions of the knee joint were harvested and the samples were immediately placed into sterile
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DMEM culture medium and transported to the laboratory for further processing. Samples were
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divided into two parts; OA group where cartilage was severely damaged or fibrillated with OARSI
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score of 15–18; and relatively normal group, where the cartilage is non-fibrillated with OARSI
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scores of 0–3. The cartilage and subchondral bone explants were cut from OA or normal sites of the
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OA joint (Fig. 1A) were cut into the size of 1.5×0.5 cm full-thickness and subjected to future
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experiments.
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Bone marrow MSCs culture Human fetal bone marrow stem cells (hBM-MSCs) were obtained from the Stem Cell Bank
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at the Prince of Wales Hospital of the Chinese University of Hong Kong. Ethical approval was
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obtained from the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical
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Research Ethics Committee (ethical approval code: CRE-2011.383). Informed written consent form
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was approved by the Clinical Research Ethics Committee and signed by the donor before sample
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collection. All experiments were carried out in accordance with the research guidelines of the
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Chinese University of Hong Kong. These cells were cultured in the complete alpha-minimum
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essential medium (α-MEM, Manassas, Virginia, USA) supplemented with 10% fetal bovine serum
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(FBS) and 1% penicillin-streptomycin-neomycin (complete culture medium; all from Invitrogen
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Corporation, Carlsbad, CA, USA) in a 5% CO2 humidified incubator at 37 0 C.
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In MSCs osteogenic induction, the MSCs were trypsinized and seeded in 6-well plate at a
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concentration of 1x105 cells per well. These cells were incubated in the ɑ-MEM for two or three days,
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the medium was then replaced by osteogenic induction medium (OIM) which contains100 nmol/L
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dexamethasone, 10 mmol/L beta-glycerophosphate and 0.05 mmol/Ll-ascorbic acid-2-phosphate.
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To block the SDF-1 signaling in primary MSCs, cells were incubated with AMD3100, a
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CXCR4 antagonist, which selectively binds to CXCR4 and prevents the binding of SDF-1[24]. The
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working concentration of AMD3100(Sigma, StLouis, MO, USA) in this study was 400 µM, which
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has been shown to effectively inhibit SDF-1 signaling without toxicity[22-24].
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Animal experiments All experiments were approved by the Animal Research Ethics Committee, the Chinese
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ACCEPTED MANUSCRIPT University of Hong Kong. 16-week old Male Sprague-Dawley (SD) rats, with the weight of 450-500
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g, were used in this study. All rats received anterior cruciate ligament transaction plus medial
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meniscus resection (ACLT + MMx) at the right knee as previously described[25]. In brief, each rat
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was anesthetized by administrating 0.2%(vol/vol) xylazine and 1% (vol/vol) ketamine, after being
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shaved and disinfected, the right knee joint was exposed through a medial parapatellar arthrotomy
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approach. The patella was dislocated laterally and the knee was placed in full flexion followed by
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ACL and MCL transection with micro-scissors and resection of the medial meniscus.
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Rats were allocated randomly into three groups: via osmotic mini-pump, the first group received
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a continuous infusion of the AMD3100 (Sigma, St.Louis, MO, USA) (n=5) and the second group
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received PBS(n=5). Just after the ACLT+MMx surgery, the Mini-osmotic pump (model 2006; Alza
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Corporation, Mountain View, CA, USA.) was inserted into the small subcutaneous pocket over the
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dorsolateral thorax, and the pocket is created by blunt dissection with a small incision (~1 cm).
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Before insertion, the 200 µl pump reservoir was filled with PBS containing 22.3 mg/ml AMD3100 or
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just PBS. The average pumping rate of the mini-osmotic pump is 0.15 µl/per hour, thus AMD3100 or
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PBS was systemically administrated to the rats during the entire experiment. For the last group, rats
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were left untreated as the OA control (n = 5). After 6 weeks of treatment, all animals were
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euthanized and their right knees were harvested for further examinations.
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ELISA examination of the clinical specimens
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Subchondral bone explants were cut off (0.5x0.5cm3) from the OA region and the relatively
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normal region of the same specimen; they were then immediately placed on dry ice. The explant was
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weighed, mechanically homogenized, ground into powder with addition of liquid nitrogen, and then
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ice-cold tissue protein extraction reagents (Life Technologies, Pleasanton, CA, USA) were added.
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Homogenate was centrifuged for 10 minutes at 14,000 rpm at 4°C and used for SDF-1 ELISA
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examination according to the instruction of the kit(Sigma, StLouis, MO, USA).
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Western blot The lysates of subchondral bone and the cultured MSCs were subject to Western blot
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examination. Before tested by following assays, MSCs were pretreated with AMD3100 at 400 µM
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for 2 hours at 37 °C prior to culture in osteogenic inductive media (OIM); the controls were cultured
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for 15 minutes in either normal culture medium (ɑ-MEM) or OIM. The cells were then washed with
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cold PBS twice and harvested by scraping in cell extraction buffer (Invitrogen, Cat. no.FNN0011).
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Protein concentration was determined using Bradford method (Biorad, Richmond, CA, USA). An
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equal amount of proteins was loaded onto 10%Tris/glycine gels for electrophoresis and then
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transferred to a PVDF membrane (Millipore, Bedford, MA, USA). It was then blocked in 5% nonfat
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milk (Biorad, Richmond, CA, USA) for 1h at room temperature with rocking. Reagents such as the
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primary antibody, anti-p-ERK (1:1000, BD Biosciences, USA), anti-total Erk1/2(1:8000, BD
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Biosciences, USA), anti-GAPDH (1:10000, Santa Cruz, USA) was respectively added and incubated
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for 2 hours room temperature or at 40C overnight. After washing in TBS for three times (5 minutes
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for each washing), the membrane was incubated with horseradish peroxidase-linked secondary
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antibodies.
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Alkaline phosphatase (ALP) activity assays
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MSCs were cultured in osteogenic induced medium (OIM) or in the normal culture medium as
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acontrol. In parallel, cells were pretreated with AMD3100 at400 µM for 2 hours at 37 °C prior to
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culture in OIM. The ALP staining was performed using alkaline phosphatase (alp)-amp (Biosystems,
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Foster City, CA, USA), according to the instructions from the manufacturer. ALP staining assays
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were observed under light microscope (Leica DMRB, Leica Cambridge Ltd., UK).
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Micro-CT Assessment Subchondral bone explants from the clinical specimens and the rat specimens were fixed over
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night in 10% formalin. These were then analyzed using high-resolution µCT (µCT40, Scanco
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Medical, Basserdorf, Switzerland). Three-dimensional (3D) reconstructions of mineralized tissues
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were performed by an application of a global threshold (180 for theclinical specimens and 158 for
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the animalspecimens, in mg hydroxyapatite/cm3), and a Gaussian filter (sigma = 0.8, support = 2)
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was used to suppress noise. For the clinical sample, the 3D images showed the whole subchondral
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bone explants; and for the animal study, 100 sagittal images of the tibiae subchondral bone were used
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to perform the 3D histomorphometric analysis. 3D structural parameters analysis included: bone
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mineral density (BMD), bone volume/total tissue volume (BV/TV), Tb.Th (trabecular thickness),
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Tb.Sp (trabecular separation), SMI (structure model index).
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Histological and immunochemical examinations
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The right knee explants of the clinical specimen and the rat specimen were fixed in 10%
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formalin for 48 hours, followed by decalcification in 10% EDTA for 21 days before embedding them
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in paraffin. In animal study, serial sagittal-oriented sections of the knee joint (medial compartment)
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were cut at intervals of 0 µm, 100 µm, and 200 µm before mounted onto glass slides. Five-
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micrometer-thick sagittal-oriented sections of the sample were processed for Safranin-O/fast green
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and H&E staining.
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Immunostaining was performed using a standard protocol as previously reported[26, 27]. We
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incubated sections with primary antibodies to rabbit Nestin (Sigma, 1:300, N5413), Osterix (Abcam,
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ACCEPTED MANUSCRIPT 1:600, ab22552),CXCR4 (R&D, 1:200, MAB171), ALP (Abcam, 1:200, ab54778), Col I (Santa Cruz,
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1:200, sc-8784), overnight at 4°C. For immunohistochemical staining, a horse radish peroxidase–
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streptavidin detection system (Dako,Carpinteria, CA, USA) was used, followed by counterstaining
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with hematoxylin. Photographs of the selected areas were taken under a light microscope. We
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counted the number of positively stained cells in three randomized areas of the sections in the human
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specimens; or in the whole subchondral bone area in three sequential sections (0 µm, 100 µm, and
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200 µm) per specimen, and the numbers were compared statistically.
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For immunofluorescent staining, the MSCs were fixed in 4% paraformaldehyde for
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10minutes and then washed with PBS twice. The treated MSCs were incubated with primary
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antibodies to rabbit Nestin (Sigma, 1:300, N5413) and mouse CXCR4 (R&D, 1:300, MAB171)
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overnight at 4°C.
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CY3(LifeTechnologies, Pleasanton, CA, USA. 1:800)or goat anti-rabbit Alex 488(LifeTechnologies,
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Pleasanton, CA, USA. 1:300) were added, and slides were incubated at room temperature for 1 hour
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in the dark. Photographs of the selected areas were taken under a microscope.
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Statistical analysis
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In accordance with the ARRIVE guidelines[28], we have reported measures of precision,
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confidence, and n number to provide an indication of significance. All statistical analyses were
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performed using the statistical software SPSS15.0. The data of rat samples were analyzed using one-
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way ANOVA, while data of clinical samples studies were tested by Pair sample T-test. In one-way
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ANOVA tested, the assumptions of the analysis were assessed by the Shapiro-Wilk test of normality
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and Levene’s test for homogeneity of variance. The result of Levene's test was used to determine the
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post hoc testing strategy. If the result is significant, Dunnett’s T3 post hoc test for unequal variance
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was used, while LSD-t post hoc test was employed if there is no significant result. Values of p< 0.05
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were considered significant. Data was reported as mean±standard deviation. The graphs were
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generated in GraphPad Prism 6(GraphPad Software, San Diego, CA, USA).
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RESULTS
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Elevated SDF-1 levels in subchondral bone in the severe OA part of the clinical specimen
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The gross macroscopic observation showed that in the severe OA region of the samples (on the
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left, Fig. 1a.), the cartilage was almost worn out on the left side and the surrounding area showed
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rough articular cartilage surfaces. In the adjacent relatively normal (RN) region(on the right, Fig. 1a.),
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the amount of cartilage damage was much less pronounced and the articular surface was relatively
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smooth. Moreover, inSafranin-O/fast green and H&E staining showed that proteoglycan loss was
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aggravated inthe severe OA region rather than inthe RN region(Fig. 1b.). H&E staining showed that
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the thickness of the hyaline cartilage zone in the RN region was much greater than that in the severe
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OA part (Fig. 1b.).
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The 3D reconstructed images of µCT showed that the micro-architecture of the subchondral
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bone changed significantly at end-stage of OA. There was also an obvious difference between the
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worn sides of the OA region compared to that of the RN region(Fig. 2a.). Bone mineral density
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(BMD) in the OA region(282.3 ± 75.7 mg/cm3, n=5) had significantly increased than that of the RN
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region(144.9± 35.8 mg/cm3, n=5, p=0.023.)(Fig. 2b.). Similarly, bone volume/total tissue volume
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(BV/TV) up-regulated distinctly in the OA region(0.38± 0.10, n=5) when compared with the RN
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region(0.19± 0.05, n=5, p=0.017) (Fig. 2c.). Moreover, trabecular bone thickness (Tb.Th.) in the
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OA region(0.27 ± 0.07 mm, n=5) was higher than that of the RN region(0.16 ± 0.04 mg/cm3, n=5,
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p=0.074.) (Sup. Fig. 1a.), though no statistically significantdifference was found. Trabecular bone
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space (Tb.Sp.)(0.36 ± 0.14, n=5) and structure model index (SMI)(-0.04± 1.7, n=5) in the OA
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region showed stronger decrease than the RN region(0.55± 0.10, n=5, p=0.028; 0.91± 0.67, n=5,
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p=0.027) (Sup. Fig. 1b,c.). The result of immunohistochemistry staining with Nestin, a marker of adult bone marrow
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MSCs[29, 30], showed a significant increase in the numbers of Nestin-positive cells in the
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subchondral bone marrow cavity at the severe OA region(137.3± 27.6, n=5) when compared with
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the RN region(55.0±9.6, n=5, p=0.009) (Sup.Fig. 2a,b.). Osterixis a marker of osteoprogenitors and
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the number of Osterix-positive osteoprogenitors in the subchondral bone in the OA side(176.3±12.0,
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n=5) was significantly higher than the RN side(60.6±16.8,n=5, p=0.019)(Sup. Fig. 2a,c.). However,
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the type I collagen was highly expressed in the trabecular bone in both groups(Sup. Fig.3b.) and the
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number of ALP-positive cells in the subchondral bone showed no statistical difference between OA
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side(91.33±8.212, n=5) and RN side.(88±8.712, n=5, p>0.05)(Sup. Fig. 4b,d.) In addition, we also
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found that the number of the CXCR4-positive cells was significantly higher in the OA side of the
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subchondral bone(Sup. Fig.5.).
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ELISA assay showed that SDF-1 was elevated in the severe OA region of the clinical samples.
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The level of SDF-1in the severe OA region was 424.3 ± 244.1 pg/ml, (n=7); which was
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significantly increased when compared with the RN region (328.6 ± 251.7 pg/ml, n=7,
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p=0.025)( Fig. 3a.) Since the phosphorylation of Erk1/2 can be activated by SDF-1 in the osteogenic
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differentiated MSCs, we examined whether the high level of SDF-1 may activate the
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phosphorylation of Erk in the severe OA part of clinical specimens. The results from western blot
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showed that the amount of pErk1/2 was significantly higher in the severe OA region(Fig. 3b.).
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In summary, the elevated SDF-1 signaling in the subchondral bone is associated with the changes in the subchondral bone structure in the clinical OA specimens.
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SDF-1 plays an important role in MSCs osteogenic differentiation Immunofluorescent staining showed that Nestin positive (green) MSCs expressed CXCR4
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(red), a receptor of SDF-1(Fig. 4.). After culturing the MSCs in OIM for 5 days, stainings showed
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significantly increased ALP activity, while it was not obvious in the control cells cultured in the
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normal medium. AMD3100 is a CXCR4 antagonist which blocks the SDF-1 signal and pretreating it
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to the cells could reduce the OIM-induced ALP activity significantly when compared with the
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untreated cells (Fig. 5a.). The results from western blot also showed that level of pErk1/2 was down-
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regulated only in the AMD3100-pretreated group and this suggests that perturbing the MSCs with
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SDF-1 signal could inhibit intracellular Erk phosphorylation in osteogenic differentiation of
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MSCs(Fig. 5b.).
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Inhibition of SDF-1 signaling reduces aberrant subchondral bone formation and attenuates
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articular cartilage degeneration in ACLT + MMx rat
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Safranin-O/fast green staining showed that there were significantly more degenerative features
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in the PBS-treated and the OA groups than the AMD3100-treated group (Fig. 6). Three-dimensional
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µCT images of cross-sectional, sagittal views of tibia subchondral bone from rats showed that the
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abnormal bone formation feature was more down regulated in the subchondral bone of the
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AMD3100 group (Fig. 7a). Structural parameters of subchondral bone were tested by µCT as a
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quantitative assay. BMD in the AMD3100 group (545.9±9.5 mg/cm3, n=5) was significantly lower
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than that of the PBS group (619.8±19.5 mg/cm3, n=5, p=0.001.) and the OA group(607.7 ± 33.5
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mg/cm3, n=5, p=0.003.)(Fig. 7b.). Moreover, BV/TV was distinctly down regulated in the AMD3100
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group (0.31± 0.02, n=5) when compared with the PBS group (0.45±0.03, n=5, p=0.001.) and the
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(0.16± 0.02 mm, n=5) than the PBS group (0.19±0.03 mg/cm3, n=5, p=0.124.) and the OA group
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(0.19± 0.03 mg/cm3, n=5, p=0.088.) (Sup. Fig.6a.), though no statistically significant difference was
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found. Tb.Sp.(0.40± 0.02, n=5 ) and SMI(1.4±0.16, n=5) of the AMD3100 group increased more
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significantly than those of the PBS group ( 0.30± 0.03, n=5, p=0.01; 0.067± 0.34, n=5, p=0.01,
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respectively) and the OA group (0.32±0.03, n=5, p=0.018; 0.074±0.38, n=5, p=0.018)(Sup. Fig. 6b,
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c).
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In addition, the result of immunohistochemistry staining with Nestin revealed a significant decreased
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in the numbers of Nestin positive cells in the subchondral bone marrow in the AMD3100 group
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(165.5 ± 23.9, n=5)when compared with the PBS group (237.3 ± 22.5, n=5, p=0.001) and the OA
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group (239.2 ± 14.9, n=5, p=0.001)(Fig. 8a,b).The number of Osterix-positive osteoprogenitors was
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also significantly more down regulated in the subchondral bone marrow in the AMD3100 group
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(221.1 ± 17.7, n=5) that the PBS group (347.7 ± 29.8,n=5, p<0.001) and the OA group (360.7 ±
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16.8,n=5, p<0.001)( Fig. 8a,c). However, the Col I was highly expressed in trabecular bone in all
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three groups (Sup. Fig. 3a) and the number of ALP-positive cells in the subchondral bone in the
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AMD3100 group (116.7 ± 7.535, n=5) showed no statistical differences with the PBS groups (122
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± 7.638, n=5, p>0.05) and the OA group (119.3 ± 9.528, n=5, p>0.05)(Sup. Fig. 4a,c.).
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DISCUSSION
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OA is a common and disabling degenerative disease and the pathological mechanisms
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involved in the progress of OA are not fully understood. In this study, we first found that the level of
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SDF-1 was elevated within the subchondral bone of the severe OA region(Fig. 3a). High level of
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SDF-1 would activate the Erk signaling and associate with abnormal changes of subchondral bone
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ACCEPTED MANUSCRIPT structure in clinical specimens (Fig. 1-3 and Sup. Fig 1-2,5). In our in-vitro studies, we have
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demonstrated that the activation of SDF-1via the Erk signaling pathway is critical to the osteogenic
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differentiation of MSCs (Fig 4-5). Finally, this study showed that inhibiting the SDF-1 signaling with
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AMD3100 could reduce the aberrant subchondral bone formation and attenuated articular cartilage
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degeneration in the rat OA model(Fig 6-8 and Sup. Fig 6).These results suggested that SDF-1 is an
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important regulator of subchondral bone remodeling and the resultant abnormal bone formation
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during the progress of OA and it is expected Inhibition of SDF-1may attenuate further progress of
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OA.
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In previous studies, it has been shown that SDF-1 could activate the Erk signaling and hence
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promote the osteogenic differentiation of MSCs [22, 23]. In this study, we found that the expression
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of SDF-1 was elevated in the subchondral bone of severe OA part clinical specimens. High level of
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SDF-1 in the subchondral bone may play a role in attracting stem cells and progenitor cells to the
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abnormally formed bone. In animal study, we showed that blocking the expression of SDF-1 would
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relieve cartilage degeneration which is consistent with a previous study[18], in which Wei. et al.
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showed that treating the guinea pigs OA model with using AMD3100 could reduce the secondary
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inflammation and attenuate cartilage damage. However, their group did not test whether this
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treatment could is beneficial to the subchondral bone. Subchondral bone is believed to play a crucial
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role in OA pathogenesis as it provided mechanical support to the overlying hyaline cartilage.
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Abnormal changes in the subchondral bone have been shown to be related to subchondral
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micro-fractures or micro-damages [31]. Subchondral sclerosis is commonly considered as an
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indisputable sign of OA[32, 33]. In the OA animal model, researchers showed that the BV/TV, BMD,
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and osteophytes were significantly elevated in the subchondral bone 6 weeks after the ACLT and
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MMx surgery[26, 34]. In the guinea pig spontaneous OA model and at the late stage of OA clinical
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ACCEPTED MANUSCRIPT specimens, the common micro-architectural characteristics of subchondral bone are: elevated BMD;
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increased BV/TV; increased Tb.Th.; decreased Tb.Sp.; thickened subchondral bone plateand
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switched SMI of trabecular transform from rod-like into plate-like[35, 36]. It is also found that in OA,
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the overlying calcified cartilage is thickened, with advancement and duplication of the tidemark,
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which contributes to articular cartilage thinning and deterioration[37]. In the present study, the high
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level of SDF-1 in the subchondral bone from the severe OA part was associated with abnormal
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changes including increased BMD, BV/TV, Tb.Th., while the Tb.Sp. was significantly decreased.
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Using the rat OA model, the abnormal formation of subchondral bone was attenuated in the
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AMD3100 treatment group, suggesting that SDF-1 could be a therapeutic target for OA treatment.
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Zhen and his colleagues demonstrated that transforming growth factor β1 (TGF-β1) was
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activated in the subchondral bone of OA mouse model in response to abnormal mechanical loading.
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High levels of TGF-β1 induced the formation of Nestin+ MSCs clusters, which leads to the
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formation of marrow osteoid islets accompanied by high levels of angiogenesis[38]. In our study, the
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immunohistochemical staining showed that the number of Nestin and the Osterix positive cells in the
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clinical specimens were significantly higher in the severe OA region than the RN region, and the
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AMD3100 treatments significantly reduced the number of Nestin and the Osterix positive cells in the
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subchondral bone of the rat OA model. Clustering of Osterix positive cells in the subchondral bone
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marrow, and consequently increased bone formation, have also been observed during the pathogens
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is knee OA in previous studies[39]. Therefore, our findings indicated that high level of SDF-1 in the
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subchondral bone contributes to the abnormal bone formation and blocking SDF-1 signal has
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therapeutic implication for OA.
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In this study, we tested the hypothesis that SDF-1 is activated in the subchondral bone in
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response to abnormal mechanical loading. High level of SDF-1 increased the numbers of 15
ACCEPTED MANUSCRIPT osteoprogenitors in the subchondral bone region, which may be responsible for the aberrant bone
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formation in OA. However, we did not exam whether relieving the abnormal mechanical loading
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could down regulate the level of SDF-1 using OA animal model [26]. Lastly, in the current study, we
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only applied ADM3100 to inhibit the SDF-1 signaling, and we did not test whether or not
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administrating the monoclonal antibody of SDF-1 to block SDF-1could also cause a similar result.
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Future researches should consider investigating these two models mentioned above to confirm the
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hypothesis.
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In conclusion, the results showed that high level of SDF-1 in the subchondral bone may induce
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abnormal changes of the subchondral bone in OA. Inhibition of SDF-1 signaling could be a potential
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new therapeutic approach for OA treatment.
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ACKNOWLEDGMENTS
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The work was partially supported by grants from National Natural Science Foundation of China
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(NSFC No.81371946) to Gang Li; Hong Kong Government Research Grant Council, General
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Research Fund (CUHK470813 and 14119115) and project grants from China Shenzhen City Science
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and Technology Bureau (GJHZ20140419120051680 and GJHZ20130418150248986) to Gang Li and
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Xiaohua Pan. This study was also supported in part by SMART program, Lui Che Woo Institute of
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Innovative Medicine, The Chinese University of Hong Kong and the research was made possible by
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resources donated by Lui Che Woo Foundation Limited.
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CONFLICTS OF INTEREST
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No conflicts of interest were stated.
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AUTHOR’S CONTRIBUTIONS
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Chen YF carried out the animal experiments, data collection, analysis and manuscript preparation.
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Lin S and Sun YX carried out the animal experiments; Guo J, Lu YF provided materials. Suen CW
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has contributed to editor the manuscript. Zhang JF and Zha ZG has contributed to data analysis. Ho
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KW, Pan XH and Li G have contributed to the funding for supporting this research project and
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supervised all the experiments. All authors reviewed and approved the manuscript.
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reporting: the ARRIVE guidelines for reporting animal research. Osteoarthritis Cartilage 2012; 20: 256-260. Mendez-Ferrer S, Michurina TV, Ferraro F, Mazloom AR, Macarthur BD, Lira SA, et al. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 2010; 466: 829-834. Wiese C, Rolletschek A, Kania G, Blyszczuk P, Tarasov KV, Tarasova Y, et al. Nestin expression--a property of multi-lineage progenitor cells? Cell Mol Life Sci 2004; 61: 25102522. Li G, Yin J, Gao J, Cheng TS, Pavlos NJ, Zhang C, et al. Subchondral bone in osteoarthritis: insight into risk factors and microstructural changes. Arthritis Res Ther 2013; 15: 223. Wang Y, Wluka AE, Pelletier JP, Martel-Pelletier J, Abram F, Ding C, et al. Meniscal extrusion predicts increases in subchondral bone marrow lesions and bone cysts and expansion of subchondral bone in osteoarthritic knees. Rheumatology (Oxford) 2010; 49: 997-1004. Wen C, Lu WW, Chiu KY. Importance of subchondral bone in the pathogenesis and management of osteoarthritis from bench to bed. Journal of Orthopaedic Translation 2014; 2: 16-25. Hayami T, Pickarski M, Zhuo Y, Wesolowski GA, Rodan GA, Duong LT. Characterization of articular cartilage and subchondral bone changes in the rat anterior cruciate ligament transection and meniscectomized models of osteoarthritis. Bone 2006; 38: 234-243. Ding M. Microarchitectural adaptations in aging and osteoarthrotic subchondral bone issues. Acta Orthop Suppl 2010; 81: 1-53. Ding M, Odgaard A, Danielsen CC, Hvid I. Mutual associations among microstructural, physical and mechanical properties of human cancellous bone. J Bone Joint Surg Br 2002; 84: 900-907. Goldring SR. Role of bone in osteoarthritis pathogenesis. Med Clin North Am 2009; 93: 2535, xv. Zhen G, Wen C, Jia X, Li Y, Crane JL, Mears SC, et al. Inhibition of TGF-beta signaling in mesenchymal stem cells of subchondral bone attenuates osteoarthritis. Nat Med 2013; 19: 704-712. Wang T, Wen CY, Yan CH, Lu WW, Chiu KY. Spatial and temporal changes of subchondral bone proceed to microscopic articular cartilage degeneration in guinea pigs with spontaneous osteoarthritis. Osteoarthritis Cartilage 2013; 21: 574-581.
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FIGURE LEGENDS
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Fig. 1. (a) Gross appearance of tibial plateau of the samples obtained from patients at the time of
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total joint arthroplasty. On the OA part, the cartilage was severely fibrillated or damaged and from
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themore affected compartment; and on the relatively normal part (RN), the cartilage was relatively
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normal or nonfibrillated which was from the uninvolved compartment. (b) Safranin-O / fast green
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and H&E staining of sagittal sections of the subchondral tibia in the OA and RN compartments of the 19
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same OA patient. Scale bar, 200 µm (in top), 50 µm (in bottom).
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Fig. 2.(a) 3D µCT images of the tibia subchondral bone compartment of the OA (left)and RN (right)
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area from the same OA patient. Scale bar, 1 mm. Quantitative analysis of structural parameters of
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subchondral bone by µCT test. (b) Bone mineral density (BMD), (c) bone volume/total tissue volume
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(BV/TV). The results showed that subchondral bone had significantly higher BMD, BV/TVin the OA
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compartment compared to RN compartment. n= 5 per group. *p < 0.05: the OA compartment
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compared to the RN compartment.
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Fig. 3. (a) ELISA assay showed the SDF-1 level in subchondral bone in the severe OA part and the
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RN part from the same patient (n = 7). The result showed that SDF-1 in the severe OA part was
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significantly higher than that of the RN part. The statistical significance was determined by pair
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sample t-test.*p < 0.05: the OA part vs. the RN part.
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(b)The phosphorylated (p)Erk1/2and GAPDH in subchondral bone lysates were detected by Western
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blotting using appropriate antibodies. The result showed that pErk1/2 was up-regulated in OA side
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cartilage compared to the RN side.
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Fig. 4. Immunofluorescent staining showed that MSCs which were Nestin-positive (green) and
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CXCR4-positive (red).Their nuclei were stained by DAPI (blue).Scale bars25 µm.
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Fig. 5.SDF-1 plays an important role in MSCs osteogenic differentiation. (a)Blocking the SDF-1
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signal reduced the ALP activity during MSCs osteogenic differentiation. MSCs were cultured in OIM
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or in normal culture medium as control. In parallel, cells were pretreated with AMD3100 at 400 µM
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ACCEPTED MANUSCRIPT for 2h at 37 °C prior to culture in OIM. The ALP staining assay in MSCs was detected at 5 days after
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cultured in OIM. The number of ALP positive cells (violet) were significantly reduced in the
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AMD3100 treated group compared to the OIM treated group. Scale bars, 200 µm.
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(b) Blocking SDF-1 signal inhibited intracellular Erk phosphorylation in MSCs osteogenic
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differentiation. The MSCs were pretreated with CXCR4 antagonist AMD3100 at400 µM for 2 h at
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37 °C prior to culture in OIM for 15 min; the controls were culturedin normal culture media or OIM. .
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Total Erk1/2, the phosphorylated (p) Erk1/2, and GAPDH were detected in cell lysates by Western
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blot. Level of phosphorylated (p) Erk1/2 was down-regulated in the AMD3100 treated group
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compared to the OIM group.
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Fig.6. AMD3100 attenuates articular cartilage degeneration in ACLT + MMx rat. Safranin O and fast
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green staining of sagittal sections of the subchondral femoramedial compartment in the AMD3100
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treatment group, PBS group and OA group. Scale bar, 400 µm (in top), 200 µm (in bottom). The
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result showed that cartilage degeneration was less severe in the AMD3100 group, compared to the
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PBS or OA group.
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Fig.7. AMD3100 reduces aberrant subchondral bone formation in a rat OA model. (a)3D µCT
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images of the tibia subchondral bone compartment (sagittal view) of rats in the AMD3100 group,
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PBS group and OA group. Scale bar, 1 mm.
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Quantitative assay of structural parameters of subchondral bone by µCT. (b)BMD, (c) BV/TV. The
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results showed that subchondral bone had significantly reduce BMD, BV/TV in the AMD3100
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treated group compared to PBS group or OA group. n= 5 per group. *p < 0.05: the AMD3100 group
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compared to the PBS or OA group.
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Fig.8.(a) Immunohistochemical analysis and quantification of Nestin(b) and Osterix (c) positive cells
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(brown) in the tibial subchondral region. The result showed that AMD3100 treatment would
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significantly reduced Nestin and Osterix positive cells compared to the other two groups. Scale bars,
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50 µm. *p < 0.05, the AMD3100 group compared to the PBS or OA group.
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SUPPLEMENTARY FIGURE LEGENDS
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Sup. Fig.1. Quantitative assay of structural parameters of subchondral bone by µCT.(a) trabecula
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bone thickness (Tb.Th.), (b) trabecula bone space (Tb.Sp.) and (c) structure model index (SMI) in
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subchondral bone determined by µCT. n= 5 per group. The results showed that subchondral bone had
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significantly higher Tb.Th. in the OA compartment compared to RN compartment, while Tb.Sp. and
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SMI had reduce significantly. *p<0.05: the OA compartment compared to RN compartment.
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Sup. Fig.2.(a) Immunohistochemical analysis and quantification (b,c) of Nestin and Osterix positive
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cells (brown) in the subchondral region of clinical specimen. The result showed that in OA part the
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number of Nestin and Osterix positive cells were significantly reduced compared to the RN part.
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Scale bars, 50 µm. *p < 0.05: the OA part compared to the RN part. SB = subchondral bone; BM
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=subchondral bone marrow.
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Sup. Fig.3. Immunohistochemical analysis of Type I collagen in the subchondral region of animal (a)
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and clinical specimen (b). The result showed that Type I collagen was highly expressed in trabecula
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bone. Scale bar in a. is 100µm; in b. is 200µm.
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(brown) in the subchondral region of animal and clinical specimen. The results showed that ALP
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positive cells were found in subchondral bone in both the clinical (b) and rat samples (a), but not
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statistical differences between the OA and the control samples (c, d). Scale bar in a. is 100µm; in b.
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is 200µm.
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Sup. Fig.5. Immunohistochemical analysis of CXCR4 positive cells (brown) in the subchondral
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region of clinical specimen. The result showed that in OA part the number of CXCR4 positive cells
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was significantly increased compared to the RN part. Scale bar in a. is 400µm; in b. is 200µm.
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Sup. Fig.6.Quantitativeassay of structural parameters of subchondral bone by µCT. (a) Tb.Th., (b)
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Tb.Sp.and (c) SMI.in subchondral bone determined by µCT. n= 5 per group. The results showed that
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subchondral bone had significantly reducedTb.Th. in the AMD3100 treated group compared to PBS
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group or OA group, while Tb.Sp. and SMI had increased significantly.*p < 0.05: the AMD3100
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group compared to the PBS or OA group
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S1063-4584(17)30034-1
DOI:
10.1016/j.joca.2017.01.008
Reference:
YJOCA 3940
To appear in:
Osteoarthritis and Cartilage
Received Date: 21 July 2016 Revised Date:
28 December 2016
Accepted Date: 17 January 2017
Please cite this article as: Chen Y, Lin S, Sun Y, Guo J, Lu Y, Suen CW, Zhang J, Zha Z, Ho KW, Pan X, Li G, Attenuation of subchondral bone abnormal changes inosteoarthritis by inhibition of SDF-1 signaling, Osteoarthritis and Cartilage (2017), doi: 10.1016/j.joca.2017.01.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Attenuation of subchondral bone abnormal changes inosteoarthritis by
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inhibition of SDF-1 signaling
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Chen Yuanfeng1,2,3, Lin Sien2,3, Sun Yuxin2,3, Guo Jia2, Lu Yingfei2, Suen Chun Wai2,3, Zhang
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* * * Jinfang2,3, Zha Zhengang1, Ho Ki Wai2 , Pan Xiaohua4 , Li Gang2,3,5
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1. Institute of Orthopedic Diseases and Department of Orthopedics, the First Affiliated Hospital,
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Jinan University, Guangzhou, China
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2. Department of Orthopaedics& Traumatology, Li Ka Shing Institute of Health Sciences and Lui
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Che Woo Institute of Innovative Medicine, Faculty of Medicine, The Chinese University of Hong
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Kong, Prince of Wales Hospital, Shatin, Hong Kong SAR, PR China.
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3. The CUHK-ACC Space Medicine Centre on Health Maintenance of Musculoskeletal System, The
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Chinese University of Hong Kong Shenzhen Research Institute, Shenzhen, PR China.
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4. Department of Orthopaedics and Traumatology, Bao-An District People’s Hospital, Shenzhen, PR
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China.
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5. Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences,
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Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, PR China.
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* Correspondence: [email protected]; [email protected] or [email protected]
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Abstract: Background: Current conservative treatments for osteoarthritis (OA) are largely
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symptoms control therapies. Further understanding on the pathological mechanisms of OA is crucial
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for new pharmacological intervention. Objective: In this study, we investigated the role of Stromal
29
cell-derived factor-1(SDF-1) in regulating subchondral bone changes during the progression of
30
osteoarthritis. Methods: Clinical samples of different stages of OA severity were analyzed by
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histology staining, micro-CT, enzyme-linked immunosorbent assay (ELISA) and western blotting, to
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compare SDF-1 level in subchondral bone. The effects of SDF-1 on human mesenchymal stem cells
33
(MSCs) osteogenic differentiation were evaluated. In vivo assessment was performed in an anterior
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cruciate ligament transaction plus medial meniscus resection in the SD rats. The OA rats received
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continuous infusion of AMD3100 (SDF-1 receptor blocker) in osmotic mini-pump implanted
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subcutaneously for 6 weeks. These rats were then terminated and subjected to the same in vitro
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assessments as human OA samples. Results: SDF-1 level was significantly elevated in the
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subchondral bone of human OA samples. In the cell studies, the results showed SDF-1 plays an
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important role in osteogenic differentiation of MSCs. In the OA animal studies, there were less
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cartilage damage in the AMD3100-treated group; microCT results showed that the subchondral bone
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formation was significantly reduced and so did the number of positive Nestin or Osterix cells in the
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subchondral bone region. Conclusions: Higher level of SDF-1 may induce the subchondral bone
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abnormal changes in osteoarthritis and inhibition of SDF-1 signaling could be a potential therapeutic
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approach for OA.
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Keywords: Stromal cell-derived factor-1, osteoarthritis, subchondral bone.
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INTRODUCTION Osteoarthritis (OA) is one of the leading cause of physical disability affecting nearly 80% of the
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individuals aged 75 years or over[1]. Current pharmacologic therapies mainly target at symptoms
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controlling and their efficacy in altering the progression of OA are disappointing. Further
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understanding of the pathological mechanisms of OA development is crucial for the design of the
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new pharmacological intervention.
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Various inflammatory cytokines including interleukin-1β(IL-1β), tumor necrosis factor-α (TNF-
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α)and NF-κB signal, attribute to the development and progress of OA[2-8]. Articular cartilage
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degeneration is the main feature in OA and many factors such as matrix metalloproteinase 13
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(MMP13)[9], collagen fragments (C2C)[10] as well asnerve growth factor (NGF)[11] are involved.
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The homeostasis and integrity of articular cartilage rely on its biochemical and biomechanical
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interplays with the subchondral bone and the surrounding soft tissues[12]. Subchondral bone
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provides mechanical support for the articular cartilage and constantly undergoes bone
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remodeling[13]. Bone marrow lesions are closely associated the severity of cartilage damage and
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pain in OA [14]. However, the relationship between the abnormal formation of subchondral bone and
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progression of OA still remains largely unknown.
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The role of stromal derived factor-1 (SDF-1) in the pathogenesis of OA or rheumatoid arthritis
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(RA) has drawn increasing attention in recent years[4, 15-17]. A dramatic elevation of SDF-1 is
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found in the knee synovium from RA and OA patients[4]. SDF-1 has been shown that it could
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regulate chondrocyte catabolic activities by stimulating the release of MMP-3 and MMP-13[4].
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Synovectomy could significantly reduce the concentrations of SDF-1, MMP-9, and MMP-13 in
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blood serum[15]. In animal study, blockage of SDF-1 signaling pathway using AMD3100, a CXCR4
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antagonist of the SDF-1 receptor, attenuated the cartilage degeneration [18]. SDF-1 is a well-known
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osteogenic differentiation of MSCs[22, 23]. Furthermore, in 2006 Lisignoli G and colleagues
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demonstrated that SDF-1 could significantly induce proliferation and collagen type I expression in
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osteoblasts from OA patients[17]. This study implies that SDF-1 may play a role in the abnormal
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changes in subchondral bone with OA. SDF-1 influences cartilage degeneration through stimulating
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the release of MMPs from chondrocytes, however, whether SDF-1 contributes to subchondral bone
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changes in OA is still not fully understood. Therefore, the objective of this study is to investigate the
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role of SDF-1 in subchondral bone changes during OA development.
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MATERIALS AND METHODS
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Clinical sample collection
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This study was approved by the Joint Chinese University of Hong Kong-New Territories East
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Cluster Clinical Research Ethics Committee and informed consent was obtained from each donor. All
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experiments were carried out in accordance with the research guidelines of the Chinese University of
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Hong Kong. The clinical specimens were obtained from patients with OA at the time of total knee
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arthroplasty surgery (n = 12; 8 women and 4 men; age 65.8±6.5 years, range 49–76 years). Various
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regions of the knee joint were harvested and the samples were immediately placed into sterile
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DMEM culture medium and transported to the laboratory for further processing. Samples were
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divided into two parts; OA group where cartilage was severely damaged or fibrillated with OARSI
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score of 15–18; and relatively normal group, where the cartilage is non-fibrillated with OARSI
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scores of 0–3. The cartilage and subchondral bone explants were cut from OA or normal sites of the
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OA joint (Fig. 1A) were cut into the size of 1.5×0.5 cm full-thickness and subjected to future
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experiments.
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Bone marrow MSCs culture Human fetal bone marrow stem cells (hBM-MSCs) were obtained from the Stem Cell Bank
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at the Prince of Wales Hospital of the Chinese University of Hong Kong. Ethical approval was
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obtained from the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical
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Research Ethics Committee (ethical approval code: CRE-2011.383). Informed written consent form
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was approved by the Clinical Research Ethics Committee and signed by the donor before sample
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collection. All experiments were carried out in accordance with the research guidelines of the
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Chinese University of Hong Kong. These cells were cultured in the complete alpha-minimum
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essential medium (α-MEM, Manassas, Virginia, USA) supplemented with 10% fetal bovine serum
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(FBS) and 1% penicillin-streptomycin-neomycin (complete culture medium; all from Invitrogen
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Corporation, Carlsbad, CA, USA) in a 5% CO2 humidified incubator at 37 0 C.
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In MSCs osteogenic induction, the MSCs were trypsinized and seeded in 6-well plate at a
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concentration of 1x105 cells per well. These cells were incubated in the ɑ-MEM for two or three days,
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the medium was then replaced by osteogenic induction medium (OIM) which contains100 nmol/L
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dexamethasone, 10 mmol/L beta-glycerophosphate and 0.05 mmol/Ll-ascorbic acid-2-phosphate.
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To block the SDF-1 signaling in primary MSCs, cells were incubated with AMD3100, a
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CXCR4 antagonist, which selectively binds to CXCR4 and prevents the binding of SDF-1[24]. The
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working concentration of AMD3100(Sigma, StLouis, MO, USA) in this study was 400 µM, which
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has been shown to effectively inhibit SDF-1 signaling without toxicity[22-24].
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Animal experiments All experiments were approved by the Animal Research Ethics Committee, the Chinese
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ACCEPTED MANUSCRIPT University of Hong Kong. 16-week old Male Sprague-Dawley (SD) rats, with the weight of 450-500
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g, were used in this study. All rats received anterior cruciate ligament transaction plus medial
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meniscus resection (ACLT + MMx) at the right knee as previously described[25]. In brief, each rat
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was anesthetized by administrating 0.2%(vol/vol) xylazine and 1% (vol/vol) ketamine, after being
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shaved and disinfected, the right knee joint was exposed through a medial parapatellar arthrotomy
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approach. The patella was dislocated laterally and the knee was placed in full flexion followed by
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ACL and MCL transection with micro-scissors and resection of the medial meniscus.
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Rats were allocated randomly into three groups: via osmotic mini-pump, the first group received
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a continuous infusion of the AMD3100 (Sigma, St.Louis, MO, USA) (n=5) and the second group
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received PBS(n=5). Just after the ACLT+MMx surgery, the Mini-osmotic pump (model 2006; Alza
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Corporation, Mountain View, CA, USA.) was inserted into the small subcutaneous pocket over the
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dorsolateral thorax, and the pocket is created by blunt dissection with a small incision (~1 cm).
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Before insertion, the 200 µl pump reservoir was filled with PBS containing 22.3 mg/ml AMD3100 or
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just PBS. The average pumping rate of the mini-osmotic pump is 0.15 µl/per hour, thus AMD3100 or
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PBS was systemically administrated to the rats during the entire experiment. For the last group, rats
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were left untreated as the OA control (n = 5). After 6 weeks of treatment, all animals were
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euthanized and their right knees were harvested for further examinations.
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ELISA examination of the clinical specimens
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Subchondral bone explants were cut off (0.5x0.5cm3) from the OA region and the relatively
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normal region of the same specimen; they were then immediately placed on dry ice. The explant was
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weighed, mechanically homogenized, ground into powder with addition of liquid nitrogen, and then
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ice-cold tissue protein extraction reagents (Life Technologies, Pleasanton, CA, USA) were added.
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Homogenate was centrifuged for 10 minutes at 14,000 rpm at 4°C and used for SDF-1 ELISA
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examination according to the instruction of the kit(Sigma, StLouis, MO, USA).
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Western blot The lysates of subchondral bone and the cultured MSCs were subject to Western blot
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examination. Before tested by following assays, MSCs were pretreated with AMD3100 at 400 µM
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for 2 hours at 37 °C prior to culture in osteogenic inductive media (OIM); the controls were cultured
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for 15 minutes in either normal culture medium (ɑ-MEM) or OIM. The cells were then washed with
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cold PBS twice and harvested by scraping in cell extraction buffer (Invitrogen, Cat. no.FNN0011).
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Protein concentration was determined using Bradford method (Biorad, Richmond, CA, USA). An
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equal amount of proteins was loaded onto 10%Tris/glycine gels for electrophoresis and then
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transferred to a PVDF membrane (Millipore, Bedford, MA, USA). It was then blocked in 5% nonfat
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milk (Biorad, Richmond, CA, USA) for 1h at room temperature with rocking. Reagents such as the
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primary antibody, anti-p-ERK (1:1000, BD Biosciences, USA), anti-total Erk1/2(1:8000, BD
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Biosciences, USA), anti-GAPDH (1:10000, Santa Cruz, USA) was respectively added and incubated
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for 2 hours room temperature or at 40C overnight. After washing in TBS for three times (5 minutes
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for each washing), the membrane was incubated with horseradish peroxidase-linked secondary
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antibodies.
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Alkaline phosphatase (ALP) activity assays
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MSCs were cultured in osteogenic induced medium (OIM) or in the normal culture medium as
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acontrol. In parallel, cells were pretreated with AMD3100 at400 µM for 2 hours at 37 °C prior to
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culture in OIM. The ALP staining was performed using alkaline phosphatase (alp)-amp (Biosystems,
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Foster City, CA, USA), according to the instructions from the manufacturer. ALP staining assays
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were observed under light microscope (Leica DMRB, Leica Cambridge Ltd., UK).
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Micro-CT Assessment Subchondral bone explants from the clinical specimens and the rat specimens were fixed over
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night in 10% formalin. These were then analyzed using high-resolution µCT (µCT40, Scanco
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Medical, Basserdorf, Switzerland). Three-dimensional (3D) reconstructions of mineralized tissues
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were performed by an application of a global threshold (180 for theclinical specimens and 158 for
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the animalspecimens, in mg hydroxyapatite/cm3), and a Gaussian filter (sigma = 0.8, support = 2)
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was used to suppress noise. For the clinical sample, the 3D images showed the whole subchondral
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bone explants; and for the animal study, 100 sagittal images of the tibiae subchondral bone were used
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to perform the 3D histomorphometric analysis. 3D structural parameters analysis included: bone
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mineral density (BMD), bone volume/total tissue volume (BV/TV), Tb.Th (trabecular thickness),
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Tb.Sp (trabecular separation), SMI (structure model index).
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Histological and immunochemical examinations
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The right knee explants of the clinical specimen and the rat specimen were fixed in 10%
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formalin for 48 hours, followed by decalcification in 10% EDTA for 21 days before embedding them
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in paraffin. In animal study, serial sagittal-oriented sections of the knee joint (medial compartment)
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were cut at intervals of 0 µm, 100 µm, and 200 µm before mounted onto glass slides. Five-
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micrometer-thick sagittal-oriented sections of the sample were processed for Safranin-O/fast green
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and H&E staining.
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Immunostaining was performed using a standard protocol as previously reported[26, 27]. We
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incubated sections with primary antibodies to rabbit Nestin (Sigma, 1:300, N5413), Osterix (Abcam,
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ACCEPTED MANUSCRIPT 1:600, ab22552),CXCR4 (R&D, 1:200, MAB171), ALP (Abcam, 1:200, ab54778), Col I (Santa Cruz,
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1:200, sc-8784), overnight at 4°C. For immunohistochemical staining, a horse radish peroxidase–
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streptavidin detection system (Dako,Carpinteria, CA, USA) was used, followed by counterstaining
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with hematoxylin. Photographs of the selected areas were taken under a light microscope. We
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counted the number of positively stained cells in three randomized areas of the sections in the human
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specimens; or in the whole subchondral bone area in three sequential sections (0 µm, 100 µm, and
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200 µm) per specimen, and the numbers were compared statistically.
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For immunofluorescent staining, the MSCs were fixed in 4% paraformaldehyde for
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10minutes and then washed with PBS twice. The treated MSCs were incubated with primary
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antibodies to rabbit Nestin (Sigma, 1:300, N5413) and mouse CXCR4 (R&D, 1:300, MAB171)
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overnight at 4°C.
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CY3(LifeTechnologies, Pleasanton, CA, USA. 1:800)or goat anti-rabbit Alex 488(LifeTechnologies,
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Pleasanton, CA, USA. 1:300) were added, and slides were incubated at room temperature for 1 hour
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in the dark. Photographs of the selected areas were taken under a microscope.
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Statistical analysis
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In accordance with the ARRIVE guidelines[28], we have reported measures of precision,
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confidence, and n number to provide an indication of significance. All statistical analyses were
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performed using the statistical software SPSS15.0. The data of rat samples were analyzed using one-
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way ANOVA, while data of clinical samples studies were tested by Pair sample T-test. In one-way
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ANOVA tested, the assumptions of the analysis were assessed by the Shapiro-Wilk test of normality
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and Levene’s test for homogeneity of variance. The result of Levene's test was used to determine the
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post hoc testing strategy. If the result is significant, Dunnett’s T3 post hoc test for unequal variance
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was used, while LSD-t post hoc test was employed if there is no significant result. Values of p< 0.05
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were considered significant. Data was reported as mean±standard deviation. The graphs were
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generated in GraphPad Prism 6(GraphPad Software, San Diego, CA, USA).
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RESULTS
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Elevated SDF-1 levels in subchondral bone in the severe OA part of the clinical specimen
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The gross macroscopic observation showed that in the severe OA region of the samples (on the
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left, Fig. 1a.), the cartilage was almost worn out on the left side and the surrounding area showed
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rough articular cartilage surfaces. In the adjacent relatively normal (RN) region(on the right, Fig. 1a.),
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the amount of cartilage damage was much less pronounced and the articular surface was relatively
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smooth. Moreover, inSafranin-O/fast green and H&E staining showed that proteoglycan loss was
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aggravated inthe severe OA region rather than inthe RN region(Fig. 1b.). H&E staining showed that
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the thickness of the hyaline cartilage zone in the RN region was much greater than that in the severe
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OA part (Fig. 1b.).
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The 3D reconstructed images of µCT showed that the micro-architecture of the subchondral
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bone changed significantly at end-stage of OA. There was also an obvious difference between the
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worn sides of the OA region compared to that of the RN region(Fig. 2a.). Bone mineral density
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(BMD) in the OA region(282.3 ± 75.7 mg/cm3, n=5) had significantly increased than that of the RN
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region(144.9± 35.8 mg/cm3, n=5, p=0.023.)(Fig. 2b.). Similarly, bone volume/total tissue volume
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(BV/TV) up-regulated distinctly in the OA region(0.38± 0.10, n=5) when compared with the RN
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region(0.19± 0.05, n=5, p=0.017) (Fig. 2c.). Moreover, trabecular bone thickness (Tb.Th.) in the
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OA region(0.27 ± 0.07 mm, n=5) was higher than that of the RN region(0.16 ± 0.04 mg/cm3, n=5,
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p=0.074.) (Sup. Fig. 1a.), though no statistically significantdifference was found. Trabecular bone
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space (Tb.Sp.)(0.36 ± 0.14, n=5) and structure model index (SMI)(-0.04± 1.7, n=5) in the OA
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region showed stronger decrease than the RN region(0.55± 0.10, n=5, p=0.028; 0.91± 0.67, n=5,
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p=0.027) (Sup. Fig. 1b,c.). The result of immunohistochemistry staining with Nestin, a marker of adult bone marrow
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MSCs[29, 30], showed a significant increase in the numbers of Nestin-positive cells in the
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subchondral bone marrow cavity at the severe OA region(137.3± 27.6, n=5) when compared with
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the RN region(55.0±9.6, n=5, p=0.009) (Sup.Fig. 2a,b.). Osterixis a marker of osteoprogenitors and
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the number of Osterix-positive osteoprogenitors in the subchondral bone in the OA side(176.3±12.0,
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n=5) was significantly higher than the RN side(60.6±16.8,n=5, p=0.019)(Sup. Fig. 2a,c.). However,
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the type I collagen was highly expressed in the trabecular bone in both groups(Sup. Fig.3b.) and the
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number of ALP-positive cells in the subchondral bone showed no statistical difference between OA
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side(91.33±8.212, n=5) and RN side.(88±8.712, n=5, p>0.05)(Sup. Fig. 4b,d.) In addition, we also
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found that the number of the CXCR4-positive cells was significantly higher in the OA side of the
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subchondral bone(Sup. Fig.5.).
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ELISA assay showed that SDF-1 was elevated in the severe OA region of the clinical samples.
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The level of SDF-1in the severe OA region was 424.3 ± 244.1 pg/ml, (n=7); which was
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significantly increased when compared with the RN region (328.6 ± 251.7 pg/ml, n=7,
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p=0.025)( Fig. 3a.) Since the phosphorylation of Erk1/2 can be activated by SDF-1 in the osteogenic
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differentiated MSCs, we examined whether the high level of SDF-1 may activate the
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phosphorylation of Erk in the severe OA part of clinical specimens. The results from western blot
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showed that the amount of pErk1/2 was significantly higher in the severe OA region(Fig. 3b.).
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In summary, the elevated SDF-1 signaling in the subchondral bone is associated with the changes in the subchondral bone structure in the clinical OA specimens.
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SDF-1 plays an important role in MSCs osteogenic differentiation Immunofluorescent staining showed that Nestin positive (green) MSCs expressed CXCR4
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(red), a receptor of SDF-1(Fig. 4.). After culturing the MSCs in OIM for 5 days, stainings showed
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significantly increased ALP activity, while it was not obvious in the control cells cultured in the
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normal medium. AMD3100 is a CXCR4 antagonist which blocks the SDF-1 signal and pretreating it
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to the cells could reduce the OIM-induced ALP activity significantly when compared with the
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untreated cells (Fig. 5a.). The results from western blot also showed that level of pErk1/2 was down-
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regulated only in the AMD3100-pretreated group and this suggests that perturbing the MSCs with
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SDF-1 signal could inhibit intracellular Erk phosphorylation in osteogenic differentiation of
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MSCs(Fig. 5b.).
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Inhibition of SDF-1 signaling reduces aberrant subchondral bone formation and attenuates
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articular cartilage degeneration in ACLT + MMx rat
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Safranin-O/fast green staining showed that there were significantly more degenerative features
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in the PBS-treated and the OA groups than the AMD3100-treated group (Fig. 6). Three-dimensional
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µCT images of cross-sectional, sagittal views of tibia subchondral bone from rats showed that the
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abnormal bone formation feature was more down regulated in the subchondral bone of the
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AMD3100 group (Fig. 7a). Structural parameters of subchondral bone were tested by µCT as a
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quantitative assay. BMD in the AMD3100 group (545.9±9.5 mg/cm3, n=5) was significantly lower
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than that of the PBS group (619.8±19.5 mg/cm3, n=5, p=0.001.) and the OA group(607.7 ± 33.5
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mg/cm3, n=5, p=0.003.)(Fig. 7b.). Moreover, BV/TV was distinctly down regulated in the AMD3100
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group (0.31± 0.02, n=5) when compared with the PBS group (0.45±0.03, n=5, p=0.001.) and the
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(0.16± 0.02 mm, n=5) than the PBS group (0.19±0.03 mg/cm3, n=5, p=0.124.) and the OA group
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(0.19± 0.03 mg/cm3, n=5, p=0.088.) (Sup. Fig.6a.), though no statistically significant difference was
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found. Tb.Sp.(0.40± 0.02, n=5 ) and SMI(1.4±0.16, n=5) of the AMD3100 group increased more
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significantly than those of the PBS group ( 0.30± 0.03, n=5, p=0.01; 0.067± 0.34, n=5, p=0.01,
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respectively) and the OA group (0.32±0.03, n=5, p=0.018; 0.074±0.38, n=5, p=0.018)(Sup. Fig. 6b,
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c).
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In addition, the result of immunohistochemistry staining with Nestin revealed a significant decreased
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in the numbers of Nestin positive cells in the subchondral bone marrow in the AMD3100 group
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(165.5 ± 23.9, n=5)when compared with the PBS group (237.3 ± 22.5, n=5, p=0.001) and the OA
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group (239.2 ± 14.9, n=5, p=0.001)(Fig. 8a,b).The number of Osterix-positive osteoprogenitors was
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also significantly more down regulated in the subchondral bone marrow in the AMD3100 group
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(221.1 ± 17.7, n=5) that the PBS group (347.7 ± 29.8,n=5, p<0.001) and the OA group (360.7 ±
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16.8,n=5, p<0.001)( Fig. 8a,c). However, the Col I was highly expressed in trabecular bone in all
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three groups (Sup. Fig. 3a) and the number of ALP-positive cells in the subchondral bone in the
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AMD3100 group (116.7 ± 7.535, n=5) showed no statistical differences with the PBS groups (122
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± 7.638, n=5, p>0.05) and the OA group (119.3 ± 9.528, n=5, p>0.05)(Sup. Fig. 4a,c.).
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DISCUSSION
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OA is a common and disabling degenerative disease and the pathological mechanisms
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involved in the progress of OA are not fully understood. In this study, we first found that the level of
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SDF-1 was elevated within the subchondral bone of the severe OA region(Fig. 3a). High level of
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SDF-1 would activate the Erk signaling and associate with abnormal changes of subchondral bone
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demonstrated that the activation of SDF-1via the Erk signaling pathway is critical to the osteogenic
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differentiation of MSCs (Fig 4-5). Finally, this study showed that inhibiting the SDF-1 signaling with
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AMD3100 could reduce the aberrant subchondral bone formation and attenuated articular cartilage
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degeneration in the rat OA model(Fig 6-8 and Sup. Fig 6).These results suggested that SDF-1 is an
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important regulator of subchondral bone remodeling and the resultant abnormal bone formation
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during the progress of OA and it is expected Inhibition of SDF-1may attenuate further progress of
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OA.
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In previous studies, it has been shown that SDF-1 could activate the Erk signaling and hence
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promote the osteogenic differentiation of MSCs [22, 23]. In this study, we found that the expression
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of SDF-1 was elevated in the subchondral bone of severe OA part clinical specimens. High level of
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SDF-1 in the subchondral bone may play a role in attracting stem cells and progenitor cells to the
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abnormally formed bone. In animal study, we showed that blocking the expression of SDF-1 would
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relieve cartilage degeneration which is consistent with a previous study[18], in which Wei. et al.
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showed that treating the guinea pigs OA model with using AMD3100 could reduce the secondary
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inflammation and attenuate cartilage damage. However, their group did not test whether this
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treatment could is beneficial to the subchondral bone. Subchondral bone is believed to play a crucial
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role in OA pathogenesis as it provided mechanical support to the overlying hyaline cartilage.
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Abnormal changes in the subchondral bone have been shown to be related to subchondral
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micro-fractures or micro-damages [31]. Subchondral sclerosis is commonly considered as an
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indisputable sign of OA[32, 33]. In the OA animal model, researchers showed that the BV/TV, BMD,
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and osteophytes were significantly elevated in the subchondral bone 6 weeks after the ACLT and
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MMx surgery[26, 34]. In the guinea pig spontaneous OA model and at the late stage of OA clinical
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increased BV/TV; increased Tb.Th.; decreased Tb.Sp.; thickened subchondral bone plateand
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switched SMI of trabecular transform from rod-like into plate-like[35, 36]. It is also found that in OA,
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the overlying calcified cartilage is thickened, with advancement and duplication of the tidemark,
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which contributes to articular cartilage thinning and deterioration[37]. In the present study, the high
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level of SDF-1 in the subchondral bone from the severe OA part was associated with abnormal
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changes including increased BMD, BV/TV, Tb.Th., while the Tb.Sp. was significantly decreased.
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Using the rat OA model, the abnormal formation of subchondral bone was attenuated in the
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AMD3100 treatment group, suggesting that SDF-1 could be a therapeutic target for OA treatment.
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Zhen and his colleagues demonstrated that transforming growth factor β1 (TGF-β1) was
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activated in the subchondral bone of OA mouse model in response to abnormal mechanical loading.
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High levels of TGF-β1 induced the formation of Nestin+ MSCs clusters, which leads to the
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formation of marrow osteoid islets accompanied by high levels of angiogenesis[38]. In our study, the
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immunohistochemical staining showed that the number of Nestin and the Osterix positive cells in the
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clinical specimens were significantly higher in the severe OA region than the RN region, and the
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AMD3100 treatments significantly reduced the number of Nestin and the Osterix positive cells in the
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subchondral bone of the rat OA model. Clustering of Osterix positive cells in the subchondral bone
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marrow, and consequently increased bone formation, have also been observed during the pathogens
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is knee OA in previous studies[39]. Therefore, our findings indicated that high level of SDF-1 in the
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subchondral bone contributes to the abnormal bone formation and blocking SDF-1 signal has
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therapeutic implication for OA.
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In this study, we tested the hypothesis that SDF-1 is activated in the subchondral bone in
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response to abnormal mechanical loading. High level of SDF-1 increased the numbers of 15
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formation in OA. However, we did not exam whether relieving the abnormal mechanical loading
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could down regulate the level of SDF-1 using OA animal model [26]. Lastly, in the current study, we
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only applied ADM3100 to inhibit the SDF-1 signaling, and we did not test whether or not
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administrating the monoclonal antibody of SDF-1 to block SDF-1could also cause a similar result.
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Future researches should consider investigating these two models mentioned above to confirm the
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hypothesis.
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In conclusion, the results showed that high level of SDF-1 in the subchondral bone may induce
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abnormal changes of the subchondral bone in OA. Inhibition of SDF-1 signaling could be a potential
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new therapeutic approach for OA treatment.
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ACKNOWLEDGMENTS
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The work was partially supported by grants from National Natural Science Foundation of China
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(NSFC No.81371946) to Gang Li; Hong Kong Government Research Grant Council, General
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Research Fund (CUHK470813 and 14119115) and project grants from China Shenzhen City Science
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and Technology Bureau (GJHZ20140419120051680 and GJHZ20130418150248986) to Gang Li and
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Xiaohua Pan. This study was also supported in part by SMART program, Lui Che Woo Institute of
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Innovative Medicine, The Chinese University of Hong Kong and the research was made possible by
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resources donated by Lui Che Woo Foundation Limited.
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CONFLICTS OF INTEREST
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No conflicts of interest were stated.
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AUTHOR’S CONTRIBUTIONS
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Chen YF carried out the animal experiments, data collection, analysis and manuscript preparation.
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Lin S and Sun YX carried out the animal experiments; Guo J, Lu YF provided materials. Suen CW
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has contributed to editor the manuscript. Zhang JF and Zha ZG has contributed to data analysis. Ho
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KW, Pan XH and Li G have contributed to the funding for supporting this research project and
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supervised all the experiments. All authors reviewed and approved the manuscript.
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FIGURE LEGENDS
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Fig. 1. (a) Gross appearance of tibial plateau of the samples obtained from patients at the time of
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total joint arthroplasty. On the OA part, the cartilage was severely fibrillated or damaged and from
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themore affected compartment; and on the relatively normal part (RN), the cartilage was relatively
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normal or nonfibrillated which was from the uninvolved compartment. (b) Safranin-O / fast green
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and H&E staining of sagittal sections of the subchondral tibia in the OA and RN compartments of the 19
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same OA patient. Scale bar, 200 µm (in top), 50 µm (in bottom).
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Fig. 2.(a) 3D µCT images of the tibia subchondral bone compartment of the OA (left)and RN (right)
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area from the same OA patient. Scale bar, 1 mm. Quantitative analysis of structural parameters of
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subchondral bone by µCT test. (b) Bone mineral density (BMD), (c) bone volume/total tissue volume
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(BV/TV). The results showed that subchondral bone had significantly higher BMD, BV/TVin the OA
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compartment compared to RN compartment. n= 5 per group. *p < 0.05: the OA compartment
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compared to the RN compartment.
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Fig. 3. (a) ELISA assay showed the SDF-1 level in subchondral bone in the severe OA part and the
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RN part from the same patient (n = 7). The result showed that SDF-1 in the severe OA part was
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significantly higher than that of the RN part. The statistical significance was determined by pair
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sample t-test.*p < 0.05: the OA part vs. the RN part.
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(b)The phosphorylated (p)Erk1/2and GAPDH in subchondral bone lysates were detected by Western
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blotting using appropriate antibodies. The result showed that pErk1/2 was up-regulated in OA side
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cartilage compared to the RN side.
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Fig. 4. Immunofluorescent staining showed that MSCs which were Nestin-positive (green) and
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CXCR4-positive (red).Their nuclei were stained by DAPI (blue).Scale bars25 µm.
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Fig. 5.SDF-1 plays an important role in MSCs osteogenic differentiation. (a)Blocking the SDF-1
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signal reduced the ALP activity during MSCs osteogenic differentiation. MSCs were cultured in OIM
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or in normal culture medium as control. In parallel, cells were pretreated with AMD3100 at 400 µM
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cultured in OIM. The number of ALP positive cells (violet) were significantly reduced in the
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AMD3100 treated group compared to the OIM treated group. Scale bars, 200 µm.
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(b) Blocking SDF-1 signal inhibited intracellular Erk phosphorylation in MSCs osteogenic
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differentiation. The MSCs were pretreated with CXCR4 antagonist AMD3100 at400 µM for 2 h at
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37 °C prior to culture in OIM for 15 min; the controls were culturedin normal culture media or OIM. .
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Total Erk1/2, the phosphorylated (p) Erk1/2, and GAPDH were detected in cell lysates by Western
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blot. Level of phosphorylated (p) Erk1/2 was down-regulated in the AMD3100 treated group
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compared to the OIM group.
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Fig.6. AMD3100 attenuates articular cartilage degeneration in ACLT + MMx rat. Safranin O and fast
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green staining of sagittal sections of the subchondral femoramedial compartment in the AMD3100
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treatment group, PBS group and OA group. Scale bar, 400 µm (in top), 200 µm (in bottom). The
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result showed that cartilage degeneration was less severe in the AMD3100 group, compared to the
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PBS or OA group.
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Fig.7. AMD3100 reduces aberrant subchondral bone formation in a rat OA model. (a)3D µCT
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images of the tibia subchondral bone compartment (sagittal view) of rats in the AMD3100 group,
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PBS group and OA group. Scale bar, 1 mm.
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Quantitative assay of structural parameters of subchondral bone by µCT. (b)BMD, (c) BV/TV. The
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results showed that subchondral bone had significantly reduce BMD, BV/TV in the AMD3100
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treated group compared to PBS group or OA group. n= 5 per group. *p < 0.05: the AMD3100 group
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compared to the PBS or OA group.
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Fig.8.(a) Immunohistochemical analysis and quantification of Nestin(b) and Osterix (c) positive cells
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(brown) in the tibial subchondral region. The result showed that AMD3100 treatment would
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significantly reduced Nestin and Osterix positive cells compared to the other two groups. Scale bars,
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50 µm. *p < 0.05, the AMD3100 group compared to the PBS or OA group.
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SUPPLEMENTARY FIGURE LEGENDS
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Sup. Fig.1. Quantitative assay of structural parameters of subchondral bone by µCT.(a) trabecula
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bone thickness (Tb.Th.), (b) trabecula bone space (Tb.Sp.) and (c) structure model index (SMI) in
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subchondral bone determined by µCT. n= 5 per group. The results showed that subchondral bone had
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significantly higher Tb.Th. in the OA compartment compared to RN compartment, while Tb.Sp. and
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SMI had reduce significantly. *p<0.05: the OA compartment compared to RN compartment.
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Sup. Fig.2.(a) Immunohistochemical analysis and quantification (b,c) of Nestin and Osterix positive
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cells (brown) in the subchondral region of clinical specimen. The result showed that in OA part the
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number of Nestin and Osterix positive cells were significantly reduced compared to the RN part.
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Scale bars, 50 µm. *p < 0.05: the OA part compared to the RN part. SB = subchondral bone; BM
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=subchondral bone marrow.
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Sup. Fig.3. Immunohistochemical analysis of Type I collagen in the subchondral region of animal (a)
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and clinical specimen (b). The result showed that Type I collagen was highly expressed in trabecula
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bone. Scale bar in a. is 100µm; in b. is 200µm.
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(brown) in the subchondral region of animal and clinical specimen. The results showed that ALP
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positive cells were found in subchondral bone in both the clinical (b) and rat samples (a), but not
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statistical differences between the OA and the control samples (c, d). Scale bar in a. is 100µm; in b.
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is 200µm.
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Sup. Fig.5. Immunohistochemical analysis of CXCR4 positive cells (brown) in the subchondral
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region of clinical specimen. The result showed that in OA part the number of CXCR4 positive cells
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was significantly increased compared to the RN part. Scale bar in a. is 400µm; in b. is 200µm.
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Sup. Fig.6.Quantitativeassay of structural parameters of subchondral bone by µCT. (a) Tb.Th., (b)
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Tb.Sp.and (c) SMI.in subchondral bone determined by µCT. n= 5 per group. The results showed that
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subchondral bone had significantly reducedTb.Th. in the AMD3100 treated group compared to PBS
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group or OA group, while Tb.Sp. and SMI had increased significantly.*p < 0.05: the AMD3100
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group compared to the PBS or OA group
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