Bone morphogenetic protein-2-induced Wnt/β-catenin signaling pathway activation through enhanced low-density-lipoprotein receptor-related protein 5 catabolic activity contributes to hypertrophy in osteoarthritic chondrocytes
© Papathanasiou et al.; licensee BioMed Central Ltd. 2012
Received: 14 November 2011
Accepted: 18 April 2012
Published: 18 April 2012
Events normally taking place in the terminal chondrocyte differentiation in the growth plate are also observed during osteoarthritis (OA) development, suggesting that molecules, such as Wnts and bone morphogenetic proteins (BMPs) regulating chondrocyte activity in the growth plate, may play a key role in osteoarthritis pathogenesis. The aim of the study was to investigate the possible cross-talk between BMP-2 and Wnt/β-catenin pathways in OA progression.
Low-density-lipoprotein receptor-related protein 5 (LRP-5) and 6, BMP-2, -4, and -7, bone morphogenetic protein receptor-IA and IB (BMPR-IA and BMPR-IA), lymphoid enhancer factor-1 (LEF-1), and transcription factor 4 (TCF-4) expression levels were investigated in normal and osteoarthritic chondrocytes. LRP-5, β-catenin (phospho and active form), matrix metalloproteinases (MMPs) 7, 9, 13, 14, ADAMTS-4, 5, as well as collagen X (COL10A1) expression levels were evaluated after LRP-5 silencing in BMP-2-treated chondrocytes. The investigation of Smad1/5/8 binding to LRP-5 promoter was assessed with chromatin immunoprecipitation (ChIP). Furthermore, we evaluated the effect of experimental activation of the Wnt/β-catenin pathway with LiCl and LEF-1 silencing, in LiCl-treated chondrocytes, on matrix metalloproteinases (MMPs) 7, 9, 13, 14, ADAMTS-4, 5, and collagen X (COL10A1) expression, as well as possible interactions between LEF-1 and MMPs and COL10A1 promoters by using a ChIP assay.
LRP-5, BMP-2, BMP-4, BMPR-IA, and LEF-1 mRNA and protein expression levels were found to be significantly upregulated in osteoarthritic chondrocytes compared with normal. We showed that treatment of cultured chondrocytes with BMP-2 resulted in increased β-catenin nuclear translocation and LRP-5 expression and that the BMP-2-induced LRP-5 upregulation is mediated through Smad1/5/8 binding on LRP-5 promoter. LRP-5 silencing reduced nuclear β-catenin protein levels, MMPs and collagen X expression, whereas increased phospho-β-catenin protein levels in BMP-2-treated chondrocyte. Furthermore, we demonstrated that activation of the Wnt/β-catenin signaling pathway by LiCl and LEF-1 downregulation by using siRNA regulates MMP-9, 13, 14, ADAMTS-5, and COL10A1 expression, evidenced by the observed strong binding of LEF-1 to MMP-9, 13, 14, ADAMTS-5 and COL10A promoters.
Our findings suggest, for the first time to our knowledge, that BMP-2-induced Wnt/β-catenin signaling activation through LRP-5 may contribute to chondrocyte hypertrophy and cartilage degradation in osteoarthritis.
Osteoarthritis (OA) is a progressively degenerative joint disorder characterized by extracellular matrix degradation, articular cartilage loss, and osteophyte formation . It is considered a major health problem worldwide, causing chronic disability in elderly people . However, the molecular mechanisms underlying OA pathogenesis are poorly understood, and no disease-modifying therapy is currently available [1, 2].
Osteoarthritis involves mainly the dysfunction of articular chondrocytes, which leads to cartilage degradation through chondrocyte maturation and MMPs production [3, 4]. In growth-plate chondrogenesis, chondrocytes become hypertrophic, expressing collagen X, remove the collagen matrix through the production of MMP-13, and finally die by apoptosis and are replaced by bone [5, 6]. Alternatively, chondrocytes of permanent cartilage reside at the ends of the long bones and do not mature into the hypertrophic state, preventing terminal differentiation by an unknown mechanism . However, during osteoarthritis, chondrocytes lose the stable phenotype and undergo changes that occur in terminal differentiated growth-plate chondrocytes, such as high expression of MMP-13 and collagen X [8–10].
The function of articular chondrocytes is regulated by different growth factors, including Wnt ligands and BMPs, which have been shown to play a critical role in chondrocyte proliferation, differentiation, and apoptosis [11–13]. The canonic or Wnt/β-catenin pathway signals through Frizzled family receptors and coreceptors LRP-5/LRP-6 and leads to stabilization of β-catenin, which in turn interacts with transcription factors, such as LEF-1/TCF-4 proteins, and activates specific genes, as c-myc and cyclin D1 . The role οf Wnt/β-catenin signaling pathway in OA development has been previously suggested, as an association has been reported between hip OA susceptibility in women and two functional genetic variants in FRZB, which encode frizzled-related protein, a soluble antagonist of the Wnt canonic signaling pathway [15–17]. Additional evidence for the involvement of Wnt/β-catenin signaling in OA comes from the observation that frzd knockout mice are more susceptible to chemically induced OA . Besides FRZB, another antagonist of the pathway, dickkopf-related protein 1 (Dkk-1), has been also shown to be associated with reduced progression of OA in elderly women when it is present in elevated levels in the serum . A recent study showed the implication of SOST, a potent inhibitor of canonic Wnt signaling by binding to LRP5/6 in OA disease processes with opposing effects by promoting disease-associated subchondral bone sclerosis and inhibiting degradation of cartilage . Recently, we showed that coreceptor of the Wnt/β-catenin signaling pathway, LRP-5, may have a catabolic role in osteoarthritis, as we observed significant upregulation of LRP-5 expression in osteoarthritic chondrocytes . Moreover, the involvement of the Wnt/β-catenin signaling pathway in the regulation of cartilage development and homeostasis has been confirmed, as increased expression levels of several Wnt proteins and Frizzled receptors have been found in the synovial tissue of arthritic cartilage , and the conditional activation of the β-catenin gene in articular chondrocytes in adult mice leads to premature chondrocytes differentiation and the development of an OA-like phenotype . Increased levels of β-catenin have been reported in chondrocytes within areas of degenerative cartilage, and its accumulation and transcriptional activity has been shown to stimulate chondrocyte matrix catabolic action, to induce the expression of different MMPs in articular chondrocytes, and to promote hypertrophic differentiation of chondrocytes, evidenced by increased expression of collagen X [24–26].
Besides the Wnt/β-catenin pathway, BMPs also play a significant role in chondrocyte differentiation. BMP signaling is initiated by BMPs binding to their receptor (BMPR-I and II), resulting in increased phosphorylation of downstream signaling molecules, including Smad1, Smad5, and Smad8 (R-Smads). The R-Smads form complexes with Smad-4 and translocate into the nucleus, where they bind to regulatory regions of target genes regulating their expression . Although BMPs are considered to have a protective effect in articular cartilage, it has been proposed that they are also involved in chondrocyte hypertrophy and matrix degradation [28–31]. Chen et al.  reported BMP-2, 4-6, and 11 mRNA expression in normal and osteoarthritic adult human cartilage, whereas Steinert et al.  showed that BMP-2 and BMP-4 induce hypertrophy during the in vitro chondrogenic differentiation of human mesenchymal stem cells. Moreover, in preosteoblastic cells, BMP-2 was found to increase nuclear β-catenin protein levels and induce the expression of different Wnts, suggesting the interaction between Wnt and BMP-2 signaling during osteoblastic differentiation .
As activation of Wnt/β-catenin and BMP-2 signaling pathways may signify a causal relation to chondrocyte differentiation and matrix degradation, we sought to investigate the possible cross-talk between BMP-2 and Wnt/β-catenin pathways in the catabolic action and hypertrophy of osteoarthritic chondrocytes.
Materials and methods
Promoter sequences of MMP-7, 9, 13, 14, ADAMTS-5, 4, and COL10A1 genes were obtained from CHIP Bioinformatics Tools . For each of these genes, we scanned the region from 1,500 base pairs upstream of the transcript start to 100 base pairs downstream of the coding-sequence start to find putative LEF-1 binding elements (5'-CTTTGWW-3'). In addition, LRP-5 promoter was tested for Smad binding sites (SBEs, 5'-GCCGnGCG-3').
Patients and cartilage samples
Articular cartilage samples were obtained from femoral condyles and tibial plateaus of patients with primary osteoarthritis undergoing knee-replacement surgery at the Orthopaedics Department of University Hospital of Larissa. In total, 18 patients were included in this study (15 women/three men; mean age, 64.7 ± 9.7 years). All osteoarthritic specimens had Mankin scores of 10 to 14. Radiographs were obtained before surgery and graded according to the Kellgren-Lawrence system. All patients had K/L scores ≥ 2. The radiographs were assessed by two independent observers who were blinded to all data of the individuals. Normal articular cartilage was obtained from nine individuals (four women/five men; mean age, 44.6 ± 7.6 years) with 0 Mankin score, undergoing fracture-repair surgery, with no history of joint disease. Patients with rheumatoid arthritis and other autoimmune diseases, as well as chondrodysplasias, infection-induced OA, and posttraumatic OA, were excluded from the study. Written informed consent was obtained from all individuals in the study. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected in a priori approval by the Local Ethical Committee of the University Hospital of Larissa.
Primary cultures of normal and osteoarthritic human articular chondrocytes
Articular cartilage was dissected and subjected to sequential digestion with 1 mg/ml proteinase mixture (Pronase; Roche Applied Science, Mannheim, Germany) and 1 mg/ml collagenase P (Roche Applied Science, Mannheim, Germany). Chondrocytes were counted and checked for viability by using trypan blue staining. More than 95% of the cells were viable after isolation. Isolated chondrocytes from individual specimens were separately cultured with Dulbecco Modified Eagle Medium/Ham F-12 (DMEM/F-12) (GIBCO BRL, Paisley, UK) plus 5% fetal bovine serum (FBS; Invitrogen, Life Technologies, Paisley, UK) at 37οC under a humidified 5% CO2 atmosphere until reaching confluence for 4 to 6 days.
RNA extraction and quantification of mRNA expression
Oligonucleotide primers used in real-time PCR assay
Forward primer sequence
Reverse primer sequence
Protein extraction and Western blot analysis
Chondrocytes were lysed by using RIPA buffer and a cocktail of protease and phosphatase inhibitors. Protein concentration was quantified by using the Bio-Rad Bradford protein assay (Bio-Rad Protein Assay; BioRad, Hercules, CA, USA) with bovine serum albumin as standard. Cell lysates from normal and OA chondrocytes were electrophoresed and separated on a 4% to 20% Tris-HCl gel (Bio-Rad, Hercules, CA, USA) and transferred to a Hybond-ECL nitrocellulose membrane (Amersan Biosciences, Piscataway, NJ, USA). The membrane was probed with anti-LRP-5 (sc-21389; Santa Cruz Biotechnology, Santa Cruz, CA, USA), BMP-2 (sc-57040; Santa Cruz Biotechnology, Santa Cruz, CA, USA), BMP-4 (AF757, R&D Systems, Minneapolis, MN, USA) BMPR-IA (sc-134285; Santa Cruz Biotechnology, Santa Cruz, CA, USA), LEF-1 antibody (sc-8591; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and phospho-β-catenin (9561; Cell Signaling Technology, Boston, MA, USA), and signals were detected by using immunoglobulin (IgG) conjugated with horseradish peroxidase (1:10,000 dilution). The results were normalized by using anti-β-actin polyclonal antibody (Sigma-Aldrich, St. Louis, MO, USA). The nitrocellulose membranes were then exposed to photographic film, which was scanned, and the intensities of the protein bands were determined with computerized densitometry.
Cartilage specimens were fixed in 10% formalin overnight and decalcified in 13% EDTA (Sigma-Aldrich, St. Louis, MO, USA) for 3 weeks. Paraffin-embedded sections were deparaffinized in xylene, dehydrated through graded alcohols, and placed in 3% H2O2 to block endogenous peroxidase. Nonspecific staining was blocked with TBS containing 2% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) for 1 hour at room temperature, followed by incubation with primary antibody BMP-2 (c-57040; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:20 dilution with TBS containing 2% BSA) overnight at room temperature. Normal serum of the same species as the primary antibody was used as a control for the primary antibody. After extensive washing with TBS, the sections were incubated with horseradish peroxidase-conjugated secondary antibody (Goat anti-Mouse; Invitrogen, Life Technologies, Paisley, UK; 1:250 dilution with TBS containing 2% BSA) for 1 hour at room temperature in a humid chamber. Finally, color reaction was performed by using the substrate reagent 3,3'-diaminobenzidine tetrahydrochloride (DAB; Thermo Scientific, Rockford, IL, USA). After being washed, the sections were incubated with DAB and coverslipped with mounting medium.
Chondrocyte treatment with BMPs
Normal and osteoarthritic chondrocytes were seeded on six-well plates at 3 × 105 cells/well, and 3 days after seeding, cells were treated with 50 ng/ml of BMP-2 or BMP-4 for 12, 24, and 48 hours; each experiment was conducted in triplicate wells. RNA was extracted, and real-time PCR analysis for osteocalcin, LRP-5, and LRP-6 was performed, as described earlier. All primers used are shown in Table 1. Cell lysates were extracted, and LRP-5 (sc-21389; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-β-catenin (9561; Cell Signaling Technology, Boston, MA, USA), and total β-catenin protein levels (9582; Cell Signaling Technology, Boston, MA, USA) were evaluated by using Western blot analysis, as described earlier.
Chondrocyte treatment with LiCl
Normal and osteoarthritic chondrocytes were seeded on six-well plates at 3 × 105 cells/well, and 3 days after seeding, cells were treated with 20 mM LiCl (Sigma-Aldrich, St. Louis, MO, USA) for 12, 24, and 48 hours; each experiment was conducted in triplicate wells. RNA was extracted, and real-time PCR analysis for MMP-7, 9, 13, 14, ADAMTS-4, 5, and collagen X was performed as described earlier. All primers used are shown in Table 1. Cell lysates were extracted, and phospho-β-catenin protein levels were evaluated by using Western blot analysis, as described earlier.
Chromatin immunoprecipitation (ChIP) assay
Oligonucleotide primers used for ChIP assay
Normal and osteoarthritic chondrocytes were seeded in six-well plates in DMEM/F-12 containing 5% FBS. After overnight incubation, cells were transfected with specific siRNA against LRP-5 (Ambion, Life Technologies, Paisley, UK), LEF-1 (Invitrogen, Life Technologies, Paisley, UK), and scrambled siRNA (used as a control; Invitrogen, Life Technologies, Paisley, UK) by using LipofectAMINE 2000 (Invitrogen, Life Technologies, Paisley, UK), as described by the manufacturer. No cell toxicity was detected from the transfection agent. Four hours after siRNA transfection, the medium was changed, and cells were treated with 50 ng/ml BMP-2 (cultures with siRNA against LRP-5) or 20 mM LiCl (cultures with siRNA against LEF-1) for 48 hours. In each experiment, the results from three of the six-well plates were averaged and considered as n = 1. No significant variance was observed among the individual wells in each averaged group. RNA was extracted, and real-time PCR analysis for MMP-7, 9, 13, 14, ADAMTS-4, 5, and collagen X was performed, as described earlier. Moreover, cell lysates were extracted, and phospho- as well as total β-catenin protein levels were evaluated in BMP-2-treated chondrocytes after LRP-5 siRNA transfection by using Western blot analysis, as described earlier.
Statistical significance was determined by using the Student t test with a confidence level of 95% (P < 0.05).
COL2A1, COL10A1, aggrecan, and MMP-13 expression in normal and osteoarthritic chondrocytes
LRP-5, BMP-2, BMP-4, BMP-7, BMPR-IA, BMPR-IB, LRP-6, phospho-β-catenin, LEF-1, and TCF-4 expression in normal and osteoarthritic chondrocytes
BMP-2 modulates Wnt/β-catenin signaling pathway through stimulation of LRP-5 mRNA and protein expression in chondrocytes
LRP-5 upregulation by BMP-2 contributed to catabolic activity and hypertrophy of osteoarthritic chondrocytes
Effect of activation of the Wnt/β-catenin signaling pathway by LiCl on the expression of genes implicated in catabolic action and hypertrophy of chondrocytes
Articular chondrocyte proliferation, expression of hypertrophy markers, and remodeling of the cartilage matrix by proteases are among the main characteristics of osteoarthritis . Recent studies have shown that events normally taking place in terminal chondrocyte differentiation in the growth plate are also observed during OA development, suggesting that signaling molecules, such as Wnts and BMPs, regulating chondrocytes activity in the growth plate may play a key role in osteoarthritis pathogenesis [7, 8].
In the present study, we provide for the first time, to our knowledge, evidence for a cross-talk between BMP-2 and Wnt/β-catenin signaling pathways in osteoarthritic chondrocytes. Although BMPs are involved in all phases of chondrogenesis affecting chondrocyte differentiation and cartilage anabolism [28–30], recent studies have shown that BMPs can also have harmful effects on articular cartilage . However, their role in osteoarthritis is not completely elucidated. We evaluated BMP-2, 4, and 7, as well as their receptors, BMPR-IA and BMPR-IA expression levels in osteoarthritic and normal chondrocytes and found that osteoarthritic chondrocytes exhibited significantly higher BMP-2, 4, and BMPR-IA mRNA and protein levels, suggesting the involvement of BMP signaling in osteoarthritis progression. Previous studies have shown that BMP-2 can be activated by IL-1 and TNF-α in normal and osteoarthritic chondrocytes  and that it can stimulate the synthesis of matrix molecules and MMPs expression by modulation of chondrocyte differentiation [37, 38]. In addition, mice deficient in type 1 receptors Bmpr1a or Bmpr1b in cartilage develop severe generalized chondrodysplasia, demonstrating that BMP signaling is required for chondrocyte proliferation, survival, and differentiation .
It has been suggested that BMP-2 modulates β-catenin signaling through stimulation of Wnts, LRPs, and Frizzled receptors expression in mesenchymal cells and osteoblasts [34, 40]. To investigate the role of BMP-2 on the Wnt/β-catenin signaling-pathway activation in osteoarthritic chondrocytes, we evaluated β-catenin and LRP-5 levels after treatment of normal and osteoarthritic chondrocytes with BMP-2. We observed that BMP-2 enhances the nuclear-active form of β-catenin protein levels, decreases phospho-β-catenin protein levels, and increases LRP-5 mRNA and protein levels, which were found significantly upregulated in osteoarthritic chondrocytes compared with normal. In addition, blocking LRP-5 expression in osteoarthritic chondrocytes resulted in a significant decrease in MMP-13 expression, the basic catabolic enzyme of osteoarthritis . All of these findings suggest LRP-5 involvement in the increased activation of Wnt/β-catenin signaling and cartilage degradation in osteoarthritis. No difference was observed in the nuclear-active form of β-catenin levels in BMP-4-treated chondrocytes, providing evidence on the Wnt/β-catenin signaling-pathway activation by BMP-2 and not by BMP-4.
To investigate further the molecular mechanism involved in the BMP-2-induced LRP-5 upregulation, we examined the effect of BMP-2 through Smads binding on LRP-5 promoter activity in chondrocytes. Chromatin immunoprecipitation indicated that BMP-2 directly modulates LRP-5 expression, as we found, for the first time, that Smads complexes bind to SBEs on the LRP-5 promoter, suggesting the BMP-2 induced-Wnt/β-catenin signaling-pathway activation through direct modulation of LRP-5 expression. Previous studies have shown that osteoblast differentiation and new-bone formation require the interaction of BMP-2 and the Wnt/β-catenin signaling pathway [41, 42]. BMP-2-enhanced LRP-5 expression has a strong catabolic activity in chondrocytes, as LRP-5 silencing inhibited BMP-2-induced-β-catenin protein levels, MMPs, and collagen X expression, whereas increased phospho-β-catenin protein levels, providing evidence on the involvement of BMP-2-modulated β-catenin signaling in OA progression.
The Wnt/β-catenin signaling pathway participates in normal adult bone and cartilage biology and seems to be involved in cartilage degeneration and subsequent OA progression [43, 44]. To determine whether changes in the Wnt-signaling pathway are associated with osteoarthritis, we evaluated the expression levels of Wnt transcription factors, LEF-1 and TCF-4, and phospho-β-catenin in osteoarthritic and normal articular chondrocytes. We observed that LEF-1 mRNA and protein-expression levels were significantly increased in osteoarthritic chondrocytes, whereas phospho-β-catenin protein levels were significantly decreased in osteoarthritic chondrocytes, suggesting the excessive activation of canonic the Wnt-signaling pathway in osteoarthritis.
To test for a possible association between the LEF-1 transcription factor of the Wnt/β-catenin signaling pathway and catabolic action, as well as hypertrophy in osteoarthritic chondrocytes, we activated Wnt/β-catenin signaling by using LiCl in cultured chondrocytes. LiCl is often used to mimic canonic Wnt signaling, as it inhibits GSK-3β, therefore stimulating downstream components of the Wnt-signaling pathway in an LRP-5-independent manner . The activation of the canonic Wnt-β-catenin signaling by LiCl is not modulated by LRP-5 phosphorylation, and until now, differences in phospho-LRP-5 protein levels between OA and normal cartilage have not been reported. We observed that experimental activation of Wnt/β-catenin signaling induced significant upregulation of catabolic enzymes such as MMP-9, 13, 14, aggregenases, as ADAMTS-5 and hypertrophic marker, collagen X. The upregulation of the above genes takes place in a direct manner, as we demonstrated, conserved LEF-binding sites in MMP-9, 13, 14, ADAMTS-5, and COL10A1 promoters, responsible for their promoter activity and is associated directly with the β-catenin/LEF-1 complex. Moreover, LEF-1 downregulation using siRNA reduced MMPs, ADAMTS-5, and collagen X mRNA expression, whose levels increased after treatment with LiCl, providing strong evidence of gene-expression regulation of catabolic factors by LEF-1. No upregulation was observed in MMP-7 and ADAMTS-4 levels, as no conserved LEF-binding sites were found on their promoters. It has been shown that Lef-1 binding to the 3' region of mmp-13 is involved in the transcriptional regulation of the mmp-13 gene in mouse chondrocytes . It can be suggested that the Wnt/β-catenin signaling pathway promotes chondrocytes catabolic activity, degrading not only collagen II, as evidenced by the observed increased expression of MMP-13 and 14, but also other extracellular matrix molecules, such as fibronectin and aggrecan, through enhancement of MMP-9 and ADAMTS-5 expression, respectively. It has been shown that MMP-9, a gelatinase with broad substrate specificity, contributes to fibronectin degradation and increases the fibronectin fragments, which have been shown to be elevated in OA [47, 48], whereas ADAMTS-5, one of the most efficient aggrecanases, degrades aggrecan, the major proteoglycan in cartilage . In additional studies in a murine model of osteoarthritis, deletion of ADAMTS-5 provided significant protection against proteoglycan degradation ex vivo and decreased the severity of osteoarthritis . We also found that the Wnt/β-catenin signaling pathway contributes to the hypertrophic differentiation of chondrocytes, increasing collagen X expression, the basic hypertrophic marker of chondrocytes. We therefore showed that the Wnt/β-catenin signaling pathway plays a major role in OA progression, as articular chondrocytes, after experimental activation of the Wnt/β-catenin signaling pathway, cannot maintain the characteristics of the permanent cartilage but instead enhance extracellular matrix degradation, evidenced by increased MMPs and ADAMTS-5 expression, and mature to hypertrophic through stimulation of collagen X expression.
In conclusion, we demonstrated, for the first time to our knowledge, that the BMP-2-induced Wnt/β-catenin signaling pathway activation through LRP-5 induces chondrocyte catabolic action and hypertrophy, providing thus novel and direct evidence on the role of BMP-2 mediated by Wnt/β-catenin signaling in osteoarthritis progression.
bone morphogenetic proteins
bone morphogenetic protein receptor-IA
bovine serum albumin
Dickkopf-related protein 1
Dulbecco Modified Eagles Medium/Ham F-12
fetal bovine serum
glyceraldehyde 3-phosphate dehydrogenase
lymphoid enhancer factor-1
low-density-lipoprotein receptor-related protein 5
The authors thank Prof. GK Koukoulis and Assist. Prof. M Ioannou from the Department of Pathology, University Hospital of Larissa, for the kind provision of histologic analysis of articular cartilage tissues, and Assist. Prof. of Pharmacology, A Vassilaki for her valuable assistance with ICH experiments. The present work was supported by the research-financed project, "Inhibition of gene's expression involved in hypertrophy and calcification of osteoarthritic cartilage," from the Hellenic Society of Orthopaedic Surgery and Traumatology.
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