Regulation of senescence associated signaling mechanisms in chondrocytes for cartilage tissue regeneration

Accepted Manuscript Regulation of senescence associated signaling mechanisms in chondrocytes for cartilage tissue regeneration Sajjad Ashraf, Ph.D, Byung-Hyun Cha, Ph.D, Jin-Su Kim, Ph.D, Jongchan Ahn, Ph.D, Inbo Han, MD, Ph.D., Associate Professor, Hansoo Park, Ph.D., Assistant Professor, Soo-Hong Lee, Ph.D., Associate Professor PII:

S1063-4584(15)01238-8

DOI:

10.1016/j.joca.2015.07.008

Reference:

YJOCA 3541

To appear in:

Osteoarthritis and Cartilage

Received Date: 15 February 2015 Revised Date:

11 June 2015

Accepted Date: 9 July 2015

Please cite this article as: Ashraf S, Cha B-H, Kim J-S, Ahn J, Han I, Park H, Lee S-H, Regulation of senescence associated signaling mechanisms in chondrocytes for cartilage tissue regeneration, Osteoarthritis and Cartilage (2015), doi: 10.1016/j.joca.2015.07.008. 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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Regulation of senescence associated signaling mechanisms in chondrocytes for

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cartilage tissue regeneration

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Sajjad Ashraf, Byung-Hyun Cha, Jin-Su Kim, Jongchan Ahn, Inbo Han, Hansoo Park,

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*Soo-Hong Lee

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Sajjad Ashraf, Ph.D.

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School of Integrating Engineering, Chung-Ang University, Seoul, Korea

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Department of Biomedical Science, CHA University, Seoul Korea

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Email: [email protected],

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Byung-Hyun Cha, Ph.D.

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Department of Biomedical Science, CHA University, Seoul Korea

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Email: [email protected]

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Jin-Su Kim, Ph.D.

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Department of Biomedical Science, CHA University, Seoul Korea

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Email ID: [email protected]

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Jongchan Ahn, Ph.D.

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Department of Biomedical Science CHA University, Seoul Korea

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Email: [email protected]

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Inbo Han, MD, Ph.D., Associate Professor

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Department of Neurosurgery,CHA University, CHA Bundang Medical Center,59, Yatap-ro

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Bundang-gu, Seongnam-si, Kyeunggi-do, 463-712,Korea

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Email: [email protected]

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Hansoo Park, Ph.D., Assistant Professor

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School of Integrative Engineering, Chung-Ang University, Seoul, Korea

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Email ID: [email protected], Cell No. 00821033070817

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* Corresponding Author: Soo-Hong Lee, Ph.D., Associate Professor

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Department of Biomedical Science CHA University,

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Email ID: [email protected], Phone No. 0082-031-881-7143. Fax. 0082-031-881-7249

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Running Head:

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Senescence and Cartilage Regeneration

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Abstract

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Adult articular chondrocytes undergo slow senescence and dedifferentiation during in vitro

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expansion, restricting successful cartilage regeneration. A complete understanding of the

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molecular signaling pathways involved in the senescence and dedifferentiation of chondrocytes

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is essential in order to better characterize chondrocytes for cartilage tissue engineering

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applications. During expansion, cell fate is determined by the change in expression of various

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genes in response to aspects of the microenvironment, including oxidative stress, mechanical

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stress, and unsuitable culture conditions. Rapid senescence or dedifferentiation not only results in

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the loss of the chondrocytic phenotype but also enhances production of inflammatory mediators

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and matrix-degrading enzymes. This review focuses on the two groups of genes that play direct

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and indirect roles in the induction of senescence and dedifferentiation. Numerous degenerative

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signaling pathways associated with these genes have been reported. Upregulation of the genes

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interleukin 1 beta (IL-1β), p53, p16, p21, and p38 mitogen-activated protein kinase (MAPK) is

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responsible for the direct induction of senescence, whereas downregulation of the genes

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transforming growth factor-beta (TGF-β), bone morphogenetic protein-2 (BMP-2), SRY (sex

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determining region Y)-box 9 (SOX9), and insulin-like growth factor-1 (IGF-1), indirectly

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induces senescence. In senescent and dedifferentiated chondrocytes, it was found that TGF-β,

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BMP-2, SOX9, and IGF-1 are downregulated, while the levels of IL-1β, p53, p16, p21, and p38

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MAPK are upregulated followed by inhibition of the normal molecular functioning of the

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chondrocytes. This review helps to elucidate the underlying mechanism in degenerative cartilage

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disease, which may help to improve cartilage tissue regeneration techniques.

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Keywords: Senescence, Dedifferentiation, Genes, Chondrocytes, Cartilage

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Introduction Articular cartilage is a flexible, thin, smooth, connective tissue that covers the surfaces of

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all of the synovial joints in the human body. Chondrocytes are the primary cellular component of

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articular cartilage and are required for the production of extracellular matrix (ECM) and for

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maintaining cartilage structure and function1. Articular cartilage injuries due to accidents or

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trauma affect more than one million people worldwide annually, and surgery is currently the best

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treatment option2. Additionally, osteoarthritis resulting from genetic disorders, obesity, and

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excessive mechanical loading affects more than 100 million people globally3.

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Articular cartilage tissue has limited ability to self-repair due to the lack of blood vessels

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and the paucity of resident progenitor stem cells4. Although progenitor cells have been found in

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different zones of the adult cartilage, the origin and functions of these cells are not completely

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understood (for review see Candela et al.4; Jiang and Tuan5). Currently there is a lack of medical

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treatments that can be used to regenerate articular cartilage. Mediations that aim to re-establish

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the articular tissue have been developed such as autologous chondrocyte implantation (ACI),

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marrow stimulation techniques (such as abrasion arthroplasty, drilling, and microfracture), and

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mosaicplasty6.

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Treatment via chondrocyte transplantation does not always guarantee a good prognosis

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because it is difficult to isolate and maintain the normal replicative physiological activity of

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human chondrocytes in long-term culture7. Successful cartilage regeneration or transplantation

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requires a sufficient quantity of clinical-grade chondrocytes. In vitro monolayer cultures

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requiring serial cell passages to expand chondrocytes have been extensively adopted for

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transplantation8. The donor age also influences the yield, capacity for expansion in monolayer 4

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culture, and redifferentiation ability of chondrocytes. Cell density and proliferation potential of

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chondrocytes decreases as the age of the donor increases9,10. Barbero et al.10 reported that

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chondrocyte yield was similar up to the donor age of 40, and a profound decline was observed in

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older donors. Additionally, chondrocytes acquired from donors older than 30 years of age

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exhibited significantly decreased proliferation and showed a greater tendency towards

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senescence.

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Native cartilage architecture has zonal differences in its distribution of ECM11. Zonal

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cells lose specific characteristics during expansion in monolayer culture. In pellet culture,

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however, deep zone cells produce higher amount of glycosaminoglycans (GAGs) than the

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superficial zone cells12. Surprisingly, another study reported rapid dedifferentiation of the

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chondrocyte subpopulation after the first passage during monolayer culture and in 3D

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encapsulation13.

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When chondrocytes isolated from native cartilage undergo in vitro culture as a monolayer,

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they lose their phenotype and a variety of senescence and dedifferentiation-mediated genes are

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turned on14. It was found that after four passages, the morphology of chondrocytes changed from

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polygonal or round to a flattened, amoeboid-like shape, and the cells started synthesizing

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collagen type X (Col X), as shown in Fig. 1A and 1B (unpublished data). This result is

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coincident with previous reports8,14. Senescence and dedifferentiation of chondrocytes during

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monolayer culture is also accompanied by decreased expression of chondrocyte-specific proteins

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such as collagen type II (Col II), proteoglycans, and glycoproteins15. The senescence-associated

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beta-galactosidase assay results shown in Fig. 1C reveal that more senescent cells are observed

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in late passages compared to early passages (unpublished data). Transplantation of such

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senescent and dedifferentiated cells results in the formation of undesirable fibrous cartilage at the

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transplantation site16. Loss of chondrocyte-specific properties is due to so-called “stress

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responses” triggered by in vitro culture conditions. Various kinds of stress such as oxidative,

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mechanical, damage, aging, and exposure to ionizing radiation are responsible for the senescence

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of chondrocytes17 and a large number of genes are involved, e.g., Raf-1/MEK/ERK, p53, and p38

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mitogen activated protein kinase (MAPK)18. The regulation of genes/transcription factors, which

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are capable of controlling the native characteristics of chondrocytes using gene therapy, is a

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promising approach for cartilage regeneration8,19.

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This review focuses on the genes required to maintain the normal phenotypic

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characteristics of chondrocytes as well as direct/indirect roles of senescence and

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dedifferentiation-mediated genes in chondrocytes and summarizes the correlation between those

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genes.

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Change of Chondrocyte Phenotype during In Vitro Culture Maintaining cellular morphology is important for the regeneration of cartilage tissue and

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during in vitro monolayer culture chondrocytes show significant phenotypic or morphological

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changes depending on the culture period1,7,8. Various proteins are involved in the phenotypic

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change from polygonal to fibroblastic. The integrity of normal chondrocytes depends on the

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synthesis of Col II. Various genes related to proliferation, viability, apoptosis, and the synthesis

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of Col II change as the passage number increases20. With increasing passage number, the gene

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expression of apoptotic or senescent pathways increases while the cell proliferation and viability

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decreases8,16. For example, passage 2 chondrocytes exhibit high expression of Col II and low

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expression of Col X; however, passage 6 chondrocytes exhibit low expression of Col II and high

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expression of Col X (Fig. 1B). In contrast, passage 5 chondrocytes exhibit low expression of Col

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II and high expression of collagen type I (Col I), indicating a chondrocyte dedifferentiation state7.

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Together with Col II, GAGs also contribute to the mechanical stability of chondrocytes via a

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structure of an interwoven meshwork21. Another core protein, aggrecan, plays an important role

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in maintaining the chondrocyte phenotype by linking covalently with GAGs, resulting in the

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production of matrix proteoglycans (PGs). This complex model of ECM involving Col II helps

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chondrocytes to maintain vital interactions with one another. Therefore, cells deprived of ECM

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molecules rapidly undergo senescence or apoptosis22.

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Chondrocytes undergoing senescence actively produce cartilage-degrading enzymes such

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as matrix metalloproteinase 13 (MMP13) and aggrecanases, which promote the degradation of

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aggrecan and Col II23,24. One important factor influencing the change in chondrocyte behavior is

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the formation of Col X, which indicates that chondrocytes are in a dedifferentiated stage8,25. The

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function of Col X is still not clear, but its expression in chondrocytes is considered the main

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reason for the dedifferentiation phenomenon26.

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The exact molecular mechanism of senescence in chondrocytes is still not elucidated, but

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it is highly associated with several genes, including p53, p16, osteopontin, osteocalcin, Indian

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Hedgehog, vascular endothelial growth factor (VEGF), HtrA1, and transglutaminase-2 (TG-2)27.

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To recover chondrogenic properties, a number of senescence-related genes have been applied to

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chondrocytes in vitro without significant disruption in the cellular morphology28.

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Several alternatives to conventional monolayer culture have been explored including

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growth factor treatment29, pellet culture1, and bioreactor treatment30. Techniques such as

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functionalization of the dynamic culture surfaces with ECM31, culture on a continuously 7

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expanding surface32, and surface coating with Col I33 have shown great potential for inhibiting

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dedifferentiation and recovering chondrogenicity. Other reports have also revealed the role of

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mechanical loading and shear stress in the regulation of phenotypic characteristics of

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chondrocytes during in vitro culture34,35. Alternately, excessive mechanical loading and high

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fluid shear also recapitulated the genes associated with OA36. Most importantly, these treatments

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were relatively effective in maintaining the phenotypic characteristics of chondrocytes but

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sufficient expansion of chondrocytes was not achieved37. According to Mandl et al.38, a twenty

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fold minimum multiplication of donor chondrocytes usually obtained is required to recover the

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average cartilage defect of 4 cm2. Chondrocyte expansion is critical for clinical applications and

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depends on multiple factors including the site of biopsy, cell seeding density, supplements in the

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culture medium, and the cultivation period39.

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Despite promising outcomes using these various techniques, the molecular mechanisms

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involved in the process of re-differentiation of chondrocytes are not well-understood40. Another

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approach that has been widely studied is the delivery of genes related to growth factors, i.e.,

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transforming growth factor beta-1 (TGF-ß1), BMP, insulin-derived growth factor (IGF), and

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fibroblast growth factor (FGF); transcription factors, i.e., SOX9, RUNX2, and SMAD;

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microRNAs, and Col II promoter-binding proteins19.

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We believe this could be a novel approach for obtaining a sufficient quantity of normal

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chondrocytes, but further studies are required to sort out common genes that control most of the

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signaling pathways involved in senescence. Further in this review, we focus on the multiple

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genes that are directly or indirectly involved in senescence and try to determine common genes

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that control multiple pathways.

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Genes Required for the Maintenance (Lineage Determination) of Chondrogenic Properties

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and are Indirectly Involved in Senescence

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TGF-β

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TGF-β is a cytokine and plays an important role in the induction of chondrogenesis.

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There are three isoforms of TGF-β, and among them TGF-β1 has been the most widely studied

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in chondrocytes. TGF-β1 follows a complex signaling mechanism that ultimately helps to

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maintain the chondrocytic characteristics during in vitro culture via promoting cell proliferation,

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ECM protein synthesis, and protecting normal morphology by inhibiting MMPs41.

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Since Skonier et al.42 isolated the TGF-β gene from human adenocarcinoma cells and

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identified the function of the TGF-β protein, it has been reported that many cell types, including

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chondrocytes, produce TGF-β as a high-molecular weight macromolecule in association with the

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latent homodimer with latent TGF-β-binding protein (LTBP). The LTPB complex mediates the

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deposition of the latent complex to the ECM43. Release of the mature TGF-β from the latent

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complex is a multifaceted process involving proteolytic cleavage and deglycosylation of latency

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associated peptide (LAP)44. In chondrocytes, TGF-β signaling involves the heterotetrameric

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complex including two type I receptors also known as activin receptor like kinases (ALKs) and

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two type II receptors (TβR-II)45. The type I receptors are ultimately phosphorylated by type II

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receptors when dimeric TGF-β molecules bind to the tetrameric complex46. ALK5 activation is

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crucial in TGF-β signaling that leads to phosphorylation of SMAD2 and SMAD347. TGF-β1

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promotes proliferation of chondrocytes through β-catenin signaling48 and maintains the

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polygonal phenotype of chondrocytes by enhancing Col II synthesis through SMAD3 activation

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of SOX9 together with the SMAD2 and SMAD4 complex (Fig. 2)49. The SMAD2/3 signaling

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pathway also inhibits maturation and hypertrophy of chondrocytes during in vitro culture.

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Mutant mice with SMAD3 deficiency develop cartilage degenerative disease and those with

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TGF-β1 deficiency develop osteoarthritis50-52.

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TGF-β1 has variable roles in chondrogenesis in vivo and in vitro. Introducing TGF-β1

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into the periosteum of a femur causes chondrocyte differentiation and cartilage formation, while

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TGF-β1 inhibits and slows down chondrocyte maturation (hypertrophy) in vitro41,53,54. TGF-β1 is

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a major regulator of molecular and physiological characteristics of chondrocytes. Therefore, in

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the TGF-β1 signaling pathway, any gene inhibiting TGF-β1 may result in senescence and

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dedifferentiation.

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BMPs

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BMP signaling pathways are also crucial regulators of chondrogenesis and potent

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inducers of cartilage formations19,55. More than 20 types of BMP-related proteins comprising the

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BMP family have been described, which are categorized into three main groups depending on

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their function and sequence homology: the BMP2/4 group, the osteogenic protein 1 (OP1,

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BMP7) group, and the growth and differentiation factor 5 (GDF5) group56. BMP-2 is a cytokine

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required for in vitro chondrocyte culture. Like TGF-β1, BMP-2 follows a complex downstream

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signaling mechanism for the activation and transcription of target genes. BMPR1A (also known

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as ALK3), BMPRIB (also known as ALK6), and various SMAD are activated by BMP-2,

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expressed independently, and essential for in vitro maintenance of chondrocyte morphology, in

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vivo chondrogenesis, and cartilage growth development57. Combinational therapy of BMP-2 with

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bFGF in three-dimensional culture significantly enhances the expression of Col II and aggrecan;

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however, similar treatments to monolayer culture result in no effect on Col II and aggrecan58.

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BMP-2 repairs damaged cartilage via boosting PG synthesis59. BMP-4, -6, -7, -9, and -13 also

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help to maintain chondrocytes and induce chondrogenesis of MSCs in vitro60,61. However, there

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is still debate about the relative potencies of BMPs due to variation in the model systems and

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culture conditions used.

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SOX9

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SOX9 is a transcriptional regulator for chondrogenesis confirmed by multiple in vitro and

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in vivo studies on mice and humans19. SOX9 also has a significant role in mesenchymal

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condensation following chondroblast differentiation. Genetic studies using Sox9–ires–Cre

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knock-in mouse models showed that SOX9-presenting cells are the progenitors of

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chondroblasts61.

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Recently, mouse genetic analysis revealed that Wnt/β-catenin signaling plays a strong

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role in the regulation of both the Sox9 and Runx2 genes and, in particular, Wnt signaling

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suppresses chondrogenic differentiation while increasing osteoblastic differentiation62. In

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addition to Wnt signaling, Notch signaling and HES-1 are also potent negative regulators of

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SOX9 that ultimately lead to decreased expression of Col II63. On the other hand, retinoic acid

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enhances SOX9 expression and ultimately activates the type II procollagen gene via the Sry/Sox

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consensus sequence64.

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SOX9 is known as a master regulator of the chondrocyte phenotype and one of the potent

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transcription factors for Col II regulation65. SOX9 has a specific binding site at the enhancer 11

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region in intron 1 of the pro-α1 Col II gene and SOX9 binding to the enhancer region ultimately

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upregulates Col II synthesis66. SOX9 is very important for synthesizing aggrecan and core proteins, which are also

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required to maintain the normal chondrocyte phenotype (Fig. 2)67. Overexpression of SOX9

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leads to aggrecan synthesis via binding to the promoter or enhancer region of the aggrecan

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coding gene68.

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SOX9 is also involved in the redifferentiation of chondrocytes via a novel post-

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translational regulatory mechanism. SOX9 is able to re-express chondrogenic markers even in

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degenerated chondrocytes, implying that SOX9 has great potential to recover chondrogenic

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function in degenerated cartilages69. As shown in Fig. 1D, early-passage (P2) degenerated

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chondrocytes isolated from damaged cartilage show similar fibroblastic morphology to late-

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passaged chondrocytes (P6), and these degenerated chondrocytes also exhibit higher expression

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of COL I compared to normal chondrocytes (Fig. 1E). SOX9 is ultimately activated by almost all

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the growth factors associated with chondrogenic lineage maintenance. Furthermore, SOX9 alone

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can regulate almost all of the genes required for COL II, core protein, and aggrecan production.

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IGF-1

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IGF-1 is one of the significant growth factors present in cartilage and has a unique

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endocrine, paracrine, and autocrine signaling mechanism. Principally, IGF-1 increases cell

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proliferation, enhances synthesis of matrix-associated proteins, and inhibits apoptosis by

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regulating downstream signals via phosphoinositide 3-kinase (PI3K) and extracellular signal-

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regulated kinase (ERK), a member of the MAPK cascade. PI3K activates cell survival and 12

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protein synthesis through downstream signaling of Akt and p70S6 kinase, respectively70. On the

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other hand, the ERK signaling cascade is engaged in downstream activation by RAS (mostly

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stimulated by IGF-1) and growth-factor-receptor bound protein 2 (GRB2). IGF-1 stimulates PG

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synthesis in human articular chondrocytes during in vitro culture by activating the

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PI3K/Akt/mTOR/p70S6 kinase pathway71. PG synthesis, as well as Akt phosphorylation was

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inhibited by using the IGF-1 antagonist. Significant downregulation of PG synthesis was

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observed when mTOR and p70S6 kinase were inhibited71. Thus, IGF-1 is involved in the

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activation of the PI3K/Akt pathway followed by the mTOR and p70S6 kinase pathways (Fig. 2).

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In chondrocytes, the primary role of IGF-1 in PG synthesis is to stimulate translational

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activity rather than transcription activity70-72. IGF-1 induces the differentiation of mesenchymal

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cells into chondrocytes through the PI3K pathway as opposed to the ERK pathway because

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inhibition of ERK is not able to block the synthesis of collagen and PG71. In chondrocytes, the pathway of IGF-1 to PI-3 kinase to Akt signaling also promotes

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matrix synthesis (including Col II) and survival through increased SOX9 expression70. Recently,

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it has been observed that stress in the endoplasmic reticulum stimulates the expression of tribble

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homology 3 (TRB3), and TRB3 is also known as a strong inhibitor of Akt. TRB3 inhibits Akt

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phosphorylation, and the resulting stress leads to a decrease in the survival of chondrocytes73.

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Induction of oxidative stress in normal chondrocytes resulted in the same change in signaling

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pathway that is found in degenerated nucleus pulposus chondrocytes (dNPCs) isolated from

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osteoarthritis patients (Fig. 1F), presumably because of inhibition of the IGF-1 pathway via Akt

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inhibition. dNPCs also show low GAG matrix formation as shown by alcian blue staining when

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compared to normal NPCs (Fig. 1G). These findings have also been observed in normal

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chondrocytes undergoing oxidative stress8,18. Fig. 2 illustrates the brief signaling pathway of

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IGF-1 in chondrocytes.

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Genes Directly Involved in Cellular Senescence and Dedifferentiation of

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Chondrocytes

Oxidative stress or reactive oxygen species (ROS) are considered a major regulator of

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senescence in chondrocytes. ROS activate multiple genes that induce senescence,

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dedifferentiation, and even apoptosis via initiation of multiple downstream signals as shown in

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Fig. 3. The genes and their interconnection pathways are discussed in detail as follows.

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IL-1β

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The cytokine IL-1β works as a negative regulator for chondrogenicity, inhibits

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proliferation, and often induces dedifferentiation in chondrocytes. There is direct evidence that

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there is an increase in the IL-1β level, associated with dedifferentiation, during monolayer

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expansion of human chondrocytes after four passages74. IL-1β-associated dedifferentiation of

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chondrocytes involves multiple mechanisms such as the repression of SOX9 and VEGF and

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induction of nitric oxide synthase and MMPs75. IL-1β activates B-Raf in turn causing MEK1/2

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and ERK1/2 phosphorylation, but fails to induce expression of SOX9 and VEGF, and inhibited

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the proliferation of articular chondrocytes76. IL-1β induces aggrecan degradation by activating

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aggrecanase through the MAPK and ERK1/2 signaling mechanism. IL-1β is also associated with

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the induction of other signals such as phosphorylation of p38 and activating transcription factor-

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2 (ATF-2), C-JNK, c-fos, activating protein (AP-1), and nuclear factor kappa B (NF-κB), that

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ultimately activates aggrecanase77. IL-1β is also associated with the induction of nitric oxide

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(NO) synthase, which has the ability to activate JNK, and JNK is a well-known inducer of

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senescence and mediator of osteoarthritis78.

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As IL-1β induces dedifferentiation of chondrocytes, SOX9 and Col II are downregulated

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through α, β, and γ-catenin in the Wnt signaling pathway79. The inhibition of catenin signaling

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induced by IL-1β via a low dosage of γ-radiation can inhibit dedifferentiation and inflammation

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of chondrocytes80. Chondrocytes with high expression of IL-1β may undergo apoptosis because

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IL-1β enhances ROS and p53. Chondrocytes treated with IL-1β for five days in vitro became

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senescent and exhibited a decrease in expression of aggrecan with upregulation of p1681. On the

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other hand, inhibition of IL-1β with resveratrol decreased p53 and inhibited apoptosis82.

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Moreover, in dedifferentiated chondrocytes, IL-4 inhibited catabolic events such as NO

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production, a release of collagenase by downregulating IL-1β, and increased proliferation of

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chondrocytes83. Further, ERK1/2, also involved in IL-1β, enhanced MMP3 and MMP13

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expression and inhibited COL II and aggrecan expression in chondrocytes. Inhibition of either

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ERK1 or ERK2 reverted the altered gene expression of MMP13, MMP3, COL II, and aggrecan

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induced by IL-1β in human chondrocytes84.

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p53, p16, and p21

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In chondrocytes, two different mechanisms of senescence have been suggested, including

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replicative senescence and stress-induced premature senescence (SIPS). Replicative senescence 15

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is due to telomere shortening, while the mechanism for SIPS may involve oxidative stress and

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DNA damage without a change in telomere length85. p53, p21, and pRb genes follow the

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telomere shortening pathway to induce senescence as well as apoptosis in chondrocytes.

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However, p16 and pRb play a synergistic role in senescence through SIPS86. In senescent cells,

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the expression of p53, p21, p16, and pRb increases and results in cell cycle arrest through the

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inhibition of multiple cyclin-dependent kinases. Articular chondrocytes with high expression of

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p21, p16, and pRb also exhibit more positive senescent cells with SA-β gal staining87. Additional

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studies reveal that nitric oxide increases the expression of p53 by phosphorylating p38 MAPK

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and p53, further upregulating BAX, a pro-apoptotic gene88. Chondrocytes expressing p53 show

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similar morphology to osteoarthritic cartilage cells and exhibit apoptosis, whereas

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downregulation of p53 expression can prevent chondrocytes from undergoing apoptosis or

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senescence89. A more important gene associated with senescence is p16, and chondrocytes

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expressing p16 start losing the normal phenotype rapidly, expressing more inflammatory

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cytokines (IL-1β and IL-6) and MMPs (MMP1 and MMP13)24. ROS are direct mediators of p16

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that promote senescence and dedifferentiation90. Mainly, p16 engages in cell cycle arrest at the

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G1 stage by blocking CDK4/6 and maintaining retinoblastoma (pRb), p107, and p130 in their

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active un-phosphorylated forms91. Similarly, siRNA inhibition of p16 recovers osteoarthritic

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chondrocytes to normal functioning92. Another in vitro study revealed that growth arrest in old

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(more passages) articular chondrocytes was due to downregulation of proliferating cell nuclear

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antigen (PCNA) and upregulation of p21. PCNA and Col II expression are negatively correlated

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with p21 expression93. p21 is also involved in senescence through activation by DNA damage-

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inducible protein 45 β (GADD45 β) and CCAAT/enhancer binding protein β (C/EBP β). Another

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suggested mechanism is that GADD45β enhances C/EBPβ and C/EBPβ regulates p21

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expression94.

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p38 MAPKs

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p38 is a crucial member of the MAPK group and in chondrocytes, changes in p38 MAPK

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have been associated with variations in phenotype due to changes in metabolism and matrix

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degradation. Two upstream signals, ERK and JNK, regulate p38 MAPK. For instance, p38

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MAPK inhibition causes significant upregulation of Col II as well as downregulation of Col I.

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p38 knockdown significantly improves chondrocyte morphology even in monolayer culture95.

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Inhibition of p38 MAPK converts hypertrophic chondrocytes to pre-hypertrophic chondrocytes.

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p38 MAPK inhibition by PTH and protein kinase C is responsible for reducing the expression of

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hypertrophic markers such as Col X and is associated with BCL2 upregulation96. p38 MAPK is

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not only involved in senescence and dedifferentiation of chondrocytes but also plays a role in

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apoptosis. According to Takebe et al.97, heat stress or mechanical stress induces apoptosis and

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phosphorylation of p38 in normal chondrocytes. The proliferation and differentiation of

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chondrocytes were significantly decreased in transgenic mice upon overexpression of MKK6, an

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upstream activator of p38 MAPK98. The levels of pJNK and p38 were higher in chondrocytes

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from human osteoarthritis cartilage compared to normal tissue99. p38 MAPK is also involved in

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matrix associated proteins and Col II degradation in articular chondrocytes via MMP and

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aggrecanases100.

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Future Research Prospective In the last decade, the major goal for researchers has been to develop novel therapeutic

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strategies for cartilage defects. Existing therapeutic treatments are symptomatic and only

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alleviate the pain instead of controlling disease progression. Recently, autologous chondrocyte

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implantation has been considered a successful treatment. Therefore, to develop effective

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therapies, an understanding of the molecular signaling mechanisms of chondrocytes is essential.

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For autologous transplantation, immune rejection is not the major obstacle but rather the lack of

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availability of large numbers of chondrocytes and maintaining chondrocyte viability during in

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vitro expansion is a deterrent.

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There are numerous genes responsible for the direct induction of senescence, which also

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change the phenotypic and molecular characteristics of chondrocytes. Among them, IL-1β and

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p38 MAPK are the most critical genes which play direct role in the induction of senescence.

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Similarly, there are certain genes that induce senescence indirectly via down regulation or

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inhibition. SOX9 is one of the vital genes regulating chondrocyte biology and its downregulation

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is involved in the indirect induction of senescence and dedifferentiation.

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Chondrocytes change their morphology and typical molecular characteristics after the

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fourth passage because of senescence and dedifferentiation and lose their cartilage regeneration

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ability. Therefore, future in vitro and in vivo research must take this into account for effective

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cartilage regeneration therapies.

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A majority of the published results are obtained from in vitro data, therefore it is

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necessary to replicate the results in vivo using animal models to confirm the role of the above

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mentioned signaling pathways. Animal models are an important tool in developmental and

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regenerative studies. A brief schematic diagram (Fig. 4) explains the overall change in the behavior of genes,

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growth factors, and cytokines from normal phenotypic chondrocytes towards senescent

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chondrocytes. Future studies can be designed to better understand the signaling mechanism in

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animal models.

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Conclusion

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Despite numerous studies on chondrocytes and cartilage regeneration, a comprehensive

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understanding of chondrocyte behavior, growth, death, matrix remodeling, and interaction

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between signaling pathways is lacking. Multiple complex signaling pathways are involved in the

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senescence and dedifferentiation of chondrocytes, therefore, a systematic approach is needed to

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sort out the key genes exhibiting crucial roles in regulating the fate of chondrocytes. From the

14

review of numerous studies, it has been made clear that oxidative stress is one of the major

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factors in the induction of senescence in chondrocytes through multiple signaling pathways.

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Specifically, oxidative stress involved in the degeneration of chondrocytes occurs via

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upregulation of IL-1β and p38 MAPK. On the other hand, down regulation of SOX9 significantly

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reduces the chondrocytes’ cartilage tissue regeneration potential. If we can control the expression

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of SOX9, IL-1β, and p38 MAPK, then senescence and dedifferentiation in chondrocytes can be

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overcome to regenerate proper cartilage tissue.

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Signaling pathways and molecular processes are complex; therefore, further in vitro and

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in vivo animal studies are required to achieve our goal of controlling senescence in chondrocytes

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by manipulating a single master gene.

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This study was supported by the National Research Foundation of Korea (NRF) funded

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Acknowledgements

by the Ministry of Science, ICT & Future Planning (NRF-2013R1A2A1A09013980).

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Author Disclosure Statement No competing financial interests exist.

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Authors Contributions

The idea of this study was conceived and brought in to write up form by Sajjad Ashraf.

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All other authors contributed to make the final form of manuscript. Byung-Hyun Cha helped to

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draw the figures used in the manuscript. Jin-Su Kim and Jongchan Ahn contributed for the

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experimental data used as an example to strengthen the study. Inbo Han and Hansoo Park

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critically reviewed and edited the manuscript. Whole study was done under the supervision of

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Soo-Hong Lee.

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Figure Legends

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Figure 1. Cellular senescence and dedifferentiation in chondrocytes. (A) Chondrocytes

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serially cultured in monolayer and their morphology changed from polygonal in passage 2 (P2)

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to fibroblastic in passage 6 (P6) that reveal the dedifferentiated cells. (B) mRNA expression

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analysis of collagen type II (Col II) and collagen type X (Col X) in P2 and P6 chondrocytes by

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RT-PCR. (C) Senescence observed by SA-β Galactosidase assay also shows more positive

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senescent cells in P6. The blue color indicates the positive senescent cells. (D) DIC images of

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normal chondrocytes and degenerated chondrocytes isolated from normal cartilage and OA

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cartilage respectively. (E) Collagen type I (Col 1) observation by western blotting in normal (N)

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and degenerated (D) chondrocytes. (F) DIC images of normal nucleus pulpous chondrocytes

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(NPC) and degenerated nucleus pulpous chondrocytes (dNPC) isolated from normal cartilage

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and OA cartilage respectively. (G) Left panel shows Alcian blue staining of NPC and dNPC. The

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right panel shows Alcian blue staining extraction of NPC and dNPC. (Scale bar = 100 µm, n=3,

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*p<0.05).

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Figure 2. Genes involved in the maintenance of normal phenotypic chondrocytes and can

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regenerate cartilage. Chondrocytes isolated from normal articular cartilage can regenerate

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cartilage again after expansion in monolayer culture if followed the signaling mechanism of IGF-

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1, TGB-β and BMP-2. IGF-1, TGB-β and BMP-2 ultimately involved in the formation of GAG

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matrix required for maintaining chondrocytes normal phenotype and molecular functions.

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Inhibition or down regulation of IGF-1, TGF-β, BMP-2 and especially Sox9 cause senescence in

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the chondrocytes. Sox9 seems to be a major regulator of controlling senescence in chondrocyte

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via maintaining chondrocytes normal phenotype/morphology through Col2a1, core protein and

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aggrecan (For further details please see the text).

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Figure 3. Genes directly involved in cellular senescence and dedifferentiation of

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chondrocytes during prolonged in vitro monolayer culture. Reactive oxygen species (ROS)

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are a main cause of senescence and regulator of genes responsible for senescence. Among all

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genes, IL-1β and p38MAPK are two main genes which further activate multiple pathways to

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induce senescence and apoptosis (For more details please see the text).

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Figure 4. Schematic illustration of discrete genes expression in normal and senescent

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chondrocytes. Genes mentioned in upper green arrow responsible for maintenance of normal

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phenotypic chondrocytes and their down regulation lead towards senescent chondrocytes.

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Genes mentioned in red arrow responsible for senescence and their expression up regulated

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when normal chondrocytes shift towards senescent chondrocytes.

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