OPEN
`
`Citation: Bone Research (2017) 5, 16044; doi:10.1038/boneres.2016.44
`www.nature.com/boneres
`
`REVIEW ARTICLE
`Osteoarthritis: toward a comprehensive understanding of
`pathological mechanism
`
`Di Chen1, Jie Shen2, Weiwei Zhao1,3, Tingyu Wang4, Lin Han5, John L Hamilton1 and Hee-Jeong Im1
`
`Osteoarthritis (OA) is the most common degenerative joint disease and a major cause of pain and disability in
`adult individuals. The etiology of OA includes joint injury, obesity, aging, and heredity. However, the
`detailed molecular mechanisms of OA initiation and progression remain poorly understood and, currently,
`there are no interventions available to restore degraded cartilage or decelerate disease progression. The
`diathrodial joint is a complicated organ and its function is to bear weight, perform physical activity and
`exhibit a joint-specific range of motion during movement. During OA development, the entire joint organ is
`affected, including articular cartilage, subchondral bone, synovial tissue and meniscus. A full understanding
`of the pathological mechanism of OA development relies on the discovery of the interplaying mechanisms
`among different OA symptoms, including articular cartilage degradation, osteophyte formation, subchondral
`sclerosis and synovial hyperplasia, and the signaling pathway(s) controlling these pathological processes.
`Bone Research (2017) 5, 16044; doi:10.1038/boneres.2016.44; published online: 17 January 2017
`
`INTRODUCTION
`Osteoarthritis (OA) is the most common degenerative joint
`disease, affecting more than 25% of the population over 18
`years-old. Pathological changes seen in OA joints include
`progressive loss and destruction of articular cartilage,
`thickening of the subchondral bone, formation of osteo-
`phytes, variable degrees of inflammation of the synovium,
`degeneration of ligaments and menisci of the knee and
`hypertrophy of the joint capsule.1 The etiology of OA is
`multi-factorial and includes joint injury, obesity, aging, and
`heredity.1–5 Because the molecular mechanisms involved
`in OA initiation and progression remain poorly understood,
`there are no current interventions to restore degraded
`cartilage or decelerate disease progression. Studies using
`genetic mouse models
`suggest
`that growth factors,
`including transforming growth factor-β (TGF-β), Wnt3a and
`Indian hedgehog, and signaling molecules,
`such as
`Smad3, β-catenin and HIF-2α,6–10 are involved in OA
`development. One feature common to several OA animal
`models is the upregulation of Runx2.7–8,11–13 Runx2 is a key
`
`transcription factor directly regulating the transcription of
`genes encoding matrix degradation enzymes in articular
`chondrocytes.14–17 In this review article, we will discuss the
`etiology of OA,
`the available mouse models for OA
`research and current techniques used in OA studies. In
`addition, we will also summarize the recent progress on
`elucidating the molecular mechanisms of OA pain. Our
`goal is to provide readers a comprehensive coverage on
`OA research approaches and the most up-to-date
`progress on understanding the molecular mechanism of
`OA development.
`
`ETIOLOGY
`OA is the most prevalent joint disease associated with pain
`and disability. It has been forecast that 25% of the adult
`population, or more than 50 million people in the US, will be
`affected by this disease by the year 2020 and that OA will
`be a major cause of morbidity and physical
`limitation
`among individuals over the age of 40.18–19 Major clinical
`symptoms include chronic pain, joint instability, stiffness and
`
`1Department of Biochemistry, Rush University Medical Center, Chicago, IL, USA; 2Department of Orthopaedic Surgery, Washington University, St
`Louis, MO, USA; 3Department of Orthopaedics & Traumatology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China;
`4Department of Pharmacy, Shanghai Ninth People’s Hospital, Shanghai JiaoTong University School of Medicine, Shanghai, China and 5School of
`Biomedical Engineering, Science, and Health Systems, Drexel University, Philadelphia, PA, USA
`Correspondence: Di Chen (di_chen@rush.edu)
`Received: 4 August 2016; Revised: 2 September 2016; Accepted: 8 September 2016
`
`

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`2
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`Osteoarthritis
`D Chen et al
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`radiographic joint space narrowing.20 Although OA primar-
`ily affects the elderly, sports-related traumatic injuries at all
`ages can lead to post-traumatic OA. Currently, apart from
`pain management and end stage surgical
`intervention,
`there are no effective therapeutic treatments for OA. Thus,
`there is an unmet clinical need for studies of the etiology
`and alternative treatments for OA. In recent years, studies
`using the surgically induced destabilization of the medial
`meniscus (DMM) model and tissue or cells from human
`patients demonstrated that genetic, mechanical, and
`environmental factors are associated with the develop-
`ment of OA. At the cellular and molecular level, OA is
`characterized by the alteration of the healthy homeostatic
`state toward a catabolic state.
`
`Aging
`One of the most common risk factors for OA is age. A
`majority of people over the age of 65 were diagnosed with
`radiographic changes in one or more joints.21–25 In addition
`to cartilage, aging affects other
`joint tissues,
`including
`synovium, subchondral bone and muscle, which is thought
`to contribute to changes in joint loading. Studies using
`articular chondrocytes and other cells suggest that aging
`cells show elevated oxidative stress that promotes cell
`senescence and alters mitochondrial function.26–29 In a
`rare form of OA, Kashin-Back disease, disease progression
`was associated with mitochondrial dysfunction and cell
`death.30 Another hallmark of aging chondrocytes
`is
`reduced repair response, partially due to alteration of the
`receptor expression pattern. In chondrocytes from aged
`and OA cartilage, the ratio of TGF-β receptor ALK1 to ALK5
`was increased, leading to down-regulation of the TGF-β
`pathway and shift from matrix synthesis activity to cata-
`bolic matrix metalloproteinase (MMP) expression.31–32
`Recent studies also indicate that methylation of the entire
`genomic DNA displayed a different signature pattern in
`aging cells.33–34 Genome-wide sequencing of OA patients
`also confirmed that this epigenetic alteration occurred in
`OA chondrocytes,35–37 partially due to changes in expres-
`sion of Dnmts (methylation) and Tets (de-methylation)
`enzymes.38–40
`
`Obesity
`In recent years, obesity has become a worldwide epi-
`demic characterized by an increased body composition
`of adipose tissue. The association between obesity and OA
`long been recognized.41–42 Patients with obesity
`has
`develop OA earlier and have more severe symptoms,
`higher risk for infection and more technical difficulties for
`total
`joint replacement surgery. In addition to increased
`biomechanical loading on the knee joint, obesity is thought
`to contribute to low-grade systemic inflammation through
`
`Bone Research (2017) 16044
`
`secretion of adipose tissue-derived cytokines, called
`adipokines.43–45 Specifically,
`levels of pro-inflammatory
`cytokines, including interleukin (IL)-1β, IL-6, IL-8, and tumor
`necrosis factor alpha (TNF-α) were elevated46–50 in high-fat
`diet-induced mouse obesity models51–54 and in obese
`patients.55–57 These inflammatory factors may trigger the
`nuclear factor-κB (NF-κB) signaling pathway to stimulate an
`articular chondrocyte catabolic process and lead to
`extracellular matrix (ECM) degradation through the upre-
`gulation of MMPs.58–60
`
`Sport injury
`Knee injury is the major cause of OA in young adults,
`increasing the risk for OA more than four times. Recent
`clinical reports showed that 41%–51% of participants with
`previous knee injuries have radiographic signs of knee OA
`in later years.61 Cartilage tissue tear, joint dislocation and
`ligament strains and tears are the most common injuries
`seen clinically that may lead to OA. Trauma-related sport
`injuries can cause bone, cartilage, ligament, and meniscus
`damage, all of which can negatively affect
`joint
`stabilization.62–66 Signs of inflammation observed in both
`patients with traumatic knee OA and in mouse injury
`models include increased cytokine and chemokine pro-
`duction, synovial tissue expansion, inflammatory cell infiltra-
`tion, and NF-κB pathway activation.67
`
`Inflammation
`the chronic low-grade
`It has been established that
`inflammation found in OA contributes to disease develop-
`ment and progression. During OA progression, the entire
`synovial joint, including cartilage, subchondral bone, and
`synovium, are involved in the inflammation process.68 In
`aging and diabetic patients, conventional
`inflammatory
`factors, such as IL-1β and TNF-α, as well as chemokines,
`were reported to contribute to the systemic inflammation
`that leads to activation of NF-κB signaling in both synovial
`cells and chondrocytes. Innate inflammatory signals were
`also involved in OA pathogenesis,
`including damage
`associated molecular patterns (DAMPs), alarmins (S100A8
`and S100A9) and complement.69–71 DAMPs and alarmins
`were reported to be abundant in OA joints, signaling
`through either toll-like receptors (TLR) or the canonical
`NF-κB pathway to modulate the expression of MMPs and a
`disintegrin and metalloprotease with thrombospondin motif
`in chondrocytes.72–76 Complement can be
`(ADAMTS)
`activated in OA chondrocytes and synovial cells by
`DAMPs, ECM fragments and dead-cell debris.77–78 Recent
`studies further clarified that systemic inflammation can re-
`program chondrocytes through inflammatory mediators
`toward hypertrophic differentiation and catabolic
`responses through the NF-κB pathway,9–10,79 the ZIP8/Zn+/
`
`

`

`MTF1 axis,80 and autophagy mechanisms.81–85 Indeed, the
`recent Kyoto Encyclopedia of Genes and Genomes
`(KEGG) pathway analyses of OA and control samples
`provide evidence that inflammation signals contribute to
`OA pathogenesis through cytokine-induced mitogen-acti-
`vated protein (MAP) kinases, NF-κB activation, and oxida-
`tive phosphorylation.86
`
`Genetic predisposition
`An inherited predisposition to OA has been known for
`many years from family-based studies.87–89 Although the
`genetics of OA are complex, the genetic contribution to
`OA is highly significant. Over the past decade, the roles of
`genes and signaling pathways in OA pathogenesis have
`been demonstrated by ex vivo studies using tissues derived
`from OA patients and in vivo studies using surgically
`induced OA animal models and genetic mouse models.
`For example, alterations in TGF-β, Wnt/β-catenin,
`Indian
`Hedgehog (Ihh), Notch and fibroblast growth factor (FGF)
`pathways have been shown to contribute to OA
`development and progression by primarily inducing cata-
`bolic responses in chondrocytes.8,90–95 Such responses
`converge on Hif2α, Runx2, and inflammatory mediators
`that
`lead to cartilage ECM degradation through the
`increased
`expression
`of MMPs
`and
`ADAMTS
`activity.80,96–99 Recent studies of genome-wide association
`screens (GWAS) that have been performed on large
`numbers of OA and control populations throughout the
`world have confirmed over 80 gene mutations or single-
`nucleotide polymorphisms (SNPs) involved in OA patho-
`genesis. Some of the genes are important structural and
`ECM-related factors (Col2a1, Col9a1, and Col11a1), and
`critical
`signaling molecules
`in the Wnt
`(Sfrp3), bone
`morphogenetic protein (BMP) (Gdf5), and TGF-β (Smad3)
`signaling pathways; most of
`these genes have been
`previously implicated in OA or articular cartilage and joint
`maintenance by studies using mouse models of induced
`genetic alteration- or surgically induced OA.100–106 A
`recent arcOGEN Consortium genome-wide screen
`study107 identified new SNPs in several genes, including
`GNL3, ASTN2, and CHST11. These findings need to be
`verified by further studies.
`
`MOUSE MODELS FOR OA RESEARCH
`DMM model
`DMM was developed 10 years ago and is a well
`established surgical OA model in mice and rats. It is widely
`used to study OA initiation and progression in combination
`with transgenic mouse models and aging and obesity
`models. DMM surgery was performed by transection of the
`medial meniscotibial ligament (MMTL).26–27 Briefly, following
`the initial incision, the joint capsule on the medial side was
`
`Osteoarthritis
`D Chen et al
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`3
`
`incised using scissors to expose either the intercondylar
`region or the MMTL, which anchors the medial meniscus
`(MM) to the tibial plateau. The MMTL was visualized under a
`dissection microscope and the MMTL was cut using micro-
`surgical scissors,
`releasing the ligament
`from the tibia
`plateau thus destabilizing the medial meniscus. Closure of
`the joint capsule and skin was with a continuous 8–0
`tapered Vicryl suture. As a control for DMM studies, sham
`surgery was performed by only exposing the medial side of
`knee joint capsule. Because of the medial displacement of
`the meniscus tissue, greater stress occurred on the posterior
`femur and central tibia, especially on the medial side.108
`Histology demonstrated the severity of OA lesions at
`4-weeks post-surgery with fibrillation of
`the cartilage
`surface. Cartilage destruction and subchondral bone
`sclerosis developed 8 weeks post-surgery and osteophyte
`formation was seen 12-weeks post- surgery.98,109–111
`
`Aging model
`in
`As a degenerative disease, OA always occurs
`elderly populations; thus, aging is a major risk factor for
`the most common form in humans, spontaneous OA.
`Several
`laboratory
`animals
`develop
`spontaneous
`OA, which approximates
`the stages of human OA
`progression. These animal models are valuable tools for
`studying natural OA pathogenesis.112–113 The most com-
`monly used inbred strain of laboratory mouse is C57/BL6;
`these mice usually develop knee OA at about 17 months
`of age.112 The STR/ort mouse is one strain that easily
`It requires 12–20 weeks for
`develops spontaneous OA.
`STR/ort mice
`to
`develop
`articular
`cartilage
`destruction.114–116 This may be partially due to their heavier
`body weight compared with other mouse strains. Given
`the background genetic consistency, although aging OA
`models have many advantages,
`it normally requires at
`least one year for mice to model the disease. Therefore,
`surgically induced OA models107,117 and genetic mouse
`models are preferred in recent decades for their relatively
`fast induction for use as aging models for the study of OA
`lesions.
`In addition to the mouse, the Dunkin Hartley guinea
`pig provides an aging model widely used to study OA
`development.118 The Dunkin Hartley guinea pig can
`develop a spontaneous, age-related OA phenotype
`within 3 months. The severity of OA lesions increases with
`age, and moderate to severe OA is observable in
`18-month-old animals. Histological analysis demonstrated
`that the spontaneous OA progression in Dunkin Hartley
`guinea pig resembles that of humans. Thus, the Dunkin
`Hartley guinea pig is a useful animal
`to study the
`pathogenesis and evaluation of potential treatments for
`human OA.
`
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`4
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`Osteoarthritis
`D Chen et al
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`Obesity model
`It has become evident that obesity contributes to a variety
`of musculoskeletal diseases, particularly OA, because of
`inflammatory and metabolic responses.119 Together with
`surgically induced injury and genetic models, mouse
`obesity models are widely used to explore the mechanisms
`of obesity-induced OA. The obese mouse model is induced
`by a high-fat diet, in which 60% of calories are derived from
`fat as opposed to the normal 13%.120 The entire joint tissue,
`but especially synovium tissue, is affected by the high-fat
`diet. A synovial
`inflammation phenotype has been inde-
`pendently reported by different laboratories.54 An ele-
`vated systemic inflammation was observed in obese mice
`following DMM surgery. Serum levels of pro-inflammatory
`including interleukin-12p70,54 interleukin-6, TNFα
`factors,
`and several other chemokines, were increased, suggesting
`a role for obesity in the development of post-traumatic OA
`(PTOA).
`
`Genetic mouse models
`Genetic mouse models have recently become widely
`used to investigate the cellular and molecular mechanisms
`of OA development. Based on the GWAS studies of human
`patients, mutant mouse strains were generated carrying
`either mutant genes or SNPS. For example, Del1+/- mice
`carried a mutation in the collagen II gene. Both Del1+/-
`mice and Col9a1−/− mice developed spontaneous OA.121
`Because cartilage functions as a skeletal architect, con-
`ventional gene deletion approaches have the drawback
`of causing embryonic lethality or severe skeletal deforma-
`tion. To overcome embryonic lethality and bypass the limits
`of constitutive gene knockout (KO), inducible conditional
`KO technology has been widely used. This usually com-
`bines Cre-loxP gene targeting with tamoxifen-induced
`nuclear translocation of CreER fusion protein driven by
`tissue-specific promoters. The Col2a1-CreERT2, Agc1-CreERT2
`and Prg4-CreERT2 transgenic mice122–124 have become
`powerful
`tools
`for
`targeting joint
`tissue to study the
`mechanism of OA development. Based on the gene
`expression pattern, both Col2a1 and Agc1 can efficiently
`target chondrocytes in the growth plate cartilage, articular
`cartilage and temporomandibular joint. Because Agc1 is
`expressed more robustly than Col2a1 in adult cartilage
`tissue, Agc1 is expected to better target chondrocytes in
`adult mice.123 In addition to chondrocytes, Agc1 were also
`reported to target nucleus pulposus tissue in the interver-
`tebral disc.123 Prg4 only targets the superficial
`layer of
`articular chondrocytes.124 It needs to be emphasized that
`all of
`these genetic tools are used to address
`the
`importance of cartilage tissue in OA development. Addi-
`tional CreER transgenic mice need to be developed to
`
`Bone Research (2017) 16044
`
`Table 1. Available transgenic mouse models for osteoarthritis
`research
`Gene
`
`Targeting tissue
`
`Pathway
`
`Del1125
`Col9a1126
`Tgfbr290
`Smad36
`Smurf2127
`Tgfbr291
`Frzb128
`β-catenin7
`Rbpjk93
`Fgfr1129
`Smo8
`Runx211
`Hif2a9
`Mmp1397
`Mmp13130
`Adamts598
`
`Global
`Global
`4xMRE
`Global
`Col2-Cre
`Col2-CreER
`EIIaCre
`Col2-CreER
`Prx1Cre
`Col2-CreER
`Col2-Cre
`Global
`Global
`Global
`Col2-CreER
`Global
`
`ECM
`ECM
`TGF-β
`TGF-β
`TGF-β
`TGF-β
`Wnt
`Wnt
`Notch
`FGF
`Ihh
`
`Abbreviations: ECM, extracellular matrix; FGF, fibroblast growth factor; Ihh, Indian
`Hedgehog; TGF-β, transforming growth factor-β.
`
`efficiently target subchondral bone, synovial tissue and
`meniscus.
`specific genes have
`Using these transgenic mice,
`been studied in chondrocyte-specific experiments
`to
`dissect their role in OA. In vivo studies employing mutant
`mice suggest that pathways involving (i) receptor ligands,
`such as TGF-β1, Wnt3a, and Indian hedgehog, (ii) signaling
`β-catenin, Runx2 and
`molecules,
`such as
`Smads,
`HIF-2α and,
`(iii) peptidases,
`such as MMP13 and
`ADAMTS4/5, have some degree of involvement in OA
`development.
`Table 1 summarizes
`the mutant
`lines
`available for OA study.
`TGF-β and its downstream molecules have important
`roles in OA pathogenesis. Mutations of Smad3, a central
`molecule in TGF-β
`signaling, have been found in
`patients with early-onset OA.131–133 It has been known for
`years that TGF-β promotes mesenchymal progenitor cell
`differentiation
`and matrix
`protein
`synthesis
`and
`inhibits chondrocyte hypertrophy. TGF-β signaling may
`play differential roles in joint tissues during OA develop-
`ment. For example, global deletion of Smad3 causes
`chondrocyte hypertrophy and OA-like articular cartilage
`damage.6 The deletion of Tgfbr2, encoding for type II TGF-β
`receptor,91 or Smad312 in articular chondrocytes also
`led to an OA-like phenotype. In contrast, the activation
`of TGF-β signaling in mesenchymal progenitor cells of
`subchondral bone also caused OA-like lesions.134 These
`findings suggest that TGF-β signaling may have differential
`tissues135 and that
`therapeutic
`roles
`in various
`joint
`interventions targeting TGF-β signaling may require a
`tissue-specific approach.
`
`

`

`TECHNIQUES FOR OA STUDIES
`In vitro studies
`To
`In vitro articular chondrocyte isolation and culture.
`investigate signaling mechanisms in articular cartilage,
`primary human articular chondrocytes will be obtained
`from surgically discarded cartilage tissues. Briefly,
`full-
`thickness sections of cartilage are excised from the
`subchondral bone. The cartilage pieces will be digested
`for about 15 h using a digestion buffer. The isolated cells
`will be then collected and filtered to remove undigested
`tissue and debris, and washed with Hanks' buffered salt
`solution. The cells will be then re-suspended in chondro-
`cyte basal medium and plated in high density monolayer
`cultures as shown in Table 2.136–137 Human articular
`chondrocytes can also be cultured in three dimensions.
`Briefly, 4 × 106 freshly isolated human articular chondro-
`cytes will be re-suspended in alginate solution and the cell
`suspension is added drop-wise into 102 mmol·L − 1 CaCl2 to
`form beads. After washing the beads with 0.15 mol·L − 1
`NaCl and basal medium, the chondrocytes encapsu-
`lated in alginate beads will be cultured in three dimen-
`sions with basal medium.138–139
`
`In vitro human articular cartilage explant culture. Osteo-
`chondral tissues from radiographically and anatomically
`normal joints will be obtained from patients with different
`surgeries, such as oncologic surgical procedures, menis-
`cal
`tear
`repair or
`total knee joint
`replacement. The
`collected osteochondral tissues will be first washed with
`sterile phosphate-buffered saline (PBS). Fresh cartilage
`samples will be harvested from the femoral condyle using
`a 6 mm diameter biopunch. The cartilage explants will be
`cultured in chondrocyte basal medium.140
`
`Histology/histomorphometry
`Knee cartilage samples to be used for histological and
`histomorphometric analyses will be fixed in 10% neutral
`buffered formalin (NBF), decalcified in 14% EDTA for 10 days
`and embedded in paraffin.
`The paraffin-embedded
`samples will be cut into 5 μm sections and stained with
`Alcian blue/Hematoxylin-Orange G (ABH) or Safranin
`O/Fast green to determine changes in architectures of
`cartilage, bone, and synovial
`tissues
`throughout OA
`progression. Quantitative histomorphometric analyses of
`
`Table 2. Monolayer culture conditions for human primary
`articular chondrocytes
`Plate type
`Volume per well
`
`No. of cells per well
`
`6-well
`12-well
`24-well
`
`2.5 mL
`1 mL
`0.5 mL
`
`1 × 106
`4 × 105
`2 × 105
`
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`D Chen et al
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`5
`
`ABH-stained sections can be performed using a Visiopharm
`analysis system.141 Using this system, high resolution digital
`images of histology slides can be obtained. Cartilage
`thickness will be measured from the middle of the femoral
`and tibial condyles. Cartilage area will be traced from
`both articular cartilage surfaces. The tidemark will be used
`to delineate the upper and deep zone of articular
`cartilage.91,93
`
`OARSI score system
`Several scoring systems have been developed to semi-
`quantify the severity of OA lesions of the knee. A scoring
`system recommended by the Osteoarthritis Research
`Society International (OARSI) society is based on contin-
`uous histological staining of the knee joint. A 0–6 subjective
`scoring system, as shown in Table 3, is applied to all four
`quadrants through multiple step sections of
`the joint.
`Sagittal sections obtained every 80 μm across the medial
`femoral-tibial joint will be used to determine the maximal
`and cumulative scores.142
`
`Nanoindentation
`It is necessary to understand changes in mechanical
`properties of OA cartilage across multiple length scales
`because they directly reflect cartilage functional changes
`during degradation.143 Atomic force microscopy (AFM)-
`based nanoindentation is well-suited for evaluating
`changes at a nm-to-μm scale that is comparable to the
`sizes of matrix molecules and cells.144
`For AFM-
`nanoindentation measurement, a microspherical or a
`pyramidal tip is programmed to indent the sample tissues,
`cells or tissue sections to a pre-set force or depth. An
`effective indentation modulus can be calculated by fitting
`the loading portion of each indentation force versus depth
`curve to the elastic Hertz model.145 The use of nanoinden-
`tation over the past decade has uncovered many new
`aspects of cartilage structure-mechanics relationships and
`OA pathomechanics. Highlights among these include
`micromechanical anisotropy and heterogeneity of healthy
`and OA cartilage146 or meniscus,147 cartilage weakening in
`spontaneous148–149
`post-traumatic150–152
`and
`OA,
`mechanics of individual chondrocytes,151,153 and quality
`evaluation of engineered neo-tissues.154–156
`Notably, AFM-nanoindentation has made it possible to
`study the mechanical properties of murine cartilage.
`the ~ 100 μm thickness of murine cartilage
`Previously,
`prevented such attempts. Because in vivo OA studies are
`largely dependent on murine models,157 nanoindentation
`provides a critical bridge across two crucial fields of OA
`research: biology and biomechanics. The benefit of
`nanoindentation for murine model
`studies has been
`demonstrated by a number of recent studies. For example,
`
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`6
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`Osteoarthritis
`D Chen et al
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`Table 3. The recommended semi-quantitative scoring system143
`Grade
`Osteoarthritic damage
`
`0
`0.5
`1
`2
`3
`4
`5
`6
`
`Normal
`Loss of Safranin O without structural changes
`Small fibrillations without loss of cartilage
`Vertical clefts down to the layer immediately below the superficial layer and some loss of surface lamina
`Vertical clefts/erosion to the calcified cartilage extending to o25% of the articular surface
`Vertical clefts/erosion to the calcified cartilage extending to 25%–50% of the articular surface
`Vertical clefts/erosion to the calcified cartilage extending to 50%–75% of the articular surface
`Vertical clefts/erosion to the calcified cartilage extending to 475% of the articular surface
`
`cartilage in mice lacking collagen IX (Col9a1−/−)148
`showed abnormally higher moduli, while those lacking
`lubricin (Prg4−/−)158 or chondroadherin (Chad−/−)159
`showed lower moduli. Col9a1−/− and Prg4−/− mice also
`developed macroscopic signs of OA,148,158 underscoring
`the high correlation between abnormalities in cartilage
`biomechanics and OA. Li et al. also recently demonstrated
`the applicability of nanoindentation to the murine
`meniscus.160 Further applications of nanoindentation to
`clinically relevant OA models, such as the DMM model,110
`hold the potential of assessing OA as an entire joint disease
`through biomechanical
`symptoms
`in multiple murine
`synovial tissues.
`Two other recent technological advances provide paths
`in-depth studies. First, Wilusz et al.161 stained
`to further
`cartilage cryosections with immunofluorescence antibo-
`dies of the pericellular matrix signature molecules, type VI
`collagen and perlecan.162 Using immunofluorescence
`guidance, nanoindentation was used to delineate the
`mechanical behavior of cartilage pericellular matrix and
`ECM,161–163 and to reveal the role of type VI collagen in
`each matrix by employing Col6−/− mice.164 Therefore, it is
`now possible to directly examine the relationships across
`micro-domains between biochemical content and biome-
`chanical properties of cartilage,161 meniscus165 or other
`synovial tissues in situ. Second, Nia et al.166 converted the
`AFM to a high-bandwidth nanorheometer.
`This
`tool
`enabled separation of the fluid flow-driven poroelasticity
`and macromolecular frictional
`intrinsic viscoelasticity that
`govern cartilage energy-dissipative mechanics.166–168
`Hydraulic permeability, the property that regulates poroe-
`lasticity, was found to be mainly determined by aggrecan
`rather than collagen169 and to change more drastically
`than modulus upon depletion of aggrecan.166,170 This new
`tool provides a comprehensive approach beyond the
`scope of elastic modulus for assessing cartilage functional
`changes in OA.
`
`MOLECULES MEDIATING OA PAIN
`The perception of OA pain is a complex and dynamic
`process involving structural and biochemical alterations at
`
`Bone Research (2017) 16044
`
`the joint as well as in the peripheral and central nervous
`systems. While there have been extensive studies of
`mediators of OA joint degeneration, only recently have
`studies begun to characterize biochemical
`influences on
`and in the peripheral and central nervous systems in OA. In
`this regard, OA appears to show similarities and differences
`with other conditions causing pain.171–172 There are a wide
`variety of signaling pathways linked to joint destruction
`and/or pain. In this section we will discuss three emerging
`and highly relevant pathways that provide insight into the
`mechanisms underlying OA pain.
`
`Chemotactic cytokine ligand 2/chemokine (C–C motif)
`receptor 2
`Chemotactic cytokine ligand 2 (CCL2), also known as
`monocyte chemoattractant protein 1 (MCP-1),
`is well-
`known to mediate the migration and infiltration of mono-
`cytes and macrophages by signaling through chemokine
`(C–C motif)
`receptor 2 (CCR2).173
`In arthritis, CCL2
`the joint.174 Evidence also
`promotes
`inflammation of
`suggests
`that CCL2 is an important mediator of
`neuroinflammation.175–176
`In neuropathic pain, CCL2
`expression is increased in microglia and in sensory neurons
`in the dorsal root ganglia (DRGs), where CCL2 can be
`further transported and released into central spinal nerve
`terminals.
`Increased CCL2/CCR2 signaling has been
`correlated with direct excitability of nociceptive neurons
`and microglial activation, leading to persistent hyperalge-
`sia and allodynia.177–178
`In a DMM mouse OA model, CCL2 and CCR2 levels were
`elevated in DRGs at 8 weeks post surgery, correlating with
`increased OA-associated pain behaviors. Increased CCL2
`and CCR2 levels in the DRG were thought to mediate pro-
`nociceptive effects both by increasing sensory neuron
`excitability through CCL2/CCR2 signaling directly in DRG
`sensory neurons and through CCL2/CCR2-mediated
`recruitment of macrophages in the DRG. Compared with
`wild-type mice, Ccr2-null mice showed reduced pain
`behaviors
`following DMM with similar
`levels of
`joint
`damage.179 Although CCR2 antagonists are currently
`being assessed in clinical studies, no clinical studies have
`targeted CCL2 or CCR2 in OA pain.180
`
`

`

`Osteoarthritis
`D Chen et al
`
`7
`
`Nerve growth factor/tropomyosin receptor kinase A
`In both clinical and animal studies, the targeted inhibition
`of nerve growth factor (NGF) and inhibition of its cognate
`receptor, tropomyosin receptor kinase A (TrkA), reduced
`OA pain. Clinically, the systemic administration of NGF
`caused persistent whole-body muscle hyperalgesia in
`healthy human subjects,174,177 while anti-NGF antibody,
`tanezumab, therapy significantly reduced OA pain.181–184
`There are a number of potential mechanisms through
`which NGF mediates pain. Over-expressed NGF in periph-
`eral tissues can bind directly to TrkA at sensory neuron
`nerve terminals and be retrogradely transported to the
`DRG. There it
`stimulates
`sensory neurons
`to activate
`mitogen-activated protein kinase (MAPK)/extracellular
`signal-regulated kinase (ERK) signaling.185 The activation of
`the NGF-MAPK/ERK axis upregulates the expression of pain-
`related molecules, including transient receptor potential
`cation channel subfamily V member I (TRPV1), substance P,
`calcitonin gene-related peptide (CGRP), brain-derived
`neurotrophic factor (BDNF), and nociceptor-specific ion
`channels, such as Cav 3.2, 3.3, and Nav1.8.186–188
`In addition to direct signaling of sensory neurons, NGF
`promotes algesic effects by targeting other cell types. For
`example, NGF/TrkA signaling occurs in mast cells, triggering
`release of pro-inflammatory and pain mediators, including
`in addition to NGF.186,189
`histamine and prostaglandins,
`NGF
`signaling is upregulated by pro-inflammatory
`mediators, and NGF promotes
`leukocyte chemotaxis
`and
`vascular
`permeability,
`further
`stimulating
`inflammation.190–192 NGF/TrkA signaling further promotes
`angiogenesis and nerve growth. The process of angiogen-
`esis is not only inflammatory, but also serves as a track for
`nerve growth into the joint.193
`Given the high efficacy of targeting NGF in a clinical study
`on reducing OA pain, it is of great interest to further define
`NGF/TrkA pain signaling mechanisms and to find additional
`therapeutic targets in this pathway. Recent evidence
`indicates that loss of PKCδ signaling significantly increases
`both NGF and TrkA in the DRG and synovium, is associated
`with increased MAPK/ERK signaling at the innervating DRGs,
`and is associated with OA hyperalgesia.194 However,
`in
`recent clinical studies, a small population of patients treated
`with systemic anti-NGF therapy exhibited rapid progression
`of OA and were more prone to bone fractures.195
`Considering the analgesic effects by anti-NGF therapy on
`OA-associated pain, understanding of the precise roles of
`the NGF/TrkA pathway in different joint tissues in OA and
`OA-associated pain is of great interest.
`
`ADAMTS5
`The use of Adamts5 KO mice and therapeutic treatment
`with anti-ADAMTS5 antibody in wild-type mice produce
`
`inhibition of ADAMTS5 signaling/expression in the DMM
`model, resulting in reduction of both joint degeneration
`and pain.98,196–197 ADAMT