Osteoarthritis: toward a comprehensive understanding of pathological mechanism

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 catabolic 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 expression of Dnmts (methylation) and Tets (de-methylation) enzymes.^38–40

Obesity

In recent years, obesity has become a worldwide epidemic characterized by an increased body composition of adipose tissue. The association between obesity and OA has long been recognized.^41,42 Patients with obesity 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 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 elevated^46–50 in high-fat diet-induced mouse obesity models^51–54 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 upregulation 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 production, synovial tissue expansion, inflammatory cell infiltration, and NF-κB pathway activation.⁶⁷

Inflammation

It has been established that the chronic low-grade inflammation found in OA contributes to disease development 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 (ADAMTS) in chondrocytes.^72–76 Complement can be 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-activated protein (MAP) kinases, NF-κB activation, and oxidative 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 catabolic 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 pathogenesis. 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.

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 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

As a degenerative disease, OA always occurs in 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 commonly 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 develops spontaneous OA. It requires 12–20 weeks for 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 models^107,117 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.

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 independently reported by different laboratories.⁵⁴ An elevated systemic inflammation was observed in obese mice following DMM surgery. Serum levels of pro-inflammatory factors, including interleukin-12p70,⁵⁴ interleukin-6, TNFα 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, conventional gene deletion approaches have the drawback of causing embryonic lethality or severe skeletal deformation. To overcome embryonic lethality and bypass the limits of constitutive gene knockout (KO), inducible conditional KO technology has been widely used. This usually combines Cre-loxP gene targeting with tamoxifen-induced nuclear translocation of CreER fusion protein driven by tissue-specific promoters. The Col2a1-Cre^ERT2, Agc1-Cre^ERT2 and Prg4-Cre^ERT2 transgenic mice^122–124 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 intervertebral 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. Additional CreER transgenic mice need to be developed to efficiently target subchondral bone, synovial tissue and meniscus.

Using these transgenic mice, specific genes have 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 molecules, such as Smads, β-catenin, Runx2 and 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 development. 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 roles in various joint tissues¹³⁵ and that therapeutic interventions targeting TGF-β signaling may require a tissue-specific approach.

In vitro studies

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 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 continuous 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 nanoindentation 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 spontaneous^148,149 and post-traumatic^150–152 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. Previously, the ~100 μm thickness of murine cartilage 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, 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 to further in-depth studies. First, Wilusz et al.¹⁶¹ stained cartilage cryosections with immunofluorescence antibodies 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 biomechanical 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 poroelasticity, 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.

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 monocytes and macrophages by signaling through chemokine (C–C motif) receptor 2 (CCR2).¹⁷³ In arthritis, CCL2 promotes inflammation of the joint.¹⁷⁴ Evidence also 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 hyperalgesia 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

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 peripheral 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 Ca_v 3.2, 3.3, and Na_v1.8.^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 histamine and prostaglandins, in addition to NGF.^186,189 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 angiogenesis 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 ADAMTS5 is a major aggrecanase, and because aggrecan is a major component of the proteoglycans in cartilage that provides compressive resistance, ADAMTS5 is thought to be a critical mediator of cartilage degeneration during the development of OA.¹⁹⁸ Although variations in pain signaling can be independent from the degree of joint degeneration, the use of Adamts5 KO mice and direct inhibition of joint degeneration with anti-ADAMTS5 antibody may provide insight into how joint degeneration produces OA pain. For example, hyalectan fragments generated by ADAMTS5 have been suggested to directly stimulate nociceptive neurons as well as glial activation, promoting increased pain perception.^196,199 Furthermore, inhibition of ADAMTS5 following DMM resulted in reduced levels of CCL2 in DRG neurons, thus suggesting a role for CCL2 in OA-specific pain.¹⁹⁷

Pain-related behavior tests

Pain is the most common reason patients seek medical treatment and is a major indication for joint replacement surgery.^200,201 Therefore, evaluating pain in pre-clinical animal models is of critical importance to better understand mechanisms of and to develop treatments for OA pain. The evaluation of OA pain in animals involves indirect and direct measures.

Recognizing pain as a clinical sign and quantitatively assessing pain intensity are essential in research for effective OA pain management. Rodent animal models are routinely used for basic and pre-clinical studies because of the relatively low cost of animal maintenance, the abundance of historical data for comparison, and smaller amounts of drugs required for experimental studies. For pain measurements, rodents have advantages over other small animal models, such as rabbits, which present challenges to obtain a pain response and are immobile if startled by an unfamiliar observer. Mice are usually used for the development of genetically engineered strains to enable molecular understanding of OA progression and pain in vivo.²⁰² Larger animals, including dogs, sheep, goats, and horses are also sometimes used for modeling OA pain.^202,203

A wide range of direct and indirect measures of pain are used in small animal models of OA. Indirect and/or direct measures of pain include static or dynamic weight bearing, foot posture, gait analysis, spontaneous activity, as well as sensitivity to mechanical allodynia, mechanical hyperalgesia, and thermal, and cold stimuli.^202,203 Among indirect tests involving pain-evoked behaviors, mechanical stimuli may be the most correlated with OA pain. A commonly used measure of indirect pain is the von Frey test for mechanical allodynia using filaments to assess referred pain.^{186,194,196,202,204} Direct mechanical hyperalgesia is performed using an analgesymeter for paw pressure pain threshold. Additional direct measures of OA pain include the hind limb withdrawal test, vocalization evoked by knee compression on the affected knee, the struggle reaction to knee extension, and ambulation and rearing spontaneous movements.^194,202,203 Weight-bearing and gait analyses may have important translational relevance for assessing OA pain because these tests are also used to assess clinical OA pain.²⁰³ However, obtaining clear pain responses from weight bearing or gait is challenging when using the unilateral DMM mouse model because the nature of OA pain is a dull pain unlike that of, for example, sharp inflammatory pain.

In large animals, pain behavior testing is more challenging and there is no consensus for the best method of evaluating pain.²⁰² However, dogs, the most commonly used large animal, have been suggested to provide the best predictive modeling for OA pain translated into the clinical setting.²⁰⁵ Methods used for assessing pain in large animals are restricted to assessing degree of lameness, gait analysis, and subjective rating scales, which assess descriptors of pain similar to those of humans.

Overall, there is a wide range of pain-behavior tests for small and large animal models. Although no animal model or pain behavior test perfectly translates to OA-associated pain in patients, these tests yield a valuable understanding of the mechanisms of OA pain and allow assessment of treatments for relief from OA-associated pain. Rodents will continue to be widely used for basic OA pain research, but large animals continue to be important because of their greater potential for modeling clinical OA pain.