key: cord-0922160-f8r2barc authors: Vincent, Tonia L title: Of mice and men: converging on a common molecular understanding of osteoarthritis date: 2020-09-23 journal: Lancet Rheumatol DOI: 10.1016/s2665-9913(20)30279-4 sha: 367c093dfa40e8b704eeb01d87351c3aaab3bf73 doc_id: 922160 cord_uid: f8r2barc Despite an increasing burden of osteoarthritis in developed societies, target discovery has been slow and there are currently no approved disease-modifying osteoarthritis drugs. This lack of progress is due in part to a series of misconceptions over the years: that osteoarthritis is an inevitable consequence of ageing, that damaged articular cartilage cannot heal itself, and that osteoarthritis is driven by synovial inflammation similar to that seen in rheumatoid arthritis. Molecular interrogation of disease through ex-vivo tissue analysis, in-vitro studies, and preclinical models have radically reshaped the knowledge landscape. Inflammation in osteoarthritis appears to be distinct from that seen in rheumatoid arthritis. Recent randomised controlled trials, using treatments repurposed from rheumatoid arthritis, have largely been unsuccessful. Genome-wide studies point to defects in repair pathways, which accords well with recent promise using growth factor therapies or Wnt pathway antagonism. Nerve growth factor has emerged as a robust target in osteoarthritis pain in phase 2–3 trials. These studies, both positive and negative, align well with those in preclinical surgical models of osteoarthritis, indicating that pathogenic mechanisms identified in mice can lead researchers to valid human targets. Several novel candidate pathways are emerging from preclinical studies that offer hope of future translational impact. Enhancing trust between industry, basic, and clinical scientists will optimise our collective chance of success. The global impact of osteoarthritis, the most common form of joint disease in developed societies, is predicted to rise steadily as obesity and longevity increase. 1 Osteo arthritis is a substantial societal burden, associated with increased mortality and frequently complicated by multimorbidity and polypharmacy. [2] [3] [4] The recent accep tance of osteoarthritis as a serious disease has helped to drive the therapeutic agenda forward, to garner support from academia and industry, and to influence healthcare prioritisation. 5 The market for symptomatic and disease modifying treatments is huge, and yet relatively little progress has been made thus far in bringing new treat ments to patients. Osteoarthritis research can be broadly divided into clinical and basic categories. Clinical research includes pathology, epidemiology, and interventional studies in humans, whereas basic research encompasses the study of molec ular pathogenesis through invitro systems, preclinical models, and largescale omics (ie, genomics, transcrip tomics, proteomics, and metabolomics) studies. Osteo arthritis is a mechanically driven disease. This notion is compellingly described in the epidemiological literature 6 and confirmed in basic science studies, which have shown the highly mechano sensitive nature of joint tissues, 7-12 the activation of inflammatory signalling by mechanical injury, 12,13 the dependence on mechanics in preclinical osteoarthritis, 14, 15 and the involvement of mechanosens ing mechanisms in invivo pathogenesis. 16, 17 Several other important causal factors-such as obesity, age, and genetics-affect the ability of joint tissues to withstand mechanical stress over a lifetime and affect the ability to repair damaged tissues. These factors might also increase the risk of osteoarthritis in ways that are indepen dent of mechanics. For example, osteoarthritis in non weightbearing joints is increased in obese individuals, 18 possibly due to lowgrade systemic inflammation, 19, 20 which might be linked to the gut microbiome. 21 Various impediments are recognised in osteoarthritis drug development. Osteoarthritis is an insidious and heterogeneous disease. These qualities inevitably mean that clinical trials are often prohibitively expensive, and raise the possibility that one target might not work for all. Molecular pathogenesis also has its challenges. Molecular tools have needed to be refined to work in pauci cellular, matrixrich tissues, such as articular cartilage. Low access to human tissue at early stages of disease has necessi tated a reliance on preclinical models, which has also required substantial refinement, largely involv ing moving away from disease models involving chemical induction methods (eg, monosodium iodoacetate, papain, and col lagenase injection) in favour of those induced by surgi cal desta bilisa tion of the joint. 22 In the past 15 years, target discovery in osteoarthritis has increased substantially, particularly through large, agnostic omic studies using endstage human disease tissue and through molecular validation facilitated by preclinical mouse models and clinical trials. There has also been considerable research into methodological tools for improving clinical outcome measures and osteo arthritis trial design. 23 In this Review, recent successes and failures in osteoarthritis clinical trials are considered in parallel with preclinical advances. Together, these different types of research are helping to unravel the complexities of osteoarthritis pathogenesis and to provide future target ing strategies with a higher chance of translational success. Support for the involvement of inflammation in osteo arthritis comes from clinical observation (joint line tender ness, synovial thickening, and episodic joint effu sion) and radiographic evidence of synovial hypertrophy and bone e634 www.thelancet.com/rheumatology Vol 2 October 2020 Review marrow oedema (by MRI and ultrasound) that are associated with clinical outcome. [24] [25] [26] Additionally, various inflammatory molecules-including cytokines, chemo kines, and metalloproteinases-have been mea sured in osteoarthritis cartilage and synovium. 27, 28 Clinicians distinguish between inflammatory arthritis and osteoarthritis through a relative paucity of leucocytes in osteoarthritis synovial fluid, which are predominantly monocytes (in osteoarthritis) rather than neutrophils (in rheumatoid arthritis). Patients with osteoarthritis typically complain of less than 30 min early morning stiffness and show a modest systemic inflammatory response. 29 These features are used clinically to aid the diagnosis of osteo arthritis. Whether lowgrade inflammation contributes to osteoarthritis pathogenesis, both in terms of pain and structural disease, has been subject to heated debate over the years. Several randomised controlled trials that address different aspects of inflammation have been recently conducted. All the tested drugs derive from experience in rheumatoid arthritis where there is proven efficacy for such therapies. Intraarticular corticosteroids are widely used in clinical practice in osteoarthritis, although few historical studies have applied stringent placebocontrolled, randomised, and doubleblind assessments. In hand osteoarthritis, a randomised controlled trial 30 of intraarticular triamcina lone hexacetomide (a longacting steroid prepara tion) plus lidocaine (a local anaesthetic) showed clinical improve ment up to 12 weeks following injection of the drug compared with lidocaine alone. This result met the primary outcome of the study, albeit in only two of eight coprimary endpoints. 30 For both groups, the injected joint was splinted for 48 h immediately after treatment. A phase 2b trial 31 of an extendedrelease intraarticular steroid showed greater efficacy than placebo in pain outcomes for knee osteoarthritis at several time points, even though the primary endpoint (pain at 12 weeks) was not met. Combined phase 2-3 studies of this preparation have showed an acceptable safety profile and a reduc tion in use of rescue pain medication (paracetamol or acetaminophen). 32 In 2017, this prepara tion received approval by the US Food and Drug Administration (FDA) for clinical use under the trade name Zilretta. Oral prednisolone has also been tested in hand osteo arthritis. The first study by Wenham and colleagues, 33 in which 70 patients were randomly assigned to receive 5 mg of prednisolone or placebo daily, found no statistically significant improvement in pain at 4 or 12 weeks. How ever, a 2019 study, 34 in which patients were randomly assigned to receive 10 mg of prednisolone or placebo daily for 6 weeks, showed significant improvement in patient reported pain and function at the primary endpoint of 6 weeks. Symptoms returned rapidly after withdrawal of the drug. The study found a reduction in synovial thickness, but no improvement in synovitis by MRI or power doppler assessment, making the primary target tissue of the drug unclear. 34 Few studies have attempted to examine the longterm effects of corticosteroids on joints. In a randomised controlled trial by McAlindon and colleagues, 35 140 patients with knee osteoarthritis were randomly assigned to receive intraarticular injections of triamcinolone or saline once every 3 months for 2 years. Clinical outcomes were assessed every 3 months, and cartilage damage was mea sured by MRI at 2 years. No clinical benefit was seen for any of the outcome measures compared with placebo, although it is possible that the periodicity of followup caused transient responses to be missed (ie, if responses returned to baseline by 3 months). Importantly, this study showed a small but statistically significant increase in cartilage volume loss, raising concerns about the effect of repeated and longterm corticosteroid use on joint health. 35 Similar findings were also shown using data derived from the Osteoarthritis Initiative. 36 A cautious approach to intraarticular steroid is indicated by a "conditional type 1B recommendation" for this treatment in the 2019 guidelines from the Osteoarthritis Research Society International for nonsurgical treatment of hip and knee osteoarthritis. 37 Both hydroxychloroquine and methotrexate are used in patients with rheumatoid arthritis and, less com monly, on an individualpatient basis in osteoarthritis. Two random ised controlled trials using oral hydroxy chloroquine in hand osteoarthritis have been published, neither of which met the primary study endpoint of reduction in pain. 38, 39 Additionally, no clinical response was seen in a predefined substudy in which patients were stratified by the presence or absence of power doppler signal, which is indicative of a more inflammatory phenotype. 38 The PROMOTE study 40 has reported by abstract 41 a small difference in pain in those with knee osteoarthritis taking methotrexate, although the effect size was not deemed clinically meanin gful. A small random ised controlled trial 42 of 64 patients with hand osteoarthritis taking 10 mg of methotrexate failed to show a beneficial effect on pain, the primary out come, although some changes to the evolution of joint remodelling were suggested in the reported abstract. A metaanalysis 43 has concluded no efficacy of conventional synthetic diseasemodifying antirheumatic drugs across all joint osteoarthritis. An absence of efficacy was also evident in four random ised controlled trials in hand osteoarthritis that targeted either tumour necrosis factor or interleukin (IL)1 [44] [45] [46] [47] and two trials in knee osteoarthritis targeting IL1, 48, 49 one of which used an intraarticular approach. Despite promise from various small openlabel studies, none of the random ised controlled trials met their primary study end points, suggesting that classical cytokinedriven inflam mation is www.thelancet.com/rheumatology Vol 2 October 2020 e635 Review at the root of neither pain nor structural damage in osteo arthritis. These results are in accordance with preclinical data in which gene deletion of IL1β, 50 the IL1converting enzyme, IL1R (Vincent, unpublished data), tumour necro sis factor, 51 or inflammasome pathway components (which lead to processing of IL1family cytokines) 52 does not confer pro tec tion from osteoarth ritis after surgical joint destabilisa tion. Despite a strong rationale based on invitro studies, evidence to support a direct pathogenic role for IL1 in osteoarthritis pathogenesis appears, in retrospect, to have been weak. 53 These studies force us to conclude that classical inflam mation, of the type that is pathogenic in rheumatoid arthritis, does not drive osteoarthritis. One exception to this notion might be IL6. Although the osteoarthritis phenotype has been inconsistently reported in IL6 knock out mice, 54, 55 therapeutic studies suggest that neutralisa tion of IL6 modifies disease in murine osteoarthritis. 56 A clinical trial using tocilizumab, an IL6receptor neutralising antibody, in hand osteoarthritis completed in 2019 but has not yet been reported (registered with ClinicalTrials.gov, NCT02477059). Targeting the proteases that degrade the articular cartilage extracellular matrix has long been regarded as an attractive approach to disease modification in osteoarthritis. A disintegrin and metalloproteinase with thrombo spondin motif (Adamts)5 was identified as the princi pal aggrecan degrading enzyme in mice, 57 and in humans ADAMTS5 also mediates proteolytic activity in osteo arthritis chondro cytes 58 (possibly also involv ing ADAMTS4). 59 Aggrecanase inhibition is being reexplored, after companies had aban doned earlier studies at the preclinical phase because of adverse cardio vascular events, using an antiAdamts5 monoclonal antibody. 60 A good safety profile and evidence of target engagement with a small molecule inhibitor 61 is now being followed by phase 2 studies in knee osteo arthritis, with struc tural disease as the primary outcome (registered with ClinicalTrials.gov, NCT03595618). Activation of other components of the innate immune system might have more important pathogenic roles in disease, and some of these components have been examined in preclinical osteoarth ritis (table 1) . Several chemo kine family members have been explored after joint destabilisation, with some having diseasemodifying effects in murine osteoarthritis (table 1) . These proteins are expressed by chondrocytes and have chondroprotective and diseasecausing roles, not always correlating with cell infiltration of the joint. They therefore probably act in both canonical and noncanonical ways. [68] [69] [70] CC motif chemo kine 2 (Ccl2) and its receptor, CC chemokine receptor type 2 (Ccr2), are the best validated of these targets. Miotla Zarebska et al, 63 Miller et al, 64 Raghu et al, 65 Constitutive deletion of Ccl2 or Ccr2 delays and suppresses pain severity in preclinical osteoarthritis but has little effect on cartilage damage when induced in animals that are 10 weeks old. 63, 64 However, a reduction in structural disease has been seen when older (aged 20 weeks) Ccr2 knockout mice are subjected to joint destabilisation, 65 and when pharmaco logical Ccr2 inhibition is delivered. 67 In another study, 66 structure modification was observed when a Ccr2 antagonist was given either between 1-4 or 4-8 weeks after joint destabilisation, but not when given between 8-12 or 1-8 weeks after. A reduction in pain behaviour was observed over short (3 week) and long (12 week) periods of treatment at all stages of disease. Blocking trans forming growth factor α (Tgfα) signalling, a strong inducer of Ccl2 in the rodent osteoarthritis joint, also reduced structural disease after joint destabilisation in the rat. 67 TGFα is of particular interest because it has been identified as a candidate gene for determining cartilage thickness and osteoarthritis risk in humans. 77, 78 These results point towards a role for Ccl2 and Ccr2 in murine osteoarth ritis pain and a possible role in structural progres sion. Two mRNA studies of human synovium have been done in individuals stratified by having painful or nonpainful osteoarthritis. 27, 79 One of these studies 27 identified CCL2 as being signi ficantly upregulated in painful disease. CCR2 antagonism in osteoarthritis pain has been explored clinically (regis tered with ClinicalTrials.gov, NCT00689273), although the results of the study do not appear to have been reported. A clinical study examining TGFα blockade is currently recruiting (regis tered with ClinicalTrials.gov, NCT04456686). All current osteoarthritis diseasemodifying drug trials are summarised in table 2. Other types of innate immune activation might be important in osteoarthritis pathogenesis but have as yet only been explored as targets in preclinical models (table 1). Components of the common terminal pathway of complement activation are strongly upregulated in the synovial fluid of individuals with osteoarthritis, 80 with evidence of the formation of membrane attach complex within human osteoarthritis cartilage. 62 Deletion of C5 (an upstream activator of the common pathway) in mice led to reduced disease severity after joint destabilisation, whereas deletion of an inhibitor of terminal activation (Cd59a) led to increased disease severity. 62 The same research group also identified mastcell activity as a pathogenic mediator in murine osteoarthritis. 76 Mastcell activation has previously been described in the osteoarth ritis joint, 81 and is associated with structural disease. 25 Inflammasome activation is purported to have a role in osteoarthritis, especially when disease is complicated by a crystal arthropathy. However, studies in mice in which components of the inflammasome pathway (activated by crystals) were genetically deleted failed to show a role for inflammasome in surgically induced osteo arthritis. 82, 83 Several groups have examined the role of alarmins in osteoarthritis, through deletion of Tolllike receptors, S100 proteins, or advanced glycosylation end productspecific receptors. Collectively, these studies do not support a role for these molecules in surgically induced murine osteo arthritis. 82, 84 Many preclinical studies in this area of research remain unpublished, and this reporting bias has been unhelpful for research over the years. 85 My own work, and work arising from the Centre of Osteoarthritis Pathogenesis at the Kennedy Institute of Rheumatology (Oxford, UK) has highlighted an important role for what has been termed mechanoflammation, 86 showing that mechanical injury directly drives inflam matory signalling and inflammatory genes in joint tissues, including the articular cartilage and synovium. 87, 88 Joint immobilisation after destabilisation surgery attenuates the induction of pathogenic proteases and prevents osteoarthritis development. 14 Mechanoflammation drives TGFβactivated kinase (TAK1) and downstream activation of the inflammatory mitogenactivated protein kinases (JNK and p38) and nuclear factor κB (NFκB). 12 NFκB signalling pathway has long been considered an important inducer of inflammatory gene regulation in osteoarthritis. It is a complex pathway with canonical and noncanonical pathways that mediate antiapoptotic and proinflammatory functions. This characterisation has been confirmed in vivo in a dosedependent manner, in which heterozygous deletion of RelA (p65), a transcription factor activated upon canonical NFκB activation, resulted in chondroprotection, whereas homozygous deletion led to accelerated disease through the suppression of apoptosis. 73 Accelerated disease resulting from homozygous deletion of p65 was mediated through decreased expression of the antiapoptoic gene Pik3r1, itself a candidate gene arising from a genomewide association study for cartilage thickness. 89 Deletion of Ikkα (which inhibits κBkinaseα, an upstream NFκB pathway activ ator) leads to disease protection and antiapoptotic effects in vivo. 74 Although NFκB might be important in trans criptional regulation of proteases in osteoarthritis, JNK activation controls the bioavailability of aggrecanase acti vity in vitro 58 and in vivo, 75 by a mechanism that appears to involve reuptake of aggrecanases by the cell surface scavenger receptor, lowdensity lipoprotein receptorrelated protein. 90, 91 Targeting protease activity through metal cation symporter Zip8, a zinc transporter, has also been shown in murine osteoarthritis. 92 Zip8 is regulated by the hypoxia transcription factor Hif2α, 93 which has also been shown to be diseasemodifying in preclinical osteoarthritis models. 94 The inability of articular cartilage to repair is famously attributed to William Hunter who stated in 1743 that "…ulcerated Cartilage is universally allowed to be a very troublesome disease…and when destroyed, it is never recovered". 95 The essence of this statement has been reiterated in textbooks for decades, but recent years have seen a paradigm shift. Improved MRI imaging indicates that asymptomatic focal defects in the joint surface are much more common than previously suggested, and prospective studies conclude that around 30% of focal Review cartilage defects spontaneously regress over time. 96 Regres sion of osteoarthritis, as measured by Kellgren and Lawrence xray score during a 14year period, has been documented in the Chingford Women's cohort, 97 and preclinical studies show evidence of intrinsic repair of focal cartilage defects in a mouse strain (genetic) and agedependent manner. 98, 99 Load-altering procedures 103 The procedure results in a reduction of compressive load through the joint and complete prevention of surface shear stress (ie, no joint flexion). These concepts fit well with observations in mice, in which immobilising the knee joint in a fully extended position prevents pro tease regulation and protects the mouse from osteo arthritis after joint destabilisation. Maintaining some compressive force is likely to be more effective than complete joint immobilisation because it promotes the release of matrixbound chondroprotective growth factors, such as fibroblast growth factor (FGF) 2. 14, 16 When the synovial fluid levels of candidate molecules were examined over the course of joint distraction, out of ten analytes exam ined, only two, FGF2 and TGFβ (both proregenerative growth factors), predicted a good clinical response. 104 High tibial osteotomy, whereby a wedge of bone is removed from the top of the tibia (usually) to correct valgus-varus joint malalignment, is also associated with clinical improvement. 105 Moreover, when studies have examined the cartilage macroscopically through arthro tomy, histologically, or by MRI, evidence of cartilage regeneration is observed in the now offloaded part of the joint. [106] [107] [108] Sperifermin is a truncated form of FGF18. The FGFs form a large family of pleiotropic growth factors implicated in a range of physiological and pathologi cal processes, including embryonic development, tissue repair, and cancer. 109 Whereas FGF2 is promiscuous, binding to all four FGF receptors (FGFRs), FGF18 is thought to be more selective for FGFR3, which is the chondroprotective FGFR in murine osteoarthritis studies. [110] [111] [112] Of note, polymorphic variants in FGFR3 have been identified in two genome wide association studies: a population study 77 associating a polymorphic variant with articular cartilage thickness, and another study 113 that identified it as an atrisk allele in osteoarthritis. The latter study also identified FGF18 as a candidate gene associated with osteoarthritis risk. 113 In 2014, a proofofconcept study 114 was reported in which 192 individuals with osteoarthritis were randomly assigned to receive three doses of intraarticular sprifermin (recombinant truncated form of FGF18) or placebo, with followup at 6 months and 12 months. The study failed to meet its primary endpoint (a difference in articular cartilage thickness in the central medial femorotibial compartment), but it did show delayed loss of cartilage overall and thickening in the lateral compartment. 114 In 2019, the FORWARD trial, 115 in which 549 participants received intraarticular sprifermin every 6 months or 12 months, or placebo, reported a significant increase in total femorotibial cartilage volume compared with placebo at 2year followup, albeit without significant clinical improve ment. In a recent posthoc analysis 116 of the original trial data (thus far reported in abstract form), sprifermin treatment showed a statistically significant clinical and structural improvement over among a subgroup of 161 patients who were defined as being at high risk of progression. Although these studies do not specifically show reversal of cartilage damage (ie, true repair), they do show that damage can be arrested and therefore indicate a structuremodifying osteoarthritis drug. Whether these drugs turn out to be true diseasemodifying osteoarthritis drugs is not yet clear. The apparent discordance between structure and symptoms in osteoarthritis is discussed later in this Review. Wnts are a complex family of cellular signalling molecules that direct a broad range of cellular responses, particularly regarding bone development. Wnts are activated upon mechanical stress of articular cartilage 117, 118 and are thought to drive the dedifferentiated chondrocyte phenotype, bone remodelling, and induction of catabolic enzymes seen in osteoarthritis. [119] [120] [121] Canonical Wnt signalling involves stabilisa tion of the signalling molecule betacatenin within the cell. Interfering with betacatenin has shown conflict ing outcomes in experimental osteoarthritis, indicating that this molecule is not readily amenable to therapeutic translation. 122 Interfering with natural inhibitors of Wnt signalling in mice, such as Dkk1 and Dot1l, reveals the diseasemodifying potential of this pathway. [123] [124] [125] [126] SM04690 is a synthetic Wnt inhibitor with an undisclosed (unknown) primary mechanism of action that has shown success in murine models of osteoarthritis. 127, 128 A phase 1 study of a single intraarticular dose of SM04690 in 61 participants with moderate osteoarthritis showed acceptable safety, with exploratory clinical endpoints that showed a positive trend towards improvement in pain and joint space narrowing. 129 A phase 2 study of 455 individuals with unilateral knee osteoarthritis, although not reaching its primary endpoint (improvement of WOMAC pain at week 13), showed improvement in pain and an increase in joint space indicative of disease modification, which was especi ally evident in people with unilateral disease. 130 In May 2019, the phase 3 studies were launched (registered on ClinicalTrials.gov, NCT04385303). Recent studies in murine osteoarthritis identify the transcriptional coactivator Yap and WW domain containing transcription regulator protein 1 (Taz) pathway as a strong chondroprotective mechanism. Yap and Taz are both transcription factors that are activated by Hippo signalling, a highly conserved pathway thought to be involved in cellular mechanotransduction. 131 Genetic and pharmacological enhancement of this pathway protects joints from osteoarthritis after joint destabilisation, 132 which might in part be due to it controlling the generation of chondroprogenitor cells arising from the synovium. 133 The Yap-Taz pathway also reciprocally controls Tak1 132 (strongly induced by cartilage injury), and this might be an important mechanism by which inflammation sup presses repair (figure 1). Nerve growth factor (NGF) has long been known to sensitise pain fibres and, in doing so, enhance the firing rate of nociceptors in response to mechanical and thermal stimuli. NGF is also known to be a neurotrophic factor, directing the growth of new nerves. 134 The use of antiNGF neutralising antibodies to inhibit osteoarthritis pain has been heralded as a huge breakthrough for osteoarthritis patients who have struggled for years with inade quate pain relief. Several biological drugs targeting NGF, all delivered systemically (intravenously or subcutaneously), have been tested in phase 2 studies, with a metaanalysis showing efficacy across the different studies. 135 Two companies have now published phase 2-3 studies using NGF neutralising antibodies, [136] [137] [138] with fasinumab and tanezumab showing efficacy over placebo. Concerns over patients developing rapidly pro gressive osteoarthritis in index and nonindex joints (ranging from 2 to 10% according to dose and study) forced the FDA to introduce mitigation strategies, which included reducing highest doses and prohibiting the use of concomitant nonsteroidal antiinflammatory drugs. The community now awaits a decision from the FDA on whether this class of drug, which was designated as fast track in 2017, will be approved for patient use. Other strategies to target NGF signalling have also been tested. In 2019, two randomised controlled trials targeting high affinity nerve growth factor receptor (TrkA), the receptor through which NGF signals, were published. 139, 140 In one study, 139 215 participants were randomly assigned to receive twice daily oral dosing with ASP7962, placebo or naproxen for 4 weeks. The study failed to meet its primary endpoint (WOMAC pain subscore). 139 A second study 140 randomly assigned 104 participants to intraarticular GZ389988A or placebo. This study did show improved pain outcomes of the drug compared with placebo, although the effect size was small and of question able clinical value. 140 Neither study was accompanied by evidence of target tissue drug bioavailability. AntiNGF clinical trials align well with evidence of NGF mediated painlike behaviour in rodent osteoarthritis. Painlike behaviour can be measured by evoked or non evoked methods. Like humans, rodents will spontaneously offload the damaged joint when experiencing pain, and this behaviour can be measured by assessing the amount of weight transmitted through each hind limb. Using this technique, mice have been shown to display two phases of pain behaviour after joint destabilisation: an initial post operative phase that resolves after 1 week, and a later phase that starts only once there is significant joint damage (10 weeks after destabilisation of the medial meniscus or 8 weeks after partial meniscectomy). [141] [142] [143] Late osteoarthritis pain in rodents is Ngfdependent 144,145 and tumour necrosis factorindependent. 145 The driver of NGFdependent late osteoarthritis pain is unclear, but Ngf mRNA upregulation occurs in the articular cartilage rather than bone or meniscus, and there is very little inflammatory gene regulation in the joint during this time. 146 Although this observation might be surprising in view of broadly held views that osteoarthritis pain originates from inflam matory processes in the synovium or subchondral bone, emerging molecular data from Review human tissue also support the notion that cartilage is the principal source of NGF in the osteoarthritis joint. Using agnostic approaches, NGF was not regulated in the synovium of individuals with painful compared with non painful osteoarthritis, 27, 79 and it was not found in bone marrow lesions from samples taken at the time of arthroplasty. 147 NGF was found to be regulated in damaged articular cartilage in early micro array studies of osteo arthritis cartilage, 148 and it defines one of seven subsets of chondrocytes identified by singlecell sequencing of human osteoarthritis cartilage. 149 NGF is regulated by direct cartilage injury (mechanoflammation) in a TAK1 dependent manner, and it is tempting to speculate that damage to chondrocytes near the osteo chondral junction is an important trigger for the NGFdriven neoinnervation of the articular cartilage that is seen late in both murine and human disease. 150, 151 Neoinnerva tion of this region also requires a permissive subchondral bone to support axonal extension. This neoinnervation has recently been shown to be dependent upon Netrin1, secreted by osteoclasts during the course of murine osteoarthritis. 152 An overall model for the development of pain in osteoarthritis has been proposed. 153 There are many reasons to be optimistic about new therapeutic developments in osteoarthritis. Although it is true that much of what has been learned in the past few years from clinical studies is what not to use in disease, these negative studies have been highly informative in reminding the medical community that osteoarthritis is distinct from inflammatory arthritidies, such as rheuma toid arthritis. Research has shown that inflammation in osteoarthritis is nuanced and that classical immuno modulatory pathways are not good targets, but that there are several other inflammatory pathways awaiting clinical exploration, including those driven by direct mechanical injury of the cartilage (socalled mechanoflammation), complement, and mast cells. The nature and role of inflammation in osteoarthritis pathogenesis thus remains unclear. Clarification is crucially important, not only so that we can develop appropriate targeted therapies for patients, but also to decide whether patients require stratification before treat ment. There has been a popular move to try to phenotype patients, with a view to personalising their treatment to improve the efficacy of a given drug. However, these phenotypes currently lack cohesion; some are defined by clinical features (eg, inflammatory osteo arthritis), and others by comorbidity (eg, metabolic osteoarthritis), precipitating factor (eg, posttraumatic osteoarthritis), or anatomical site (eg, hand osteoarthritis, hip osteoarthritis). There is little or no evidence that stratification by any of these features changes the response to treatment. Further carefully considered phenotypes that take into considera tion molecular pathways are probably required. Large scale molecular endotyping of patient samples is currently in its infancy, but will probably help. Clinical successes point towards a focus on regenerative or anabolic pathways rather than inflammatory ones (figure 1). This suggestion fits well with preclinical studies, although the reciprocal relationship between repair and inflammation in the chondrocyte suggests that targeting one will probably affect the other. 132 Recent large genomewide association studies in osteoarthritis also support the concept that osteoarthritis is a failure of repair. Several atrisk loci have been attributed to genes in the TGFβ and FGF pathways, and there is a notable absence of loci that predict the regulation of classical inflammatory genes. 113, 154 Newer targets identified by genome studies, including the retinoic acid pathway, also look promising. 155 NGFtargeting for pain relief is the target closest to being ready to use in osteoarthritis. Clinical success in late osteoarthritis indicates that analgesia occurs largely as a result of nociceptor desensitisation. It remains to be seen whether interfering with this pathway at earlier stages of the disease could affect the neoinnervation of the cartilage Figure 2 : Concordance between tested targets in mouse and human osteoarthritis studies Several pathways have been or are being tested in human osteoarthritis, having also been tested in murine surgical models. Yellow indicates therapies that show treatment success or target engagement in each study. Grey shows therapies that have failed to modify symptomatic or structural disease. Note that there is 100% concordance between mouse (outer ring) and human (inner ring) studies. FGF=fibroblast growth factor. FGFR=fibroblast growth factor receptor. DMOAD=disease-modifying osteoarthritis drug. GWAS=genome-wide association study. NGF=nerve growth factor. IL=interleukin. TNF=tumour necrosis factor. ICE=capsase-1/interleukin-1 converting enzyme. CCL=C-C motif chemokine. CCR=C-C chemokine receptor. ADAMTS=A disintegrin and metalloproteinase with thrombospondin motif. This type of strategy would need to be considered in the context of current safety concerns around the development of rapidly progressive osteo arthritis, which remains a real concern. Other molecules that appear to have a role in the neoinnervation of the osteochondral junction in osteo arthritis models include Netrin1, 152 a molecule secreted by osteoclasts that guides axonal growth through the subchondral bone. Blocking bone remodelling with a bisphospho nate early in murine osteoarthritis develop ment appears to block pain without affecting structural disease, according well with clinical studies in osteo arthritis in which bisphosphonates are not disease modifying when given in established disease. 156, 157 One major outstanding issue remains the apparent discordance between structural and symptomatic disease, which raises questions about whether validated drugs need to be able to, or indeed could ever, target both. Whether different joint pathologies give rise to different types of symptoms at different stages of disease is currently unknown, as is the relative contribution of factors that drive central sensitisation of pain. Of the few examples available at this stage, cartilage structure modify ing drugs (eg, sprifermin) mainly arrest disease progression rather than regenerating the cartilage, so perhaps symptoms could not be expected to reverse. Where structural damage appears to reverse (eg, after joint distraction), symptoms also appear to improve (albeit with no placebo control). Targeting pain alone is unlikely to improve structure in the short term and might worsen damage through mechanical overuse. In preclinical models, there tends to be better accordance between struc tural damage and painlike behaviour, 153 with some clear examples emerging that might identify true disease modifying osteoarthritis drugs of the future, such as those involving the YAP-TAZ pathway. Finally, it is reassuring to conclude that, where there is overlap, research in surgical preclinical osteoarth ritis models aligns well with findings in clinical trials ( figure 2) . This concordance provides valuable validation of the models and will help develop mutual trust between the different osteoarthritis research disciplines. It is increasingly difficult to claim that mouse osteoarthritis is fundamentally different to human osteoarthritis, or that posttraumatic osteoarthritis does not inform agerelated disease in humans. Part of this reassurance has emerged through improved awareness of bias mitigation in clinical and preclinical studies. 158 It is also partly due to the acceptance that osteoarthritis has diseasespecific molecular targets. Regardless, this is an important time for osteoarthritis research, with tangible translational benefits within reach. TLV has consulted for GlaxoSmithKline, Mundipharma, and Union Chimique Belge in the past 3 years on an adhoc basis. She directs the STEpUP osteoarthritis consortium, which has financial support from Samumed, Fidia, and Galapagos. This is a narrative Review based on clinical trials done in hand and knee or hip osteoarthritis by searching PubMed with the terms "Phase" with "Trial" and "Osteoarthritis" in the title from Jan 1, 2012, to July 31, 2019. Further information was sought through Clinicaltrials.gov, by searching for "osteoarthritis" studies in which the intervention was "drug". Preclinical studies were interrogated through Skeletalvis.ncl.ac.uk. This Review is not intended to be a comprehensive review of all clinical trials in osteoarthritis or all pathways identified through murine studies. Rather, its intention is to focus on those targets for which there is overlap between murine and human studies. Additionally, the Review highlights a few emerging pathways that have strong preclinical evidence for a role in pathogenesis and which could be amenable to clinical targeting. Inevitably, an exercise of this sort reflects the author's personal views on pathogenesis, based on 20 years of working with preclinical surgical models, human tissue, and patients with osteoarthritis. Mechanical regulation of mitogenactivated protein kinase signaling in articular cartilage Joint immobilization prevents murine osteoarthritis and reveals the highly mechanosensitive nature of protease expression in vivo Matrix crosslinkingmediated mechanotransduction promotes posttraumatic osteoarthritis Fibroblast growth factor 2 is an intrinsic chondroprotective agent that suppresses ADAMTS5 and delays cartilage degradation in murine osteoarthritis Increased susceptibility of Trpv4deficient mice to obesity and obesityinduced osteoarthritis with very highfat diet Adiposity and hand osteoarthritis: the Netherlands Epidemiology of Obesity study The role of leptin and adiponectin as mediators in the relationship between adiposity and hand and knee osteoarthritis Both systemic and local lipopolysaccharide (LPS) burden are associated with knee OA severity and inflammation Intestinal microbiome composition and its relation to joint pain and inflammation Mapping pathogenesis of arthritis through small animal models OARSI clinical trials recommendations: design and conduct of clinical trials of lifestyle diet and exercise interventions for osteoarthritis Bone marrow lesions and synovitis on MRI associate with radiographic progression after 2 years in hand osteoarthritis Pain in knee osteoarthritis patients associates with distinct pattern of synovitis Association of joint inflammation with pain sensitization in knee osteoarthritis: the multicenter osteoarthritis study Molecular expression patterns in the synovium and their association with advanced symptomatic knee osteoarthritis Anabolic and catabolic gene expression pattern analysis in normal versus osteoarthritic cartilage using complementary DNAarray technology Effectiveness of triamcinolone hexacetonide intraarticular injection in interphalangeal joints: a 12week randomized controlled trial in patients with hand osteoarthritis Brief report: a phase iib trial of a novel extendedrelease microsphere formulation of triamcinolone acetonide for intraarticular injection in knee osteoarthritis Rescue analgesic medication use by patients treated with triamcinolone acetonide extendedrelease for knee osteoarthritis pain: pooled analysis of three phase 2/3 randomized clinical trials A randomized, doubleblind, placebocontrolled trial of lowdose oral prednisolone for treating painful hand osteoarthritis Results of a 6week treatment with 10 mg prednisolone in patients with hand osteoarthritis (HOPE): a doubleblind, randomised, placebocontrolled trial Effect of intraarticular triamcinolone vs saline on knee cartilage volume and pain in patients with knee osteoarthritis: a randomized clinical trial Intraarticular corticosteroids and the risk of knee osteoarthritis progression: results from the Osteoarthritis Initiative OARSI guidelines for the nonsurgical management of knee, hip, and polyarticular osteoarthritis Hydroxychloroquine effectiveness in reducing symptoms of hand osteoarthritis: a randomized trial Efficacy of hydroxychloroquine in hand osteoarthritis: a randomized, double blind, placebocontrolled trial Pain reduction with oral methotrexate in knee osteoarthritis, a pragmatic phase iii trial of treatment effectiveness (PROMOTE): study protocol for a randomized controlled trial Significant pain reduction with oral methotrexate in knee osteoarthritis; results from a randomised controlled phase III trial of treatment effectiveness Methotrexate in patients with hand osteoarthritis refractory to analgesics: a randomised, doubleblind, placebo controlled trial Conventional and biologic diseasemodifying antirheumatic drugs for osteoarthritis: a metaanalysis of randomized controlled trials Etanercept in patients with inflammatory hand osteoarthritis (EHOA): a multicentre, randomised, doubleblind, placebocontrolled trial Phase IIa, placebocontrolled, randomised study of lutikizumab, an antiinterleukin1α and antiinterleukin1β dual variable domain immunoglobulin, in patients with erosive hand osteoarthritis Adalimumab in patients with hand osteoarthritis refractory to analgesics and NSAIDs: a randomised, multicentre, doubleblind, placebocontrolled trial Tumour necrosis factor blockade for the treatment of erosive osteoarthritis of the interphalangeal finger joints: a double blind, randomised trial on structure modification A phase ii trial of lutikizumab, an antiinterleukin1α/β dual variable domain immunoglobulin, in knee osteoarthritis patients with synovitis Intraarticular injection of anakinra in osteoarthritis of the knee: a multicenter, randomized, doubleblind, placebocontrolled study Gene deletion of either interleukin1beta, interleukin1betaconverting enzyme, inducible nitric oxide synthase, or stromelysin 1 accelerates the development of knee osteoarthritis in mice after surgical transection of the medial collateral ligament and partial medial meniscectomy Lack of a chondroprotective effect of cyclooxygenase 2 inhibition in a surgically induced model of osteoarthritis in mice Stressinduced cartilage degradation does not depend on the NLRP3 inflammasome in human osteoarthritis and mouse models IL1 in osteoarthritis: time for a critical review of the literature Interleukin6 plays an essential role in hypoxiainducible factor 2αinduced experimental osteoarthritic cartilage destruction in mice Male IL6 gene knock out mice developed more advanced osteoarthritis upon aging Systemic inhibition of IL6/Stat3 signalling protects against experimental osteoarthritis Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis Interleukin1 acts via the JNK2 signaling pathway to induce aggrecan degradation by human chondrocytes Aggrecan degradation in human articular cartilage explants is mediated by both ADAMTS4 and ADAMTS5 The highs and lows of translational drug development: antibodymediated inhibition of ADAMTS5 for osteoarthritis disease modification A safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) study with increasing oral doses of GLPG1972 administered daily for 29 days shows a strong biomarker effect in patients with knee and/or hip OA Identification of a central role for complement in osteoarthritis CCL2 and CCR2 regulate painrelated behaviour and early gene expression in post traumatic murine osteoarthritis but contribute little to chondropathy CCR2 chemokine receptor signaling mediates pain in experimental osteoarthritis CCL2/CCR2, but not CCL5/CCR5, mediates monocyte recruitment, inflammation and cartilage destruction in osteoarthritis Role of the CC chemokine receptor2 in a murine model of injuryinduced osteoarthritis Reduction in disease progression by inhibition of transforming growth factor αCCL2 signaling in experimental posttraumatic osteoarthritis The chemokine receptor CCR5 plays a role in posttraumatic cartilage loss in mice, but does not affect synovium and bone Chemokine receptor7 (CCR7) deficiency leads to delayed development of joint damage and functional deficits in a murine model of osteoarthritis A homeostatic function of CXCR2 signalling in articular cartilage SDF1/CXCR4 axis coordinates crosstalk between subchondral bone and articular cartilage in osteoarthritis pathogenesis Alleviation of murine osteoarthritis by cartilagespecific deletion of IkappaBzeta Biphasic regulation of chondrocytes by Rela through induction of antiapoptotic and catabolic target genes Inducible knockout of CHUK/IKKα in adult chondrocytes reduces progression of cartilage degradation in a surgical model of osteoarthritis Brief report: JNK2 controls aggrecan degradation in murine articular cartilage and the development of experimental osteoarthritis IgEmediated mast cell activation promotes inflammation and cartilage destruction in osteoarthritis Novel genetic variants for cartilage thickness and hip osteoarthritis Genomewide analyses using UK Biobank data provide insights into the genetic architecture of osteoarthritis Painassociated transcriptome changes in synovium of knee osteoarthritis patients Proteomic analysis of synovial fluid from the osteoarthritic knee: comparison with transcriptome analyses of joint tissues Mast cell subpopulations in the synovial tissue of patients with osteoarthritis: selective increase in numbers of tryptasepositive, chymasenegative mast cells Dispensable role of myeloid differentiation primary response gene 88 (MyD88) and MyD88dependent tolllike receptors (TLRs) in a murine model of osteoarthritis Revisiting the role of interleukin1 pathway in osteoarthritis: interleukin1α and 1β, and NLRP3 inflammasome are not involved in the pathological features of the murine menisectomy model of osteoarthritis Active involvement of alarmins S100A8 and S100A9 in the regulation of synovial activation and joint destruction during mouse and human osteoarthritis Time to be positive about negative data? Osteoarthritis and cartilage/OARS Mechanoflammation in osteoarthritis pathogenesis Induction of interleukin1 in articular cartilage by explantation and cutting Src and fibroblast growth factor 2 independently regulate signaling and gene expression induced by experimental injury to intact articular cartilage Novel genetic variants for cartilage thickness and hip osteoarthritis Low density lipoprotein receptorrelated protein 1 (LRP1)mediated endocytic clearance of a disintegrin and metalloproteinase with thrombospondin motifs4 (ADAMTS4): functional differences of noncatalytic domains of ADAMTS4 and ADAMTS5 in LRP1 binding LRP1mediated endocytosis regulates extracellular activity of ADAMTS5 in articular cartilage Regulation of the catabolic cascade in osteoarthritis by the zincZIP8MTF1 axis Reciprocal activation of hypoxiainducible factor (HIF)2α and the zincZIP8MTF1 axis amplifies catabolic signaling in osteoarthritis Transcriptional regulation of endochondral ossification by HIF2alpha during skeletal growth and osteoarthritis development Joint surface defects: clinical course and cellular response in spontaneous and experimental lesions The natural history of radiographic knee osteoarthritis: a fourteenyear populationbased cohort study A novel in vivo murine model of cartilage regeneration. Age and straindependent outcome after joint surface injury Heritability of articular cartilage regeneration and its association with ear wound healing in mice Sustained clinical and structural benefit after joint distraction in the treatment of severe knee osteoarthritis Tissue structure modification in knee osteoarthritis by use of joint distraction: an open 1year pilot study Fiveyear followup of knee joint distraction: clinical benefit and cartilaginous tissue repair in an open uncontrolled prospective study Functional articular cartilage repair: here, near, or is the best approach not yet clear? The molecular profile of synovial fluid changes upon joint distraction and is associated with clinical response in knee osteoarthritis High tibial osteotomy: a sevenyear clinical and radiographic followup Regeneration of degenerated articular cartilage after high tibial valgus osteotomy for medial compartmental osteoarthritis of the knee Articular cartilage changes in patients with osteoarthritis after osteotomy Immunolocalization of carboxyterminal type II procollagen peptide in regenerated articular cartilage of osteoarthritic knees after reduction of mechanical stress The FGF family: biology, pathophysiology and therapy Defects in articular cartilage metabolism and early arthritis in fibroblast growth factor receptor 3 deficient mice Fibroblast growth factor receptor 3 inhibits osteoarthritis progression in the knee joints of adult mice A novel fibroblast growth factor receptor 1 inhibitor protects against cartilage degradation in a murine model of osteoarthritis Identification of new therapeutic targets for osteoarthritis through genomewide analyses of UK Biobank data Intraarticular sprifermin (recombinant human fibroblast growth factor 18) in knee osteoarthritis: a randomized, doubleblind, placebocontrolled trial Effect of intraarticular sprifermin vs placebo on femorotibial joint cartilage thickness in patients with osteoarthritis: the FORWARD randomized clinical trial OP0010 cartilage thickness modification with sprifermin in knee osteoarthritis patients translates into symptomatic improvement over placebo in patients at risk of further structural and symptomatic progression: posthoc analysis of the phase II FORWARD trial Identification of the molecular response of articular cartilage to injury, by microarray screening: Wnt16 expression and signaling after injury and in osteoarthritis Activation of WNT and BMP signaling in adult human articular cartilage following mechanical injury Wntbetacatenin signaling in the pathogenesis of osteoarthritis Roles of βcatenin signaling in phenotypic expression and proliferation of articular cartilage superficial zone cells Transient activation of Wnt/ betacatenin signaling induces abnormal growth plate closure and articular cartilage thickening in postnatal mice Regulation of osteoarthritis development by Wntbetacatenin signaling through the endochondral ossification process Dkk1mediated inhibition of Wnt signaling in bone ameliorates osteoarthritis in mice Articular cartilage and biomechanical properties of the long bones in Frzbknockout mice WNT16 antagonises excessive canonical WNT activation and protects cartilage in osteoarthritis DOT1L safeguards cartilage homeostasis and protects against osteoarthritis A smallmolecule inhibitor of the Wnt pathway (SM04690) as a potential disease modifying agent for the treatment of osteoarthritis of the knee Modulation of the Wnt pathway through inhibition of CLK2 and DYRK1A by lorecivivint as a novel, potentially diseasemodifying approach for knee osteoarthritis treatment A novel Wnt pathway inhibitor, SM04690, for the treatment of moderate to severe osteoarthritis of the knee: results of a 24week, randomized, controlled, phase 1 study Lorecivivint, a novel intraarticular CLK/DYRK1A inhibitor and Wnt pathway modulator for treatment of knee osteoarthritis: a phase 2 randomized trial The biology of YAP/TAZ: hippo signaling and beyond Reciprocal inhibition of YAP/TAZ and NFκB regulates osteoarthritic cartilage degradation Joint morphogenetic cells in the adult mammalian synovium Nerve growth factor and pain mechanisms The efficacy of nerve growth factor antibody for the treatment of osteoarthritis pain and chronic lowback pain: a metaanalysis The efficacy, tolerability, and joint safety of fasinumab in osteoarthritis pain: a phase iib/iii doubleblind, placebocontrolled, randomized clinical trial Pooled analysis of tanezumab efficacy and safety with subgroup analyses of phase III clinical trials in patients with osteoarthritis pain of the knee or hip Effect of tanezumab on joint pain, physical function, and patient global assessment of osteoarthritis among patients with osteoarthritis of the hip or knee: a randomized clinical trial Tropomyosinrelated kinase A (TrkA) inhibition for the treatment of painful knee osteoarthritis: results from a randomized controlled phase 2a trial Efficacy and safety of intraarticular injection of tropomyosin receptor kinase A inhibitor in painful knee osteoarthritis: a randomized, doubleblind and placebocontrolled study Regulation of pain sensitivity in experimental osteoarthritis by the endogenous peripheral opioid system Active immunisation targeting nerve growth factor attenuates chronic pain behaviour in murine osteoarthritis Partial medial meniscectomy produces osteoarthritis painrelated behaviour in female C57BL/6 mice Nerve growth factor inhibition with tanezumab influences weightbearing and subsequent cartilage damage in the rat medial meniscal tear model Treatment of murine osteoarthritis with TrkAd5 reveals a pivotal role for nerve growth factor in noninflammatory joint pain nociceptive sensitizers are regulated in damaged joint tissues, including articular cartilage, when osteoarthritic mice display pain behavior Microarray analysis of bone marrow lesions in osteoarthritis demonstrates upregulation of genes implicated in osteochondral turnover, neurogenesis and inflammation Comparative analysis of gene expression profiles in intact and damaged regions of human osteoarthritic cartilage Singlecell RNAseq analysis reveals the progression of human osteoarthritis The nociceptive innervation of the normal and osteoarthritic mouse knee Angiogenesis and nerve growth factor at the osteochondral junction in rheumatoid arthritis and osteoarthritis Subchondral bone osteoclasts induce sensory innervation and osteoarthritis pain Peripheral pain mechanisms in osteoarthritis Metaanalysis of Icelandic and UK data sets identifies missense variants in SMO, IL11, COL11A1 and 13 more new loci associated with osteoarthritis Following the genetic clues towards treatment of hand OA Are bisphosphonates efficacious in knee osteoarthritis? A metaanalysis of randomized controlled trials Bisphosphonates therapy for osteoarthritis: a metaanalysis of randomized controlled trials Can animal models of disease reliably inform human studies Acknowledgments TLV's lab is supported by the Centre for Osteoarthritis Pathogenesis, Versus Arthritis (grant numbers 20205 and 21621). I would like to thank Dr Elizabeth Thompson for help with figures.