M.D Francis Berenbaum
email: francis.berenbaum@aphp.fr

Ph.D Timothy M Griffin
email: tim-griffin@omrf.org

Ph.D Ru Liu-Bryan

Francis Pr

Berenbaum

Metabolic Regulation of Inflammation in Osteoarthritis

Keywords: Metabolic Syndrome, Obesity, AMPK, Sirtuins, Adipokines, Energy metabolism

Introduction

Osteoarthritis (OA) has long been considered the unique consequence of a tear and wear process leading to cartilage degradation. Indeed, it is true that an excessive mechanical stress on any joint leads to cartilage loss, with the production of osteophytes being considered a reactive process of the bone to protect and stabilize the altered joint [START_REF] Felson | Osteoarthritis as a disease of mechanics[END_REF]. This initial paradigm has been slowly but deeply modified in the past 20 years due to critical discoveries [START_REF] Berenbaum | Osteoarthritis as an inflammatory disease (osteoarthritis is not osteoarthrosis!)[END_REF]. First, the enzymes involved in cartilage degradation have been identified as belonging to metalloproteinases and ADAMTS families, with pro-inflammatory cytokines regulating all of these enzymes. It was the first time that the pathophysiology of OA was associated with a role of soluble mediators better known in the pathophysiology of many other classical inflammatory diseases. Second, later on, whereas synovitis was considered as pathognomonic of RA, some groups demonstrated that a mild to severe synovitis reaction was frequently present in OA, its intensity being correlated to prognosis [START_REF] Ayral | Synovitis: a potential predictive factor of structural progression of medial tibiofemoral knee osteoarthritis --results of a 1 year longitudinal arthroscopic study in 422 patients[END_REF][START_REF] Sellam | The role of synovitis in pathophysiology and clinical symptoms of osteoarthritis[END_REF]. Third, the role of obesity in OA shifted from an exclusive deleterious mechanical effect on load-bearing joints to its capacity to play a role in hand OA too. Indeed, epidemiological studies demonstrated that obese patients have a 2-fold increase in hand OA compared to a non-obese population [START_REF] Yusuf | Association between weight or body mass index and hand osteoarthritis: a systematic review[END_REF]. The interpretation of these unexpected results has been related to a well-known systemic release of pro-inflammatory cytokines primarily by abdominal adipose tissue (called adipocytokines or adipokines), which cause a low-grade inflammatory state involved in damage to many peripheral tissues, including joint tissues [START_REF] Berenbaum | Osteoarthritis as an inflammatory disease (osteoarthritis is not osteoarthrosis!)[END_REF]. More recently, the intra-articular fat pads, adipose tissue present inside some joints, have been considered as a new potential source of adipokines [START_REF] Ioan-Facsinay | An emerging player in knee osteoarthritis: the infrapatellar fat pad[END_REF]. All these steps demonstrating the role of inflammation in OA opened the door to a novel and exciting area of research trying to understand the molecular interactions between local articular tissues/cells and distant organs/tissues and how these metabolic processes are disturbed in OA.

Epidemiological and prospective clinical studies linking metabolic syndrome and OA

Puenpatum et al. were the first to suggest that, on top of obesity, the metabolic syndrome (MetS) (defined as the association of components that independently increase the risk of cardiovascular events: abdominal adiposity, diabetes or insulin resistance, high cholesterol, and high blood pressure) could be an independent risk factor for OA [START_REF] Puenpatom | Increased prevalence of metabolic syndrome in individuals with osteoarthritis: an analysis of NHANES III data[END_REF]. They found in the NHANES III cohort that 59% of OA patients had MetS compared to 23% in the general population. Furthermore, they calculated that having OA at 44 years old increases 5.26-fold the risk of having a concomitant MetS, suggesting that the presence of knee OA should inform the practitioner on a need to screen for MetS. Then, other cross-sectional studies confirmed this association [START_REF] Yoshimura | Association of Knee Osteoarthritis with the Accumulation of Metabolic Risk Factors Such as Overweight, Hypertension, Dyslipidemia, and Impaired Glucose Tolerance in Japanese Men and Women: The ROAD Study[END_REF][START_REF] Shin | Association between metabolic syndrome, radiographic knee osteoarthritis, and intensity of knee pain: results of a national survey[END_REF][START_REF] Michishita | The possible influence of osteoarthritis of the knee on the accumulation of coronary risk factors in postmenopausal obese women[END_REF][START_REF] Lee | Obesity, metabolic abnormality, and knee osteoarthritis: a cross-sectional study in Korean women[END_REF][START_REF] Calvet | High prevalence of cardiovascular co-morbidities in patients with symptomatic knee or hand osteoarthritis[END_REF], even if obesity could sometimes be a strong confounding factor longitudinally [START_REF] Engström | C-reactive protein, metabolic syndrome and incidence of severe hip and knee osteoarthritis. A populationbased cohort study[END_REF]. Nevertheless, the presence of MetS is also associated with hand OA. Taking advantage of an increased prevalence of metabolic syndrome in HIV patients, Tomi et al. looked at the prevalence of hand OA in sex and age-matched HIV patients according to their metabolic status.

Interestingly, hand OA prevalence was higher for those with MetS than those without it (64.7% vs 46.3%, p=0.002) and the severity was greater [START_REF] Tomi | Increased prevalence and severity of radiographic hand osteoarthritis in patients with HIV-1 infection associated with metabolic syndrome: data from the cross-sectional METAFIB-OA study[END_REF]. In the Netherlands Epidemiology of Obesity population cohort, hand OA was associated with MetS, adjusted for weight (OR 1.46 (95% CI 1.06 to 2.02)) [START_REF] Visser | The relative contribution of mechanical stress and systemic processes in different types of osteoarthritis: the NEO study[END_REF]. But a breakthrough in this field comes from the recent publication of several prospective cohorts showing that obese patients with MetS have an increased risk of incidence and severity of knee and hand OA (Table 1) [START_REF] Engström | C-reactive protein, metabolic syndrome and incidence of severe hip and knee osteoarthritis. A populationbased cohort study[END_REF][START_REF] Sowers | Knee osteoarthritis in obese women with cardiometabolic clustering[END_REF][START_REF] Yoshimura | Accumulation of metabolic risk factors such as overweight, hypertension, dyslipidaemia, and impaired glucose tolerance raises the risk of occurrence and progression of knee osteoarthritis: a 3-year follow-up of the ROAD study[END_REF][START_REF] Hussain | Incidence of total knee and hip replacement for osteoarthritis in relation to the metabolic syndrome and its components: A prospective cohort study[END_REF][START_REF] Schett | Diabetes is an independent predictor for severe osteoarthritis: results from a longitudinal cohort study[END_REF][START_REF] Davies-Tuck | Total cholesterol and triglycerides are associated with the development of new bone marrow lesions in asymptomatic middle-aged women -a prospective cohort study[END_REF][START_REF] Hoeven | Association of atherosclerosis with presence and progression of osteoarthritis: the Rotterdam Study[END_REF]. Based on a sample of 482 women with knee OA, the prevalence of knee OA in obese women with less than 2 cardiometabolic parameters (low levels of high-density lipoprotein cholesterol; elevated levels of low-density lipoprotein cholesterol, triglycerides, blood pressure, C-reactive protein, waist:hip ratio, or glucose; or diabetes mellitus) was 12.8% compared to 23.2% in obese women with more or equal to 2 cardiometabolic parameters [START_REF] Sowers | Knee osteoarthritis in obese women with cardiometabolic clustering[END_REF].

Interestingly, some of these studies show that incidence and severity were positively correlated to the number of cardiometabolic parameters. In a multivariate analysis based on a cohort of 1384 Japanese individuals followed 3 years (ROAD cohort), the incidence of knee OA significantly increased according to the number of MetS components (OR vs no parameters: one parameters, 2.33; two components, 2.82; ≥ three parameters, 9.83) [START_REF] Yoshimura | Accumulation of metabolic risk factors such as overweight, hypertension, dyslipidaemia, and impaired glucose tolerance raises the risk of occurrence and progression of knee osteoarthritis: a 3-year follow-up of the ROAD study[END_REF]. Similarly, progression of knee OA significantly increased according to the number of parameters present (OR vs no component: one component, 1.38; two components, 2.29; ≥ three components: 2.80). In a large Australian population cohort followed during 2003-2007 (the Melbourne Collaborative Cohort Study), 660 patients had a total knee replacement (TKR) for OA [START_REF] Hussain | Incidence of total knee and hip replacement for osteoarthritis in relation to the metabolic syndrome and its components: A prospective cohort study[END_REF]. The accumulation of MetS components was associated with TKR, independently of BMI: one component, OR 2.12 (1.15-3.91); two components, 2.92 (1.60-5.33) and three or more components, 3.09 (1.68-5.69).

However, the weight of each of these factors for increasing OA risk remains largely unknown. Suffering from diabetes mellitus is associated with an independent increase of incident, prevalent and severe OA, although discrepancies exist in the literature [START_REF] Schett | Diabetes is an independent predictor for severe osteoarthritis: results from a longitudinal cohort study[END_REF] (review in [START_REF] King | The adverse effects of diabetes on osteoarthritis: update on clinical evidence and molecular mechanisms[END_REF]).

Recent studies have shown an increased risk of knee or hand OA in hypertensive and/or atherosclerotic patients, giving strength to the hypothesis that an abnormal perfusion of the subchondral bone due to atherosclerosis could participate into the OA process [START_REF] Hussain | Incidence of total knee and hip replacement for osteoarthritis in relation to the metabolic syndrome and its components: A prospective cohort study[END_REF][START_REF] Hoeven | Association of atherosclerosis with presence and progression of osteoarthritis: the Rotterdam Study[END_REF][START_REF] Conaghan | Is progressive osteoarthritis an atheromatous vascular disease?[END_REF].

Interestingly, this hypothesis fits with a recent study showing an association of serum cholesterol and triglyceride levels with the incidence of bone marrow lesions on knee MRI at 2 years in an asymptomatic middle-aged women population [START_REF] Davies-Tuck | Total cholesterol and triglycerides are associated with the development of new bone marrow lesions in asymptomatic middle-aged women -a prospective cohort study[END_REF]. Moreover, in a population of 809 OA patients, hypercholesterolemia (OR 1.61; 95% CI 1.06-2.47) and high serum cholesterol levels (3rd versus 1st tertile: OR 1.73; 95% CI 1.02-2.92) were independently associated with generalized OA [START_REF] Stürmer | Serum cholesterol and osteoarthritis. The baseline examination of the Ulm Osteoarthritis Study[END_REF]. Such an independent association has also been shown in hand OA [START_REF] Addimanda | Clinical associations in patients with hand osteoarthritis[END_REF].

Some groups looked at the prognostic value of plasma concentration of adipokines in OA cohorts. An association between leptin levels (but not adiponectin) and knee OA occurrence and progression was demonstrated in a 5-year cohort but this association disappeared after adjustment for BMI [START_REF] Berry | Temporal relationship between serum adipokines, biomarkers of bone and cartilage turnover, and cartilage volume loss in a population with clinical knee osteoarthritis[END_REF]. In the CHECK cohort, there is a weak association between resistin levels (but not leptin) and disease progression [START_REF] Van Spil | Cross-sectional and predictive associations between plasma adipokines and radiographic signs of early-stage knee osteoarthritis: data from CHECK[END_REF]. Finally, hand OA patients with high adiponectin levels (but not leptin nor resistin) had a decrease disease progression compared to normal/low levels in a Dutch cohort [START_REF] Yusuf | Association between leptin, adiponectin and resistin and long-term progression of hand osteoarthritis[END_REF]. Taken together, these epidemiological studies show clear associations between OA, the MetS, and its components, with some of them being clearly independent of the overload effect of obesity (Figure 1). However, there is a high heterogeneity in the design of all these crosssectional and longitudinal epidemiological studies (definition of the MetS, gender selection, record of medication, etc.), which prevents a definitive conclusion on the real impact of the MetS on knee OA initiation and progression. Recent experimental in vitro and animal studies have helped to begin deciphering the potential roles of each of these factors in the initiation and progression of OA.

Perspectives on the role of metabolic inflammation in OA from obese animal models

Beginning in the 1940s, Drs. Martin and Ruth Silberberg studied the role of increased dietary fat on the development of OA in mice. They showed that supplementing rodent chow with lard (30% by weight) increased the incidence of knee OA in C57BL mice by 2-fold [START_REF] Silberberg | Effects of a high fat diet on the joints of aging mice[END_REF].

Recent studies replicated these early observations in both spontaneous and post-traumatic models of OA (Table 2), focusing primarily on changes in inflammatory cytokines and adipokines as mediators of the increase in OA pathology [START_REF] Griffin | Dietinduced obesity differentially regulates behavioral, biomechanical, and molecular risk factors for osteoarthritis in mice[END_REF][START_REF] Griffin | Induction of osteoarthritis and metabolic inflammation by a very high-fat diet in mice: Effects of short-term exercise[END_REF][START_REF] Collins | Relationship between inflammation, the gut microbiota, and metabolic osteoarthritis development: studies in a rat model[END_REF][START_REF] Louer | Diet-induced obesity significantly increases the severity of posttraumatic arthritis in mice[END_REF][START_REF] Hamada | Insulin suppresses TNF-dependent early osteoarthritic changes associated with obesity and type 2 diabetes[END_REF]. In spontaneous models, knee OA severity was positively correlated with total body fat, and numerous cytokines and adipokines were also elevated with the percentage of body fat [START_REF] Griffin | Dietinduced obesity differentially regulates behavioral, biomechanical, and molecular risk factors for osteoarthritis in mice[END_REF][START_REF] Griffin | Induction of osteoarthritis and metabolic inflammation by a very high-fat diet in mice: Effects of short-term exercise[END_REF][START_REF] Collins | Relationship between inflammation, the gut microbiota, and metabolic osteoarthritis development: studies in a rat model[END_REF]. Statistically adjusting for adiposity using multivariable modeling negated most of the associations between systemic inflammatory mediators and knee OA, except for leptin and adiponectin [START_REF] Griffin | Dietinduced obesity differentially regulates behavioral, biomechanical, and molecular risk factors for osteoarthritis in mice[END_REF][START_REF] Griffin | Induction of osteoarthritis and metabolic inflammation by a very high-fat diet in mice: Effects of short-term exercise[END_REF]. These findings are notable since both leptin and adiponectin are implicated in OA pathogenesis [START_REF] Berry | Temporal relationship between serum adipokines, biomarkers of bone and cartilage turnover, and cartilage volume loss in a population with clinical knee osteoarthritis[END_REF][START_REF] Yusuf | Association between leptin, adiponectin and resistin and long-term progression of hand osteoarthritis[END_REF]. They are also of interest because mice with loss of function mutations in the leptin gene or the leptin receptor gene are protected from developing knee OA despite an extreme obesity phenotype, with body weights in excess of 80 grams and body fat levels >50% [START_REF] Griffin | Extreme obesity due to impaired leptin signaling in mice does not cause knee osteoarthritis[END_REF]. Mice with impaired leptin signaling become obese primarily by hyperphagia, and in the study by Griffin and colleagues (35), leptin deficient mice were fed a normal chow-based diet.

Increasing body weight by chemically inducing hyperphagia in mice fed a normal chow diet also does not increase knee OA [reviewed in [START_REF] Griffin | Why is obesity associated with osteoarthritis? Insights from mouse models of obesity[END_REF]]. This finding may be explained by the antiinflammatory and anti-oxidative actions of aurothioglucose, the chemical agent used to induce hyperphagy. However, it also raises the possibility that an increase in dietary fat or the composition of fat itself contribute to OA pathogenesis.

Mice fed high-fat lard diets supplemented with polyunsaturated fatty acids (PUFAs) or diets composed of vegetable oils (e.g., cottonseed oil) develop less severe knee OA [START_REF] Griffin | Why is obesity associated with osteoarthritis? Insights from mouse models of obesity[END_REF]. Fatty acids are well known to modulate inflammation positively or negatively, and recent studies have tested the effect of different types of dietary fatty acids on inflammation in rodent models of idiopathic and post-traumatic OA. Two central classes of lipid mediators of inflammation are omega-6 (n-6) and omega-3 (n-3) PUFAs. n-6 PUFAs, such as arachidonic acid, are precursors of pro-inflammatory eicosanoids, including prostaglandins, thromboxanes, and leukotrienes.

Conversely, n-3 PUFAs, such as eicosapentaenoic and docosahexaenoic acids, inhibit inflammation and accelerate its resolution. Obesogenic diets of middle and high-income countries, commonly referred to as Western Diets, contain a high ratio of n-6:n-3 PUFAs.

Two recent studies in mice have shown that decreasing the ratio of n-6:n-3 PUFAs, either by diet or genetically through the introduction of the fat-1 transgene that endogenously converts n-6 to n-3 PUFAs, reduces the development of post-traumatic knee OA [START_REF] Huang | Enhancement of the synthesis of n-3 PUFAs in fat-1 transgenic mice inhibits mTORC1 signalling and delays surgically induced osteoarthritis in comparison with wild-type mice[END_REF][START_REF] Wu | Dietary fatty acid content regulates wound repair and the pathogenesis of osteoarthritis following joint injury[END_REF]. This reduction was associated with a decrease in serum pro-inflammatory cytokines and adipokines (e.g., IL-1β, TNF, leptin, and resistin), joint synovitis, synovial macrophage infiltration, and heterotrophic joint ossifications. Intriguingly, wound healing in an ear-punch model was also improved by reducing the ratio of n-6:n:3 PUFAs [START_REF] Wu | Dietary fatty acid content regulates wound repair and the pathogenesis of osteoarthritis following joint injury[END_REF]. In contrast, the development of early-stage idiopathic knee OA in 1 year-old fat-1 transgenic mice was not reduced compared to control littermates despite lower serum IL-6 and TNF levels [START_REF] Cai | Metabolic enrichment of omega-3 polyunsaturated fatty acids does not reduce the onset of idiopathic knee osteoarthritis in mice[END_REF]. These studies suggest that the ratio of n-6:n-3

PUFAs has the greatest impact on OA progression under conditions of more acute activation of cellular and humoral immune responses, such as following tissue damage or joint trauma. It is not yet clear how n-6 and n-3 PUFAs regulate joint inflammation and repair. There is evidence that the ratio of n-6:n-3 PUFAs directly modulate cytokine-induced catabolic responses in joint connective tissues, such as cartilage [START_REF] Zainal | Relative efficacies of omega-3 polyunsaturated fatty acids in reducing expression of key proteins in a model system for studying osteoarthritis[END_REF]; however, the effect likely involves heterogeneous cell populations involved in the regulation of wound healing and resolution of inflammation. A recent randomized clinical trial of fish oil supplementation in knee OA patients showed improvement in OA-related pain and function at 2 years, albeit without altering cartilage loss [START_REF] Hill | Fish oil in knee osteoarthritis: a randomised clinical trial of low dose versus high dose[END_REF], suggesting that n-3 PUFAs may also reduce inflammation by improving physiologic joint use and function.

Another way in which high-fat diets may stimulate joint inflammation and OA is through activation of innate immune pathways. Obesogenic high-fat diets and alcohol consumption impair the gut mucosal barrier, resulting in increased intestinal permeability and elevated circulating levels of lipopolysaccharide. In addition, long-chain saturated fatty acids have also been implicated in promoting low-grade inflammation through activation of the Toll-like receptor (TLR) pathway, specifically TLR4. Mice lacking functional lipopolysaccharide receptor CD14 and TLR4 are partially protected from high-fat diet induced inflammation and related metabolic impairments, such as insulin resistance. Two recent animal studies support a model of systemic lipopolysaccharide inflammation in OA [START_REF] Collins | Relationship between inflammation, the gut microbiota, and metabolic osteoarthritis development: studies in a rat model[END_REF][START_REF] Kc | Induction of Osteoarthritislike Pathologic Changes by Chronic Alcohol Consumption in an Experimental Mouse Model[END_REF]. However, this work is at an early stage, and there are many questions about how TLR4 or lipopolysaccharide may mediate inflammation in OA [START_REF] Huang | Does lipopolysaccharide-mediated inflammation have a role in OA?[END_REF].

A growing body of work has implicated dyslipidemia in innate immune pathway activation and OA pathophysiology. Activated synovial macrophages are present in OA joints, especially in diabetic patients [START_REF] Hamada | Insulin suppresses TNF-dependent early osteoarthritic changes associated with obesity and type 2 diabetes[END_REF]. One potential mediator of macrophage activation is lowdensity lipoprotein (LDL) cholesterol. LDL is elevated with obesity and when oxidized stimulates the production of pro-inflammatory mediators, matrix degrading proteinases, and growth factors involved in OA pathogenesis [START_REF] De Munter | High systemic levels of low-density lipoprotein cholesterol: fuel to the flames in inflammatory osteoarthritis?[END_REF]. Furthermore, obesity and MetS also suppress the production of high-density lipoprotein (HDL) cholesterol, which exerts anti-inflammatory effects on macrophages through down-regulation of TLR-induced pro-inflammatory cytokines.

Mouse models of dietary and genetic-induced hypercholesterolemia increase knee OA pathology, even without increasing body weight [START_REF] Gierman | Metabolic stress-induced inflammation plays a major role in the development of osteoarthritis in mice[END_REF][START_REF] Triantaphyllidou | Perturbations in the HDL metabolic pathway predispose to the development of osteoarthritis in mice following long-term exposure to western-type diet[END_REF][START_REF] De Munter | Cholesterol accumulation caused by low density lipoprotein receptor deficiency or a cholesterolrich diet results in ectopic bone formation during experimental osteoarthritis[END_REF][START_REF] Gierman | Osteoarthritis development is induced by increased dietary cholesterol and can be inhibited by atorvastatin in APOE*3Leiden.CETP mice--a translational model for atherosclerosis[END_REF][START_REF] De Munter | High LDL levels lead to increased synovial inflammation and accelerated ectopic bone formation during experimental osteoarthritis[END_REF]. Moreover, statin treatment diminished OA severity in these models, supporting a direct role for cholesterol homeostasis in OA pathogenesis [reviewed in [START_REF] De Munter | High systemic levels of low-density lipoprotein cholesterol: fuel to the flames in inflammatory osteoarthritis?[END_REF]]. Recent evidence also shows a role for cholesterol homeostasis in chondrocyte hypertrophy through regulation of hedgehog signaling [START_REF] Ali | Regulation of Cholesterol Homeostasis by Hedgehog Signaling in Osteoarthritic Cartilage[END_REF], indicating multiple potential pathways linking dyslipidemia and OA pathophysiology.

Infrapatellar fat pad as a local regulator of knee joint inflammation

Abdominal adipose tissue inflammation is a central feature of dysregulated metabolism that occurs with obesity. This finding has piqued interest in the infrapatellar fat pad (IFP) as a local paracrine mediator of knee joint inflammation and OA pathogenesis. Indeed, the IFP is a source of numerous soluble factors, including cytokines (eg, IL-1, IL-4, IL-5, IL-6, IL-8, IL-13, IFNγ, TNF), chemokines (eg, CCL2), adipokines (eg, leptin, adiponectin, adipsin, resistin, and visfatin/eNAMPT), growth factors (eg, basic fibroblast growth factor and vascular endothelial growth factor), free fatty acids, and lipid derivatives (eg, arachidonic acid and prostaglandin E2)

[reviewed in [START_REF] Ioan-Facsinay | An emerging player in knee osteoarthritis: the infrapatellar fat pad[END_REF]]. These factors are derived from adipocytes as well as stromal cells, including resident and infiltrating macrophages and T cells present within the IFP.

The role of IFP-derived soluble factors in OA initiation and progression is unclear because the majority of studies have evaluated IFP cell populations and secreted factors in tissue harvested from joints with end-stage OA during joint replacement surgery. At this late stage of disease, the IFP contains more anti-inflammatory M2/CD206+ macrophages than proinflammatory M1/CD86+ macrophages and exerts an anti-catabolic effect on cartilage in vitro using IFP-conditioned medium [START_REF] Bastiaansen-Jenniskens | Infrapatellar fat pad of patients with end-stage osteoarthritis inhibits catabolic mediators in cartilage[END_REF]. It is not known if these findings are unique to IFP tissue from end-stage OA joints or if these are general characteristics of IFP tissue.

Clinically, alterations in IFP magnetic resonance imaging signal intensity are significantly and positively associated with knee pain in cross-sectional and longitudinal analyses [START_REF] Hill | Synovitis detected on magnetic resonance imaging and its relation to pain and cartilage loss in knee osteoarthritis[END_REF][START_REF] Han | Signal intensity alteration in the infrapatellar fat pad at baseline for the prediction of knee symptoms and structure in older adults: a cohort study[END_REF]. Peripatellar synovitis scores are positively associated with synovial neovascularization, hyperplasia, and villi [START_REF] De Lange-Brokaar | Degree of synovitis on MRI by comprehensive whole knee semi-quantitative scoring method correlates with histologic and macroscopic features of synovial tissue inflammation in knee osteoarthritis[END_REF], and IFP-conditioned medium stimulates the expression of pro-inflammatory mediators and matrix metalloproteinases in autologous fibroblast-like synoviocytes, suggesting a role for the IFP in synovial inflammation [START_REF] Eymard | Induction of an inflammatory and prodegradative phenotype in autologous fibroblast-like synoviocytes by the infrapatellar fat pad from patients with knee osteoarthritis[END_REF]. Surprisingly, though, IFP size is negatively associated with knee OA prevalence and risk of progression [START_REF] Pan | A longitudinal study of the association between infrapatellar fat pad maximal area and changes in knee symptoms and structure in older adults[END_REF]. These findings imply that a larger healthy IFP protects against OA, possibly by helping to better distribute joint biomechanical stresses [START_REF] Pan | A longitudinal study of the association between infrapatellar fat pad maximal area and changes in knee symptoms and structure in older adults[END_REF]. Alternatively, IFP size could be indirectly associated with knee OA risk.

In a recent analysis of the IFP in an aging rodent model of adult-onset obesity and knee OA [START_REF] Fu | Effect of Aging on Adipose Tissue Inflammation in the Knee Joints of F344BN Rats[END_REF], IFP mass significantly decreased with age despite increasing total body mass. This decrease occurred independent of changes in adipocyte size, suggesting that the reduction in fat pad size with age is due to an imbalance in the rates of adipogenesis and cell death. Although the basal expression of markers for adipogenesis did not change with age, they were significantly reduced with aging following IL-1β stimulation, suggesting that inflammation negatively regulates IFP adipogenesis and potentially IFP size. In this model, aging increased the production of TNF and IL-13 and decreased the expression of anti-inflammatory M2 macrophage markers. Thus, joint inflammation may mediate the negative association between IFP size and OA risk. Intriguingly, IL-1β inhibited leptin production in an age-dependent manner. This suggests that aging may protect against OA when joint inflammation is elevated (eg, post injury) by downregulating IFP adipogenesis and adipocyte-derived inflammatory mediators such as leptin.

Although the clinical evidence is growing that links alterations in IFP size or quality to OA risk and pain, the mechanistic understanding of these relationships is poor. Additional studies are needed at the early stages of disease to better evaluate the cause-effect relationship between IFP inflammation, size, and OA pathogenesis. Testing in multiple pre-clinical models of OA (eg, post-traumatic, diet-induced obesity, aging, genetic) will help to identify shared and unique mediators of IFP remodeling and inflammation by OA sub-type. Furthermore, the IFP is highly innervated with nociceptive fibers, and little is known about how changes in IFP remodeling and inflammation regulate OA pain and joint function.

Energy and nutrient sensors, metabolic syndrome, and OA

Evolutionarily conserved cellular energy and nutrient sensors, such as AMP-activated protein kinase (AMPK), sirtuins (SIRTs), and mammalian target of rapamycin (mTOR), determine how cells respond to excess or insufficient nutrients and changes in cellular energy balance. With the development of obesity or MetS-conditions of chronic excess nutrients-these sensors become impaired and further contribute to MetS, including abdominal adiposity, diabetes, and dyslipidemia [START_REF] Steinberg | AMPK in Health and Disease[END_REF][START_REF] Salminen | AMP-activated protein kinase (AMPK) controls the aging process via an integrated signaling network[END_REF][START_REF] Chalkiadaki | Sirtuins mediate mammalian metabolic responses to nutrient availability[END_REF][START_REF] Jia | Overnutrition, mTOR signaling, and cardiovascular diseases[END_REF]. These changes include a decrease in AMPK and sirtuin activities and an increase in mTOR activity. Understanding how obesity and conditions that contribute to MetS also lead to aberrant AMPK, SIRT, and mTOR function provide potential clues into how metabolic derangements increase OA.

AMPK is a serine/threonine protein kinase that exists as a heterotrimeric complex consisting of a catalytic α-subunit and two regulatory β-and γ-subunits [START_REF] Steinberg | AMPK in Health and Disease[END_REF]. Phosphorylation of a conserved threonine residue within the activation loop of the kinase domain (Thr172) is required for the kinase activity of AMPK [START_REF] Steinberg | AMPK in Health and Disease[END_REF]. AMPK is activated in response to decreases in cellular energy state by stimulating processes that generate ATP (e.g., fatty acid oxidation and glucose transport), and inhibiting others that consume ATP (e.g., fatty acid and protein synthesis) [START_REF] Steinberg | AMPK in Health and Disease[END_REF]. In this manner, AMPK allows cells to adjust to changes in energy demand. Sirtuins, a conserved family composed of seven members (SIRT1-7), possess nicotinamide adenine dinucleotide (NAD + )-dependent protein deacetylase, deacylase, and ADP-ribosyltransferase activities [START_REF] Giblin | Sirtuins: guardians of mammalian healthspan[END_REF]. They guard against cellular metabolic stress by modifying the activity of metabolic enzymes and gene transcription to balance nutrient supply and demand [START_REF] Giblin | Sirtuins: guardians of mammalian healthspan[END_REF]. mTOR is a serine-threonine kinase that serves as a converging point for signals controlling cellular growth, energy metabolism, nutrient availability, and stress. The mTOR signaling pathway is frequently activated in various tissues during conditions of excessive nutrient intake [START_REF] Giblin | Sirtuins: guardians of mammalian healthspan[END_REF]. Chronic activation of mTOR signaling can lead to the development insulin resistance [START_REF] Giblin | Sirtuins: guardians of mammalian healthspan[END_REF]. These energy and nutrient sensors become dysfunctional when cells are exposed to obesity-associated metabolic derangements, such as high concentrations of blood glucose and fatty acids (e.g., palmitate), low-grade metabolic inflammatory mediators (e.g., TNF), and low concentrations of adiponectin [START_REF] Salminen | AMP-activated protein kinase (AMPK) controls the aging process via an integrated signaling network[END_REF][START_REF] Weikel | Unraveling the actions of AMP-activated protein kinase in metabolic diseases: Systemic to molecular insights[END_REF].

Recent studies have implicated dysfunctional AMPK, sirturin and mTOR activity in OA development and progression. Phosphorylation of AMPKα Thr172 is decreased in human knee OA chondrocytes/cartilage [START_REF] Terkeltaub | Chondrocyte AMP-activated protein kinase activity suppresses matrix degradation responses to proinflammatory cytokines interleukin-1β and tumor necrosis factor α[END_REF] and in mouse knee OA cartilage, compared with their respective normal counterparts [START_REF] Petursson | Linked decreases in liver kinase B1 and AMP-activated protein kinase activity modulate matrix catabolic responses to biomechanical injury in chondrocytes[END_REF]. In addition, mouse knee cartilage exhibits an aging-associated reduction in phosphorylation of AMPKα [START_REF] Petursson | Linked decreases in liver kinase B1 and AMP-activated protein kinase activity modulate matrix catabolic responses to biomechanical injury in chondrocytes[END_REF]. Moreover, in vitro studies demonstrate that the inflammatory cytokines IL-1β and TNFα, as well as biomechanical injury, can cause dephosphorylation of AMPKα in normal articular chondrocytes. This is correlated with increased catabolic responses (e.g. increased MMP-3 and MMP-13 release). All of these effects can be inhibited by AMPK pharmacological activators [START_REF] Terkeltaub | Chondrocyte AMP-activated protein kinase activity suppresses matrix degradation responses to proinflammatory cytokines interleukin-1β and tumor necrosis factor α[END_REF][START_REF] Petursson | Linked decreases in liver kinase B1 and AMP-activated protein kinase activity modulate matrix catabolic responses to biomechanical injury in chondrocytes[END_REF]. Furthermore, recent in vivo studies show that activation of AMPK by berberine, a natural plant product that is known to activate AMPK, limits both surgical knee instability-induced and aging-related OA in mice [START_REF] Bryan | Activation of AMP-Activated Protein Kinase (AMPK) By Berberine Limits Both Surgical Knee Instability-Induced and Aging-Related Osteoarthritis in Mice[END_REF]. Collectively, sustained AMPK activity in articular chondrocytes appears to be chondroprotective. Three members of sirtuins (SIRT1, SIRT3 and SIRT6) have been studied in articular chondrocytes. Among them, SIRT1 is best characterized so far. Similar to AMPKα phosphorylation, SIRT1 expression is decreased in both human and mouse knee OA cartilage and in aged mouse knee cartilage [START_REF] Dvir-Ginzberg | Towards elucidating the role of SirT1 in osteoarthritis[END_REF][START_REF] Fujita | Potential involvement of SIRT1 in the pathogenesis of osteoarthritis through the modulation of chondrocyte gene expressions[END_REF][START_REF] Matsuzaki | Disruption of Sirt1 in chondrocytes causes accelerated progression of osteoarthritis under mechanical stress and during ageing in mice[END_REF]. Consequently, adult heterozygous Sirt1 mice and mice with a Sirt1 point mutation lacking SIRT1 enzyme activity exhibit increased OA progression [START_REF] Gabay | Increased apoptotic chondrocytes in articular cartilage from adult heterozygous SirT1 mice[END_REF][START_REF] Gabay | Sirtuin 1 enzymatic activity is required for cartilage homeostasis in vivo in a mouse model[END_REF]. Moreover, cartilage-specific Sirt1 knockout (KO) mice develop accelerated OA progression [START_REF] Matsuzaki | Disruption of Sirt1 in chondrocytes causes accelerated progression of osteoarthritis under mechanical stress and during ageing in mice[END_REF]. Sustained SIRT1 expression and activity in articular chondrocytes also appears to be important for cartilage homeostasis. Indeed, SIRT1 promotes cartilage-specific gene expression [START_REF] Dvir-Ginzberg | Regulation of cartilage-specific gene expression in human chondrocytes by SirT1 and nicotinamide phosphoribosyltransferase[END_REF], and activation of SIRT1 in a mouse OA model using an intra-articular injection of resveratrol significantly reduced cartilage destruction [START_REF] Li | Intra-articular resveratrol injection prevents osteoarthritis progression in a mouse model by activating SIRT1 and thereby silencing HIF-2α[END_REF]. Cartilage SIRT3 expression was recently shown to also decrease with age, and SIRT3 deficiency increases acetylation of the antioxidant enzyme SOD2 resulting in impaired SOD2 activity in chondrocytes [START_REF] Fu | Aging Promotes SIRT3-dependent Cartilage SOD2 Acetylation and Osteoarthritis[END_REF]. In addition, whole-body deletion of SIRT3 accelerates the development of knee OA [START_REF] Fu | Aging Promotes SIRT3-dependent Cartilage SOD2 Acetylation and Osteoarthritis[END_REF]. Expression of SIRT6 is significantly decreased in human OA chondrocytes as well [START_REF] Wu | Overexpression of Sirtuin 6 suppresses cellular senescence and NF-κB mediated inflammatory responses in osteoarthritis development[END_REF]. Depletion of SIRT6 in human chondrocytes causes increased DNA damage, telomere dysfunction, and premature senescence, which are processes implicated in cartilage degeneration in OA [START_REF] Nagai | Depletion of SIRT6 causes cellular senescence, DNA damage, and telomere dysfunction in human chondrocytes[END_REF].

Activation of AMPK stimulates the expression and activity of SIRT1 in articular chondrocytes [START_REF] Ruderman | AMPK and SIRT1: a long-standing partnership?[END_REF]. Activated AMPK-SIRT1 signaling increases cellular stress resistance through downstream targets peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α, a master regulator of mitochondrial biogenesis and function) and forkhead box O 3a (FOXO3a, a transcription factor that regulates oxidative stress response). In this context, mitochondrial function is preserved and excessive oxidative stress and inflammation-mediated matrix catabolism are inhibited [START_REF] Wang | Mitochondrial biogenesis is impaired in osteoarthritic chondrocytes but reversible via peroxisome proliferator-activated receptor-γ coactivator 1α[END_REF][START_REF] Zhao | Peroxisome Proliferator-Activated Receptor γ Coactivator 1α and FoxO3A Mediate Chondroprotection by AMP-Activated Protein Kinase[END_REF]. Activated AMPK also protects housekeeping cellular quality control in articular chondrocytes by limiting endoplasmic reticulum (ER) stress ( 81) and maintains autophagy capacity [START_REF] Bohensky | Chondrocyte autophagy is stimulated by HIF-1 dependent AMPK activation and mTOR suppression[END_REF]. Activation of AMPK can promote SIRT3 expression in articular chondrocytes as well (unpublished observation). Age-related loss of SIRT3 expression in cartilage may be a consequence of loss of AMPK activity. Loss of mitochondrial function is a hallmark of aging and age-related diseases, which is associated with reduced activity of AMPK and sirtuins [START_REF] Salminen | AMP-activated protein kinase (AMPK) controls the aging process via an integrated signaling network[END_REF][START_REF] Giblin | Sirtuins: guardians of mammalian healthspan[END_REF]. Since loss of SIRT3 activity results in profound aberrations in mitochondrial function [START_REF] Giblin | Sirtuins: guardians of mammalian healthspan[END_REF], AMPK-SIRT3 signaling could play an important role in maintaining normal mitochondrial function in chondrocytes. mTOR is a potent inhibitor of autophagy, which is a cellular degradative process that maintains fundamental biological activities during cellular stresses, especially nutrient starvation [START_REF] Salminen | AMP-activated protein kinase (AMPK) controls the aging process via an integrated signaling network[END_REF]. Activation of AMPK and SIRT1 promotes autophagy by inhibiting activity of mTOR complex (e.g. mTORC1) and enhancing the later steps in autophagosome formation through deacetylation of several autophagy-related proteins, respectively [START_REF] Salminen | AMP-activated protein kinase (AMPK) controls the aging process via an integrated signaling network[END_REF]. Chondrocyte autophagy is reduced with a linked increase in apoptosis in human knee OA, mouse knee OA, and aged mouse knee cartilage [START_REF] Caramés | Autophagy is a protective mechanism in normal cartilage, and its aging-related loss is linked with cell death and osteoarthritis[END_REF]. In mice, cartilage-specific genetic deletion of mTOR [START_REF] Zhang | Cartilage-specific deletion of mTOR upregulates autophagy and protects mice from osteoarthritis[END_REF] and pharmacologic inhibition of mTOR signaling by rapamycin (85) upregulate autophagy, which reduces the severity of post-traumatic OA.

Cellular energy and nutrient sensors, in particular AMPK and sirtuins (SIRTs), are potential mediators of the systemic (plasma) and local (synovial fluid) effects of adipokines on cartilage degeneration, osteophyte formation and synovial inflammation [START_REF] Poonpet | Adipokines: Biomarkers for osteoarthritis?[END_REF][START_REF] Neumann | Adipokines in bone disease[END_REF]. AMPK and sirturins regulate inflammatory responses, cartilage homeostasis, and bone metabolism [reviewed in [START_REF] Liu-Bryan | Inflammation and intracellular metabolism: new targets in OA[END_REF]]. Since dysregulation of AMPK and sirtuins are seen in experimental animal models of obesity and MetS, AMPK and sirtuins could be critical mediators in the cross talk between dysregulated metabolic tissues, such as adipose tissue, and joint tissues. A recent study showed significantly increased mRNA expression of inflammatory cytokines and adipokines, including TNFα, IL-6 and leptin, in the IFP of SIRT6 haploinsufficient (SIRT6 +/-) mice compared to WT mice [START_REF] Ailixiding | Pivotal role of Sirt6 in the crosstalk among ageing, metabolic syndrome and osteoarthritis[END_REF]. Following high-fat diet feeding, mRNA expression levels of all these cytokines were enhanced in WT and SIRT6 +/-mice, with IL-6 mRNA expression further enhanced in SIRT6 +/- mice [START_REF] Ailixiding | Pivotal role of Sirt6 in the crosstalk among ageing, metabolic syndrome and osteoarthritis[END_REF]. Moreover, SIRT6 +/-mice exhibited accelerated cartilage degradation when fed a normal diet, and they displayed greater osteophyte formation and synovial inflammation when fed a high-fat diet [START_REF] Ailixiding | Pivotal role of Sirt6 in the crosstalk among ageing, metabolic syndrome and osteoarthritis[END_REF]. These results suggest that impaired SIRT6 links metabolic dysfunction with multiple aspects of OA, including cartilage degradation, synovial inflammation, and bone remodeling. Further study is warranted to investigate how energy sensors in joint tissues become dysregulated in response to abnormal systemic and/or local metabolism associated with MetS.

AMPK, sirtuins and mTOR as therapeutic targets for OA

The pathways of AMPK, sirturins and mTOR are tightly cross-linked and share many downstream targets that regulate cell processes involved in aging, including mitochondrial biogenesis, cellular metabolism, autophagy, and DNA repair [START_REF] Steinberg | AMPK in Health and Disease[END_REF]. Increasing evidence indicates that aging, joint injury, low-grade inflammation, and possibly nutritional overload, obesity and MetS impair normal function of energy and nutrient sensors in articular cartilage. Reduced activities of AMPK and sirtuins and increased mTOR activity in articular chondrocytes trigger significant metabolic stress, manifested as mitochondrial dysfunction, oxidative stress, and inflammation. As a result, cell survival and tissue integrity and function are compromised, ultimately leading to OA development and progression (Figure 2). Interestingly, activation of AMPK and sirtuins and inhibition mTOR signaling are implicated in the beneficial effects of dietary restriction [START_REF] Solon-Biet | Macronutrients and caloric intake in health and longevity[END_REF]. In addition, some drugs already in the clinic (e.g. metformin for type 2 diabetes and methotrexate for rheumatoid arthritis), and some natural plant products used as dietary supplements (e.g. berberine, resveratrol) activate the AMPK/SIRT1 and/or AMPK/mTOR signaling pathways. As such, AMPK, sirtuins, and mTOR are potentially attractive and achievable therapeutic targets for OA. A systemic treatment (e.g. activation of AMPK) may be an effective means to combat both metabolic diseases and OA.

Conclusions

Exciting new discoveries are revealing a vast network of inter-connected pathways that regulate metabolism and inflammation. These pathways exist at the molecular level in the form of cellular energy sensors (e.g. AMPK, sirtuins, mTOR) that regulate enzyme activity, gene transcription, and proteostasis to coordinate cell survival mechanisms. They also exist at the cellular level, where changes in energy metabolism and nutrient utilization impact the activation and resolution of inflammation via effects on both immune and non-immune cells. At the organismal level, excess caloric intake, increased saturated and n-6 dietary fat, and insufficient metabolic energy expenditure can impair organ and tissue function, resulting in systems-level dysfunction (e.g. diabetes) and pathologic paracrine or endocrine pro-inflammatory signaling (e.g. intestinal or adipose-tissue derived inflammatory mediators). The increasingly recognized role of inflammation in OA, together with the high comorbidity of obesity and OA, set an exciting new path for trying to understand how disrupted metabolic signaling in distant organs or in local articular tissues contribute to an increase in OA risk.

Current prospective epidemiological studies show clear associations between OA, the MetS, and its components, even when controlling for obesity in some studies. Animal models that recapitulate some aspects of the MetS also support a role for disrupted metabolic and inflammatory signaling in OA risk independent of obesity itself. Many questions remain, however, about the nature of these interactions. Despite a growing body of clinical evidence on sex-specific associations between components of the MetS and OA, few animal studies have

M = male; F = female; PTOA = Post-traumatic osteoarthritis; MetS = Metabolic Syndrome; supp. = supplement.

•

  tested sex-dependent outcomes in obese OA models. Additional questions remain about the relative role of systemic versus local factors in regulating articular joint tissue inflammation and metabolism. Therefore, we propose the following research agenda to address these questions and additional unmet needs to better understand how metabolic dysfunction mediates joint inflammation and the pathophysiology of OA. Research Agenda To design an epidemiological prospective study looking at the risk of hand OA in presence of MetS • To design an epidemiological prospective study looking at the risk of knee/hip/hand OA in presence of hypertension • To design studies and develop model systems evaluating sex-dependent differences in OA risk in the presence of MetS and its underlying components • To design studies and develop model systems that isolate the effects of body weight, MetS components, and aging on tissue-specific cellular and molecular mediators of articular joint inflammation and metabolism • To develop methods and model systems to evaluate in vivo cellular energy metabolism and metabolic signaling networks within and between articular joint tissues under physiologic and pathophysiologic conditions • To design clinical and preclinical studies that test the efficacy of metabolic-based therapies (e.g., dietary, natural products, pharmaceutical) on OA risk in the presence of obesity and MetS

Figure 1 .

 1 Figure 1. Cardiometabolic factors and associated mediators of increased OA

Figure 2 .

 2 Figure 2. AMPK and sirtuins as potential therapeutic targets for OA. Aging, joint injury,

Table 1 :

 1 See separate file (landscape orientation)

Table 2 . Obese rodent models of spontaneous and induced OA and Metabolic Syndrome-related outcomes

 2

	Species (Sex)	Obesity-related manipulation	OA model	OA phenotype(s)	OA & MetS-related outcomes	Ref.
	Rat	Dietary (high fat, sucrose)	Spontaneous	Cartilage pathology, synovial	Body fat; serum cytokines and	(42)
	(M)	Fat source: Soybean oil, lard		fluid inflammation	adipokines
	Rat	Spontaneous (adult-onset)	Spontaneous	Cartilage and meniscus	Infra-patellar fat pad cytokines	(75)
	(M)	Fat source: Soybean oil, fish		pathology, synovial	and adipokines
		meal		thickening
	Mouse	Dietary (high fat)	Spontaneous	Cartilage pathology	Body fat; serum cytokines and	(30)
	(F)	Fat source: Soybean oil, lard			adipokines
	Mouse	Dietary (high fat, sucrose)	Spontaneous	Cartilage pathology,	Infra-patellar fat pad	(91)
	(M)	Fat source: Safflower oil, beef		osteophytes, synovial	cytokines; xanthine
		tallow		thickening	oxidoreductase inhibition
	Mouse	Dietary (very high fat)	Spontaneous	Cartilage pathology,	Glucose intolerance; serum	(31)
	(M)	Fat source: Soybean oil, lard		subchondral bone thickness	cytokines and adipokines
	Mouse	Dietary (very high fat)	Spontaneous	Osteophytes, synovial	Glucose intolerance; systemic	(34)
	(M)	Fat source: Soybean oil, lard		inflammation	and synovial TNF
	Mouse	Genetic (H-CRP) and dietary	Spontaneous	Cartilage pathology (male	Serum CRP, statin treatment	(46)
	(M/F)	(high fat, sucrose)		only)	(protective)
		Fat source: Soybean oil, lard
	Mouse	Genetic (Lcat -/-, ApoA-I -/-) and	Spontaneous	Cartilage pathology	Serum dyslipidemia	(47)
	(M)	dietary (high fat, sucrose)
		Fat source: Milk fat
	Mouse	Genetic	Spontaneous	Cartilage pathology	Serum dyslipidemia, serum E-	(49)
	(F)	(APOE*3Leiden.CETP) and			selectin, statin treatment
		dietary (cholesterol supp.)			(protective)
	Mouse	Dietary (very high fat)	PTOA	Cartilage pathology, synovial	Body fat; serum cytokines and	(33)
	(M)	Fat source: Soybean oil, lard	(Intra-articular	inflammation, reduced	adipokines
			fracture)	cancellous bone fraction
	Mouse	Dietary (very high fat)	PTOA	Cartilage pathology,	Glucose intolerance	(92)
	(M)	Fat source: Soybean oil, lard	(Meniscus-	osteophytes
			ligament injury)
	Mouse	Dietary (very high fat, varied	PTOA	Cartilage pathology, synovial	Body fat; dietary fatty acid	(38)
	(M)	type) Fat source: varied	(Medial	inflammation, heterotopic	composition (saturated and ω-
		(Soybean, safflower, olive,	meniscus	ossification, altered	6 fatty acids); serum
		coconut, menhaden, and lard)	destabilization)	cancellous bone fraction	adipokines; hyperinsulinemia
	Mouse	Genetic (Ldlr -/-) and dietary	Intra-articular	Osteophytes, heterotopic	Serum dyslipidemia	(48)
	(F)	(cholesterol supp.)	collagenase	ossification, synovial
				inflammation
	Mouse	Genetic (ApoE -/-, S100a9 -/-)	Intra-articular	Osteophytes, heterotopic	Serum dyslipidemia
	(F)	and dietary (cholesterol supp.)	collagenase	ossification, synovial
				inflammation

Table 1 : Prospective studies on the risk association of OA with metabolic syndrome and/or its components

 1 HR 1.59, 95% CI 1.25-2.01) and hypertension (1.24, 1.05-1.48) associated with increased risk of knee OA. Risk of knee OA with accumulation of MetS components , independent of BMI: one component, 2.12 (1.15-3.91); two, 2.92 (1.60-5.33) and three or more, 3.09(1.68-5.69). No statistically significant associations for hip OA. 95% CI 1.3-3.3) After adjustment for age, sex,smoking, physical activity and CRP but nonsignificant after adjustment for BMI (RR: 1.1, 95% CI 0.7-1.8). MetS not associated with incidence of hip OA. In women, CRP associated with knee OA in the ageadjusted analysis but no significant relationship between CRP and incidence of knee or hip OA after risk factor adjustments OA: osteoarthritis; BMI: Body Mass Index; DM: diabetes mellitus; SBP: systolic blood pressure; DBP: diastolic blood pressure; trigly: triglycerides; HDL-C: HDL cholesterol; LDL-C: LDL cholesterol; CAC: coronary artery calcification; BML: bone marrow lesions; OR: odds ratio; HR: hazard ratio; CI: confidence interval; MetS: metabolic syndrome; RR: relative risk; SD: standard deviation;

		Partici-	M/F	OA	follow-	Overweight	Diabetes	Hypertension	Dyslipidemia	Athero-	Results
		pants			up					sclerosis
					(year)
	Sowers (16)	482	0/482	K	10	Waist:hip ratio	Self-reported	SBP> 130mm	HDL-C ≤45	ND	Prevalence:
						>0.81 cm	DM OR	Hg	mg/dl		Nonobese women without cardiometabolic clustering
							medications	Or DBP>85mm	OR LDL-C>		(at least 2 metabolic parameters including CRP
							OR glucose level >126mg/dL	Hg OR medication	160 mg/dl		≥2mg/dL) 4.7%, compared with 12.8% in obese women without cardiometabolic clustering and 23.2% in obese women with cardiometabolic clustering
	Yoshimura (17)	1384	466/918	K	3	BMI > 25 kg/m2 HbA1C> 5.5% Or medication	SBP≥130mm Hg Or DBP≥85mm Hg OR medication	HDL-C <40 mg/dL OR medication	ND	Occurrence: OR vs no component: one component, 2.33; two, 2.82; three, 9.83 Progression: OR vs no component: one component, 1.38; two, 2.29;
											three: 2.80).
	Monira-Hussain (18) Central obesity (Schett (19) 1222 413/809 K H 6.8 SD=1.5 Waist circonference: Men: ≥94 cm Women: ≥80cm Fasting glucose ≥5.6mM/L OR history of type 2 diabetes SBP≥130mmHg Or DBP ≥85mmHg OR medication Trigly ≥1.7mM/L AND/OR Men: HDL-C <1.03 Women: <1.29mM/L OR medication ND 927 431/427 K 20 years ND Fasting glucose ND ND ND Risk for arthroplasty with type 2 diabetes : HR
				H	Follow-up		≥7 mmol/L Or medication				(95%CI), 2.1 (1.1-3.8) after adjustment for age, BMI, and other risk factors for OA
	Davies-Tuck	148	0/176	K	2.2	ND	ND	ND	ND	ND	Risk for incident BML associated with higher levels of
	(20)				SD=0.12						total cholesterol (OR 1.84, 95% CI 1.01-3.36) and
											triglycerides (OR 8.4, 95% CI 1.63-43.43), but not
											HDL, LDL or total cholesterol/HDL ratio.
	Hoeven (21)	3465	1501/1964	K	4.5 SD =0.5	ND	ND	SBP≥140mmH Or medication 90mmHg Or DBP ≥	ND	CD40L, CAC VEGF, VCAM-1,	CD40L and VCAM-1 higher in in women with knee OA compared with those without knee OA.
	Engstrom (13)	209	70/139	K H	12.4	Waist circonference: Men: ≥102 cm Women: ≥88cm	Fasting glucose ≥5.6 mmol/L	Or medication SBP> 130mm Hg Hg Or DBP>85mm	Or medication Trigly≥ 1.7 mmol/L	ND	MetS associated with increased risk of knee OA (RR: 2.1,

Acknowledgments

This work has been supported by grants from the ROAD-Foundation Arthritis Network and the French Society of Rheumatology (FB), the NIH (National Institute of Arthritis and Musculoskeletal and Skin Diseases grant R03AR066828 and National Institute on Aging grant R01AG049058 to TMG), and the Department Veterans Affairs grant 1I01BX002234 (RLB). The

Author Contributions

All authors were involved in drafting the article and revising it critically for important intellectual content, and all authors approved the final version to be published. content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.


Weichao Shi

Glenn H Fredrickson

Edward J Kramer

Christos Ntaras

Apostolos Avgeropoulos

Quentin Demassieux

Costantino Creton

Mechanics of an Asymmetric Hard-Soft Lamellar Nanomaterial

published or not. The documents may come

INTRODUCTION

Fabrication of nano-layered materials is an important and increasingly popular topic in nanoscience and nanotechnology. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] The materials are broadly used as coatings, adhesives, membranes, electronic devices and bio-sensors with tunable functionalities, such as solar cell devices (typically multi-nanolayers of conducting materials), [7][8][9] polyelectrolyte membranes (usually through a layer-by-layer technique), [13][14][15]18 artificial skins 16,17 and so on. Meanwhile, those materials need to meet various mechanical requirements for different purposes. For example, extensibility and resistance to fracture is critical to develop deformable electronic devices; 8,9 nanomechanical properties and domain features are also sensitive to tune photonic response in polymer multilayers. 10 Recent research has developed to bio-related applications, such as strain sensors for artificial skins. 16 In spite of the broad current and future applications, the fundamental mechanics of lamellar nanomaterials has not yet been well elucidated. [19][START_REF] Strobl | The Physics of Polymers[END_REF][21][22][23][24][25][26][27][28][29][30][31][32] From a polymer mechanics perspective, nano-layers of two hard polymers (modulus over ~1 GPa) exhibit high modulus but cannot deform to large strains without fracture; nano-layers of two soft polymers are usually extensible but lack stiffness; a combination of hard-soft polymer nano-layers could synergetically improve the mechanical properties over a broad range. [19][START_REF] Strobl | The Physics of Polymers[END_REF][21][22][23] Yet the mechanical properties of these structures, in a soft-hard configuration for example, are nearly unexplored and are a prerequisite to expand the applications. Hard-soft block copolymers (BCPs) are good prospects to achieve nano-layered structures by microphase separation. [33][34][35][36] Most current nano-layered materials obtained by traditional block copolymers are however only thermodynamically stable in a nearly symmetric layer thickness, although nanotechnology nowadays requires lamellae with flexibly tunable thickness.

In linear block copolymers (such as AB diblocks or ABA triblocks), the available composition range for lamellar structures is usually from 35% to 65% (by volume) in the strong segregation regime. 33 Nano-layered lamellar structures are not stable at higher or lower compositions. How to achieve highly asymmetric lamellae has long been a challenging question in polymer science. Due to this limitation, current understanding on lamellar mechanics is limited in symmetric case, where the inclusion of a high content of soft component improves the toughness and meanwhile results in a considerable loss of modulus and strength. Highly asymmetric hard-soft lamellae, with a high content (above 65%) of a hard component, are expected to manifest a promising balance between toughness and stiffness. [37][38][39][START_REF] Herman | Mark Encyclopedia of Polymer Science and Technology[END_REF][START_REF] Adhikari | [END_REF][42][43] However, almost nothing has been achieved in this direction. In addition, the mechanics of lamellar structures is highly dependent on the molecular architecture. For hard-soft nano-layers, hard blocks are expected to behave as anchoring points at both ends to provide stiffness, while the middle soft blocks supply extensibility. 37,38,[START_REF] Herman | Mark Encyclopedia of Polymer Science and Technology[END_REF] The number of hard and soft blocks within one molecule is a key parameter to tune mechanical properties. So, hard-soft diblock copolymers exhibit poor mechanical properties; hard-soft-hard triblock copolymers have improved mechanical properties, but the number of anchoring points per molecules is still low. 35,36 Guided by self-consistent field theory simulations, [START_REF] Fredrickson | The Equilibrium Theory of Inhomogeneous Polymers[END_REF] we find that the limiting factors described above are addressed with a carefully designed miktoarm architecture A 1 -(B-b-A 2 ) n (n>2, A is the hard block and B is the soft block, as illustrated in Scheme 1). 37 Driven by the nonlinear molecular architecture at the A 1 B n junctions and the bi-dispersity of A blocks within the molecule, the miktoarm star block copolymer reinforces curvature towards A and stabilizes lamellar structures up to a remarkably high volume fraction f A =80% for n=3 and 1 1 2 ( ) 0.9

A A A N N N    
, where N is the number of repeating units in each block. The specific realization of this model system was achieved with a PS 1 -(PI-b-PS 2 ) 3 system (PS denotes polystyrene and PI poly(isoprene)). 38 Furthermore, blending of PS 1 -(PI-b-PS 2 ) 3 with homo-polystyrene (hPS) leads to even more extreme morphologies. The upper boundary of an extremely asymmetric lamellar structure was realized for up to 97 wt% of PS segments in total. 39 Another key aspect of the PS 1 -(PI-b-PS 2 ) 3 architecture is that the junctions between PI blocks and the short PS 2 tails supply multiple anchoring points in the glassy PS domains so that stress is more uniformly distributed, compared with fewer anchoring points in linear-chain BCP counterparts.

Scheme 1. Schematic illustration of an A 1 -(B-b-A 2 ) 3 miktoarm block copolymer.

Based on this molecular design, we would expect the mechanical properties to be tunable over a broad range, combining high extension from the soft rubbery component and high modulus from the hard glassy component. The deformation mechanism is investigated in details using transmission electron microscop y and in situ real-time tensile-SAXS synchrotron facility. Notably, although the study is carried out with PS and PI hard-soft systems, the mechanism should be more general and guide the design of other functional materials (such as electric, photonic, permeable) based on lamellar nanostructures with promising mechanical properties. In addition, this study demonstrates the significant advantages of using miktoarm block copolymers as a structuring unit relative to traditional linear block copolymers, resulting in new perspective to design advanced nanomaterials.

RESULTS AND DISCUSSION

The neat miktoarm block copolymer PS 1 -(PI-b-PS 2 ) 3 with 70 wt% PS exhibits an asymmetric lamellar structure. Even more asymmetric lamellae are stable with 90 wt% hPS, which is equivalent to 97 wt% PS in total. For this set of materials, the rubbery PI layer thickness is constant (~13 nm) while the PS layer thickness can be varied from 26 nm to over 300 nm. 39 The monotonic tensile testing results are shown in Figure 1 for miktoarm block copolymer blends with different hPS weight fractions. The neat miktoarm copolymer already exhibits a high stress and strain at break. But due to the significant rubbery content (30 wt% of PI), the modulus, yield stress and nominal strength at break are relatively low as shown in Figure 1. For neat miktoarm and the blend with 10 wt% of hPS, clear plateau regions of nearly constant nominal stress after yield, followed by a mild strain hardening, are observed. The plateau region was accompanied by necking, which indicates that shear yielding is the main deformation mechanism. For blends with hPS content above 30 wt% (rubbery domain volume fraction is now less than 20 wt%), the yield point shifts to larger stresses and pronounced strain hardening is observed after yielding. The plateau region disappears from the stress-strain curves and necking is no longer observed. Remarkably, the material remains ductile with nearly 150% strain at break at an hPS content of 50 wt% (equivalent to 15 wt% rubbery phase). For blends with even more hPS, the nominal strength at break keeps increasing until 80 wt% hPS, while the strain at break continuously decreased. The Young's moduli of these nano-layered materials increased from ~300 MPa to ~900 MPa (Figure 1b). Compared with 2~3 GPa and ~2% ultimate strain for pure hPS thermoplastics, 19,20 the unusual rubber configuration in these blends is remarkably effective at giving ductility while resulting in only a moderate decrease in modulus relative to the hard PS glassy component. The drop of the modulus will be discussed later. void formation in the rubbery phase. Image (f) is another magnified image of (d) highlighting crazes in the glassy PS domains.

To investigate the fracture mechanism, we studied the structure after tensile deformation (post failure). Figure 2 (a, b, c) show the corresponding TEM images for the sample containing 50 wt% hPS (More evidence was presented as supplementary information: part B). One can clearly indentify five different regions. Region 1 is an intact region where lamellae were well maintained after stretching. Region 2 shows the formation of kinks, which include the original defects and deformed lamellae in the tensile stress direction. [START_REF] Cohen | [END_REF][46][47] It is notable that the lamellae near the kinks exhibit minor damage. In region 3, we observe weakly deformed lamellae with undulating interfaces which manifest a stress-induced instability. In these regions, the lamellae are relatively well aligned with the tensile stress direction. We denote region 3 as a "pre-damage grain." Region 4, neighboring region 3, contains lamellae that were more severely damaged resulting in homogenized grains with greatly reduced orientational order. The originally sharp PS/PI interfaces are smeared and distorted within such grains. We denote region 4 as a "damage grain." V-shaped boundaries between the regions 3 and 4 can be clearly identified (Figure 2b). The deformation behavior of regions 3 and 4 are very similar to the shear deformation of semi-crystalline polymers under tensile stress. 32 Finally, we consider regions (5) where the lamellae are oriented perpendicular to the tensile stress direction. As shown in Figure 2c, the PS glassy layers remained largely intact while voids grew within the PI rubbery domains. The PI domains form fibrils between neighboring PS layers and can extend to several times their original thickness before breaking. The original PI layer thickness is 13 nm. From the TEM images, we observe that the PI fibrils break when the extension is ~100 nm, which indicates an extension ratio of approximately 8. Further tensile deformation evidently leads to fibril breakdown and coalescence of neighboring voids into cracks. It appears, therefore, that the fracture of the material is initiated from region 5. Local shear deformation is still a dominant factor under tensile deformation, in spite of the fact that brittle PS constitutes the majority phase (85 wt% PS in total).

When the hPS fraction was increased to 80 wt% (94 wt% PS in total), we found that the fracture mechanism changed considerably. Figure 2 (d,e, f) show the corresponding TEM images for the 80 wt% hPS sample after monotonic tensile testing to break. The average PS layer thickness is now approximately 170 nm while the PI layer thickness is 13 nm. In Figure 2d, we observed significant void formation in the rubbery layers; there are also clear indications of voids in the glassy PS domains. We can also observe a crack tip (right bottom) expanding into both PS and PI regions. In Figure 2e, the rubbery fibrils seem to remain intact at small extension, and fail when the thickness of the fibrillar region was larger than ~100 nm. Crazes, consisting of PS fibrils and voids between two interfaces, can be identified in Figure 2f. The size of the crazes is about ~50 nm. The craze phenomenon of the PS nano-layers indicates a similar mechanical response than that of the bulky PS. Notably, the interfaces between glassy and rubbery domains remain almost intact, a significantly different behavior from that observed for the blends with lower (50 wt%) hPS content.

From the above comparison, we conclude that the tensile deformation of the blends with relatively low hPS content is characterized by two features: 1) the extension of the rubbery nanolayers and 2) the significant deformation of the interfacial regions, which leads to the formation of pre-damage and damage grains. Both factors dissipate energy and assist the release of local stress during stretching, which cause irreversible damage in the material. In contrast, for the blends with high hPS content (above 80 wt%), the interface regions remain largely intact, while crazing is observed in the relatively thick PS domains (> 100 nm). Although PS crazes can still sustain a load at small strains, the nano-fibrils will break at larger deformation and result in catastrophic coalescence of voids into growing cracks (Figure 2d). The void formation in both PS and PI layers acts potentially as 3-dimensional channels for crack propagation. So, in the high hPS content blends, the observed toughness mainly relies on the extension of the PI rubbery lamellae (8~10 times of extension). shows the domain spacing variation with nominal strain. The domain spacing was calculated from the primary peak positions in the SAXS curves shown in image (b).

To further investigate the void formation during stretching, we carried out in situ real-time tensile-SAXS experiments, which provide instantaneous scattering patterns under deformation. Figure 3 shows the scattering patterns for miktoarm/hPS (50 wt%)

blends at different strains. The original sample showed isotrop ic scattering rings, indicating lamellar structures with random orientation (Figure 3a). Upon stretching, the intensity grew strongly in the tensile direction and exhibited "butterfly" patterns.

The anisotropic scattering patterns are the result of increased correlation and contrast in the tensile direction, a sign of void formation. The 2-D SAXS patterns were plotted as 1-D curves in the tensile and transverse directions in Figures 3b andc, respectively.

The intensity in the tensile direction is observed to be about 10 times stronger than that in the transverse direction near q=0.02 nm -1 . In Figure 3b, the primary peak position is seen to shift toward smaller scattering wavenumber q with growing intensity; the slope in the high q region followed a power la w index -4, indicative of scattering from sharp interfaces. We postulate the primary peak is ascribed to the increasing domain spacing associated with substantial extension of PI layers oriented perpendicular to the tensile direction (filled with PI fibrils and voids). The initial domain spacing is 73 nm, where the thicknesses of the PS and PI layers are 60 and 13 nm, respectively. As shown in Figure 3d, the domain spacing increased significantly upon stretching, with the increment slowing after yielding, and finally reaching 182 nm before break. We infer that the PI fibrils can extend to 122 nm until fracture of the sample. These results are consistent with the TEM images (Figure 2) showing void formation in the rubbery phase and PI fibrils stretched between glassy domains with extension ratios of 8~10. In Figure 3c, we observe only weak, broad peaks in the transverse scattering, which suggests low contrast or smeared lamellar order; the slope in the high q region shows a power law index -3, which is characteristic of scattering from diffuse/fractal interfaces.

The scattering patterns generally exhibited similar features for other blends at lower or higher hPS contents (supplementary information: part C). Notably, for a range of hPS compositions, a weak, broad peak always appeared at ~0.12 nm -1 in the transverse direction during stretching (Figure 3c), which corresponds to a correlation length of about 50 nm. Since TEM analysis reveals that the domain features are quite different in high and low hPS content miktoarm/hPS blends and the only common feature arises from the void formation, we postulate that the transverse scattering peak reflects the correlations between neighboring voids (not the form factor of individual voids, since the peak position did not move with strain). The correlation length is ~50 nm, which is consistent with TEM images. Using the well established three-phase model, 48,49 we can quantitatively calculate the volume fraction of voids during tensile stretching with the full 2-D scattering data (Figure 4). The neat miktoarm block copolymer had low void fraction around 5% in a large range of strains. The void content increased significantly when hPS was blended with the miktoarm copolymer. For 30 wt% hPS blends, the fraction could reach 17% before rupture. For blends with even higher hPS content, the void content followed a similar increasing tendency with strain but the material broke at smaller strains. One common feature can be clearly identified for all blends: the void formation follows a similar strain-dependence before yielding, which indicates that the void formation mechanism is similar for all blends at small strains. From Figure 3d, we found that the domain spacing increased quickly before yielding. So, cavitation in the rubbery nano-layers was the major contribution to the total void fraction for all miktoarm/hPS blends.

To gain further insight into the deformation mechanism, we carried out SAXS experiments during step-cycle tensile testing. For the neat miktoarm copolymer, the strain increment in each cycle was 0.5; that is, the sample was stretched to a strain of 0.5 and then the force was reduced to 0 in the first cycle; in the second cycle, the sample was stretched to a strain of 1.0 and then the force was reduced to 0; the maximum strain is 1.5 in the third cycle; and the process was repeated until the rupture of the specimen. For blends with 50 wt% and 80 wt% hPS, the strain increment was 0.12 for each cycle. The volume fraction of voids was calculated throughout the repeated load-unload processes and the results are shown in Figure 5.

Figure 5. The void content during step-cycle tensile tests for miktoarm/hPS blends at different hPS weight fractions. For neat miktoarm copolymer (a), the strain increment was 0.5 for each cycle; for blends with 50 wt% (b) and 80 wt% hPS (c), the strain increment was 0.12 for each cycle.

In the neat miktoarm copolymer (Figure 5a), the voids were significantly initiated in the first loading cycle, and then almost fully disappeared after the first unloading.

In the next multiple loading-unloading cycles, the maximum volume fraction of voids kept decreasing. In the seventh cycle, the maximum void content was about 2% and could be fully recovered after unloading. From the lamellae "melting" under deformation (damage grains in Figure 2), we infer that the local stress inhomogeneity was significantly released by breaking the lamellar domains therefore the voids cannot be initiated in the already homogenized grains. When the hPS content is increased, it becomes difficult to reversibly deform hard PS layers and the readily formed voids do not close upon unloading. Void formation in both PS and PI domains becomes prominent as the amount of hPS increases. For blends with 50 wt% (Figure 5b) and 80 wt% hPS (Figure 5c), we found that although the voids could almost be fully recovered in the first loading-unloading cycle (10% strain), the void content kept increasing with strain for each subsequent tensile cycle and only part of the voids closed after each unloading. By further analysis of the SAXS data for each cycle (Supplementary information: part D), we found evidence of a strong effect of the mechanical history on the structure along the tensile direction, while the mechanical history had a much weaker effect on the transverse structure. Notably, the void correlation peak at 0.12 nm -1 is clearly visible upon loading but is attenuated after unloading. Here we conclude two points: the non-recovery of miktoarm/hPS blends reflects both plastic deformation and residual voids; voids can be partially kept open under hydrostatic stress in the tensile direction, while shear deformation dominates in the transverse direction. The latter point is also supported by TEM images (Figure 2).

As stated above, PS layers tend to be destroyed rather than forming crazes when the PS layers are thin; crazing in PS domains becomes prominent when the thickness of the PS layers is large enough. A key question remains: what is the critical thickness separating the two regimes? In different, but related systems, researchers have proposed that glassy lamellae should be thin enough to avoid brittle fracture by crazing. [START_REF] Adhikari | [END_REF][42][43] They suggested a critical ligament thickness around 20 nm, which is approximately the craze fibril thickness in neat hPS. We do not find evidence supporting this 20 nm value in our systems. For the asymmetric miktoarm block copolymer blended with 50 wt% hPS, the thickness of the PS layers is 60 nm, which is a value three times higher than the proposed critical thickness, yet the material exhibits substantial extension with no PS crazing detected. In contrast, PS crazing was clearly observed in blends containing 80 wt% hPS, with PS domain thickness of ~170 nm. Based on these observations, we propose that the PS critical thickness in asymmetric lamellar systems defining a ductile to brittle transition is roughly about 100 nm.

Combining all the above results and analysis, we can classify five categories of lamellar deformation under tensile stretching:

Ⅰ. Elastic deformation. Elastic deformation is only maintained at small strains before any irreversible damage occurs.

Ⅱ. Orientational reorganization. Under stretching, the domains will adapt to the local shear or compression/extension forces by changing orientation. The observed kinks are typical features associated with orientational reorganization of lamellar structures. [START_REF] Cohen | [END_REF][46][47] Ⅲ. Mechanical homogenization. If the chain density at the interface is low and a lamellar region is oriented poorly, short PS blocks can be pulled out from their domains 50,51 and PS layers can be fragmented, leading to the "melting" of a lamellar structure into homogenized state. In this study, regions 3 and 4 in Figure 2a show evidence of this kind of deformation.

Ⅳ. Void formation in rubbery domains. Since the rubbery phase has a low modulus and is confined between hard PS layers, it is easy to deform it under stress. A void can "nucleate" where the local hydrostatic tensile stress is sufficiently high.

Once nanovoids are formed, the rubbery phase can continue sustain load until the fibrils break. 19,48,49,52 An open question left here is: where does the nucleation of voids primarily occur? A recent simulation work demonstrates that the primary positions of voids should be at the interface between soft and hard domains, 53 which still need experimental confirmation.

Ⅴ. Void formation in the glassy domains. Because of the high modulus of the glassy domains, a high local stress (above the crazing stress of PS) is needed to form crazes in the glassy domains. 19 For small PS domain thicknesses, the local stress can be relieved by "melting" of PS layers; but crazes in the glassy domains dominate the fracture mechanism when the PS domains are thick enough. From our study, the critical thickness is in the vicinity of 100 nm.

As a final note, compared with neat PS, the asymmetric PS-PI lamellae exhibit a smaller modulus even with 3 wt% soft PI component (90 wt% hPS in this case). The relatively small modulus, we postulate, is due to the presence of voids initiated at small strains (<1%, see Figure 3) in the soft PI nanolayers deformed in tension. Such early damage gives an apparent modulus smaller than neat PS.

CONCLUSION

In

EXPERIMENTAL SECTIION

Materials. The PS 1 -(PI-b-PS 2 ) 3 miktoarm star block copolymers were synthesized by anionic polymerization, which was reported elsewhere. 38,39 The long PS 1 block has a molecular weight of ~ 80 kg/mol and the short PS 2 blocks have a molecular weight of ~ 10 kg/mol. The length of the middle PI block was adjusted to control and vary the soft/hard volume fraction. In this study, for the highly asymmetric lamellar thermoplastics, we used a miktoarm copolymer with an overall PS volume fraction of 0.67 (70 wt% PS) and hPS with a molecular weight of 81.7 kg/mol. The detailed molecular characterization is provided in supplemental information (Part A).

Sample preparation.

Polymer blends with various hPS fractions were prepared in toluene and stirred at room temperature overnight. A small amount of BHT (< 0.5%) was added to prevent the oxidation of PI blocks. The mixtures were poured on Teflon films and further dried at a fume hood for one week to evaporate the solvent. The thermal annealing was carried out at 150 o C for 48 h in a high vacuum chamber (10 -8 mbar).

Mechanical tensile testing. The annealed films were punched with a metal die to produce dog-bone shaped specimens. Each specimen had 7 mm gauge length and a cross-section of 2 mm wide by ~ 0.5 mm thick. Monotonic and step-cycle tensile tests were carried out with 5 specimens on a home-made tensile apparatus. The load cell had capacity of 40 N. The bottom clamp was stationary while the upper one was movable. The crosshead speed was maintained at 5 mm/min for all the specimens, producing an initial strain rate of 0.012 s -1 .

Transmission electron microscopy (TEM).

After the tensile testing, the broken specimens were collected and stained in osmium tetroxide aqueous solution (2 wt%) for one week. The OsO 4 selectively stains PI (due to cross-linking of the vinyl bonds with the oxygen atoms of the OsO 4 molecule), so the structures were fixed and prevented relaxation or mechanical deformation during sectioning by microtome. The stained specimens were subsequently cut on an ultra-microtome machine at room temperature to produce ultrathin (~100 nm) slices. The thin slices were collected on copper grids for TEM observation. The PI domains appear dark in the TEM images.

Real-time tensile-SAXS experiments. The synchrotron small angle x-ray scattering (SAXS) experiments were carried out using the Advanced Photon Source beamline 5-ID at Argonne National Laboratory. The wavelength of the source was 0.124 nm. The sample to detector distance was set to 7496 mm so that the lowest wave vector was 0.0155nm -1 , which is equivalent to 400 nm. The beamline was adapted to an Instron 850 tensile tester. The load cell capacity was 200 N. Both upper and bottom clamps were movable to create symmetric tensile stretching. The strain rate was kept at 0.012 s -1 for all tests.

Corresponding Authors

*E-mail: ghf@mrl.ucsb.edu (GHF); aavger@cc.uoi.gr (AA)

Notes

The authors declare no competing financial interest.

Acknowledge ments

This research was supported by the Institute for Collaborative Biotechnologies through

Figure 1 .

 1 Figure 1. Monotonic tensile testing results for miktoarm block copolymer blends with different weight fractions of hPS: (a) stress-strain curves and (b) Young's moduli.

Figure 2 .

 2 Figure 2. TEM images for the miktoarm block copolymer blended with 50 wt% hPS (a, b, c) and 80 wt% hPS (d, e, f) after stretching until break. The arrow indicates the stretching direction. Image (a) was taken at low magnification showing 5 distinct regions. Image (b) is a magnified image of (a). Image (c) highlights the void formation of a region 5. Image (d) was taken at low magnification. The arrow indicates the stretching direction. Image (e) is a magnified image of (d) highlighting

Figure 3 .

 3 Figure 3. Tensile-SAXS experiments for the miktoarm block copolymer blended with 50 wt% hPS. The 2-D SAXS patterns are shown in image (a) at different nominal strains. The arrow indicates the tensile direction. Images (b) and (c) are scattering curves in the tensile direction and transverse directions, respectively. The arrow in image (c) marks the scattering peak associated with correlation of voids. Image (d)

Figure 4 .

 4 Figure 4. The volume fraction of voids during monotonic tensile stretching for miktoarm/hPS blends at different hPS weight fractions.

  conclusion, we demonstrate that well designed PS 1 -(PI-b-PS 2 ) 3 miktoarm star block copolymers can be remarkably effective at toughening PS homopolymer, even when the blend has a very low rubber concentration (3 to 15 wt% PI). The ductility arises from highly asymmetric lamellar morphologies perfused with continuous rubbery PI sheets and represents a new paradigm for creating tough, hard, and strong material from intrinsically brittle glassy polymers. The toughening mechanism for these novel thermoplastics primarily relies on the extensive deformation of the rubbery domains and mechanical homogenization. Deformation of the rubbery layers leads to significant void formation, but the rubbery fibrils sustain significant stress until break. Mechanical homogenization of the glassy and rubbery nano-layers leads to the formation of pre-damage and damage grains, which dissipate energy and prevent the formation of crazes in the glassy domains. However, when the PS nano-layers are sufficiently thick (> 100 nm), crazing in PS domains becomes dominant and leads to catastrophic fracture of the material. The specific hard-soft asymmetric nano-layered polymer systems in this study present a powerful protocol for revealing the structure-mechanics relationship. The mechanism should be ubiquitous to direct other nanomaterials with desired mechanical properties.

  grant W911NF-09-0001 from the U.S. Army Research Office. The content of the information does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred. Extensive use was made of the MRL Shared Experimental Facilities supported by the MRSEC Program of the NSF under Award No. DMR 1121053; a member of the NSF-funded Materials Research Facilities Network (www.mrfn.org). This work was performed at the DuPont-Northwestern-Dow Collaborative Access Team (DND-CAT) located at Sector 5 of the Advanced Photon Source (APS). DND-CAT is supported by E.I. DuPont de Nemours & Co., The Dow Chemical Company and Northwestern University. Use of the APS, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357.

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