New developments in osteoarthritis. Sex differences in magnetic resonance imaging-based biomarkers and in those of joint metabolism

Sex differences in the prevalence, incidence, and severity of osteoarthritis (OA) have long been known. Some differences in the evaluation of this issue across studies may be related to differences in study design, sampling, study size, study populations, targeted joint sites, and definitions of OA. This report highlights recent studies of sex differences in individual joint components imaged by magnetic resonance imaging and in systemic biomarkers of joint metabolism. Particularly important are those studies that examine this issue in young unaffected adults and children before the development of disease. Despite some variation across studies, women appear for the most part to have a thinner and more reduced volume of cartilage in the knee than men, and this may occur from early childhood. It is not clear whether women have a more accelerated rate of cartilage volume loss than men. Few data exist on sex differences in systemic biomarkers of joint metabolism. In these studies, it is critically important to characterize the total body burden of OA and the presence of comorbid conditions likely to influence a given biomarker. Lastly, future research should dovetail studies of sex differences in imaging and biochemical biomarkers with genetics to maximize insight into the mechanisms behind observed sex differences.

OA or even the examination of normal children. With increasing attention to risk factors for multiple diseases across the life course [15,16], this approach could expose potential mechanisms behind sex diff erences in OA and identify high-risk people in advance of disease.
Reviews of the epidemiology of sex diff erences in OA have recently been published [5][6][7]17]. Th is review will instead highlight novel study designs or studies of unaff ected and younger individuals, including children, to focus on sex diff erences in (a) structural joint components using MRI and (b) biomarkers of joint metabolism.

Cartilage thickness, volume, and defects
Since men and women vary in body size, one might assume that men have greater cartilage volume. Cicuttini and colleagues [18] were among the fi rst teams to test this hypothesis in knee cartilage volume using MRI. In 17 Australian men and 11 women who had normal knee radiographs and who were having knee MRI because of knee pain of less than 3 months in duration, men had larger femoral and patellar cartilage volumes than women, independently of age, height, weight, and bone volume. Sex diff erences in patellar cartilage volume were magnifi ed with increasing age [18]. In a study of nine healthy German men and nine women in their early 20s without a history of athletic or heavy physical activity, Faber and colleagues [19] confi rmed lower cartilage volumes in women than men and showed that this sex diff erence was related primarily to diff erences in joint surface area or bone size rather than cartilage thickness, where diff erences were less pronounced and not statistically signifi cant.
Otterness and Eckstein [20] hypothesized that smaller joint surfaces in women might explain sex diff erences in knee OA because of higher articular pressures with smaller surface area. Using healthy men and women, the authors confi rmed that men have greater knee sub chondral bone area, cartilage thickness, and cartilage volume compared with women, after adjustment for height and weight [20]. Estimated tibial or patellar pressures, using the metric of body weight/joint surface area, however, were equivalent in men and women, suggesting that smaller joint surfaces in women were not a likely explanation for sex diff erences in knee OA [20].
Th ese authors found that total subchondral bone area and cartilage volume were strongly associated in young healthy men and women. However, while cartilage volume and bone area were strongly related to height in women, their associations with height in men were weak and inconsistent, leading the authors to suggest the possibility that diff erent factors are responsible for bone and cartilage growth in men and women [21].
Longitudinally, Australian women have been shown to have a higher rate of cartilage loss than men [22,23], whereas men may have a higher rate of cartilage loss than women in studies in the US [24,25]. In 135 Australian men and 190 women from 26 to 61 years old (mean age of 45 years), Ding and colleagues [22] reported that, over an average of 2.3 years, women had a higher annual rate of cartilage volume loss than men in all knee compartments, although only tibial cartilage loss was statistically signifi cantly diff erent by sex. Th ese sex diff erences fi rst appeared at age 40 and increased with age [22]. Importantly, there were no signifi cant sex diff erences in the crude annual percentage change or in the annual percentage change adjusted for age, BMI, and off spring/control status in cartilage volume in any plates; sex diff erences were evident only after further adjustment for baseline cartilage volume and bone size, and this could have infl ated the diff erence. Th e composition of this con venience sample was intriguing; the sample consisted of off spring of people who had undergone knee arthroplasty for knee OA and the rest were from the general popu lation. Interestingly, the magnitude of cartilage loss was higher in off spring than the general population, suggest ing a high risk for the development of cartilage loss and presumably, later, for the development of knee OA [22]. Women were also three times more likely than men to have increases in tibial cartilage defects over time [22,23].
A diff erent result was obtained from the Osteoarthritis Initiative (OAI) [24,25], an ongoing multi-center study in which a 3-Tesla MRI of the knee is obtained annually in approximately 4,800 individuals from 45 to 79 years old at baseline either with symptomatic radiographic knee OA (progression cohort) or with risk factors to develop knee OA (incidence cohort) [26]. An early study of the progression subcohort evaluated individuals (79 women and 77 men, mean age of 61 years) with frequent knee symptoms and radiographic knee OA in at least one knee. After 1 year, modest cartilage thick ness loss occurred, more in the medial compartment than in the lateral, more in the medial femur than in the medial tibia, and more in the lateral tibia than in the lateral femur. Th ere were no statistically signifi cant diff er ences in the rate of change of cartilage volume or thick ness by age, sex, BMI, frequent symptoms, or radio graphic Kellgren-Lawrence grade [24]. In the OAI, in contrast to the Australian studies, there was a non-statistically signifi cant trend for men to have a greater rate of change in cartilage volume and thickness than women. Th e authors conceded that statistical power was limited, the period of observation was short, and only one knee (which may not have been the symptomatic knee) for imaging with Coronal FLASHwe (fast low angle shot with water excitation) was studied [24,25]. When only some plates demonstrate diff erences, it is unclear whether this illuminates potential mechanisms, perhaps biomechanical, or represents a chance occurrence. None of these studies accounted for multiple comparisons inherent in the analysis of detailed MRI data, and it remains to be seen whether diff erences will be replicated in larger samples in which such multiple comparison testing is considered.

Hormonal associations with cartilage metrics by magnetic resonance imaging
Sex hormones and HRT after menopause have received considerable attention in the assessment of radiographic knee and hip OA, symptomatic OA, and joint replacement but with confl icting results [8][9][10][11][27][28][29]. Using MRI outcomes, Wluka and colleagues [30] reported that healthy women who had no knee pain and who were taking HRT had greater knee cartilage volume than women not on HRT, suggesting a chondroprotective role for HRT. Such promising cross-sectional results were not borne out on longitudinal assessment [31]. Serum testosterone levels in such healthy women were not associated with cartilage thickness, cartilage defects, bone surface area, or large bone marrow lesions [32].
In healthy men without knee pain, on the other hand, cartilage volume was directly related to serum testosterone levels, but testosterone was not associated with change in cartilage volume [33]. Th e authors of these studies acknowledged that serum measures of androgens may not accurately refl ect levels and activity at the site of interest, and so the implication of these fi ndings is unclear [32,33].
Leptin, a 16-kDa non-glycosylated hormone encoded by the obese gene and secreted by adipocytes, osteoblasts, and chondrocytes, has received some attention in OA and may be related to sex diff erences in OA [34]. Leptin is elevated in OA cartilage and osteophytes [35,36], and higher concentrations have been found in synovial fl uid in OA [37]. Women and those with higher BMI have higher leptin levels as well [38]. Ding and colleagues [39] evaluated cross-sectional associations between serum leptin levels and cartilage volume and carti lage defects in a subsample (n = 190, 48% were women, and mean age was 63 years) of the Tasmanian Older Adult Cohort, a population-based cohort of incidence and progression of OA and osteoporosis. In multi-variable analyses controlling for sex, age, BMI, smoking, radiographic knee OA, bone size, and other diseases (rheumatoid arthritis, cardiovascular disease, asthma, and diabetes), log-transformed leptin levels were signifi cantly associated with knee cartilage volume but not with cartilage defects. Importantly, for the purposes of this review on sex diff erences in cartilage volume, leptin levels partially mediated the relationship between sex and cartilage volume, with a decrease in the R 2 of the multi-variable model from 51% to 30% with additional adjustment for leptin levels [39]. Leptin did not mediate sex associations with cartilage defects. Th ese studies suggest that leptin may mediate some sex diff erences in OA.

Imaging of cartilage in children
In keeping with the premise that sex diff erences in cartilage may be intrinsic or present many years in advance of OA onset, Jones and colleagues [40] performed a cross-sectional study of knee cartilage thickness and volume and bone surface area in 49 boys and 43 girls from 9 to 18 years old. One might expect boys to have larger cartilage volume than girls, and the authors controlled for multiple factors -such as age, BMI, bone area, number and type of sports participated in, vigorous physical activity, and lower limb muscle strength -that could infl uence these relationships. After adjustment, boys had greater cartilage thickness and volume than girls in all Tanner stages, with sex accounting for 20% of patellar volume, 26% of medial tibial volume, and 8% of lateral tibial cartilage volume. Th ere was no diff erence in cartilage volume between pre-and post-menarchical girls.
Although many factors are related to the sex diff erence in cartilage parameters, these results imply that sex diff erences exist from early stages in the life course and that OA is likely determined or at least infl uenced by events in early life, even in the absence of joint injury. Th is principle is consistent with murine mesenchymal stem cell studies in which cells from male animals produced a 'richer extra-cellular matrix' [41] and larger culture pellet than cells from female animals. Furthermore, the regenerative potential of male cells was superior to that of female cells, with male cells providing better cartilage repair in nude mice than female cells did [41]. Koelling and Miosge [42] recently described sex diff erences in chondrogenic pro geni tor cells in cartilage from men and women under going knee joint replacement for OA. Th e authors observed that gene expression patterns diff ered by sex for ESR-1 and -2 genes, the transcription factor Sox9, and types I and II collagen [42]. Th ere were also sex diff er ences in the eff ect of sex hormones upon collagen II gene expression and in regulatory eff ects independently of Sox9 and Runx2. Th ese studies showed that sex diff er ences in cartilage occur from early development and persist through end-stage OA, suggesting that therapies might need to be tailored to men and women.

Meniscus and ligaments
Girls and women have diff erent biomechanics, gait, and structural and morphometric properties of tendons and ligaments than boys and men [43][44][45][46][47]. It is likely that these intrinsic diff erences contribute to the increased frequency of ligamentous injury in young female athletes [43][44][45][46][47]. Whether these issues potentially contribute to the sex disparity in OA in later life is unclear, and surprisingly little attention has been devoted to sex diff erences in these structures using MRI in adults [48]. Fayad and colleagues [48] described diff erences in anterior cruciate ligament bundle volumes in 33 men and 30 women (mean age of 43, range of 15 to 70 years) referred for clinical MRI. Although there was a sex diff erence in anterior cruciate ligament volume, this was explained entirely by diff erences in height [48].
In a study that was mostly of asymptomatic individuals in Australia, women were over four times more likely than men to have meniscal tears by 1.5-Tesla MRI [49]. In contrast, 32% of the men and 19% of the women in the Framingham cohort (n = 991, 57% were women, and mean age was 62.3 years) had meniscal tears or destruction by 1.5-Tesla MRI, with the prevalence in both sexes increasing with age [50]. In a study of women who were older than 40 years of age, had knee OA, and had been screened into a clinical trial for OA, meniscal tears were present in 73%, and not surprisingly, these were associated with signifi cant impairment in walking endurance and balance after OA duration, symp toms, disability, body composition, and relevant clinical characteristics were controlled for [51].
Finally, Stehling and colleagues [52] recently reported associations between various knee lesions on 3-Tesla MRI and physical activity in 236 individuals from 45 to 55 years old in the asymptomatic incidence subcohort of the OAI. Although assessment of sex diff erences in MRI features was not the purpose of the paper, ligamentous abnormalities were more likely in men than women (23% versus 12.5%) and meniscal lesions were more common and more likely to be severe in men (54% versus 42%). In contrast, cartilage abnormalities were slightly more common in women (76.5% versus 72%) but full-thickness cartilage defects were more common in men (24% versus 14.7%). Th e prevalence of some features was higher than in previous studies, especially since these were asymptomatic individuals, and this is likely related to increased sensitivity from the stronger magnetic fi eld used in this study.
Th ese few studies show widely disparate results, likely because of diff erent study designs, statistical power, MRI protocols, and study populations varying by geographic location, age distribution, clinical characteristics, and source of participants. Th ese factors make interpretation impossible and mandate further research to determine whether sex diff erences exist in these parameters and to understand the mechanisms behind such diff erences.

Bone marrow lesions and bone cysts
In OA, bone marrow lesions are common and are associated with knee OA progression and pain [53][54][55]. In the fi rst description of these in healthy men and women free of knee pain, Davies-Tuck and colleagues [56] reported that sex was not associated with the presence, development, or persistence of bone marrow lesions on knee 1.5-Tesla MRI over 2 years in the Melbourne Collaborative Cohort Study. Studies evaluating the coexistence of these lesions with cartilage loss, meniscal abnormalities, and bone cysts have for the most part not focused on sex diff erences in these relationships [54]. Tanamas and colleagues [57] reported that bone cysts were more common in men than women in a study of the relationship between bone cysts and subsequent knee replacement 4 years later, but further investigation into the role of sex diff erences in these lesions was not conducted.

Sex diff erences in biomarkers of joint metabolism
Another way to understand the etiopathogenesis of sex diff erences in OA is to examine factors representative of joint metabolism. In order for synovial and systemically measured biomarkers to be used to identify high-risk individuals before OA occurs or before it becomes clinically manifest, normative data in various populations, including subgroups by sex, are required. For some markers, such as type I collagen N-telopeptide (NTX-I) and osteocalcin (which are markers of bone resorption and synthesis, respectively), much is known about sex diff erences and (within women) the eff ects of menopause and HRT, but for other markers, much less is known. Reports using markers for OA frequently control for sex but do not describe sex diff erences specifi cally [58,59]. Critically important is knowing what other factors -such as the body burden of OA, BMI, hormonal status, or other medical conditions -might confound a sex diff erence in a specifi c marker, especially when considering a biomarker that is ubiquitous in connective tissue, such as hyaluronan (HA) [60]. Th is report will examine sex diff erences in several of the more frequently used markers in OA, targeting presumably diff erent processes.

Type II collagen degradation
Mouritzen and colleagues [61] described a marker of type II collagen turnover, cartilage-derived urinary collagen type II C-telopeptide degradation products (CTX-II), in 615 healthy men and women from 20 to 87 years old. Levels were similar in men and women from 30 to 45 years old and then increased in both men and women, with the levels of women being slightly higher than those of men (Figure 1 from [61]). Levels were also higher in post-menopausal women compared with premenopausal women; and in post-menopausal women, those taking HRT had lower levels than those not taking HRT. Furthermore, those taking HRT for a longer time had lower levels than those taking HRT for a shorter duration [61].
Kojima and colleagues [62] described serum levels of C2C, a marker of intra-helical type II collagen cleavage, in 69 Japanese men (mean age of 43 years) and 71 Japanese women (mean age of 44 years; 34% of the women were post-menopausal) who did not have joint or spinal pain or major medical conditions and who were not taking medications aff ecting bone metabolism. In individuals younger than 50 years, C2C was higher in women than men, and the reverse was the case in those older than 50 years. C2C levels were unrelated to menopausal status. Since CTX-II and C2C are both markers of type II collagen cleavage, why would results be diff erent for each marker? Th e authors propose that diff erences might be because the markers are the product of diff erent areas in the type II collagen molecule that get degraded, and the markers' diff erent locations in the joint [62].

Matrix protein degradation
Cartilage oligomeric matrix protein (COMP) is a 64-kDa pentameric matrix protein found in most joint tissues, including cartilage, bone, tendon, ligament, synovium, and vascular smooth muscle. It is elevated in OA [63][64][65][66][67], predicts incidence of radiographic hip OA [65,66], and is higher with increasing body burden of OA-aff ected large joints [63,64,67]. Clark and colleagues [63] described the fi rst and largest population-based assessment of serum COMP using competitive enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody 17-C10 in Caucasians in the Johnston County Osteoarthritis Project (JoCo OA). COMP increased with age and was higher in OA than controls but did not signifi cantly vary by sex. Later, in the same study population, Jordan and colleagues [64] used a sandwich ELISA with monoclonal antibodies 16-F12 and 17-C10 and reported that serum COMP levels among Caucasians but not African-Americans were higher in men than women (Figure 1, page 679 from [64]). Serum COMP levels were associated with a 30% increased risk of hip OA development in older Caucasian women in the Study of Osteoporotic Fractures [65]; those in the highest three quartiles of change in this marker had a fi vefold increase in the risk of incident hip OA compared with those in the lowest quartile of change [66]. No direct comparison of these results to men was possible in this study. No further specifi c evaluations of sex diff erences in COMP in relation to OA have been conducted.

Synovial infl ammation
HA is a ubiquitous glycosaminoglycan formed from alternating units of glucosamine and glucuronic acid. It is a constituent of synovium and cartilage and is indicative of synovial infl ammation and has been shown to be an important marker of systemic burden of OA in women [60]. Elliott and colleagues [60] reported that men had higher serum HA levels than women did in the JoCo OA; importantly, this eff ect was independent of diff erences in age, race/ethnicity, OA burden in knees or hips, BMI, or comorbidities. Th is was confi rmed in a recent study of mitochondrial DNA haplogroups and their eff ect upon serum levels of multiple biomarkers in Spanish patients with knee and hip OA [68].

Systemic and synovial cytokines
Pagura and colleagues [69] examined systemic and synovial measures of cytokines (interleukin [IL]-1-alpha/ beta, tumor necrosis factor-alpha, and IL-6) and growth factors (insulin-like growth factor-1 [IGF-1], transform ing growth factor-beta [TGF-β], and interleukin 1 receptor antagonist [IRAP]) in a small study of 9 Canadian men and 8 women awaiting knee replacement and compared them with 21 age-and sex-matched controls recruited from the local community. Men had higher levels of serum and synovial IGF-1, but there were no sex diff erences in any of the other markers. However, the very small sample size and undetectable levels of cytokines, except for IL-6, render the impact of this study questionable [69].

Growth factor
Lastly, in the largest study of serum TGF-β to date, Nelson and colleagues [70] reported that this marker was higher in women than men in the JoCo OA and that the associations between this marker and prevalent radiographic knee and hip OA, osteophytes, and joint space narrowing were similar in men and women. A single measure of this marker was unable to predict incidence or progression of radiographic knee or hip OA, osteophytes, or joint space narrowing in either men or women [71].

Summary and suggestions for future research
Sex diff erences in prevalence, incidence, and severity of radiographic and clinical OA have been described, but specifi c examination of sex diff erences in MRI biomarkers and in biomarkers of joint metabolism are few, and results vary considerably. Despite some variation across study designs and study populations, women appear to have a thinner and more reduced volume of cartilage in the knee than men, and this may occur from early childhood. Th e relationship between cartilage volume and bone area cannot be ignored in analyses of these issues. Whether women have a more accelerated rate of cartilage volume loss than men remains unsettled. Few data exist on sex diff erences in other tissues of the knee by MRI and in systemic biomarkers of joint metabolism, and those that do exist frequently vary in the assessment of potential mediators of sex diff erences.
Most studies of OA have been limited to Caucasians. Future studies should examine these relationships in other race/ethnic groups and, perhaps more importantly, delve into aspects that are likely to shed light upon mechanisms behind sex diff erences. In particular, studies of imaging and biochemical biomarkers, rather than merely control for sex, should specifi cally examine whether sex diff erences exist in that biomarker. Additionally, studies should establish whether risk factors act similarly or diff erently in men and women, with an eye to deter mining whether sex-specifi c therapies make sense. Studies of mesenchymal stem cells, outlined above [41], suggest that this may not be such a far-fetched idea.
Another question deserving attention is whether height, weight, and bone area can adequately serve as proxies for body size, a crucial issue in understanding sex diff erences in imaging biomarkers. Future studies might evaluate other potential proxies, such as height × weight and others [19], in relation to sex diff erences in joint structures by MRI. Finally, sex diff erences in the genetics of OA have been noted for multiple genes and joint sites [72][73][74], and studies of sex diff erences in these genetic eff ects should dovetail with those using advanced imaging and biochemical biomarkers for maximal mechanistic insight.