Oestrogen is important for maintenance of cartilage and subchondral bone in a murine model of knee osteoarthritis

Osteoarthritis (OA) of the knee is a common, disabling and expensive disease [1, 2]. The knee is a complex joint consisting of three compartments: medial and lateral tibiofemoral joint (TFJ) compartments and the patellofemoral joint (PFJ). Although clinical studies of osteoarthritis of the knee have tended to focus on the tibiofemoral compartment alone, the patellofemoral compartment is often affected as well. An investigation in people with knee pain revealed that the most common radiographic pattern is combined TFJ and PFJ disease (40%), followed by isolated PFJ OA (24%) [3]. Isolated TFJ OA occurred in only 4% of subjects [3]. The patellofemoral joint is an important source of symptoms associated with knee OA, such as pain, stiffness and disability [4].

It has been suggested in the literature that oestrogen depletion plays a role in the onset or progression of OA. Men are known to have a higher prevalence of OA than women before the age of 50 [5], but after this age the prevalence is higher in women [6, 7]. The prevalence increases with age in both men and women, but in women, it increases dramatically around the age of 50 [5, 8, 9], which coincides with menopause.

Also, in animal models, a link between oestrogen and OA has been found. A number of animal studies have been performed to investigate the effect of oestrogen depletion and oestrogen replacement on articular cartilage (reviewed in [10]). In several animal models, ovariectomy leads to osteoarthritic changes, and oestrogen replacement therapy reduces cartilage degradation [11‐13]. Oestrogen acts via oestrogen receptors (ERs), which have been found in articular cartilage, bone, synovial tissue and ligaments [14‐17], which may all be involved in OA. Several studies have reported associations between OA and polymorphisms in ERα and ERβ [18‐20]. Also, low serum estradiol levels have been found to be associated with OA [21]. Taken together, these findings argue for a role of oestrogen in the development of osteoarthritis. However, patella OA has not yet been included in studies on oestrogen and OA.

The exact mechanism by which oestrogen affects OA is not known. Apart from a direct effect of oestrogen on cartilage, bone may also be involved. Oestrogen is known to affect bone metabolism and to regulate the balance between bone formation and resorption [22]. Subchondral bone changes have been reported in OA patients [23‐25] and in animal models for OA [26‐28]. It has been suggested that subchondral bone changes are important in the aetiology of OA [29]. Alteration in subchondral bone remodelling, and subsequently in bone structure, may lead to changes in load distribution. This may in turn cause or accelerate cartilage damage. Therefore, bone changes induced by oestrogen depletion may play a role in OA development.

Although the prevalence increases after the age of 50, not all postmenopausal women get osteoarthritis [30], indicating that hormonal changes alone are not enough to cause OA. We hypothesize that oestrogen depletion increases the susceptibility of tissues in the joint for changes, but an additional trigger is needed to develop osteoarthritic changes. This concurs with the idea that OA is a multifactorial disease. We addressed this hypothesis by investigating bone and cartilage changes in the proximal tibia and patella of knee joints of ovariectomized mice and ovariectomized mice receiving oestrogen replacement, combined with a mild osteoarthritis trigger induced by iodoacetate, an inhibitor of glycolysis that is an accepted model for osteoarthritis [31‐34]. The impact of bone changes was investigated by using bisphosphonates to inhibit bone resorption after ovariectomy and iodoacetate.

The current study demonstrates that loss of oestrogen increases cartilage damage in the patella and increases susceptibility for cartilage damage in the tibia and subchondral bone loss at both sites, while oestrogen supplementation appears to dampen these effects. This observation corroborates clinical and epidemiological observations of increased OA incidence after menopause [5, 8, 9]. It also illustrates the interplay between hormonal changes and external triggers in the aetiology of osteoarthritis.

This study was designed to investigate the impact of oestrogen loss on articular cartilage and subchondral bone and the role of oestrogen in susceptibility of the patella and tibia for osteoarthritis, by combining a mild osteoarthritis trigger with oestrogen depletion. IA has been widely used in animal models of OA [31‐34]. In both patella and tibia, injection of 6 μl of 0.5% IA did not cause a significant increase in cartilage damage. However, OVX together with IA injection led to the severest cartilage damage in the patella, indicating an increased susceptibility. At the patella, OVX alone also increased cartilage damage and supplementation with estradiol after OVX abolished this effect. Our findings are in line with previous studies that reported more cartilage damage in the tibiofemoral compartments of the knee after OVX alone [11, 13, 44] or after OVX in combination with OA induction [13, 45, 46].

In contrast to our patella data and data from the literature, in our study, tibial cartilage damage was not increased after OVX alone. However, when OVX was combined with IA injection, tibial cartilage damage was more severe than after either OVX or IA injection alone. This is reflected in the significant interaction between OVX and IA injection and further supports the hypothesis of increased susceptibility for osteoarthritis after oestrogen loss. Our data are supported by previous studies using more severe methods of OA induction in combination with OVX [13, 45]. However, the effect of oestrogen supplementation was not investigated in these studies [13, 45].

Subchondral cortical thickness in both the patella and tibia plateaus only decreased in the OVX situation when the osteoarthritis trigger had been applied. The effects of OVX and IA treatment were additive, resulting in a significantly decreased subchondral cortical thickness. This provides further support for a role of estradiol in osteoarthritis and adds to the concept of the involvement of bone, specifically the subchondral bone plate, in osteoarthritis. It remains unknown whether these changes are directly caused by the osteoarthritis process triggered by IA or are caused by unloading of the joint (due to pain); it has been suggested that oestrogen deficiency may exacerbate pain perception [47]. However, cortical bone in the metaphysis was not affected, suggesting that changes in subchondral cortical bone of the tibia are not caused by unloading, but are an intrinsic part of the osteoarthritic pathological process.

The decrease in subchondral plate thickness is in line with findings from previous OA animal studies evaluating the early disease stage [26, 27, 48‐50]. In some of these studies, this was followed by plate thickening [26, 49]. This also explains the discrepancy with sclerosis described in human studies [51‐53], which often concern patients with late osteoarthritis, whereas our present study reflects an early phase of osteoarthritis. Comparison of patella and medial and lateral tibia demonstrated an as yet unexplained site specificity. At the patella and tibial medial side after 3 weeks of OVX-IA, the subchondral plate showed a progressive restoration of thickness, while at the lateral tibia side a progressive thinning was observed (compare OVX+IA in Figures 3a, c and 3e).

Loss of estradiol did not cause changes in patellar trabecular bone, and compared to the metaphysis, caused only mild changes in epiphyseal trabecular bone. However, at all three sites, supplementation with estradiol strongly increased bone volume fraction, suggesting that these sites are sensitive but that patellar and epiphyseal bone are less dependent on circulating estradiol than metaphyseal bone. This may be due to differences in oestrogen receptor levels or in local estradiol production. These as yet unknown differences between epiphyseal and patellar bone on one side and metaphyseal bone on the other side could be important for understanding the functional link between estradiol loss, bone turnover and development of osteoarthritis.

To further investigate the relationship of bone changes with cartilage damage, we studied bone and cartilage changes in OVX mice in the absence or presence of BP. In this way, we attempted to assess the impact of OVX on the IA effect on cartilage while the changes in bone would be blocked. In the tibia, treatment with BP prevented subchondral plate thinning after IA injection and increased bone volume fraction in both saline-injected and IA-injected epiphyses. Both medial and lateral cartilage damage appeared to be less when OVX-IA mice were treated with BP. In the patella, the effect of BP treatment was different. In OVX+BP+Sal patellae, no bone changes occurred, while in OVX+BP+IA patellae, subchondral cortical thickness and bone volume fraction were strongly increased (Figures 7a and 7c). Because this occurred only in IA-injected knees, IA may have activated bone turnover (increased activity of both osteoblasts and osteoclasts), of which BP only reduced osteoclastic resorption, resulting in a net increase. In contrast, estradiol is also directly bone anabolic, explaining the increase in bone volume in both OVX+E+Sal and OVX+E+IA groups (compare with Figures 5b, d and 5f). Interestingly, cartilage damage was less in both BP groups (OVX+BP+Sal and OVX+BP+IA), which is in line with reports suggesting that BPs may have an OA protective effect [54‐56]. Since the decrease in patellar cartilage damage occurred independently of the observed bone changes in both BP groups, BP may have a direct effect on cartilage. This precludes drawing definitive conclusions on the relation between bone changes and cartilage changes.

It has been suggested in the literature that patella OA is distinct from tibiofemoral OA [4]. When comparing patellar changes to tibial changes, we notice several differences between these compartments. First, cartilage damage is more severe in the patella than in the tibia, which is in line with previous reports [57, 58]. Second, oestrogen depletion increases cartilage damage in the patella, but increases the susceptibility for cartilage damage in the tibia. Third, subchondral cortical bone became thinner in the first 3 weeks in both areas (patella and tibia) after OVX+IA, but in the tibia the thickness continued to be decreased, while in the patella the thickness subsequently increased to values similar to Sham patellae. This may reflect different dynamics or a different mechanism, which is supported by differences in biochemical and mechanical properties of articular cartilage of patella and tibia [59‐61] and by differences in biomechanics between the patellofemoral and tibiofemoral joint [4].

These data demonstrate the significance of oestrogen for articular cartilage and subchondral bone and the maintenance of healthy joints. Depending on the site in the joint, oestrogen depletion may directly increase cartilage damage and subchondral bone loss or increase susceptibility for an additional trigger. The current data support an etiological role for altered oestrogen signalling in osteoarthritis and thereby substantiates the link between estradiol and the development of osteoarthritis. The current data strongly add to the concept of the involvement of bone, specifically the subchondral bone plate, in osteoarthritis.