To assess longitudinal changes, the values were calculated for each person and expressed as the percentage of the initial baseline value. The data of the creatinine-corrected values of CTXI and CTXII, and the relative changes were logarithmically transformed to obtain normality and symmetry of variances. The trapezoidal method was applied for calculation of the time-averaged mean change from baseline. Analysis of variance (ANOVA) was used for comparison of baseline data between treatment groups. The two-tailed Student's t-test was applied for pairwise comparison of data from the active treatment groups with placebo.

For all tests p < 0.05 was considered significant. All statistical calculations were performed using the SAS software package (release 9.1, SAS Institute Inc., Cary, NC, USA).

Bone resorption was investigated by measurement of urinary CTX-I during the 2 years of therapy every 3^rd month in the groups receiving 2.5 mg or 1.25 mg of tibolone, or placebo. Tibolone inhibited bone resorption highly significant with a plateau after 6, as seen in figure 1, upper panel. The relative change from baseline in urinary CTX-I during 2 years of therapy with 1.25 and 2.5 mg of tibolone is presented in figure 1, lower panel. Values shown are placebo-corrected time-averaged mean change during the treatment period and given as mean ± 1 SEM. The level of significance denotes difference from the placebo group: *** p < 0.001. Data are modified with permission from 21.

Cartilage degradation was measured by measurement of urinary CTX-II during the 2 years of therapy every 3^rd month in the groups receiving 2.5 mg or 1.25 mg of tibolone, or placebo. Tibolone did not affect cartilage degradation, as presented in figure 2, upper panel. The relative change from baseline in urinary CTXII during 2 years of therapy with 1.25 and 2.5 mg of tibolone is presented in figure 2, lower panel. Values shown are placebo-corrected time-averaged mean change during the treatment period and given as mean ± 1 SEM.

An increasing amount of attention has been devoted to the role of the bone in the pathogenesis of osteoarthritis. Subchondral bone sclerosis, alterations in the trabecular structure, lesions (previously called development of bone marrow edema) and osteophytes are important features of the pathology of OA 21029373839. The present understanding remains on an observational level, in which an increasing range of experimental evidence is emerging. In support of the important role of bone turnover in the pathogenesis of OA, several independent lines of experimental evidence are found 329303132. In brief, examinations of peri-articular bone in knees and hips with OA have confirmed that the subchondral bone is abnormal in OA joints, which altered trabecular structure, sclerosis of the subchondral plate 40, as well increased bone turnover 4142. Cross-sectional studies have also established that women with advanced knee or hip OA have a higher bone mineral density (BMD) near or at the site of joint OA 43. Plausible proof of a link between bone and cartilage recently came from an animal model of OA, where extensive inhibition of bone resorption resulted in a 50% decrease in cartilage pathology score assessed by Mankin score 2938. In addition, accelerated bone turnover has in both traumatic and estrogen deficiency models (ovariectomy (OVX)) been shown to augment articular cartilage erosion 711124445, in which increased bone resorption alone results in increased articular cartilage damage 103746.

Thereby, interventions that may positively affect bone turnover, may in theory have secondary positive effects on progression of OA. The present data strongly suggest that these processes can be uncoupled. This might in part be mediated through the androgenic properties of tibolone, as androgens in contrast to that of estrogens might have deleterious effects on cartilage health 45. However the effects of androgens and the direct on articular cartilage compared to that of estrogens needs additional attention.

The putative positive of some estrogens of cartilage health may both be though indirect and direct effects on cartilage. Estrogens are anti-resorptives that directly and indirectly attenuates osteoclastogenesis and osteoclastic resorption 47. At the same time, chondrocytes express estrogen receptors, and respond to estrogen 114849505152.

The positive long-term beneficial effect of estrogen for prevention of OA was recently demonstrated in 180 female cynomolgus monkeys 10, receiving estrogen replacement therapy (ERT) treatment for 3 years. Significant less cartilage lesions of OA were seen in the ERT group compared to the control group. In addition, several preclinical models have provided evidence for protective effects of estrogen 111246.

In clinical settings a number of studies have suggested positive effects on cartilage degradation. A SERM was shown to protect against both bone and cartilage degradation 53, and cartilage degradation and was found to be significantly lower in women using hormone replacement therapy (HRT) compared to control 61254, and the cartilage degradation was significantly higher in postmenopausal women when compared to an age-matched group of pre-menopausal women. These data are in alignment with the recent analysis from the WHI studies, which documented that women taking estrogen had 45% less total joint surgery compared to of placebo 55.

However in the present study Tibolone did not result in secondary positive effects on cartilage degradation. This may in part be described by the complicated pharmacology of Tibolone on testosterone, estrogen and progesterone receptors. This is in contrast to others SERM and estrogen like molecules that to a much larger extent is selective for the estrogen receptor.

The present study has some limitations. Cartilage degradation was evaluated by the biochemical markers u-CTX-II. Levels of u-CTX-II have been shown to correlate with cartilage damage in both animal models and clinical trials 7112756, however long term randomized clinical trials studies are needed to further investigate and document the putative positive effects of estrogens on the pathogenesis of osteoarthritis.

Biochemical markers of cartilage degradation measured in the systemic fluids, serum and urine, are the net results of the biological activity of all joints and tissues in which collagen type II present. CTX-II is generated by MMP activity 57 have been shown to be produced by catabolically stimulated articular cartilage, and present in damaged articular cartilage 2556. However, CTX-II may in addition be generated by the cartilage of non-synovial joints. The main contributors of cartilage degradation biomarkers have been shown to be; knees, hips, hands, vertebral facet joints in addition to spinal disc degeneration (DD) 5859. In addition, a smaller contribution of CTX-II may originate from the calcified cartilage in the subchondral bone area 28. The contribution of each cartilage compartment to the total pool of cartilage degradation measured is important to further understand, for the interpretation of the effect on the total pool of any biochemical marker. Some insights into the relative contribution of the different joints to the total amount of CTX-II have been provided 58. CTX-II was shown to be related to the number of joint affected, evaluated by radiological OA, in which generalised resulted in an approximately 100% increase in CTX-II 58. In addition, CTX-II was very recently shown to predict medial knee articular cartilage loss evaluated by quantitative MRI 60. With respect to the current study, each compartment may have contributed differently to the pool of CTX-II, also in response to therapy. Further research is needed to understand the effects of estrogen and estrogen related compounds on the individual joints.

The analysis was performed by re-analysis on stored samples, which to some extend may have influenced that measurements. However, in house data suggest that u-CTX-II is stable for more than 3 years under the appropriate conditions.