Bone turnover and bone collagen maturation in osteoporosis: effects of antiresorptive therapies
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- Byrjalsen, I., Leeming, D.J., Qvist, P. et al. Osteoporos Int (2008) 19: 339. doi:10.1007/s00198-007-0462-5
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Bone collagen maturation may be important for anti-fracture efficacy as the reduction in risk is only partly explained by a concomitant increase in BMD during anti-resorptive therapy. Different treatments caused diverse profiles in bone collagen degradation products, which may have implications for bone quality.
The aim of the present study was to evaluate the effect of different anti-resorptive treatments on bone collagen maturation measured as the ratio between the degradation products of newly synthesized and mature isomerized C-telopeptides of type I collagen.
Participants were from cohorts of healthy postmenopausal women participating in double blind, placebo-controlled 2-year studies of alendronate, ibandronate, intranasal hormone replacement therapy (HRT), oral HRT, transdermal HRT, or raloxifene (n = 427). The non-isomerized ααCTX and isomerized ββCTX were measured in urine samples obtained at baseline, and after 6, 12, and 24 months of therapy.
Bone collagen maturation measured as the ratio between ααCTX and ββCTX showed that bisphosphonate treatment induced a collagen profile consistent with an older matrix with a 52% (alendronate) and 38% (ibandronate) reduction in the ratio between the two CTX isoforms vs. 3% and 15% with HRT or raloxifene, respectively.
Anti-resorptive treatments had different effects on the endogenous profile of bone collagen maturation. Whether that effect on bone collagen has an impact on bone strength independent on the treatment-dependent effect on BMD should be investigated.
KeywordsBone qualityBone turnoverCollagenFracture riskMatrix proteins
Bone is a dynamic tissue being continuously remodeled throughout life not only to maintain the calcium homeostasis but also to maintain mechanical strength by replacing fatigued bone by new, mechanically sound bone .
Bone formation and resorption are two highly coupled metabolic processes [2–6], although the balance between resorption and formation differs in different phases of life. The loss of ovarian sex steroids at menopause results in accelerated bone turnover with a predominance of bone resorption over bone formation . The related negative calcium balance promotes bone loss, increases bone fragility, and thereby the risk of future fractures . A rational approach to counter this altered balance is the inhibition of bone resorption, although also bone formation is inhibited due to coupling between these cellular events [9–11]. Accordingly, pronounced inhibition of osteoclast function can be expected to impair the dynamic renewal of skeletal tissue leading to alterations in the biochemical composition of bone and changed mechanical properties . The inorganic phase of the bone provides the stiffness, i.e., the ability to resist compression, whereas the organic phase, mainly constituted of type I collagen, provides bone its flexibility, i.e., the ability to absorb energy and undergo deformation. How biochemical changes in the bone collagen are associated with fracture and bone quality remains to be investigated and understood.
The bone collagen is continuously renewed and post-translationally modified, including enzymatic cross-linking (pyridinoline and deoxypyridinoline), non-enzymatic glycation (pentosidine), and β-isomerization occurring in the DG motif of the CTX epitope (1207EKAHDGGR1214). It has been shown that the degree of isomerization reflects the skeletal age [13, 14]. Studies on in vitro aging of fetal bovine cortical bone recently demonstrated that the bone mechanical properties changed as a function of time, as did the amount of cross-linking and pentosidine, as well as the degree of β-isomerization . The biochemical changes of collagen were associated with a 30% decrease in bending and compressive yield stress and a 2.5-fold increase in compressive post-yield energy absorption. In another recent study of human lumbar vertebrae the amount of pentosidine and degree of β-isomerization was related to the biomechanical properties of bone after adjustment for BMD .
BMD is considered the most important determinant of fracture risk with the World Health Organization (WHO) criteria of osteoporosis defined as BMD-measurements falling 2.5 standard deviation below the average of young adults (T-score ≤−2.5). However, recent studies have shown that up to one-half of patients with incident fractures have BMD above the WHO diagnostic threshold criteria [17–19], and it has also been reported that the risk of an osteoporotic fracture is approximately tenfold higher in old compared with young individuals at the same BMD level . Additionally, recent data demonstrate that during anti-resorptive therapy the reduction in fracture risk is only vaguely explained by the concomitant increase in BMD . Even though drugs have become more and more effective in terms of increasing BMD, achievements in terms of anti-fracture efficacy have not followed correspondingly, e.g., the mean increase in spinal BMD to alendronate is sevenfold higher compared to that of calcitonin, yet the reductions in vertebral fracture risk are fairly comparable being 44% and 36%, respectively [21, 22] .
The discrepancies in BMD response and fracture risk protection may be due to the different mode of action of different anti-resorptive treatments. To look into this aspect of anti-resorptive therapy, we investigated the effect on bone collagen age, measured as the ratio of ααCTX to ββCTX in urine samples during different types of anti-resorptive treatment regimens of bisphosphonates, hormone replacement therapy (HRT), and raloxifene (RLX).
Subjects and study design
All studies were conducted in accordance with Helsinki Declaration II, and approved by local ethical committees. Written informed consent was obtained for all participants.
Bisphosphonate - Alendronate
The participants were part of a double blind, placebo-controlled, randomized 3-year study on the effects of oral alendronate in the prevention of postmenopausal osteoporosis . Study participants who completed the first 2-year study period and who received daily 10 mg of alendronate (n = 14), 20 mg of alendronate (n = 13), or placebo (n = 13) were included.
Bisphosphonate – Ibandronate
The participants were part of a double blind, placebo-controlled, randomized 2-year study on the effects of oral ibandronate in the prevention of postmenopausal osteoporosis . A randomly selected subgroup of study participants receiving daily continuous oral 2.5 mg of ibandronate daily (n = 36), intermittent oral 20 mg of ibandronate every 2nd day for 24 days every 3 months (n = 36), or placebo (n = 26) was included. After the first year of therapy, the placebo group was crossed-over to receive active treatment.
HRT - intranasal estradiol
The participants were part of a double blind, placebo-controlled, randomized 2-year study on the effects of daily intranasal 17β-estradiol for prevention of bone loss in early postmenopausal women . A randomly selected subgroup of study participants receiving daily 300 μg of 17β-estradiol (n = 50), or placebo (n = 25) was included.
HRT - oral estradiol
The participants were part of a double blind, placebo-controlled, randomized 2-year study on the effects of 17β-estradiol continuously combined with drospirenone on the safety and efficacy for prevention of postmenopausal osteoporosis . A randomly selected subgroup of study participants receiving daily 1 mg of 17β-estradiol continuously combined with 1 mg or 2 mg of drospirenone (n = 49), or placebo (n = 33) was included.
HRT - transdermal estradiol
The participants were part of a double blind, placebo-controlled, randomized 2-year study on the effects of daily transdermal 17β-estradiol continuously combined with transdermal levonorgestrel on the safety and efficacy for prevention of postmenopausal osteoporosis . A randomly selected subgroup of study participants receiving daily 45 μg of 17β-estradiol continuously combined with 40 μg of levonorgestrel (n = 35), or placebo (n = 20) was included.
The participants were part of a double blind, placebo-controlled, randomized 2-year study on the effects of raloxifene on bone mineral density in postmenopausal women . A randomly selected subgroup of study participants receiving daily 60 mg of raloxifene (n = 30), or placebo (n = 47) was included.
From all studies second void fasting urine samples were obtained at baseline, after 6 months, 12 months, and 24 months of therapy.
Biochemical markers and bone mineral density
Urine samples were stored at −20°C until analysis. The urinary excretion of ααCTX was measured using the ALPHA CrossLaps ELISA , and the urinary excretion of ββCTX was measured using the Serum CrossLaps One Step ELISA (urine samples were pre-dilution 1:10 in assay buffer before measurement)(Nordic Bioscience Diagnostics, Herlev, Denmark) [14, 28]. Urinary creatinine was measured by routine chemistry method and used for calculation of creatinine-corrected marker levels. The urine samples were measured at same time in all subgroups in present study.
Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry at the spine (BMDspine), and hip (BMDhip) with Hologic QDR-1000, or QD-R2000 densitometers (Hologic, Waltham, MA, USA).
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 ααCTX and ββCTX and the relative changes were logarithmically transformed to obtain normality and symmetry of variances. Analysis of variance (ANOVA) was used for comparison of baseline data between study groups with Tukey–Kramer adjusted significance levels in the multiple comparisons. The two-tailed Student’s t-test was applied for pair-wise comparisons of data from the active treatment group and placebo within each study and for comparison of selected subgroup with non-selected subgroup within each study. Regression analysis was used to assess relation of baseline characteristics with age, and to calculate the changes in BMD during treatment.
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).
Characteristics of initial study populations at baseline and subgroup comparison
Characteristics of subgroups at baseline
n = 40
n = 98
n = 75
n = 82
n = 55
n = 77
Ratio of ααCTX to ββ CTX changes
ααCTX/ββCTX Ratio (mean and 1 SEM) at baseline and at 24 months
We have investigated if different anti-resorptive treatments cause diverse age profiles in excreted bone collagen degradation products in face of similar effects on BMD. We measured urine degradation products of collagen type I (both the non-isomerized younger ααCTX isoform and the older mature ββCTX isoform), which are released during osteoclastic resorption of bone.
The main findings were 1) treatment with bisphosphonate, HRT, or RLX was associated with a statistically highly significant suppression in bone resorption of both newly synthesized (ααCTX) and mature (ββCTX) collagen type I degradation with the most pronounced suppression observed in the bisphosphonate groups, and 2) treatment with bisphosphonate induced a lower ratio between ααCTX and ββCTX compared to that of HRT and RLX.
Bisphosphonates are potent agents that target the hydroxyapatite bone mineral surfaces in vivo at sites of active bone remodeling, inhibiting osteoclast activity . Other agents for prevention of osteoporosis include HRT and RLX with a different mode of action. HRT co-administrated with progesterone is a rational approach to counter the consequences of estrogen deficiency in postmenopausal women, whereas RLX acts as an estrogen agonist on skeletal tissue. The new generation of drugs are Zoledronic acid, a bisphosphonate, and Denosumab, which is a high affinity, fully human monoclonal antibody binding to RANKL and blocking the interaction between RANKL and RANK, mimicking the endogenous effects of osteoprotegerin [30, 31].
The bone collagen maturation should preferably be measured in the bone matrix, but the degree of isomerization of bone collagen can non-invasively be measured as the ratio between ααCTX and ββCTX in urine samples. Using the surrogate marker in urine samples, one can assess the dynamic changes in the bone collagen occurring during treatment. In the present study we found that all six anti-resorptive treatment regimens were highly efficient in reducing the level of bone resorption as measured by urinary ααCTX and ββCTX although to different levels. The most suppressed levels were found in the bisphosphonate groups of alendronate and ibandronate. This higher suppression seems to be related to the drugs rather than caused by differences in the study cohorts as the bisphosphonate treatment groups represented both the youngest and the eldest subjects among the studies. The decreases in levels of ααCTX and ββCTX mirrored each other, although ααCTX was slightly more suppressed than ββCTX. This higher reduction in ααCTX is likely the result of attenuated bone turnover caused by the anti-resorptives (2–6). This is in line with the increase in the ratio between ααCTX and ββCTX of 1.9% per year since menopause in untreated postmenopausal women reflecting the general increase in bone turnover caused by the menopause leading to a decrease in skeletal age and to the paradigm that older postmenopausal women may have on average a less mature skeleton than younger postmenopausal women. However, we have not specifically investigated the difference between pre- and postmenopausal women. In particular the bisphosphonate treatments induced a pronouncedly lower ratio between ααCTX and ββCTX with a reduction of 52% in the alendronate group and 38% in the ibandronate group compared to 3 to 15% reduction in the HRT and raloxifene groups. The treatment-dependent effect on the ratio could not be explained by the study-specific placebo-adjustments used in the calculations as the time-averaged mean used for adjustments were 116% in the alendronate, 77% for ibandronate, 117% for nasal HRT, 91% for oral HRT, 101% for transdermal HRT and raloxifene.
Several lines of evidence suggest that BMD gains only to a certain extent explain fracture reduction. In a study on the relationship between change in BMD and vertebral fracture risk in women treated with risedronate, BMD increases of 2–4% were correlated to a decrease in fracture risk, whereas an increase above this threshold presented no additional benefit . Accordingly, others have demonstrated that improvement of BMD explained only a small part of the risk reduction in RLX, bisphosphonate, and calcitonin-treated patients [33, 34]. These discrepancies may partly be accounted for by the fact that treatment effect on bone matrix quality is important to anti-fracture efficacy and a certain level of remodeling is needed to sustain an optimal quality of the skeleton .
We recognize the limitations of our study. The analysis is based on randomly selected subgroups from six individual double-blind, placebo-controlled studies. The subgroups differed in size both within the studies and across studies, but the selection is assumed not to affect the reliability of the data based on the randomness in the selection process. Furthermore the urine samples were analyzed without knowledge of the treatment group. All studies included healthy postmenopausal female volunteers, but inclusion criteria differed between studies. However, even though the two bisphosphonate groups represented the youngest and the oldest cohorts in mean age, the two bisphosphonate treatments were associated with a comparable and most pronounced decrease in the ααCTX to ββCTX ratio. The observed differences in treatment effects are, therefore, most probably a consequence of the treatments and not caused by differences between populations. Furthermore, one may speculate whether storage of the urine samples for prolonged time at −20°C affects the results. To our knowledge we have not been able to detect any changes in the measured parameters caused by storage time at this temperature. Also, the data presented are placebo-adjusted values (adjusted within the individual study), if any change occurred it has been corrected by the placebo-adjustment. Finally one may speculate whether differences in the baseline bone turnover rate could explain the observed treatment differences. However, the data of the bone resorption markers of ααCTX and ββCTX, measured at exactly the same time in all the subgroups, were at comparable levels e.g., in the alendronate and the nasal HRT group, yet the treatment response differed between these groups.
In conclusion, we have for the first time demonstrated that different anti-resorptive therapies induce differences in the maturation profile of bone collagen measured as the ratio between ααCTX and ββCTX.
Conflict of Interest
Inger Byrjalsen, Diana J Leeming, and Per Qvist are employees of Nordic Bioscience A/S, and Per Qvist , Claus Christiansen, and Morten A Karsdal are stock owners of Nordic Bioscience A/S.