Fragility fractures in men with idiopathic osteoporosis are associated
with undermineralization of the bone matrix
Nadja Fratzl-Zelman1*, Paul Roschger1, Barbara M. Misof1, Kamilla NawrotWawrzyniak1, Sarah Pötter-Lang2#, Christian Muschitz2, Heinrich Resch2,
Klaus Klaushofer1 and Elisabeth Zwettler1
1
Ludwig Boltzmann Institute of Osteology at Hanusch Hospital of WGKK and AUVA
Trauma Centre Meidling, 4th Med. Dept. Hanusch Hospital, A-1140 Vienna, Austria
2
#
Medical Department II, St. Vincent Hospital Vienna, A-1060 Vienna, Austria
Now at: Department of Radiology, Medical University of Vienna,
Vienna General Hospital, A-1090 Vienna, Austria
Short title: Bone mineralization in male idiopathic osteoporosis
Keywords: Male idiopathic osteoporosis; Bone biopsies; quantitative Backscattered Electron
Imaging (qBEI); Bone Mineralization Density Distribution; Bone Histomorphometry
Word count of main text: 3860
*Corresponding author:
Dr. Nadja Fratzl-Zelman Ph.D
Ludwig Boltzmann Institute of Osteology, UKH Meidling,
Kundratstrasse 37, A-1120 Vienna, Austria.
Tel: +43-(0)1-60150-2652, Fax: +43-(0)1-60150-2651
Email: [email protected]
Disclosures: All authors state that they have no conflicts of interest.
Bone biopsies from men with idiopathic osteoporosis showed abnormally low matrix
mineralization densities, paucity of osteoblasts suggesting an inherent mineralization defect
contributing to bone fragility.
1
1
Structured abstract:
2
3
Context: The pathogenesis of primary osteoporosis in younger individuals is still elusive. An
4
important determinant of the biomechanical competence of bone is bone material quality.
5
6
Objective, Design and Patients: In this retrospective study we evaluated bone material
7
quality based on quantitative backscattered electron imaging (qBEI) to assess bone
8
mineralization density distribution (BMDD) in bone biopsies of 25 male patients (aged 18 to
9
61 years) who sustained fragility fractures but were otherwise healthy. BMDD of cancellous
10
bone was compared with previously established adult reference data. Complementary
11
information was obtained by bone histomorphometry.
12
13
Results: The BMDD analyses of cancellous bone revealed a significant shift of BMDD to
14
lower mineralization densities. CaMean (weighted mean Ca-content), CaPeak (mode of Ca
15
content) and CaHigh (portion of full mineralized bone) were decreased compared to
16
normative reference, CaWidth (heterogeneity in mineralization) and CaLow (portion of low
17
mineralized bone) were significantly increased. The histomorphometric results showed a
18
paucity of osteoblasts and osteoclasts on the bone surface in the large majority of our patients.
19
20
Conclusion: The shift towards lower mineral content in the bone matrix in combination with
21
reduced indices of bone formation and resorption suggest an inherent mineralization defect
22
leading to undermineralized bone material which might be contributing to the susceptibility to
23
fragility fractures of our patients. The alteration in bone material might be related to
24
osteoblastic dysfunction and seem fundamentally different to high bone turnover osteoporosis
25
with a negative bone balance.
2
1
Introduction
2
3
Osteoporosis is a systemic skeletal disorder characterized by increased bone fragility
4
associated with decreased bone mass and deterioration of bone quality (microarchitecture and
5
intrinsic material properties) (1-3). While postmenopausal osteoporosis seems to be
6
predominantly based on increased bone turnover with a negative net balance in bone
7
deposition, the etiology of osteoporosis in younger individuals is quite diverse. In fact, these
8
patients are very often found to have secondary causes like hyperparathyroidism (4, Khosla,
9
1994 #246), hyperthyroidism (5, 6), hypogonadism (7, 8), glucocorticoid excess (9, 10),
10
hypercalciuria (11-13) diabetes mellitus (14) or alcohol abuse (reviewed by (15-20)).
11
Nevertheless, it remains a cohort of patients, which seems otherwise healthy but has sustained
12
fragility fractures. Khosla and coworkers reported already 1994 that 0.04% of individuals of
13
both sexes have primary osteoporosis of unknown etiology and therefore considered to have
14
“idiopathic” osteoporosis (21). Until now it remains a considerable lack of knowledge
15
regarding the pathophysiology of the disease since it is widely accepted that idiopathic
16
osteoporosis is in fact a heterogeneous entity with different underlying causes.
17
Moreover, there might be inherent differences between men and women as well. Idiopathic
18
osteoporosis is only diagnosed in young premenopausal women (22) (since postmenopausal
19
women are usually estrogen deficient which plays a major role in the pathogenesis of bone
20
loss), whereas adult men until the age of 70 can be concerned (13).
21
Reduced serum levels of IGF-1 were found in males with idiopathic osteoporosis (23, 24) but
22
not in affected women (22). Also, elevated levels of serum sex hormone binding globulin
23
leading to decreased free estradiol and androgen indices were reported (8, 25, 26).
24
Interestingly, these disturbances appeared to be associated with high bone remodeling rates
25
leading to a negative bone balance similar as in postmenopausal osteoporosis (20, 27). In
26
contrast, several other researchers report on low bone formation and osteoblasts dysfunction
27
in idiopathic osteoporosis in both genders (23, 28-32).
28
Bone fragility does not only depend on bone mass but also on the material properties of the
29
bone tissue (reviewed by (17, 33, 34)). An important determinant of the biomechanical
30
competence of bone at the material level is the bone mineralization density distribution
31
(BMDD). It reflects the amount and distribution of mineral within the bone matrix and can be
32
determined in bone biopsy samples by quantitative backscattered electron imaging (qBEI)
33
(35). The BMDD depends on bone turnover (average tissue age) and mineralization kinetics
34
and was found to have little biological variance in trabecular bone of healthy adult individuals
3
1
(36). In contrast, a number of bone diseases have been shown to alter the BMDD (35). In
2
particular, in non-treated postmenopausal osteoporosis a decrease of the mean mineral content
3
of the bone matrix was observed (37-40) which is caused by increased bone turnover leading
4
to a decrease in average tissue age. It is well accepted, that abnormal bone remodeling can
5
have a profound impact on bone structural as well as material properties (41). For example, a
6
deterioration of trabecular microarchitecture has been observed in male osteoporosis (42, 43)
7
and in osteoporosis of premenopausal women with fragility fractures (44).
8
9
The present study focuses on bone material quality in male idiopathic osteoporosis (mIOP).
10
We conducted a retrospective study on patients who presented at two Viennese centers after
11
sustaining fragility fractures but appeared otherwise healthy. Bone biopsies were taken as part
12
of the clinical evaluation and BMDD was assessed in each patient and compared with
13
previously established normative reference values (36). Complementary information on bone
14
structure was obtained by evaluation of bone histomorphometric parameters.
15
16
Materials and Methods
17
18
Subjects
19
In this retrospective study, we screened bone biopsies from male patients who presented at
20
two Viennese centers (Hanusch Hospital of WGKK and St. Vincent Hospital) after sustaining
21
fragility fractures but appeared otherwise healthy. The transiliac bone biopsies were obtained
22
from each patient as a part of clinical evaluation and all patients provided written informed
23
consent. The criteria for being selected in the present study were:
24
1) At least one fragility fracture documented by radiographs and being defined as a fracture
25
occurred after a fall from standing height or less excluding skull or digit fractures.
26
2) Detailed history, clinical examination and laboratory findings for excluding known
27
secondary causes of osteoporosis, like glucocorticoid excess, alcohol abuse, inflammatory
28
diseases, hyperthyroidism, hypogonadism, diabetes mellitus or hyperparathyroidism (although
29
PTH was not available in all patients, severe primary hyperparathyroidism could be excluded
30
by serum-calcium levels within the normal range) and serum 25-hydroxyvitamin D levels
31
above 20ng/ml (45).
32
3) Patients with previous osteoporosis treatment (for instance with bisphophonates) were
33
excluded as well.
4
1
According to these criteria 25 biopsies from male patients between age 18 and 61 years could
2
be selected. Seven of the study patients are known to have received Ca and vitamin D for
3
different periods of time.
4
Bone mineral density was measured by means of dual-energy x-ray absorptiometry at the
5
lumbar spine (L1-L4) and right or left femur with a Lunar Prodigy Densitometer equipped
6
with software containing data for an adult male and female reference population from
7
Germany (Dicom enCore Prodigy Version 8.50.093). Osteopenia was defined in accordance
8
with the World Health Organization guidelines as a T score <–1Standard Deviations (SD),
9
osteoprosis as a T-Score less or equal -2,5SD.
10
L1-L4 T-scores were available in all but 4 patients, femoral neck T-score in all but 1 patient
11
(however, from each patient at least one T-score was available). Some of the biochemical
12
variables were not available for each of the 25 patients: PTH was measured in all but 3
13
patients, CTX and OC was available from 60% of the study cohort. Also urinary calcium was
14
not measured in all patients, however those available were within normal range (data not
15
shown).
16
17
Quantitative backscattered electron imaging (qBEI)
18
The transiliac bone biopsies were fixed in 70% ethanol, dehydrated in a graded series of
19
ethanol, and embedded undecalcified in polymethylmethacrylate (PMMA). The block
20
samples containing cancellous and cortical bone were prepared by grinding and polishing to
21
receive plane and parallel surfaces. Subsequently, the surface plane containing bone tissue
22
was coated by carbon (using vacuum evaporation) for qBEI analysis in the scanning electron
23
microscope. Trabecular (Cn.) and cortical (Ct.) bone mineralization density distribution
24
(BMDD) was determined by qBEI using a digital scanning electron microscope (DSM 962,
25
Zeiss, Oberkochen, Germany) equipped with a four-quadrant semiconductor backscattered
26
electron (BE) detector as described previously (46). The accelerating voltage of the electron
27
beam was adjusted to 20 kV, the probe current to 110 pA, and the working distance to 15 mm.
28
The entire cancellous and cortical bone areas were imaged at x50 nominal magnification
29
(corresponding to a pixel resolution of 4 µm/pixel), using a scan speed of 100 sec/frame,
30
resulting in digital calibrated BE images of 512x512 pixels. From these digital images, grey
31
level histograms were deduced, displaying the percentage of bone area occupied by pixels of a
32
certain grey level. The transformation of these into calcium wt% histograms led to a bin width
33
of 0.17wt% calcium. A technical precision of 0.3% was achieved. The BMDD parameters like
34
the mean (weighted mean) CaMean and the most frequent calcium concentration CaPeak
5
1
(peak position of the BMDD) in the sample, the width of the distribution CaWidth (full width
2
at half maximum) reflecting the heterogeneity in matrix mineralization, the fraction of low
3
mineralized bone (CaLow), which is the percentage of the area below 17.68 wt% Ca
4
(corresponding to the 5th percentile of reference BMDD) and the fraction of fully mineralized
5
bone (CaHigh), which is the portion of the area above 25.30 wt% Ca (corresponding to the
6
95th percentile of reference BMDD) (35, 36) were derived from the histogram. The BMDD
7
values obtained from the patients were compared to those of the reference BMDD of normal
8
adults (see Figure 1) (36).
9
10
Bone histomorphometric evaluation
11
From the PMMA embedded block samples, 3 µm thick sections were cut with a hard
12
microtome (Leica SM2500, Nussloch, Germany). The sections were deplastified with 2-
13
methoxyethyl-acetate before being stained with a modified Goldner's Trichrome method.
14
Histological analysis was performed according to Parfitt et al. (47) on the whole area of the
15
bone sections. A light microscope (Axiophot, Zeiss, Oberkochen, Germany) equipped with a
16
Zeiss AxioCam videocamera was used to obtain digital images of the sections. The images
17
were analyzed using standard procedures (NIH Image software versions 1.63, Wayne
18
Rasband, National Institutes of Health, Bethesda, MD) on a Power Macintosh G4. Structural
19
parameters and static parameters of bone formation and resorption were obtained from all
20
patients and compared with published normative data from men aged between 41 and 50 years
21
(48).
22
23
Statistical evaluations
24
Statistical analysis was performed using SigmaStat for Windows Version 2.03 (SPSS Inc.).
25
Assumption of normality was based on the Kolmogorov-Smirnov test. Throughout the
26
manuscript, normally distributed data are presented by mean (SD), non-normally distributed
27
data by median [25%; 75%]. Comparison of the mIOP cancellous BMDD variables with
28
reference values (published previously)(36) and comparison between vertebral-fracture versus
29
no-vertebral-fracture subgroups was done using t-tests or Mann-Whitney rank sum tests.
30
Within mIOP group comparison of Cn. versus Ct. BMDD variables was done using paired t-
31
tests or Wilcoxon signed rank tests. For this comparison, only biopsy samples with both
32
cortical plates present were included (3 of our patients had to be excluded from this statistical
33
analysis).
6
1
Dependency on age was analyzed using Spearman rank order correlations and is presented by
2
the correlation coefficient (r) and corresponding p-value. Statistical significance was
3
considered at p<0.05.
4
5
Results
6
7
Clinical characteristics of mIOP patients
8
A total of 25 male subjects with fragility fractures were selected, 9 of which had a history of
9
up to 6 vertebral fractures and 17 had up to 7 non-vertebral fractures (rib, femur, tibia, ulna,
10
radius, scapula). Only one of these patients had sustained both one vertebral and 2 non-
11
vertebral fractures. BMD data from L1-L4 vertebrae or femoral neck revealed T-Scores
12
corresponding to either osteopenia (-2.5 ≤T-Score <-1) or osteoporosis (T-Score <-2.5) (for 4
13
patients, no L1-L4 T-score was available).
14
Table 1 shows the biochemical parameters measured in our patients. Normal serum levels of
15
25-hydroxyvitamin D and testosterone, as well as no signs of primary hyperparathyroidism
16
were a prerequisite for the inclusion of the patients to the study. Mean levels of phosphate,
17
calcium, carboxy-terminal collagen crosslinks (CTX), and osteocalcin were also within
18
normal limits. A decrease with age was found for the levels of testosterone (r= -0.55,
19
p=0.004), CTX (r= -0.60, p=0.008), and osteocalcin (r= -0.59, p=0.01).
20
21
BMDD from mIOP patients
22
The BMDD of cancellous bone from our mIOP patients showed a significant shift towards
23
lower mineralization densities compared to normal adult reference values (see Figure 1) (36).
24
As shown in Table 2, the mean calcium concentration, Cn.CaMean (-5.9%), as well as the
25
most frequent calcium content Cn.CaPeak (-5.6%) and the portion of fully mineralized bone
26
areas, Cn.CaHigh (-76.8%) were markedly reduced. Additionally, there was an increase in the
27
portion of low mineralized bone areas Cn.CaLow (+68.8%) and in the heterogeneity of
28
mineralization, Cn.CaWidth (+18.5%).
29
The BMDD parameters for cortical bone could not be compared to normal reference values,
30
since BMDD reference values have not been established yet for cortical bone. However, for
31
22 patients in our study, BMDD parameters have been compared pairwise between cortical
32
and cancellous bone (in 3 patients total cortex was not available). CaMean, CaPeak and
33
CaLow were found similar for both compartments while, CaWidth and CaHigh were
34
significantly increased in cortical bone (Ct.CaWidth +6.9%, p<0.05 and Ct.CaHigh +58.9%,
7
1
p<0.001). The mean or median values of cortical and for cancellous bone are shown in Table
2
2. We also plotted the degree of matrix mineralization as a function of serum 25-
3
hydroxyvitamin D and found no significant correlation between these variables in our patients
4
(Figure 2C).
5
Comparing the BMDD parameters between the groups with vertebral fractures to those
6
without vertebral fractures, we found only one slight association for Cn.CaPeak, which was
7
decreased in the patient group with vertebral fractures compared to those without vertebral
8
fractures: 21.54 [20.80; 21.68] wt%Ca versus 22.01 [21.52; 22.36] wt%Ca, respectively
9
(p=0.048).
10
11
Structural and static indices of bone formation and resorption in mIOP patients
12
Mean values and standard deviations for all measured histomorphometric indices are given in
13
Table 3. For comparison, we added previously published data from healthy men of
14
comparable mean age (48). While a good agreement was found for some parameters, such as
15
BV/TV, TbN or Tb.Th (Table 3 and Figure 3), several others were outside the normal range
16
of ± 1SD of the mean value for the controls from Rehman et al. (48) (see Table 3). The
17
amount of osteoblasts (ObS/BS) was below the normal range in 24 of our mIOP patients. In
18
more than half of the total cohort, no active cuboidal osteoblasts seams could be viewed on
19
the trabecular surface. The amount of osteoclasts (OcS/BS) was below normal in 14 mIOP
20
patients and the eroded surface per bone surface (ES/BS) showed a broader range of values (it
21
was normal in 8 and increased in 10 of our patients). However, the osteoid thickness (O.Th.)
22
was in the normal range or slightly increased and the osteoid surface per bone surface
23
(OS/BS) was normal or lower in most of our patients. For the present study cohort, BV/TV
24
decreased with age (which was near to significance, r= -0.39, p=0.055), whereas trabecular
25
thickness (Tb.Th.) and trabecular number (Tb.N) did not reveal such an age-dependency.
26
There was also a highly significant decrease of cortical thickness (Ct. Th) with the patients’
27
age (r= -0.76, p<0.001), attaining levels well below control reference range for the older
28
patients (see Figure 3).
29
Parameters characterizing the amount of osteoid (O.Th. and OS/BS) were also plotted as a
30
function of serum 25-hydroxyvitamin D levels in our patients (see Figure 2A and 2B). These
31
data clearly did not show any correlation with vitamin D levels. In particular, osteoid
32
formation was not increased in subjects with lower 25-hydroxyvitamin D serum levels.
33
34
8
1
Discussion
2
3
The present retrospective study assesses bone mineralisation density distribution (BMDD) by
4
backscattered electron imaging (qBEI) on transiliac bone biopsies to address the effects of
5
mIOP on bone material quality. Additionally, histomorphometric analysis was used for
6
complementary information. The outcomes revealed, that the investigated group of mIOP
7
patients showed a significant shift in BMDD towards lower bone matrix mineralization
8
compared to a normative reference cohort.
9
10
In general, idiopathic osteoporosis does not seem to represent a homogenous entity neither
11
clinically nor pathogenetically as mirrored by differences in diagnostic methods and inclusion
12
criteria between different study designs. In the present work, we report data on a population
13
comprised of 25 male patients aged 18 to 61 years with no identifiable causes of bone disease,
14
according to clinical and laboratory investigations. The diagnosis of idiopathic osteoporosis
15
was based on the presence of non-traumatic fractures and not on decreased BMD. In
16
consequence, we found a rather large variation of BMD values, ranging from T-scores
17
between -6 and -1 SD. In fact, only half of our patients were osteoporotic (T-score <-2.5).
18
This emphasizes that BMD alone has only a limited power to predict fracture risk, as shown
19
by the Rotterdam (49, 50) study and the Minos study (51), where a significant percentage of
20
low trauma fractures was reported in individuals with osteopenic or even normal BMD values.
21
In view of the aforementioned heterogeneity of male idiopathic osteoporosis, it is remarkable,
22
that the decrease in calcium concentration was found in the entire mIOP cohort. Moreover, it
23
has to be emphazised that this decrease in mineralization seems to have another origin than
24
the one observed in postmenopausal osteoporosis. It is well documented that this latter
25
decrease of mineralization density is attributed to increased bone remodeling (33, 35, 37-40,
26
52-54), whereas in the present study cohort higher bone turnover can be excluded as the
27
histomorphometric outcomes indicate (see below). Generally, it has been shown that the
28
specific shape of the BMDD results from two different processes (54, 55), the kinetics of
29
mineralization of the osteoid matrix (the speed of mineral deposition into the organic matrix)
30
and bone turnover. Indeed, once mineralization has started in a newly formed osteoid, the
31
mineral content increases at first rapidly up to 70% of the total value within days (56) - a
32
process referred as primary mineralization - followed by a much slower process of secondary
33
mineralization, which lasts years to reach full mineralization (35). Provided that there are no
34
changes in the kinetics of mineralization, increased bone turnover is expected to decrease the
9
1
average mineralization density of the bone matrix, because of lack of time for secondary
2
mineralization, whereas reduced bone-turnover is expected to increase average mineralization
3
density, because of prolonged time of secondary mineralization (55). Consistently, an
4
antiresorptive therapy shifts the BMDD of postmenopausal women towards normal (33, 35,
5
37-40, 52-54). However, in the present patient cohort of mIOP the situation appears exactly
6
reversed: we found a decrease of mineralization density, while the bone turnover was low
7
indicating alterations in the mineralization kinetics. A slow down of the mineral deposition
8
process might to be responsible for it.
9
underlying mechanism, for the reduced bone matrix mineralization, because none of our
10
patients was VitD deficient. Further, we did not find any correlation between serum 25-
11
hydroxyvitamin D levels and the mineral content of the matrix. However, abnormalities in the
12
secreted organic bone matrix might be an alternative source of decreased bone matrix
13
mineralization. This assumption is supported by the findings of osteoblastic dysfunction and
14
reduced bone formation in mIOP (30-32). Clearly more studies are needed to find clinical
15
determinants including genetic factors leading to those alterations. For example an impaired
16
ability of osteoblast-like cells to form mineralized bone nodules in vitro and decreased bone
17
mineralization in vivo were reported for patients with the COLIA1 Sp1 binding site
18
polymorphism (57).
Insufficient vitamin D levels are not likely the
19
20
Since there is no normative reference BMDD data for cortical bone available, we can only
21
relate the outcomes to that of the cancellous bone. The cortical compartment has similar
22
CaMean and CaPeak, but a larger CaWidth and CaLow compared to cancellous bone. For
23
these differences, the osteonal architecture is likely to be responsible, due to its higher
24
variation in matrix mineralization caused by the coexsistence of young and old osteons as well
25
as highly mineralized interstitial bone matrix. Further, a comparison between the subgroup of
26
patients who sustained at least one vertebral fracture with the remaining cohort revealed no
27
differences in any BMDD variables in cortical or cancellous bone, with the exception of
28
Cn.CaPeak which was somewhat lower in the vertebral fracture subgroup. This result reached
29
significance (p=0.048) however in light of the low sample size its clinical relevance remains
30
unclear.
31
32
As mentioned above, for the correct interpretation of the BMDD outcomes complementary
33
information on the bone turnover situation is necessary. For this reason, data on bone
34
histomorphometry have been obtained from the patients’ biopsies. The most striking
10
1
histomorphometric finding in our study cohort was the alteration of static indices of bone
2
formation and bone resorption indicative for low bone turnover. The lack of information on
3
bone formation rates from dynamic histomorphometric indices is a limitation of this
4
retrospective analysis. Nevertheless, nearly all patients showed a dramatic drop in the values
5
of osteoblast surface compared to controls and half of them did not display any cuboidal
6
osteoblasts at all, suggesting an arrest of bone formation. Also, the amount of osteoclasts
7
(NOc/BPm) was lower in all but one patient and osteoclast surface was lower than normal in
8
more than half of the study cohort. The concomitant reduction in osteoid surface on the
9
trabecular bone and increase in osteoid thickness in nearly 40% of our patients are consistent
10
with a deficit in osteoblastic function, as previously reported for mIOP (30). Osteomalacia
11
resulting from severe vitamin D deficiency can be excluded, since all our patients had serum
12
25-hydroxyvitamin D levels of at least 20ng/ml. While there is increasing evidence that this
13
concentration might be suboptimal for fracture prevention (58-60), our data clearly indicate
14
no correlation between osteoid indices and 25-hydroxyvitamin D serum levels, which ranged
15
up to 85ng/ml. Moreover, none of the patients showed signs of reactive hyperparathyroidism
16
and/or enhanced osteoclastogenesis. Hence, the decrease of the amount of osteoblasts mirrors
17
most probably a situation of low bone formation which supports previous findings in men and
18
women that idiopathic osteoporosis is related to an alteration of osteoblastic function (21, 23,
19
24, 29, 30, 32, 61). Interestingly, this low bone turnover status does not seem to be mirrored
20
by the biochemical variables of bone formation (osteocalcin) and resorption (CTX), which
21
were both found to be within normal range and even increased in single patients. However,
22
this is not necessarily in contradiction to the observations obtained from histomorphometry,
23
since the latter reflects the situation at the biopsy site, whereas serum markers for bone
24
turnover reflect a more generalized metabolic situation. Especially, these markers were found
25
to be increased in patients in the year following fractures (62-66). Since all patients of our
26
study had their biopsy taken after sustaining at least one bone fracture, it seems reasonable to
27
assume that their bone turnover markers mirror postfracture metabolic processes in addition to
28
“normal” bone cell activity.
29
30
Considering the structural bone indices of the iliac crest biopsies from our patient cohort, it is
31
remarkable, that there is a large variance of cancellous bone volume (BV/TV) among our
32
patients resulting in a mean value similar to healthy age-related adult men (48). This is
33
consistent with the high variability found in the BMD measurements ranging from -6 to -1 of
34
T-scores in our patients. Others studies reported a marked decrease of BV/TV in mIOP in
11
1
comparison to age-matched controls (21, 24, 30, 61). Again, this can be explained by the fact
2
that in these former studies only patients with a BMD strictly within osteoporotic range were
3
selected (21, 24, 30, 61). In line with our study, normal bone volume was also observed in a
4
cohort of premenopausal women with idiopathic osteoporosis selected on the basis of fragility
5
fractures (29).
6
7
Since the age range was rather wide in our study group, we assessed age dependency of the
8
different histomorphometric indices and found that BV/TV showed a weak (near to
9
significance) decrease, while cortical width (Ct.Wi) had a strong (highly significant) decrease
10
with increasing age. While BV/TV comprised values below as well as above normal, Ct.Wi.
11
values were normal in all younger mIOP patients but within lower normal range or below
12
from the age of 40 years onwards. The observed decrease with age might indicate a premature
13
decline of trabecular bone volume and cortical thickness in our study population.
14
15
In conclusion, this study provides strong evidence that in mIOP the intrinsic material
16
properties of bone are changed by reduced mineral content compared to normal bone matrix.
17
This is in contrast to postmenopausal osteoporosis, where the increase in bone turnover is
18
responsible for the reduction in mineral content. These findings for mIOP suggest an inherent
19
mineralization defect, which is likely contributing to the bone fragility in these patients.
20
Further studies are needed to elucidate the origin of impaired osteoblastic function and to
21
characterize abnormalities of the organic matrix in mIOP.
22
23
24
Acknowledgements:
25
26
We thank Gerda Dinst, Phaedra Messmer, Daniela Gabriel and Sonja Lueger for careful
27
sample preparations and qBEI measurements. This study was supported by the AUVA
28
(Research funds of the Austrian workers compensation board) and by the WGKK (Viennese
29
sickness insurance funds).
30
12
1
References:
2
3
1.
4
5
Kanis JA 2002 Diagnosis of osteoporosis and assessment of fracture risk. Lancet
359:1929-36
2.
6
Kanis JA, McCloskey EV, Johansson H, Oden A, Melton LJ, 3rd, Khaltaev N
2008 A reference standard for the description of osteoporosis. Bone 42:467-75
7
3.
Poole KE, Compston JE 2006 Osteoporosis and its management. Bmj 333:1251-6
8
4.
Eriksen
9
10
EF
2002
Primary
hyperparathyroidism:
lessons
from
bone
histomorphometry. J Bone Miner Res 17 Suppl 2:N95-7
5.
Bassett JH, Nordstrom K, Boyde A, Howell PG, Kelly S, Vennstrom B, Williams
11
GR 2007 Thyroid status during skeletal development determines adult bone structure
12
and mineralization. Mol Endocrinol
13
6.
Bassett JH, O'Shea PJ, Sriskantharajah S, Rabier B, Boyde A, Howell PG, Weiss
14
RE, Roux JP, Malaval L, Clement-Lacroix P, Samarut J, Chassande O, Williams
15
GR 2007 Thyroid hormone excess rather than thyrotropin deficiency induces
16
osteoporosis in hyperthyroidism. Mol Endocrinol 21:1095-107
17
7.
18
19
Riggs BL, Khosla S, Melton LJ, 3rd 2002 Sex steroids and the construction and
conservation of the adult skeleton. Endocr Rev 23:279-302
8.
Van Pottelbergh I, Goemaere S, Zmierczak H, Kaufman JM 2004 Perturbed sex
20
steroid status in men with idiopathic osteoporosis and their sons. J Clin Endocrinol
21
Metab 89:4949-53
22
9.
23
24
and treatment options. Minerva Med 99:23-43
10.
25
26
De Nijs RN 2008 Glucocorticoid-induced osteoporosis: a review on pathophysiology
Sinigaglia L, Mazzocchi D, Varenna M 2008 Bone involvement in exogenous
hypercortisolism. J Endocrinol Invest 31:364-70
11.
Perry HM, 3rd, Fallon MD, Bergfeld M, Teitelbaum SL, Avioli LV 1982
27
Osteoporosis in young men: a syndrome of hypercalciuria and accelerated bone
28
turnover. Arch Intern Med 142:1295-8
29
12.
Resch H, Pietschmann P, Woloszczuk W, Krexner E, Bernecker P, Willvonseder
30
R 1992 Bone mass and biochemical parameters of bone metabolism in men with
31
spinal osteoporosis. Eur J Clin Invest 22:542-5
32
13.
Bilezikian JP 1999 Osteoporosis in men. J Clin Endocrinol Metab 84:3431-4
33
14.
Janghorbani M, Van Dam RM, Willett WC, Hu FB 2007 Systematic review of
34
type 1 and type 2 diabetes mellitus and risk of fracture. Am J Epidemiol 166:495-505
13
1
15.
2
3
50
16.
4
5
Painter SE, Kleerekoper M, Camacho PM 2006 Secondary osteoporosis: a review
of the recent evidence. Endocr Pract 12:436-45
17.
6
7
Seeman E 2002 Pathogenesis of bone fragility in women and men. Lancet 359:1841-
Seeman E, Delmas PD 2006 Bone quality--the material and structural basis of bone
strength and fragility. N Engl J Med 354:2250-61
18.
Peris P, Martinez-Ferrer A, Monegal A, Martinez de Osaba MJ, Alvarez L, Ros
8
I, Muxi A, Reyes R, Guanabens N 2008 Aetiology and clinical characteristics of
9
male osteoporosis. Have they changed in the last few years? Clin Exp Rheumatol
10
11
26:582-8
19.
12
Kanis JA, Johansson H, Oden A, McCloskey EV 2009 Assessment of fracture risk.
Eur J Radiol 71:392-7
13
20.
Khosla S 2010 Update in male osteoporosis. J Clin Endocrinol Metab 95:3-10
14
21.
Khosla S, Lufkin EG, Hodgson SF, Fitzpatrick LA, Melton LJ, 3rd 1994
15
Epidemiology and clinical features of osteoporosis in young individuals. Bone 15:551-
16
5
17
22.
Rubin MR, Schussheim DH, Kulak CA, Kurland ES, Rosen CJ, Bilezikian JP,
18
Shane E 2005 Idiopathic osteoporosis in premenopausal women. Osteoporos Int
19
16:526-33
20
23.
Johansson AG, Eriksen EF, Lindh E, Langdahl B, Blum WF, Lindahl A,
21
Ljunggren O, Ljunghall S 1997 Reduced serum levels of the growth hormone-
22
dependent insulin-like growth factor binding protein and a negative bone balance at
23
the level of individual remodeling units in idiopathic osteoporosis in men. J Clin
24
Endocrinol Metab 82:2795-8
25
24.
Kurland ES, Rosen CJ, Cosman F, McMahon D, Chan F, Shane E, Lindsay R,
26
Dempster D, Bilezikian JP 1997 Insulin-like growth factor-I in men with idiopathic
27
osteoporosis. J Clin Endocrinol Metab 82:2799-805
28
25.
Legrand E, Hedde C, Gallois Y, Degasne I, Boux de Casson F, Mathieu E, Basle
29
MF, Chappard D, Audran M 2001 Osteoporosis in men: a potential role for the sex
30
hormone binding globulin. Bone 29:90-5
31
26.
Lapauw B, Taes Y, Goemaere S, Toye K, Zmierczak HG, Kaufman JM 2009
32
Anthropometric and skeletal phenotype in men with idiopathic osteoporosis and their
33
sons is consistent with deficient estrogen action during maturation. J Clin Endocrinol
34
Metab 94:4300-8
14
1
27.
Tuck SP, Scane AC, Fraser WD, Diver MJ, Eastell R, Francis RM 2008 Sex
2
steroids and bone turnover markers in men with symptomatic vertebral fractures. Bone
3
43:999-1005
4
28.
5
6
Khosla S 1997 Idiopathic osteoporosis--is the osteoblast to blame? J Clin Endocrinol
Metab 82:2792-4
29.
Donovan MA, Dempster D, Zhou H, McMahon DJ, Fleischer J, Shane E 2005
7
Low bone formation in premenopausal women with idiopathic osteoporosis. J Clin
8
Endocrinol Metab 90:3331-6
9
30.
Ciria-Recasens M, Perez-Edo L, Blanch-Rubio J, Marinoso ML, Benito-Ruiz P,
10
Serrano S, Carbonell-Abello J 2005 Bone histomorphometry in 22 male patients
11
with normocalciuric idiopathic osteoporosis. Bone 36:926-30
12
31.
13
14
Pernow Y, Granberg B, Saaf M, Weidenhielm L 2006 Osteoblast dysfunction in
male idiopathic osteoporosis. Calcif Tissue Int 78:90-7
32.
Ruiz-Gaspa S, Blanch-Rubio J, Ciria-Recasens M, Monfort J, Tio L, Garcia-
15
Giralt N, Nogues X, Monllau JC, Carbonell-Abello J, Perez-Edo L Reduced
16
Proliferation and Osteocalcin Expression in Osteoblasts of Male Idiopathic
17
Osteoporosis. Calcif Tissue Int
18
33.
Chavassieux P, Seeman E, Delmas PD 2007 Insights into material and structural
19
basis of bone fragility from diseases associated with fractures: how determinants of the
20
biomechanical properties of bone are compromised by disease. Endocr Rev 28:151-64
21
34.
22
23
Science 52:1263-1334
35.
24
25
Fratzl P, Weinkamer R 2007 Nature´s hierarchical materials. Progress in Materials
Roschger P, Paschalis EP, Fratzl P, Klaushofer K 2008 Bone mineralization
density distribution in health and disease. Bone 42:456-66
36.
Roschger P, Gupta HS, Berzlanovich A, Ittner G, Dempster DW, Fratzl P,
26
Cosman F, Parisien M, Lindsay R, Nieves JW, Klaushofer K 2003 Constant
27
mineralization density distribution in cancellous human bone. Bone 32:316-23
28
37.
Roschger P, Rinnerthaler S, Yates J, Rodan GA, Fratzl P, Klaushofer K 2001
29
Alendronate increases degree and uniformity of mineralization in cancellous bone and
30
decreases the porosity in cortical bone of osteoporotic women. Bone 29:185-91
31
38.
Zoehrer R, Roschger P, Paschalis EP, Hofstaetter JG, Durchschlag E, Fratzl P,
32
Phipps R, Klaushofer K 2006 Effects of 3- and 5-year treatment with risedronate on
33
bone mineralization density distribution in triple biopsies of the iliac crest in
34
postmenopausal women. J Bone Miner Res 21:1106-12
15
1
39.
Fratzl P, Roschger P, Fratzl-Zelman N, Paschalis EP, Phipps R, Klaushofer K
2
2007 Evidence that Treatment with Risedronate in Women with Postmenopausal
3
Osteoporosis Affects Bone Mineralization and Bone Volume. Calcif Tissue Int 81:73-
4
80
5
40.
Roschger P, Manjubala I, Zoeger N, Meirer F, Simon R, Li C, Fratzl-Zelman N,
6
Misof B, Paschalis E, Streli C, Fratzl P, Klaushofer K 2009 Bone Material Quality
7
in Transiliac Bone Biopsies of Postmenopausal Osteoporotic Women After 3 Years
8
Strontium Ranelate Treatment. J Bone Miner Res
9
41.
10
11
Seeman E 2008 Bone quality: the material and structural basis of bone strength. J
Bone Miner Metab 26:1-8
42.
Legrand E, Chappard D, Pascaretti C, Duquenne M, Krebs S, Rohmer V, Basle
12
MF, Audran M 2000 Trabecular bone microarchitecture, bone mineral density, and
13
vertebral fractures in male osteoporosis. J Bone Miner Res 15:13-9
14
43.
Legrand E, Audran M, Guggenbuhl P, Levasseur R, Chales G, Basle MF,
15
Chappard D 2007 Trabecular bone microarchitecture is related to the number of risk
16
factors and etiology in osteoporotic men. Microsc Res Tech 70:952-9
17
44.
Cohen A, Liu XS, Stein EM, McMahon DJ, Rogers HF, Lemaster J, Recker RR,
18
Lappe JM, Guo XE, Shane E 2009 Bone microarchitecture and stiffness in
19
premenopausal women with idiopathic osteoporosis. J Clin Endocrinol Metab
20
94:4351-60
21
45.
22
23
van Schoor NM, Visser M, Pluijm SM, Kuchuk N, Smit JH, Lips P 2008 Vitamin
D deficiency as a risk factor for osteoporotic fractures. Bone 42:260-6
46.
Roschger P, Fratzl P, Eschberger J, Klaushofer K 1998 Validation of quantitative
24
backscattered electron imaging for the measurement of mineral density distribution in
25
human bone biopsies. Bone 23:319-26
26
47.
Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott
27
SM, Recker RR 1987 Bone histomorphometry: standardization of nomenclature,
28
symbols, and units. Report of the ASBMR Histomorphometry Nomenclature
29
Committee. J Bone Miner Res 2:595-610
30
48.
Rehman MT, Hoyland JA, Denton J, Freemont AJ 1994 Age related
31
histomorphometric changes in bone in normal British men and women. J Clin Pathol
32
47:529-34
33
34
49.
Schuit SC, van der Klift M, Weel AE, de Laet CE, Burger H, Seeman E, Hofman
A, Uitterlinden AG, van Leeuwen JP, Pols HA 2004 Fracture incidence and
16
1
association with bone mineral density in elderly men and women: the Rotterdam
2
Study. Bone 34:195-202
3
50.
Schuit SCE, an der Klift M, Weel AEAM, de Laet CEDH, Burger H, Seeman E,
4
Hofman A, Uitterlinden AG, van Leeuwen JPTM, Pols HAP 2006 Corrigendum to
5
“Fracture incidence and association with bone mineraldensity in elderly men and
6
women: The Rotterdam Study” [Bone 34 (2004) 195–202]. Bone 38:603
7
51.
Szulc P, Munoz F, Duboeuf F, Marchand F, Delmas PD 2005 Bone mineral density
8
predicts osteoporotic fractures in elderly men: the MINOS study. Osteoporos Int
9
16:1184-92
10
52.
11
12
Musculoskelet Neuronal Interact 2:538-43
53.
13
14
Boivin G, Meunier PJ 2002 Effects of bisphosphonates on matrix mineralization. J
Boivin G, Meunier PJ 2002 Changes in bone remodeling rate influence the degree of
mineralization of bone. Connect Tissue Res 43:535-7
54.
Ruffoni D, Fratzl P, Roschger P, Phipps R, Klaushofer K, Weinkamer R 2008
15
Effect of temporal changes in bone turnover on the bone mineralization density
16
distribution: a computer simulation study. J Bone Miner Res 23:1905-14
17
55.
Ruffoni D, Fratzl P, Roschger P, Klaushofer K, Weinkamer R 2007 The bone
18
mineralization density distribution as a fingerprint of the mineralization process. Bone
19
40:1308-19
20
56.
Misof BM, Roschger P, Cosman F, Kurland ES, Tesch W, Messmer P, Dempster
21
DW, Nieves J, Shane E, Fratzl P, Klaushofer K, Bilezikian J, Lindsay R 2003
22
Effects of intermittent parathyroid hormone administration on bone mineralization
23
density in iliac crest biopsies from patients with osteoporosis: a paired study before
24
and after treatment. J Clin Endocrinol Metab 88:1150-6
25
57.
Stewart TL, Roschger P, Misof BM, Mann V, Fratzl P, Klaushofer K, Aspden R,
26
Ralston SH 2005 Association of COLIA1 Sp1 alleles with defective bone nodule
27
formation in vitro and abnormal bone mineralization in vivo. Calcif Tissue Int 77:113-
28
8
29
58.
Bischoff-Ferrari HA, Giovannucci E, Willett WC, Dietrich T, Dawson-Hughes B
30
2006 Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple
31
health outcomes. Am J Clin Nutr 84:18-28
32
59.
Lips P, Bouillon R, van Schoor NM, Vanderschueren D, Verschueren S, Kuchuk
33
N, Milisen K, Boonen S 2009 Reducing fracture risk with calcium and vitamin D.
34
Clin Endocrinol (Oxf)
17
1
60.
Adams JS, Hewison M 2010 Update in vitamin D. J Clin Endocrinol Metab 95:471-8
2
61.
Pernow Y, Hauge EM, Linder K, Dahl E, Saaf M 2009 Bone histomorphometry in
3
4
male idiopathic osteoporosis. Calcif Tissue Int 84:430-8
62.
5
6
turnover following ankle fracture. Osteoporos Int 10:408-15
63.
7
8
Ingle BM, Hay SM, Bottjer HM, Eastell R 1999 Changes in bone mass and bone
turnover following distal forearm fracture. Osteoporos Int 10:399-407
64.
Yu-Yahiro JA, Michael RH, Dubin NH, Fox KM, Sachs M, Hawkes WG, Hebel
JR, Zimmerman SI, Shapiro J, Magaziner J 2001 Serum and urine markers of bone
9
10
11
Ingle BM, Hay SM, Bottjer HM, Eastell R 1999 Changes in bone mass and bone
metabolism during the year after hip fracture. J Am Geriatr Soc 49:877-83
65.
Veitch SW, Findlay SC, Hamer AJ, Blumsohn A, Eastell R, Ingle BM 2006
12
Changes in bone mass and bone turnover following tibial shaft fracture. Osteoporos
13
Int 17:364-72
14
66.
Ivaska KK, Gerdhem P, Akesson K, Garnero P, Obrant KJ 2007 Effect of fracture
15
on bone turnover markers: a longitudinal study comparing marker levels before and
16
after injury in 113 elderly women. J Bone Miner Res 22:1155-64
17
18
19
18
1
Figure legends:
2
3
Figure 1:
4
A - Backscattered electron images and corresponding BMDD of a transiliac bone biopsy of a
5
mIOP patient: overview of the entire biopsy core and detail of cancellous bone.
6
B – Definition of BMDD variables and BMDD curve of normal human adult trabecular bone
7
(white dotted line) and of a mIOP patient (black line), which is shifted to lower calcium
8
concentrations with respect to normal. The grey band indicates the 95% confidence interval
9
(C.I.) of the normal adults reference population as described previously (35).
10
11
Figure 2:
12
Scatterplots of Cn.CaMean (Cancellous CaMean) (A), O.Th, (osteoid Thickness) (B), and
13
OS/BS (osteoid surface/bone surface) (C) versus 25-hydroxyvitamin D (25OHVitD) levels in
14
our mIOP cohort. The white dotted line and the grey bar indicate the normal mean and the
15
mean ± 1SD range, respectively. For Cn.CaMean, normal levels are independent of age for
16
adult, healthy individuals, as published previously (36). For O.Th and OS/BS, normal levels
17
drawn in the figure are from healthy men aged 41-50 yrs published by Rehman et al. (48)(see
18
also Table 2). None of the plotted variables Cn.CaMean, O.Th, or OS/BS was found
19
correlated with 25OHVitD.
20
21
Figure 3:
22
Age dependency of structural parameters in our mIOP cohort compared to those of normal
23
morphometric results of British men published by Rehman and coworkers (48). The lines
24
represent mean and mean ± 1SD. The decrease of BV/TV (Bone Volume/Tissue Volume) as
25
indicated by the dotted line is near to statistical significance (Spearmen rank order correlation
26
p=0.055). The decrease of Ct.Th (Cortical Thickness, dotted line) with age in our mIOP
27
cohort is highly significant (Spearman rank order correlation p<0.001). For Tb.N (Trabecular
28
Number) and Tb.Th (Trabecular Thickness) no age dependency was observed. Tb.N in the
29
mIOP cohort was found in the lower range or below normal for a large portion of our mIOP
30
patients.
31
19
1
Table 1: mIOP Patients’ Characteristics
2
3
mIOP patients
Normal range
(n=25)
Age [a]
41.0 (12.2)
BMD neck [g/cm2]1
0.782 (0.117)
BMD L1-L4 [g/cm2]1
0.895 (0.099)
T-score neck1
-2.16 (0.92)
T-score L1-L41
-2.20 [-2.96; -1.94]
PTH [pg/mL]1
31.42 (10.15)
12 - 72
Serum Phosphate [mg/dL]
3.26 (0.52)
2.5 – 4.8
Serum Ca [mval/L]
4.82 (0.25)
4.00 – 5.35
25OHVitD [ng/mL]
36.62 [29.44; 46.20]
> 20
4.69 (1.19)
1.81 - 7.72
0.34 [0.30; 0.44]
0.000 - 0.400
Testosterone [µg/l]
Serum CTX [ng/mL]
Serum OC [ng/mL]1
1
15.75 [13.75; 22.41]
5.0 – 30.5
4
5
Data are mean (SD) or median [25th; 75th percentile].
6
1
These variables were not available for each of the 25 patients.
20
1
Table 2: BMDD in mIOP
2
mIOP patients
Normal adult reference
values (36)
Cancellous Bone (n=25)
Cn. CaMean [wt%]
20.90 (0.75)***
22.20 (0.45)
Cn. CaPeak [wt%]
21.68 (0.80)***
22.96 [22.70; 23.14]
Cn. CaWidth [∆wt%]
3.90 (0.44)***
3.29 [3.12; 3.47]
Cn. CaLow [%]
7.63 [6.14; 9.21]***
4.52 [3.87; 5.79]
Cn. CaHigh [%]
1.07 [0.64; 2.42]***
4.62 [3.52; 6.48]
Ct. CaMean [wt%]
21.13 (0.66)
n.a.
Ct. CaPeak [wt%]
21.92 (0.66)
n.a.
Ct. CaWidth [∆wt%]
4.17 (0.48)#
n.a.
Ct. CaLow [%]
8.27 (3.21)
n.a.
Ct. CaHigh [%]
1.70 [0.80; 3.86]###
n.a.
Cortical Bone (n=22)§
3
4
Data are mean (SD) or median [25th; 75th percentile].
5
n.a. = not available
6
***p<0.001 versus normal reference values
7
#
8
§
p<0.05, ###p<0.001 versus cancellous bone (paired t-test or signed rank test)
In 3 of the biopsy samples total cortex was not available.
9
21
1
Table 3: Histomorphometric Characteristics of the mIOP cohort
2
Cohort average
(n=25)
Controls:
Men 41-50 yrs
(48)
BV/TV [%]
20.93 (7.13)
21.9 (5.3)
Tb.Th [µm]
148 (32)
136 (25)
Tb.N [1/mm]
1.40 (0.37)
1.7 (0.4)
OV/BV [%]
1.39 [0.79; 2.45]
n.a
O.Th. [µm]
12.6 (5.5)
9.4 (3.9)
OS/BS [%]
11.3 (6.3)
16.5 (5.4)
ObS/BS [%]
0.00 [0.00; 1.21]
5.2 (2.1)
OcS/BS [%]
0.30 [0.00; 0.85]
0.6 (0.2)
ES/BS [%]
5.14 (3.72)
4.1 (1.8)
0.10 [0.00; 0.15]
1.1 (0.5)
0.97 (0.34)
1.159 (0.297)
5.79 [4.04; 7.97]
n.a.
NOc/BPm [1/mm]
Ct.Wi. [mm]1
Ct.Po. [%]1
3
4
Data are mean (SD) or median [25th; 75th percentile].
5
1
6
one cortex was available).
Data shown are mean of external and internal cortex for n=22 patients (in 3 biopsies, only
22
1
2
A
B
3
4
Figure 1
5
23
1
24
1
2
3
4
5
240
40
Tb.Th. (µm)
BV/TV (%)
35
30
25
20
15
200
160
120
10
80
5
10
20
30
40
50
60
10
70
20
30
50
60
70
Age [a]
Age [a]
1.8
Ct.Wi. (mm)
2.2
Tb.N (mm-1)
40
1.8
1.4
1.0
0.6
1.4
1.0
0.6
0.2
10
20
30
40
50
Age [a]
60
70
10
20
30
40
50
60
Age [a]
6
7
8
9
10
11
12
13
Figure 3
14
25
70