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ORIGINAL ARTICLE
Trabecular Bone Microstructure and Local Gene
Expression in Iliac Crest Biopsies of Men With
Idiopathic Osteoporosis
Janina M Patsch, 1,2 Thomas Kohler, 3 Andrea Berzlanovich, 4 Christian Muschitz, 5 Christian Bieglmayr, 6
Paul Roschger , 7 Heinrich Resch , 5 and Peter Pietschmann 1
1
Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Immunology and Infectiology,
Medical University of Vienna, Vienna, Austria
2
Division of Muskuloskeletal Radiology and Neuroradiology, Department of Radiology, Medical University of Vienna, Vienna, Austria
3
B-cube AG, Schlieren-Zürich, Switzerland
4
Department of Forensics, Medical University of Vienna, Vienna, Austria
5
Medical Department II, St Vincent Hospital Vienna, Vienna, Austria
6
Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria
7
Ludwig-Boltzmann Institute of Osteology, Vienna, Austria
ABSTRACT
Male idiopathic osteoporosis (MIO) is a metabolic bone disease that is characterized by low bone mass, microstructural alterations, and
increased fracture risk in otherwise healthy men. Although the detailed pathophysiology of MIO has yet to be clarified, evidence
increasingly suggests an osteoblastic defect as the underlying cause. In this study we tested the hypothesis that the expression profile of
certain osteoblastic or osteoblast-related genes (ie, WNT10B, RUNX2, Osterix, Osteocalcin, SOST, RANKL, and OPG) is different in iliac crest
biopsies of MIO patients when compared with healthy controls. Furthermore, we investigated the relation of local gene expression
characteristics with histomorphometric, microstructural, and clinical features. Following written informed consent and diligent clinical
patient characterization, iliac crest biopsies were performed in nine men. While RNA extraction, reverse-transcription, and real-time
polymerase chain reactions (PCRs) were performed on one biopsy, a second biopsy of each patient was submitted for histomorphometry
and micro–computed tomography (mCT). Age-matched bone samples from forensic autopsies served as controls. MIO patients displayed
significantly reduced WNT10B, RUNX2, RANKL, and SOST expression. Performing mCT for the first time in MIO biopsies, we found
significant decreases in trabecular number and connectivity density. Trabecular separation was increased significantly, but trabecular
thickness was similar in both groups. Histomorphometry revealed decreased BV/TV and osteoid volume and fewer osteoclasts in MIO. By
providing evidence for reduced local WNT10B, RUNX2, and RANKL gene expression and histomorphometric low turnover, our data
support the osteoblast dysfunction model discussed for MIO. Further, MIO seems to lead to a different microstructural pathology than
age-related bone loss. ß 2011 American Society for Bone and Mineral Research.
KEY WORDS: MALE IDIOPATHIC OSTEOPOROSIS; MICRO–COMPUTED TOMOGRAPHY; GENE EXPRESSION
Introduction
O
steoporosis is a skeletal disorder characterized by compromised bone strength predisposing a person to an increased
risk of fracture.(1) Owing to major efforts in basic, translational,
and clinical research, disease awareness and therapeutic options
for postmenopausal and age-related osteoporosis have
increased significantly over the last decades.(2–4) However,
osteoporosis not only affects postmenopausal and elderly
women but also can occur in men.(5) Aged males who have
sustained a hip fracture display excess mortality compared with
women.(6) Men with osteoporosis frequently suffer from an
underlying pathology such as endocrine disorders, hematooncologic diseases, or drug-induced bone loss.(7) In case of
absence of secondary causes of bone loss, male idiopathic
osteoporosis (MIO) is diagnosed.(8) The term male idiopathic
Received in original form September 7, 2010; revised form December 11, 2010; accepted January 19, 2011. Published online February 1, 2011.
Address correspondence to: Peter Pietschmann, MD, Medical University of Vienna, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Immunology and Infectiology, Währinger Gürtel 18-20, A-1090 Vienna, Austria. E-mail: [email protected]
Journal of Bone and Mineral Research, Vol. 26, No. 7, July 2011, pp 1584–1592
DOI: 10.1002/jbmr.344
ß 2011 American Society for Bone and Mineral Research
1584
osteoporosis appears to cover a disease of multifactorial etiology
with a strong genetic background. MIO has been associated
with subclinical alterations of sex hormones,(9) as well as changes
in the growth hormone insulin-like growth factor 1 (IGF-1)
axis.(10,11) Several studies based on histomorphometry(12) and
explant cell cultures(13) provide evidence for an underlying
osteoblast defect, but the detailed pathophysiology of MIO
remains to be elucidated. Expanding on the concept of
osteoblast deficiency, we used iliac crest biopsies of clinically
homogeneous MIO patients to detect alterations in the local
expression of genes involved either in osteoblast differentiation
and regulation or with an association with osteoblast activity.
Second, we intended to relate WNT10B, RUNX2, OSX, Osteocalcin,
RANKL, OPG, and SOST expression to histomorphometric and
microstructural biopsy characteristics. Stressing the translational
aspect of this study, we aimed at interpreting gene expression
and microstructural findings in a clinical context.
Patients and Methods
Clinical evaluation, densitometry, and X-ray imaging
Physical examination was performed, and body weight and
patient height were assessed using a stadiometer with
integrated weighing scale (BWB 700; Tanita, Tokyo, Japan).
Fasting blood samples were drawn from all patients between
8:00 and 10:00 a.m. Routine blood and urine analyses were
performed at the Vienna-based Central Routine Laboratory of the
St Vincent Group. Routine blood tests included a whole blood
count and determinations of serum potassium, sodium, calcium,
and phosphate, as well as parathyroid hormone (PTH), 25hydroxyvitamin D, thyroid-stimulating hormone (TSH), kidney
and liver function parameters, and total testosterone measurements. Calcium and phosphate excretion was measured from a
24-hour urine collection. Moreover, patients underwent dualenergy X-ray absorptiometry (DXA) scanning of the hip and spine
(Lunar iDXA; GE Healthcare, Piscataway, NJ, USA). To complete
the clinical workup, vertebral fracture status was analyzed by
anteroposterior and lateral X-ray studies of the thoracic and
lumbar spine. The images were read by an experienced physician
trained for Genant scoring.
Patients
Following study approval by the local ethics committee, MIO
patients were recruited at the outpatient clinic of the Medical
Department II, St Vincent Hospital Vienna, Vienna, Austria. All
patients signed a written informed consent prior to any studyrelated procedures. Medical history details, including the
previous use of antiosteoporotic drugs, current medications,
lifestyle habits (eg, smoking), fractures (including trauma history),
and parental fractures were recorded in detail. Inclusion criteria
were adapted from major interventional trials in male
osteoporosis(14,15) and consisted of either (1) a femoral neck
T-score of 2 or less and a lumbar spine T-score of 1 or less or
(2) a femoral neck T-score of 1 or less and at least one prevalent
osteoporotic fracture (ie, at least one moderate vertebral fracture
according to the Genant criteria or one low-trauma peripheral
fracture).(16)
Exclusion criteria included the previous use of any specific
antiosteoporotic substances other than vitamin D and calcium
supplements (eg, bisphosphonates). Moreover, hypogonadism,
thyroid disorders, any malignoma history, or the use of
corticosteroids, thyroid hormones, or antiepileptic substances
within the last 5 years excluded patient enrollment. Agematched control bone samples were obtained from male
sudden-death or accident victims examined at the Department
of Forensic Medicine of the Ludwig-Maximilian University,
Munich, Germany. The procedure was approved by the
university’s institutional review board. A control patient was
considered eligible for bone sampling if autopsy revealed no
evidence of major chronic diseases such as malignancy or severe
kidney or hepatic disease. Moreover, medical records and
postmortem family inquiries were not allowed to reveal
prevalent fragility fractures in the donor or his relatives. The
use of either antiosteoporotic drugs or medications leading to
secondary osteoporosis (eg, corticosteroids) further excluded
donor sampling. To protect data privacy, patients and controls
received an anonymous study code that was used throughout
the study and its statistical analysis.
STRUCTURE AND GENE EXPRESSION IN MALE OSTEOPOROSIS
Bone markers
For each patient, 2 mL of serum was stored in 1-mL voids at
708C for batched bone marker analyses at the Department of
Laboratory Medicine of the Medical University of Vienna. Bone
markers were measured using electrochemiluminescence
immunoassays [ECLIA; b-CrossLaps (CTX), N-MID Osteocalcin
(OCN), total N-terminal type 1 procollagen propeptide (P1NP); all
Roche Diagnostics, Mannheim, Germany) on a Modular Analytics
E170 device (Roche Diagnostics). Because of autopsies being the
source of control bone samples, serum was unavailable from
these individuals. Instead, patients were compared with
preexisting in-house data from marker studies in healthy
controls. The data set was age-matched (n ¼ 152), and the normal
range was defined as the 5th to 95th percentile of each parameter.
Transiliac bone biopsy
In order to perform histomorphometry and structural analyses,
as well as gene expression testing, each patient underwent two
parallel-oriented left transiliac bone biopsies. Using a trephine
needle, all biopsies were carried out under sterile conditions at
the local operating theater. At the ward, patients received an
analgesic premedication (lornoxicam). Before local anesthesia,
sedation was achieved using midazolam. No biopsy-related
complications occurred. Both biopsy cylinders (inner diameter ¼ 7 mm) were examined visually. The larger, more intact
sample was selected for subsequent histomorphometric and
structural analyses and was placed in 70% ethanol. The second
biopsy was submerged immediately in RNA-Later (Ambion,
Warrington, UK) and stored as instructed by the manufacturer.
With a median postmortem interval of 23 hours, control samples
were obtained as soon as possible during forensic autopsies.
Identical to patient samples, one specimen was placed in 70%
ethanol, and the other was submerged in RNA-Later. The RNA
sample was stored at 208C and shipped on dry ice to the
Medical University of Vienna.
Journal of Bone and Mineral Research
1585
Histology and histomorphometry
Specimen processing for histology and histomorphometry was
performed according to Roschger and colleagues.(17,18) Briefly, all
structure biopsies were fixed in ethanol, dehydrated, and
embedded in polymethyl methacrylate-(PMMA). Histologic
sections were cut from the blocks and stained with Goldner
stain. In order to exclude malignant causes of osteoporosis such
as lymphoma or mastocytosis, a certified pathologist performed
routine reading in patients.
Histomorphometry was performed by the BIOQUANT Image
Analysis Corporation (Nashville, TN, USA) using the BIOQUANT
OSTEO Bone Biology Research System, Version 8.40.10. For each
section, 25 systematically random fields of view were imaged
with a 20 objective from within the trabecular compartment.
Histomorphometric data are reported according to the standardized nomenclature.(19) In addition, marrow volume (Ma.V) was
calculated as tissue volume minus bone volume. Fat volume
(FatV) was calculated as the total adipocyte volume within bone
marrow volume.
Micro–computed tomography (mCT)
The mCT imaging system (mCT40, Scanco Medical AG, Brüttisellen, Switzerland) used in this study was equipped with a 5-mm
focal-spot X-ray tube as a source. A 2D charge coupled device
(CCD) coupled with a thin scintillator as a detector permitted
acquisition of 206 tomographic images in parallel. The long axis
of the PMMA-embedded biopsy specimen was oriented along
the rotation axis of the scanner. The X-ray tube was operated at
70 kVp and 114 mA with an integration time set to 200 ms. Scans
were performed at an isotropic nominal resolution of 10 mm
(high-resolution mode). The image data were filtered using a
Gaussian filter (s ¼ 1.2, support ¼ 1) to partially suppress noise in
the volume. The mineralized tissue was segmented from soft
tissue by a global thresholding procedure,(20) with a threshold
value set to 22% of the maximum grayscale value. A special
contouring algorithm was used to automatically detect the
envelope of the biopsy, followed by a 3D erosion algorithm to
define the trabecular bone volume of interest (VOI) within the
biopsy and to exclude any cortical bone. Morphometric indices
were determined for the trabecular bone compartment using a
direct 3D approach(21) and included bone volume (BV/TV),
trabecular thickness (Tb.Th), trabecular separation (Tb.Sp),
trabecular number (Tb.N), connectivity density (Conn.D), and
the structure model index (SMI).
Invitrogen, Carlsbad, CA, USA).(22) Apart from double precipitation and centrifugation at the isopropanol stage (12,000g,
10 minutes, 48C), the manufacturer’s protocol was followed.
RNA quality and quantity were checked by electrophoresis
and photometry at 260 and 280 nm. The 260/280-nm
absorbance ratios ranged from 1.5 to 2. cDNA was sythesized
from 4 mg of total RNA using a cDNA synthesis kit (High
Capacity cDNA Reverse Transcription Kit; Applied Biosystems,
Warrington, UK). Real-time PCR was performed using assay-ondemand primers and probes following the manufacturer’s
instructions. For each reaction well, the amplification
mixture (20 mL) consisted of 9 mL of cDNA (dilution 1:10),
10 mL of mastermix buffer (TaqMan Universal PCR Mastermix,
Applied Biosystems, Warrington, UK), and 1 mL of probe, that
is,. Wnt10b (Hs00559664_m1), RUNX2 (Hs00231692_m1),
OSX
(Hs00541729_m1),
Osteocalcin
(Hs00609452_g1),
SOST (Hs00228830_m1), RANKL (Hs00243519_m1), OPG
(Hs00171068_m1), and GAPDH (Hs99999905_m1). Using a
thermal cycler (ABI Prism Sequence Detection System 7700;
Applied Biosystems), cycler conditions were 508C for 2 minutes
and 948C for 2 minutes, followed by 40 cycles at 948C for
15 seconds and 608C for 30 seconds. Amplification curves were
checked visually for exponentiality, and thresholds were set at
0.15 unit for RANKL, OPG, OSX, and RUNX2. The threshold for
GAPDH was 0.03 unit, whereas the threshold for WNT,
Osteocalcin, and SOST was set at 0.07 unit. All experiments
were performed in triplicate and were normalized to the
housekeeping gene GAPDH. GAPDH was chosen as the
housekeeping gene because of its repeated use in human
gene expression studies addressing osteoporosis research
questions.(23) The results were calculated applying the DDCt
method and are presented as fold increase relative to GAPDH
expression.
Statistics
Parametric data were reported as means SEM. For group
comparisons of parametric data, t tests were calculated.
Nonparametric data were reported as median values and the
25th to 75th percentile range. Nonparametric data were
compared using Kruskal-Wallis tests (PASW 18.0 for Mac; SPSS,
Inc., Chicago, IL, USA). For correlation analyses, Spearman
coefficients were calculated. The critical value for data
significance was set at p < .05.
Results
RNA extraction, cDNA, and real-time PCR
Patient characteristics
Using RNAse-free instruments, a small cube of trabecular bone
(approximately 5 5 5 mm) was cut out of the middle part of
the intact biopsy at a laminar flow bench. To reduce marrow
content, the cube was rinsed repeatedly with RNA-Later.
Together with two small steel beads, the bone sample was
transferred to an RNAse-free Eppendorf tube and subsequently
flash frozen in liquid nitrogen. For tissue homogenization, the
cooled tubes were placed in a grinding mill (3 minutes, 30 Hz).
Subsequently, RNA extraction was performed on the basis of a
guanidinium thiocyanate–phenol–chloroform protocol (Trizol;
Nine patients with the clinical diagnosis of male idiopathic
osteoporosis (MIO) were included in the study. Their mean age
was 53 years, ranging between 38 and 68 years. The mean age of
control donors was 58 years (33 to 78 years, n ¼ 9, p ¼ 0.38). All
MIO patients had a positive fracture history, including both
adequate and inadequate traumas. Peripheral low-trauma
fractures were present in two-thirds of patients. A single patient
even reported four peripheral low-trauma fractures, whereas
three others had two peripheral low-trauma fractures. The
remaining third had prevalent vertebral fractures confirmed by
1586
Journal of Bone and Mineral Research
PATSCH ET AL.
the inclusion X-ray. The highest number of vertebral fractures in a
single patient was three. In addition, 56% had previous fractures
following adequate traumas (eg, from skiing or other sportrelated accidents). Hip fractures were not prevalent in any
participant. One-third reported a positive family history, defined
as the occurrence of osteoporotic fractures in either of the
parents. Patients with a positive family history had a 10.5% lower
bone density at the lumbar spine ( p ¼ .025), but all other
parameters investigated throughout this study were similar.
One-third had never smoked; two-thirds were former (50%) or
current smokers (50%). The mean body mass index (BMI) was
27 kg/m2 (19.7 to 33.3 kg/m2). In control patients, the autopsy
protocols did not reveal major chronic diseases (eg, cancer).
Owing to the nature of sample procurement (ie, forensic
autopsies), a fracture history was not available.
Histology and histomorphometry
Neither osteomalacia nor malignant infiltration was reported
from routine readings. Bone surface (BS), bone volume (BV), and
bone volume fraction (BV/TV) were reduced significantly in
MIO patients. Osteoid volume (OV) also was lower. In MIO
biopsies, significantly fewer osteoclasts were found. The
marrow parameters (Ma.V and Ma.V/TV) were inversely related
to BV/TV. Fat volume within the marrow (FatV/Ma.V) did not
differ between the two groups. BS and OV were associated with
the serum bone-formation marker P1NP (r ¼ 0.733, p ¼ .025;
r ¼ 0.794, p ¼ .011). Confirming the absence of histomorphometric evidence of osteoporosis in the control group, we found
similar BV/TV values in our autopsy samples and published data
on healthy males.(24,25) Histomorphometric data are given in
Table 2.
Densitometry, serum analyses, and bone markers
Micro–computed tomography (mCT)
The patients’ mean T-scores for total hip, femoral neck, and
lumbar spine (L1–L4) were 2.32 (0.22), 2.33 (0.22), and
2.5 (0.19), respectively. Based on mere densitometry, 8 of 9
men met the International Society for Clinical Densitometry
(ISCD) criteria for the diagnosis of male osteoporosis (Zscore < 2.0). According to World Health Organization (WHO)
criteria (T-score < 2.5), two-thirds of our patients were
osteoporotic; the rest were osteopenic, with a T-score of at
least 2.1 at any of the sites. Mean 25-hydroxyvitamin D levels
were relatively low (32.5 ng/mL), but serum calcium, serum
phosphate, parathyroid hormone (PTH), total alkaline phosphatase, and urinary calcium excretion were normal in all patients. In
a single patient, a minimal increase in phosphate excretion was
found, but his serum phosphate level was normal. The biopsy of
that patient revealed no evidence of osteomalacia. TSH, total
testosterone, serum potassium and sodium, and electrophoresis
of all patients were normal. Routine blood chemistry showed no
evidence of kidney dysfunction or hepatic disease. Age-matched
healthy males 52 years of age, on average, served as in-house
controls for marker reference ranges. Metabolic parameters and
bone markers are presented in Table 1.
Three-dimensional (3D) microstructural assessment showed a
strong trend toward reduced trabecular bone volume (BV/TV) in
MIO patients (29%, p ¼ .096). MIO patients revealed a
significantly reduced number of trabeculae (Tb.N), a significantly
increased trabecular separation (Tb.Sp), and a significantly
reduced connectivity density (Conn.D) of the microstructure
compared with nonosteoporotic controls (Fig. 1). Interestingly,
we found increases in trabecular separation to be associated
with elevated but still normal urinary phosphate excretion
(r ¼ 0.810, p ¼ .015). Trabecular thickness (Tb.Th) was similar in
both groups. The structure model index (SMI) showed a slight
increase in MIO patients. Family history did not have any
influence on bone microstructure. The results of mCT analyses are
given in Table 3.
Table 1. Biochemical Markers of Bone Metabolism in MIO
Patients
Parameter
Serum calcium (mmol/L)
Serum phosphate (mmol/L)
Total alkaline phosphatase (U/L)
25-(OH)-vitamin D (ng/mL)
PTH (pg/mL)
24-Hour calcium excretion (mmol)
24-Hour phosphate excretion (mmol)
P1NP (ng/mL)
Osteocalcin (ng/mL)
CTX (ng/mL)
MIO
Reference
range
2.27 ( 0.03)
0.99 ( 0.04)
79 ( 5)
32.5 ( 5.4)
44.6 ( 4.0)
3.7 ( 0.6)
24.1 ( 4.5)
46.8 ( 4.9)
19.0 ( 1.6)
0.34 ( 0.4)
2.10–2.58
0.6–1.55
40–129
30
11.1–79.5
2.5–7.5
12.0–42.0
23.3–64.8
14.1–34.5
0.08–0.38
Note: Data are presented as mean SEM.
STRUCTURE AND GENE EXPRESSION IN MALE OSTEOPOROSIS
Table 2. Histomorphometric Parameters
Parameter
2
BV (mm )
BS (mm)
OS (mm)
OV (mm2)
Ma.V (mm2)
FatV (mm2)
BV/TV (%)
OV/BV (%)
OS/BS (%)
Ma.V/TV (%)
FatV/TV (%)
FatV/Ma.V (%)
N.Ob (n)
N.Ob/OS (n/mm)
N.Oc (n)
Oc.S (mm)
Oc.S/BS (%)
MIO
Controls
p Value
0.47 (0.29–0.52)
6.25 (4.17–7.98)
1.56 (0.69–3.49)
0.01 (0.00–0.01)
2.85 (2.76–3.00)
1.1 ( 0.1)
12.9 ( 1.5)
1.4 (1.1–2.8)
25.1 (20.1–44.5)
87.1 ( 1.5)
34.5 ( 3.1)
42.9 (31.4–47.8)
19 (8–25)
10.8 (8.2–15.8)
0.0 (0.0–0.8)
0.00 (0.00–0.04)
0.0 (0.0–0.6)
0.79 (0.61–0.97)
9.8 (8.55–13.9)
3.3 (2.09–5.23)
0.02 (0.01–0.03)
2.5 (2.33–2.64)
1.1 ( 0.1)
23.9 ( 1.8)
2.3 (1.2–3.2)
35.0 (19.6–50.1)
76.1 ( 1.8)
32.0 ( 4.2)
44.2 (28.6-53.0)
17 (14–24)
6.8 (3.9-8.5)
0.3 (0.0–1.4)
0.01 (0.00–0.08)
0.1 (0.0–0.8)
.002
.006
n.s.
.027
.006
n.s.
.000
n.s.
n.s.
.007
n.s.
n.s.
n.s.
n.s.
.04
n.s.
n.s.
Note: Parametric data are given as mean SEM; nonparametric data are
represented as median and 25th to 75th percentile.
Journal of Bone and Mineral Research
1587
Table 3. Micro–Computed Tomography (mCT)
Parameter
MIO
Controls
p Value
BV/TV (%)
15.44 (11.65–17.60) 21.60 (17.14–23.76) n.s.
Tb.Th (mm)
0.18 ( 0.04)
0.17 ( 0.03)
n.s.
Tb.N (1/mm)
1.08 (0.96–1.15)
1.33 (1.23–1.40)
.005
Tb.Sp (mm)
0.89 (0.83–0.99)
0.75 (0.68–0.80)
.004
Conn.D (1/mm3) 3.35 (2.41–5.38)
6.66 (4.80–7.17)
.038
SMI (1)
0.83 (0.70–1.30)
0.39 (0.15–1.06)
n.s.
Data are presented as mean SEM or as median and the 25th to 75th
percentile.
Gene expression
The expression of WNT10B, RUNX2, and RANKL was decreased
significantly in MIO patients. Osterix, Osteocalcin, and OPG
expression did not differ between the two groups (Fig. 2). MIO
patients also had a reduced RANKL/OPG ratio (62%, p ¼ .007).
Despite single outliers, the statistical range of the expression
profile of all genes except Osteocalcin was smaller in MIO
biopsies. Considering all biopsies, WNT10B expression correlated
significantly with RUNX2 (r ¼ 0.554, p ¼ .017), RANKL (r ¼ 0.617,
p ¼ .006), and local Osteocalcin (r ¼ 0.509, p ¼ .031), as well as BV/
TV (r ¼ 0.540, p ¼ .025). In addition, RUNX2 was associated with
Osterix (r ¼ 0.499, p ¼ .035), RANKL (r ¼ 0.509, p ¼ .031), and local
Osteocalcin expression (r ¼ 0.517, p ¼ .028). Moreover, RUNX2
correlated with PTH serum levels (r ¼ 0.683, p ¼ .042). Positive
correlations also were found for RANKL and BV/TV (r ¼ 0.489,
p ¼ .046), trabecular number (r ¼ 0.506, p ¼ .046), and osteoid
volume (r ¼ 0.519, p ¼ .033). However, RANKL was negatively
associated with Tb.Sp (r ¼ 0.576, p ¼ .019). Osterix and local
Osteocalcin expression correlated with osteoblast number
(r ¼ 0.594, p ¼ .012; r ¼ 0.709, p ¼ .001). OPG was positively
associated with age (r ¼ 0.520, p ¼ .027). SOST expression was
significantly lower in MIO patients (97%, p ¼ .002). In all
biopsies, SOST was strongly correlated with BV/TV (r ¼ 0.807,
p ¼ .000) and inversely related to marrow volume (Ma.V/TV;
r ¼ 0.807, p ¼ .000). Like trabecular microstructure, gene expression was independent of family history.
Discussion
To the best of our knowledge, this is the first MIO biopsy study
reporting reductions of WNT10B, RUNX2, and RANKL at the tissue
level. Being aware of the largely unknown and presumably
multifactorial etiology of MIO, we put major effort into recruiting
Fig. 1. Representative 3D reconstructions of MIO and control biopsies.
1588
Journal of Bone and Mineral Research
a clinically homogeneous patient cohort. The double-biopsy
approach enabled us to assess histomorphometric data and 3D
microstructure in addition to gene expression analyses. When
compared with other recent MIO studies,(26,27) our patient ages
were comparable. Since all participants had a history of
peripheral and/or vertebral low-trauma fractures, we are
confident to have examined patients with a clinically relevant
disease profile.
Other research groups used explant cell cultures from iliac
crest biopsies of MIO patients. In line with our study, they found
decreased osteoblastic DNA synthesis, impaired cell proliferation, and lower Osteocalcin gene expression on vitamin D
stimulation.(26,27) Supporting the model of osteoblast dysfunction, Pernow and colleagues reported a decreased proliferative
response to exogenous parathyroid hormone–related protein
[PTHrP(1–34)] but also increased basal PTHrP expression in
cultured MIO osteoblasts.(26) Demonstrating decreased gene
expression of WNT10B and RUNX2, our PCR data point to a
potential disturbance in osteoblast differentiation.
Osteoblast lineage cells arise from mesenchymal stem cells
(MSCs). In the presence of specific factors, including bone
morphogenetic protein 2 (BMP-2), insulin-like growth factor 1
(IGF-1), 1,25-hydroxyvitamin D, or PTH, MSCs differentiate into
preosteoblasts. Differentation into osteoblasts further requires
Wnt pathway–dependent intracellular accumulation of bcatenin, as well as the expression of certain osteoblast-specific
genes, namely, RUNX2 and Osterix.2 Wnt10b transgenic mice
display major increases in bone mass and mechanical
competence, as well as a resistance to aging- and ovariectomy-induced bone loss.(28) Since Wnt10b seems to enhance
osteoblastogenesis and to inhibit osteoblast apoptosis, the
osteoblastic transcription factors runx2, dlx5, and osterix were
found to be upregulated in these transgenic mice. In reverse,
Wnt10b/ mice display substantial bone loss after only a few
weeks of life.(29) Somewhat surprisingly, prior to these changes,
young Wnt10b/ mice exhibit a rich trabecular microarchitecture.(30) Stevens and colleagues concluded that this high-to-low
bone quality sequence could provide evidence for an early
depletion of the osteoprogenitor pool. Pointing at the potential
importance of WNT10B in human medicine, genetic association
analyses revealed a relation of WNT10B polymorphisms with hip
bone mineral density (BMD) in Afro-Caribbean offspring.(31)
Although such associations were not detectable in a cohort of
Spanish postmenopausal women,(32) we found reduced WNT10B
expression in iliac crest biopsies of MIO patients. The potential
relevance of reduced WNT10B expression in MIO bone tissue is
supported by a significant correlation with decreased local
trabecular bone volume (BV/TV). Positive correlations of WNT10B
with RUNX2, Osteocalcin, and RANKL indicate potential inhibitory
downstream effects on osteoblastic transcriptional activity, but
expression profiles have been studied in tissue samples and not
pure human osteoblasts. Therefore, it would be of great interest
to confirm the downregulation of RUNX2, Osteocalcin, and RANKL
expression by low WNT10B via cell culture experiments.
RUNX2 and Osterix are key osteogenic transcription factors
that are required for osteoblastic differentiation of MSCs.(33)
Owing to the entire absence of osteoblasts, Runx2/ and Osx/
mice are lacking bone from birth.(34,35) Leading to preosteoblast
PATSCH ET AL.
Fig. 2. Gene expression (DDCt) of WNT10B, RUNX2, Osterix (OSX), RANKL, OPG, and Osteocalcin (OC). The asterisk () indicates p < .05. Data are shown as
box-and-whisker plots.
formation, RUNX2 is upregulated by BMP-2, IGF-1, and Wnt
signaling.(36,37) Despite its osteoinductive role in preosteoblasts
and immature cells, RUNX2 was shown to inhibit the late stage of
osteoblast maturation.(38,39) Osterix is considered to act downstream of RUNX2.(35,40) In our study, MIO patients exhibited
significantly decreased RUNX2 levels that were correlated with
low Osterix, Osteocalcin, and RANKL expression. However, Osterix
only displayed a trend decrease in osteoporotic patients. It has
been published that Wnt signaling and RUNX2 inhibit adipogenesis.(41) Moreover, elderly osteopenic and osteoporotic patients
seem to exhibit a shift toward increased fat in their marrow.(42,43)
Despite these reports, we did not observe such marrow changes
STRUCTURE AND GENE EXPRESSION IN MALE OSTEOPOROSIS
in our study. We speculate that this could be due to the relatively
young age of MIO patients enrolled.
RANKL belongs to the tumor necrosis factor (TNF) ligand
superfamily and is of pivotal importance for the generation,
activity, and survival of osteoclasts.(44,45) Acting as a decoy
receptor, its endogenous opponent, osteoprotegerin (OPG),
blocks RANKL action and thereby balances bone turnover.(46) In
bone, RANKL is expressed most abundantly by MSCs, osteoblasts,
and T cells.(47) Since osteoblast-osteoclast coupling is exerted
mainly by immature cells of the osteoblast lineage, RANKL
expression is inversely related to osteoblast differentiation.(48)
Similarly, OPG is produced by MSCs and osteoblasts. Besides PTH
Journal of Bone and Mineral Research
1589
and 1,25-hydroxyvitamin D, RANKL expression is stimulated by
interleukin 6 (IL-6)–type ligands. Wnt signaling seems to have
inhibitory effects on RANKL, but Wnts enhance OPG.(49) RANKL
and OPG are opposedly regulated by RUNX2, which matches the
reductions in RANKL expression and osteoclast number that we
found in MIO patients.(50–52) Increased expression of RANKL and
IL-6 was reported from female hip fracture patients,(53,54)
suggesting major pathophysiologic differences between MIO
and postmenopausal osteoporosis.
Sclerostin, the SOST gene product, is a strong inhibitor of
osteoblastogenesis and enhancer of osteoblast apoptosis.(55)
Specific loss-of-function mutations are associated with Van
Buchem disease and sclerosteosis, two human sclerosing bone
disorders.(56) Contrasting the literature, SOST expression was
lower in patients with osteoporosis. Further, SOST correlated
positively with trabecular number and inversely with trabecular
separation. The strongest correlations were found with marrow
volume, a parameter that inversely depends on bone volume.
Since osteocytes are the main source of sclerostin, we assume
that the reduction of SOST expression is due to reduced
trabecular volume. Hypothetically, the SOST reduction in MIO
also could indicate an autoregulatory rescue mechanism, but
these assumptions were not further tested and thus remain
speculative.
In our study, MIO patients displayed a much smaller overall
range of gene expression than controls. These findings overlap
with a study from Balla and colleagues investigating tissuespecific gene expression in elderly postmenopausal women.
These authors analyzed the expression of 96 selected genes in
femoral head bone samples obtained during hip arthroplasty
and found a decreased expression pattern of almost all genes.(57)
The mean age of the study participants was about 70 years. At
that stage, postmenopausal high turnover is mostly replaced by
an age-related low-bone-turnover pattern. Likewise, Marie and
colleagues reported decreased DNA synthesis in cultured MIO
osteoblasts.(13) These findings, together with our results, provoke
speculation that low-turnover osteoporosis could be a disease
set off, maintained, or at least partially expressed by insufficient
transcriptional activity of osteoblasts.
The histomorphometric pattern that we found in the biopsies
resembled that of previous MIO studies.(24,58,59) Low trabecular
bone volume (BV/TV) was accompanied by decreased amounts
of osteoid. Osteoclast number was reduced, further suggesting
low bone turnover. Since high-turnover patterns were found in
hypercalciuric MIO patients,(24) the low number of osteoclasts
matches the normocalciuria of our patients. The number of
osteoblasts was similar among the two groups. Osteoporotic
alterations of bone microstructure seem to be genderdependent. Like postmenopausal women, osteoporotic men
were found to lose trabecular bone volume. Trabecular thinning
that is reminiscent of glucocorticoid-induced osteoporosis is
considered to predominate in men. Trabecular perforations are
classified as a more female than male disease characteristic.(60)
Although age-related changes in men do not seem to involve
major connectivity loss, males with vertebral fractures display
this feature.(61) Using mCT for the first time in a clinically
homogeneous MIO cohort, we found significant decreases in
trabecular number combined with increases in trabecular
1590
Journal of Bone and Mineral Research
separation. Distinguishing the MIO pattern from age-related
changes, we also observed major losses of trabecular connectivity. However, microstructural properties of MIO patients
with vertebral fractures did not differ from those of patients with
other fractures (data not shown). Trabecular thickness was
unchanged, and plate-to-rod transitions were evident only as a
nonsignificant trend.
Further interpreting our study from a clinical point of view, it is
interesting that neither gene expression nor trabecular microstructure differed in patients with or without a positive family
history. Not entirely matching the low-turnover pattern observed
at the tissue level, mean serum CTX levels were close to the
upper limit of the age- and sex-matched normal range. Providing
additional evidence for potentially inadequate osteoclastosteoblast coupling, P1NP and osteocalcin were centered within
the normal range. However, when interpreting the marker
results, it should be restated that age- and sex-matched but still
individually different controls were used here rather than for the
rest of the study. In accordance with the presumptive osteoblast
pathology observed throughout our study, serum P1NP levels
were associated with osteoid volume and bone surface, which
per se were decreased.
A major strength of our study is the diligent definition of
inclusion and exclusion criteria, which aimed at optimization of
patient homogeneity. However, since MIO is considered to be a
strongly heterogeneous entity, a homogeneous group of
patients is difficult to define. Another limitation of our study
was the decision for static histomorphometry. We opted against
tetracycline labeling because it is unknown whether gene
expression would have remained unbiased. Although our control
data were stable, and Kulibawa and colleagues provided
evidence for adequate quality of postmortem bone RNA, control
sampling from forensic autopsies may be technically challenging.(62) Nevertheless, we believe that performing biopsies in
healthy individuals raises ethical concerns. Finally, it remains
arguable whether the iliac crest, as a non-weight-bearing site,
provides representative tissue information. Similar to sitespecific characteristics in bone microstructure, gene expression
patterns also may depend on location and bone compartment.
In conclusion, our data strongly support the hypothesis of
osteoblast dysfunction in MIO. Regarding this potential
pathomechanism, osteoanabolic drugs could provide a specifically promising therapeutic option for MIO patients with low
bone turnover.
Disclosures
JMP has received speaker honoraria from Amgen. HR serves as a
paid consultant for Eli Lilly, Amgen, Roche, Novartis, Nycomed,
and Servier. He is a speaker for Merck (MSD), Lilly, Servier, Roche,
and Nycomed and has received educational grants/research
support from Lilly and Roche. CB is a paid consultant for
Roche Diagnostics. PP has received research support and/or
honoraria from Amgen GmbH, Eli Lilly GmbH, Leo Pharma,
Merck, Sharp and Dohme GmbH, Novartis Pharma, Nycomed
Pharma, Roche Austria, Servier Austria, and Sanofi-Aventis. CM is
speaker for Eli Lilly, Amgen, Nycomed, Servier, Roche, Daichi
PATSCH ET AL.
Sankyo, Novartis, and Sanofi-Aventis. He has received research
grants from Roche Austria and is member of the national
advisory boards for Amgen Austria, Eli Lilly Austria, and Novartis
Austria. TK is stock owner of B-cube AG and a member of the
board of directors. All the other authors state that they have no
conflicts of interest.
Acknowledgments
We are grateful to Prof Philippe Zysset, Dr Thomas Woegerbauer,
Dr Peter Varga, Mag Julia Deutschmann, Mrs Katharina Wahl, Mrs
Gerda Dinst, and Mrs Daniela Gabriel for medical, technical, and
logistic support. This research was supported by the Austrian
Federal Bank (Grant No. 12544 to PP) and educational grants
from VINFORCE, Roche Diagnostics, Roche Austria, and Eli Lilly
Austria.
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