Introduction
Introduction
Osteoporosis is a complex systemic disease [1 2
Pigs as well as sheep are among the most frequently used large animal species in osteoporosis
research. Although there are a lot of similarities of diverse porcine organ systems
to their human analogues, the pig's usefulness as an osteologic model species is still
not entirely clear. However, even though porcine femoral compact bone is predominantly
plexiforme, it is converted to well-developed osteonal bone earlier than in sheep
[3 2+ ), which had been started already at an age of 4 months, led to a 10% reduction in
vertebral BMD and significant increases in final erosion depth and vertebral marrow
star volume [4 2+ ) showed moderate and transient increases in plasma PTH, calcitriol and bone-specific
alkaline phosphatase (BAP) levels over a time span of 1 year after OVX [5 6
Materials and Methods
Materials and Methods
Animals and group assignment
32 multiparous Large White sows aged 33.5±9.6 months in the mean and all of them approximately
2 months post partum and thus not lactating for over 5 weeks were allocated equally
to 4 groups to compare the effects of OVX and dietary calcium shortage: (I) OVX, 0.75%
Ca2+ , (II) OVX, 0.3% Ca2+ , (III) sham-OVX, 0.75% Ca2+ , and (IV) sham-OVX, 0.3% Ca2+ . Ovariectomy was performed from the left flank under full anesthesia with an IV bolus
of ketamine and azaperone. Post-surgical treatment was done with enrofloxacine, metamizole,
and local oxytetracycline spray. Sows were housed as groups of 5 or 6 in a separate
stable under standard conditions. Diets were mainly based on barley, soy, and lignocellulose
and produced by a farm animals’ feed company (Biomin, Herzogenburg, Austria). One
diet contained the usually supplemented calcium levels for nongestating sows, that
is, 0.75%, and the other one was a low calcium diet containing 0.3%. For detailed
composition of diets see [Table 1
Table 1 Composition of diets (per kg)
<TD VALIGN="TOP">
</TD><TD VALIGN="TOP">
Low-calcium diet
</TD><TD VALIGN="TOP">
Physiological diet
</TD>
<TD VALIGN="TOP">
ME (MJ)
</TD><TD VALIGN="TOP">
11.1
</TD><TD VALIGN="TOP">
11.1
</TD>
<TD VALIGN="TOP">
Crude protein (%)
</TD><TD VALIGN="TOP">
13.0
</TD><TD VALIGN="TOP">
13.0
</TD>
<TD VALIGN="TOP">
Crude fat (g)
</TD><TD VALIGN="TOP">
22.0
</TD><TD VALIGN="TOP">
22.0
</TD>
<TD VALIGN="TOP">
Crude fiber (g)
</TD><TD VALIGN="TOP">
102.0
</TD><TD VALIGN="TOP">
102.0
</TD>
<TD VALIGN="TOP">
Vit A (IU)
</TD><TD VALIGN="TOP">
9 900
</TD><TD VALIGN="TOP">
9 900
</TD>
<TD VALIGN="TOP">
Vit D3 (IU)
</TD><TD VALIGN="TOP">
1 320
</TD><TD VALIGN="TOP">
1 320
</TD>
<TD VALIGN="TOP">
Vit E (mg)
</TD><TD VALIGN="TOP">
70
</TD><TD VALIGN="TOP">
70
</TD>
<TD VALIGN="TOP">
Ca (g)
</TD><TD VALIGN="TOP">
3.0
</TD><TD VALIGN="TOP">
7.5
</TD>
<TD VALIGN="TOP">
P (g)
</TD><TD VALIGN="TOP">
3.8
</TD><TD VALIGN="TOP">
6.0
</TD>
<TD VALIGN="TOP">
Na (g)
</TD><TD VALIGN="TOP">
1.7
</TD><TD VALIGN="TOP">
1.7
</TD>
<TD VALIGN="TOP">
Cu (mg)
</TD><TD VALIGN="TOP">
16.5
</TD><TD VALIGN="TOP">
16.5
</TD>
Biochemical bone markers and hormones
Serum levels of following bone metabolism markers were determined by commercially
available ELISA kits, which have been validated also for pigs [7 8 ® BAP ELISA, Qidel Corporation, San Diego, CA, USA), osteocalcin (Metra® Osteocalcin ELISA, Qidel Corporation), CICP (Metra® CICP ELISA, Qidel Corporation), pyridinoline (Metra® Serum PYD ELISA, Qidel Corporation), and crosslaps (Serum CrossLaps® ELISA, Immunodiagnostic Systems, Frankfurt/Main, Germany). For measurement of serum
estrogens, a home made enzyme immunoassay for the detection of estron and 17β-estradiol
was applied [9
Hematological and immunological analyses
Hematological analyses were performed out of EDTA-blood using an ADVIA® 120 (Siemens Health Care Diagnostics, Deerfield, IL, USA) with veterinary software
adapted for pigs. Cytokine levels were determined at both the genomic as well as the
protein levels. For preparation of total RNA out of peripheral blood mononuclear cells
(PBMCs), the pellet was suspended in 1.2 ml of hemolysis buffer (140 mM NH4 Cl, 17 mM Tris, pH 7.2) and incubated at 37°C for 15 min. Samples were then centrifuged
and supernatant was removed. Pelleted white blood cells were resuspended in 1 ml of
TriReagent® (Molecular Research Center, Inc., Cincinnati, USA) and frozen at −80°C until analysis.
Total RNA was extracted in accordance to the manufacturers’ protocol. Integrity, quantity,
and contamination with genomic DNA were analyzed on the Agilent BioAnalyzer 2100 (Agilent
Technologies, Palo Alto, USA) using the RNA6000 Nano LabChip® kit (Agilent). 1 μg of total RNA was used to synthesize cDNA using SuperscriptTM II RNAse H-reverse transcriptase (200 U/reaction; Invitrogen, Carlsbad, USA) and
anchored oligo-dT-primers (3.5 μM final concentration). To check the generation of
amplifiable cDNA in the reverse transcription, a conventional PCR step was performed
using GAPDH specific primers. The profiling of the expressions of the cytokine genes
interleukin (IL)-1, IL-2, IL-4, IL-6, IL-10, interferon (IFN)-γ, tumor necrosis factor
(TNF)-α, as well as inducible NO synthase (iNOS) and heme oxygenase (HO)-1 and the
3 housekeeping genes cyclophilin, GAPDH, and β-actin was performed by means of real-time
triplex PCR, using TaqMan® probes and specific primer pairs on the iCycler iQ® (Bio-Rad, Hercules, USA) as described by others [10
Intracellular cytokine expressions of IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p35, TNF-α,
and IFN-γ were measured in PBMCs as described previously using a FACSAria® flow cytometer (Becton Dickinson, San Jose, CA, USA) [11 11
μCT and DXA analyses of selected bone sites
The following bone sites were considered for μCT imaging and analysis: sixth lumbar
vertebra (L6), proximal tibia, and distal radius. Bones were frozen at −80°C until
preparation. After freeing bones from soft tissue, a transverse section was obtained
from each site of interest and from each animal (diamond band saw, 300 CP, Exakt GmbH,
Germany). Then, one trabecular bone cylinder of 8 mm in nominal diameter and 12 mm
in nominal length was extracted from each section using a diamond coated coredrill.
The biopsies were immersed in saline solution, freed of air bubbles by application
of vacuum and scanned with 12 μm resolution using a μCT 40 system (ScancoMedical AG,
Switzerland) with 70 kV, 114 mA, and 200 ms integration time. The region of interest
was restricted to an inner cylinder with 7 mm diameter and 11.5 mm length via contouring
to exclude peripheral artefacts. Morphological analysis of each biopsy was performed
with the inbuilt software tools provided by the manufacturer (IPL). The images were
filtered using a Gaussian filter (sigma=0.7, support=1) and segmented using a global
threshold value corresponding to 20% of the maximum gray value intensity. A standard
evaluation protocol was used to compute the histomorphometric parameters from the
segmented images. 2 biopsies containing a significant part of the growth plate were
excluded from the analysis.
DXA scans of femoral neck, trochanter, and the total region including head, neck,
and trochanter were performed using a Lunar iDXA® bone densitometer with prepared femora being placed in a water bath for mimicking
soft tissue.
Bone ash analysis
The volume of each trabecular bone biopsy used for μCT analysis was calculated out
of 5 consecutive measurements of height and diameter. Ashing was performed for 24 h
at 650°C and ash weight was measured after a consecutive cooling down phase at room
temperature for another 24 h.
Statistics
Statistical evaluation was performed by PASW-Statistic Software, version 17.0. 2.
All variables were tested for normal distribution. As most of them fulfilled the requirements
for parametric evaluation, ANOVA was applied for group comparisons. In cases where
normal distribution was excluded, a Kruskal-Wallis test was used. For post hoc analysis,
a Bonferroni test was applied.
Results
Results
Hematology and cellular immune parameters
Hematological analyses including FACS-based analysis of the distribution of antigen-presenting
cell and lymphocyte subpopulations as well as cytokine profile kinetics revealed no
meaningful trends over the course of the trial. Also, no differences could be observed
between the 4 groups at either time point. Instead, cytokine titers or cell numbers
of immunocyte subpopulations were found within the respective reference ranges [11
Biochemical bone markers and hormones
Mean values and standard deviations of investigated biochemical bone markers of the
last sampling session are given in [Table 2
Table 2 Biochemical bone marker levels acquired at the termination of the experiment (10
months post OVX)
<TD VALIGN="TOP">
</TD><TD VALIGN="TOP" COLSPAN="2">
Group 1 (OVX)
</TD><TD VALIGN="TOP" COLSPAN="2">
Group 2 (OVX, low Ca)
</TD><TD VALIGN="TOP" COLSPAN="2">
Group 3 (Sham)
</TD><TD VALIGN="TOP" COLSPAN="2">
Group 4 (Sham, low Ca)
</TD><TD VALIGN="TOP">
</TD>
<TD VALIGN="TOP">
</TD><TD VALIGN="TOP">
Mean
</TD><TD VALIGN="TOP">
SD
</TD><TD VALIGN="TOP">
Mean
</TD><TD VALIGN="TOP">
SD
</TD><TD VALIGN="TOP">
Mean
</TD><TD VALIGN="TOP">
SD
</TD><TD VALIGN="TOP">
Mean
</TD><TD VALIGN="TOP">
SD
</TD><TD VALIGN="TOP">
p-Value
</TD>
<TD VALIGN="TOP">
sRANKL (pmol/l)
</TD><TD VALIGN="TOP">
0.7
</TD><TD VALIGN="TOP">
0.6
</TD><TD VALIGN="TOP">
0.6
</TD><TD VALIGN="TOP">
0.7
</TD><TD VALIGN="TOP">
0.5
</TD><TD VALIGN="TOP">
0.5
</TD><TD VALIGN="TOP">
0.4
</TD><TD VALIGN="TOP">
0.6
</TD><TD VALIGN="TOP">
0.89
</TD>
<TD VALIGN="TOP">
BAP (E/l)
</TD><TD VALIGN="TOP">
13.5
</TD><TD VALIGN="TOP">
4.2
</TD><TD VALIGN="TOP">
16.5
</TD><TD VALIGN="TOP">
2.3
</TD><TD VALIGN="TOP">
14.7
</TD><TD VALIGN="TOP">
5.7
</TD><TD VALIGN="TOP">
15.7
</TD><TD VALIGN="TOP">
5.2
</TD><TD VALIGN="TOP">
0.55
</TD>
<TD VALIGN="TOP">
OC (ng/ml)
</TD><TD VALIGN="TOP">
194.7
</TD><TD VALIGN="TOP">
139.3
</TD><TD VALIGN="TOP">
304.2
</TD><TD VALIGN="TOP">
88.6
</TD><TD VALIGN="TOP">
236.2
</TD><TD VALIGN="TOP">
150.4
</TD><TD VALIGN="TOP">
192.7
</TD><TD VALIGN="TOP">
106.8
</TD><TD VALIGN="TOP">
0.34
</TD>
<TD VALIGN="TOP">
CICP (ng/ml)
</TD><TD VALIGN="TOP">
40.0
</TD><TD VALIGN="TOP">
38.3
</TD><TD VALIGN="TOP">
71.2
</TD><TD VALIGN="TOP">
109.7
</TD><TD VALIGN="TOP">
32.9
</TD><TD VALIGN="TOP">
53.2
</TD><TD VALIGN="TOP">
88.7
</TD><TD VALIGN="TOP">
74.2
</TD><TD VALIGN="TOP">
0.54
</TD>
<TD VALIGN="TOP">
PYD (nmol/l)
</TD><TD VALIGN="TOP">
8.5
</TD><TD VALIGN="TOP">
0.9
</TD><TD VALIGN="TOP">
8.0
</TD><TD VALIGN="TOP">
0.8
</TD><TD VALIGN="TOP">
8.5
</TD><TD VALIGN="TOP">
0.4
</TD><TD VALIGN="TOP">
8.4
</TD><TD VALIGN="TOP">
0.6
</TD><TD VALIGN="TOP">
0.55
</TD>
<TD VALIGN="TOP">
Crosslaps (ng/ml)
</TD><TD VALIGN="TOP">
1.5
</TD><TD VALIGN="TOP">
0.9
</TD><TD VALIGN="TOP">
2.0
</TD><TD VALIGN="TOP">
1.3
</TD><TD VALIGN="TOP">
0.9
</TD><TD VALIGN="TOP">
0.7
</TD><TD VALIGN="TOP">
0.9
</TD><TD VALIGN="TOP">
0.6
</TD><TD VALIGN="TOP">
0.16
</TD>
<TD VALIGN="TOP">
PTH (pg/ml)
</TD><TD VALIGN="TOP">
16.3
</TD><TD VALIGN="TOP">
30.6
</TD><TD VALIGN="TOP">
19.5
</TD><TD VALIGN="TOP">
37.1
</TD><TD VALIGN="TOP">
23.2
</TD><TD VALIGN="TOP">
19.1
</TD><TD VALIGN="TOP">
34.6
</TD><TD VALIGN="TOP">
33.8
</TD><TD VALIGN="TOP">
0.40
</TD>
<TD VALIGN="TOP">
VitD3 (nmol/l)
</TD><TD VALIGN="TOP">
170.1
</TD><TD VALIGN="TOP">
101.8
</TD><TD VALIGN="TOP">
157.5
</TD><TD VALIGN="TOP">
106.6
</TD><TD VALIGN="TOP">
212.9
</TD><TD VALIGN="TOP">
19.3
</TD><TD VALIGN="TOP">
123.7
</TD><TD VALIGN="TOP">
92.9
</TD><TD VALIGN="TOP">
0.47
</TD>
Serum estrogens (estron and 17β-estradiol) were measured at baseline, 1 month after
OVX, and at the termination of the experiment. At baseline, estrogens were detectable
in only 13 out of 32 animals, giving evidence of 13 sows being in estrus at that time
point. 1 month post OVX, estrogens were detectable in 6/16 ovariectomized sows as
compared to 10/16 non-ovariectomized animals. At the end of the experiment, only 1
ovariectomized sow had a detectable estrogen titer (4.6 pg/ml), whereas estrogen titers
of 7 non-ovariectomized sows were exceeding the detection limit.
Morphological and densitometrical analyses
Also morphological as well as densitometrical analyses gave no evidence of any impact
of OVX or calcium shortage on bone microstructure or density ([Table 3 4 Fig. 1
Fig. 1 2-dimensional μCT images of trabecular bone of distal radius, proximal tibia, and
sixth lumbar vertebra of representative sows of each group. Measurements were started
at a predefined distance from the end of the samples to avoid inclusion of growth
plates. Different anatomical sites exhibited characteristic micromorphological features
but were uniform between groups.
Table 3 Bone mineral content at 10 months post OVX
<TD VALIGN="TOP">
</TD><TD VALIGN="TOP" COLSPAN="2">
Group 1 (OVX)
</TD><TD VALIGN="TOP" COLSPAN="2">
Group 2 (OVX, low Ca)
</TD><TD VALIGN="TOP" COLSPAN="2">
Group 3 (Sham)
</TD><TD VALIGN="TOP" COLSPAN="2">
Group 4 (Sham, low Ca)
</TD><TD VALIGN="TOP">
</TD>
<TD VALIGN="TOP">
</TD><TD VALIGN="TOP">
Mean
</TD><TD VALIGN="TOP">
SD
</TD><TD VALIGN="TOP">
Mean
</TD><TD VALIGN="TOP">
SD
</TD><TD VALIGN="TOP">
Mean
</TD><TD VALIGN="TOP">
SD
</TD><TD VALIGN="TOP">
Mean
</TD><TD VALIGN="TOP">
SD
</TD><TD VALIGN="TOP">
p-Value
</TD>
<TD VALIGN="TOP" COLSPAN="10">
DXA (g/cm2 )
</TD>
<TD VALIGN="TOP">
Neck
</TD><TD VALIGN="TOP">
1.47
</TD><TD VALIGN="TOP">
0.08
</TD><TD VALIGN="TOP">
1.56
</TD><TD VALIGN="TOP">
0.16
</TD><TD VALIGN="TOP">
1.48
</TD><TD VALIGN="TOP">
0.11
</TD><TD VALIGN="TOP">
1.43
</TD><TD VALIGN="TOP">
0.15
</TD><TD VALIGN="TOP">
0.34
</TD>
<TD VALIGN="TOP">
Trochanter
</TD><TD VALIGN="TOP">
1.59
</TD><TD VALIGN="TOP">
0.14
</TD><TD VALIGN="TOP">
1.58
</TD><TD VALIGN="TOP">
0.09
</TD><TD VALIGN="TOP">
1.76
</TD><TD VALIGN="TOP">
0.2
</TD><TD VALIGN="TOP">
1.6
</TD><TD VALIGN="TOP">
0.34
</TD><TD VALIGN="TOP">
0.51
</TD>
<TD VALIGN="TOP">
Total
</TD><TD VALIGN="TOP">
1.67
</TD><TD VALIGN="TOP">
0.09
</TD><TD VALIGN="TOP">
1.67
</TD><TD VALIGN="TOP">
0.05
</TD><TD VALIGN="TOP">
1.76
</TD><TD VALIGN="TOP">
0.09
</TD><TD VALIGN="TOP">
1.62
</TD><TD VALIGN="TOP">
0.26
</TD><TD VALIGN="TOP">
0.54
</TD>
<TD VALIGN="TOP" COLSPAN="10">
Bone ash values (mg/cm3 )
</TD>
<TD VALIGN="TOP">
Radius
</TD><TD VALIGN="TOP">
380.0
</TD><TD VALIGN="TOP">
61.0
</TD><TD VALIGN="TOP">
393.7
</TD><TD VALIGN="TOP">
108.9
</TD><TD VALIGN="TOP">
393.7
</TD><TD VALIGN="TOP">
49.1
</TD><TD VALIGN="TOP">
346.2
</TD><TD VALIGN="TOP">
45.5
</TD><TD VALIGN="TOP">
0.65
</TD>
<TD VALIGN="TOP">
Tibia
</TD><TD VALIGN="TOP">
344.3
</TD><TD VALIGN="TOP">
95.4
</TD><TD VALIGN="TOP">
339.3
</TD><TD VALIGN="TOP">
113.8
</TD><TD VALIGN="TOP">
312.0
</TD><TD VALIGN="TOP">
94.5
</TD><TD VALIGN="TOP">
282.7
</TD><TD VALIGN="TOP">
78.9
</TD><TD VALIGN="TOP">
0.65
</TD>
<TD VALIGN="TOP">
Lumbar vertebra
</TD><TD VALIGN="TOP">
394.7
</TD><TD VALIGN="TOP">
55.3
</TD><TD VALIGN="TOP">
374.9
</TD><TD VALIGN="TOP">
60.7
</TD><TD VALIGN="TOP">
394.3
</TD><TD VALIGN="TOP">
65.3
</TD><TD VALIGN="TOP">
385.2
</TD><TD VALIGN="TOP">
39.4
</TD><TD VALIGN="TOP">
0.91
</TD>
Table 4 μCT data of respective bone sites at 10 months post OVX
<TD VALIGN="TOP">
Bone site
</TD><TD VALIGN="TOP" COLSPAN="2">
Group 1 (OVX)
</TD><TD VALIGN="TOP" COLSPAN="2">
Group 2 (OVX, low Ca)
</TD><TD VALIGN="TOP" COLSPAN="2">
Group 3 (Sham)
</TD><TD VALIGN="TOP" COLSPAN="2">
Group 4 (Sham, low Ca)
</TD><TD VALIGN="TOP">
</TD>
<TD VALIGN="TOP">
</TD><TD VALIGN="TOP">
Mean
</TD><TD VALIGN="TOP">
SD
</TD><TD VALIGN="TOP">
Mean
</TD><TD VALIGN="TOP">
SD
</TD><TD VALIGN="TOP">
Mean
</TD><TD VALIGN="TOP">
SD
</TD><TD VALIGN="TOP">
Mean
</TD><TD VALIGN="TOP">
SD
</TD><TD VALIGN="TOP">
p-Value
</TD>
<TD VALIGN="TOP" COLSPAN="10">
Radius
</TD>
<TD VALIGN="TOP">
BMD apparent (mg/cm3 )
</TD><TD VALIGN="TOP">
357.5
</TD><TD VALIGN="TOP">
68.4
</TD><TD VALIGN="TOP">
404.3
</TD><TD VALIGN="TOP">
111.0
</TD><TD VALIGN="TOP">
376.0
</TD><TD VALIGN="TOP">
58.2
</TD><TD VALIGN="TOP">
341.2
</TD><TD VALIGN="TOP">
43.1
</TD><TD VALIGN="TOP">
0.50
</TD>
<TD VALIGN="TOP">
BMD tissue (mg/cm3 )
</TD><TD VALIGN="TOP">
1 048.1
</TD><TD VALIGN="TOP">
40.9
</TD><TD VALIGN="TOP">
1 093.3
</TD><TD VALIGN="TOP">
120.6
</TD><TD VALIGN="TOP">
1 047.8
</TD><TD VALIGN="TOP">
19.9
</TD><TD VALIGN="TOP">
1 075.6
</TD><TD VALIGN="TOP">
24.6
</TD><TD VALIGN="TOP">
0.58
</TD>
<TD VALIGN="TOP">
BV/TV (%)
</TD><TD VALIGN="TOP">
0.327
</TD><TD VALIGN="TOP">
0.058
</TD><TD VALIGN="TOP">
0.349
</TD><TD VALIGN="TOP">
0.090
</TD><TD VALIGN="TOP">
0.339
</TD><TD VALIGN="TOP">
0.048
</TD><TD VALIGN="TOP">
0.304
</TD><TD VALIGN="TOP">
0.038
</TD><TD VALIGN="TOP">
0.63
</TD>
<TD VALIGN="TOP">
BS/BV (%)
</TD><TD VALIGN="TOP">
12.594
</TD><TD VALIGN="TOP">
2.160
</TD><TD VALIGN="TOP">
12.062
</TD><TD VALIGN="TOP">
2.374
</TD><TD VALIGN="TOP">
12.504
</TD><TD VALIGN="TOP">
1.932
</TD><TD VALIGN="TOP">
12.725
</TD><TD VALIGN="TOP">
1.050
</TD><TD VALIGN="TOP">
0.93
</TD>
<TD VALIGN="TOP">
Tb.N (/mm)
</TD><TD VALIGN="TOP">
2.019
</TD><TD VALIGN="TOP">
0.212
</TD><TD VALIGN="TOP">
2.031
</TD><TD VALIGN="TOP">
0.234
</TD><TD VALIGN="TOP">
2.087
</TD><TD VALIGN="TOP">
0.104
</TD><TD VALIGN="TOP">
1.922
</TD><TD VALIGN="TOP">
0.123
</TD><TD VALIGN="TOP">
0.52
</TD>
<TD VALIGN="TOP">
Tb. Th (mm)
</TD><TD VALIGN="TOP">
0.162
</TD><TD VALIGN="TOP">
0.024
</TD><TD VALIGN="TOP">
0.171
</TD><TD VALIGN="TOP">
0.032
</TD><TD VALIGN="TOP">
0.162
</TD><TD VALIGN="TOP">
0.024
</TD><TD VALIGN="TOP">
0.158
</TD><TD VALIGN="TOP">
0.013
</TD><TD VALIGN="TOP">
0.81
</TD>
<TD VALIGN="TOP">
Tb. Sp (mm)
</TD><TD VALIGN="TOP">
0.337
</TD><TD VALIGN="TOP">
0.061
</TD><TD VALIGN="TOP">
0.327
</TD><TD VALIGN="TOP">
0.079
</TD><TD VALIGN="TOP">
0.317
</TD><TD VALIGN="TOP">
0.028
</TD><TD VALIGN="TOP">
0.363
</TD><TD VALIGN="TOP">
0.040
</TD><TD VALIGN="TOP">
0.57
</TD>
<TD VALIGN="TOP">
Degree of anisotropy
</TD><TD VALIGN="TOP">
2.303
</TD><TD VALIGN="TOP">
0.286
</TD><TD VALIGN="TOP">
2.184
</TD><TD VALIGN="TOP">
0.439
</TD><TD VALIGN="TOP">
2.142
</TD><TD VALIGN="TOP">
0.301
</TD><TD VALIGN="TOP">
2.429
</TD><TD VALIGN="TOP">
0.198
</TD><TD VALIGN="TOP">
0.44
</TD>
<TD VALIGN="TOP">
ConnDens (/mm3 )
</TD><TD VALIGN="TOP">
5.818
</TD><TD VALIGN="TOP">
1.547
</TD><TD VALIGN="TOP">
7.635
</TD><TD VALIGN="TOP">
3.236
</TD><TD VALIGN="TOP">
6.118
</TD><TD VALIGN="TOP">
1.021
</TD><TD VALIGN="TOP">
5.837
</TD><TD VALIGN="TOP">
1.254
</TD><TD VALIGN="TOP">
0.33
</TD>
<TD VALIGN="TOP">
Structure model index
</TD><TD VALIGN="TOP">
−0.439
</TD><TD VALIGN="TOP">
0.276
</TD><TD VALIGN="TOP">
−0.448
</TD><TD VALIGN="TOP">
0.693
</TD><TD VALIGN="TOP">
−0.472
</TD><TD VALIGN="TOP">
0.289
</TD><TD VALIGN="TOP">
−0.053
</TD><TD VALIGN="TOP">
0.351
</TD><TD VALIGN="TOP">
0.34
</TD>
<TD VALIGN="TOP" COLSPAN="10">
Tibia
</TD>
<TD VALIGN="TOP">
BMD apparent (mg/cm3 )
</TD><TD VALIGN="TOP">
320.2
</TD><TD VALIGN="TOP">
98.9
</TD><TD VALIGN="TOP">
298.1
</TD><TD VALIGN="TOP">
108.3
</TD><TD VALIGN="TOP">
294.3
</TD><TD VALIGN="TOP">
101.5
</TD><TD VALIGN="TOP">
255.9
</TD><TD VALIGN="TOP">
85.1
</TD><TD VALIGN="TOP">
0.69
</TD>
<TD VALIGN="TOP">
BMD tissue (mg/cm3 )
</TD><TD VALIGN="TOP">
1 073.9
</TD><TD VALIGN="TOP">
44.2
</TD><TD VALIGN="TOP">
1 044.3
</TD><TD VALIGN="TOP">
55.6
</TD><TD VALIGN="TOP">
1 086.0
</TD><TD VALIGN="TOP">
25.8
</TD><TD VALIGN="TOP">
1 094.5
</TD><TD VALIGN="TOP">
54.5
</TD><TD VALIGN="TOP">
0.24
</TD>
<TD VALIGN="TOP">
BV/TV (%)
</TD><TD VALIGN="TOP">
0.291
</TD><TD VALIGN="TOP">
0.086
</TD><TD VALIGN="TOP">
0.282
</TD><TD VALIGN="TOP">
0.096
</TD><TD VALIGN="TOP">
0.264
</TD><TD VALIGN="TOP">
0.090
</TD><TD VALIGN="TOP">
0.238
</TD><TD VALIGN="TOP">
0.068
</TD><TD VALIGN="TOP">
0.70
</TD>
<TD VALIGN="TOP">
BS/BV (%)
</TD><TD VALIGN="TOP">
15.078
</TD><TD VALIGN="TOP">
4.272
</TD><TD VALIGN="TOP">
15.877
</TD><TD VALIGN="TOP">
4.196
</TD><TD VALIGN="TOP">
15.872
</TD><TD VALIGN="TOP">
4.047
</TD><TD VALIGN="TOP">
16.874
</TD><TD VALIGN="TOP">
3.638
</TD><TD VALIGN="TOP">
0.88
</TD>
<TD VALIGN="TOP">
Tb. N (/mm)
</TD><TD VALIGN="TOP">
2.040
</TD><TD VALIGN="TOP">
0.175
</TD><TD VALIGN="TOP">
2.074
</TD><TD VALIGN="TOP">
0.235
</TD><TD VALIGN="TOP">
1.969
</TD><TD VALIGN="TOP">
0.200
</TD><TD VALIGN="TOP">
1.920
</TD><TD VALIGN="TOP">
0.228
</TD><TD VALIGN="TOP">
0.54
</TD>
<TD VALIGN="TOP">
Tb. Th (mm)
</TD><TD VALIGN="TOP">
0.141
</TD><TD VALIGN="TOP">
0.035
</TD><TD VALIGN="TOP">
0.134
</TD><TD VALIGN="TOP">
0.039
</TD><TD VALIGN="TOP">
0.133
</TD><TD VALIGN="TOP">
0.035
</TD><TD VALIGN="TOP">
0.123
</TD><TD VALIGN="TOP">
0.030
</TD><TD VALIGN="TOP">
0.84
</TD>
<TD VALIGN="TOP">
Tb. Sp (mm)
</TD><TD VALIGN="TOP">
0.351
</TD><TD VALIGN="TOP">
0.067
</TD><TD VALIGN="TOP">
0.352
</TD><TD VALIGN="TOP">
0.079
</TD><TD VALIGN="TOP">
0.379
</TD><TD VALIGN="TOP">
0.072
</TD><TD VALIGN="TOP">
0.403
</TD><TD VALIGN="TOP">
0.081
</TD><TD VALIGN="TOP">
0.55
</TD>
<TD VALIGN="TOP">
Degree of anisotropy
</TD><TD VALIGN="TOP">
1.820
</TD><TD VALIGN="TOP">
0.376
</TD><TD VALIGN="TOP">
1.880
</TD><TD VALIGN="TOP">
0.307
</TD><TD VALIGN="TOP">
1.917
</TD><TD VALIGN="TOP">
0.315
</TD><TD VALIGN="TOP">
1.841
</TD><TD VALIGN="TOP">
0.424
</TD><TD VALIGN="TOP">
0.96
</TD>
<TD VALIGN="TOP">
ConnDens (/mm3 )
</TD><TD VALIGN="TOP">
8.610
</TD><TD VALIGN="TOP">
2.261
</TD><TD VALIGN="TOP">
9.138
</TD><TD VALIGN="TOP">
3.024
</TD><TD VALIGN="TOP">
8.103
</TD><TD VALIGN="TOP">
0.698
</TD><TD VALIGN="TOP">
8.287
</TD><TD VALIGN="TOP">
2.700
</TD><TD VALIGN="TOP">
0.87
</TD>
<TD VALIGN="TOP">
Structure model index
</TD><TD VALIGN="TOP">
0.144
</TD><TD VALIGN="TOP">
0.530
</TD><TD VALIGN="TOP">
0.132
</TD><TD VALIGN="TOP">
0.727
</TD><TD VALIGN="TOP">
0.299
</TD><TD VALIGN="TOP">
0.641
</TD><TD VALIGN="TOP">
0.450
</TD><TD VALIGN="TOP">
0.622
</TD><TD VALIGN="TOP">
0.77
</TD>
<TD VALIGN="TOP" COLSPAN="10">
L6
</TD>
<TD VALIGN="TOP">
BMD apparent (mg/cm3 )
</TD><TD VALIGN="TOP">
367.3
</TD><TD VALIGN="TOP">
65.1
</TD><TD VALIGN="TOP">
347.9
</TD><TD VALIGN="TOP">
80.2
</TD><TD VALIGN="TOP">
399.8
</TD><TD VALIGN="TOP">
53.2
</TD><TD VALIGN="TOP">
383.6
</TD><TD VALIGN="TOP">
38.2
</TD><TD VALIGN="TOP">
0.53
</TD>
<TD VALIGN="TOP">
BMD tissue (mg/cm3 )
</TD><TD VALIGN="TOP">
1 014.5
</TD><TD VALIGN="TOP">
49.7
</TD><TD VALIGN="TOP">
994.8
</TD><TD VALIGN="TOP">
64.0
</TD><TD VALIGN="TOP">
1 036.4
</TD><TD VALIGN="TOP">
12.7
</TD><TD VALIGN="TOP">
1 006.0
</TD><TD VALIGN="TOP">
46.6
</TD><TD VALIGN="TOP">
0.54
</TD>
<TD VALIGN="TOP">
BV/TV (%)
</TD><TD VALIGN="TOP">
0.339
</TD><TD VALIGN="TOP">
0.046
</TD><TD VALIGN="TOP">
0.327
</TD><TD VALIGN="TOP">
0.053
</TD><TD VALIGN="TOP">
0.352
</TD><TD VALIGN="TOP">
0.047
</TD><TD VALIGN="TOP">
0.348
</TD><TD VALIGN="TOP">
0.033
</TD><TD VALIGN="TOP">
0.77
</TD>
<TD VALIGN="TOP">
BS/BV (%)
</TD><TD VALIGN="TOP">
13.706
</TD><TD VALIGN="TOP">
2.656
</TD><TD VALIGN="TOP">
15.014
</TD><TD VALIGN="TOP">
1.813
</TD><TD VALIGN="TOP">
13.159
</TD><TD VALIGN="TOP">
2.135
</TD><TD VALIGN="TOP">
14.300
</TD><TD VALIGN="TOP">
1.270
</TD><TD VALIGN="TOP">
0.44
</TD>
<TD VALIGN="TOP">
Tb. N (/mm)
</TD><TD VALIGN="TOP">
2.289
</TD><TD VALIGN="TOP">
0.265
</TD><TD VALIGN="TOP">
2.421
</TD><TD VALIGN="TOP">
0.232
</TD><TD VALIGN="TOP">
2.286
</TD><TD VALIGN="TOP">
0.265
</TD><TD VALIGN="TOP">
2.477
</TD><TD VALIGN="TOP">
0.144
</TD><TD VALIGN="TOP">
0.39
</TD>
<TD VALIGN="TOP">
Tb. Th (mm)
</TD><TD VALIGN="TOP">
0.149
</TD><TD VALIGN="TOP">
0.024
</TD><TD VALIGN="TOP">
0.134
</TD><TD VALIGN="TOP">
0.016
</TD><TD VALIGN="TOP">
0.155
</TD><TD VALIGN="TOP">
0.029
</TD><TD VALIGN="TOP">
0.140
</TD><TD VALIGN="TOP">
0.014
</TD><TD VALIGN="TOP">
0.36
</TD>
<TD VALIGN="TOP">
Tb.Sp (mm)
</TD><TD VALIGN="TOP">
0.292
</TD><TD VALIGN="TOP">
0.045
</TD><TD VALIGN="TOP">
0.281
</TD><TD VALIGN="TOP">
0.049
</TD><TD VALIGN="TOP">
0.286
</TD><TD VALIGN="TOP">
0.041
</TD><TD VALIGN="TOP">
0.263
</TD><TD VALIGN="TOP">
0.023
</TD><TD VALIGN="TOP">
0.66
</TD>
<TD VALIGN="TOP">
Degree of anisotropy
</TD><TD VALIGN="TOP">
2.196
</TD><TD VALIGN="TOP">
0.241
</TD><TD VALIGN="TOP">
2.065
</TD><TD VALIGN="TOP">
0.169
</TD><TD VALIGN="TOP">
2.118
</TD><TD VALIGN="TOP">
0.170
</TD><TD VALIGN="TOP">
2.041
</TD><TD VALIGN="TOP">
0.135
</TD><TD VALIGN="TOP">
0.33
</TD>
<TD VALIGN="TOP">
ConnDens (/mm3 )
</TD><TD VALIGN="TOP">
3.902
</TD><TD VALIGN="TOP">
1.354
</TD><TD VALIGN="TOP">
4.332
</TD><TD VALIGN="TOP">
1.358
</TD><TD VALIGN="TOP">
4.027
</TD><TD VALIGN="TOP">
1.247
</TD><TD VALIGN="TOP">
3.989
</TD><TD VALIGN="TOP">
1.297
</TD><TD VALIGN="TOP">
0.93
</TD>
<TD VALIGN="TOP">
Structure model index
</TD><TD VALIGN="TOP">
−1.258
</TD><TD VALIGN="TOP">
0.385
</TD><TD VALIGN="TOP">
−1.259
</TD><TD VALIGN="TOP">
0.364
</TD><TD VALIGN="TOP">
−1.405
</TD><TD VALIGN="TOP">
0.452
</TD><TD VALIGN="TOP">
−1.418
</TD><TD VALIGN="TOP">
0.365
</TD><TD VALIGN="TOP">
0.80
</TD>
Discussion and Conclusions
Discussion and Conclusions
To date, mostly minipigs are used as large animal biomedical model species. Pure bred
minipigs are expensive and have distinct ontogenetic and physiologic features when
compared to conventional pigs [12 4
Interestingly, OVX and calcium shortage alone as well as in combination did not influence
bone metabolism and microstructure. This was unexpected, as other authors observed
increasing PTH, calcitriol, and BAP plasma levels, albeit transient and moderate,
in sows of the same age group as the ones investigated here, although those were ovariectomized
but fed a standard diet [5 13
In other ungulates investigated so far, that is, sheep, OVX alone reduces bone mass,
which can be enforced by an additional nutritive calcium shortage. BMD of L5 and distal
radius as evaluated by DXA was significantly changed after 6 months and the one of
L4 1 year after OVX, whereas the proximal parts of femur, humerus, and tibia did not
exhibit alterations to that extent [14 15 16 17 18 19 20
Sows seem to differently handle loss of ovarian function and nutritive calcium shortage
with respect to bone morphology, but also their cellular immune system gives no evidence
of being affected by these measures. This is another difference to postmenopausal
women, whose T cells have been shown to be activated as a consequence of increased
IFN-γ, TNF-α, and RANKL synthesis [21 + CD57+ cells and to suffer from a proinflammatory state [22 23
Another way to possibly achieve an osteoporosis-like phenotype in adult sows could
be glucocorticoid treatment with all the disadvantages discussed above. Glucocorticoid-induced
osteoporosis has already been shown as an option in the porcine model, albeit again
minipigs have been used for these experiments. Scholz-Ahrens and colleagues [24 3 from baseline, whereas in the control group reduction was 12 mg/cm3 . These changes were also evidenced by plasma BAP levels, which decreased significantly
in the glucocorticoid group. In the long term, the loss of BMD became more pronounced,
and bone mineral content, trabecular thickness, and mechanical stability tended to
be lower compared with the control group. There was a negative association between
the cumulative dose of glucocorticoids and BMD, which could be traced back to impaired
osteoblastogenesis.
The present study has 2 major limitations. First, we analyzed only 8 sows per group.
This small number is a consequence of the animals’ dimension with each sow weighing
over 300 kg. On the other hand, comparable large animal studies included even smaller
numbers of pigs [4 25
In conclusion, the skeleton of adult conventional sows is seemingly protected from
effects of OVX and calcium shortage, which is a very interesting finding. Hence, these
animals do not appear to be a suitable model for investigations concerning postmenopausal
osteoporosis but could be used to identify factors that protect bone from calcium
or sex hormone deficiency. Moreover, this study additionally provides valuable information
about yet unknown physiological data on bone metabolism parameters in adult sows.
At the moment we have to accept that bone of some species such as laboratory rodents
reacts to generally accepted osteoporosis-inducing stimuli such as OVX and calcium
shortage as would human bone, whereas the skeleton of other species, such as the adult
pig, seems to be resistant to the development of osteopenia or osteoporosis. The skeleton
of bears may serve as another contradictory example, as it remains unaltered in the
context of immobilization during hibernation [26
Acknowledgements
Acknowledgements
This study was supported by a grant from the Austrian Science Fund, Project No: P20337-B13.
We thank Katharina Wahl for her excellent assistance.
