Page 1 of 38
Accepted Preprint first posted on 23 September 2014 as Manuscript JOE-14-0486
1 2 3 4
Bone quality is affected by food restriction and by nutrition-induced catch up growth. Rakefet Pando1,2, Majdi Masarwi1,2, Biana Shtaif1,2, Anna Idelevich3, Efrat Monsonego-Ornan3, Ron Shahar4, Moshe Phillip1,2,5, Galia Gat-Yablonski1,2,5
5 6
1
Felsenstein Medical Research Center, Petach Tikva; 2Sackler School of Medicine,
7
Tel Aviv University, Tel Aviv; 3Institute of Biochemistry and Nutrition, Robert H. Smith
8
Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem; 4Koret
9
School of Veterinary Medicine, Faculty of Agricultural, Food and Environmental Quality
10
Sciences, The Hebrew University of Jerusalem, Israel, 5The Jesse Z and Sara Lea Shafer
11
Institute for Endocrinology and Diabetes, National Center for Childhood Diabetes, Schneider
12
Children’s Medical Center of Israel, Petach Tikva
13 14
Correspondence (address for reprint requests ):
15
Galia Gat-Yablonski
16
The Jesse Z and Sara Lea Shafer Institute for Endocrinology and Diabetes
17
National Center for Childhood Diabetes,
18
Schneider Children’s Medical Center of Israel
19
14 Kaplan Street
20
Petach Tikva 49202
21
Israel
22
Email: [email protected]; Tel: 972-3-9376133; Fax: 972-3-9211478
23 24
Short Title: Bone quality in catch up growth.
25
Key words: catch-up growth, food restriction, micro CT, mechanical testing
26
Word count: 5763, 5 Figures, 4 Tables
27 28 1
Copyright © 2014 by the Society for Endocrinology.
Page 2 of 38
29
Abstract
30 31
Growth stunting constitutes the most common effect of malnutrition. When the
32
primary cause of malnutrition is resolved, catch-up (CU) growth usually occurs. In this study
33
we wanted to explore the effect of food restriction (RES) and re-feeding on bone structure
34
and mechanical properties. 24 days old SD male rats were subjected to 10 days of 40% RES,
35
followed by re-feeding for one (CU) or 26 days (LTCU). Rats fed ad libitum served as
36
controls. The growth plates were measured, osteoclast were identified using TRAP staining,
37
Micro-CT scanning and mechanical testing were used to study structure and mechanical
38
properties. Micro-CT analysis showed that RES led to a significant reduction in trabecular
39
BV/TV and Tb. N, concomitant with an increase in Tb. Sp. Trabecular BV/TV and Tb. N
40
were significantly greater in the CU group than in the RES in both short and long term
41
experiments. Mechanical testing showed that RES led to weaker and less compliant bones;
42
interestingly, bones of the CU group were also more fragile after one day of CU. Longer term
43
of re-feeding enabled correction of the bone parameters, however LTCU did not achieve full
44
recovery. These results suggest that RES in young rats attenuated growth and reduced
45
trabecular bone parameters. While nutrition-induced CU growth led to an immediate increase
46
in EGP height and active bone modeling, it was also associated with a transient reduction in
47
bone quality. This should be taken into consideration when treating children undergoing CU
48
growth.
49 50
List of abbreviations used:
51
AL- ad libitum; RES-food restricted; CU- catch up; LTAL- long term ad libitum; LTRES-
52
long term food restricted; LTCU- long term catch up; EGP- Epiphyseal growth plate; Ct.Ar-
2
Page 3 of 38
53
Cortical area; Tt. Ar- Total area; Tb.N- trabecular number; Tb.Th- trabecular thickness;
54
Tb.Sp- trabecular separation;
55
3
Page 4 of 38
56 57
1.1
Introduction Although there are numerous genetic and environmental factors that may affect
58
growth, malnutrition, marked by various nutrient deficiencies, is considered to be a leading
59
cause of low weight and short stature. In the developing countries an average 33% of all
60
children younger than 5 years of age suffer from linear growth retardation or stunting due to
61
chronic malnutrition, caused by food shortage as well as by infectious diseases (Walker, et al.
62
2007) . Malnutrition may also occur in developed countries, mostly due to chronic disease.
63
Catch up (CU) growth occurs after the resolution of a growth-inhibiting condition and has
64
been found to be characterized by a growth velocity above the normal statistical limits for age
65
and/or maturity (Boersma, et al. 2002). Some excellent examples for nutrition-induced CU
66
growth are in childhood celiac disease (Boersma et al. 2002) as remarkable CU growth has
67
been documented shortly after the onset of a gluten-free diet (Boersma et al. 2002).
68
The skeleton undergoes rapid structural adaptations to the demands of the growing
69
body, manifested by elongation and bone modeling, together with changes in the bone
70
mineral composition, cortical and trabecular architecture and altered mechanical properties.
71
Required for this process is an adequate supply of numerous nutritional factors some of
72
which, such as proteins, lipids, and carbohydrates, constitute the "building materials" while
73
others play regulatory roles.
74
To study the effect of food restriction and CU growth on bones we used young,
75
rapidly growing rats. In this experimental model, 40% food restriction was initiated on 24
76
days old rats and continued during the linear growth period for 10 days. In our previous
77
studies we have shown that the most dramatic changes in weight gain and a significant
78
increase in epiphyseal growth plate (EGP) height were observed after only one day of re-
79
feeding (Even-Zohar, et al. 2008). The changes in the EGP height in response to nutritional
80
manipulation were found to result from differences in both cell number and extracellular 4
Page 5 of 38
81
matrix production, as exemplified by changes in gene expression, with the most affected
82
genes being those associated with matrix formation (Even-Zohar et al. 2008). It was therefore
83
reasoned that these changes are likely to have an effect on the architecture and material
84
properties of the developing bones. In the present study we aimed to analyze the changes occurring in the long bones
85 86
during food restriction and nutrition-induced-CU growth, using micro-CT analysis
87
(Bouxsein, et al. 2010) and mechanical testing (Shipov, et al. 2010).
88 89
1.2
90
1.2.1 Animals
91
Methods and Materials
Male Sprague-Dawley (SD) rats, 21 days old, were purchased from Harlan
92
(Jerusalem, Israel) and housed individually at the animal care facility of the Felsenstein
93
Medical Research Center in separate cages to facilitate the monitoring of their food
94
consumption. Animals were allowed to adapt to these conditions for 3 days before the
95
beginning of the experiment. One group of animals was sacrificed at the beginning of the
96
experiment (24 days old; BC; n=6). The data obtained from these young animals served to
97
assess the effect of age and weight on bone quality. Other rats were divided into three groups:
98
one was given an unlimited amount of food (complete diet for rats and mice, 3.4 Kcal/gr,
99
provided by Teklad [South Easton, MA]), (ad libitum group; AL), and two received 60% of
100
the same chow (food-restricted group; RES; catch-up group; CU); all animals had unlimited
101
access to water. The 40% restriction was calculated on the basis of a previous study wherein
102
animals were housed individually and the amount of food consumed each day was measured,
103
together with the animals' weight and weight gain (Even-Zohar et al. 2008). The food
104
restriction was maintained for 10 days. At that point, while the RES group was kept
105
restricted, the CU group was given normal chow ad libitum. To study the effect of long term 5
Page 6 of 38
106
re-feeding the CU period was extended to 26 days (Long term CU (LTCU)); AL and RES
107
groups were monitored throughout this extended period as well (LTAL; LTRES,
108
respectively) (Fig.1). After one or 26 days of re-feeding, animals were sacrificed by CO2
109
inhalation. Throughout the study, animals were observed daily, and all remained bright, alert
110
and active, with no evidence of any disorder. The Tel Aviv University Animal Care
111
Committee approved all procedures.
112
At sacrifice, tibias and one humerus of each animal were carefully removed,
113
measured, and fixed in 4% neutral buffered formalin for 24 hours, followed by
114
demineralization in 20% EDTA for six weeks for histological and morphological studies. The
115
other humerus was cleaned and stored at -200C until analyzed by micro-CT and three point
116
bending.
117 118
1.2.2 Micro Computed Tomography (CT)
119
Bones were stored in saline-soaked gauze, at -200C. They were thawed on the day of
120
scanning and scanned in high humidity (moistened and wrapped in ceram-wrap then placed
121
in the holder for scanning).
122
The region of the proximal metaphysis to mid-diaphysis of all humeri were scanned
123
with a Skyscan 1174 X-ray-computed microtomograph scanner (Skyscan, Aartselaar
124
Belgium), equipped with a CCD detector. Scanner source parameters used were 50 kV and
125
800 µA. Bone samples were scanned using an 0.25 mm aluminum filter, 4000 ms exposure
126
time, and at 11.1 µ pixel size resolution. For each specimen, a series of 900 projection images
127
was obtained, with a rotation step of 0.4°, 2 frames averaging, for a total 360° rotation. Flat
128
field correction was performed at the beginning of each scan for a specific zoom and image
129
format. A stack of 2-D X-ray shadow projections was reconstructed to obtain images using
130
NRecon software (Skyscan), and subjected to morphometric analysis using CTAn software 6
Page 7 of 38
131
(CTAn software, v.1.11, SkyScan, Belgium). During reconstruction, dynamic image range,
132
post-alignment value, beam hardening and ring-artifact reduction were optimized for each
133
experimental set. Analysis of the diaphyseal cortical region was performed on 200 slices,
134
similarly selected for all samples, corresponding to a section of mid-diaphysis 2.764 mm
135
long. The following cortical parameters were measured: volumetric BMD (mg/mm3), polar
136
moment of inertia (µm 4), mean cortical thickness (µ), cortical cross-sectional area (µ2).
137
Cortical cross-sectional area (µ2) was calculated by the software individually for each slice,
138
and mean values are presented.
139
In addition, two phantoms of known mineral density (0.25 g/cc and 0.75 g/cc)
140
supplied by the manufacturer of the scanner (SkyScan, Belgium) were also scanned, using the
141
same scanning parameters. This allowed calibration of the attenuation levels directly to bone
142
mineral density values. For the trabecular region, a total of 150 slices, corresponding to a
143
segment 2.073 mm long, were selected. The selection of region of interest (ROI) was
144
consistent in all bones analyzed. Adaptive threshold was used (this is a software option that
145
helps overcome errors resulting from partial volume effects, it calculates density gradients to
146
determine if a particular pixel is bone or not). Morphometric analysis was performed using
147
SkyScan software (CTAn software, v.1.11, SkyScan, Belgium). 3-D images (CTM file
148
format) were constructed from the cortical and trabecular regions of interest, utilizing
149
Marching Cubes 33 algorithm, using SkyScan software (CTVol, SkyScan, Belgium)
150
(Idelevich, et al. 2011; Reich, et al. 2010).
151 152
1.2.3 Mechanical testing
153
Following micro-CT scanning, mechanical tests were performed on the diaphyseal
154
region of the humeri. The loading system included a stainless-steel test chamber so that
155
mechanical testing could be performed on samples immersed in physiologic solution. An 7
Page 8 of 38
156
axial-motion DC motor (PIM-235.2DG, Physik Instrumente, GmbH, Germany) moved a
157
metal shaft into the testing chamber in small sub-micron steps while being able to apply
158
substantial force (>100 N). The metal shaft was connected in series to a load cell (AL311,
159
Sensotec, Honeywell, USA, ± 0.4 N), which was attached in turn to a movable anvil that was
160
designed to contact the bone sample. The bones were tested by the three-point bending
161
method while being fully immersed in physiologic saline. Each bone was placed on two
162
supports having rounded profiles (0.5 mm radius) to limit stress concentration at the point of
163
load application, so that the supports were equidistant from the ends of the bone, and both
164
contacted the posterior aspect of the diaphysis. The distance between the supports was 8 mm.
165
Each bone was loaded on its anterior aspect, by a moving prong with rounded profile, at the
166
mid-point between the bottom supports, and at the mid-point along its length.
167
Force and displacement data were collected at 50 Hz, using custom-written software
168
(NI Labview, Austin, TX, USA). The resulting load-displacement curves were used to
169
calculate whole-bone stiffness (slope of the linear portion of the load-displacement curve),
170
ultimate load and load to fracture (Lanyon, et al. 1982).
171
172 173
1.2.4 Tartrate-resistant acid phosphatase (TRAP) staining for osteoclasts Tibias were fixed overnight in 4% formalin (Sigma Chemical, St Louis, MO, USA) in
174
PBS at 4°C, dehydrated in graded ethanol solutions, cleared in chloroform, and embedded in
175
Paraplast. Thin (6 µm) sections were prepared. Staining for osteoclasts was done with PNPP
176
(Leukocyte Acid Phosphatase Kit, Sigma Chemical, St. Louis, MO, USA). Quantitation of
177
TRAP-stained sections was performed on digital images in Image-Pro Plus software (Media
178
Cybernetics, Silver Spring, MD). Only cells at the lower border of the EGP were counted
179
since dramatic differences were observed at this area. At least six sections from each animal
8
Page 9 of 38
180
were analyzed. The sections were visualized using an Olympus Bx52 light microscope and
181
photographed using Camera Olympus DP71, Japan.
182
183
1.2.5 Histology Humeri were fixed as described for tibias. Thin (6 µm) Horizontal cross-sectional
184 185
sections were prepared and stained with H&E or Masson's trichrome staining (Sigma
186
Chemical, St Louis, MO, USA). The sections were visualized as described above.
187 188
1.2.6 Chemical analysis of serum samples Blood was collected at euthanization by cardiac puncture, serum was separated and
189 190
kept at -200C until analyzed using an Immunolite automated analyzer. Serum Leptin and total
191
IGF-I levels were measured using commercial ELISA kits (rat Leptin kit; Millipor, Billerica,
192
USA & rat IGF-I kit (cat # MG100), Quantikine; R&D systems, MN, USA, respectively);
193
bone specific ALP was measured using a commercial Rat C-terminal Peptide of Bone-
194
specific Alkaline Phosphates ELISA kit (Cusabio Biotech co. LTD).
195 196
1.2.7 Statistical Analysis Results are expressed as mean ± SD. Differences between groups were analyzed by
197 198
one-way analysis of variance (ANOVA) with Tukey's post hoc test. A P value below 0.05
199
was considered significant
200 201
1.3
202
1.3.1 Effect of food restriction on Morphological parameters
203 204
Results
The effects of food restriction and short term nutrition–induced CU growth on weight, bone and EGP length are presented in Table 1 (and see also (Even-Zohar et al. 2008)). 9
Page 10 of 38
205
The height of the EGP measured from the reserve zone to the ossification front of the
206
metaphyseal bone, which was significantly smaller in the RES group (P
Accepted Preprint first posted on 23 September 2014 as Manuscript JOE-14-0486
1 2 3 4
Bone quality is affected by food restriction and by nutrition-induced catch up growth. Rakefet Pando1,2, Majdi Masarwi1,2, Biana Shtaif1,2, Anna Idelevich3, Efrat Monsonego-Ornan3, Ron Shahar4, Moshe Phillip1,2,5, Galia Gat-Yablonski1,2,5
5 6
1
Felsenstein Medical Research Center, Petach Tikva; 2Sackler School of Medicine,
7
Tel Aviv University, Tel Aviv; 3Institute of Biochemistry and Nutrition, Robert H. Smith
8
Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem; 4Koret
9
School of Veterinary Medicine, Faculty of Agricultural, Food and Environmental Quality
10
Sciences, The Hebrew University of Jerusalem, Israel, 5The Jesse Z and Sara Lea Shafer
11
Institute for Endocrinology and Diabetes, National Center for Childhood Diabetes, Schneider
12
Children’s Medical Center of Israel, Petach Tikva
13 14
Correspondence (address for reprint requests ):
15
Galia Gat-Yablonski
16
The Jesse Z and Sara Lea Shafer Institute for Endocrinology and Diabetes
17
National Center for Childhood Diabetes,
18
Schneider Children’s Medical Center of Israel
19
14 Kaplan Street
20
Petach Tikva 49202
21
Israel
22
Email: [email protected]; Tel: 972-3-9376133; Fax: 972-3-9211478
23 24
Short Title: Bone quality in catch up growth.
25
Key words: catch-up growth, food restriction, micro CT, mechanical testing
26
Word count: 5763, 5 Figures, 4 Tables
27 28 1
Copyright © 2014 by the Society for Endocrinology.
Page 2 of 38
29
Abstract
30 31
Growth stunting constitutes the most common effect of malnutrition. When the
32
primary cause of malnutrition is resolved, catch-up (CU) growth usually occurs. In this study
33
we wanted to explore the effect of food restriction (RES) and re-feeding on bone structure
34
and mechanical properties. 24 days old SD male rats were subjected to 10 days of 40% RES,
35
followed by re-feeding for one (CU) or 26 days (LTCU). Rats fed ad libitum served as
36
controls. The growth plates were measured, osteoclast were identified using TRAP staining,
37
Micro-CT scanning and mechanical testing were used to study structure and mechanical
38
properties. Micro-CT analysis showed that RES led to a significant reduction in trabecular
39
BV/TV and Tb. N, concomitant with an increase in Tb. Sp. Trabecular BV/TV and Tb. N
40
were significantly greater in the CU group than in the RES in both short and long term
41
experiments. Mechanical testing showed that RES led to weaker and less compliant bones;
42
interestingly, bones of the CU group were also more fragile after one day of CU. Longer term
43
of re-feeding enabled correction of the bone parameters, however LTCU did not achieve full
44
recovery. These results suggest that RES in young rats attenuated growth and reduced
45
trabecular bone parameters. While nutrition-induced CU growth led to an immediate increase
46
in EGP height and active bone modeling, it was also associated with a transient reduction in
47
bone quality. This should be taken into consideration when treating children undergoing CU
48
growth.
49 50
List of abbreviations used:
51
AL- ad libitum; RES-food restricted; CU- catch up; LTAL- long term ad libitum; LTRES-
52
long term food restricted; LTCU- long term catch up; EGP- Epiphyseal growth plate; Ct.Ar-
2
Page 3 of 38
53
Cortical area; Tt. Ar- Total area; Tb.N- trabecular number; Tb.Th- trabecular thickness;
54
Tb.Sp- trabecular separation;
55
3
Page 4 of 38
56 57
1.1
Introduction Although there are numerous genetic and environmental factors that may affect
58
growth, malnutrition, marked by various nutrient deficiencies, is considered to be a leading
59
cause of low weight and short stature. In the developing countries an average 33% of all
60
children younger than 5 years of age suffer from linear growth retardation or stunting due to
61
chronic malnutrition, caused by food shortage as well as by infectious diseases (Walker, et al.
62
2007) . Malnutrition may also occur in developed countries, mostly due to chronic disease.
63
Catch up (CU) growth occurs after the resolution of a growth-inhibiting condition and has
64
been found to be characterized by a growth velocity above the normal statistical limits for age
65
and/or maturity (Boersma, et al. 2002). Some excellent examples for nutrition-induced CU
66
growth are in childhood celiac disease (Boersma et al. 2002) as remarkable CU growth has
67
been documented shortly after the onset of a gluten-free diet (Boersma et al. 2002).
68
The skeleton undergoes rapid structural adaptations to the demands of the growing
69
body, manifested by elongation and bone modeling, together with changes in the bone
70
mineral composition, cortical and trabecular architecture and altered mechanical properties.
71
Required for this process is an adequate supply of numerous nutritional factors some of
72
which, such as proteins, lipids, and carbohydrates, constitute the "building materials" while
73
others play regulatory roles.
74
To study the effect of food restriction and CU growth on bones we used young,
75
rapidly growing rats. In this experimental model, 40% food restriction was initiated on 24
76
days old rats and continued during the linear growth period for 10 days. In our previous
77
studies we have shown that the most dramatic changes in weight gain and a significant
78
increase in epiphyseal growth plate (EGP) height were observed after only one day of re-
79
feeding (Even-Zohar, et al. 2008). The changes in the EGP height in response to nutritional
80
manipulation were found to result from differences in both cell number and extracellular 4
Page 5 of 38
81
matrix production, as exemplified by changes in gene expression, with the most affected
82
genes being those associated with matrix formation (Even-Zohar et al. 2008). It was therefore
83
reasoned that these changes are likely to have an effect on the architecture and material
84
properties of the developing bones. In the present study we aimed to analyze the changes occurring in the long bones
85 86
during food restriction and nutrition-induced-CU growth, using micro-CT analysis
87
(Bouxsein, et al. 2010) and mechanical testing (Shipov, et al. 2010).
88 89
1.2
90
1.2.1 Animals
91
Methods and Materials
Male Sprague-Dawley (SD) rats, 21 days old, were purchased from Harlan
92
(Jerusalem, Israel) and housed individually at the animal care facility of the Felsenstein
93
Medical Research Center in separate cages to facilitate the monitoring of their food
94
consumption. Animals were allowed to adapt to these conditions for 3 days before the
95
beginning of the experiment. One group of animals was sacrificed at the beginning of the
96
experiment (24 days old; BC; n=6). The data obtained from these young animals served to
97
assess the effect of age and weight on bone quality. Other rats were divided into three groups:
98
one was given an unlimited amount of food (complete diet for rats and mice, 3.4 Kcal/gr,
99
provided by Teklad [South Easton, MA]), (ad libitum group; AL), and two received 60% of
100
the same chow (food-restricted group; RES; catch-up group; CU); all animals had unlimited
101
access to water. The 40% restriction was calculated on the basis of a previous study wherein
102
animals were housed individually and the amount of food consumed each day was measured,
103
together with the animals' weight and weight gain (Even-Zohar et al. 2008). The food
104
restriction was maintained for 10 days. At that point, while the RES group was kept
105
restricted, the CU group was given normal chow ad libitum. To study the effect of long term 5
Page 6 of 38
106
re-feeding the CU period was extended to 26 days (Long term CU (LTCU)); AL and RES
107
groups were monitored throughout this extended period as well (LTAL; LTRES,
108
respectively) (Fig.1). After one or 26 days of re-feeding, animals were sacrificed by CO2
109
inhalation. Throughout the study, animals were observed daily, and all remained bright, alert
110
and active, with no evidence of any disorder. The Tel Aviv University Animal Care
111
Committee approved all procedures.
112
At sacrifice, tibias and one humerus of each animal were carefully removed,
113
measured, and fixed in 4% neutral buffered formalin for 24 hours, followed by
114
demineralization in 20% EDTA for six weeks for histological and morphological studies. The
115
other humerus was cleaned and stored at -200C until analyzed by micro-CT and three point
116
bending.
117 118
1.2.2 Micro Computed Tomography (CT)
119
Bones were stored in saline-soaked gauze, at -200C. They were thawed on the day of
120
scanning and scanned in high humidity (moistened and wrapped in ceram-wrap then placed
121
in the holder for scanning).
122
The region of the proximal metaphysis to mid-diaphysis of all humeri were scanned
123
with a Skyscan 1174 X-ray-computed microtomograph scanner (Skyscan, Aartselaar
124
Belgium), equipped with a CCD detector. Scanner source parameters used were 50 kV and
125
800 µA. Bone samples were scanned using an 0.25 mm aluminum filter, 4000 ms exposure
126
time, and at 11.1 µ pixel size resolution. For each specimen, a series of 900 projection images
127
was obtained, with a rotation step of 0.4°, 2 frames averaging, for a total 360° rotation. Flat
128
field correction was performed at the beginning of each scan for a specific zoom and image
129
format. A stack of 2-D X-ray shadow projections was reconstructed to obtain images using
130
NRecon software (Skyscan), and subjected to morphometric analysis using CTAn software 6
Page 7 of 38
131
(CTAn software, v.1.11, SkyScan, Belgium). During reconstruction, dynamic image range,
132
post-alignment value, beam hardening and ring-artifact reduction were optimized for each
133
experimental set. Analysis of the diaphyseal cortical region was performed on 200 slices,
134
similarly selected for all samples, corresponding to a section of mid-diaphysis 2.764 mm
135
long. The following cortical parameters were measured: volumetric BMD (mg/mm3), polar
136
moment of inertia (µm 4), mean cortical thickness (µ), cortical cross-sectional area (µ2).
137
Cortical cross-sectional area (µ2) was calculated by the software individually for each slice,
138
and mean values are presented.
139
In addition, two phantoms of known mineral density (0.25 g/cc and 0.75 g/cc)
140
supplied by the manufacturer of the scanner (SkyScan, Belgium) were also scanned, using the
141
same scanning parameters. This allowed calibration of the attenuation levels directly to bone
142
mineral density values. For the trabecular region, a total of 150 slices, corresponding to a
143
segment 2.073 mm long, were selected. The selection of region of interest (ROI) was
144
consistent in all bones analyzed. Adaptive threshold was used (this is a software option that
145
helps overcome errors resulting from partial volume effects, it calculates density gradients to
146
determine if a particular pixel is bone or not). Morphometric analysis was performed using
147
SkyScan software (CTAn software, v.1.11, SkyScan, Belgium). 3-D images (CTM file
148
format) were constructed from the cortical and trabecular regions of interest, utilizing
149
Marching Cubes 33 algorithm, using SkyScan software (CTVol, SkyScan, Belgium)
150
(Idelevich, et al. 2011; Reich, et al. 2010).
151 152
1.2.3 Mechanical testing
153
Following micro-CT scanning, mechanical tests were performed on the diaphyseal
154
region of the humeri. The loading system included a stainless-steel test chamber so that
155
mechanical testing could be performed on samples immersed in physiologic solution. An 7
Page 8 of 38
156
axial-motion DC motor (PIM-235.2DG, Physik Instrumente, GmbH, Germany) moved a
157
metal shaft into the testing chamber in small sub-micron steps while being able to apply
158
substantial force (>100 N). The metal shaft was connected in series to a load cell (AL311,
159
Sensotec, Honeywell, USA, ± 0.4 N), which was attached in turn to a movable anvil that was
160
designed to contact the bone sample. The bones were tested by the three-point bending
161
method while being fully immersed in physiologic saline. Each bone was placed on two
162
supports having rounded profiles (0.5 mm radius) to limit stress concentration at the point of
163
load application, so that the supports were equidistant from the ends of the bone, and both
164
contacted the posterior aspect of the diaphysis. The distance between the supports was 8 mm.
165
Each bone was loaded on its anterior aspect, by a moving prong with rounded profile, at the
166
mid-point between the bottom supports, and at the mid-point along its length.
167
Force and displacement data were collected at 50 Hz, using custom-written software
168
(NI Labview, Austin, TX, USA). The resulting load-displacement curves were used to
169
calculate whole-bone stiffness (slope of the linear portion of the load-displacement curve),
170
ultimate load and load to fracture (Lanyon, et al. 1982).
171
172 173
1.2.4 Tartrate-resistant acid phosphatase (TRAP) staining for osteoclasts Tibias were fixed overnight in 4% formalin (Sigma Chemical, St Louis, MO, USA) in
174
PBS at 4°C, dehydrated in graded ethanol solutions, cleared in chloroform, and embedded in
175
Paraplast. Thin (6 µm) sections were prepared. Staining for osteoclasts was done with PNPP
176
(Leukocyte Acid Phosphatase Kit, Sigma Chemical, St. Louis, MO, USA). Quantitation of
177
TRAP-stained sections was performed on digital images in Image-Pro Plus software (Media
178
Cybernetics, Silver Spring, MD). Only cells at the lower border of the EGP were counted
179
since dramatic differences were observed at this area. At least six sections from each animal
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were analyzed. The sections were visualized using an Olympus Bx52 light microscope and
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photographed using Camera Olympus DP71, Japan.
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1.2.5 Histology Humeri were fixed as described for tibias. Thin (6 µm) Horizontal cross-sectional
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sections were prepared and stained with H&E or Masson's trichrome staining (Sigma
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Chemical, St Louis, MO, USA). The sections were visualized as described above.
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1.2.6 Chemical analysis of serum samples Blood was collected at euthanization by cardiac puncture, serum was separated and
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kept at -200C until analyzed using an Immunolite automated analyzer. Serum Leptin and total
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IGF-I levels were measured using commercial ELISA kits (rat Leptin kit; Millipor, Billerica,
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USA & rat IGF-I kit (cat # MG100), Quantikine; R&D systems, MN, USA, respectively);
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bone specific ALP was measured using a commercial Rat C-terminal Peptide of Bone-
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specific Alkaline Phosphates ELISA kit (Cusabio Biotech co. LTD).
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1.2.7 Statistical Analysis Results are expressed as mean ± SD. Differences between groups were analyzed by
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one-way analysis of variance (ANOVA) with Tukey's post hoc test. A P value below 0.05
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was considered significant
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1.3
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1.3.1 Effect of food restriction on Morphological parameters
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Results
The effects of food restriction and short term nutrition–induced CU growth on weight, bone and EGP length are presented in Table 1 (and see also (Even-Zohar et al. 2008)). 9
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The height of the EGP measured from the reserve zone to the ossification front of the
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metaphyseal bone, which was significantly smaller in the RES group (P
