This document compares two dual-energy X-ray absorptiometry (DXA) systems - PIXImus and Insight - for skeletal phenotyping in mice. It finds that while both systems can measure bone mineral density (BMD) and content (BMC) non-invasively and rapidly, they produce somewhat different quantitative results. PIXImus is a portable unit that uses low X-ray energies and high-resolution pixels, allowing measurements in low-density bone, while Insight requires a longer scanning time. Overall, the document evaluates the two DXA systems for analyzing skeletal changes in mouse models of bone disease.

1. INSiGHT & PIXImus:
A Tale of Two Systems
David Maridas, PhD
Instructor in
Developmental Biology,
Harvard School of Dental Medicine

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Outline
• Dual X-ray Absorptiometry (DXA) in skeletal studies.
• IGFBP4 knockout mice skeletal phenotype.
• Preliminary results of Alk4-FC treatment in mice.
• Comparison between two DXA systems.
2

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Skeletal phenotyping in mice
3
16 wks
8 wks
4 wks 22 wks
Female
Male
14 wks 52 wks
1mm

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Skeletal phenotyping in mice
4
16 wks
8 wks
4 wks 22 wks
Female
Male
14 wks 52 wks
1mm

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Microcomputed tomography (µCT) limitations
5
• Requires sacrifice of mice to isolate bones.
• Very slow, days worth of work to process a cohort.
• Measurement thresholds (contouring, segmentation …) set by user.
• Expensive.
• In vivo systems available but expensive and time-consuming.

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• Using 2 X-Rays beams with different energy to measure bone mineral density
(and other parameters).
Strengths:
Non-invasive.
Minimal dose of radiation.
Fast
Cheap
Weaknesses:
No assessment of trabecular bone.
No 3D reconstruction and microarchitecture assessment.
Dual X-ray Absorptiometry (DXA)
6

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Dual X-ray Absorptiometry (DXA)
7
DXA scan in clinics
DXA (PIXImus) for small animals
• Using 2 X-Rays beams with different energy to measure bone mineral density
(and other parameters).
Strengths:
Non-invasive.
Minimal dose of radiation.
Fast
Cheap
Weaknesses:
No assessment of trabecular bone.
No 3D reconstruction and microarchitecture assessment.

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Dual X-ray Absorptiometry (DXA)
8
DXA scan in clinics
DXA (PIXImus) for small animals
• Using 2 X-Rays beams with different energy to measure bone mineral density
(and other parameters).
• Strengths:
• Non-invasive.
• Minimal dose of radiation.
• Fast
• Cheap
Weaknesses:
No assessment of trabecular bone.
No 3D reconstruction and microarchitecture assessment.

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The PIXImus system
• The PIXImus densitometer is a portable unit.
• It uses a stationary anode X-ray tube that generates a cone beam X-ray.
• PIXImus uses low X-ray energies (80/35 kVp at 500 µA).
• The stationary area detector uses ultrahigh resolution pixels (0.18 x 0.18 mm).
• Achieves contrast in extremely low-density bone and for tissue comparison analysis.
9
30 x 63 x 33 cm, ~ 60lbs

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PhD Thesis
Growth Hormone
Hypothalamus
Anterior Pituitary
GHRH Somatostatin
Liver
IGF-I
Metabolism
• Adipose Tissue
• Bone
• Skeletal muscle
Adapted from Thorner MO, et al. In: Williams Textbook of Endocrinology. 9th ed. 1998:249.*
*Clemmons D, Cook D, Kleinberg D

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Mesenchymal
Progenitor
IGF-I: mitogenic and differentiating factor
IGF-I

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Mesenchymal
Progenitor
Adipocyte
Chondrocyte
IGF-I: mitogenic and differentiating factor
Osteoblast
IGF-I
IGF-I
IGF-I
IGF-I

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Mesenchymal
Progenitor
Adipocyte
Chondrocyte
IGF-I: mitogenic and differentiating factor
Osteoblast
IGF-I
IGF-I
IGF-I
IGF-I
IGFBP

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IGFBP4
Growth
IGFBP1
Glucose
Metabolism
IGFBP2
Fat
Bone
IGFBP3
Growth
IGFBP5
Cell
Proliferation
IGFBP6
Proliferation
Migration
IGFBPs regulate IGF actions locally

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IGFBP4
Growth
IGFBP1
Glucose
Metabolism
IGFBP2
Fat
Bone
IGFBP3
Growth
IGFBP5
Cell
Proliferation
IGFBP6
Proliferation
Migration
IGFBPs regulate IGF actions locally

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Actions of IGFBP4
• IGFBP4 inhibits IGF actions in almost all in vitro models:
• Bone cells
• Vascular smooth muscle cells
• Colon cancer cells
• In vivo overexpression of IGFBP4 in bone reduced bone formation.
• Injections of IGFBP4 in mice induced bone formation.
• Surprisingly, IGFBP4 KO mice a growth delay but the knockout was
generated on a mixed background.
IGFBP4
Growth
?

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Hypothesis:
IGFBP4 regulates growth by affecting skeletal postnatal
acquisition

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Hypothesis:
IGFBP4 regulates growth by affecting skeletal postnatal
acquisition
Investigate postnatal growth of IGFBP4 knockout mice on C54BL6/J background.

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Hypothesis:
IGFBP4 regulates growth by affecting skeletal postnatal
acquisition
Investigate postnatal growth of IGFBP4 knockout mice on C54BL6/J background.
Females
Males
Igfbp4+/+
Igfbp4-/-
*
Igfbp4+/+
Igfbp4-/-
0.0
0.5
1.0
1.5
Igfbp4
RNA
expression
/
Actin
Females Males

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Hypothesis:
IGFBP4 regulates growth by affecting skeletal postnatal
acquisition
Investigate postnatal growth of IGFBP4 knockout mice on C54BL6/J background.
10
20
30
40
Body
Weight
(g)
8wk
**
**
16wk
Females
Males
Igfbp4+/+
Igfbp4-/-
**
**
Igfbp4+/+
Igfbp4-/-
Females
Males
Igfbp4+/+
Igfbp4-/-
*
Igfbp4+/+
Igfbp4-/-
0.0
0.5
1.0
1.5
Igfbp4
RNA
expression
/
Actin
Females Males

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Hypothesis:
IGFBP4 regulates growth by affecting skeletal postnatal
acquisition
Investigate postnatal growth of IGFBP4 knockout mice on C54BL6/J background.
10
20
30
40
Body
Weight
(g)
8wk
**
**
16wk
Females
Males
Igfbp4+/+
Igfbp4-/-
**
**
Igfbp4+/+
Igfbp4-/-
100
120
140
160
180
Body
Length
(mm)
* **
16wk
Females
Males
Igfbp4+/+
Igfbp4-/-
*
Igfbp4+/+
Igfbp4-/-
0.0
0.5
1.0
1.5
Igfbp4
RNA
expression
/
Actin
Females Males

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Igfbp4
+/+
Igfbp4
-/-
Females Males

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40
Females Males
Igfbp4+/+
Igfbp4-/-
**
Igfbp4+/+
Igfbp4-/-
0
Females Males
Igfbp4+/+
Igfbp4-/-
**
Igfbp4+/+
Igfbp4-/-
0.2
0.3
0.4
0.5
aBMC
(g)
*
Females
Males
Igfbp4
Igfbp4
Igfbp4
Igfbp4
8wk 16wk
0.035
0.040
0.045
0.050
aBMD
(g/cm
2
)
** Females
Males
Igfbp4+/+
Igfbp4-/-
Igfbp4+/+
Igfbp4-/-
8wk 16wk
DXA revealed that female Igfbp4-/- mice had decreased BMD at 16wks

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Igfbp4
+/+
Ig
fb
p4
-/-
Females Males
Igfbp4
+/+
Ig
fb
p4
-/-
Females Males
Females Males
Igfbp4+/+
Igfbp4-/-
**
Igfbp4+/+
Igfbp4-/-
Females Males
Igfbp4+/+
Igfbp4-/-
**
Igfbp4+/+
Igfbp4-/-
BV/TV
(%)
0
5
10
15
20
*
Females Males
36
40
44
48
52
Ct.A/TA
(%)
*
Females Males
MicroCT results confirmed our DXA data

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Igfbp4-/- mice are protected against ovariectomy-induced bone loss

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Model: Igfbp4 is regulated by estrogen and enhances IGFI availability

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Not enough to graduate!

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Igfbp4-/- mice have decreased adiposity
**
Females Males
Igfbp4+/+
Igfbp4-/-
**
**
Igfbp4+/+
Igfbp4-/-
**
Females Males
Igfbp4+/+
Igfbp4-/-
**
**
Igfbp4+/+
Igfbp4-/-

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Igfbp4 deletion inhibits adipogenesis in vitro.

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Igfbp4 deletion inhibits adipogenesis in vitro.

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Igfbp4 deletion inhibits adipogenesis in vitro.

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I could graduate!

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I could graduate!
HSDM
And move on

34. Audience Poll

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Alk2
Acvr1
Alk3
Bmpr1a
Alk6
Bmpr1b
Acvr2a Acvr2b Bmpr2
BMP signaling
Type I receptors Type II receptors
Smad1/5/8
Acvr2a Acvr2b
Activin signaling
Alk4
Acvr1b
Smad2/3
TGFβr2
TGFβ signaling
Alk5
TGFβr1
Smad2/3
BMPs, activins and TGFβ receptors in skeletal progenitors

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Alk2
Acvr1
Alk3
Bmpr1a
Alk6
Bmpr1b
Acvr2a Acvr2b Bmpr2
BMP signaling
Type I receptors Type II receptors
Smad1/5/8
Acvr2a Acvr2b
Activin signaling
Alk4
Acvr1b
Smad2/3
TGFβr2
TGFβ signaling
Alk5
TGFβr1
Smad2/3
BMPs, activins and TGFβ receptors in skeletal progenitors

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Activin signaling
Alk4
Acvr1b Smad2/3

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Activin signaling
Alk4
Acvr1b Smad2/3

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Activin signaling
Alk4
Acvr1b Smad2/3
Female
2 6
0
5
10
15
20
BV/TV
(%)
*
*
2 6
0
10
20
30
40
BV/TV
(%)
*
Male
Alk4fl/fl
Alk4fl/fl, Prx1-Cre
Age (months)

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Systemic approach!
:Alk4 binding ligands
Alk4
Alk4-FC
Smad2/3
:Alk4 binding ligands
Alk4
Smad2/3 Smad2/3
Overarching hypothesis
Circulating Alk4-binding ligands regulate bone homeostasis in mice

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Systemic approach!
:Alk4 binding ligands
Alk4
Alk4-FC
Smad2/3
:Alk4 binding ligands
Alk4
Smad2/3 Smad2/3
Overarching hypothesis
Circulating Alk4-binding ligands regulate bone homeostasis in mice
Grant deadline: needed pilot data FAST!

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Inject 12-week-old C57BL6J (n=3) with 3mg/kg BW of Alk4-FC for 2 weeks

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Inject 12-week-old C57BL6J (n=3) with 3mg/kg BW of Alk4-FC for 2 weeks
660 x 581 x 1127 mm, ~ 570lbs

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Inject 12-week-old C57BL6J (n=3) with 3mg/kg BW of Alk4-FC for 2 weeks
95.0
95.5
96.0
96.5
97.0
Ct.
area
/
Total
area
(%)
0.180
0.185
0.190
0.195
0.200
Ct.
Thickness
(mm)
95
100
105
110
Weight
(%
from
Day
0)
p =0.05
65
70
75
80
85
Lean
Mass
(%
from
Day
0)
p =0.03
95
100
105
110
Fat
Mass
(%
from
Day
0)
A B
0.00
0.02
0.04
0.06
BMD
(g/cm
2
)
0.0
0.1
0.2
0.3
0.4
0.5
BMC
(g)
0
5
10
15
20
BV/TV
(%) p =0.05
95.0
95.5
96.0
96.5
97.0
CA/TA
(%)
660 x 581 x 1127 mm, ~ 570lbs

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Inject 12-week-old C57BL6J (n=3) with 3mg/kg BW of Alk4-FC for 2 weeks
95.0
95.5
96.0
96.5
97.0
Ct.
area
/
Total
area
(%)
0.180
0.185
0.190
0.195
0.200
Ct.
Thickness
(mm)
95
100
105
110
Weight
(%
from
Day
0)
p =0.05
65
70
75
80
85
Lean
Mass
(%
from
Day
0)
p =0.03
95
100
105
110
Fat
Mass
(%
from
Day
0)
A B
0.00
0.02
0.04
0.06
BMD
(g/cm
2
)
0.0
0.1
0.2
0.3
0.4
0.5
BMC
(g)
0
5
10
15
20
BV/TV
(%) p =0.05
95.0
95.5
96.0
96.5
97.0
CA/TA
(%)
Secured funding! Project ongoing...
660 x 581 x 1127 mm, ~ 570lbs

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Comparison of DXA systems
PIXImus
Insight Inalyzer

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Comparison of DXA systems
* Significantly different from MRI

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Comparison of DXA systems
* Significantly different from MRI

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Comparison of DXA systems
* Significantly different from MRI
Scanning time was different too!

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Acknowledgements
Rosen Lab
Vicki Rosen, PhD
Emily Van Doren, DMD
Laura Gamer, PhD
Emily Moore, PhD
Bouxsein Lab
Jennifer Coulombe, PhD
Daniel Brooks, PhD
Mary Bouxsein, PhD

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