CORTICAL BONE POROSITY, MECHANICAL PROPERTIES, AND CROSS-SECTIONAL PROPERTIES DO NOT
SHOW LOSS DURING DISUSE (HIBERNATION) OR WITH AGE IN GRIZZLY AND BLACK BEAR FEMURS
*McGee, ME; *Miller DL; *Maki, AJ; **Auger J; **Black HL;***Nelson, OL; ***Robbins, CT; +*Donahue, SW
+*Michigan Technological University, Houghton, MI
[email protected]
Methods
Effects of Hibernation on Grizzly Bear Femurs
Femurs were obtained from six grizzly bears with approval from the
Washington State University Institutional Animal Care and Use
Committee. Three bears were 17-18 weeks into hibernation and the
others had been active following hibernation for at least 14 weeks. The
bears were administered an IV solution of calcein on days 15 and 5
before euthanization. One femur from each bear was used for histology
(n=6), and the contralateral was used for mechanical testing (n=5).
A cortical section 20 mm distal to midshaft was removed and thin
sections were stained with basic fuchsin. Porosity (Por) was quantified
at 40x magnification with image analysis software (BIOQUANT). The
maximum moment of inertia (Imax) and cross-sectional area (CSA) were
determined with a custom-written macro in Scion Image.
Another cortical section was removed 35 mm distal to midshaft and
processed for dynamic histomorphometry. Inter-label width was
measured at 400x magnification in 5 µm intervals between calcein label
midpoints; a maximum of 50 osteons per anatomical quadrant were
measured and averaged. The total number of labeled osteons was
quantified at 250x magnification, and intracortical mineral apposition
rate (MAR) and labeled osteon density (L.On.Dn) were calculated.
The contralateral femurs (n=5) were loaded to failure in three-point
bending on an Instron to determine the ultimate stress (σU) and energy to
failure (U). A cortical section 7.5 mm proximal to midshaft was ashed
in a furnace to determine the mineral content (n=6).
Cumulative Effects of Annual Disuse Periods in Black Bears
Femurs were obtained from hunter-killed black bears and black bears
that had died a natural death in Utah and Alaska. Twelve were from
females (ages 2-20 yrs) and 15 were from males (ages 1-17). A
midshaft section was removed, and the cross-section was imaged with a
digital camera. Cross-sectional area, section modulus, moments of
inertia, and porosity were quantified with Scion Image. ANCOVA was
used compare males and females, treating age as the covariate. Males
and females were significantly (p < 0.05) different for all properties, and
were therefore separated for regression analyses.
Results
Intracortical remodeling decreased in hibernating grizzly bears
compared to active bears (i.e., lower mineral apposition rate and labeled
osteon density), which significantly decreased porosity (Table 1). Ash
fraction, cross-sectional, and mechanical properties were not
significantly (p > 0.3) different between hibernating and active bears.
In the male black bears, all cross-sectional properties significantly
(p ≤ 0.003) increased with age (R2 ≥ 0.52). None of the cross-sectional
properties changed with age in female bears (p > 0.159). Cortical
porosity did not change with age in male (p < 0.122) or female
(p < 0.131) bears (Figure 1).
Discussion
In most animals, disuse uncouples bone formation and resorption,
leading to an increase in porosity and a decrease in whole bone
geometrical and mechanical properties. Conversely, grizzly bear femurs
had lower porosity during hibernation compared to summer, probably
because the activation of osteonal remodeling decreased and bone
formation remained coupled to resorption during hibernation. The
structural and mechanical properties of hibernating grizzly bear femurs
were not significantly different compared to active bears. Our findings
suggest that grizzly bears do not experience a transient increase in
cortical bone remodeling and porosity or a temporary loss of mechanical
properties during hibernation. Additionally, black bears do not
accumulate losses of cross-sectional properties or increases in porosity
with age despite the relatively short remobilization period following
hibernation. Research on the biological mechanism by which bears
maintain bone integrity during disuse and with age may provide insight
regarding pharmaceutical treatments for human metabolic bone diseases.
Table 1: Bone properties for active and hibernating grizzly bears.
Property
Por
(%)
MAR
(µm/day)
L.On.Dn
(# / mm2)
Imax
(mm4)
CSA
(mm2)
σU
(MPa)
U
(J)
Ash fraction
(g/g)
Active
Hibernating
pvalue
Percent
Difference
7.5
5.3
.003
-29.3%
1.0
0.8
.038
-20.0%
.081
-62.3%
.949
-5.1%
7.7
3.9 x 10
2.9
4
3.7 x 10
4
344
358
.959
4.1%
184
213
.308
15.8%
47.1
57.0
.827
21.0%
.664
.675
.407
1.7%
Figure 1: Cortical porosity did not change with age in bears.
Cortical Porosity With Age
Male
Female
12
10
Porosity (%)
Introduction
Disuse typically causes an imbalance in bone formation and
resorption, leading to bone loss and reduced mechanical properties. The
remobilization time required to recover lost bone is typically 2-3 times
longer than the length of the disuse period. Bears from northern
climates, experience annual periods of disuse (hibernation) and activity
that are approximately equal in length (6 months), yet black bear bone
mechanical properties do not decline with age. Bears may maintain bone
mass despite annual disuse because bone formation remains balanced
with resorption during hibernation. It is not known if bears prevent bone
loss during hibernation or experience transient bone loss and make
complete recoveries during remobilization. Thus, we investigated
cortical bone properties in hibernating and active grizzly bears (Ursus
arctos horribilis) and in black bears (Ursus americanus).
Male
R2 = 0.174
p=.122
8
Female
R2 = 0.214
p=.131
6
4
2
0
0
5
10
15
20
25
Age (yrs)
Acknowledgements
This work was supported by grants from NIH (NIAMS AR050420) and
the Michigan Space Grant Consortium, and a gift from Timothy Floyd,
M.D.
Affiliated Institutions for Co-Authors
** Brigham Young University, Provo, UT
*** Washington State University, Pullman, WA
52nd Annual Meeting of the Orthopaedic Research Society
Paper No: 1608