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Abstract |
Osteoporosis is a disease characterized by low bone mass and structural
deterioration of bone tissue, which leads to bone fragility and increased
fracture risk. The major factor contributing to the structural
deterioration of cortical bone is increased porosity caused by bone
remodeling. Consequently, the strength and stiffness of bones are
reduced. For prolonged bed rest, immobilization, and space flight, disuse
atrophy of bone is prevalent and increases fracture risk when normal bone
loading is resumed.18,52 Disuse osteopenia occurs in many animals including
rats,9,39,53,54 roosters,29 hibernating ground squirrels,71 sheep,57,58
dogs,34 monkeys,36,69,70 and humans.4,18,46,56,61 On the contrary, black
bears may not develop osteopenia during long periods of disuse (i.e.,
hibernation) to the extent that other animals do.23,27 During hibernation
osteoclastic resorption increases as it does in other animals. However,
osteoblastic formation levels remain normal, a metabolic bone feature
unique to bears.23,27 Additionally, black bears may have a mechanism for
more rapid and complete bone recovery from disuse than other animals do.23
Therefore, we hypothesize that black bears minimize the degradation of bone
material properties, which result from annual periods of disuse, because
they maintain normal bone formation during disuse and make a rapid and
complete recovery of bone mass during remobilization. The impetus for this
project is to understand the effects of annual periods of disuse on bone
mechanical properties, histologic features, and mineral composition, and
the biological mechanisms that may be involved in bone recovery during
remobilization. The goals of the proposed project are (1) to quantify
age-related changes in black bear bone mechanical properties, mineral
content, and histologic features, and (2) to quantify the effects of black
bear serum on mechanically stimulated intracellular calcium signaling in
osteoblastic cells in vitro.
Hypothesis 1: Due to increased bone resorption caused by annual periods of
disuse (i. e., hibernation), the activation frequency of bone remodeling is
higher in black bears than in other species and Haversian canals accumulate
more rapidly with age in black bears than in other species.
Aim 1: Black bear bones will be obtained from hunter-killed
bears. Histologic cross-sections from black bears tibias will be prepared
to quantify the activation frequency and Haversian canal density. The
bones will be stained with basic fuchsin and embedded in methyl
methacrylate. Cross-sectional wafers will be cut from each specimen on a
precision saw and mounted on glass microscope slides. Cross-sections will
be viewed at 100x magnification using a light microscope and digital
imaging system. An overlaid grid will be used to count the total number of
Haversian canals. Cross-sectional area will be measured with a digital
imaging system and image analysis software. The activation frequency and
Haversian canal density will be calculated and regressed against age. To
calculate the remodeling activation frequency we will use an equation
derived by Martin.42 The activation frequency of bone remodeling and the
rate of change of Haversian canal density with age will be used to evaluate
how annual periods of disuse affect these rates in black
bears. Age-related changes in these parameters will be compared with
values for other species including humans.
Hypothesis 2: Because of their ability to maintain osteoblastic function
during disuse and rapidly increase bone formation during remobilization,
cortical bone porosity, mineral content, and mechanical properties do not
decrease with age at faster rates in bears than in humans, despite
increased bone resorption in black bears during annual periods of disuse.
Aim 2: For mechanical testing, bone specimens will be milled into dumbbell
shaped testing coupons. The testing coupons will be loaded to failure in
uniaxial tension in a materials testing machine at strain rates used in
previous studies on human bone. The elastic modulus, yield stress and
strain, ultimate stress and strain, and energy to fracture will be
calculated for each specimen. Histologic slides will be prepared as
described in aim 1 for one half of the fractured testing coupons. A grid
containing 121 points will be used to measure porosity by counting the
fraction of points that fall within a void space on the bone section. The
other half of the testing coupon will be ashed in a furnace to quantify
mineral content. Regression analyses will be used to correlate the
porosity, mineral content, and mechanical parameters with age. The slopes
of these regression analyses will be compared to the values for human bone
from previous studies.
Hypothesis 3: Osteoblastic MC-3T3 cells are more sensitive to mechanical
stimulation in the presence of black bear serum collected during
remobilization, than in the presence of fetal bovine serum or black bear
serum collected during hibernation.
Aim 3: MC-3T3 cells will be cultured to confluency and seeded onto quartz
microscope slides. On the day of experimentation, the cell-seeded
microscope slides will be mounted into a parallel plate flow chamber and a
custom built pumping apparatus will expose the cells to fluid flow induced
shear stress. This system is designed to load the cells on the stage of a
fluorescent microscope so that real-time intracellular calcium
concentration can be quantified in individual cells using ratiometric dye
methodology. We will use a custom written computer program to quantify the
peak values, rise and fall times, overall duration, and the time-averaged
magnitude of the oscillation in intracellular calcium concentration for
each individual bone cell. ANOVA will be used to compare these oscillation
parameters and the percentage of cells responding between 3 groups: fluid
flow with hibernating bear serum, active bear serum, or fetal bovine serum.
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Discipline |
Keywords |
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Osteoporosis, bone mass, bone, black bear, bone miscrostucture, microcracks
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