Osteogenic Potential of Bone
Cylinders in Alcohol and ControlFed Rats
Student: Cyndi Trevisiol
Sponsor: Russell Turner
Nutrition and Exercise Sciences
Impact
• 1 in 3 women and 1 in 5 men over the
age of 50 worldwide are estimated to
have osteoporosis
• Once a skeleton is osteopenic, it is very
difficult to restore lost bone mass
• Musculoskeletal disorders account for
over $125 billion in health costs every
year in the US alone
http://www.answers.com/osteoporosis&r=67
Alcohol and Bone
• Chronic alcohol abuse is a risk factor for
osteoporosis
• Exact impact of alcohol depends on:
• Consumption patterns
– Amount, Duration, and Frequency
– Moderate, Binge or Chronic alcoholism
• Age of the consumer
Our Focus
• Alcohol-induced damage that
precedes osteoporosis
• Therapies that may
counteract the detrimental
effects of alcohol
• Alcohol’s potential benefits
for skeletal health at different
stages of life
• Alcohol-induced
impairment of bone healing
Alcohol stunts bone growth in a rat model
Summer Research Focus
What changes occur when bone
repairs in an alcoholic
environment?
Fracture Healing
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Wound bleeds, clot forms at fracture site
Clot is infiltrated by blood vessels which bring fibroblasts
Fibroblasts produce collagen and a rubbery matrix is formed
Mineralization occurs – calcium sticks to the adhesive matrix
http://www.badgers.org.uk/badgerpages/pictures/meles-xray-1a.jpg
Osteoinductive Bone Cylinders:
Models for Fracture Healing
• Bone cylinders
– Less pain for animals
– Prepared from femurs
– Demineralized bone cylinders
are a matrix of collagen, growth
factors, and cytokines
– Stimulate osteogenesis
Fracture Healing
and Osteoinduction
• Osteoinduction
– A process that induces
osteogenesis
– Osteogenesis
• Endochondral Ossification
occurs after fracture
• Occurs on a cartilage
base
• Osteoprogenitors are
incited to differentiate into
osteoblasts
Osteoprogenitors are found
along bone surfaces
Study Design
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Photo taken by Cyndi Trevisiol
Male Sprague Dawley rats
Rats were labeled on their tails with a marker
Rats received either a control or an alcohol liquid diet
Animals were administered Calcein 9 days apart
Methods
• Preparing demineralized bone cylinders:
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Fascia is removed from femurs
Femurs soak in cold deionized water
Both ends of the long bone are removed
Marrow is flushed out with deionized water
Bones soak at 4°C in 0.6 N HCl overnight (to
demineralize)
– Long bone shafts are extracted in 70% and then
100% ethanol (1 hour each)
– Bone cylinders soak in a 1:1 chloroform/methanol
solution (overnight, to remove fats)
Progression
Frozen rat legs
(m & f) ordered
from Pel-Freez
Biologicals
Six weeks later,
rats (n=20)
sacrificed and
implants excised
Femurs
demineralized &
cut into implants
(n = 80)
Implants
analyzed on the
μCT & select
ones embedded
Bone cylinders
implanted
abdominally (4
per rat)
Other cylinders
ashed for mineral
content
Bone Cylinders Post-Implantation
Whole femur
Demineralized
bone cylinder
Postimplantation
cylinder
• Six weeks after implantation, the rats were
sacrificed and the implants collected
μ-CT analysis
• Micro - Computed
Tomography
• Fires an X-ray beam at a
rotating specimen
• Produces 3D images for
structural measurements
• Used to determine total
bone volume
Histomorphometry
• Representative
bone cylinders
embedded, cut,
and fixed on
slides
• Samples are
digitized by hand
by tracing visible
labels
• Measurements
yield:
– Bone Volume
– Total Volume
– Mineral
Apposition Rate
– Mineralizing
Surface
Photo taken by Cyndi Trevisiol
UV Histomorphometry Image
Fluorescent labels show mineralizing surfaces under UV light
Histology Photos
Stained section showing mineralized
bone, fatty marrow and unmineralized
cartilage
An osteoclast resorbing bone
matrix
μ-CT Results
5
3
*P < 0.000
*P < 0.000
3
SMI
BONE VOLUME (mm3)
4
2
2
1
0
1
Control
Alcohol
DIET
Control
Alcohol
DIET
5
50
4
40
3
30
Ash Percent
Ash Weight (mg)
Ashing Results
2
20
1
10
0
0
Control
Alcohol
DIET
Control
Alcohol
DIET
Conclusions
• Bone volume, structural integrity, and
overall bone mineralization are impaired in
the presence of alcohol in a rat model
• We can conclude that the consumption of
alcohol during the healing process
reduces the quality of newly formed bone
• The alcoholic skeleton is prone to a higher
risk of re-injury at healed fracture sites
On the horizon…
• We now know that chronic alcohol consumption
significantly suppressed proper bone healing in
a long-term rat model
• Is there a biological “memory” of alcohol
consumption in bone – will previously alcoholic
bone heal and remodel properly in an alcoholfree environment?
• Possible therapies to reverse alcohol-induced
osteopenia in the presence of alcohol? (PTH)
Acknowledgements
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Howard Hughes Medical Institute
Russell Turner
Urszula Iwaniec
Jaime Hunter
Jill Pfaff
Phil Menagh
Uli’s
Kevin Ahern
http://www.jdrf.org/images/Chapters_and_Affiliates/minnesota_chapter/thank%20you%20kids.jpg