The Effect of Decalcification
On Chicken Bone’s Young’s Modulus
and Fracture Energy
04/25/2007
Zheng Lin
BE-210-101
I. BACKGROUND:
As the primary component of the endoskeleton system, bones are composed of a
collagen fiber matrix, which is hardened by calcium hydroxyapatite mineral crystals
(Ca10(PO4)6(OH)2). Osteoporosis is a disease of bone in which the rate of breaking down
the bone is faster than its formation, and causes overall loss of the hydroxypatite
minerals.1 Thus, a patient with osteoporosis has fragile bones that are more susceptible to
injuries that result in fracturing, which is closely related to bone’s energy-absorbing
capability. In the previous experiment (EXP#4), the fracturing property of the chicken
bone was quantitatively characterized by determining its failure energy and Young’s
modulus. The Young’s modulus was calculated by using the area moment of inertia
formula, E = PL3 / 48yI, where y is the bending displacement, P is the ultimate failure
strength, L is the distance between the two supports, and I is the area second moment of
inertia, which is approximated by assuming an oval shape cross-sectional area (Figure A1,
Appendix). The fracture energy was calculated by integrating force over bending
displacement graph until ultimate failure point occurs (Figure A2, Appendix).
As an expansion of the previous experiment, this proposed experiment will utilize
the already-determined oval shape cross-sectional area approximation method to
calculate and compare the Young’s modulus between normal chicken bones and
decalcified chicken bones. Additionally, their required failure energy will also be
calculated and compared. To decalcify the bone, two general methods are used: acid
method and chelating method. Acids will directly dissolve and ionize calcium mineral
inside the bones, while chelating agents bind the calcium and slowly depletes the exposed
area of the mineral crystal.2, 3 However, due to limited time in the lab, only the acid
method will be used, because of its relatively rapid decalcifying rate. The required
decalcifying solution will be purchased in a form of Decal Stat™ solution, which extracts
calcium by reacting hydrochloric (HCl) acid with the calcium hydroxyapatite present in
the bones.
II. HYPOTHESIS AND OBJECTIVES:
Objectives:
 To determine the failure energy and Young’s modulus of both normal and decalcified chicken bone by using an Instron Model 4444 materials testing machine.
 To simulate and study the osteoporosis conditions by comparing the fracturing
properties between the normal and decalcified chicken bone.
Hypothesis:
 The fracture energy and Young’s modulus of the decalcified chicken bones will be
significantly less than those of the normal chicken bones.
III. EQUIPMENT:
Major Equipment

Instron Model 4444 Benchtop Materials Testing Machine
The Instron Model 4444 will be used to apply uni-axial load to the chicken bone samples.
The crosshead speed and sampling rate of the machine will be configured to take proper
readings of the displacement of samples in response to the monitored applied force.

Computer with LabView Program
The LabView Program will be needed to monitor the data output of the Instron Model
4444. The program will construct force-displacement graphs for each bone sample.

An mini-incubator
It is used to mimic the in-vivo conditions of the chicken bones as accurately as possible
by keeping the harvested bones at approximately the body temperature of a chicken.
Lab Equipment


Cutting Board, Dissecting Knives, Scissors, Tongs
Calipers and Rulers
The scalpel, scissors and cutting board will be used to isolate the chicken bones from
their attached fleshes. The calipers and ruler are use to measure the dimensions of the
bones, such as their vertical and horizontal diameter, which are important in determining
the cross-sectional area of the bones.
Supplies

Paper Towel and Soap
The supplies will be used for clean-up and to avoid messes.
Newly Purchased Equipment

Raw Chicken Legs
Chicken legs will be used as the source of chicken bones for the experiment, and can be
purchased from any major supermarket, suck as Fresh Grocer and Costco Warehouse.

Decal StatTM solution
Decal StatTM solution can be used to decalcify the chicken bones and can be purchased
from Decal Chemical Corp.
IV. PROPOSED METHODS AND ANALYSIS:
A. Sample Dissection
1. Use a dissecting knife to carefully remove the tabiotarsus from each
chicken leg and making sure all skins and fleshes are removed completely
off the bones. Repeat this step for all 10 chicken legs.
2. Separate the harvested bones into two groups – five into the D-group (or
the de-calcified group) and five into the C-group (or the control group).
3. Take any relevant measurement of the bones (i.e. vertical and horizontal
diameters and circumference).
4. For the bones in the C-group, wrap them around with a dampened paper
towel and store inside a 37ºC incubator.
5. For the bones in the D-group, de-calcify the them by completely
submerging them in 400 ml of Decal StatTM solution for exactly 30
minutes and store them inside a 37ºC incubator as well.
B. Setup and calibrate the Instron Model 4444 Machine
a. Set the Instron Model 4444 to “SI” mode and calibrate the machine
according to part A of the protocol in Experiment #4, “Bending: Bone
Fracture”.
b. Specify the speed and direction of movement of the crosshead.
C. Testing C-group Bones
a. While the bones in D-group are still in the solution, take the C-group
bones and begin fracturing them on the instron.
b. Make sure the bone samples are lying as flat as possible between the
supports to prevent them from sliding off.
c. Run the Instron at 5 cm/min with a sampling rate of 20 points per second.
d. After fracture, measure thickness of the bone and calculate inner diameter
by subtracting it from the outer diameter.
e. Using the force and displacement data determine failure point and find
failure force and displacement. Use MatLab to calculate failure energy by
integrating from zero to the point of failure.
f. Calculate the Young’s Modulus using the area moment of inertia formula,
E = PL3 / 48yI, where P is the failure force, L is the distance between the
supports, y is the bending displacement, and I is the area moment of
inertia of an oval,
.
D. Testing D-group Bones
a. Once bones have undergone de-calcification, follow the same procedure to
test the D-Group bones as the one used to test the C-Group bones in
Section C of this experiment.
b. Using the same method mentioned above to determine the failure energy
and the Young’s modulus for the D-group bones.
E. Data Analysis
a. Compare the facture energy using a paired one-tailed t-test, alpha = 0.05,
to determine whether the average fracture energy of the D-group bones is
significantly less than that of the C-group bones.
b. Compare the Young’s modulus using a same paired one-tailed t-test, alpha
= 0.05, to determine whether the average Young’s modulus of the D-group
bones is significantly less than that of the C-group bones.
V. POTENTIAL PITFALLS
A major problematic area of the experiment has to do with the decalcification
solution, Decal StatTM. The rate of decalcification is affected by the surface area of bone
exposed. The problem arises because not all of the bones have the same surface area.
This means that the surface area of all chicken bones used must not be statistically
different in order for the solution to decalcify consistently, allowing for the experiment to
give appropriate results. If the bones have not been decalcified equally, then the fracture
energy and Young’s modulus of decalcified bones will be inconsistent even though they
have been kept in solution for the same amount of time.
Additionally, the possibility of over-decalcification is another major concern.
Although the manufacturer of Decal StatTM solution recommends two hours for a
complete decalcification of a similarly sized bone, the exact amount of time required to
achieve a desire decalcification is still unclear. Thus, it is possible to remove more than
enough calcium to render the bones too flexible to break in the Instron machine, because
the overexposing of its underlying collagen-matrix structure. Besides over-decalcification,
under-decalcification could also present a problem. Assuming the Decal StatTM supplier
was correct in stating that the time required to completely decalcify the bone is two hours,
then thirty minutes should be enough time to notice a significant decalcification in the Dgroup bones, as compared to the C-group bones. However, there is also a possibility that
the thirty minutes may be too little time to achieve enough decalcification to notice a
significant difference between the two groups. To solve these problems, three or four
extra chicken bones should be purchased and tested prior to the actual experiment to
determine if thirty minutes is an adequate time for desired decalcification.
Moreover, random errors are also major parts of concern in this experiment. The
first major concern is the small sampling size of bones, which was also a problem in the
prior experiment. With such a limited sampling size of chicken bones, random errors
such as chicken’s health, age and dieting habit are hard to be eliminated and may even be
magnified during the data analysis step. Similarly, the inconsistent fracturing pattern of
each individual chicken bone also poses the problem for data analysis. For example, if
one bone out of five breaks awkwardly, the average obtained value can then drastically
influenced if the sampling size is small. Therefore, in order to rid of these random errors,
a larger sample size of at least 20 bones for each group should be used, but this might be
hard to do because of restriction in time.
VI. BUDGET
Listed below are the purchases required to complete this lab. Prices include the
approximate costs of shipping for the equipment to be delivered to Philadelphia. All
prices and product information were taken from the listed suppliers.

2 Quarts of Decal StatTM Solution
 Price: $38.00
 Supplier: Decal Chemical Corp
 Specifications: Rapid de-calcifying solution, active ingredient is
hydrocholoric acid ( <3% HCl )
This decalcifying solution was chosen, as opposed to the many others available, for its
speed in reacting with bone (approximately 1.5 – 2.0 hour for full decalcification) and for
its relatively low cost. Although this solution, like most other decalcifying solutions, is
intended to be used for bone biopsies, its effect on the chicken bones will still be to
reduce calcium content and therefore the stiffness and brittleness of the bones.
According to the manufacturer (http://www.decal-bone.com), the solution will also
reduce calcium content without affecting the bone’s mineral content in any other way and
without compromising the bone’s structural integrity.

3 Farm Fresh Brand 6-Chicken Drumstick Package
 Price: $30.00
 Supplier: Fresh Grocery
 Specifications: 2 – 4 pound quarter uncooked chicken
These specific chickens were selected for their relatively large size in comparison to
other whole chickens available. Larger chickens will make the process of removing the
femurs easier, since it would not be necessary to deal with smaller ligaments or other
connective tissue.
VII. APPENDEX
E = PL3/48yI
for cylinder cross-section:

I
* (ro,average2 – ri,average2)
4
for oval cross-section:

I
* (ro,long * ro,short3 – ri,long* ri,short3)
4
rshort,inner rlong,outer
rshort,inner
rshort,outer
Figure A1: Equations used to calculate Young’s modulus. P is ultimate force, y is
ultimate displacement, and L is length of the sample from clamp-to-clamp.
Ultimate failing force
&displacement
Figure A2: Identifying the point of ultimate failing force and bending displacement from
the prior experiment.
VIII. REFERENCE
Stewart, Susan C. “Taking Osteoporosis Out of your Future – A Major Challenge for
Women.” 2004. 18 Apr. 2005. <http://www.thedoctorwillseeyounow.com/articles
/womens_health/osteoporosis_1>.
1
Woods and Ellis, “Bone,” Laboratory Histopathology: A Complete Reference, 1994.
http://home.primus.com.au/royellis/bone.htm
2
3
M. Shahnazari, D. H. Lang, G. J. Fosmire, N. A. Sharkey, A. D. Mitchell, R. M. Leach.
“Strontium Administration in Young Chickens Improves Bone Volume and Architecture
but Does not Enhance Bone Structural and Material Strength” Calcified Tissue
International 2007 80:160