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 de-calcified 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 Toweland 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:

  1. Sample Dissection
  2. 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.
  3. 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).
  4. Take any relevant measurement of the bones (i.e. vertical and horizontal diameters and circumference).
  5. For the bones in the C-group, wrap them around with a dampened paper towel and store inside a 37ºC incubator.
  6. 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.
  1. Setup and calibrate the Instron Model 4444 Machine
  2. 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”.
  3. Specify the speed and direction of movement of the crosshead.
  1. Testing C-group Bones
  2. While the bones in D-group are still in the solution, take the C-group bones and begin fracturing them on the instron.
  3. Make sure the bone samples are lying as flat as possible between the supports to prevent them from sliding off.
  4. Run the Instron at 5 cm/min with a sampling rate of 20 points per second.
  5. After fracture, measure thickness of the bone and calculate inner diameter by subtractingit from the outer diameter.
  6. 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.
  7. 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, .
  1. Testing D-group Bones
  2. 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.
  3. Using the same method mentioned above to determine the failure energy and the Young’s modulus for the D-group bones.
  1. Data Analysis
  2. 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.
  3. 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 D-group 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 ( 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.

  • 3Farm 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:

* (ro,average2 – ri,average2)

for oval cross-section:

* (ro,long * ro,short3 – ri,long* ri,short3)

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.

Figure A2: Identifying the point of ultimate failing force and bending displacement from the prior experiment.

VIII. REFERENCE

1Stewart, Susan C. “Taking Osteoporosis Out of your Future – A Major Challenge for Women.” 2004. 18 Apr. 2005. < /womens_health/osteoporosis_1>.

2 Woods and Ellis, “Bone,” Laboratory Histopathology: A Complete Reference, 1994.

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