4/25/2007

Antonios Ioannou

Instron Tensile Testing: Structural and Material Properties of Skeletal and Cardiac muscle

Background Information

Myosin not only causes the contraction of the muscle by forcing the thick and thin filaments slide together but also, consisting a major part of the basic unit of muscle tissue (sarcomere),it structurally holds it together. The thick filaments (myosin complexes) bind together but also bind to the thin filaments (actin filaments), providing strength to the structure of the muscle (Appendix: Figure 1)[i]. It could be assumed that in different types of muscle, myosin levels would differ. In a related experiment skeletal and cardiac pig muscle where tested for protein analysis. Using the methods of gel electropohoresis and MAT LAB image analysis it was found that the ratio of myosin in skeletal versus cardiac muscle was 3.02/1 (Appendix: Table 2). The difference is significant and could lead to form the suggestion that due to higher myosin levels, skeletal pig muscle would show greater strength than cardiac muscle when tested with the Instron4444. This kind oftesting will provide the data to calculate and compare the failure strength and failure force between the two groups. In this way combining two kinds of experiments (Muscle Protein Detection & Instron Tensile Testing) it’s made possible to test if higher myosin level in muscle tissue can be related to higher strength.

Hypothesis/Objective and Aim(s)

In this experiment it is hypothesized that skeletal pig muscle is going to show higher failure strength and force than cardiac pig muscle when tested with the Instron4444. From previous testing this hypothesis is based on the difference in myosin levels between the two muscle groups.

Equipment

  • Major Equipment:
  1. Instron4444 : This machine is needed as its purpose is to test materials by applying force (pulling apart) and collecting data at the same time (displacement, force applied).The clamps/specimen holders that would be ideal for this experiment are the 2712-003 Pneumatic Action Grips whose price ($3,390) unfortunately overcomes the budget provided. The grips used are going to be the ones already on the Instron4444 which have the area of contact needed to grip on the muscle without destroying it in any way but also can exert the adequate force to keep the samples in place.
  • Lab Equipment:
  1. Scalpel, Scissors and Cutting board: Needed for the cardiac and skeletal muscle to be extracted and form uniform samples from each of the groups.
  2. Length measuring instruments (calipers & rulers): Needed for the samples to be measured and as precision is important, the instruments need to provide a small scale of good quality.
  3. Pens and Markers: Needed for assistance while forming the samples.
  4. Weight set (500g, 1kg, 2kg): Needed to calibrate the Instron4444
  • Supplies:
  1. Foam: Needed for initial testing and familiarizing with the Instron4444 (one roll of 1/2 inch thick Uline Polyurethane Foam).
  2. Pig Hearts: Two pig hearts will be provided for each group (40 pig hearts totally).
  3. PigFeet: 8 pig feet will be provided for each group (160pig feet totally).
  • Newly Purchased Equipment: None

Proposed Methods & Analysis

Safety Issues: See Safety Issues in the Lab Manual for Experiment #3

A. Specimen Harvest and Preparation

Extract muscle from each of the eight feet and the two pig hearts. You will need to have 8 samples from each group. One sample from each foot and four from each heart (the size of a pig heart is almost the size of a human heart which hasapproximately the size of a clenched human fist and weighs about 11 ounces/310 grams)[ii]. The samples should be 1.5×0.75×0.3 inches. Make sure you note down the exact dimensions of the samples extracted, in case error has occurred.It is recommended that you examine the clamp geometry for gripping the specimen and how much vertical space is available between the clamps for your specimen’s length to confirm that your plan is adequate.

B. Instron4444 Set-Up and Surrogate Tensile Testing

1-2. Follow steps 1-2 in the Lab Manual for Experiment #3

3.Foam is provided to serve as surrogate samples for tensile testing to familiarize your group with the testing apparatus, data acquisition, geometric limitations, and overall testing procedure variables, such as loading rate. It is suggested you cut the foam provided to approximate the geometry of your muscle samples and determine how many surrogate samples you need to practice running the Instron4444 and acquiring data. Use these tests to determine the appropriate rate of loading and sampling rate for data acquisition. You may wish to vary the crosshead speed to examine the effects of loading rate on failure properties if you have enough specimens. Regardless, use the surrogate sample testing to establish your group’s protocol for testing pig muscle groups in tension.

4. Follow step 4 in the Lab Manual for Experiment #3

C. Instron Specimen Tensile Testing

  1. Follow step 1 in the Lab Manual for Experiment #3
  2. Follow step 2 in the Lab Manual for Experiment #3, applying everything that is being said for chicken skin to muscle samples.
  3. Follow step 3 in the Lab Manual for Experiment #3

D. Protocol

Calibrate Instron4444 according to the instructions in the lab manual.(15 min)

Test on the Instron4444 a piece of Foam (size similar to your samples), figuring out at what crosshead speed and what sampling rate is more appropriate.(10 min)

Extract the 8 skeletal and 8 cardiac muscle(one sample from each foot and four from each heart) from the given pig organs and cut them into 1.5×0.75×0.3 inch samples. Keep the muscle samples moist for the duration of the experiments.(40-60 min)

Load the skeletal and cardiac muscle samples into the Instron444 and after recording the sample width, gage length and thickness, apply stress with the Instron4444 until the sample tears. Save electronic data files for later analysis. (60-90 min)

Using MATLAB, plot force-displacement and stress-strain curves for the two sample groups. Calculate using MATLAB tools the failure force and failure strain from the two plots.

Use an unpaired t-test assuming equal or unequal variances depending on the descriptive statistics find any significance of failure force and failure strength between the cardiac and skeletal muscle samples.

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Potential Pitfalls & Alternative Methods/Analysis

Having to deal with muscle tissue could be a source of pitfalls and error. The texture of the muscle itself turns the task of getting uniform samples into a risky and difficult procedure. The markers will not always be clear on the tissue, while the slicing process could lead to error in the dimensions of the samples due to: the scalpel/scissors not being sharp enough and inaccuracy of the individual carrying out this procedure. Performing descriptive statistics on the same group samples would reveal how close the dimensions of the samples are and how reliable they are. It would be preferred if this part of the experiment was completed by the group member with the most steady hands (ideally practice on a piece of tissue sample that is not going to be used) and who has a plan on how to physically approach this situation.

In addition to the above, while trying to keep the muscle samples moist, different levels of wetness could be reached. This could alter the properties of the sample in ways that could interfere with the results and what is being tested in the experiment. Damping the samples uniformly and carefully can be crucial here.

Placing the samples on the Instron4444 is one of the trickiest parts as it’s challenging to manipulate tissue samples of such small size. The space between the clamps after the specimen is placed on the Instron4444 should be as similar as possible between the trials. Otherwise, by varying the distance, what is examined varies (stiffness will vary). Position of specimen placement should be recorded and again descriptive statistics are needed to show how close the samples of each group are. The specimens should be clamped always in the middle in order to maintain consistency.

The position of the split varies most of the time, something that cannot be controlled. The ideal split would be at the middle. Comments about the geometry of the split sample should be made and be taken into consideration for the lab repot.

Budget

The budget of the experiment is not even approaching the limits given, as there is no new purchased equipment. Needed are only supplies such as: pig hearts (40 pieces), pig feet (160 pieces) and a roll of polyurethane foam. Forty pig hearts willcost $266.56, purchased online from Sargent-Welch ( Hundred and sixty pig feet will cost $176.8 (as the price of 2 feet is $2.21), purchased from Fresh Grocer (40th and Walnut).

The polyurethane foam costs $97 (one roll of 24’’×27’ is a sufficient amount) purchased online from Uline (

Totally the money spent for the experiment is $540.36.

References:

[i] Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, Peter Walter

Molecular Biology of the Cell(4th edition), New York: Garland Science 2002

[ii] Through the website links of the online Britannica Encyclopedia, “The Matter of the Heart”, The Franklin Institute,

Appendix

Figure 1: The Sarcomere: shows how the basic unit of muscle tissue is organized. It is obvious how myosin thick filaments are the basic part that holds the sarcomere together at the M line and at Cap Z but also while it is bounded to the actin thin filaments.

Table 2:The concentrations of the heavy chain of myosin found in the muscle digest solutions(six samples from each group). There was a significantly higher concentration of the heavy chain of myosin found in the skeletal muscle than in the cardiac muscle (p=0.001).