Mentor: Roeder
Location of Research: ND
Category of Research: Bioengineering
Working Title: Influence of Bone Adaptation and Loading on Stress Fracture Suseptibility
Topic/problem:
Stress fractures and fatigue fractures affect a large number of people in society. These fractures range from insufficiency fractures, due to diseases such as osteoporosis, to fatigue fractures, due to strenuous physical activity such as distance running. Osteoporotic fractures can be fatal. Fatigue or stress fractures are not typically fatal, still present a burden on those afflicted with them. The factors that cause these fatigue fractures and methods of prevention are not yet well understood. Ex vivo studies, while not directly comparable to an in vivo model, can still shed light on the mechanisms and possible causes for fatigue and insufficiency fractures. The main function of the skeleton is to support and provide protection for the body. Different bones in the skeleton are adapted to different modes and levels of mechanical loading. For example, the human tibia is primarily loaded in compression and must bear greater loads than the ulna, which is primarily loaded in torsion and bending. Animal models can provide a useful surrogate for impractical or unethical human experiment, as well as the limited supply of cadaver tissue.

Research Plan:
Purpose: Stress fractures and fatigue fractures affect a large number of people in society. These fractures range from insufficiency fractures, due to diseases such as osteoporosis, to fatigue fractures, due to strenuous physical activity such as distance running. Osteoporotic fractures can be fatal. Fatigue or stress fractures are not typically fatal, still present a burden on those afflicted with them. The factors that cause these fatigue fractures and methods of prevention are not yet well understood. Ex vivo studies, while not directly comparable to an in vivo model, can still shed light on the mechanisms and possible causes for fatigue and insufficiency fractures. The main function of the skeleton is to support and provide protection for the body. Different bones in the skeleton are adapted to different modes and levels of mechanical loading. For example, the human tibia is primarily loaded in compression and must bear greater loads than the ulna, which is primarily loaded in torsion and bending. Animal models can provide a useful surrogate for impractical or unethical human experiment, as well as the limited supply of cadaver tissue.

Hypothesis: The rat tibia will exhibit a greater fatigue life and different damage patterns compared to the ulna/radius when loaded in cyclic uniaxial compression ex vivo due to being more adapted to mechanical loading.

Specific Aims:

1) Develop an ex vivo loading protocol to apply controlled cyclic uniaxial compressive loads to the tibia in whole rat hind limbs and the radius/ulna in whole rat forelimbs.

2) Measure differences in the fatigue life and mechanical degradation of the tibia and radius/ulna for equal load levels, as well as physiological load levels.

3) Measure differences in the location, extent and morphology of damaged tissue and/or stress fractures in the tibia and radius/ulna using contrast enhanced micro-computed tomography.

Methods:

Aim 1: Develop an ex vivo loading protocol to apply controlled cyclic uniaxial compressive loads to the tibia in whole rat hind limbs and the radius/ulna in whole rat forelimbs.

1) Acquire whole rat forelimbs and hind limbs.

2) Place the limbs into the load frame fixtures for cyclic uniaxial compression.

3) Investigate and tune the instrument for different levels of applied load magnitude (N) and frequency (Hz) in order to determine appropriates levels for Aim 2. For example, a load level that is too high will prematurely fracture the bone while a level that is too low will not produce enough damage in the tissue.

Aim 2: Measure differences in the fatigue life and mechanical degradation of the tibia and radius/ulna for equal load levels, as well as physiological load levels.

1) Load both the tibia and ulna/radius in cyclic uniaxial compression at fixed load and frequency levels.

2) Measure displacement of the bone to calculate stiffness.

3) After calculating stiffness, determine fatigue life of each bone.

4) After fatigue life has been determined, expose tibia and ulna/radius to 5% and 10% stiffness loss as well as the failure level (determined by calculating fatigue life) to achieve fracture. Also, set multiple samples of tibia and ulna/radius aside to be used for the control. Use several samples of both bone types to ensure accuracy.

Aim 3: Measure differences in the location, extent and morphology of damaged tissue and/or stress fractures in the tibia and radius/ulna using contrast enhanced micro-computed tomography.

1) Stain bones from both the variable and control groups that were used during Aim 2 with a BaSO4 precipitation. Soak the bones in a solution of equal parts acetone, PBS, and 0.5 M BaCl2 in DI water for 3 days under vacuum. Soak the bones in a solution of equal parts acetone, PBS, and 0.5 M Na2SO4 in DI water for 3 days under vacuum. After each soaking, rinse the bones with DI water to prevent build up of foreign particles and ions on the surfaces of the bones.

2) Image the entire bone by micro-computed tomography (µCT-80, Scanco Medical AG, Brüisellen, Switzerland). Adjust scanner settings (resolution, voltage, current, and integration time) as needed in order to obtain a high quality image. Observe areas of the bone highlighted by BaSO4 in order to locate damage on the bones.

3) Measure damage by using the equation d=SV/TV where d is damage, SV is stain volume, and TV is total volume. Stain volume is the total volume of the stained tissue. Total volume is the volume of the entire bone segment. Both volumes can be measured using the micro-CT scanner.

Potentially Hazardous Tissue:

I will be working with the tibia and ulna/radius of rats. I will be using whole bone limbs in my test. The bones are being acquired from animals that are sacrificed for other purposes. The hazard level is low as long as proper sanitation techniques are observed. I will not be directly disposing of the tissues. Those who will be will follow the methods set forth by the University of Notre Dame.

Comments:
11/16/11-So I am really interested in pursuing a career in Biomedical Engineering and I was able to find Dr. Roeder over at ND who is in bioengineering aka biomed engineering. If you read my research plan you should be able to follow what I'll be doing here. I will be taking whole rat forelimbs (arms) and whole rat hindlimbs (legs) and subjecting them to uniaxial cyclic loading. This loading causes compression in the bones and after several thousand cycles the bones are weakened and evetually fracture at the failure point. I will be looking at the different effects this loading causes on the bones. The bones are adapted differently. On the left is the unla/radius from the forelimb. On the right is the tibia from the hindlimb. You can see that the main adaptation of the ulna/radius is the curvature (correct my spelling) whereas the tibia is much more straight up and down. One thing I will be looking at is how this curvature affects the compression loading of the bone. The bone is slightly curved like this paranthese ) so when it is compressed, it will bend more severly to she shape of its curvature like this arrow >. This is just one of the many aspects of bones I will be looking at. Ok lates guys I have to eat my dinner now. NOM NOM NOM!

ulna-1933.jpg0199210896_tibia_1.jpg
11/17/11-What's up guys I'm back again. So as I was saying yesterday the curvature of the bones is one of the key adaptation I will be looking at. Another one is the types of loading each bone is adapted to withstand for. In humans, the ulna/radius is designed for torsion. Torsion is sort of a twisting motion. Try putting your right arm straight out so your palm is face down. Now turn your arm clockwise so that your palm is face up. Notice how you did not have to stand on your head in order to complete this task without breaking your forearm. This is because the bone there (the unla/radius) is designed to move in that way. In rats, however, torsion may not be the case. You don't see many rats walking upright and completing tasks similar to the ones that humans complete. The ulna/raidus may be adapted for more compression, sort of like the human tibia. Humans walk upright and a lot of pressure is put on their legs. One of the bones that bears quite as sum of this weight is the tibia. It is located in the shin. Rats walk on both their front and back legs so I would expect the tibiae of both humans and the rodents to be designed to bear similar forms of compression. The link below shows an animation of torsion. The other link shows an animation depicting compression. Actually its a YouTube video but it still explains it.
Torsion
Compression/Tension
Also included here are a couple of reports my mentor, Dr. Roeder, had me read. They led me to my idea for a project.





1/2/12-Whaddup wikiNATION? so i finally started my testing now. kinda. On Friday, aka, last year, my grad student Travis and I began my experiment. We were just finallizing the details of my research plan and right as we were about to load some rat hindlimbs, Travis dropped the top half of the load frame onto the bottom half which busted the 300 lb. sensitive load cell. We couldnt do any more testing that day so I went back today. He told me he had to order a new 300 lb. load cell but it cost $800. Shoot man thats some serious cash. Today, Travis and I installed the 500 lb. load cell and we had to adjust the computer and load frame accordingly and we started some testing. We loaded 8 specimens today. The first four, we ran to see where the failure level is. The two male limbs failed around 35 pounds. but wait. That was the knee! See, the way i am loading these specimen puts the top of the knee above the lower part. This is really hard to explain without photos which are coming tomorrow. Anyway its an issue. The tendon in the knee is snapping at 35 pounds, not the tibia. That didnt fail until closer to 40 pounds. And theres another issue. The loading we were doing today was all manual. When the actual automated testing starts to simulate fatigue loading, the load level, aka pounds of pressure, will be much lower the failure rate of around 40 lbs that we found today. However we also discovered that the ankle (which fails around 10 pounds...WEAKSAUCE) and the knee fail first. The computer will detect this change in stiffness of what is compressing and stop the test! OH NO! We need a way to make sure the computer only detects the tibia failure. I suggested pre-breaking the ankle and knee. not a bad idea but it would be difficult, not impossible, and time comsuming to find a uniform way to pre-break the bones of all the specimens. So we had another idea. Why not just hack of the femur and the foot. so i took a scalpel and cut down the middle of the knee where the two bones meet and at the ankle. We loaded those specimens, only two, and broke the tibia clean and nothing else. our first success. #thatsJUSTICE So we found a few different failure levels for 2 male and 2 female specimens. Here are some graphs:



Sorry about that I couldnt figure out how to save it as a picture file. The following is not a picture file either. It is the first graph but really enlarged with my writing on it to point out some key areas of the graph and teach all of you n00bs how to read them: by the way the B-15 just denotes which animal it is. The B stands for breeder. Basically this rat's only purpose in life was to keep the colony going.





Again, pictures would make this a lot easier but I don't have any yet. Oh and those outliers, imagine like the top and bottom parts of the load frame pushing on the specimen from the top and bottom. They keep pushing and pushing and eventually its gets harder to push because the resistive force from the bone becomes greater and greater. When it finally breaks, the points of compression crash together, then bounce off one another and bob up and down before coming to rest. This is a load frame similar to the one i use except the one i use is made by bose and actually operates by sound. Not gonna talk about that though.

external image WDW-100E%20FT1.jpg
So check out the graphs and see if you can figure out where the ankle/foot and knee are failing in the first four, then try and see whats going on in the second four. The titles of the graphs should help. For instance, where it says tibia+tissue only, that means that i have lopped off the femur and foot but not the rest of the surrounding muscle and fat tissue.

1/3/12-So today I spent 2.5 hours disecting rat limbs. Yesterday it was decided that I can easily disect out the tibiae in all the limbs to make sure the knees and ankles don't fail first and ruin all of my data. I prepared 28 tibiae today and 2 ulnae as well to begin testing on those. Here is my process for preparing the tibiae.
1. Determine if the hind limb is whole. For nearly half of the specimens I prepared, the femur was missing.
2. If the hind limb is whole, bend the knee several times to determine where the point of bending is. A small dimple will be visible here.
3. Using a scalpel, cut at the point of bending. If the point has been correctly located, no bone will be needed to cut through, only a few tendons/ligaments.
4. Save the femur in case it can be used in future research.
5. Using pliers, cut through the foot right near the ankle. This will not cut through the skin so use the scalpel again to get through the skin.
6. Cut off excess calf muscle and fat in back of the tibia while being careful to not to damage fibula.
7. Place extracted tibia into individual containers.
8. Store in fridge or freezer depending on when the next planned use is.