The overall goal of this technique is to use indentation and tensile testing to characterize the mechanical properties of human soft tissues. This method can help answer key questions in the tissue engineering field, such as how stiff should I make an implant to replace a failing or injured human organ. The main advantages of these techniques are that they are non-destructive, and simple to implement.
We first had the idea for this method when we were designing synthetic ear and nose implants, and wondered how stiff we should make the material. Generally, individuals new to this method will struggle due to incorrect sample preparation and difficulties in setting up the tests correctly. To begin, obtain a skin sample, or other tissue, from consenting patients undergoing surgical procedures, or cadaveric bodies consented for research purposes.
Place the skin sample into a Petri dish, and cover it with PBS to keep the tissue moist. Use a scalpel blade and forceps to manually dissect the adipose tissue and the thin layer of deep dermis from the sample. Cut the resulting sheet of split thickness skin into one-by-five centimeter strips.
Then, dispose of the use scalpel blades into a sharps bin. Finally, use calipers to measure the thickness of the skin. The thickness will also need to be measured after mechanical testing.
Test skin samples in uniaxial tension using a calibrated materials testing machine at room temperature. Start by orienting the skin samples so that the natural orientation of the collagen fibers are all in the same direction, according to the Langer lines. Next, load the sample into the grips, so that the direction of the applied force will either be in line with the Langer lines, or perpendicular to them.
Immobilize the sample by finger-tightening the grips so that approximately 0.5 centimeters of the sample is placed into each grip. Ensure that one of the grips of the testing machine is attached to an appropriate load cell, and the other to an immovable base plate. Once secured, cover the sample area on both sides with petroleum jelly to prevent specimen desiccation.
Now, program the tensile loading and relaxation testing regime into the software so that the sample will be loaded at one millimeter per second, to a tension of 29.42 newtons. Once the tissue reaches a tension of 29.42 newtons, set the program to hold the displacement constant for one and a half hours, to allow the tissue to relax, while continuing to monitor the change in tension. When finished, use calipers again to measure the thickness of the sample.
After obtaining a cartilage specimen from a consenting individual, place it into a Petri dish, and cover it with PBS. Use a scalpel blade and forceps to remove the skin and fascia from the specimen. Then, cut the cartilage specimens into 1.5 centimeter squares.
Once cut, measure the thickness of the cartilage to be loaded using calipers. The final sample size should be at least eight times larger than the diameter of the indenter. Center the cartilage sample under the indenter on a large impermeable base, and orient it so the surface is perpendicular to the indenter.
This allows the compression to be uniaxial, and limits any sheer. Then, cover the cartilage in PBS, and keep the sample hydrated throughout the test. Program the compressive loading and relaxation testing regime into the software, so that the sample is loaded at one millimeter per second to a compressive force of 2.94 newtons.
After the 2.94 newton load is reached, maintain the indenter at the same position, and allow the cartilage to relax for 15 minutes. Once finished, release the sample, and again measure the sample's thickness. To evaluate the viscoelasticity of skin tissue after tensile testing, all of the values in the stress strain plot are included for the Young's modulus calculation, until the line curve fit has a minimum R value of 0.98.
The stress versus time plot is used to evaluate the relaxation properties of the skin. The rate of relaxation is calculated from the last 200 seconds of the collected data, which for this sample is 3.1 times ten to the five megapascals per second. The graph shown here is examples stress versus strain data, showing the results of indentation testing on human cartilage.
As with the Young's modulus calculation for the skin sample, the values at the end of the curve are used for cartilage, as long as they fit in the minimum R value of 0.98. The cartilage has a rate of relaxation of 8.78 times ten to the negative six megapascals per second and an absolute final level of relaxation of 0.028 megapascals. Once mastered, this technique can be done in seven hours if it is performed properly.
While attempting this procedure, it's important to remember to have a repeatable dissection protocol and sample dimensions. Following this procedure, other methods, like cyclical loading, can be formed in order to answer additional questions, like how do tissues behave under physiological, dynamic loads. This technique paves the way for researchers in the field of biomechanics to explore using this non-destructive technique for non-invasive mechanical testing in vivo.
Don't forget that working with human tissues can be extremely hazardous, and precautions such as gloves should always be taken while performing this procedure.
