This method can provide a better understanding of heart valve biomechanics which can be extremely useful in computational modeling and in the refinement of treatment methods for heart valve disease. This protocol is advantageous compared to other established testing protocols because it can be used to perform mechanical characterizations of heart valve tissues using a unified testing scheme. This biaxial testing protocol with the unified testing scheme will be beneficial to the soft tissue biomechanics research of mechanical quantifications such as full characterization of arterial vessels and skin tissue.
Begin by using forceps to remove the leaflet specimen of interest from PBS storage. Lay the leaflet flat on a cutting mat with the radial direction aligned to the Y direction and the circumferential direction aligned to the X direction. Identify the central region of the leaflet as the testing section and align a tissue cutter so that the desired tissue testing region is within the boundaries of the razor blades.
Make one horizontal and one vertical cut to form a square region of the desired dimensions and use a surgical pen to label the radial orientation of the tissue. Then, use the forceps to stretch the chordae from the leaflet and use a razor blade to trim any chordal attachments, taking care not to damage the leaflet. Using forceps, lay the tissue specimen flat on a spatula and use a digital caliper to measure the thickness of the spatula-tissue pair at three different leaflet locations.
Next, mount the tissue to the biaxial testing system, ensuring that the circumferential and radial directions of the specimen are aligned with the machine's X and Y directions. For the fiducial marker placement, place glass beads into one small open-faced container and add a small pool of superglue to another container. Coat the tip of a fine-tipped tool with a small amount of superglue and stick an individual bead to the tip of the tool.
Then, carefully use the tool to transfer the bead to one corner of the middle third of the tissue testing region, repeating this placement until a square array of four beads is formed. On the computer connected to the biaxial testing system, create a preconditioning protocol so that the tissue will undergo 10 loading/unloading cycles at the forces associated with the peak membrane tension and a loading rate of 4.42 newtons per minute including a preload of 2.5%of the maximum force. Create a new arbitrary testing directory to temporarily store the preconditioning data, and establish a loading rate of 4.42 newtons per minute for subsequent testing.
Create a new set of testing parameters and set the name of the protocol as Preconditioning0. For the X and Y axes, set the Control Mode to Force, and the Control Function to Step. Set the Load Magnitude as the force associated with the targeted peak membrane tension, and the Preload Magnitude as 2.5%of the maximum force for the first repetition only.
Set both the Stretch Duration and Recovery Duration to 25 seconds and set the number of Repetitions to 10. When the preconditioning step finishes, make a note of the deformation of the tissue in the X and Y directions and prepare a protocol to move the specimen to the maximum force, beginning from the recorded size. Next, begin the maximum force loading protocol, starting from the post-preconditioning deformation while simultaneously starting a stopwatch when the machine begins actuation.
Stop the stopwatch when the actuation stops as indicated by auditory cues. Then, record the post-preconditioning peak tissue deformation, along with the time from the stopwatch representing the optimal tissue stretch time. For biaxial mechanical testing, prepare a force-controlled protocol at a loading rate of 4.42 newtons per minute as demonstrated and open a new testing directory.
Name the test and set the data to save to a known location for use in later stress and strain calculations. Move the specimen back to the original mounting configuration and create a protocol set titled First Image. Set the X and Y axis Control Mode to Force and the Control Function to Step.
Set the Load Magnitude to zero millinewton and set both the Stretch Duration and Recovery Duration to one second. Set the number of Repetitions to one and both the Data Output Frequency and Image Output Frequency to one hertz. Instruct a new testing set and name it Preconditioning A, establishing the testing parameters such that the tissue will undergo 10 repetitions of cyclic loading/unloading to the targeted force for the desired membrane tension as demonstrated.
Instruct another testing set named Preconditioning B with testing parameters identical to the Preconditioning A testing set, but with the Image Output Frequency set to 15 hertz and with no applied preload. After the preconditioning protocols, create the testing protocols so that the tissue is loaded to the peak membrane tension in the indicated circumferential to radial loading ratios at a loading rate of 4.42 newtons per minute. Retrieve the data from the last two cycles of each loading ratio for subsequent data processing and analyses described.
Prepare a displacement-controlled biaxial stretching testing protocol at a loading rate of 4.42 newtons per minute in the X and Y directions for the displacements associated with the peak circumferential and radial stretches respectively. Prepare a pure shear testing protocol along the X direction, stretching in the X direction associated with the peak circumferential stretch and shortening in the Y direction while keeping the dashed area constant under deformation. Prepare a constrained uniaxial stretching testing protocol along the X direction.
Then, prepare a pure shear testing protocol along the Y direction and a constrained uniaxial stretching testing protocol along the Y direction. Between each of these protocols, construct a rest cycle of one minute that holds the tissue at the original mounted configuration and retrieve the data from the last two cycles of each loading ratio for data processing and analyses. Then, prepare a stress relaxation protocol so that the tissue is loaded in each direction at a loading rate of 4.42 newtons per minute to the displacements associated with the peak membrane tensions and held at that displacement for 15 minutes.
After 15 minutes, the protocol should be set to recover the tissue to its original mounting configuration. Stress stretch data from a representative force-controlled biaxial mechanical testing reveals a non-linear curve with some resemblance to an exponential curve, with the material behavior curves transversely isotropic and the radial stretch greater than the circumferential deformation. In some cases, the direction of the anisotropy may flip, with the circumferential direction exhibiting greater compliance than the radial direction.
From displacement-controlled testing, stress stretch data follows a non-linear response for the principal direction undergoing tension. In the constrained uniaxial tension protocol, an increasing stress stretch response is exhibited in the constrained direction, demonstrating the coupling of applied stretching in the other principal direction. From the stress relaxation testing, normalized membrane tension time data follows a non-linear decaying curve.
Both the mitral and tricuspid valve leaflet tissues exhibit a greater stress reduction in the radial direction compared to that in the circumferential direction. Representative histologic analysis of mitral valve and tricuspid valve anterior leaflet tissue sections using Masson's trichrome staining demonstrates typical constituents found in atrioventricular heart valves such as collagen fibers and valvular interstitial cells. It's important that the glass beads are not accidentally glued together to avoid significant errors in the tissue deformation calculation during the post-processing step.
The acquired data can be later used in heart valve computational modeling that can better inform how the valve functions and for improvements in the surgical procedures for treating valvular heart diseases. This protocol opens doors in the field of soft tissue biomechanics for comparing the mechanical behaviors between healthy and diseased tissues as well as for designing biomimetic materials.
