This protocol describes the biaxial mechanical characterization, polarized spatial frequency domain imaging-based collagen quantification, and microdissection of tricuspid valve leaflets. The provided method elucidates how the leaflet layers contribute to the holistic leaflet behaviors.
This method can isolate the contributions of tissue layers to the overall tissue behavior, providing new insight into the tissue microstructure. Our microdissection and testing framework allows us to directly compare the mechanical and microstructural properties of heart valve leaflets to their own composite layers. The microdissection can be difficult to master, hence, it is highly recommended to practice as much as possible before launching your study.
First, gather the required materials, wax board, DI water, pipette, scalpel, micro scissors, thin forceps, curved forceps, thick forceps, and pins. Unmount the tissue from the biaxial tester and measure its thickness using the laser displacement sensor. Place the tissue on the wax board.
Examine the ventricularis side of the tissue for large chorda insertions. Take a photograph for reference. Spread the tissue flat on the wax board with the atrialis facing up.
To affix the tissue to the board, place one pin angled away from the tissue in each corner so that it slightly pulls the tissue taut. Ensure that the pins are outside the holes created by the tines when mounting the tissue and the tissue is square and does not shift during the layer microdissection. If necessary, place pins along the side of the tissue during the dissection to stretch the tissue more.
Remove the glass bead fiducial markers. Place DI water on the surface of the tissue with a pipette so that it completely covers the tissue in a bubble-like manner. To make the initial corner, select a corner of the pinned specimen, avoiding large chorda insertions in fragile areas.
Then, cut the A/S layer by lightly dragging the scalpel over the tissue surface along the mounting holes for mechanical testing. The edges of the cut start to pull apart, revealing the F/V layer underneath. With blunt, thin forceps, firmly rub along the cut to further separate its edges.
If the cut in the A/S layer does not start to pull apart, lightly trace over the same cut again with a scalpel. Make a second cut perpendicular to the first cut. If the two cuts are not connected, run the thin tweezers under the small area of tissue separating the two cuts.
Then, carefully use the scissors to cut the tissue. Rub along the cuts of the corner with a thin forceps until the tissue separates from the F/V layer. As soon as a small piece of tissue is separated, grasp it with the large tweezers and gently pull it to separate the composite layers further.
Continue to peel the tissue while grasping it with larger tweezers to prevent undesired ripping and tearing of the A/S composite layer, and rub the seam until it reaches the end of the two cuts made for the corner. If the first corner has major issues with separation, try a different corner as a starting point. Then, to make a second corner, extend the two cuts made for the first corner by placing the scalpel tip at the bottom of each cut and lightly dragging it along the tissue surface so that the cut extensions connect to the original cuts and continue to follow the tine or suture holes.
Continue extending the cuts and peeling the top composite A/S layer back while rubbing the seam until one side is finished. Observe that the tissue will be entirely separated along one cut. Next, create a second corner perpendicular to the end of the fully peeled side.
Extend the remaining cuts while avoiding large chorda insertions. Exclude the chorda insertions from the A/S separation area only when this exclusion will allow for an A/S specimen large enough for experimental characterizations. Continue separating the A/S and F/V layers by using the rubbing and pulling techniques employed for the first corner.
Stop separating the tissue immediately if a tear or hole forms. To prevent tweezers from being caught, place the scissors in any hole that forms and cut the tissue away from the center. If the defect forms along the seam of separation, then begin separating the tissue along another edge.
Look for interlayer connections that may appear when separating the tissue and prevent further tissue separation. Observe that these are thin but strong strands that must be carefully cut using scissors. Avoid creating a hole in the A/S layer or cutting downwards into the F/V layer.
Continue this process until the largest possible sample of the A/S layer has been separated. Mark the orientation of the sample using the surgical pen. Finally, cut along the seam of separation for the remaining tissue edge with the scissors.
Place the separated A/S composite layer flat on the cutting mat. If necessary, use the scalpel to straighten the edges of the tissue and create a square tissue shape suitable for biaxial mechanical testing. Place the A/S layer in DI water until it is ready to be tested.
Mark the orientation of the F/V layer that remains on the wax board. Cut the largest square possible out of the area where the A/S layer was removed. Then, place it in DI water.
Histological analysis of the intact leaflet and the two dissected layers verified if the tissue was correctly separated along the border between the spongiosa and fibrosa. The histology micrographs were used to determine the tissue layer thickness and constituent mass fractions using ImageJ software. The displacement controlled mechanical testing and post-processing produced membrane tension versus stretch data of the tricuspid valve anterior leaflet, tricuspid valve posterior leaflet, and tricuspid valve septal leaflet, describing the nonlinear mechanical behavior of the tissue.
The polarized spatial frequency domain imaging data yielded color maps of the collagen fiber orientation and degree of optical anisotropy. Specifically, these color maps provided a comprehensive understanding of the collagen fiber architecture across the entire tissue specimen. It is important to note that the composite layer shrinks once they are separated from the pinned tissue.
Collect the largest possible sample to ensure there is enough tissue for the subsequent testing.
