The overall goal of this protocol is to characterize the local surface strain field on vascular tissue samples. This is accomplished using a digital image correlation technique with uniaxial tensile testing. This method can help answer key questions in the field of soft tissue mechanics and specifically provide information to identify and invalidate increasingly sophisticated computational and mathematical models of vascular tissue.
The main advantage of this technique that facilitates precise quantification of a strain field heterogeinity on the sample surface, which is a function of inherent material properties, symbol geometry and employed testing modality. As soon as the tissue arrives at the lab, isolate the abdominal aorta from surrounding tissue using surgical scissors and forceps. Then, wash the vessel three times using a syringe filled with PBS.
Following the washes, use scissors and forceps to remove as much perivascular tissue as possible, without compromising the integrity of the sample. Then, onto the middle section of the vessel, position a sharp razor blade, perpendicular to the vessel's longitudinal axis and chop the vessel three times to make two ring samples. Next, position a blade onto one of the rings to make a radial cut.
Firmly slice the ring to make a strip shaped sample for uniaxial mechanical testing. Do this to both rings. Temporarily store the two samples in a 100 mm glass petri dish submerged in PBS.
To speckle the sample surface have an air brush ready to spray tissue marking dye. Connect the air brush to a pressure valve set to produce 100 psi. Next, add the tissue marking dye.
The nozzle diameter must be calibrated for speckle diameters between 60 and 100 microns. Then, spray tissue marking dye on the intimal surface of the sample for approximately five seconds. Do this three times to ensure that the speckle pattern uniformly covers the sample surface.
To begin, mount the sample to the testing apparatus. First, attach each end of the sample to a plastic strip using a tissue adhesive. Place plastic on both the top and bottom of the sample with the adhesive, making a sandwich.
Place the sample flat on a tissue cutting board then measure it's dimensions using a digital caliper. Next, initiate the system controls for the test apparatus. On the system control's home screen, select waveform on the task bar located on the set up tab.
Then, adjust the position of the upper grip of the mechanical tester to negative four mm to provide a 4 mm extension relative to the designated initial position in the system. Now, gently secure one plastic strip into the upper grip of the mechanical tester and allow the sample to hang freely. Next, adjust the position of the lower grip so that the free end of the sample can be secured without extension.
Then, gently secure the plastic strip attached to the free end, into the lower grip of the tester. With the sample mounted, zero the system load cell. Then measure the length of the sample and use this as the reference length when calculating global circumferential strains.
Now, in the software control, enter the mechanical testing protocol. Here, four uniaxial displacement cycles are applied. Each cycle extends the sample length by 18 percent at a displacement rate of 0.01 mm per second.
To document the test, take a photo every few seconds. Position a camera 1.5 m from the loading frame, perpendicular to the sample and focus it on the sample. In the image capture software, in the option select system"select PGR2, then set the project path to save the images to be analyzed.
Click the time square icon and specify the acquisition interval as 5 seconds. Now, import the image obtained. Zoom in on an individual speckle and then count the number of pixels within this individual speckle.
After verifying the quality of the speckle pattern, simultaneously click the run"icon in the system and the start"icon in the image capture software to start the test. Throughout the protocol, intermittently spray PBS on the sample so it does not dry out. However, do this whenever it is in the unloaded state not during data acquisition.
After opening the image analysis software, click the speckle images"tab and select all images which need to be analyzed. Then, click on the rectangle tool and select the area of interest in the first image. Next, enter a 31 by 31 pixel subset size and a step size of five pixels.
Now click the start analysis"tab in the software and set the interpolation to be an optimized 8 trap. Also, set the criterion as zero normalized square differences and the subset weights option as Gaucian. Check the threshold options.
They should be on the default. Next, click on the post processing sub tab under the start analysis tab. Select the option, string computation"and leave filter size and type of filter as the default.
Set the tensor type to Lagrange. Finally, select the data tab and then select an analyzed image to visualize the surface strain field. 2D DIC in conjunction with uniaxial mechanical testing, was used to measure the surface strain field of a vascular tissue specimen, at various deformed states.
The viscoelastic nature of the tissue was notable by the degree of hysteresis in the low displacement curves prior to the mechanical preconditioning. However, with preconditioning, the hysteresis was gradually diminished. As expected, the local circumferential strain values increased with applied sample displacement.
Circumferential strain was generally less near the center of the sample as compared to near the sample grip interface. In the longitudinal direction, the resultant, non-uniform compressive strain on the sample intimal surface increased as the sample was gradually extended. The color changes within the contours for the full field strain measurements, clearly show the high heterogeneous nature of the aorta when subjected to nominally, uniaxial tensile loading.
The coefficients of variation of the surface strain fields in orthogonal directions were then calculated in each experimental state. The variation in the surface strain was found to have an inverse relationship with the samples extension. The high level of heterogeneity in the material response is quantified in the table by the coefficent of variation, or CV.After watching this video, you should have good understanding of how to quantify the local surface mechanical response using digital image correlation coupled with uniaxial tensile testing of vascular tissue samples.
After it's development, this technique paved the way for researchers in the field of soft tissue mechanics to explore physiological, pathological and modulated mechanical response of vascular tissue.
