Biaxial tensile tester under a microscope
The basic mechanism of the biaxial tensile tester under a microscope was similar to that used for the biaxial stretching of cells[18, 19]. Figure1a shows a schematic illustration of the tester. The tester was designed for translucent specimens, which are 15 × 15 mm2. Very thin specimens were glued and sandwiched between two 20 × 20 mm2 polyethylene terephthalate (PET) film sheets with holes 10 mm in diameter at their centers to ease specimen handling. The specimen was then glued onto a stainless steel frame with a hole, which was 10 mm in diameter. A stainless steel hollow cylinder 6 and 8 mm in inner and outer diameters, respectively, was placed above the center of the specimen in the hole. The metal frame was then moved toward the cylinder to stretch the specimen biaxially. Figure1b is a schematic illustration of the entire experimental system. To control its position, the cylinder was fixed on a manually operated XYZ stage (TSDS255S; Sigma Koki, Tokyo, Japan). The frame was fixed on two Z stages (SGSP80-20ZF; Sigma Koki) through cantilevers. By synchronously moving these Z stages upwards, the specimen was pushed onto the cylinder. These XYZ and Z stages were fixed on a XY stage (BIOS-225TWI; Sigma Koki) to allow observation of any part of the specimen, and the XY stage was set under an inverted microscope (IX71; Olympus, Tokyo, Japan). The Z and XY stages were controlled with software (SGTERM and Software Joystick; Sigma Koki) on a personal computer (PC). Specimen images were captured on a charge-coupled device (CCD) camera (Abrio-LS; CRi, Woburn, MA, USA) through a 2× objective lens (PLAPON2×; Olympus).
To measure the force F applied by the cylinder to the specimen, strain gauges were bound on the cantilevers. Data measured with the strain gauges were recorded with a data acquisition system (LabView 2010; National Instruments, Austin, TX, USA) on the PC through a bridge box (DB120A; Kyowa Electronic Instruments, Tokyo, Japan), a strain amplifier (DPM911A; Kyowa Electronic Instruments), and an analog–digital converter (NI USB-6289; National Instruments). We calibrated the load cell and found that this device could measure the force acting on specimens with a resolution of 12 mN and that the relationship between the force and the measured voltage was linear with a correlation coefficient of 0.999.
Equibiaxial tensile test for a homogeneous and isotropic specimen
To confirm that the specimen could be stretched equibiaxially with this tester, a polydimethylsiloxane (PDMS) sheet was used as a homogeneous and isotropic specimen. PDMS prepolymer (Sylgard 184; Dow–Corning, Midland, MI, USA) was mixed with curing reagent at 10:1 (w/w), spread on a dish, and cured at 75°C for 4 h. For strain markers, black lacquer was sprayed on the surface of the approximately 50-μm thick PDMS sheet. The sheet was then cut into 15 × 15 mm squares, and a square sheet was glued on the metal frame with modified silicone adhesive (Super X; Cemedine, Tokyo, Japan). PET films were not used for this test. The specimen was then stretched biaxially with the tester. Since precise determination of the origin was important for the analysis of stress–strain curves, the height of the Z stage h was taken as 0 when 12 mN (0.01 V) of the force F was applied to the specimen. The Z stages were moved stepwise by Δh = 0.5 mm while measuring the force F and capturing images until specimen failure. The Z stage was moved after the force F became stabilized; i.e., changes in the force F became smaller than 12 mN/min. This normally occurred in approximately 10 min, but sometimes took 50 min.
The images obtained during the biaxial tensile test were analyzed with the image analysis software ImageJ 1.42i (National Institutes of Health, Bethesda, MD, USA). The black lacquer markers located at the center of the specimen and eight arbitrary surrounding points located at almost equal intervals in a circumferential direction were selected and tracked with the particle tracking tool MTrackJ[20]. The distances between the center marker and the surrounding eight markers were measured, and the nominal strains were calculated for the eight points. We also constructed a finite element model to simulate an equibiaxial tensile test for a homogeneous and isotropic specimen. Material parameters were chosen to simulate the PDMS sheet and aortic specimen (see Additional file1 for details).
Preparation of aortic slices thinned at their centers
Figure2 shows schemata and photographs of the process used to locally thin a specimen at its center. Porcine thoracic aortas (PTAs) obtained from a local slaughterhouse were used as specimens. After loose connective tissues were removed, the PTAs were cut into rectangular specimens (15 mm in the longitudinal direction and 20 mm in the circumferential direction). These specimens were sandwiched between two metal plates having center holes 3 mm in diameter (Figure2b). The thickness of each specimen was measured four times at different locations with a dial gauge by subtracting the thickness of the metal plates from the total thickness. The sample was then compressed 0–40% (Figure2c) and frozen at −80°C for 10 min to fix the specimen shape. After the metal plates were removed (Figure2d), the frozen sample was embedded in Tissu Mount (Chiba Medical, Saitama, Japan), frozen in liquid nitrogen, and sectioned with a cryostat (CM3050SIV; Leica Microsystems, Wetzlar, Germany) into 50-μm sections. The sections were obtained sequentially from the intimal to the adventitial sides of the aorta.
To measure the thickness of the specimens, some of the sample was thawed on a glass slide at room temperature (Figure2e) and cut with a surgical knife to produce a sample of approximately 1 mm width that included the thinned area. This cut sample was mounted on a glass slide and rotated 90°, and its image was captured under the microscope. The captured image was binarized using the Otsu method[21], and the thickness was measured with ImageJ. For determining the thicknesses at the thin center area and peripheral areas, 1-mm wide regions were selected, and the average thickness was obtained for each region.
Biaxial tensile test of aortic slices
To determine whether cracks initiated at the center of the specimens, the PTA slices prepared in the previous section were glued between two PET sheets with cyanoacrylate adhesive, and a biaxial test was performed. Z stages were elevated 0.1 mm/s while specimen images were captured with a high speed camera (Exilim EX-F1; Casio Computer, Tokyo, Japan) at 300 frames/s to determine the crack initiation site. The location where the crack initiated was divided into areas inside and outside the hollow cylinder, and the area inside the cylinder was further divided into three areas: the center (thin) area, the edge of the thin area, and other areas inside the cylinder (thick area). Biaxial tests were performed for slices obtained sequentially from the intimal to the adventitial sides, and these specimens were grouped into three categories of equal intervals in a radial direction: sub-intima, mid-media, and sub-adventitia.