# Tensile Test of Human Lumbar Ligamentum Flavum: Age-Related Changes of Stiffness
## 2 Materials and Methods  
This study was conducted after approval was obtained from the Ethics Committee of our institute, and written informed consent was received from each patient (H27-009, H28-054). Patients visiting our institute with lumbar disease showing nerve root symptoms or cauda equina symptoms treated with surgery (decompression or decompression-fusion) were included. Exclusion criteria were patients under 20 years of age, patients with infection (syphilis, hepatitis B, hepatitis C, acquired immune deficiency syndrome) considering the examiner's safety, patients with ossification of the ligaments, patients undergoing secondary operations, and patients in whom adhesion of the ligament and the dura was strong and en bloc excision of the ligament was impossible. A total of 44 patients (23 men, 21 women) with a mean age of 66.5 (range, 33-85) years were included in the study. The ligamentum flavum was harvested during the operation from the site of decompression as a part of a necessary procedure for decompression. This tissue is normally disposed of. At every surgery, we aimed to completely separate the ligamentum flavum from the dura mater en bloc, including the deep layer. The harvested ligaments were brought to the department of mechanical engineering of our university wrapped in gauze that had been soaked in 36 \({}^{\circ}\)C saline, and tensile testing was conducted within 24 h after harvesting. All tests were practiced in the same room with the room temperature at 25 \({}^{\circ}\)C. After the test, the samples were brought back to our institute and were disposed of in the general method of infectious waste. The harvested ligamentum flavum samples were sliced to 1.5 mm in width, 1.5 mm in thickness, and 10 mm in length with a surgical knife. Samples not reaching 10 mm in length were excluded. A total of 152 sample specimens were obtained. To fix the sample to the tensile testing machine, we made a plastic tab with a 3D-printer (Creator Pro; APPLE TREE Co., Ltd., Osaka, OS, Japan). The samples were glued to the tabs by sandwiching the sample with two tabs with instant glue at both ends (Figure 1). The tabs were fixed to the tab fastener of the tensile testing machine. The gauge distance of the samples was set at 5 mm. Saline (36 \({}^{\circ}\)C) was frequently sprayed on the samples in order to keep them wet. Figure 2 shows the tensile testing machine used in this experiment. The lower and upper tab fasteners, where the samples glued to the tabs were fixed, are attached to the load cell (LTS-1 KA; KYOWA Co., Ltd., Tokyo, TY, Japan) and the sliding part, respectively. As the actuator (directly below the sliding part) moves upward, the sliding part to which the upper tab fastener is attached is pushed up, and load is given to the fixed samples monoaxially. The signal measured by the load cell is converted into a voltage by a dynamic strain measuring device (DPY-713B; KYOWA Co., Ltd., Tokyo, TY, Japan) and displayed on the personal computer (PC) via a data logger (PCD-320A; KYOWA Co., Ltd., Tokyo, TY, Japan). An aluminum foil as an electrode was installed on the actuator side of the contact part of the actuator and slide part; this is connected to a power supply device (PR18-3A; TEXIO TECHNOLOGY Co., Ltd., Yokohama, KN, Japan). As a result, a voltage change occurs when the slide part and the actuator come into contact, and by reading with a PC via the data logger, the time at which the slide part starts to move is known and the time at which the tensile load of the sample starts can be measured. Tensile rate was set to 0.1 mm/s for all tests. The tabs on both ends of the specimen were fixed to the upper and lower tab fasteners without applying any load to the specimen. After that, the distance between the tabs is gradually increased, and the point where the load started to apply was set as the starting point. We confirmed that the distance between the tabs of the sample was 5 mm, the same as the gauge distance of the samples. Further load was applied until fracture of the specimen.