# Artificial Heart Valve Scaffold Based on Electrospun PCL/PU Three-Layer Composite Fibers
## 2 Experimental Section
### Characterization of Composite Fiber Heart Valves  
The morphology of electrospun valve scaffold was characterized by scanning electron microscope (SEM, Quanta 250 FEG, FEI, Shanghai, China), and fiber diameter distributions were quantitatively assessed using NanoMeasurer software Version 1.2.5. The composition of the sample was analyzed by a Raman spectrometer (HR 800, Jobin-Yvon, Hangzhou, China). The Raman spectrometer used a laser with a wavelength of 532 nm and an exposure time of 10 s. We processed Raman data using LabSpec 5 software and used baseline function to remove Raman background signals. By removing background signals, the actual Raman peaks can be clearer and more accurate, facilitating subsequent peak position identification and quantitative analysis. To judge the hydrophilicity of the material, an experimental test was needed. We chose the contact angle measurement test; each layer of the three-layer structure sample was separately prepared as a nanofibus film to explore the hydrophilicity of each layer. Water drops were dropped on the surface of different layers of the fibrous film of the sample, and the contact image between the droplet and the film surface was taken by the measuring equipment. The hydrophilicity of the sample was characterized by a contact angle tester (Theta-flex, Biolin, Shanghai, China). A microcomputer-controlled electrohydraulic servo universal testing machine was used to measure and generate data along the circumferential direction of the valve. The mechanical properties of the samples were tested by an electronic universal tensile testing machine (HD-B612-S, Haida Instrument Co., Ltd., Dongguan, China). We set the universal tensile testing machine with an initial length of 2 cm, a temperature of 25 \({}^{\circ}\)C, and a tensile speed of 10 mm/min. After the valve scaffold was characterized, biological experiments were continued. Human valve interstitial cells are an important cell type in heart valves. They are involved in the tissue architecture of the valve and maintain the morphological stability of the valve. They have the potential of directional differentiation, and can participate in the repair process via proliferation and differentiation when the valve is damaged [21]. In this study, human aortic valve interstitial cells (immortalized, Xiamen Yimo Biotechnology Co., Ltd.) were used for cell growth experiments. The culture medium consisted of 90% DMEM/F-12 (DF12) + 10% Fetal bovine serum (FBS) + Penicillin Streptomycin Solution (P/S). A confocal fluorescence microscope (CFM, lica-tcs, SP8), green cell tracking agent (5-chloromethylfluorescein diacetate, Invitrogen, Waltham, MA, USA), and a multifunctional microplate reader (Opticsmate U1, Youi Instrument Co., Ltd., Hangzhou, China) were also used in the biological experiments. Multimode reader is an instrument used for cck-8 cell survival monitoring. The enzyme-linked immunosorbent assay (ELISA) is based on Lambert Beer's law, which detects the absorption of specific monochromatic light by a sample and converts it into an electrical signal to determine absorbance. Six days prior to cell seeding, the valve scaffold was immersed to be seeded in 37 \({}^{\circ}\)C, 5% CO\({}_{2}\) DMEM to enhance cell adhesion. After culturing human valve interstitial cells to the third generation by using normal culture methods, \(5\times 10^{4}\) cells cm\({}^{-2}\) were seeded onto the electrospun values using a 24-well plate as the carrier. The cells were divided into 5 groups and a blank control group was set up. The cells were cultivated for 1, 3, 5, 7, and 14 days, and the appropriate amount of cell proliferation was determined by using qualitative and quantitative analysis. They were incubated for 2 h using CCK-8 reagent at 37 \({}^{\circ}\)C, 5% CO\({}_{2}\), and 90% humidity. In the presence of electron-coupled reagents, WST-8 is reduced by dehydrogenase in the mitochondria to an orange-yellow product whose color is proportional to cell survival [22]. Therefore, the test of absorbance can reflect the cell survival rate.