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The objectives of this study were to prepare a new artificial eardrum patch using water-insoluble chitosan for healing the tympanic membrane perforations and to investigate biomechanical properties and cyotoxicity of the chitosan patch scaffold (CPS). Tensile strength and elongation at the rupture point of CPSs were 2.49-74.05 MPa and 0.11-107.06%, respectively. As the biomechanical properties of CPSs varied with the concentration of chitosan and glycerol, the proper conditions for the CPS were found out. SEM analysis showed very smooth and uniform surface of CPSs without pores at x1000. The result of MTT test showed that CPSs had no cytotoxicity.