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Background: Advances in regenerative medicine technologies have been strongly proposed in the management of thyroid diseases. Mechanistically, the adoption of thyroid bioengineering requires a scaffold that shares a similar three-dimensional (3D) space structure, biomechanical properties, protein component, and cytokines to the native extracellular matrix (ECM).Methods: 24 male New Zealand white rabbits were used in this experimental study. The rabbit thyroid glands were decellularized by immersion/agitation decellularization protocol. The 3D thyroid decellularization scaffolds were tested with histological and immunostaining analyses, scanning electron microscopy, DNA quantification, mechanical properties test, cytokine assay and cytotoxicity assays. Meanwhile, the decellularization scaffold were seeded with human thyroid follicular cells, cell proliferation and thyroid peroxidase were determined to explore the biocompatibility in vitro.Results: Notably, through the imaging studies, it was distinctly evident that our protocol intervention minimized cellular materials and maintained the 3D spatial structure, biomechanical properties, ECM composition, and biologic cytokine. Consequently, the decellularization scaffold was seeded with human thyroid follicular cells, thus strongly revealing its potential in reinforcing cell adhesion, proliferation, and preserve important protein expression.Conclusions: The adoption of our protocol to generate a decellularized thyroid scaffold can potentially be utilized in transplantation to manage thyroid diseases through thyroid bioengineering. [ABSTRACT FROM AUTHOR]