Despite the efforts focused on manufacturing biological engineering scaffolds for tissue engineering and regenerative medicine, a biomaterial that meets the necessary characteristics for these applications has not been developed to date. Bacterial nanocellulose (BNC) is an outstanding biomaterial for tissue engineering and regenerative medicine; however, BNC's applications have been focused on two-dimensional (2D) medical devices, such as wound dressings. Given the need for three-dimensional (3D) porous biomaterials, this work evaluates two methods to generate (3D) BNC scaffolds. The structural characteristics and physicochemical, mechanical, and cell behaviour properties were evaluated. Likewise, the effects of the pore size and surface area in the mechanical performance of BNC biomaterials and their cell response in a fibroblast cell line are discussed for the first time. In this study, a new method is proposed for the development of 3D BNC scaffolds using paraffin wax. This new method is less time-consuming, more robust in removing the paraffin and less aggressive toward the BNC microstructure. Moreover, the biomaterial had regular porosity with good mechanical behaviour; the cells can adhere and increase in number without overcrowding. Regarding the pore size and surface area, highly interconnected porosities (measuring approximately 60 μm) and high surface area are advantageous for the biomaterial's mechanical properties and cell behaviour.
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