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The present work aimed to synthesize and verify the effectiveness of the polyhydroxybutyrate and norbixin membrane as a scaffold in bone defects induced in the tibia of rats. Twenty-four male Rattus norvegicus rats were used, divided into control and membrane groups. After anesthesia, a bone defect was induced in the right tibia, followed by the implantation of the biomaterial at the site of the lesion only in the membrane group, with euthanasia after 15 and 30 days of the experiment. The deposition of organic and inorganic matrix, the quality of newly formed bone tissue and the morphology of the bone defect were measured. After 15 days of the experiment, the biomaterial significantly influenced the deposition of hydroxyapatite crystals, the formation of collagen I matrix and mineralization content in relation to the control group, in addition to the abbreviation of the inflammatory process and superior quality of the newly formed bone tissue. After 30 days, only the membrane group had fully completed its repair process. The biomaterial acted as a scaffold in the regeneration of the guided bone defect by accelerating the synthesis of collagen matrix, mineralization content, density, and maturity when compared to the control group.