The development of new bone substitutes has become an area of great interest in materials science. In fact, hydroxyapatite is the most commonly used biomaterial in defects that require bone reconstruction, and that is certainly why the discovery of new products with its formulation has been increasing continuously. The aim of this study was to analyze the biological behavior of a xenogeneic hydroxyapatite widely disclosed in the literature and a synthetic nano-hydroxyapatite/Beta tricalcium phosphate in critical defects in the calvaria of Wistar rats. For this, the groups were divided as follows: 24 adult male Wistar rats were used, weighing between 300 and 350 g, in three groups with eight animals each. In the CTRL group (control), only the clot was kept, without material insertion; in the Bioss group (bovine hydroxyapatite), Bio Oss®—Gleistlich® was introduced; and in the Blue Bone group (REG), the defect was filled in with synthetic nano-hydroxyapatite associated with betatriphosphate of calcium, Blue Bone®—Regener®. According to the results in Goldner’s Trichromics, we can observe a higher percentage of newly formed bone matrix in the REG group than in the CTRL and Bioss groups; in the VEGF, we had a more adequate cell modulation for blood vessel formation in the Blue Bone group (REG) compared to the Bioss and CTRL groups, while in osteopontin, a higher percentage of bone formation was observed in the Blue Bone group (REG) and Bioss group when compared to the CTRL group. We conclude that bone formation, mitosis-inducing cell modulation and main osteoblast activity were higher in the Blue Bone group (REG) than in the CTRL and Bioss groups.