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This study analyzed a recently developed bone substitute biomaterial made of chitosan-xanthanhydroxyapatite-graphene oxide (CXHAG). The CXHAG particles underwent in vitro structural and morphological characterization, and in vivo testing with or without osteogenic conditioned medium from mesenchymal stem cells. Aim: The aim of this study was to determine whether the CXHAG novel biomaterial, supplemented with conditioned medium from mesenchymal stem cells, could be useful for bone regeneration. Materials and Method: For the in vitro study, cells were incubated with 20mg of CXHAG granules for 24 hours and a MTT assay was performed to tests for cytotoxicity. For the in vivo study, critical size calvarial bone defects were created in twenty-five rats. One animal had the defect unfilled (Control Group-CG) and was euthanized after 42 days. Twelve rats received the CXHAG particles (Group 1-G1) and the other twelve received the CXHAG particles supplemented with the conditioned medium (Group 2-G2). All G1/G2 grafts were covered with a CXHAG membrane. G1/G2 animals were euthanized after 14 days (T1) or 42 days (T2). The specimens were processed and histologically evaluated. Results: SEM analysis of the CXHAG particles showed granules of 300-400μm, with a rough irregular surface. They were not cytotoxic to dental pulp stem cells in vitro. The CG specimen showed loose immature connective tissue and no bone formation at the center of the defect. G1 and G2 presented remnant biomaterial particles at both time points, but only G2 had bone formation at the enter of the defect. Conclusions: The conditioned medium had a positive effect on bone regeneration in rat calvarial critical size defects when associated with the novel bone substitute biomaterial., Competing Interests: The authors declare no conflicts of interest concerning the publication of this article, (SAIO.)