Copper-Substituted Calcium-Deficient Apatite: Enhancing Osteogenic and Angiogenic Synergy for Bioengineered Bone Regeneration

Optimal bone healing requires precise regulation of the spatial and temporal interplay between angiogenesis and osteogenesis. This study explored how incorporating copper (Cu) into 3D-printed calcium-deficient apatite (CDA) scaffolds influences their ability to promote osteogenesis along with angiogenesis behavior in human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs). The incorporation of Cu²⁺ ions into the CDA lattice was verified through X-ray diffraction (XRD) pattern and Fourier Transform Infrared Spectroscopy (FTIR), which revealed corresponding alterations in the phase composition and functional group characteristics. <i>In vitro</i> studies utilizing hMSCs and HUVECs indicated that a copper concentration of 0.15 M markedly enhanced osteogenic differentiation and angiogenesis. AlamarBlue, dsDNA, and BCA assays demonstrated increased cell proliferation and protein production in copper-doped scaffolds. Alkaline phosphatase (ALP) protein expression and the expression of osteogenic genes (COL1, IBSP, OCN, OPN) were significantly increased in the 0.15 Cu group, suggesting enhanced osteogenic differentiation. Matrigel assays demonstrated that the eluted media from Cu-doped scaffolds promoted HUVEC tube formation. Gene expression analysis indicated an initial upregulation of VEGF, VWF, and MMP9, succeeded by elevated expression of Ang1, Ang2, and TSP1, signifying a comprehensive angiogenic process. The obtained results suggest that 3D-printed copper-doped CDA scaffolds hold significant potential as bioactive materials for bone tissue engineering applications.