Cerium–Selenium Co-Substituted Hydroxyapatite Nanoparticles with Enhanced Biofunctional Performance for Bone Substitute Applications

Abstract Hydroxyapatite (HA) is widely used as a bone substitute; however, its limited intrinsic antimicrobial, antioxidant, and anti-inflammatory activities can constrain its performance in biologically challenging environments. In this study, cerium- and selenium-co-substituted hydroxyapatite (CS-HA) with varying substitution levels was synthesized hydrothermally to develop a multifunctional HA-based biomaterial. Pure HA and single-ion-substituted Ce-HA and Se-HA were prepared as reference materials. Energy-dispersive X-ray spectroscopy (EDX) confirmed the presence of Ce and Se, while X-ray photoelectron spectroscopy (XPS) provided further evidence of their incorporation into the substituted material. Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) demonstrated that the hexagonal crystalline structure of HA was preserved following co-substitution. Ce-Se co-substitution decreased the crystallite size and increased the surface area while maintaining moderate to high crystallinity. Biological evaluation revealed dose-dependent antimicrobial, antioxidant, and anti-inflammatory activities. CS-HA nanoparticles exhibited improved antimicrobial activity against Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. Their antioxidant activity was comparable to Se-HA and higher than that of pure HA and Ce-HA, whereas their anti-inflammatory activity exceeded that of HA and Se-HA. Cytocompatibility evaluation using human gingival fibroblasts (HGF-1) showed ˃70% cell viability under the tested conditions, meeting the cytotoxicity criteria specified in ISO 10993-5. At higher substituent levels, CS-HA also reduced the viability of CAL-27 squamous cell carcinoma cells. These findings demonstrate that Ce-Se co-substitution can modulate the physicochemical and biofunctional properties of HA, supporting further investigation of CS-HA as a multifunctional biomaterial for bone substitute applications and its potential activity against squamous cell carcinoma.