The present study aims to analyze the thermal regulation of the Ce³⁺/Ce⁴⁺ ratio on the nanonetwork titania layer over the titanium (Ti) surface developed by the alkali-mediated surface modification approach. The effect of sequential heat treatment from 200 to 800 °C was evaluated for its surface characteristics such as morphology, phase formation, roughness, hardness, hydrophilicity, etc. Surface oxidation by temperatures up to 600 °C demonstrated a progressive increase in the Ce⁴⁺ (CeO₂) content with a rutile TiO₂ network layer over the Ti surface. In contrast, a bulk reduction with increased Ce³⁺ (Ce₂O₃) content was observed at 800 °C. Heat treatment at 800 °C was also characterized by an improved nanohardness and roughness with a distorted surface network layer. The coexistence of Ce⁴⁺/Ce³⁺ (CeO₂/Ce₂O₃) on a porous titania layer displayed a noticeable enhancement in antibacterial activity, which was evaluated against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. The surface-modified sample heat-treated at 600 °C and enriched with a high Ce⁴⁺/Ce³⁺ ratio revealed enhanced cell compatibility toward MG-63 in terms of cell adhesion, noncytotoxicity, and mitochondrial membrane potential with higher extracellular matrix mineralization. Further, in vivo bone defect investigations in a rat model using additively manufactured cancellous Ti scaffolds functionalized with surface incorporation of Ce ions heat-treated at 600 °C demonstrated significant enhancement in the osseointegration potential observed from histological and micro-computed tomography analyses. The upregulation of osteogenic marker genes (ALP, OCN, OPN, OSX, and RUNX2) at the implantation site evaluated by reverse transcription polymerase chain reaction confirmed the osteogenic potential of ceria surface functionalization. Hence, the Ce-incorporated nanostructured titania layer predominantly possessing a high Ce⁴⁺/Ce³⁺ ratio or CeO₂ content on Ti metal is expected to reduce the risk of implant-related infections and improve cellular responses in orthopedic applications.

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