⁶⁸Ga-citrate visualization study on ferroptosis through transferrin receptor 1 in periodontitis

The loss of alveolar bone in periodontitis is linked to ferroptosis mediated by Transferrin Receptor 1 (TfR1). This study employed a multimethod approach integrating in vitro and in vivo experiments to investigate the utility of gallium-68 citrate (⁶⁸Ga-citrate), a high-affinity iron-mimetic tracer, for the non-invasive, dynamic positron emission tomography/computed tomography (PET/CT) imaging of ferroptosis and periodontal remodeling in vivo.

In vitro, MC3T3-E1 were stimulated with Porphyromonas gingivalis lipopolysaccharide (LPS) to model the inflammatory microenvironment. The correlation between TfR1 expression and ⁶⁸Ga-citrate uptake was validated via cellular uptake assays and plasmid-mediated TfR1 knockdown. Ferroptosis markers - TfR1, glutathione peroxidase 4 (GPX4), interleukin-1β (IL-1β), and osteocalcin (OCN), were analyzed through Western blotting (WB), quantitative PCR (qPCR) and immunohistochemistry (IHC) to characterize the ferroptotic and osteogenic profiles within periodontal tissues. In vivo, a ligature-induced periodontitis rat model was established and treated with minocycline. Periodontal inflammation and bone metabolism were longitudinally evaluated using multi-modal PET/CT ⁶⁸Ga-citrate, 2-deoxy-2-[¹⁸F]fluoro-D-glucose (¹⁸F-FDG), and[¹⁸F]sodium fluoride (¹⁸F-NaF), complemented by micro-computed tomography (micro-CT).

In vitro, LPS dose-dependently reduced MC3T3-E1 viability while upregulating TfR1 and IL1β, and downregulating GPX4 expression. ⁶⁸Ga-citrate uptake correlated positively with TfR1 expression and was significantly inhibited by TfR1 knockdown. In vivo, ⁶⁸Ga-citrate SUV values in periodontal lesions increased progressively from day 7 to 14, accompanied by upregulation of TfR1 and bone resorption. Conversely, minocycline treatment significantly suppressed TfR1 expression and attenuated ⁶⁸Ga-citrate SUV compared to periodontitis groups. The presence of active inflammation and impaired calcium metabolism were confirmed by conventional tracers ¹⁸F-FDG and ¹⁸F-NaF PET/CT.

Osteoblast exhibited increased ⁶⁸Ga-citrate uptake under inflammatory conditions, and ⁶⁸Ga-citrate PET/CT imaging demonstrates enhanced uptake in periodontitis. The expression of TfR1 in both osteoblast and periodontitis animal models is consistent with gallium absorption. These findings indicate that ⁶⁸Ga-citrate PET/CT can reflect ferroptosis-associated molecular alterations by targeting TfR1.