68Ga-citrate visualization study on ferroptosis through Transferrin Receptor 1 in periodontitis

68Ga-citrate visualization study on ferroptosis through Transferrin Receptor 1 in periodontitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article 68 Ga-citrate visualization study on ferroptosis through Transferrin Receptor 1 in periodontitis Yeungyeung Liu, Yingxin Li, Bingyu Ran, Xiaoling Cao, Qijun Cai, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9333859/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Background The loss of alveolar bone in periodontitis is linked to ferroptosis mediated by Transferrin Receptor 1 (TfR1). This study investigates the utility of gallium-68 citrate ( 68 Ga-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. Medthos MC3T3-E1 cells were stimulated with Porphyromonas gingivalis lipopolysaccharide (LPS) to model the inflammatory microenvironment. The correlation between TfR1 expression and 68 Ga-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 the periodontal tissues. A rat model of ligature-induced periodontitis was established, with minocycline serving as a therapeutic intervention. Periodontal inflammation and bone metabolism were longitudinally evaluated using multi-modal PET/CT 68 Ga-citrate, 2-deoxy-2-[ 18 F]fluoro-D-glucose( 18 F-FDG), and[ 18 F]sodium fluoride( 18 F-NaF), complemented by micro-computed tomography (micro-CT). Results In vitro, LPS dose-dependently reduced MC3T3-E1 viability while upregulating TfR1 and IL1β, and downregulating GPX4 expression. 68 Ga-citrate uptake correlated positively with TfR1 expression and was significantly inhibited by TfR1 knockdown. In vivo, 68 Ga-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 68 Ga-citrate SUV compared to periodontitis groups.The presence of active inflammation and impaired calcium metabolism were confirmed by conventional tracers 18 F-FDG and 18 F-NaF PET/CT. Conclusion Osteoblast exhibited increased 68 Ga-citrate uptake under inflammatory conditions, and 68 Ga-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. Therefore, this study suggested that 68 Ga-citrate PET/CT visualized periodontitis ferroptosis by reflecting TfR1 expression. Clinical Trial Number Not applicable 68Ga-citrate periodontitis ferroptosis TfR1 molecular imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Periodontitis is a chronic infectious disease affecting the tooth-supporting tissues, ultimately leading to periodontal attachment loss and gingival recession [1–3]. The initiation and advancement of periodontitis are closely related to abnormalities in alveolar bone metabolism. Infected periodontal tissues consistently exhibit an upregulation of iron-containing compounds, including ferritin, transferrin, and heme [4–6]. Recent studies have highlighted the association between ferroptosis and periodontitis, indicating that this iron-dependent form of cell death is actively involved in disease progression [7]. Within this inflammatory microenvironment, the elevated or dysregulated expression of TfR1 serves as a critical trigger for ferroptosis under inflammatory conditions.Wang et al reproted that human gingival fibroblasts stimulated with P.g -LPS showed increased mRNA transcription and protein expression of proinflammatory cytokines, elevated TfR1 expression, and reduced intracellular glutathione(GSH)[8]. Similarly, in vivo studies using periodontitis mouse models have revealed the downregulation of anti-ferroptotic markers (e.g., GPX4, Nrf2, and NADPH) alongside the upregulation of SLC7A11 and TfR1. This is accompanied by an exacerbated inflammatory profile characterized by increased IL6, IL1β, and IL8, and decreased IL10. The administration of ferroptosis inhibitors reversed these molecular alterations and significantly alleviated the inflammatory response in periodontal tissues [9, 10]. These findings indicated that the upregulation of TfR1 induced by LPS was a vital factor in initiating ferroptosis. 68 Ga-citrate is a radiotracer that acts as an iron mimetic in iron metabolism. It can bind to bacterial ferritin and neutrophil lactoferrin, or be directly absorbed by siderophores with a high affinity for gallium, facilitating its distribution in the circulatory system[11]. In skeletal inflammatory diseases, 68 Ga-citrate PET/CT has demonstrated promising clinical utility, offering high overall accuracy and reliable negative predictive value. 68 Ga-citrate PET/CT has been shown to be effective in accurately differentiating between infectious and aseptic inflammation[12] . Therefore, this study utilized 68 Ga-citrate PET/CT to non-invasively visualize the ferroptotic process in periodontitis by specifically targeting TfR1. We hypothesized that this dynamic molecular imaging platform would provide deeper insights into the complex in vivo dynamics of periodontal inflammation and alveolar bone remodeling. Methods Cell Culture and Treatment MC3T3-E1 Subclone 14 was acquired from Shanghai QuiCell Biotechnology. The cells were grown in α-minimum essential medium(α-MEM, Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA) with 10%fetal bovine serum (FBS, Gibco) in a humidified environment containing 5% CO 2 at 37 ° C .In preparation for subsequent experiments, cells were plated in 6-well plates at density of 2 × 10 5 cells per well. Cell viability was assessed using CCK-8 assay after treatment with different concentrations of LPS (1, 5, 10, 20, 40 and 80 mg/mL). DCFH-DA Reactive Oxygen Species (ROS) Assay MC3T3-E1 cells were plated in 24-well plates and incubated until they reached logarithmic growth phase .Intracellular ROS levels were assessed using DCFH-DA probe diluted in PBS to create a 10 mmol/L working solution .The culture medium was removed, and the cells were rinsed twice with PBS. And 1 mL of DCFH-DA working solution was added to each well, and cells were incubated in the dark at 37°C for 20-30min. Following incubation, the cells were washed three times with PBS to eliminate any excess extracellular probes. Fluorescence was detected under a fluorescence microscope (excitation: 488 nm; emission: 525 nm). TfR1 Interference Fragment Plasmid Transfection The transcript variants and CDS region of the murine TfR1 gene were acquired from the NCBI database, an additional table file shows this in more detail [see Additional file 1]. The interference plasmid was constructed using SnapGene software. The pEGFP-C1 vector was cleaved with Bgl II and EcoR I, while the pCMV-3 × FLAG-N vector was cut using BamH I. When MC3T3-E1 cells reached 70–80% confluence, the culture medium was replaced with fresh α-MEM. For transfection in a 6-well plate, a mixture containing 125 µL of MEM, 4 µL of Lipo8000™ transfection reagent, and 2 µg of plasmid DNA was prepared per well. The prepared transfection complex was thoroughly mixed, incubated at room temperature, and then added slowly to each well. The plate was gently rotated and allowed to incubate for 48 hours. The transfection efficiency was confirmed, the medium was replaced, and subsequent experiments were performed after an additional 24 hours of culture. Cellular Uptake Assay MC3T3-E1 cells were plated in 12-well culture plates at density of 4×10 5 cells per well. Following a 24-hour incubation period, cells were treated with 10 µg/mL LPS for another 24 hours. The viable cells in designated counting wells were quantified to establish a baseline.The culture medium was subsequently substituted with glucose-free α-MEM that included 68 Ga-citrate at concentration of 1 µCi/mL .The cells were cultured for 30 min and 60 min, with three replicate wells per time point. The radioactive material was disposed of, and the cells were rinsed three times with cold PBS. Then 1 mL of 1 mol/L NaOH solution was added to each well to completely lyse the cells. The lysate from each well was gathered and the radioactivity was assessed using a gamma counter. Western Blot Assay Western blotting was performed as previously described [13]. The primary antibodies used in this assay included anti-TfR1 (ab269531, Abcam, Cambridge, UK), anti-GPX4 (ab282533, Abcam), and anti-IL1β (ab315084, Abcam). Quantitative Polymerase Chain Reaction (qPCR) According to earlier report [14], total RNA was extracted from tissues and cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's guidelines. Complementary DNA (cDNA) was generated using Reverse Transcription Kit from Takara (Dalian, China). qPCR was conducted using SmartCycler® II System(Cepheid Inc., Sunnyvale, CA, USA), with GAPDH used as internal reference gene .The relative quantification of mRNA transcripts was calculated using the 2 ΔΔ Ct method. Ligature-Induced Periodontitis Model in Rats Thirty specific-pathogen-free (SPF) male Sprague-Dawley rats (200–300 g, 8 weeks old) were obtained from the Guangzhou Experimental Animal Center. All animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Jinan University (Approval No.20251209-04). Rats were weighed and anesthetized via continuous inhalation of 3% isoflurane using a small animal gas anesthesia system, followed by supine positioning. The periodontitis model was established based on previously described protocols [15, 16]. The rats were randomly assigned to five groups (n = 6 per group): a control group (Con) consisting of untreated healthy rats; an experimental periodontitis group subjected to ligature-induced periodontitis for 7 days (EP7); an experimental periodontitis group subjected to ligature-induced periodontitis for 14 days (EP14); and two minocycline therapy groups treated for 7 days (MN7) and 14 days (MN14) post-ligation. For the MN7 and MN14 groups, 0.2 mg of minocycline hydrochloride ointment (Periocline, 2%; Sunstar, Japan) was locally applied into the gingival crevices once a week. Following the experimental period, rats were humanely euthanized via an intraperitoneal injection of sodium pentobarbital (150 mg/kg) to induce deep anesthesia. Upon confirmation of complete unconsciousness and loss of reflexes, cervical dislocation was performed as a secondary method to ensure humane termination. Micro-Computed Tomography (Micro-CT) Analysis Following euthanasia, the maxillae of the rats were dissected and scanned using a Venus Micro-CT scanner (90 kV, 80 mA) at a scanning resolution of 12 µm. The furcation region of maxillary second molar was designated as region of interest (ROI). Three-dimensional images were reconstructed using Cruiser and Recon software, and bone volume (BV) and tissue volume (TV) were measured using Avatar software to calculate the BV/TV fraction. Additionally, the linear distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) was measured at both the mesial and distal aspects of the maxillary second molar, and the average value was recorded. PET/CT Imaging The 68 Ga-citrate tracer was purchased by the Atomic High-Tech Co., Ltd (Guangzhou, China). 18 F-NaF and 18 F-FDG were provided by the Department of Nuclear Medicine, The First Affiliated Hospital of Jinan University, with radiochemical purity > 99%. The IRIS small-animal PET/CT imaging system (Inviscan SAS, Strasbourg, France) was utilized to conduct PET/CT imaging. During the scanning procedure, the rats were anesthetized with 3% isoflurane, and their body temperature was maintained at 37.5°C using a temperature regulation unit (Minerve équipement vétérinaire, Marne, France). For tracer injection and scanning: 68 Ga-citrate PET/CT: After intravenous injection of 68 Ga-citrate (15–20 MBq) via the tail vein, a 10-minute static PET scan was acquired starting 45 minutes post-injection. 18 F-FDG PET/CT: After intravenous injection of 18 F-FDG (15–20 MBq) via the tail vein, a 10-minute static PET scan was acquired starting 60 minutes post-injection. 18 F-NaF PET/CT: After intravenous injection of 18 F-NaF (15–20 MBq) via the tail vein, a 10-minute static PET scan was acquired starting 60 minutes post-injection. The PET data were reconstructed utilizing 3D Ordered Subsets Expectation Maximization (3D-OSEM) algorithm that incorporates a Monte Carlo-based accurate detector model. Following the PET scan, a CT scan was conducted with the X-ray tube voltage set at 80 kV and the current at 0.9 mA. PET images were analyzed using PMOD 4.1 software (PMOD Technologies, Zurich, Switzerland). Bilateral regions of interest (ROIs) were manually delineated in the maxillary region, extending from the mesial aspect of the first molar to the distal aspect of the third molar. The average standardized uptake value (SUV) within the ROI was calculated. Immunohistochemistry Immunohistochemical staining for TfR1 and OCN was conducted on sections of paraffin-embedded maxilla. Sections were deparaffinized, rehydrated,and treated with 3% hydrogen peroxide for 25 minutes at room temperature. Following blocking with bovine serum albumin (BSA) for 30 minutesat room temperature, sections were incubated with primary antibodies against TfR1 (ab269531, 1:200; Abcam, Cambridge, UK) and OCN (GB115684- 100, 1:500; Servicebio,Wuhan, China) overnight at 4°C. Subsequently, the sections were treated with biotinylated secondary antibody (HRP-labeled goat anti-rabbit IgG, 1:200; Servicebio) for 50 minutes at room temperature. The slides were washed three times in PBS (pH 7.4 ) for 5 minutes each. A freshly prepared DAB chromogenic solution was applied for signal detection, followed by counterstaining with hematoxylin for 3 minutes. The sections were dehydrated in a sequential manner using a graded series of alcohol 75% for 5 minutes, 85% for 5 minutes, absolute ethanol twice for 5 minutes each, and n-butyl alcohol for 5 minutes, followed by a clearing step in xylene for 5 minutes. Ultimately, the sections were briefly air-dried and placed in a mounting medium. Images were taken with a light microscope (Leica DM6b, Nussloch, Germany) and analyzed through ImageJ software (NIH, Bethesda, MD, USA) . Statistical Analysis All statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA). Data are presented as the mean ± standard deviation (SD) derived from at least three independent experiments. Group comparisons were assessed using Student’s t-test (for two groups) or one-way analysis of variance (ANOVA) followed by appropriate post hoc tests (for multiple groups). A P-value of < 0.05 was considered statistically significant. Results Osteoblast ferroptosis occurs under inflammatory conditions To evaluate the impact of the inflammatory microenvironment on ferroptosis, MC3T3-E1 cells were incubated with LPS for 24 h, followed by the assessment of ferroptosis biomarkers. As demonstrated in Fig. 1 A, LPS dramatically reduced the viability of MC3T3-E1 cells in a dose-dependent manner, with a notable decrease seen at 10 µg/mL. Furthermore, the expression of TfR1 and IL1β gradually increased, whereas GPX4 expression decreased, in response to escalating LPS concentrations (0, 1, 5, and 10 µg/mL) (Fig. 1 B). A consistent trend was observed in the mRNA transcription levels of TfR1, IL1β, and GPX4 across the corresponding LPS concentrations (Fig. 1 C). At the level of 10 µg/mL LPS, cell ROS and LPS accumulation was increased (Fig. 1 D). 68 Ga-citrate reflects osteoblast ferroptosis via TfR1 Because 68 Ga-citrate serves as an imaging surrogate for iron metabolism, and TfR1 is a pivotal protein in this pathway, we evaluated 68 Ga-citrate cellular uptake following plasmid-mediated TfR1 knockdown (KD) in MC3T3-E1 cells, an additional figure file shows this in more detail [see Additional file 2]. Compared to cells treated with 10 µg/mL LPS alone, cells transfected with the TfR1-KD plasmid exhibited a significant reduction in TfR1 protein expression. Subsequent cellular uptake assays were performed at 30 and 60 minutes post- 68 Ga-citrate administration. The tracer uptake rate was considerably higher in the LPS-treated group compared to the Con group at both time points. Conversely, the KD group demonstrated the lowest 68 Ga-citrate uptake rate, confirming that the tracer's accumulation is highly dependent on TfR1 expression (Fig. 2 ). Alveolar bone defects and inflammatory changes in the periodontitis animal model A ligature-induced periodontitis rat model was developed to evaluate ferroptosis during inflammatory bone loss (Fig. 3 ). Clinical manifestations of periodontitis were evident by day 7 post-ligation (EP7), characterized by moderate inflammation of the gingival margin and interdental papillae, presenting with bright red coloration, mild edema, and bleeding upon probing confined to the gingival sulcus. By day 14 (EP14), the inflammation progressed to a severe state, exhibiting a brittle gingival texture, severe edema, and spontaneous bleeding spilling beyond the gingival sulcus (Fig. 4 A). Micro-CT analysis revealed a progressive trend of alveolar bone defects from EP7 to EP14, which was effectively attenuated by minocycline administration (Fig. 4 B). Histological evaluations confirmed the presence of inflammatory infiltration and alveolar bone loss, accompanied by suppressed osteogenesis, as indicated by low OCN expression (Fig. 4 C and Fig. 4 D). Consistent with these findings, 18 F-FDG PET/CT demonstrated intense tracer accumulation in the alveolar region under periodontitis conditions, which was subsequently reduced following antibiotic intervention (Fig. 5 ). Furthermore, calcium metabolism was evaluated using 18 F-NaF PET/CT, a bone-seeking agent that binds to hydroxyapatite crystals [17]. The inflammatory environment induced a marked reduction in local calcium metabolism within the alveolar area, an effect that was partially reversed by minocycline treatment (Fig. 6 ). 68 Ga-citrate PET/CT visualizes TfR1 expression in vivo In the rat periodontitis model, both the transcription and protein expression of TfR1 increased progressively alongside disease exacerbation. Notably, this inflammation-induced TfR1 upregulation was significantly inhibited by minocycline administration at matched time points (Fig. 7 ). In vivo PET/CT imaging acquired 45 minutes post-injection of 68 Ga-citrate provided clear visualization of the maxillary bone, with minimal background uptake in the adjacent maxillary sinus and brain tissue. The uptake of 68 Ga-citrate in the maxillae of ligature-induced periodontitis rats was markedly higher than that in the healthy control (Con) group. Quantitative analysis revealed that the mean standardized uptake values (SUV) in both the EP7 and EP14 groups were elevated compared to the Con group. Notably, a statistically significant increase in 68 Ga-citrate SUV was observed only in the EP14 group compared to the control, whereas the EP7 group showed no significant alteration. Following minocycline intervention, the MN7 group exhibited a lower mean SUV than the EP7 group, although this trend did not reach statistical significance. In contrast, the MN14 group demonstrated a significant reduction in tracer uptake compared to the EP14 group(Fig. 8 ). Discussion In the inflammatory microenvironment of periodontitis, ferroptosis is driven by multiple pathways that elevate intracellular ROS levels [4, 18]. The dysregulated overexpression of TfR1 serves as a critical catalyst for cellular ferroptosis during periodontal inflammation. Using an LPS-induced osteoblast inflammation model, we demonstrated that LPS intervention dose-dependently increased ROS accumulation in MC3T3-E1 cells. Concurrently, the protein expression and mRNA transcription of TfR1 and IL1β were upregulated, whereas the anti-ferroptotic enzyme GPX4 was progressively downregulated. These findings align with previous literature [19], further corroborating TfR1 as a pivotal mediator in the ferroptotic cascade. Previous studies have evidenced that 68 Ga-citrate, a radiotracer mimicking iron metabolism, is internalized by cells via the transferrin/TfR1 complex [20]. This mechanism provides a robust theoretical basis for utilizing 68 Ga-citrate to visualize ferroptosis. In skeletal imaging, 68 Ga-citrate has demonstrated superior specificity compared to conventional 18 F-FDG, effectively minimizing false-negative results [21] and accurately distinguishing physiological bone healing from true osteomyelitis [22, 23]. Furthermore, it exhibits broad potential in diagnosing infectious or inflammatory diseases across various organ systems [24]. In this study, we confirmed the target specificity of 68 Ga-citrate through in vitro experimentation. Following plasmid-mediated TfR1 knockdown in MC3T3-E1 cells, the cellular uptake of 68 Ga-citrate significantly decreased concurrently with TfR1 depletion, confirming its target-specific binding mechanism. A key pathological characteristic of periodontitis is the gradual loss of alveolar bone [25. 26]. As periodontal inflammation exacerbates, the homeostasis of alveolar bone remodeling is severely disrupted. This process is driven by multiple mechanisms: immune cell activation and release of inflammatory mediators hyperactivate osteoclasts while suppressing osteoblast function[27]; the RANKL/RANK signaling axis further amplifies the osteoclastogenesis [28]; and inflammatory stimuli trigger the secretion of matrix metalloproteinases, which degrade extracellular matrix components and impair osteoblast attachment [29,30]. To replicate these clinical manifestations, a rat model of periodontitis was created using a ligature-induced method. Typical clinical symptoms, including gingival marginal inflammation, bright-red coloration, localized edema, and bleeding upon probing, were observed by day 7 post-ligation [31], and these symptoms significantly worsened by day 14. Micro-CT analysis revealed that the alveolar bone defects at day 14 (EP14) were significantly more severe compared to the control and day 7 (EP7) groups, as indicated by significant bone volume loss and a decrease in alveolar crest height. These in vivo phenotypes are highly consistent with the osteoblast impairment induced by LPS in vitro. Conversely, minocycline has proven efficacy in suppressing P.gingivalis -induced inflammatory responses clinically and reducing bone abnormalities in animal models [32,33]. Histological analysis in our study demonstrated that as periodontitis progressed, inflammatory cell infiltration increased, while osteogenic activity (indicated by OCN deposition) markedly decreased. Following minocycline administration, both the inflammatory infiltration and the loss of calcium metabolism were significantly alleviated. In the context of translational molecular imaging, clinical practice traditionally utilizes technetium-99m methylene diphosphonate ( 99m Tc-methylene) for labeling autologous leukocytes, alongside 18 F-FDG, 18 F-NaF, 67 Ga, and 68 Ga to visualize inflammatory diseases. Other novel targeted tracers, including 2-deoxy-2-[ 18 F]fluoro-D-sorbitol( 18 F-FDS), [ 18 F]fluoropropyl-aminomethylene diphosphonate ( 18 F-FPTMP), and [ 11 C]para-aminobenzoic acid ( 11 C-PABA) also hold promise for characterizing specific microbial and inflammatory pathways [34–38]. Regarding periodontitis, while 18 F-FDG PET/CT PET/CT can monitor therapeutic responses, its lack of target specificity limits its clinical expectations [39]. Meanwhile, 18 F-FDG PET/CT effectively tracks hydroxyapatite metabolism, indirectly reflecting alterations in periodontal bone mass. Our dual-tracer imaging using 18 F-FDG and 18 F-NaF successfully corroborated these findings, visualizing both the heightened inflammatory metabolic burst and the impaired bone mineral density. More importantly, immunohistochemical analysis confirmed that TfR1 expression in vivo escalated parallel to the severity of local inflammation and declined upon minocycline intervention. Given that 68 Ga-citrate reliably reflects TfR1 expression in vitro, we pioneered the application of 68 Ga-citrate PET/CT to evaluate periodontal ferroptosis. The in vivo imaging revealed a marked increase in 68 Ga-citrate uptake within the maxillae of periodontitis rats. Interestingly, during the mild inflammatory stage, tracer uptake did not show statistically significant alterations compared to controls; marked tracer accumulation became evident only when the disease progressed to the severe stage. Following minocycline intervention, 68 Ga-citrate PET/CT imaging captured a corresponding reduction in signal intensity, although the therapy could not completely normalize the elevated TfR1 expression to baseline levels. Collectively, these findings underscore TfR1 as the key molecular bridge linking ferroptosis to 68 Ga-citrate accumulation. Through targeting TfR1, 68 Ga-citrate PET/CT successfully visualizes the dynamic burden of ferroptosis driven by inflammation, offering a novel and non-invasive functional imaging strategy for evaluating osteoblast ferroptosis during periodontitis. Conclusion In conclusion, osteoblast exhibited increased 68 Ga-citrate uptake under inflammatory conditions, and 68 Ga-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. Therefore, this study suggested that 68 Ga-citrate PET/CT visualized periodontitis ferroptosis by reflecting TfR1 expression. Abbreviations TfR1: Transferrin Receptor 1 Gallium-68 citrate: 68 Ga-citrate PET/CT: positron emission tomography/computed tomography GPX4: Glutathione Peroxidase 4 IL1β: interleukin 1β OCN: Osteocalcin LPS : Lipopolysaccharide ABC: Alveolar Bone Crest BV: Bone Volume CEJ: Cement-Enamel Junction CT: Computed Tomography FDG: Fludeoxyglucose GBI: Gingival Bleeding Index HE: Hematoxylin and eosin LPO: Lipid peroxide P.g: Porphyromonas gingivalis ROI : Region Of Interest ROS: Reactive Oxygen Species SPECT: Single Photon Emission Computed Tomography SUV: Standardized Uptake Value TF: Transferrin ‌TV: Tissue Volume KD: Knockown Declarations Ethics approval and consent to participate All animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Jinan University (Approval No.20251209-04). Consent for publication Not applicable Competing interests The authors declare no competing interests. Funding This study was supported by grants from Science research cultivation program of stomatological hospital, Southern medical university (PY2023042) in China. Author Contribution Liu Y and Li Y carried out the experiments, performed the analysis and drafted the manuscript. Ran B, Cao X, Cai Q, Cheng Y, Guo B and Gong J helped with data acquisition and analysis. Shang J and Hao X supervised the study, critically revised the manuscript and provided funding support. All authors reviewed the manuscript. Availability of data and materials Data and materials were provided within the manuscript and supplementary material files Acknowledgements None to declare References Eduardo B, Wagner M, Rizwan S, Lucas GA, Saira A, Fadwa NA, et al. 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Osteocyte dysregulation in periodontitis: Pathological mechanisms and therapeutic potential. Cell Signal. 2025;135:112062. AlQranei M S, Chellaiah M A. Osteoclastogenesis in periodontal diseases: Possible mediators and mechanisms. J Oral Biosci. 2020;62(2):123-30. Belibasakis G N, Bostanci N. The RANKL-OPG system in clinical periodontology. J Clin Periodontol. 2012;39(3):239-48. Elgezawi M, Haridy R, Almas K, Abdalla MA, Omar O, Abuohashish H, et al. Matrix Metalloproteinases in Dental and Periodontal Tissues and Their Current Inhibitors: Developmental, Degradational and Pathological Aspects. Int J Mol Sci. 2022;23(16):8929. Luchian I, Goriuc A, Sandu D, Covasa M. The Role of Matrix Metalloproteinases (MMP-8, MMP-9, MMP-13) in Periodontal and Peri-Implant Pathological Processes. Int J Mol Sci. 2022;23(3):1806. Tomina DC, Petruțiu ȘA, Dinu CM, Crișan B, Cighi VS, Rațiu IA. Comparative Testing of Two Ligature-Induced Periodontitis Models in Rats: A Clinical, Histological and Biochemical Study. Biology (Basel). 2022;11(5):634. Frazão DR, Matos-Souza JM, Dos Santos VRN, Nazario RMF, Chemelo VDS, Bittencourt LO, et al. Minocycline reduces alveolar bone loss and bone damage in Wistar rats with experimental periodontitis. PLoS One. 2024;19(10):e309390. Laza GM, Sufaru IG, Martu MA, Martu C, Diaconu-Popa DA, Jelihovschi I, et al. Effects of Locally Delivered Minocycline Microspheres in Postmenopausal Female Patients with Periodontitis: A Clinical and Microbiological Study. Diagnostics (Basel). 2022;12(6):1310. Kim DY, Pyo A, Ji S, You SH, Kim SE, Lim D, et al. In vivo imaging of invasive aspergillosis with (18)F-fluorodeoxysorbitol positron emission tomography. Nat Commun. 2022;13(1):1926. Mutch CA, Ordonez AA, Qin H, Parker M, Bambarger LE, Villanueva-Meyer JE, et al. [(11)C]Para-Aminobenzoic Acid: A Positron Emission Tomography Tracer Targeting Bacteria-Specific Metabolism. ACS Infect Dis. 2018;4(7):1067-72. Sellmyer MA, Lee I, Hou C, Weng CC, Li S, Lieberman BP, et al. Bacterial infection imaging with [(18)F]fluoropropyl-trimethoprim. Proc Natl Acad Sci U S A. 2017;114(31):8372-77. Koatale PC, Welling MM, Ndlovu H, Kgatle M, Mdanda S, Mdlophane A, et al. Insights into Peptidoglycan-Targeting Radiotracers for Imaging Bacterial Infections: Updates, Challenges, and Future Perspectives. ACS Infect Dis. 2024;10(2):270-86. Zhang Z, Ordonez AA, Wang H, Li Y, Gogarty KR, Weinstein EA, et al. Positron Emission Tomography Imaging with 2-[(18)F]F- p-Aminobenzoic Acid Detects Staphylococcus aureus Infections and Monitors Drug Response. ACS Infect Dis. 2018;4(11):1635-44. Arefnia B, Horina A, Nazerani-Zemann T, Seinost G, Rieder M, Wimmer G. Nuclear imaging to visualize periodontal inflammation: Findings of a randomized controlled trial. Oral Dis. 2024;30(7):4630-38. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.pdf Additionalfile2.pdf Additionalfile3.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 17 May, 2026 Reviewers invited by journal 23 Apr, 2026 Editor invited by journal 20 Apr, 2026 Editor assigned by journal 20 Apr, 2026 Submission checks completed at journal 16 Apr, 2026 First submitted to journal 16 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9333859","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":633609956,"identity":"e2e00c73-dcca-4ea6-a211-ae7cda6e8304","order_by":0,"name":"Yeungyeung Liu","email":"","orcid":"","institution":"Stomatological Hospital, School of Stomatology, Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yeungyeung","middleName":"","lastName":"Liu","suffix":""},{"id":633609957,"identity":"a553d06d-d037-4a0e-9e55-db8514ecd1c9","order_by":1,"name":"Yingxin Li","email":"","orcid":"","institution":"The first Affiliated Hospital of Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Yingxin","middleName":"","lastName":"Li","suffix":""},{"id":633609958,"identity":"81a24260-272c-47c8-b5c3-79569dd01f78","order_by":2,"name":"Bingyu Ran","email":"","orcid":"","institution":"The first Affiliated Hospital of Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Bingyu","middleName":"","lastName":"Ran","suffix":""},{"id":633609959,"identity":"3dfdd493-fb25-4d53-9b13-8d6eeee65908","order_by":3,"name":"Xiaoling Cao","email":"","orcid":"","institution":"The first Affiliated Hospital of Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoling","middleName":"","lastName":"Cao","suffix":""},{"id":633609960,"identity":"bf57bd69-16d9-43e2-9fd1-7b97e0eb0ea0","order_by":4,"name":"Qijun Cai","email":"","orcid":"","institution":"The first Affiliated Hospital of Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Qijun","middleName":"","lastName":"Cai","suffix":""},{"id":633609961,"identity":"24beae0f-55f4-4fee-af21-5dac501052e3","order_by":5,"name":"Yong Cheng","email":"","orcid":"","institution":"The first Affiliated Hospital of Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Cheng","suffix":""},{"id":633609962,"identity":"92154af9-9428-442f-a9b3-05caa755c109","order_by":6,"name":"Bin Guo","email":"","orcid":"","institution":"The first Affiliated Hospital of Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Guo","suffix":""},{"id":633609963,"identity":"cba0af88-ad89-460e-a3c0-ea3fd9be9e57","order_by":7,"name":"Jian Gong","email":"","orcid":"","institution":"The first Affiliated Hospital of Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Gong","suffix":""},{"id":633609964,"identity":"c1cb0f3b-25bf-4c2c-94ec-e5ec890cbf0d","order_by":8,"name":"Jingjie Shang","email":"","orcid":"","institution":"The first Affiliated Hospital of Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Jingjie","middleName":"","lastName":"Shang","suffix":""},{"id":633609965,"identity":"2cadf02e-4de2-45e7-8f24-ae3d7af1229e","order_by":9,"name":"Hao Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqklEQVRIiWNgGAWjYFACHsYHEmBGArEaGHiYDUjWwgbRQbQWe4ncYxWWOw4z8LPnGDD83EGMLTzn0m5InjnMINnzxoCx9wwxWth7zG5Ith1mMLiRY8DM2EaMFmYeswKQFnvitQBtYQDbIkG0ljNnjCUk29J5JM48KzjYS4wW9hk5hp8l26zl+NuTNz74SYwWEGCWAMUPEBwgUgMDA+MHopWOglEwCkbBiAQAoeItGDk2wjEAAAAASUVORK5CYII=","orcid":"","institution":"The first Affiliated Hospital of Jinan University","correspondingAuthor":true,"prefix":"","firstName":"Hao","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2026-04-06 12:23:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9333859/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9333859/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108808200,"identity":"d3415988-9472-48cf-b5b1-727abce34558","added_by":"auto","created_at":"2026-05-08 15:40:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2825255,"visible":true,"origin":"","legend":"\u003cp\u003eOsteoblast ferroptosis occurred at inflammatory status. (A) The cck8 assay was performed after MC3T3 cells treated with LPS (1 µg/mL, 5 µg/mL, and 10 µg/mL). (B) Western blot analysis was performed to detect the expression of TfR1, IL1β, and GPX4 proteins after intervention with LPS in MC3T3 cells. (C) The qPCR was performed to detect the RNA transcription of TfR1, IL1β, and GPX4 after MC3T3 cells were treated with LPS. (D) DCFH-DA reactive oxygen species (ROS) was performed after MC3T3 cells were treated with 10 µg/mL LPS. *: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, ***: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, ****: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001, ns: not significant\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9333859/v1/f385ec62ab3ad45d7c4f6ce8.png"},{"id":108808198,"identity":"b373c7a2-f30b-4e64-8ac5-288d2a4eb655","added_by":"auto","created_at":"2026-05-08 15:40:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":429169,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e68\u003c/sup\u003eGa-citrate reflexed osteoblast ferroptosis through TfR1. (A) MC3T3 cells were treated with 10 µg/ mL LPS in LPS group. TfR1 immunofluorescence staining was observed in control group(Con), knocked down group(KD) and LPS group (LPS). (B) Perform \u003csup\u003e68\u003c/sup\u003eGa-citrate uptake experiments on MC3T3 cells in each group and record the cell uptake ratio. *: \u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, **: \u003cem\u003eP\u003c/em\u003e \u0026lt;0.01, ***: \u003cem\u003eP\u003c/em\u003e \u0026lt;0.001, ****: \u003cem\u003eP\u003c/em\u003e \u0026lt;0.0001, ns: not significant\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9333859/v1/ac0e132692cc0f43112ba8e2.png"},{"id":108808156,"identity":"082e44ba-361c-44a5-99f5-7c59d980b3c1","added_by":"auto","created_at":"2026-05-08 15:40:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3534675,"visible":true,"origin":"","legend":"\u003cp\u003eLigature-Induced Periodontitis Model in Rats. a control group (Con) consisting of untreated healthy rats; an experimental periodontitis group subjected to ligature-induced periodontitis for 7 days (EP7); an experimental periodontitis group subjected to ligature-induced periodontitis for 14 days (EP14); and two minocycline therapy groups treated for 7 days (MN7) and 14 days (MN14) post-ligation. For the MN7 and MN14 groups, 0.2 mg of minocycline hydrochloride ointment (Periocline, 2%; Sunstar, Japan) was locally applied into the gingival crevices once a week\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9333859/v1/51462b7f63d1ada766e0c322.png"},{"id":108810085,"identity":"8137ca43-b0ba-48b7-b4a2-849f98391e05","added_by":"auto","created_at":"2026-05-08 15:57:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9800966,"visible":true,"origin":"","legend":"\u003cp\u003eBone defect occured in periodontitis animal model. A periodontitis rat model was established. Ligature at 7 days post-modeling was designated as EP7 group (EP7), and Ligature at 14 days post-modeling was designated as EP14 group (EP14). The minocycline group (Mino group) received intragingival injection of minocycline hydrochloride ointment into the gingiva of the second molar of the maxilla. The group receiving minocycline at 7 days post-modeling was designated as Mino7 group (MN7), and the group receiving minocycline at 14 days post-modeling was designated as Mino14 group (MN14). (A) GI index was evaluated. (B)Three-dimensional Micro-CT images and sagittal tomographic images of the maxillary bones in each group with CEJ-ABC distances, red arrows indicate the alveolar bone height. (C) HE staining of alveolar tissue in the ligated model of periodontitis rats. (D) OCN staining of alveolar tissue in the ligated model of periodontitis rats. **:\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01, ***: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, and ****: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001, ns: not significant\u003c/p\u003e","description":"","filename":"FIgure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9333859/v1/4d5c06b660a3aad5f4099985.jpg"},{"id":108808201,"identity":"4470749a-b428-43c2-8297-fc7ffd6cb6fc","added_by":"auto","created_at":"2026-05-08 15:40:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":526644,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of \u003csup\u003e18\u003c/sup\u003eF-FDG PET/CT in periodontitis animal model. \u003csup\u003e18\u003c/sup\u003eF-FDG PET/CT: After intravenous injection of \u003csup\u003e18\u003c/sup\u003eF-FDG (15 - 20 MBq) via the tail vein, a 10-minute static PET scan was acquired starting 60 minutes post-injection. (A)\u003csup\u003e18\u003c/sup\u003eF-FDG PET/CT scans were performed at corresponding time points to measure the mean SUV values of \u003csup\u003e18\u003c/sup\u003eF-FDG PET/CT in rats across all groups. (B) Statistical analysis was performed on the mean \u003csup\u003e18\u003c/sup\u003eF-FDG PET/CT SUV values of all rat groups. *: \u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, ns: not significant\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9333859/v1/51ac7d62fceee081dee1c6e8.png"},{"id":108809852,"identity":"93fab226-62d4-4f23-9c1d-d5b207253b01","added_by":"auto","created_at":"2026-05-08 15:55:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":511506,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of \u003csup\u003e18\u003c/sup\u003eF-Na PET/CT in periodontitis animal model. \u003csup\u003e18\u003c/sup\u003eF-NaF PET/CT: After intravenous injection of \u003csup\u003e18\u003c/sup\u003eF-NaF (15 - 20 MBq) via the tail vein, a 10-minute static PET scan was acquired starting 60 minutes post-injection. (A) \u003csup\u003e18\u003c/sup\u003eF-Na PET/CT scans were performed at corresponding time points to measure the mean SUV values of \u003csup\u003e18\u003c/sup\u003eF-Na PET/CT in rats across all groups.The white arrows point to the site of alveolar bone resorption at the maxillary second molar , characterized by decreased \u003csup\u003e18\u003c/sup\u003eF-NaF accumulation reflecting impaired calcium metabolism. (B) Statistical analysis was performed on the mean \u003csup\u003e18\u003c/sup\u003eF-Na PET/CT SUV values of all rat groups. **:\u003cem\u003e P\u003c/em\u003e \u0026lt;0.01, ns: not significant\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9333859/v1/0730209020e9d78fa3e2ef6e.png"},{"id":108808150,"identity":"d25bdd98-8032-459c-9872-b9ac8566163a","added_by":"auto","created_at":"2026-05-08 15:40:06","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":918738,"visible":true,"origin":"","legend":"\u003cp\u003echanges in periodontitis animal models. A periodontitis rat model was established. Ligature at 7 days post-modeling was designated as EP7 group (EP7), and Ligature at 14 days post-modeling was designated as EP14 group (EP14). The minocycline group (Mino group) received intragingival injection of minocycline hydrochloride ointment into the gingiva of the second molar of the maxilla. The group receiving minocycline at 7 days post-modeling was designated as MN7 group (MN7), and the group receiving minocycline at 14 days post-modeling was designated as MN14 group (MN14). (A)Immunohistochemical staining of TfR1 in maxillary bone tissues of rats with periodontitis. (B)Samples were collected at the corresponding time points for Western blot analysis. (C)Samples were collected at corresponding time points for qPCR analysis. **:\u003cem\u003e P\u003c/em\u003e\u0026lt;0.01, *** : \u003cem\u003eP\u003c/em\u003e \u0026lt;0.001, ****: \u003cem\u003eP\u003c/em\u003e \u0026lt;0.0001, ns: not significant\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9333859/v1/2a9ef5034db2cc1f8bcd2863.png"},{"id":108809873,"identity":"ceede7be-0ce7-4c63-8782-ea24553a02be","added_by":"auto","created_at":"2026-05-08 15:56:01","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":552340,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of \u003csup\u003e68\u003c/sup\u003eGa-citrate PET/CT in periodontitis animal model. (A) \u003csup\u003e68\u003c/sup\u003eGa-citrate PET/CT scans were performed at corresponding time points to measure the mean SUV values of \u003csup\u003e68\u003c/sup\u003eGa-citrate PET/CT in rats across all groups. (B) Statistical analysis was performed on the mean \u003csup\u003e68\u003c/sup\u003eGa-citrate PET/CT SUV values of all rat groups. **: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ****: \u003cem\u003eP\u003c/em\u003e \u0026lt;0.0001, ns: not significant\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9333859/v1/20839e614c3b1565c92450b1.png"},{"id":108814973,"identity":"51a9bb90-8767-400d-8ae5-4ec5cf7a6c97","added_by":"auto","created_at":"2026-05-08 16:21:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18752400,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9333859/v1/67ecbcf3-aaee-43b4-ae2a-98bc68702fec.pdf"},{"id":108808151,"identity":"2d993893-bcbf-493c-882d-21c5191db20d","added_by":"auto","created_at":"2026-05-08 15:40:07","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":94939,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9333859/v1/f119539b2d7d658db1ce2c26.pdf"},{"id":108808199,"identity":"6d43d184-eaea-49d6-9634-4f861a5ae829","added_by":"auto","created_at":"2026-05-08 15:40:24","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":157652,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9333859/v1/fe359038a2faf1af9d8f0344.pdf"},{"id":108808164,"identity":"118a32f5-d629-49a0-81de-d233966fb9a2","added_by":"auto","created_at":"2026-05-08 15:40:10","extension":"pdf","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":143182,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9333859/v1/f8d3d895ba55cf9c1a312781.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003csup\u003e68\u003c/sup\u003eGa-citrate visualization study on ferroptosis through Transferrin Receptor 1 in periodontitis\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003ePeriodontitis is a chronic infectious disease affecting the tooth-supporting tissues, ultimately leading to periodontal attachment loss and gingival recession [1\u0026ndash;3]. The initiation and advancement of periodontitis are closely related to abnormalities in alveolar bone metabolism. Infected periodontal tissues consistently exhibit an upregulation of iron-containing compounds, including ferritin, transferrin, and heme [4\u0026ndash;6]. Recent studies have highlighted the association between ferroptosis and periodontitis, indicating that this iron-dependent form of cell death is actively involved in disease progression [7].\u003c/p\u003e \u003cp\u003eWithin this inflammatory microenvironment, the elevated or dysregulated expression of TfR1 serves as a critical trigger for ferroptosis under inflammatory conditions.Wang et al reproted that human gingival fibroblasts stimulated with \u003cem\u003eP.g\u003c/em\u003e-LPS showed increased mRNA transcription and protein expression of proinflammatory cytokines, elevated TfR1 expression, and reduced intracellular glutathione(GSH)[8]. Similarly, in vivo studies using periodontitis mouse models have revealed the downregulation of anti-ferroptotic markers (e.g., GPX4, Nrf2, and NADPH) alongside the upregulation of SLC7A11 and TfR1. This is accompanied by an exacerbated inflammatory profile characterized by increased IL6, IL1β, and IL8, and decreased IL10. The administration of ferroptosis inhibitors reversed these molecular alterations and significantly alleviated the inflammatory response in periodontal tissues [9, 10].\u003c/p\u003e \u003cp\u003eThese findings indicated that the upregulation of TfR1 induced by LPS was a vital factor in initiating ferroptosis. \u003csup\u003e68\u003c/sup\u003eGa-citrate is a radiotracer that acts as an iron mimetic in iron metabolism. It can bind to bacterial ferritin and neutrophil lactoferrin, or be directly absorbed by siderophores with a high affinity for gallium, facilitating its distribution in the circulatory system[11]. In skeletal inflammatory diseases, \u003csup\u003e68\u003c/sup\u003eGa-citrate PET/CT has demonstrated promising clinical utility, offering high overall accuracy and reliable negative predictive value. \u003csup\u003e68\u003c/sup\u003eGa-citrate PET/CT has been shown to be effective in accurately differentiating between infectious and aseptic inflammation[12] .\u003c/p\u003e \u003cp\u003eTherefore, this study utilized \u003csup\u003e68\u003c/sup\u003eGa-citrate PET/CT to non-invasively visualize the ferroptotic process in periodontitis by specifically targeting TfR1. We hypothesized that this dynamic molecular imaging platform would provide deeper insights into the complex in vivo dynamics of periodontal inflammation and alveolar bone remodeling.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eCell Culture and Treatment\u003c/h2\u003e\n \u003cp\u003eMC3T3-E1 Subclone 14 was acquired from Shanghai QuiCell Biotechnology. The cells were grown in \u0026alpha;-minimum essential medium(\u0026alpha;-MEM, Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA) with 10%fetal bovine serum (FBS, Gibco) in a humidified environment containing 5% CO\u003csub\u003e2\u003c/sub\u003e at 37 \u0026deg; C .In preparation for subsequent experiments, cells were plated in 6-well plates at density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well. Cell viability was assessed using CCK-8 assay after treatment with different concentrations of LPS (1, 5, 10, 20, 40 and 80 mg/mL).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eDCFH-DA Reactive Oxygen Species (ROS) Assay\u003c/h3\u003e\n\u003cp\u003eMC3T3-E1 cells were plated in 24-well plates and incubated until they reached logarithmic growth phase .Intracellular ROS levels were assessed using DCFH-DA probe diluted in PBS to create a 10 mmol/L working solution .The culture medium was removed, and the cells were rinsed twice with PBS. And 1 mL of DCFH-DA working solution was added to each well, and cells were incubated in the dark at 37\u0026deg;C for 20-30min. Following incubation, the cells were washed three times with PBS to eliminate any excess extracellular probes. Fluorescence was detected under a fluorescence microscope (excitation: 488 nm; emission: 525 nm).\u003c/p\u003e\n\u003ch3\u003eTfR1 Interference Fragment Plasmid Transfection\u003c/h3\u003e\n\u003cp\u003eThe transcript variants and CDS region of the murine TfR1 gene were acquired from the NCBI database, an additional table file shows this in more detail [see Additional file 1]. The interference plasmid was constructed using SnapGene software. The pEGFP-C1 vector was cleaved with Bgl II and EcoR I, while the pCMV-3 \u0026times; FLAG-N vector was cut using BamH I. When MC3T3-E1 cells reached 70\u0026ndash;80% confluence, the culture medium was replaced with fresh \u0026alpha;-MEM. For transfection in a 6-well plate, a mixture containing 125 \u0026micro;L of MEM, 4 \u0026micro;L of Lipo8000\u0026trade; transfection reagent, and 2 \u0026micro;g of plasmid DNA was prepared per well. The prepared transfection complex was thoroughly mixed, incubated at room temperature, and then added slowly to each well. The plate was gently rotated and allowed to incubate for 48 hours. The transfection efficiency was confirmed, the medium was replaced, and subsequent experiments were performed after an additional 24 hours of culture.\u003c/p\u003e\n\u003ch3\u003eCellular Uptake Assay\u003c/h3\u003e\n\u003cp\u003eMC3T3-E1 cells were plated in 12-well culture plates at density of 4\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well. Following a 24-hour incubation period, cells were treated with 10 \u0026micro;g/mL LPS for another 24 hours. The viable cells in designated counting wells were quantified to establish a baseline.The culture medium was subsequently substituted with glucose-free \u0026alpha;-MEM that included \u003csup\u003e68\u003c/sup\u003eGa-citrate at concentration of 1 \u0026micro;Ci/mL .The cells were cultured for 30 min and 60 min, with three replicate wells per time point. The radioactive material was disposed of, and the cells were rinsed three times with cold PBS. Then 1 mL of 1 mol/L NaOH solution was added to each well to completely lyse the cells. The lysate from each well was gathered and the radioactivity was assessed using a gamma counter.\u003c/p\u003e\n\u003ch3\u003eWestern Blot Assay\u003c/h3\u003e\n\u003cp\u003eWestern blotting was performed as previously described [13]. The primary antibodies used in this assay included anti-TfR1 (ab269531, Abcam, Cambridge, UK), anti-GPX4 (ab282533, Abcam), and anti-IL1\u0026beta; (ab315084, Abcam).\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eQuantitative Polymerase Chain Reaction (qPCR)\u003c/h2\u003e\n \u003cp\u003eAccording to earlier report [14], total RNA was extracted from tissues and cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer\u0026apos;s guidelines. Complementary DNA (cDNA) was generated using Reverse Transcription Kit from Takara (Dalian, China). qPCR was conducted using SmartCycler\u0026reg; II System(Cepheid Inc., Sunnyvale, CA, USA), with GAPDH used as internal reference gene .The relative quantification of mRNA transcripts was calculated using the 2\u003csup\u003e\u0026Delta;\u0026Delta;\u003c/sup\u003eCt method.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eLigature-Induced Periodontitis Model in Rats\u003c/h3\u003e\n\u003cp\u003eThirty specific-pathogen-free (SPF) male Sprague-Dawley rats (200\u0026ndash;300 g, 8 weeks old) were obtained from the Guangzhou Experimental Animal Center. All animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Jinan University (Approval No.20251209-04). Rats were weighed and anesthetized via continuous inhalation of 3% isoflurane using a small animal gas anesthesia system, followed by supine positioning. The periodontitis model was established based on previously described protocols [15, 16]. The rats were randomly assigned to five groups (n\u0026thinsp;=\u0026thinsp;6 per group): a control group (Con) consisting of untreated healthy rats; an experimental periodontitis group subjected to ligature-induced periodontitis for 7 days (EP7); an experimental periodontitis group subjected to ligature-induced periodontitis for 14 days (EP14); and two minocycline therapy groups treated for 7 days (MN7) and 14 days (MN14) post-ligation. For the MN7 and MN14 groups, 0.2 mg of minocycline hydrochloride ointment (Periocline, 2%; Sunstar, Japan) was locally applied into the gingival crevices once a week. Following the experimental period, rats were humanely euthanized via an intraperitoneal injection of sodium pentobarbital (150 mg/kg) to induce deep anesthesia. Upon confirmation of complete unconsciousness and loss of reflexes, cervical dislocation was performed as a secondary method to ensure humane termination.\u003c/p\u003e\n\u003ch3\u003eMicro-Computed Tomography (Micro-CT) Analysis\u003c/h3\u003e\n\u003cp\u003eFollowing euthanasia, the maxillae of the rats were dissected and scanned using a Venus Micro-CT scanner (90 kV, 80 mA) at a scanning resolution of 12 \u0026micro;m. The furcation region of maxillary second molar was designated as region of interest (ROI). Three-dimensional images were reconstructed using Cruiser and Recon software, and bone volume (BV) and tissue volume (TV) were measured using Avatar software to calculate the BV/TV fraction. Additionally, the linear distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) was measured at both the mesial and distal aspects of the maxillary second molar, and the average value was recorded.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003ePET/CT Imaging\u003c/h2\u003e\n \u003cp\u003eThe \u003csup\u003e68\u003c/sup\u003eGa-citrate tracer was purchased by the Atomic High-Tech Co., Ltd (Guangzhou, China). \u003csup\u003e18\u003c/sup\u003eF-NaF and \u003csup\u003e18\u003c/sup\u003eF-FDG were provided by the Department of Nuclear Medicine, The First Affiliated Hospital of Jinan University, with radiochemical purity\u0026thinsp;\u0026gt;\u0026thinsp;99%. The IRIS small-animal PET/CT imaging system (Inviscan SAS, Strasbourg, France) was utilized to conduct PET/CT imaging. During the scanning procedure, the rats were anesthetized with 3% isoflurane, and their body temperature was maintained at 37.5\u0026deg;C using a temperature regulation unit (Minerve \u0026eacute;quipement v\u0026eacute;t\u0026eacute;rinaire, Marne, France). For tracer injection and scanning: \u003csup\u003e68\u003c/sup\u003eGa-citrate PET/CT: After intravenous injection of \u003csup\u003e68\u003c/sup\u003eGa-citrate (15\u0026ndash;20 MBq) via the tail vein, a 10-minute static PET scan was acquired starting 45 minutes post-injection. \u003csup\u003e18\u003c/sup\u003eF-FDG PET/CT: After intravenous injection of \u003csup\u003e18\u003c/sup\u003eF-FDG (15\u0026ndash;20 MBq) via the tail vein, a 10-minute static PET scan was acquired starting 60 minutes post-injection. \u003csup\u003e18\u003c/sup\u003eF-NaF PET/CT: After intravenous injection of \u003csup\u003e18\u003c/sup\u003eF-NaF (15\u0026ndash;20 MBq) via the tail vein, a 10-minute static PET scan was acquired starting 60 minutes post-injection. The PET data were reconstructed utilizing 3D Ordered Subsets Expectation Maximization (3D-OSEM) algorithm that incorporates a Monte Carlo-based accurate detector model. Following the PET scan, a CT scan was conducted with the X-ray tube voltage set at 80 kV and the current at 0.9 mA. PET images were analyzed using PMOD 4.1 software (PMOD Technologies, Zurich, Switzerland). Bilateral regions of interest (ROIs) were manually delineated in the maxillary region, extending from the mesial aspect of the first molar to the distal aspect of the third molar. The average standardized uptake value (SUV) within the ROI was calculated.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e\n \u003cp\u003eImmunohistochemical staining for TfR1 and OCN was conducted on sections of paraffin-embedded maxilla. Sections were deparaffinized, rehydrated,and treated with 3% hydrogen peroxide for 25 minutes at room temperature. Following blocking with bovine serum albumin (BSA) for 30 minutesat room temperature, sections were incubated with primary antibodies against TfR1 (ab269531, 1:200; Abcam, Cambridge, UK) and OCN (GB115684- 100, 1:500; Servicebio,Wuhan, China) overnight at 4\u0026deg;C. Subsequently, the sections were treated with biotinylated secondary antibody (HRP-labeled goat anti-rabbit IgG, 1:200; Servicebio) for 50 minutes at room temperature. The slides were washed three times in PBS (pH 7.4 ) for 5 minutes each. A freshly prepared DAB chromogenic solution was applied for signal detection, followed by counterstaining with hematoxylin for 3 minutes. The sections were dehydrated in a sequential manner using a graded series of alcohol 75% for 5 minutes, 85% for 5 minutes, absolute ethanol twice for 5 minutes each, and n-butyl alcohol for 5 minutes, followed by a clearing step in xylene for 5 minutes. Ultimately, the sections were briefly air-dried and placed in a mounting medium. Images were taken with a light microscope (Leica DM6b, Nussloch, Germany) and analyzed through ImageJ software (NIH, Bethesda, MD, USA) .\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n \u003cp\u003eAll statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA). Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) derived from at least three independent experiments. Group comparisons were assessed using Student\u0026rsquo;s t-test (for two groups) or one-way analysis of variance (ANOVA) followed by appropriate post hoc tests (for multiple groups). A P-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eOsteoblast ferroptosis occurs under inflammatory conditions\u003c/h2\u003e\n \u003cp\u003eTo evaluate the impact of the inflammatory microenvironment on ferroptosis, MC3T3-E1 cells were incubated with LPS for 24 h, followed by the assessment of ferroptosis biomarkers. As demonstrated in Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, LPS dramatically reduced the viability of MC3T3-E1 cells in a dose-dependent manner, with a notable decrease seen at 10 \u0026micro;g/mL. Furthermore, the expression of TfR1 and IL1\u0026beta; gradually increased, whereas GPX4 expression decreased, in response to escalating LPS concentrations (0, 1, 5, and 10 \u0026micro;g/mL) (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). A consistent trend was observed in the mRNA transcription levels of TfR1, IL1\u0026beta;, and GPX4 across the corresponding LPS concentrations (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). At the level of 10 \u0026micro;g/mL LPS, cell ROS and LPS accumulation was increased (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e\u0026nbsp;\u003cstrong\u003e68\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eGa-citrate reflects osteoblast ferroptosis via TfR1\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eBecause \u003csup\u003e68\u003c/sup\u003eGa-citrate serves as an imaging surrogate for iron metabolism, and TfR1 is a pivotal protein in this pathway, we evaluated \u003csup\u003e68\u003c/sup\u003eGa-citrate cellular uptake following plasmid-mediated TfR1 knockdown (KD) in MC3T3-E1 cells, an additional figure file shows this in more detail [see Additional file 2]. Compared to cells treated with 10 \u0026micro;g/mL LPS alone, cells transfected with the TfR1-KD plasmid exhibited a significant reduction in TfR1 protein expression. Subsequent cellular uptake assays were performed at 30 and 60 minutes post-\u003csup\u003e68\u003c/sup\u003eGa-citrate administration. The tracer uptake rate was considerably higher in the LPS-treated group compared to the Con group at both time points. Conversely, the KD group demonstrated the lowest \u003csup\u003e68\u003c/sup\u003eGa-citrate uptake rate, confirming that the tracer\u0026apos;s accumulation is highly dependent on TfR1 expression (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAlveolar bone defects and inflammatory changes in the periodontitis animal model\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eA ligature-induced periodontitis rat model was developed to evaluate ferroptosis during inflammatory bone loss (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Clinical manifestations of periodontitis were evident by day 7 post-ligation (EP7), characterized by moderate inflammation of the gingival margin and interdental papillae, presenting with bright red coloration, mild edema, and bleeding upon probing confined to the gingival sulcus. By day 14 (EP14), the inflammation progressed to a severe state, exhibiting a brittle gingival texture, severe edema, and spontaneous bleeding spilling beyond the gingival sulcus (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Micro-CT analysis revealed a progressive trend of alveolar bone defects from EP7 to EP14, which was effectively attenuated by minocycline administration (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Histological evaluations confirmed the presence of inflammatory infiltration and alveolar bone loss, accompanied by suppressed osteogenesis, as indicated by low OCN expression (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Consistent with these findings, \u003csup\u003e18\u003c/sup\u003eF-FDG PET/CT demonstrated intense tracer accumulation in the alveolar region under periodontitis conditions, which was subsequently reduced following antibiotic intervention (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Furthermore, calcium metabolism was evaluated using \u003csup\u003e18\u003c/sup\u003eF-NaF PET/CT, a bone-seeking agent that binds to hydroxyapatite crystals [17]. The inflammatory environment induced a marked reduction in local calcium metabolism within the alveolar area, an effect that was partially reversed by minocycline treatment (Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e\u0026nbsp;\u003cstrong\u003e68\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eGa-citrate PET/CT visualizes TfR1 expression in vivo\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eIn the rat periodontitis model, both the transcription and protein expression of TfR1 increased progressively alongside disease exacerbation. Notably, this inflammation-induced TfR1 upregulation was significantly inhibited by minocycline administration at matched time points (Fig. \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In vivo PET/CT imaging acquired 45 minutes post-injection of \u003csup\u003e68\u003c/sup\u003eGa-citrate provided clear visualization of the maxillary bone, with minimal background uptake in the adjacent maxillary sinus and brain tissue. The uptake of \u003csup\u003e68\u003c/sup\u003eGa-citrate in the maxillae of ligature-induced periodontitis rats was markedly higher than that in the healthy control (Con) group. Quantitative analysis revealed that the mean standardized uptake values (SUV) in both the EP7 and EP14 groups were elevated compared to the Con group. Notably, a statistically significant increase in \u003csup\u003e68\u003c/sup\u003eGa-citrate SUV was observed only in the EP14 group compared to the control, whereas the EP7 group showed no significant alteration. Following minocycline intervention, the MN7 group exhibited a lower mean SUV than the EP7 group, although this trend did not reach statistical significance. In contrast, the MN14 group demonstrated a significant reduction in tracer uptake compared to the EP14 group(Fig. \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the inflammatory microenvironment of periodontitis, ferroptosis is driven by multiple pathways that elevate intracellular ROS levels [4, 18]. The dysregulated overexpression of TfR1 serves as a critical catalyst for cellular ferroptosis during periodontal inflammation. Using an LPS-induced osteoblast inflammation model, we demonstrated that LPS intervention dose-dependently increased ROS accumulation in MC3T3-E1 cells. Concurrently, the protein expression and mRNA transcription of TfR1 and IL1\u0026beta; were upregulated, whereas the anti-ferroptotic enzyme GPX4 was progressively downregulated. These findings align with previous literature [19], further corroborating TfR1 as a pivotal mediator in the ferroptotic cascade. Previous studies have evidenced that \u003csup\u003e68\u003c/sup\u003eGa-citrate, a radiotracer mimicking iron metabolism, is internalized by cells via the transferrin/TfR1 complex [20]. This mechanism provides a robust theoretical basis for utilizing \u003csup\u003e68\u003c/sup\u003eGa-citrate to visualize ferroptosis. In skeletal imaging, \u003csup\u003e68\u003c/sup\u003eGa-citrate has demonstrated superior specificity compared to conventional \u003csup\u003e18\u003c/sup\u003eF-FDG, effectively minimizing false-negative results [21] and accurately distinguishing physiological bone healing from true osteomyelitis [22, 23]. Furthermore, it exhibits broad potential in diagnosing infectious or inflammatory diseases across various organ systems [24]. In this study, we confirmed the target specificity of \u003csup\u003e68\u003c/sup\u003eGa-citrate through in vitro experimentation. Following plasmid-mediated TfR1 knockdown in MC3T3-E1 cells, the cellular uptake of \u003csup\u003e68\u003c/sup\u003eGa-citrate significantly decreased concurrently with TfR1 depletion, confirming its target-specific binding mechanism.\u003c/p\u003e\n\u003cp\u003eA key pathological characteristic of periodontitis is the gradual loss of alveolar bone [25. 26]. As periodontal inflammation exacerbates, the homeostasis of alveolar bone remodeling is severely disrupted. This process is driven by multiple mechanisms: immune cell activation and release of inflammatory mediators hyperactivate osteoclasts while suppressing osteoblast function[27]; the RANKL/RANK signaling axis further amplifies the osteoclastogenesis [28]; and inflammatory stimuli trigger the secretion of matrix metalloproteinases, which degrade extracellular matrix components and impair osteoblast attachment [29,30]. To replicate these clinical manifestations, a rat model of periodontitis was created using a ligature-induced method. Typical clinical symptoms, including gingival marginal inflammation, bright-red coloration, localized edema, and bleeding upon probing, were observed by day 7 post-ligation [31], and these symptoms significantly worsened by day 14. Micro-CT analysis revealed that the alveolar bone defects at day 14 (EP14) were significantly more severe compared to the control and day 7 (EP7) groups, as indicated by significant bone volume loss and a decrease in alveolar crest height. These in vivo phenotypes are highly consistent with the osteoblast impairment induced by LPS in vitro. Conversely, minocycline has proven efficacy in suppressing \u003cem\u003eP.gingivalis\u003c/em\u003e-induced inflammatory responses clinically and reducing bone abnormalities in animal models [32,33]. Histological analysis in our study demonstrated that as periodontitis progressed, inflammatory cell infiltration increased, while osteogenic activity (indicated by OCN deposition) markedly decreased. Following minocycline administration, both the inflammatory infiltration and the loss of calcium metabolism were significantly alleviated.\u003c/p\u003e\n\u003cp\u003eIn the context of translational molecular imaging, clinical practice traditionally utilizes technetium-99m methylene diphosphonate (\u003csup\u003e99m\u003c/sup\u003eTc-methylene) for labeling autologous leukocytes, alongside \u003csup\u003e18\u003c/sup\u003eF-FDG, \u003csup\u003e18\u003c/sup\u003eF-NaF, \u003csup\u003e67\u003c/sup\u003eGa, and \u003csup\u003e68\u003c/sup\u003eGa to visualize inflammatory diseases. Other novel targeted tracers, including 2-deoxy-2-[\u003csup\u003e18\u003c/sup\u003eF]fluoro-D-sorbitol(\u003csup\u003e18\u003c/sup\u003eF-FDS), [\u003csup\u003e18\u003c/sup\u003eF]fluoropropyl-aminomethylene diphosphonate (\u003csup\u003e18\u003c/sup\u003eF-FPTMP), and [\u003csup\u003e11\u003c/sup\u003eC]para-aminobenzoic acid (\u003csup\u003e11\u003c/sup\u003eC-PABA) also hold promise for characterizing specific microbial and inflammatory pathways [34\u0026ndash;38]. Regarding periodontitis, while \u003csup\u003e18\u003c/sup\u003eF-FDG PET/CT PET/CT can monitor therapeutic responses, its lack of target specificity limits its clinical expectations [39]. Meanwhile, \u003csup\u003e18\u003c/sup\u003eF-FDG PET/CT effectively tracks hydroxyapatite metabolism, indirectly reflecting alterations in periodontal bone mass. Our dual-tracer imaging using \u003csup\u003e18\u003c/sup\u003eF-FDG and \u003csup\u003e18\u003c/sup\u003eF-NaF successfully corroborated these findings, visualizing both the heightened inflammatory metabolic burst and the impaired bone mineral density.\u003c/p\u003e\n\u003cp\u003eMore importantly, immunohistochemical analysis confirmed that TfR1 expression in vivo escalated parallel to the severity of local inflammation and declined upon minocycline intervention. Given that \u003csup\u003e68\u003c/sup\u003eGa-citrate reliably reflects TfR1 expression in vitro, we pioneered the application of \u003csup\u003e68\u003c/sup\u003eGa-citrate PET/CT to evaluate periodontal ferroptosis. The in vivo imaging revealed a marked increase in \u003csup\u003e68\u003c/sup\u003eGa-citrate uptake within the maxillae of periodontitis rats. Interestingly, during the mild inflammatory stage, tracer uptake did not show statistically significant alterations compared to controls; marked tracer accumulation became evident only when the disease progressed to the severe stage. Following minocycline intervention, \u003csup\u003e68\u003c/sup\u003eGa-citrate PET/CT imaging captured a corresponding reduction in signal intensity, although the therapy could not completely normalize the elevated TfR1 expression to baseline levels. Collectively, these findings underscore TfR1 as the key molecular bridge linking ferroptosis to \u003csup\u003e68\u003c/sup\u003eGa-citrate accumulation. Through targeting TfR1, \u003csup\u003e68\u003c/sup\u003eGa-citrate PET/CT successfully visualizes the dynamic burden of ferroptosis driven by inflammation, offering a novel and non-invasive functional imaging strategy for evaluating osteoblast ferroptosis during periodontitis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, osteoblast exhibited increased \u003csup\u003e68\u003c/sup\u003eGa-citrate uptake under inflammatory conditions, and \u003csup\u003e68\u003c/sup\u003eGa-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. Therefore, this study suggested that \u003csup\u003e68\u003c/sup\u003eGa-citrate PET/CT visualized periodontitis ferroptosis by reflecting TfR1 expression.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eTfR1: Transferrin Receptor 1\u003c/p\u003e\n\u003cp\u003eGallium-68 citrate:\u003csup\u003e68\u003c/sup\u003eGa-citrate\u003c/p\u003e\n\u003cp\u003ePET/CT: positron emission tomography/computed tomography\u003c/p\u003e\n\u003cp\u003eGPX4: Glutathione Peroxidase 4\u003c/p\u003e\n\u003cp\u003eIL1\u0026beta;: interleukin 1\u0026beta;\u003c/p\u003e\n\u003cp\u003eOCN: Osteocalcin\u003c/p\u003e\n\u003cp\u003eLPS : Lipopolysaccharide\u003c/p\u003e\n\u003cp\u003eABC: Alveolar Bone Crest\u003c/p\u003e\n\u003cp\u003eBV: Bone Volume\u003c/p\u003e\n\u003cp\u003eCEJ: Cement-Enamel Junction\u003c/p\u003e\n\u003cp\u003eCT: Computed Tomography\u003c/p\u003e\n\u003cp\u003eFDG: Fludeoxyglucose\u003c/p\u003e\n\u003cp\u003eGBI: Gingival Bleeding Index\u003c/p\u003e\n\u003cp\u003eHE: Hematoxylin and eosin\u003c/p\u003e\n\u003cp\u003eLPO: Lipid peroxide\u003c/p\u003e\n\u003cp\u003eP.g: Porphyromonas gingivalis\u003c/p\u003e\n\u003cp\u003eROI : Region Of Interest\u003c/p\u003e\n\u003cp\u003eROS: Reactive Oxygen Species\u003c/p\u003e\n\u003cp\u003eSPECT: Single Photon Emission Computed Tomography\u003c/p\u003e\n\u003cp\u003eSUV: Standardized Uptake Value\u003c/p\u003e\n\u003cp\u003eTF: Transferrin\u003c/p\u003e\n\u003cp\u003e\u0026zwnj;TV: Tissue Volume\u003c/p\u003e\n\u003cp\u003eKD: Knockown\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eAll animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Jinan University (Approval No.20251209-04).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported by grants from Science research cultivation program of stomatological hospital, Southern medical university (PY2023042) in China.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLiu Y and Li Y carried out the experiments, performed the analysis and drafted the manuscript. Ran B, Cao X, Cai Q, Cheng Y, Guo B and Gong J helped with data acquisition and analysis. Shang J and Hao X supervised the study, critically revised the manuscript and provided funding support. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eData and materials were provided within the manuscript and supplementary material files\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone to declare\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eEduardo B, Wagner M, Rizwan S, Lucas GA, Saira A, Fadwa NA, et al. Trends in the global, regional, and national burden of oral conditions from 1990 to 2021: a systematic analysis for the Global Burden of Disease Study 2021. Lancet. 2025;405(10482):897-910.\u003c/li\u003e\n \u003cli\u003eHajishengallis G, Chavakis T. 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Oral Dis. 2024;30(7):4630-38.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"68Ga-citrate, periodontitis, ferroptosis, TfR1, molecular imaging","lastPublishedDoi":"10.21203/rs.3.rs-9333859/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9333859/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe loss of alveolar bone in periodontitis is linked to ferroptosis mediated by Transferrin Receptor 1 (TfR1). This study investigates the utility of gallium-68 citrate (\u003csup\u003e68\u003c/sup\u003eGa-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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMedthos\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMC3T3-E1 cells were stimulated with Porphyromonas gingivalis lipopolysaccharide (LPS) to model the inflammatory microenvironment. The correlation between TfR1 expression and \u003csup\u003e68\u003c/sup\u003eGa-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 the periodontal tissues. A rat model of ligature-induced periodontitis was established, with minocycline serving as a therapeutic intervention. Periodontal inflammation and bone metabolism were longitudinally evaluated using multi-modal PET/CT \u003csup\u003e68\u003c/sup\u003eGa-citrate, 2-deoxy-2-[\u003csup\u003e18\u003c/sup\u003eF]fluoro-D-glucose(\u003csup\u003e18\u003c/sup\u003eF-FDG), and[\u003csup\u003e18\u003c/sup\u003eF]sodium fluoride(\u003csup\u003e18\u003c/sup\u003eF-NaF), complemented by micro-computed tomography (micro-CT).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn vitro, LPS dose-dependently reduced MC3T3-E1 viability while upregulating TfR1 and IL1β, and downregulating GPX4 expression. \u003csup\u003e68\u003c/sup\u003eGa-citrate uptake correlated positively with TfR1 expression and was significantly inhibited by TfR1 knockdown. In vivo, \u003csup\u003e68\u003c/sup\u003eGa-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 \u003csup\u003e68\u003c/sup\u003eGa-citrate SUV compared to periodontitis groups.The presence of active inflammation and impaired calcium metabolism were confirmed by conventional tracers \u003csup\u003e18\u003c/sup\u003eF-FDG and \u003csup\u003e18\u003c/sup\u003eF-NaF PET/CT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOsteoblast exhibited increased \u003csup\u003e68\u003c/sup\u003eGa-citrate uptake under inflammatory conditions, and \u003csup\u003e68\u003c/sup\u003eGa-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. Therefore, this study suggested that \u003csup\u003e68\u003c/sup\u003eGa-citrate PET/CT visualized periodontitis ferroptosis by reflecting TfR1 expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial Number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e","manuscriptTitle":"68Ga-citrate visualization study on ferroptosis through Transferrin Receptor 1 in periodontitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-07 20:14:15","doi":"10.21203/rs.3.rs-9333859/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"23114677100271150202633247265747152174","date":"2026-05-17T18:37:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-23T12:27:16+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-20T18:08:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-20T05:24:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-16T12:59:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Oral Health","date":"2026-04-16T12:38:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8a629768-0d78-44c1-8559-f9396b50df1e","owner":[],"postedDate":"May 7th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"23114677100271150202633247265747152174","date":"2026-05-17T18:37:17+00:00","index":74,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-07T20:14:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-07 20:14:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9333859","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9333859","identity":"rs-9333859","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}