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Lipid droplet accumulation impairs osseointegration by disturbing the osteogenesis-osteoclasis balance on titanium implant surface in hyperlipidemia

BMC Oral Health volume 25, Article number: 823 (2025) Cite this article

Abstract

Background

Effective osseointegration is a crucial factor for the success of implant restoration, and hyperlipidemia significantly impairs osseointegration. However, the underlying in vivo mechanisms remain unclear. This study aimed to investigate the impact and molecular mechanisms of lipid droplet accumulation on osteogenesis-osteoclasis balance on titanium (Ti) implant surface.

Methods

In vivo, hyperlipidemia mice receiving local injections of Wnt3a or N-acetyl-L-cysteine (NAC) in peri-implant tissue were used to assess osseointegration, lipid droplet accumulation, and osteogenesis-osteoclasis balance by Micro-CT, hard tissue slicing and staining, H&E staining, TRAP staining, as well as immunofluorescence (IF) staining. The in situ oxidative damage of cells obtained from the extracted implant surfaces was detected by transmission electron microscopy and enzyme-linked immunosorbent assay. In vitro, bone marrow mesenchymal stem cells (BMMSCs) were cultured on Ti sheet with intermittent treatment of NAC. The relationship between lipid droplets and oxidative damage was detected by IF staining of BODIPY, GRP78, DCFH-DA, EdU, and the quantitative assay of GSH/GSSG, MDA, NAD+/NADH, ATP.

Results

Hyperlipidemia disrupted osseointegration via downregulating the osteogenesis-related ROS/Wnt/β-catenin pathway and simultaneously upregulating the osteoclasis-related ROS/RANKL/NF-κB pathway. NAC application alleviated endoplasmic reticulum stress and mitochondrial dysfunction but failed to reduce lipid droplet accumulation. The intracellular oxidative damage is positively correlated with the accumulation of lipid droplets induced by hyperlipidemia.

Conclusion

Lipid droplet accumulation is the primary cause for inducing oxidative damage, disturbing osteogenesis-osteoclasis balance, and impairing osseointegration in hyperlipidemia. This study highlights that reducing lipid droplet accumulation may serve as a potential strategy to enhance Ti implant osseointegration in hyperlipidemia.

Peer Review reports

Background

The osseointegration of titanium (Ti) implants is the prerequisite for achieving ideal goals in dental implantation [1, 2]. Recent clinical and basic research indicated that hyperlipidemia, one of the most common systemic metabolic disease, seriously impaired the osseointegration of Ti implants [3,4,5,6]. However, the detailed mechanisms by which hyperlipidemia impaired implant osseointegration remain unclear.

Lipid droplets are composed of phospholipid monolayer and neutral hydrophobic lipid cores, serving as a hub for coordinating intracellular lipid uptake, distribution, storage, and utilization [7], as well as an important component for maintaining energy and redox homeostasis [8]. Latest research indicated that lipid droplets play an essential role in controlling the osteogenesis-osteoclasis balance in bone metabolism [9, 10]. Notably, the osseointegration process of Ti implants is constantly influenced by both osteogenesis and osteoclasis [11, 12]. From the perspective of osteogenesis, high-fat diet delayed bone regeneration of mandibular bone defect and the lingual alveolar ridge height prominently reduced around the tooth extraction wounds in hyperlipidemia mice [13]. Our previous in vitro studies also revealed that high-fat culture environment inhibited Wnt/β-catenin signaling pathway by upregulating intracellular reactive oxygen species (ROS) level, and severely damaged the biological activity of osteoblasts [14]. With respect to osteoclasis, Tintut Y et al. suggested that high-fat diet and inflammatory bioactive lipid could promote osteoclasts differentiation by inducing the production of receptor activator of nuclear factor-κ B ligand (RANKL) [15,16,17]. In addition, the osteoclast number on the surface of trabecular bone in proximal tibiae significantly increased in C57BL/6 male mice with high-fat diet [18]. Nevertheless, how lipid droplet accumulation disrupts the physiological osteogenesis-osteoclasis balance on the surface of titanium implants remains to be elucidated.

Recent researches indicated that hyperlipidemia cause excessive accumulation of intracellular lipid droplets, leading to abnormal autophagy function, lipid peroxidation, upregulation of ROS levels, and eventually cellular dysfunction [19]. Systemic and intracellular oxidant stress is implicated in mediating the effects of hyperlipidemia on osteogenesis-osteoclasis imbalance [20, 21]. Endoplasmic reticulum stress (ERS) and mitochondrial dysfunction were important cellular responses to external oxidative damage. The ERS caused by high-fat environment significantly exacerbates the progression of diseases by affecting the normal folding and expression of proteins [22]. Targeted alleviation of ERS would effectively promotes cartilage protection by regulating immune responses and reducing inflammation levels [23]. On the contrary, permanently uncontrolled ERS could stimulate osteoblast apoptosis and accelerate osteoclasis [24, 25]. Besides, the mitochondrial dysfunction caused by hyperlipidemia is closely related to fatty liver [26], acute pancreatitis [27], endothelial damage [28], and other diseases. Physiologically restoring the function of mitochondria could provide an effective therapy for bone diseases, such as periodontitis and osteoarthritis [29]. Thus, the specific role of ERS and mitochondrial dysfunction in lipid droplet accumulation-induced osteogenesis-osteoclasis imbalance on Ti implant surface is worthy of further investigation.

Therefore, the present study aimed to investigate: (1) the influence of hyperlipidemia on osteogenesis-osteoclasis balance on Ti implant surface; (2) the role of oxidant stress, especially the ERS and mitochondrial dysfunction in mediating osteogenesis-osteoclasis imbalance around Ti implants under hyperlipidemia condition; (3) whether the lipid droplets were the arch-criminal of hyperlipidemia induced oxidative damage, osteogenesis-osteoclasis imbalance and eventually impaired osseointegration under hyperlipidemia condition.

Methods

Preparation of titanium (Ti) implants and Ti plates

The methods for preparing Ti implants and Ti plates with sandblasted and acid-etched (SLA) surface were consistent with the previous literature description [14]. The Ti cylinders were used in vivo and the Ti circular sheets were utilized in vitro.

Establishment of hyperlipidemia mice and animal implant surgery

Seventy two 5-weeks aged male C57BL/6J mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd, China) were raised in Experimental Animal Center of Shandong University. The animal experimental procedures were approved by the Medical Ethics Committee of Stomatological Hospital, Shandong University, Jinan, China (Permit Number: No. 20200903) and were carried out following the ARRIVE Guidelines.

Animal experiments were divided into the following two parts.

The first part of animal experiments was designed as follows. Mice were randomly allocated to two groups: (1) the C57BL/6J normal diet group (the NC group, n = 12); (2) the C57BL/6J high-fat diet group (the HF group, n = 12). After 30 days of high-fat diet, six mice were randomly selected from each experimental group to obtain serum samples. Body weight, total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and glucose (GLU) were tested. Then, Ti implants were implanted into the distal end of bilateral femurs of the remaining mice in each experimental group, as we previously described [14, 30]. During the surgical procedure, mice were continuously anesthetized with low-flow isoflurane inhalation. The corresponding diet of each group were given throughout the experiment and mice were then euthanized using CO2 sequentially 30 days after implantation, as shown in Fig. 1A.

The second part of animal experiment was conducted as designed in Fig. 2A. Mice were randomly allocated to four groups: (1) the C57BL/6J normal diet group (the NC group, n = 12); (2) the C57BL/6J high-fat diet group (the HF group, n = 12); (3) the C57BL/6J high-fat diet with Wnt3a treatment group (the HF + Wnt3a group, n = 12); (4) the C57BL/6J high-fat diet with N-acetyl-L-cysteine (NAC) treatment group (the HF + NAC group, n = 12). After 30 days of high-fat diet, six mice were randomly selected from each experimental group to obtain serum samples. Body weight, urea nitrogen (UREA), uric acid (UA), creatinine (CREA), TC, TG, LDL-C, HDL-C, and GLU were tested. Then, Ti implants were implanted into the distal end of bilateral femurs of the remaining mice in each experimental group. Isoflurane anesthesia was administered continuously at a low flow rate to maintain sedation during the surgical intervention. After implantation, 200 µL volume of 1 µg/mL Wnt3a and 10 mmol/L NAC solution were injected into the knee joint capsule every 3 days in the HF + Wnt3a and HF + NAC groups respectively, and the corresponding diets of each group were given throughout the experiment. Furthermore, to investigate whether local injection of Wnt3a and NAC has biological toxicity to liver and kidney of mice, the paraffin tissue sections of the liver and kidney of mice were obtained after gradient ethanol dehydration and paraffin embedding. The detailed steps of H&E staining were consistent with the descriptions in previous literatures [31, 32].

Micro-computed tomography (Micro-CT) analysis

Micro-CT (Quantum GX2, PerkinElmer, Japan, located in Translational Medicine Core Facility of Advanced Medical Research Institute, Shandong University) was used to evaluate the osseointegration of the implants (90 kV, 160 µA) 30 days after implantation. The region of interest (ROI) and the 3D vertical reconstruction scope was consistent with the description in our previous literature [14]. In addition, bone volume fraction (bone volume/tissue volume, BV/TV), trabecular number (Tb. N), trabecular thickness (Tb. Th) and trabecular separation (Tb. Sp) in ROI were analyzed by CTAN software.

Hard tissue slicing and staining

The femoral with the implant was fixed, dehydrated and then embedded in a light-cured resin. 50 µM-thick slices were prepared along the coronal plane of the femur sample with the implant and stained with methylene blue-acid fuchsin. The panoramic scanning and image analysis system for high definition pathological section (VS120, located in Translational Medicine Core Facility of Advanced Medical Research Institute, Shandong University) were used to record images and calculate the bone-implant combination ratio (BIC%), which was defined as the length percentage of direct bone-implant interface to total implant circumference.

H&E and TRAP staining of paraffin tissue sections

The femoral paraffin tissue sections were obtained after decalcification of bone tissue, pulling out the Ti implants, gradient ethanol dehydration, and paraffin embedding. The detailed steps of H&E and TRAP staining were consistent with the descriptions in previous literatures [31, 32].

Immunofluorescence (IF) staining of paraffin tissue sections

The detailed steps of IF staining were consistent with the descriptions in the previous literature [31]. Primary antibodies against 8-hydroxy-2 deoxyguanosine (8-OHdG, Santa Cruz Biotechnology, sc-393871, 1:300, USA), non-phospho-β-catenin (non-p-β-catenin, CST, #8814, 1:800, USA), alkaline phosphatase (ALP, HUABIO, ET1601-21, 1:100, China), receptor activator of nuclear factor-κ B ligand (RANKL, Proteintech, 23408-1-AP, 1:300, China), receptor activator of nuclear factor-κ B ligand (RANKL, Santa Cruz Biotechnology, sc-59982, 1:300, USA), nuclear factor-kappa B (NF-κB, Proteintech, 10745-1-AP, 1:300, China), inhibitor of NF-κB (I-κB, Proteintech, 10268-1-AP, 1:300, China), CD11b (Proteintech, 65055-1-Ig, 1:300, China), F4/80 (Santa Cruz Biotechnology, sc-52664, 1:300, USA), Gr-1 (Santa Cruz Biotechnology, sc-53515, 1:300, USA), glucose-regulated protein 78 (GRP78, Proteintech, 11587-1-AP, 1:300, China), perilipin2 (PLIN2, Proteintech, 15294-1-AP, 1:300, China) were applied. Briefly, IF staining for these markers was performed, and all these antibodies mentioned above were incubated at their respective labeled optimal dilution concentrations. Cell nuclei were further stained with 4’, 6-Diamidino-2-Phenylindole (DAPI, Solarbio, China). Moreover, images were obtained under a fluorescence microscope (Leica, Germany). The mean fluorescence intensity ratio was analyzed by Image J software (National Institutes of Health, Bethesda, MD, USA). All the immunofluorescence staining images are obtained and analyzed through the above instruments and software, unless otherwise specified.

Enzyme-linked immunosorbent assay (ELISA)

Mice were euthanatized 30 days after implantation and the femoral specimens with implant were obtained. The implant was pulled out immediately. The 5 mm length distal femur tissue surrounding the implant was grinded thoroughly, lysed with RIPA (Solarbio, China) at 4 °C for 30 min and centrifuged 12,000 rpm for 30 min to obtain tissue protein solution as described by Wu et al. for further ELISA tests [33]. The concentration of interleukin-1β (IL-1β, Multi Sciences, China), interleukin-6 (IL-6, Multi Sciences, China), tumor necrosis factor-α (TNF-α, Multi Sciences, China), interleukin-17 A (IL-17 A, Multi Sciences, China), RANKL (Multi Sciences, China), and osteoprotegerin (OPG, mlbio, China) were tested according to the manufacturers’ instructions.

Meanwhile, the cells on Ti implants surface were acquired by trypsin (Biosharp, China) digestion after the implants were pulled out. After cell lysis, the supernatant were obtained for ELISA test. The concentration of GRP78 (mlbio, China), inositol-requiring enzyme 1 (IRE1, mlbio, China), X-box binding protein 1 (XBP1, mlbio, China), C/EBP homologous protein (CHOP, YISHENYUAN, China), activating transcription factor 6 (ATF6, SAIPEISEN BIOLOGY, China), and protein kinase RNA-like endoplasmic reticulum kinase (PERK, mlbio, China) were tested.

Transmission electron microscopy (TEM)

Mice were euthanatized 30 days after implantation and the femoral specimens with implant were obtained. The implant was pulled out immediately. The cells on Ti implants surface were acquired by trypsin (Biosharp, China) digestion after the implants were pulled out. After fixation of 2.5% glutaraldehyde (Solarbio, China) at 4℃ overnight, intracellular lipid droplets and typical organelles were observed by TEM as described in the previous literature [19].

Cell isolation and culture

Bone marrow mesenchymal stem cells (BMMSCs) were isolated from the femoral bone marrow of 8-week age male C57BL/6J mice as described in the previous literature [34] and were cultured in dulbecco’s modified eagle medium (DMEM, Biological Industries, Israel) supplemented with 10% fetal bovine serum (FBS, Biological Industries, Israel) at 37℃ with 5% CO2.

Measurement of the lipid droplet accumulation, endoplasmic reticulum stress (ERS), reactive oxygen species (ROS) level, and proliferation in BMMSCs on Ti surface

BMMSCs were incubated on the surface of Ti sheets for 7 days as designed in Fig. 7A. In the NC group, cells were cultured in DMEM containing 10% FBS for 7 days. In the HF group, cells were cultured in DMEM supplemented with 10% FBS, 500 µmol oleic acid and 500 µmol palmitic acid for 7 days. In the HF + NAC group, cells were cultured in the HF medium mentioned above for 3 days, HF medium supplemented with 2 mmol/L NAC for 2 days and HF medium for 2 days, respectively. The boron-dipyrromethene (Bodipy) staining (Thermo Fisher Scientific, USA) was used to detect the lipid droplet accumulation according to the previous literature [31]. The IF staining of GRP78 (Proteintech, 11587-1-AP, 1:500, China) was applied to evaluate the ERS. The 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) staining was performed to assess the intracellular total ROS level according to the methods reported in a previous literature [14]. The 5-ethynyl-2’-deoxyuridine (EdU) staining was used to evaluate the proliferation of BMMSCs based on the manufacturer’s instructions of the EdU staining kit (Ribobio, China). The above staining tests were conducted on Day 3, 5, and 7, respectively.

Detection of the glutathione (GSH) proportion, malondialdehyde (MDA) concentration, NAD+/NADH ratio and adenosine triphosphate (ATP) concentration in BMMSCs on Ti surface

BMMSCs were incubated on the surface of Ti sheets for 7 days as designed in Fig. 7A. At Day 3, 5, and 7, the cells on Ti implants surface were acquired with 0.25% trypsin (Biosharp, China) digestion and centrifugation. The GSH proportion (Beyotime Biotechnology, China), MDA concentration (Nanjing Jiancheng Bioengineering Institute, China), NAD+/NADH ratio (Beyotime Biotechnology, China) and ATP concentration (Beyotime Biotechnology, China) were detected according to manufacturer’s instructions.

Statistical analysis

All data were presented as the mean ± standard deviation (SD) of at least three independent experiments. Normal distribution of the data was assessed by the Shapiro-Wilk test. For data normally distributed, a t-test was used for comparison between two groups, and one-way ANOVA was used to analyze significant group-to-group differences using GraphPad Prism 6.0 software. Nonparametric tests were used for not normally distributed data. The P value less than 0.05 was considered to be statistically significant.

Results

Establishment of the femur implant model in hyperlipidemia mice and its effect on Ti implant surface

The hyperlipidemia mice femur implant model was successfully established as designed in Fig. 1A. As shown in Fig. 1B-F, the weight and the concentration of total cholesterol (TC), triglycerides (TG), as well as high-density lipoprotein cholesterol (HDL-C) of mice in the high-fat (HF) group was higher than mice in the negative control (NC) group after high-fat diet for 30 days. The concentration of glucose (GLU) in the HF group had no significant difference compared to the NC group (Fig. 1G). The above results suggested that the hyperlipidemia mice model without hyperglycemia had been successfully established.

No animal died or other adverse events occurred before sacrifice. After Ti implants were implanted for 30 days, mice were sacrificed and the femoral tissue specimens were obtained. Considering the critical role of oxidative stress and the Wnt/β-catenin pathway in high-fat environment in our previous in vitro study, immunofluorescence (IF) staining was used to detect the expression of 8-hydroxy-2 deoxyguanosine (8-OHdG, a marker of oxidative damage) and non-phospho-β-catenin (non-p-β-catenin, the activated form of β-catenin and the characteristic protein of activated Wnt/β-catenin pathway) around the implants in normal or diet-induced hyperlipidemia mice. As shown in Fig. 1H and I, the fluorescence intensity of 8-OHdG around the implants in the HF group was 6 times higher than that in the NC group, and the expression of non-p-β-catenin in the HF group was less than one-third of that in the NC group. These observations highlighted that hyperlipidemia induced oxidative damage and inhibited the Wnt/β-catenin signaling pathway on Ti implant surface, in vivo.

Fig. 1
figure 1

Evaluation of the levels of oxidative damage and Wnt/β-catenin pathway around the implants in hyperlipidemia mice. (A) Illustration of the first part of animal experimental design. (B-G) Various physiological and biochemical indicators in NC and HF group. (H) Fluorescent images of the nucleus (blue), 8-OHdG (green), and non-p-β-catenin (red) around the titanium implants in NC and HF group. (I) Quantitative analysis of mean fluorescence intensity of 8-OHdG and non-p-β-catenin. Data was presented as mean ± SD, n = 3 specimens/group, *P < 0.05. NC: negative control; HF: high-fat; TC, total cholesterol; TG, triglycerides; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; GLU, glucose; IM: implant; DAPI, 4’, 6-diamidino-2-phenylindole; 8-OHdG, 8-hydroxy-2 deoxyguanosine; non-p-β-catenin: non-phospho-β-catenin

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Hyperlipidemia impaired osseointegration through the osteogenesis-related ROS/Wnt/β-catenin signaling pathway in vivo

In order to further investigate the effects of the osteogenesis-related ROS/Wnt/β-catenin pathway on osseointegration under hyperlipidemia conditions, the hyperlipidemia mice model with local injection of Wnt3a (the agonist of the Wnt/β-catenin pathway) or N-acetyl-L-cysteine (NAC, the ROS antagonist) around Ti implants was established as shown in Fig. 2A. H&E staining of liver paraffin sections revealed mild lipid vacuole accumulation in the HF group, indicating high-fat diet-induced lipid metabolism dysregulation. However, compared to the NC group, hepatocyte morphology remained normal, with no pathological changes such as necrosis, inflammatory infiltration, or fibrosis. No significant differences were observed between the HF + Wnt3a or HF + NAC groups and the HF group. In kidney sections, the HF group exhibited intact glomerular and tubular structures without obvious edema, degeneration, or inflammatory cell infiltration compared to the NC group. Similarly, the HF + Wnt3a and HF + NAC groups showed no notable alterations relative to the HF group (Fig. 2C). The concentration of urea nitrogen (UREA), creatinine (CREA), and uric acid (UA) also suggested no significant difference among four groups (Fig. 2D-F). Combining with the tested biochemical indicators (Fig. 2G-K), the above data confirmed the successful establishment of hyperlipidemia mice model and the biosafety of local injection of Wnt3a or NAC.

Fig. 2
figure 2

Comprehensive evaluation of hyperlipidemia mice model with local injection of Wnt3a or NAC. (A) Illustration of the second part of animal experimental design. (B) The weight of mice in each experimental group. (C) H&E staining of the liver and kidney. (D-K) Various physiological and biochemical indicators in each experimental group. Data was presented as mean ± SD, n = 3 specimens/group, *P < 0.05. NC: negative control; HF: high-fat; Wnt3a, an activator of Wnt/β-catenin pathway; NAC: N-acetyl-L-cysteine, a ROS antagonist; Ti, titanium; UREA, urea nitrogen; CREA, creatinine; UA, uric acid; GLU, glucose; TC, total cholesterol; TG, triglycerides; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol

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Microscopic computerized tomography (Micro-CT), hard tissue section staining, and H&E staining were used to evaluate the implant osseointegration. As shown in Fig. 3A and B, the peri-implant bone trabecular structure in the HF group was sparser than that in the NC group. After local administration of Wnt3a and NAC, bone volume (BV) / tissue volume (TV) showed 20.1% and 26.1% increase separately compared to the HF group, although there was no statistical difference between the HF group and the HF + Wnt3a group. Furthermore, the number and thickness of bone trabeculae (TB. N and Tb. Th) showed similar trends, while the space gap between bone trabeculae (Tb. Sp) showed the opposite trend. The results of hard tissue section staining showed that the osseointegration in the Wnt3a and NAC group were dramatically ameliorated compared with the HF group, which was consistent with the results of Micro-CT (Fig. 3C). More fibrous tissue was observed around the implants in the HF group and the percent of bone-implant contact (BIC%) around the implants in the HF + Wnt3a group and HF + NAC group was significantly higher than that in the HF group (Fig. 3D and E). The above results indicated that local application of NAC and Wnt3a significantly promoted osseointegration in hyperlipidemia mice.

Fig. 3
figure 3

Evaluation of titanium implant osseointegration. (A-B) 3D reconstructed images and quantitative calculation of BV/TV, TB.N, Tb.Th, and Tb.Sp by Micro-CT at 4 weeks after surgery. (C) Hard tissue slicing images of the bone-implant interface. (D) Typical images of H&E staining showing bone-implant interface in different groups. (E) Quantitative calculation of BIC%. Data was presented as mean ± SD, n = 3 specimens/group, *P < 0.05. NC: negative control; HF: high-fat; Wnt3a, an activator of Wnt/β-catenin pathway; NAC: N-acetyl-L-cysteine, a ROS antagonist; BV/TV, bone volume/tissue volume; TB.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; IM: implant; BIC%, percent of bone-implant contact

Full size image

Furthermore, IF staining was used to detect the expression levels of 8-OHdG, non-p-β-catenin as well as alkaline phosphatase (ALP) in peri-implant tissue to clarify the relationship between oxidative damage and the Wnt/β-catenin pathway. As shown in Fig. 4, the mean fluorescence intensity of 8-OHdG in peri-implant tissues in the HF + NAC group was lower than that in the HF group, however, there was no significant difference between the HF + Wnt3a group and the HF group, which indicated that NAC suppressed oxidative stress in peri-implant tissue in hyperlipidemia mice, whereas Wnt3a could not. Moreover, the mean fluorescence intensity of non-p-β-catenin in peri-implant tissues were elevated in both the HF + NAC group and the HF + Wnt3a group than that in the HF group, which suggested that ROS clearance would reactivate the Wnt/β-catenin pathway.

Fig. 4
figure 4

Evaluation of the levels of oxidative damage, Wnt/β-catenin pathway, and osteogenesis around the implants after local injection of Wnt3a or NAC in hyperlipidemia mice. (A) Fluorescent images of the nucleus (blue), 8-OHdG (green) and non-p-β-catenin (red). (B) Quantitative analysis of mean fluorescence intensity of 8-OHdG and non-p-β-catenin. (C) Fluorescent images of the nucleus (blue), 8-OHdG (green) and ALP (red). (D) Quantitative analysis of mean fluorescence intensity of 8-OHdG and ALP. Data was presented as mean ± SD, n = 3 specimens/group, *P < 0.05. NC: negative control; HF: high-fat; Wnt3a, an activator of Wnt/β-catenin pathway; NAC: N-acetyl-L-cysteine, a ROS antagonist; IM: implant; DAPI, 4’, 6-diamidino-2-phenylindole; 8-OHdG, 8-hydroxy-2 deoxyguanosine; non-p-β-catenin: non-phospho-β-catenin; ALP, alkaline phosphatase

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Wnt3a is an activator of Wnt/β-catenin signaling, while NAC serves as a ROS antagonist. The Wnt/β-catenin signaling pathway represents a crucial osteogenesis-related pathway, whose proper activation is essential for maintaining osteogenic activity. ROS overproduction not only inhibits osteogenesis but also activates the osteoclasis-related RANKL/NF-κB pathway. Therefore, local treatment with Wnt3a or NAC proves crucial for clarifying the importance of osteogenesis-osteoclasis balance in osseointegration in hyperlipidemia. These above in vivo experimental results suggested that hyperlipidemia inhibited Ti implants osseointegration through osteogenesis-related ROS/Wnt/β-catenin signaling pathway.

NAC inhibited osteoclastic activity around the implant caused by oxidative damage under hyperlipidemia conditions

Apart from osteogenesis, the changes of osteoclastic activity after NAC administration under hyperlipidemia conditions were further explored. Compared with the HF group, NAC decreased the number of TRAP positive cells and howship’s lacunae around the implant significantly (Fig. 5A and B), which indicated that osteoclastic activity also played an essential role in the mechanisms of which downregulating ROS level exhibited better improvements on osseointegration under hyperlipidemia conditions.

Additionally, as precursor cells of osteoclasts, the number of mononuclear macrophages and bone marrow-derived suppressor cells (BMDSCs) in the surrounding tissue of the implant was detected using IF staining. As shown in Appendix Fig. 1C, D and E, the number of mononuclear macrophages (CD11b+ F4/80+ cells) in the NAC group was highly less than that in the HF and Wnt3a group (4 ± 1 vs. 11 ± 1 cells per section and 4 ± 1 vs. 9 ± 1 cells per section), which indicated that NAC reduced the recruitment of mononuclear macrophages in peri-implant tissue. Furthermore, the number of BMDSCs (CD11b+ Gr-1+ cells) in the NAC group was much more than that in the HF and Wnt3a group (13 ± 2 vs. 4 ± 1 cells per section and 13 ± 2 vs. 5 ± 1 cells per section), which illustrated that local application of NAC effectively inhibited the activation of osteoclasts induced by hyperlipidemia.

RANKL induced nuclear factor-kappa B (NF-κB) signaling pathway is one of the most critical regulatory mechanism of osteoclast activation. Results of IF staining and enzyme-linked immunosorbent assay (ELISA) detection suggested that the expression of RANKL and NF-κB dramatically decreased while the expression of inhibitor of NF-κB (I-κB) and osteoprotegerin (OPG) significantly increased when the oxidative damage was cutting down in the NAC group, compared with the HF group (Fig. 5C and H, Appendix Fig. 1F). Specifically, local NAC treatment suppressed the RANKL/NF-κB signaling pathway by reducing oxidative damage in peri-implant tissues, thereby inhibiting osteoclast activation and osteoclasis.

So far, we have proved that oxidative damage seriously impaired osteogenesis and promoted osteoclasis around the implants in hyperlipidemia conditions, which could be reversed by NAC. However, the arch-criminal of hyperlipidemia-induced oxidative damage still needs to be explored.

Fig. 5
figure 5

Evaluation of the levels of osteoclastogenesis, oxidative damage, and RANKL/NF-κB pathway around the implants after local injection of Wnt3a or NAC in hyperlipidemia mice. (A) Typical images of TRAP staining. (B) Quantitative analysis of TRAP positive cells and bone lacunae. (C) Fluorescent images of the nucleus (blue), 8-OHdG (green), and RANKL (red). (D) Quantitative analysis of mean fluorescence intensity of 8-OHdG and RANKL. (E) Fluorescent images of the nucleus (blue), RANKL (green), and NF-κB (red). (F) Quantitative analysis of mean fluorescence intensity of RANKL and NF-κB. (G) Fluorescent images of the nucleus (blue), RANKL (green), and I-κB (red). (H) Quantitative analysis of mean fluorescence intensity of RANKL and I-κB. Data was presented as mean ± SD, n = 3 specimens/group, *P < 0.05. NC: negative control; HF: high-fat; Wnt3a, an activator of Wnt/β-catenin pathway; NAC: N-acetyl-L-cysteine, a ROS antagonist; IM: implant; TRAP, tartrate-resistant acid phosphatase; DAPI, 4’, 6-diamidino-2-phenylindole; 8-OHdG, 8-hydroxy-2 deoxyguanosine; RANKL, receptor activator of nuclear factor-κ B ligand; NF-κB: nuclear factor-kappa B; I-κB: inhibitor of NF-κB

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Lipid droplet accumulation led to oxidative damage in peri-implant tissue under hyperlipidemia conditions in vivo

As shown in Appendix Fig. 2A and B, the accumulation of lipid droplets in bone marrow mesenchymal stem cells (BMMSCs) incubated on the Ti surface gradually increased as the concentration of oleic acid and palmitic acid in the culture environment elevated.

In order to further focus on the lipid droplet accumulation and physiological activity changes of cells on the surface of Ti implants in hyperlipidemia mice, cells on Ti implants surface were acquired after pulled out (Fig. 6A). Intracellular lipid droplets and typical organelles (such as mitochondria and endoplasmic reticulum) were observed by transmission electron microscope (TEM). As shown in Fig. 6B, a large number of lipid droplets were in contact with mitochondria (yellow arrow) and the endoplasmic reticulum (blue arrow) in HF group. Lipid droplets caused severe mitochondrial and endoplasmic reticulum damage (marked by red arrow and green arrow respectively) with decreased number of mitochondrial cristae, mitochondrial matrix dilatation, and endoplasmic reticulum swelling. After administration of NAC, mitochondrial damage and endoplasmic reticulum swelling caused by lipid droplets were ameliorated obviously. It was worth noting that the number of lipid droplets in the NAC group displayed no significant difference from the HF group, which indicated that lipid droplets were the arch-criminal to induce oxidative damage under hyperlipidemia conditions.

Endoplasmic reticulum stress (ERS) was one of the most important cellular responses to external oxidative damage. In order to further explore the manifestations of oxidative damage caused by lipid droplet accumulation at the organelle level, the key factors of ERS in the cells acquired from surface of Ti implants were detected by ELISA. As shown in Fig. 6C, the expression of glucose-regulated protein 78 (GRP78), inositol-requiring enzyme 1 (IRE1), X-box binding protein 1 (XBP1) and C/EBP homologous protein (CHOP) increased in the HF group. NAC application decreased the above ERS markers compared with the HF and Wnt3a group. Especially, the expression of XBP1 as well as CHOP were even restored to the level of the NC group. Additionally, there was no significant difference in the expression levels of activating transcription factor 6 (ATF6) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) among the four groups. Furthermore, IF staining of perilipin2 (PLIN2)/8-OHdG and 8-OHdG/GRP78 were applied to quantitatively analysis the effect of lipid droplet accumulation on oxidative damage (especially ERS). Compared with the HF group, NAC dramatically cut down the expression of 8-OHdG and GRP78 in the peri-implant tissue, whereas NAC could hardly decrease the expression of PLIN2, which suggested that NAC could reduce the oxidative damage level (especially ERS) in the peri-implant tissue in hyperlipidemia mice, but did not have the ability to reduce lipid droplet accumulation (Fig. 6D and G).

These results indicated that lipid droplets, as the critical arch-criminal, lead to severe oxidative damage, such as obvious ERS and mitochondrial dysfunction, in the peri-implants tissue and impair implant osseointegration under hyperlipidemia conditions.

Fig. 6
figure 6

Identification of the lipid droplet accumulation and its role in ERS as well as mitochondrial damage in peri-implant tissue after local injection of Wnt3a or NAC in hyperlipidemia mice. (A) Illustration of sample acquisition from the surface of titanium implant for TEM and ELISA tests. (B) Typical images of TEM detection in the HF and HF + NAC group for observation of swelling endoplasmic reticulum (green arrow), endoplasmic reticulum contacting with lipid droplets (blue arrow), damaged mitochondria (red arrow), and mitochondria contacting with lipid droplets (yellow arrow). (C) Expression of key proteins (GRP78, IRE1, XBP1, CHOP, ATF6, and PERK) in ERS detected by ELISA testing. (D) Fluorescent images of the nucleus (blue), 8-OHdG (green) and PLIN2 (red). (E) Quantitative analysis of mean fluorescence intensity of 8-OHdG and PLIN2. (F) Fluorescent images of the nucleus (blue), 8-OHdG (green) and GRP78 (red). (G) Quantitative analysis of mean fluorescence intensity of 8-OHdG and GRP78. Data was presented as mean ± SD, n = 3 specimens/group, *P < 0.05. IM: implant; TEM, transmission electron microscope; ELISA, enzyme-linked immunosorbent assay; NC: negative control; HF: high-fat; Wnt3a, an activator of Wnt/β-catenin pathway; NAC: N-acetyl-L-cysteine, a ROS antagonist; ER, endoplasmic reticulum; Mt, mitochondrial; GRP78, glucose-regulated protein 78; IRE1, inositol-requiring enzyme 1; XBP1, X-box binding protein 1; CHOP, C/EBP homologous protein; ATF6, activating transcription factor 6; PERK, protein kinase RNA-like endoplasmic reticulum kinase; DAPI, 4’, 6-diamidino-2-phenylindole; 8-OHdG, 8-hydroxy-2 deoxyguanosine; PLIN2, perilipin2

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Lipid droplet accumulation led to oxidative damage in BMMSCs on Ti surface under hyperlipidemia conditions in vitro

In order to further clarify the critical role of lipid droplets in inducing oxidative damage characterized by ERS and mitochondrial damage under hyperlipidemia conditions, BMMSCs were incubated on the surface of Ti sheets for 7 days as designed in Fig. 7A. After HF treatment for 3 days, the mean fluorescence intensity of boron-dipyrromethene (Bodipy), GRP78 and 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) surged sharply in the HF group compared with the NC group, accompanying with a significant decrease in the number of 5-ethynyl-2’-deoxyuridine (EdU) positive cells (Fig. 7B and C). Meanwhile, the mitochondrial function of redox regulation and adenosine triphosphate (ATP) supply were also disturbed in the HF group, identified by the decreasing glutathione (GSH) proportion, NAD+/NADH ratio and ATP concentration, as well as the increasing malondialdehyde (MDA) concentration (Fig. 7C). On the third day, 2 mmol/L NAC was added in the HF medium of HF + NAC group. NAC alleviated the ERS and mitochondrial damage obviously, and finally promoted the proliferation of BMMSCs in the HF medium, which were characterized by decreasing mean fluorescence intensity of GRP78 and MDA concentration, as well as increasing GSH proportion, NAD+/NADH ratio and ATP concentration (Fig. 7D and E). However, NAC did not have the ability to reduce lipid droplet accumulation. Interestingly, the ERS, mitochondrial damage and the impaired proliferation ability reappeared after the NAC treatment was removed for 2 days (Fig. 7F and G). These results suggested that the alleviating effect of NAC is temporary as long as the lipid droplet accumulation exited. So, we came to the conclusion of this part that lipid droplet accumulation led to oxidative damage in BMMSCs on Ti surface under hyperlipidemia conditions in vitro.

Fig. 7
figure 7

Identification of the lipid droplet accumulation and its role in ERS as well as mitochondrial damage in BMMSCs after treatment of NAC in high-fat medium. (A) Illustration of the experimental design in vitro. (B) Fluorescent images of the Bodipy, GRP78, DCFH-DA and EdU staining of BMMSCs cultured on titanium sheet surface for 3 d. (C) Quantitative analysis of Bodipy, GRP78, DCFH-DA and EdU positive cells, as well as detection of the proportion of GSH, MDA concentration, ratio of NAD+/NADH and ATP concentration of BMMSCs cultured on titanium sheet surface for 3 d. (D) Fluorescent images of the Bodipy, GRP78, DCFH-DA and EdU staining of BMMSCs cultured on titanium sheet surface for 5 d. (E) Quantitative analysis of Bodipy, GRP78, DCFH-DA and EdU positive cells, as well as detection of the proportion of GSH, MDA concentration, ratio of NAD+/NADH and ATP concentration of BMMSCs cultured on titanium sheet surface for 5 d. (F) Fluorescent images of the Bodipy, GRP78, DCFH-DA and EdU staining of BMMSCs cultured on titanium sheet surface for 7 d. (G) Quantitative analysis of Bodipy, GRP78, DCFH-DA and EdU positive cells, as well as detection of the proportion of GSH, MDA concentration, ratio of NAD+/NADH and ATP concentration of BMMSCs cultured on titanium sheet surface for 7 d. Data was presented as mean ± SD, n = 3 specimens/group, *P < 0.05. NC, negative control; HF, high-fat; NAC, N-acetyl-L-cysteine, a ROS antagonist; DMEM, dulbecco’s modified eagle medium; Bodipy, boron-dipyrromethene; GRP78, glucose-regulated protein 78; DCFH-DA, 2’,7’-dichlorodihydrofluorescein diacetate; EdU, 5-ethynyl-2’-deoxyuridine; GSH, glutathione; MDA, malondialdehyde; ATP, adenosine triphosphate

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Discussion

Osseointegration is a critical determinant for the success of implant restoration [35, 36], and is dynamically regulated by the bone homeostasis on titanium implant surface. Bone mass homeostasis depends on the balanced activities of osteoblasts and osteoclasts. Implant osseointegration involves woven bone formation, lamellar bone formation, and bone remodeling [37]. Woven bone bridges the implant and native bone early [38, 39], while lamellar bone increases peri-implant density. Bone remodeling, concurrent with lamellar bone formation, is lifelong [40]. Osteoclasts first resorb necrotic bone (0.5–1 mm around implants), followed by osteoblast-mediated new bone formation [41, 42]. Though remodeling enhances density, femoral implant models show acceptable osseointegration by 30 days [43, 44], with resorption stabilizing at 2–4 weeks [45, 46]. Thus, specimens were collected at 30 days. Nevertheless, this study still has some limitations in in vivo model selection. Much research has chosen the femur as their in vivo model to study the osseointegration of implant in different kinds of animals, such as rat [47, 48], mice [49], rabbits [50], and dogs [51]. However, alveolar bone is still the gold standard site for studying dental implant osseointegration. Generally, alveolar bone healing around implants follows intramembranous ossification, characterized by sequential deposition of woven bone, parallel-fibered bone, and lamellar bone [37]. The ossification pattern of the femur exhibits a heterogeneous nature, which varies depending on fracture morphology, the stability of the injury site and the displacement caused by the injury [52]. The healing process of stress fracture and stable injuries with little displacement predominantly occurs through intramembranous ossification mechanisms. The healing process of full fractures and unstable injuries with obvious displacement primarily occurs through endochondral ossification mechanisms. Therefore, the healing mechanism of implant osseointegration in femur in this study follows intramembranous ossification. Although femur model is also a common and persuasive in-vivo model to evaluate osseointegration, future studies should validate the relevant mechanisms in alveolar bone models to enhance the feasibility of clinical translation. In addition, given biological rhythms and hormonal regulation of bone metabolism [53], male models are preferred to avoid variability in female physiological cycles [32, 54]. However, using female hyperlipidemia models (induced by HFD and ovariectomy) [55], further research on hyperlipidemia’s impact on osseointegration in females remains valuable.

Clarifying the physiological processes and molecular signaling pathway changes in situ was of great value for targeted therapy [56]. Therefore, osteogenesis-osteoclasis balance was detected to observe the effects of hyperlipidemia on bone metabolism around Ti implants in vivo. From the respective of osteogenesis, the negative effects of high-fat environment on osteoblast biological activity had been confirmed by several in vitro experiments [14, 31], but there was still no direct in vivo evidence. In this study, we demonstrated that hyperlipidemia directly affected Ti implants osseointegration through the osteogenesis-related ROS/Wnt/β-catenin signaling pathway in vivo. With respect to osteoclastogenesis, we found that hyperlipidemia also impaired Ti implants osseointegration through the osteoclastogenesis-related ROS/RANKL/NF-κB signaling pathway in vivo.

The crucial role of ROS in activating osteoclast-related RANKL/NF-κB signaling pathways has been well elucidated [57]. As a potent ROS antagonist, NAC has demonstrated therapeutic efficacy in mitigating bone metabolism disorders like osteolysis induced by elevated ROS levels [58]. In this study, NAC was locally injected in peri-implant tissues in hyperlipidemia mice. The immunofluorescence staining results revealed that NAC effectively suppressed the RANKL-activated NF-κB signaling pathway (Fig. 5E and H), consistent with prior findings [59]. ROS is a core regulatory target in osteoclast differentiation and maturation [60]. NAC has been demonstrated to reduce bone resorption in diabetic periodontitis [61]. The effect of NAC on oxidative damage (8-OHdG, green fluorescence) in peri-implant tissues was particularly examined. Interestingly, NAC treatment not only significantly reduced 8-OHdG level but also decreased GRP78 expression (a key ERS marker). Previous research has shown that orthodontic pressure-induced ROS elevation led to ERS and subsequent apoptosis, while NAC treatment exhibited protective effects [62]. Consequently, our subsequent in vitro experiments focused on the relationship between ROS and ERS under hyperlipidemia conditions, as well as the potential of NAC to disrupt this vicious cycle. However, our study primarily examined broad indicators of ROS and ERS, including GRP78, DCFH-DA, GSH/GSSG, MDA, NAD+/NADH, and ATP levels following NAC intervention (Fig. 7). Further research is needed to develop therapeutic strategies targeting the ROS-ERS vicious cycle.

Although both in vivo and in vitro experimental results confirmed that lipid droplet accumulation is the primary culprit for impairing Ti implant osseointegration in hyperlipidemia, the specific type of lipid that contributes most significantly to this impairment remains unexplored. Further extensive experiments are needed to identify precise indicators that could provide more reliable references for clinical diagnosis and treatment in oral implant rehabilitation. Lipid droplets, as dynamic organelles specialized in lipid storage, could interact physically with specific proteins on the membrane of endoplasmic reticulum and mitochondria in several cell types, playing critical role in energy homeostasis and cellular function [63]. The impact of intracellular lipid droplet accumulation on biological activities such as osteogenic activity is receiving increasing attention [64]. Our study firstly demonstrated that the accumulation of lipid droplets in BMMSCs incubated on the Ti surface gradually increased as the concentration of oleic acid and palmitic acid in the culture environment elevated. More importantly, through TEM and ELISA testing of cells acquired in situ from Ti implant surface of hyperlipidemia mice, it is firmly verified that lipid droplet accumulation and the its contact with endoplasmic reticulum as well as mitochondria should be the original source of oxidant stress. These results strongly illustrated that as the critical arch-criminal, the accumulation of lipid droplets led to severe oxidative damage, disturbed the physiological osteogenesis-osteoclasis balance on Ti implant surface, and eventually impaired the osseointegration in hyperlipidemia (Fig. 8). From this perspective, comprehensive understanding of the signaling pathways at the interface between lipid droplets with other organelles, and further finding ways to reduce the accumulation level of lipid droplets are important methods for alleviating endoplasmic reticulum stress and mitochondrial damage under hyperlipidemia conditions. However, maintaining lipid droplets and bioactive lipids at a physiological level in hyperlipidemia still remains a significant challenge.

Fig. 8
figure 8

Oxidative damage induced by lipid droplet accumulation impairs the physiological osteogenesis-osteoclasis balance on titanium surface in hyperlipidemia. ERS and mitochondrial damage induced by lipid droplet accumulation in hyperlipidemia tends to form a vicious cycle network and promotes the expression of inflammatory factors in peri-implant tissues, which further dramatically drives osteoclastogenesis and inhibits osteogenesis on titanium implant surface. After local application of NAC, the ROS level is downregulated, which effectively activates the Wnt/β-catenin signaling pathway and inhibits the RANKL/NF-κB signaling pathway to alleviate the osteogenesis-osteoclasis imbalance, thereby ultimately facilitating the Ti implant osseointegration in hyperlipidemia. MDA, malondialdehyde; ROS, reactive oxygen species; ERS, endoplasmic reticulum stress; IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; RANKL, receptor activator of nuclear factor-κ B ligand; IM, implant; NAC, N-acetyl-L-cysteine, a ROS antagonist; non-p-β-catenin, non-phospho-β-catenin; DNA, deoxyribonucleic acid; ALP, alkaline phosphatase; OPG, osteoprotegerin; COLI, collagen type I

Full size image

Conclusion

Lipid droplet accumulation in peri-implant tissue directly induces intracellular oxidative damage, inhibits osteogenesis via ROS/Wnt/β-catenin pathway, and promotes osteoclassis by ROS/RANKL/NF-κB pathway, thereby dramatically impairs Ti implant osseointegration in hyperlipidemia. Reducing lipid droplet accumulation in local tissues or cells would be a promising strategy to alleviate oxidative damage long-termly, regulate the osteogenesis-osteoclasis balance in peri-implant tissue and then promote osteointegration in patients with hyperlipidemia fundamentally.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Abbreviations

TC:

Total cholesterol

TG:

Triglycerides

LDL-C:

Low-density lipoprotein cholesterol

HDL-C:

High-density lipoprotein cholesterol

GLU:

Glucose

DAPI:

4’, 6-diamidino-2-phenylindole

8-OHdG:

8-hydroxy-2 deoxyguanosine

non-p-β-catenin:

Non-phospho-β-catenin

NAC:

N-acetyl-L-cysteine

UREA:

Urea nitrogen

CREA:

Creatinine

UA:

Uric acid

BV/TV:

Bone volume/tissue volume

TB.N:

Trabecular number

Tb.Th:

Trabecular thickness

Tb.Sp:

Trabecular separation

ALP:

Alkaline phosphatase

TRAP:

Tartrate-resistant acid phosphatase

RANKL:

Receptor activator of nuclear factor-κB ligand

NF-κB:

Nuclear factor-kappa B

I-κB:

Inhibitor of NF-κB

TEM:

Transmission electron microscope

ELISA:

Enzyme-linked immunosorbent assay

ER:

Endoplasmic reticulum

Mt:

Mitochondrial

GRP78:

Glucose-regulated protein 78

IRE1:

Inositol-requiring enzyme 1

XBP1:

X-box binding protein 1

CHOP:

C/EBP homologous protein

ATF6:

Activating transcription factor 6

PERK:

Protein kinase RNA-like endoplasmic reticulum kinase

PLIN2:

Perilipin2

DMEM:

Dulbecco’s modified eagle medium

Bodipy:

Boron-dipyrromethene

EdU:

5-ethynyl-2’-deoxyuridine

GSH:

Glutathione

MDA:

Malondialdehyde

ATP:

Adenosine triphosphate

ROS:

Reactive oxygen species

ERS:

Endoplasmic reticulum stress

IL-1β:

Interleukin-1β

IL-6:

Interleukin-6

TNF-α:

Tumor necrosis factor-α

DNA:

Deoxyribonucleic acid

OPG:

Osteoprotegerin

COLI:

Collagen type I

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Acknowledgements

Not applicable.

Funding

This work was supported by the National Natural Science Foundation of China (No. 82301126, No. 82001055); China Postdoctoral Science Foundation (2024T170514, 2022M721933); Construction Engineering Special Fund of “Taishan Scholars” of Shandong Province (No. tsqn202306368, No. tsqn202408356); Shandong Provincial Natural Science Foundation (ZR2023QH207, ZR2020QH158); Basic Research Program of Jiangsu (BK20230250); Shandong Postdoctoral Science Foundation (SDCX-ZG-202303001) and the National Clinical Key Specialty (Periodontology) Construction Project.

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Authors and Affiliations

  1. Department of Implantology & Periodontology, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Research Center of Dental Materials and Oral Tissue Regeneration & Shandong Provincial Clinical Research Center for Oral Diseases, No. 44-1 Wenhua Road West, Jinan, Shandong, 250012, China

    Ya-nan Wang & Shiyue Liu

  2. Suzhou Research Institute, Shandong University, No.388 Ruoshui Road, Jiangsu, 215123, Suzhou, China

    Ya-nan Wang

Authors
  1. Ya-nan Wang
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  2. Shiyue Liu
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Contributions

Y.N. Wang, contributed to conceptualization, data curation, formal analysis, investigation, methodology, funding acquisition, visualization, and writing the original draft; S.Y. Liu, contributed to conceptualization, data curation, formal analysis, investigation, funding acquisition, project administration, supervision, validation and critically revised the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Shiyue Liu.

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The animal experimental procedures were approved by the Medical Ethics Committee of Stomatological Hospital, Shandong University, Jinan, China (Permit Number: No. 20200903).

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The authors declare no competing interests.

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Wang, Yn., Liu, S. Lipid droplet accumulation impairs osseointegration by disturbing the osteogenesis-osteoclasis balance on titanium implant surface in hyperlipidemia. BMC Oral Health 25, 823 (2025). https://doi.org/10.1186/s12903-025-06218-5

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Keywords

BMC Oral Health

ISSN: 1472-6831

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