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Alpha-minimum essential medium (α-MEM) and Dulbecco's modified Eagle’s medium (DMEM) were purchased from Gibco Co. (United States). Foetal bovine serum (FBS) was obtained from Gibco Co. (Australia). Sodium chloride (NaCl) and cetylpyridinium chloride were provided by Sigma Co., Ltd. (United States). The PrimeScriptTM RT kit and TBGreenTM PremixExTaqTM Ⅱ kit were purchased from TaKara (Japan). The RNA extraction kit was obtained from Tiangen Biochemical Technology Company (China). Thiazolyl blue (MTT), Triton X-100, 4% tissue cell fixation solution, 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI), and fluorescein isothiocyanate (FITC)-labelled ghost pen cyclic peptide were provided by Beijing Solarbio Science & Technology Co., Ltd. (China). The Bicinchoninic acid (BCA) protein concentration determination kit was purchased from Biyuntian Biotechnology Co., Ltd. (China). The Matrigel matrix adhesive was provided by Corning Co. (United States). Other chemicals were obtained from Muke Experimental Equipment Co., Ltd. (China).
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The animal study protocol was approved by the Animal Ethics Committee of Wenzhou Medical University and was in adherence to the national and local laws, as well as guidelines issued by the Animal Experimental Center of Wenzhou Medical University. Initially, clean Ti rods (diameter: 0.8 mm, length: 10 mm) were underwent a 60 s sandblasting process under 0.45 MPa. Then, these Ti rods were corroded in a mixed acid solution of 37 wt% hydrochloric acid, 98 wt% sulfuric acid, and deionized water (v/v/v = 2:1:1) at 60 °C. The final sandblasted and acid-etched (SLA) titanium was obtained after ultrasonic cleaning and drying. In this study, 18 male Sprague-Dawley (SD) rats, aged 3 weeks, were randomly assigned to 3 groups (n = 6) and fed diets containing 0.8 wt% (control group), 2 wt%, and 6 wt% NaCl. The implantation surgery was performed 2 weeks later. Briefly, rats were anaesthetized before the operation, and then the epiphyseal ends of bilateral femurs were surgically exposed. A round hole (1 mm diameter) was drilled into the centre of the femur using a surgical drill, following which sterilized SLA implants were inserted. Finally, the surgical sites were carefully sutured in layers, and all operations were performed under aseptic conditions. Penicillin was injected intramuscularly for three days post-surgery to prevent potential infection.
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To evaluate new bone formation around the implants after implantation for 8 weeks, femurs from each rat were collected and fixed with 4% paraformaldehyde. Microcomputerized tomography was used to analyze and reconstruct the three-dimensional structure around the implant, focusing on a region measuring 1 mm in thickness and 2 mm in width. Then, the percentage of bone volume to total volume ratio (BV/TV%), connectivity density, trabecular number (Tb.N), and trabecular spacing (Tb.Sp) were quantified by CTVox, CTAn and CTVol software. These metrics served as key indicators of the newly formed bone mass and its spatial distribution.
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The fixed femur specimens were decalcified with formic acid for one week, dehydrated through a series of graded alcohol solutions, embedded in paraffin, sliced, and stained with haematoxylin and eosin (HE) and a CD34 monoclonal antibody. Histological observation was performed using an optical microscope.
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Human umbilical vein endothelial cells (HUVECs) were obtained from the American type culture collection (ATCC) Biological Standard Resource Center. NaCl was dissolved in double distilled water, sterilized, and then added to DMEM under sterile conditions to create HS medium with final Na+ concentrations of 140 mmol/L, 145 mmol/L, and 150 mmol/L. The cells were subcultured twice once cell confluence reached 90%. The passages were named generations 1, 2 and 3 accordingly (Figure 3A).
Figure 3. (A) Schematic diagram of pre-treatment of HUVECs (generation 1, 2 & 3) using culture media supplemented with different concentrations of Na+; (B) Fluorescence staining images of HUVECs in each group after pretreating with different concentrations of Na+; (C) Cell viability of HUVECs (generation 1, 2 & 3) in different groups, *P < 0.05.
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To observe the cell morphology, each group of HUVECs was seeded in a 24-well plate at a density of 8,000 cells per well. After 2 days of culture, the cytoskeleton and nucleus were stained using ghost pen cyclic peptide and DAPI, respectively. Five visual fields were randomly selected for fluorescence imaging. Green fluorescence represents the cytoskeleton, and blue fluorescence represents the nucleus.
Additionally, MTT was used to assess cell viability. Cells were inoculated in 48-well plates at a density of 10,000 cells per well and cultured in HS DMEM for 1 and 3 days. The culture medium was refreshed every two days. For the MTT assay, the culture medium was first removed, and then each well was supplemented with 200 μL of serum-free DMEM containing 10% MTT. After incubation for 4 hours, the 10% MTT medium was removed, and 500 μL of dimethyl sulfoxide (DMSO) was added to each well. The plate was then placed on a shaker for 10 minutes. The absorbance at 490 nm was measured by a microplate reader.
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The horizontal migration capability of HUVECs was measured. For this purpose, cells from different groups were seeded in 24-well plates at a density of 40,000 cells per well. When the degree of convergence reached 80%–90%, a white gun head was used to cross the line in the centre of the hole plate. Cell migration was recorded at 0 (initial state), 12, and 24 hours using an optical microscope. The cell migration area was measured and calculated by Image J software.
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Different groups of HUVECs were seeded in 96-well plates, each well covered with matrix glue, at a density of 50,000 cells per well. After being cultured in varied media for 3 and 6 hours, the cells were washed 3 times with phosphate buffered solution (PBS). The resultant vascular rings were observed and analyzed using an optical microscope. Angiogenesis Analyser in Image J software was used to measure the number of computing nodes (points adjacent to three pixels) and connections (boundaries superimposed by four nodes).
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Real-time quantitative polymerase chain reaction (RT‒qPCR) was employed to detect the expression of key angiogenic genes, including VEGF, FGFR, KDR, CXCR4, and TIE-1. Different groups of HUVECs were seeded in 24-well plates at a density of 20,000 cells per well. After being cultured in different media for 3 days, the total RNA from the cells was extracted using TRIzol, and then the mRNA was reverse-transcribed into complementary DNA (cDNA) using the PrimeScriptTM RT kit. Finally, the expression of different genes was analyzed through RT‒qPCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal reference. The primer sequences are detailed in Table 1.
Table 1. The primer sequence of angiogenic genes (VEGF, FGFR, CXCR4, TIE-1)
Angiogenesis-related genes Gene sequence GAPDH F:5’- TCAAGAAGGTGGTGAAGCAGG-3’
R:5’- AGCGTCAAAGGTGGAGGAGTG-3’VEGF F:5’- AGGGCAGAATCATCACGAAGT-3’
R:5’- AGGGTCTCGATTGGATGGCA-3’FGFR F:5’- AATGAGTACGGCAGCATCAAC-3′
R:5’- ACCTCGA TGTGCTTTAGCCAC-3′CXCR4 F:5’- CACTGTTGCCTGAACCCCAT-3′
R:5’- TGTCCACCCCGTTTCCCTT-3′TIE-1 F:5’- AAGCAGACAGACGTGATCTGG-3′
R:5’- GCACGATGAGCCGAAAGAAG-3′ -
Mouse embryonic osteoblast progenitor cells (MC3T3-E1 cells) were obtained from the American ATCC Biological Standard Resource Center. α-MEM medium with Na+ concentrations of 140 mM, 145 mM, and 150 mM was prepared and then used to culture MC3T3-E1 cells for 1, 5, and 10 generations (Figure 5A). The cells were cultured at 37 °C and sub-cultured 9 times once cell confluence reached 90%.
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To observe the cell morphology, MC3T3-E1 cells from each group were seeded in 24-well plates at a density of 8,000 cells per well. After a 3-day culture, the medium was changed every two days. The staining procedure was the same as that of HUVECs, and the cell morphology was observed using a fluorescence microscope. In addition, MTT assay was utilized to assess cell viability. Different groups of cells were seeded in 48-well plates at a density of 10,000 cells per well and cultured for 4 and 7 days. The detection procedure was also the same as that of HUVECs.
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The ALP activity was used to evaluate the early osteogenic ability of MC3T3-E1 cells. Cells were seeded in 24-well plates at a density of 20,000 cells per well. After 4 and 7 days of culture, the culture medium was refreshed every two days. Triton X-100 (1%, wt/v) was used to lyse the cells. A BCA kit was used to quantify the total protein in the cell lysate (absorption at 562 nm). An ALP kit was used to measure the ALP activity in the cell lysate (absorption at 520 nm).
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To measure the mineralization level, MC3T3-E1 cells from each group were seeded in 24-well plates at a density of 20,000 cells per well. After 14 days of culture, the culture medium was changed every two days. Then, the cells were rinsed thrice with sterile PBS, fixed with 4% paraformaldehyde for 30 min, and stained with alizarin red until red calcium nodules appeared. These calcified nodules were dissolved in 10% cetylpyridinium chloride, and the absorbance was measured at 540 nm by a spectrophotometer.
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All in vitro experiments were repeated at least 3 times. All data were analyzed by one-way analysis of variance (ANOVA) and Student’s t test using SPSS Statistics 22, and presented as the mean ± standard deviation (mean ± SD). The confidence coefficient was set to 95% (*P < 0.05).
doi: 10.3967/bes2024.077
Impact of High Sodium Diet on Neovascularization and Osseointegration around Titanium Implant: An in Vivo and in Vitro Study
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Abstract:
Objective A high sodium (HS) diet is believed to affect bone metabolism processes. Clarifying its impact on osseointegration of titanium (Ti) implants holds significant implications for postoperative dietary management of implanted patients. Methods This investigation probed the impact of sodium ions (Na+) on neovascularization and osteogenesis around Ti implants in vivo, utilizing micro-computed tomography, hematoxylin and eosin staining, and immunohistochemical analyses. Concurrently, in vitro experiments assessed the effects of varied Na+ concentrations and exposure durations on human umbilical vein endothelial cells (HUVECs) and MC3T3-E1 cells. Results In vivo, increased dietary sodium (0.8%-6%) led to a substantial decline in CD34 positive HUVECs and new bone formation around Ti implants, alongside an increase in inflammatory cells. In vitro, an increase in Na+ concentration (140 mmol/L–150 mmol/L) adversely affected the proliferation, angiogenesis, and migration of HUVECs, especially with prolonged exposure. While MC3T3-E1 cells initially exhibited less susceptibility to high Na+ concentrations compared to HUVECs during short-term exposure, prolonged exposure to a HS environment progressively diminished their proliferation, differentiation, and osteogenic capabilities. Conclusion These findings suggest that HS diet had a negative effect on the early osseointegration of Ti implants by interfering with the process of postoperative vascularized bone regeneration. -
Key words:
- High-sodium /
- Implants /
- Vascularization /
- Osseointegration.
We have no conflicts of interest to declare.
注释:1) AUTHOR CONTRIBUTIONS: 2) DECLARATION OF COMPETING INTEREST: -
Figure 3. (A) Schematic diagram of pre-treatment of HUVECs (generation 1, 2 & 3) using culture media supplemented with different concentrations of Na+; (B) Fluorescence staining images of HUVECs in each group after pretreating with different concentrations of Na+; (C) Cell viability of HUVECs (generation 1, 2 & 3) in different groups, *P < 0.05.
Figure 4. (A) Migration images of HUVECs in different groups at 0, 12, and 24 h; (B) Quantitative analysis of cell migration area after 12 and 24 h of cultivation using culture media supplemented with different concentrations of Na+; (C) Images of tubule formation at 3 and 6 h in each group; Quantitative analysis of the number of nodes (D) and branches (E) formed by tubules, *P < 0.05.
Figure 6. (A) Schematic diagram of pre-treatment of MC3T3-E1 cells (generation 1, 5 & 10) using culture media supplemented with different concentrations of Na+; (B) Fluorescence staining images of MC3T3-E1 cells in each group after pretreating with different concentrations of Na+; (C) Cell viability of MC3T3-E1 cells (generation 1, 5 & 10) in different groups, *P < 0.05.
Table 1. The primer sequence of angiogenic genes (VEGF, FGFR, CXCR4, TIE-1)
Angiogenesis-related genes Gene sequence GAPDH F:5’- TCAAGAAGGTGGTGAAGCAGG-3’
R:5’- AGCGTCAAAGGTGGAGGAGTG-3’VEGF F:5’- AGGGCAGAATCATCACGAAGT-3’
R:5’- AGGGTCTCGATTGGATGGCA-3’FGFR F:5’- AATGAGTACGGCAGCATCAAC-3′
R:5’- ACCTCGA TGTGCTTTAGCCAC-3′CXCR4 F:5’- CACTGTTGCCTGAACCCCAT-3′
R:5’- TGTCCACCCCGTTTCCCTT-3′TIE-1 F:5’- AAGCAGACAGACGTGATCTGG-3′
R:5’- GCACGATGAGCCGAAAGAAG-3′ -
[1] Schwarz F, Ramanauskaite A. It is all about peri-implant tissue health. Periodontol 2000, 2022; 88, 9−12. doi: 10.1111/prd.12407 [2] Wagner J, Spille JH, Wiltfang J, et al. Systematic review on diabetes mellitus and dental implants: an update. Int J Implant Dent, 2022; 8, 1. doi: 10.1186/s40729-021-00399-8 [3] Mohammadzadeh Rad M, Saber-Samandari S, Sadighi M, et al. Macro-and micromechanical modelling of HA-Elastin scaffold fabricated using freeze drying technique. J Nanoanalysis, 2021; 8, 17−31. [4] Karlsson K, Derks J, Håkansson J, et al. Interventions for peri-implantitis and their effects on further bone loss: a retrospective analysis of a registry-based cohort. J Clin Periodontol, 2019; 46, 872−9. doi: 10.1111/jcpe.13129 [5] Lee RSB, Hamlet SM, Moon HJ, et al. Re-establishment of macrophage homeostasis by titanium surface modification in type II diabetes promotes osseous healing. Biomaterials, 2021; 267, 120464. doi: 10.1016/j.biomaterials.2020.120464 [6] Bagherifard A, Joneidi Yekta H, Akbari Aghdam H, et al. Improvement in osseointegration of tricalcium phosphate-zircon for orthopedic applications: an in vitro and in vivo evaluation. Med Biol Eng Comput, 2020; 58, 1681−93. doi: 10.1007/s11517-020-02157-1 [7] GBD 2017 Diet Collaborators. Health effects of dietary risks in 195 countries, 1990-2017: a systematic analysis for the global burden of disease study 2017. Lancet, 2019; 393, 1958−72. doi: 10.1016/S0140-6736(19)30041-8 [8] Ma PP, Zha S, Shen XK, et al. NFAT5 mediates hypertonic stress-induced atherosclerosis via activating NLRP3 inflammasome in endothelium. Cell Commun Signal, 2019; 17, 102. doi: 10.1186/s12964-019-0406-7 [9] Wu L, Luthringer BJC, Feyerabend F, et al. Increased levels of sodium chloride directly increase osteoclastic differentiation and resorption in mice and men. Osteoporosis Int, 2017; 28, 3215−28. doi: 10.1007/s00198-017-4163-4 [10] Baldisserotto J, Padilha DMP, Amenábar JM. The influence of dietary salt on the osseointegration of implants in aging rats. Int Arch Otorhinolaryngol, 2019; 23, e427−32. doi: 10.1055/s-0039-1693141 [11] Farazin A, Akbari Aghdam H, Motififard M, et al. A polycaprolactone bio-nanocomposite bone substitute fabricated for femoral fracture approaches: molecular dynamic and micromechanical Investigation. J Nanoanalysis, 2019; 6, 172−84. [12] Aghdam HA, Sanatizadeh E, Motififard M, et al. Effect of calcium silicate nanoparticle on surface feature of calcium phosphates hybrid bio-nanocomposite using for bone substitute application. Powder Technol, 2020; 361, 917−29. doi: 10.1016/j.powtec.2019.10.111 [13] Tiyasatkulkovit W, Aksornthong S, Adulyaritthikul P, et al. Excessive salt consumption causes systemic calcium mishandling and worsens microarchitecture and strength of long bones in rats. Sci Rep, 2021; 11, 1850. doi: 10.1038/s41598-021-81413-2 [14] Schröder A, Gubernator J, Leikam A, et al. Dietary salt accelerates orthodontic tooth movement by increased osteoclast activity. Int J Mol Sci, 2021; 22, 596. doi: 10.3390/ijms22020596 [15] Khandan A, Nassireslami E, Saber-Samandari S, et al. Fabrication and characterization of porous bioceramic-magnetite biocomposite for maxillofacial fractures application. Dent Hypotheses, 2020; 11, 74−85. doi: 10.4103/denthyp.denthyp_11_20 [16] Zhao YF, Xie L. Unique bone marrow blood vessels couple angiogenesis and osteogenesis in bone homeostasis and diseases. Ann N Y Acad Sci, 2020; 1474, 5−14. doi: 10.1111/nyas.14348 [17] Zeng YW, Huang C, Duan DM, et al. Injectable temperature-sensitive hydrogel system incorporating deferoxamine-loaded microspheres promotes H-type blood vessel-related bone repair of a critical size femoral defect. Acta Biomater, 2022; 153, 108−23. doi: 10.1016/j.actbio.2022.09.018 [18] Tang Y, Hu MJ, Xu Y, et al. Megakaryocytes promote bone formation through coupling osteogenesis with angiogenesis by secreting TGF-β1. Theranostics, 2020; 10, 2229−42. doi: 10.7150/thno.40559 [19] Hu XF, Xiang G, Wang TJ, et al. Impairment of type H vessels by NOX2-mediated endothelial oxidative stress: critical mechanisms and therapeutic targets for bone fragility in streptozotocin-induced type 1 diabetic mice. Theranostics, 2021; 11, 3796−812. doi: 10.7150/thno.50907 [20] Qin QZ, Lee S, Patel N, et al. Neurovascular coupling in bone regeneration. Exp Mol Med, 2022; 54, 1844−9. doi: 10.1038/s12276-022-00899-6 [21] Ramasamy SK, Kusumbe AP, Schiller M, et al. Blood flow controls bone vascular function and osteogenesis. Nat Commun, 2016; 7, 13601. doi: 10.1038/ncomms13601 [22] Vuornos K, Huhtala H, Kääriäinen M, et al. Bioactive glass ions for in vitro osteogenesis and microvascularization in gellan gum-collagen hydrogels. J Biomed Mater Res Part B Appl Biomater, 2020; 108, 1332−42. doi: 10.1002/jbm.b.34482 [23] Chen WZ, Xu K, Tao BL, et al. Multilayered coating of titanium implants promotes coupled osteogenesis and angiogenesis in vitro and in vivo. Acta Biomater, 2018; 74, 489−504. doi: 10.1016/j.actbio.2018.04.043 [24] Rath SN, Arkudas A, Lam CX, et al. Development of a pre-vascularized 3D scaffold-hydrogel composite graft using an arterio-venous loop for tissue engineering applications. J Biomater Appl, 2012; 27, 277−89. doi: 10.1177/0885328211402243 [25] Tuckermann J, Adams RH. The endothelium-bone axis in development, homeostasis and bone and joint disease. Nat Rev Rheumatol, 2021; 17, 608−20. doi: 10.1038/s41584-021-00682-3 [26] Lee EJ, Jain M, Alimperti S. Bone microvasculature: stimulus for tissue function and regeneration. Tissue Eng Part B:Rev, 2021; 27, 313−29. doi: 10.1089/ten.teb.2020.0154 [27] Burger MG, Grosso A, Briquez PS, et al. Robust coupling of angiogenesis and osteogenesis by VEGF-decorated matrices for bone regeneration. Acta Biomater, 2022; 149, 111−25. doi: 10.1016/j.actbio.2022.07.014 [28] Neal B, Wu YF, Feng XX, et al. Effect of salt substitution on cardiovascular events and death. N Engl J Med, 2021; 385, 1067−77. doi: 10.1056/NEJMoa2105675 [29] Zeng C, Rosenberg L, Li XX, et al. Sodium-containing acetaminophen and cardiovascular outcomes in individuals with and without hypertension. Eur Heart J, 2022; 43, 1743−55. doi: 10.1093/eurheartj/ehac059 [30] Ying KE, Feng WG, Ying WZ, et al. Dietary salt initiates redox signaling between endothelium and vascular smooth muscle through NADPH oxidase 4. Redox Biol, 2022; 52, 102296. doi: 10.1016/j.redox.2022.102296 [31] Cao Y, Yuan GH, Zhang Y, et al. High glucose-induced circHIPK3 downregulation mediates endothelial cell injury. Biochem Biophys Res Commun, 2018; 507, 362−8. doi: 10.1016/j.bbrc.2018.11.041 [32] Torres BM, Leal MAS, Brun BF, et al. Effects of direct high sodium exposure at endothelial cell migration. Biochem Biophys Res Commun, 2019; 514, 1257−63. doi: 10.1016/j.bbrc.2019.05.103 [33] Fu H, Chen JK, Lu WJ, et al. Inflammasome-independent NALP3 contributes to high-salt induced endothelial dysfunction. Front Pharmacol, 2018; 9, 968. doi: 10.3389/fphar.2018.00968 [34] Paddenberg E, Krenmayr B, Jantsch J, et al. Dietary salt and myeloid NFAT5 (nuclear factor of activated T cells 5) impact on the number of bone-remodelling cells and frequency of root resorption during orthodontic tooth movement. Ann Anat, 2022; 244, 151979. doi: 10.1016/j.aanat.2022.151979 [35] van der Wijst J, Tutakhel OAZ, Bos C, et al. Effects of a high-sodium/low-potassium diet on renal calcium, magnesium, and phosphate handling. Am J Physiol Renal Physiol, 2018; 315, F110−22. doi: 10.1152/ajprenal.00379.2017 [36] Müller DN, Wilck N, Haase S, et al. Sodium in the microenvironment regulates immune responses and tissue homeostasis. Nat Rev Immunol, 2019; 19, 243−54. doi: 10.1038/s41577-018-0113-4 [37] Geisberger S, Bartolomaeus H, Neubert P, et al. Salt transiently inhibits mitochondrial energetics in mononuclear phagocytes. Circulation, 2021; 144, 144−58. doi: 10.1161/CIRCULATIONAHA.120.052788 [38] Mo S, Cui Y, Sun KH, et al. High sodium chloride affects BMP-7 and 1α-hydroxylase levels through NCC and CLC-5 in NRK-52E cells. Ecotoxicol Environ Saf, 2021; 225, 112762. doi: 10.1016/j.ecoenv.2021.112762 [39] Gao P, You M, Li L, et al. Salt-induced hepatic inflammatory memory contributes to cardiovascular damage through epigenetic modulation of SIRT3. Circulation, 2022; 145, 375−91. doi: 10.1161/CIRCULATIONAHA.121.055600 [40] Xiao HR, Yan YL, Gu YP, et al. Strategy for sodium-salt substitution: on the relationship between hypertension and dietary intake of cations. Food Res Int, 2022; 156, 110822. doi: 10.1016/j.foodres.2021.110822 [41] Rizvi ZA, Dalal R, Sadhu S, et al. High-salt diet mediates interplay between NK cells and gut microbiota to induce potent tumor immunity. Sci Adv, 2021; 7, eabg5016. doi: 10.1126/sciadv.abg5016 [42] Wu L, Luthringer BJC, Feyerabend F, et al. Increased levels of sodium chloride directly increase osteoclastic differentiation and resorption in mice and men. Osteoporosis Int, 2017; 28, 3215-28. (查阅网上资料, 本条文献与第9条文献重复, 请确认) [43] Jobin K, Müller DN, Jantsch J, et al. Sodium and its manifold impact on our immune system. Trends Immunol, 2021; 42, 469−79. doi: 10.1016/j.it.2021.04.002 [44] Khandan A, Abdellahi M, Ozada N, et al. Study of the bioactivity, wettability and hardness behaviour of the bovine hydroxyapatite-diopside bio-nanocomposite coating. J Taiwan Inst Chem Eng, 2016; 60, 538−46. doi: 10.1016/j.jtice.2015.10.004 [45] Guglielmotti MB, Olmedo DG, Cabrini RL. Research on implants and osseointegration. Periodontol 2000, 2019; 79, 178−89. doi: 10.1111/prd.12254 [46] Marx RE, Carlson ER, Eichstaedt RM, et al. Platelet-rich plasma: growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endodontol, 1998; 85, 638−46. doi: 10.1016/S1079-2104(98)90029-4 [47] Kim ES, Park EJ, Choung PH. Platelet concentration and its effect on bone formation in calvarial defects: an experimental study in rabbits. J Prosthet Dent, 2001; 86, 428−33. doi: 10.1067/mpr.2001.115874 [48] Clark RAF. Fibrin and wound healing. Ann N Y Acad Sci, 2001; 936, 355−67. doi: 10.1111/j.1749-6632.2001.tb03522.x [49] Norrdin RW, Jee WSS, High WB. The role of prostaglandins in bone in vivo. Prostaglandins, Leukot Essent Fatty Acids, 1990; 41, 139−49. doi: 10.1016/0952-3278(90)90081-U [50] Simon AM, Manigrasso MB, O’Connor JP. Cyclooxygenase 2 function is essential for bone fracture healing. J Bone Miner Res, 2002; 17, 963−76. doi: 10.1359/jbmr.2002.17.6.963 [51] Cheng H, Huang HY, Guo ZK, et al. Role of prostaglandin E2 in tissue repair and regeneration. Theranostics, 2021; 11, 8836−54. doi: 10.7150/thno.63396 [52] Geusens P, Emans PJ, de Jong JJA, et al. NSAIDs and fracture healing. Curr Opin Rheumatol, 2013; 25, 524−31. doi: 10.1097/BOR.0b013e32836200b8 [53] Lisowska B, Kosson D, Domaracka K. Lights and shadows of NSAIDs in bone healing: the role of prostaglandins in bone metabolism. Drug Des Devel Ther, 2018; 12, 1753−8. doi: 10.2147/DDDT.S164562 [54] Ripamonti U, Roden LC, Renton LF. Osteoinductive hydroxyapatite-coated titanium implants. Biomaterials, 2012; 33, 3813−23. doi: 10.1016/j.biomaterials.2012.01.050 [55] Sun C, Dai XL, Zhao DL, et al. Mesenchymal stem cells promote glioma neovascularization in vivo by fusing with cancer stem cells. BMC Cancer, 2019; 19, 1240. doi: 10.1186/s12885-019-6460-0 [56] Inoue T, Sata M, Hikichi Y, et al. Mobilization of CD34-positive bone marrow-derived cells after coronary stent implantation: impact on restenosis. Circulation, 2007; 115, 553−61. doi: 10.1161/CIRCULATIONAHA.106.621714 [57] Mente A, O'Donnell M, Yusuf S. Sodium intake and health: what should we recommend based on the current evidence? Nutrients, 2021; 13, 3232. [58] Cui Y, Sun KH, Xiao YW, et al. High-salt diet accelerates bone loss accompanied by activation of ion channels related to kidney and bone tissue in ovariectomized rats. Ecotoxicol Environ Saf, 2022; 244, 114024. doi: 10.1016/j.ecoenv.2022.114024 [59] Liu ZY, Li SK, Huang CK, et al. A high-sodium diet modulates the immune response of food allergy in a murine model. Nutrients, 2021; 13, 3684. doi: 10.3390/nu13113684 [60] Amersfoort J, Eelen G, Carmeliet P. Immunomodulation by endothelial cells - partnering up with the immune system? Nat Rev Immunol, 2022; 22, 576-88. [61] Zarubova J, Hasani-Sadrabadi MM, Ardehali R, et al. Immunoengineering strategies to enhance vascularization and tissue regeneration. Adv Drug Deliv Rev, 2022; 184, 114233. doi: 10.1016/j.addr.2022.114233 [62] Lee MKS, Murphy AJ. A high-salt diet promotes atherosclerosis by altering haematopoiesis. Nat Rev Cardiol, 2023; 20, 435−6. doi: 10.1038/s41569-023-00879-x [63] Zhang LP, Yang Y, Aroor AR, et al. Endothelial sodium channel activation mediates DOCA-salt-induced endothelial cell and arterial stiffening. Metabolism, 2022; 130, 155165. doi: 10.1016/j.metabol.2022.155165 [64] Ma PP, Li G, Jiang XR, et al. NFAT5 directs hyperosmotic stress-induced fibrin deposition and macrophage infiltration via PAI-1 in endothelium. Aging (Albany NY), 2020; 13, 3661−79.