Magnesium promotes implant osseointegration by regulating LepR + stem cells

Magnesium promotes implant osseointegration by regulating LepR + stem cells | 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 Magnesium promotes implant osseointegration by regulating LepR + stem cells Linfeng Liu, Lixuan Huang, Wei Song, Danting Zhang, Shiwen Zhang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5765695/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Homing and differentiation of stem cells is a necessary step in achieving implant osseointegration. However, the specific role and mechanism about stem cells in the osseointegration process were rarely reported. Rencently, LepR + cells were identified as crucial skeletal stem cells in the long bone and alveolar bone, which participated homeostasis maintenance and damage repair. As one of the indispensable trace elements in the human body, Mg 2+ were proved to promote vascularized bone regeneration. Here, by combining tissue clearing technique and immunofluorescent mice, we proved that Mg 2+ promoted implant osseointegration by regulating LepR + stem cells aggregation and differentiation at the implant-bone interface in vivo and provided a new perspective to promote the study of implant research. Purpose The aim of the study was to explore the effects of Mg 2+ in promoting implant osseointegration in the alveolar bone. Methods The cell proliferation and osteogenic differentiation were performed to screening the optimal Mg 2+ concentration for MC3T3-E1 osteoblasts. Then the optimal Mg 2+ was loaded on a self-designed SLA implant hydrothermally. Characterization and bio-compatibility of materials were detected. In vivo, combining the PEGASOS with LepR-Cre;tdTomato;Col2.3-GFP mice, we traced the LepR positive ( LepR + ) stem cells aggregation and differentiation at the implant-bone interface. Results 5mM Mg 2+ was screened to be the optimal concentration for MC3T3-E1 osteoblasts. EDS and XRF proved that Mg 2+ was successfully corroborated onto the implants. Further detection confirmed the better stretching and proliferatio of Mg-coating implants. 3-D imaging revealed that Mg-coating implants facilitated LepR + cells aggregation and differentiation to Col2.3-GFP + cells. Conclusion Mg 2+ promoted osseointegration around implants by facilitating the LepR + cells accumulation and osteogenic differentiation. magnesium implant osseointegration LepR+ stem cells PEGASOS Figures Figure 1 Figure 2 Figure 3 Introduction Since Professor Branemark proposed the theory of osseointegration in the 1960s, with the continuous development and maturity of implant surgery technology, implant-supported restoration have been more and more clinically applied, and are becoming the preferred treatment option for missing teeth[ 1 ]. In suitable cases, satisfactory osseointegration can be achieved after implant placement. However, in clinical practice, poor osseointegration is often caused by pathological or physiological reasons such as inflammation, systemic diseases, age and hormone levels. While implant failure still occurs due to the particularity and complexity of the local oral environment[ 2 ]. Bone reconstruction around the implant exists in the whole functional dynamic cycle. Its occurrence and development require fully mobilizing the skeletal stem cells (SSCs) to function, while the homing and differentiation of SSCs determine the speed and efficiency of osseointegration. When the implant is inserted in the alveolar bone, it is surrounded by cells, cytokines, etc. As healing progresses, SSCs gradually aggregate and differentiate towards osteoblasts which accumulate, secrete osteogenic factors, form new bone tissue, and ultimately promote the formation of osseointegration. In the process of bone healing, various factors such as inflammation, immune response, hormone levels may affect the proliferation and differentiation of SSCs, thereby affecting the effect of osseointegration, and even form fibrous tissue leading to osseointegration failure[ 3 , 4 ]. As one of the indispensable mineral elements of the human body, more than half of the magnesium ion (Mg 2+ ) exists in the bones, participating in the metabolic processes[ 5 ]. Since Professor Qin first demonstrated in Nature Medicine that Mg 2+ accelerated osteogenic differentiation of stem cells by promoting the secretion of CGRP by dorsal root ganglion in 2016[ 6 ], the preferred osteogenic activity of Mg 2+ has attracted widespread attention. In 2021, Professor Kelvin W K Yeung demonstrated in Nature Communication that Mg 2+ contributes to the formation of the osteogenic immune microenvironment, which once again verified that the osteogenic activity of Mg 2+ was closely related to its regulation of the osteoblastic microenvironment[ 7 ]. At present, the role of Mg 2+ in promoting bone regeneration has been widely confirmed, but almost all in vitro and clinical studies on Mg 2+ to promote SSCs osteogenesis are derived from bone marrow mesenchymal stem cells of long bones, while it remains unknown whether Mg 2+ could affect SSCs in alveolar bone. Alveolar bones and long bones are different in origin and biological characteristics[ 8 – 10 ]. In our previous studies, we has proved that LepR + cells were osteogenic precursor cells of the alveolar bone and played an important role in the bone healing[ 11 ]. So we used LepR-Cre;tdTomato mice to label skeletal stem cells in the alveolar bone. And in order to explore the role of Mg 2+ on SSCs participating in implant osseointegration, we adopted the PEGASOS tissue clearing method[ 12 ], which realized the direct three-dimensional observation of soft and hard tissues without sectioning and provided great convenience for the phenotypic study of sketetal stem cells. Since there is no need to remove the implant for traditional tissue sectioning, the separation and destruction of the implant-osseointegration interface is avoided, which allows us to more intuitively and reliably observe the distribution, proliferation and differentiation of skeletal stem cells in tissues. Materials and Methods Cell culture MC3T3-E1 osteoblasts were cultured in alpha modified Eagle medium (α-MEM) (Hyclone, Laboratories, Logan, UT, USA) containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 U/mL penicillin (Hyclone, Cytiva, Marlborough, MA, USA) and 100µg/mL streptomycin (Hyclone, Cytiva, Marlborough, MA, USA) and changed every other day. MgSO4 (MACKLIN, Shanghai China) was added to the medium as an additional source of Mg 2+ . Cell counting kit-8 assay MC3T3-E1 osteoblasts were seeded in a 96-well plate at a density of 5000 cells per well with medium containing 2.5mM, 5mM, 10mM, or 20mM Mg 2+ . Cell counting kit-8 (CCK-8) assay was conducted after 1day and 3days separately. Optical density (OD) values were measured by a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm. Alkaline phosphatase staining MC3T3-E1 osteoblasts were cultured with osteogenic medium for 7 days and fixed with 4% PFA for 20 minutes. Then staining was performed with commercial Alkaline Phosphatase Assay Kit (Beyotime Biotechnology, Shanghai, China). ALP activity quantitation was performed with a commercial kit (Beyotime Biotechnology, Shanghai, China) and detected by spectrophotometer (Thermo Fisher Scientifific, Waltham, MA, USA) at 450 nm. Quantitative reverse-transcription PCR (qRT-PCR) MC3T3-E1 osteoblasts were cultured in normal medium and medium containing 5mM Mg 2+ for 72h. Then total RNA of MC3T3-E1 osteoblasts were extracted by Trizol Reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription complementary DNA (cDNA) from 1 µL of RNA using the PrimeScript RT Kit (TaKaRa Bio,Otsu, Japan), and the genes of interest were measured by SYBR Premix Ex Taq II (TaKaRa) in LightCycler 96 (Roche, Basel,Switzerland). Relative microRNA (mRNA) expression was analyzed using a delta delta comparative threshold cycle (2-ΔΔCt) method by normalizing with glyceraldehyde-3-phosphate dehydrogenase (GADPH). Fabrication of Mg-coating Implants For hydrothermal synthesis, an aqueous solution of MgO and NaOH (MACKLIN, Shanghai China) was prepared in ddwater. The hydrothermal autoclave was sealed and set up to 180°C for 12 h. Then the plates were washed, respectively, by ddwater and ethanol twice for a period of 10 min. The plates were placed in an oven and heated at 70°C for 1 h for drying. Materials Characterization of Samples The surface topography of the samples was scaned by a field-emission SEM (FE-SEM; JSM-7500 F, JEOL, Japan). Elemental analysis was performed by EDS and XRF (X-ray Fluorescence Spectrometer, Japan). Water contact angle test was performed by the WCA apparatus (Zhongchen Digital Technic Apparatus Co., Shanghai, China). Cell Morphology and Immunofluorescence Staining After 24h of incubation, cell cytoskeleton was stained by phalloidin (Shanghai Maokang Biotechnology Co., Ltd, China) and the nuclei stained by and DAPI (D8417, Sigma). Laser scanning confocal microscopy (FV3000; Olympus) was used to detect fluorescent imaging. For SEM observation, cells were fixed and dehydrated sequentially. Mice All protocols for animal care and experiments were reviewed and approved by the Subcommittee on Research and Animal Care of Sichuan University. All mice were C57BL/6 background and bred under a specific pathogen–free environment with a 12-h light-dark cycle. Rosa26-tdTomato (007905) were from Jackson Laboratory. Col2.3-GFP and LepR-Cre mice were kindly provided by Dr. Bo O. Zhou (Institute of Biochemistry and Cell Biology). LepR-Cre; tdTomato mice were generated by mating LepR-Cre mice with Rosa26-tdTomato mice. LepR-Cre;tdTomato;Col2.3-GFP mice were generating by crossing LepRCre;tdTomato mice with Col2.3-GFP mice. C57BL/6J wild-type mice were purchased from Chengdu Dossy Experimental Animals CO.LTD. Every effort has been made to minimize the suffering of mice, reduce the number of mice used, and use alternatives to in vivo techniques if possible. Surgery First mandibular molars were removed under anesthesia with a 26G syringe needle and forceps. For immediate implant placement, a self-designed sandblasted, large grit and acid etched (SLA) titanium implant (0.6-mmdiameter; WEGO China) was screwed in the socket after extraction under the stereomicroscope. On day 7 after extraction or implantation, mandibles were harvested. PEGASOS tissue clearing process PEGASOS tissue clearing process was performed as reported before [ 13 ]. Image acquisition and Analyses Images were acquired through a multiphoton microscope (Lecia SP8 DIVE). IMARIS software (version 9.1.2; Oxford Instruments) was used for data analysis and image processing[ 13 ]. Micro–computed tomography Image and Analyses The samples were scanned with a µCT-50 system (Scanco Medical) at a medium resolution and a voxel size of 7µm. IMARIS software (version 9.1.2; Oxford Instruments) was used for data analysis and image processing. Statistical Analyses All data were expressed as mean ± SEM. For each group, at least three independent experiments were conducted with duplicate sample. Statistical analysis was done with a one-way ANOVA followed by Bonferroni’s multiple comparison test. P < 0.05 was considered statistically significant. Results 1. Mg 2+ promoted proliferation and osteogenic differentiation of MC3T3-E1 osteoblasts in vitro To screen the optimal Mg 2+ concentration for cell survival, MC3T3-E1 osteoblasts were cultured in medium containing varying concentrations of Mg 2+ . The cell counting kit 8 (CCK-8) assay was performed to assess the proliferation on day 1 and 3. Notably, the proliferation was significantly promoted on 1 (p < 0.05) and 3 days (p < 0.001) in the medium containing 2.5mM, 5mM, and 10mM Mg 2+ compared with the control group. However, when the Mg 2+ concentration reached 20mM, cell proliferation was inhibited. This phenomenon was even more pronounced on day 3 (p < 0.001) (Figure.1A). Based on these results, medium with 20mM Mg 2+ was detrimental to cell proliferation while 2.5mM, 5mM, and 10mM Mg 2+ could promote proliferation, with no obvious difference in three groups. To further explore the optimal Mg2 + concentration for creating osteoblastic microenvironment, Alkaline phosphatase (ALP) staining was performed to evaluate osteogenic differentiation activity in vitro. ALP staining suggested that compared with the control group, Mg2 + evidently exhibited a significant effect on promoting osteogenic differentiation (Figure.1C). While 5mM and 10mM staining was more obvious and more effective in promoting osteogenic differentiation than 2.5mM. But this might be related to promoting cell proliferation. Therefore, we used ALP activity quantification and showed that 5mM and 10mM increased ALP activity without a statistically significant difference between the two groups (Figure.1B). Under the premise that 5mM and 10mM promoted osteogenesis without difference, we selected a low concentration of 5mM Mg2 + loaded onto the titanium surface which was optimal for the proliferation and osteogenic differentiation of the osteoblastic precursor cells. While qPCR results also indicated that 5mM Mg2 + could promote the expression of osteogenic-related genes like COL1A1 (p < 0.01), OSX (p < 0.01), and RUNX2 (p < 0.05) (Figure.1D). 2. Synthesis and characterization of implants Mg 2+ were hydrothermally assembled onto the titanium surface at the optimal concentration. SEM scanning suggested that the coating of the hydrothermal load retains the thread structure of the implant at the magnification of 1000 and 10000 (Figure.2A). While we could clearly observe that the surface of the implant is an accumulation of inorganic salts under 40000 (Figure.2A). To determine the elemental composition of the inorganic salts, elemental analysis was performed by EDS and XRF, which fully corroborated Mg successfully decorated onto the surface (Figure.2B, C). Then, we measured the water contact angle to detect the hydrophilicity of the samples after modification and the results showed that the water contact angle of the coating material was reduced remarkably (p < 0.001) (Figure.2D). To assess the bio-compatibility, we detected cell proliferation by cck8 experiment and SEM scanning. The results indicated that the Mg-coating group increased the cell proliferation of osteoblast precursor cells compared with the SLA group (Figure.2E), with more cells observed under scanning electron microscopy (Figure.2F). Interestingly, by hochest/pallion cytoskeleton staining, we found Mg-coating group had better surface osteoblast precursor cell stretching and more pseudopodia protrusion than the SLA group, proving that the Mg-coating group enhanced the extension of osteoblast precursor cells (Figure.2G). 3. Mg 2+ promoted the aggregation and osteogenic differentiation of LepR + cells around implants In order to verify the effects of Mg-coating implants on osseointegration in mice, SLA implants and Mg-coating implants were placed in the the first molar extraction sockets of the mandibles on both sides of the mice and as the Fig. 3 A showed, the mucosa at the implant site healed well after one week, on which day samples were harvested (Figure.3A). Visually, the CT images showed newly formed bone surrounding the implants postoperatively (Figure.3B). Micro–computed tomography analysis revealed bone-implant contact rate (BIC) and bone volume/tissue volume (BV/TV) of Mg-coating implants were remarkably higher compared with SLA implants qualitatively and quantitatively (Figure.3E), suggesting that Mg-coating implants truly enhanced osseointegration in mice, which was consistent with increased bone density, shown by the SHG signal around the implants (Figure.3C, F). In turn, LepR-Cre; tdTomato mice were harvested and treated following PEGASOS tissue clearing method to display cell distribution around the implant in three dimensions. The results indicated that more Tomato + cells were recruited around the Mg-coating implants compared with the SLA implants(P < 0.001), reflecting the ability of Mg 2+ to recruit LepR + cells. While osteoblasts differentiated from osteoblast precursor, (LepR + cells), the yellow signal was a overlap of the red (LepR + cells) and the green (Col2.3 − GFP + ) signal, indicating osteoblast precursor cells differentiated to osteoblasts. As we had seen in Fig. 3 D, the Mg-coating implants appeared to be surrounded by more of the yellow SHG signal, implying that Mg promotes differentiation of osteoblast precursor cells toward osteoblasts (Figure.3D, G). Altogether, through in vivo model validation, we observed for the first time that Mg promotes the aggregation of LepR + cells in vivo. What’s more, we demonstrated the ability of Mg-coating implants to promote osseointegration by promoting the aggregation and differentiation towards osteoblasts of osteoblast precursor cells. Discussion As the best way to restore missing teeth, dental implants are becoming more and more widely used in clinical practice. The healing response process after implant placement can be divided into three phases: the inflammatory phase, the stromal formation phase and the remodeling phase. After implant placement, the surrounding blood clot is gradually replaced by granulation tissue rich in skeletal stem cells and matrix components. Skeletal stem cells then increases and the transcription of bone-specific genes occurs under the action of transcription factors, which promotes the differentiation of stem cells into osteoblasts and forms woven bone with the deposited mineralized extracellular matrix. This forms the core of implant success: good osseointegration[ 14 ]. In this process, various factors such as inflammation, immune response, hormone levels may affect the proliferation and differentiation of sketetal stem cells (SSCs), thereby affecting the effect of osseointegration, and even forming fibrous tissue leading to osseointegration failure[ 15 ]. At the same time, the proliferation and differentiation of stem cells also regulate the host immune response by secreting various signaling molecules, effectively maintaining the osteogenesis-osteoclastic balance around the implant[ 16 , 17 ]. Massive research has been carried out on the surface modification of titanium in order to improve the biological activity of implants such as changing the surface morphology and elemental composition. As an essential element for cellular metabolic activities, Mg 2+ participates in a variety of enzymatic reactions in cells[ 5 , 18 ]. Evidence has proved that Mg 2+ can directly act on SSCs and promote osteogenic differentiation through multiple signaling pathways. S. Lin et al. found that Mg 2+ significantly enhances stem cell proliferation, migration, and osteogenic differentiation by activating the MAPK/ERK signaling pathway[ 19 ]. C. C. Hung et al. found that Mg 2+ significantly increased the protein expression of active β-catenin in SSCs and activated the classical Wnt pathway of SSCs to induce osteogenic differentiation[ 20 ]. Juan M. DÃaz-Tocados et al. investigated the role of Mg 2+ in activating Notch1 signaling to promote its proliferation and osteogenic differentiation[ 21 ]. However, previous studies have focused on the effect of Mg 2+ on long bones, but its function on the alveolar bone repair remains unknown. In terms of developmental origin and biological characteristics, the alveolar bone mainly originated from neural crest cells with intramembranous osteogenesis; the long bone originated from the mesoderm with endochondral osteogenesis. In our study, the addition of Mg 2+ has the obvious ability to hasten proliferation, and osteogenic differentiation of osteoblastic precursor cells. For screening optimal Mg 2+ in vitro, we performed cck8 test and ALP staining. The same proliferation, stretching, and osteogenesis experiments once again validate the biosafety of the Mg-coating and the osteogenesis-promoting phenotype. Furthermore, the implant-tissue interface is a key issue in implant research. Due to the limitations of traditional technical means, it was difficult to visually observe the changes of the implant-osseointegration interface in three dimensions. Drawing on the tissue clearing technology previously developed by our research group, we visually observed for the first time the change of Mg 2+ on the three-dimensional microenvironment around the implant, which clearly presented more LepR + cells recruitment and osteogenic differentiation towards Col2.3 − GFP + cells around Mg-coating implants. In conclusion, through in vitro and in vitro experiments we have demonstrated that Mg 2+ promotes the SSCs rescruitment, osteogenic differentiation and thus promotes peri-implant osseointegration. Declarations Acknowledgments We thank Dr. Qiang Guo from the State Key Laboratory of Oral Diseases, Sichuan University, for assistance with micro-CT scanning. We thank Dr. Yinghui Wen and Dr. Manlu Wang from the State Key Laboratory of Oral Diseases, Sichuan University, for assistance with the multiphoton microscope. Funding This work was supported by ITI Research Grant (No. 1477_2020) and Sichuan Science and Technology Program (No. 2022NSFSC1384). Author Contributions L. Liu contributed to the conception, design, data acquisition, analysis and interpretation, drafted and critically revised the manuscript; L. Huang, contributed to data interpretation, drafted and critically revised the manuscript; W. Song, contributed to data interpretation, drafted and critically revised the manuscript; D. Zhang, contributed to data interpretation, drafted and critically revised the manuscript; S. Zhang contributed to the conception, design, drafted and critically revised the manuscript. F. Yang contributed to the conception, design, drafted and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest concerning the research, authorship, and/or publication of this article. References Sadowsky SJ. Comprar Evidence-Based Implant Treatment Planning And Clinical Protocols | Steven J. Sadowsky | 9781119080039 | Wiley . 2017. De Angelis F, et al. Implant survival and success rates in patients with risk factors: results from a long-term retrospective study with a 10 to 18 years follow-up. Eur Rev Med Pharmacol Sci. 2017;21(3):433–7. Terheyden H, et al. Osseointegration–communication of cells. Clin Oral Implants Res. 2012;23(10):1127–35. Trindade R, Albrektsson T, Wennerberg A. Current concepts for the biological basis of dental implants: foreign body equilibrium and osseointegration dynamics. Oral Maxillofac Surg Clin North Am. 2015;27(2):175–83. de Baaij JH, Hoenderop JG, Bindels RJ. Magnesium in man: implications for health and disease. Physiol Rev. 2015;95(1):1–46. Zhang Y, et al. Implant-derived magnesium induces local neuronal production of CGRP to improve bone-fracture healing in rats. Nat Med. 2016;22(10):1160–9. Qiao W, et al. TRPM7 kinase-mediated immunomodulation in macrophage plays a central role in magnesium ion-induced bone regeneration. Nat Commun. 2021;12(1):2885. Maruyama T. Stem cells of the suture mesenchyme in craniofacial bone development, repair and regeneration. Keio J Med. 2019;68(2):42. Chai Y, Maxson RE Jr.. Recent advances in craniofacial morphogenesis. Dev Dyn. 2006;235(9):2353–75. Mavropoulos A, Rizzoli R, Ammann P. Different responsiveness of alveolar and tibial bone to bone loss stimuli. J Bone Min Res. 2007;22(3):403–10. Zhang D, et al. LepR-Expressing Stem Cells Are Essential for Alveolar Bone Regeneration. J Dent Res. 2020;99(11):1279–86. Jing D, et al. Tissue clearing of both hard and soft tissue organs with the PEGASOS method. Cell Res. 2018;28(8):803–18. Yi Y, et al. 3-dimensional visualization of implant-tissue interface with the polyethylene glycol associated solvent system tissue clearing method. Cell Prolif. 2019;52(3):e12578. Albrektsson T et al. Osseointegration of Implants: A Biological and Clinical Overview . 2017. Mavrogenis AF, et al. Biology of implant osseointegration. J Musculoskelet Neuronal Interact. 2009;9(2):61–71. Kikuiri T, et al. Cell-based immunotherapy with mesenchymal stem cells cures bisphosphonate-related osteonecrosis of the jaw-like disease in mice. J Bone Min Res. 2010;25(7):1668–79. Ma QL, et al. Bone mesenchymal stem cell secretion of sRANKL/OPG/M-CSF in response to macrophage-mediated inflammatory response influences osteogenesis on nanostructured Ti surfaces. Biomaterials. 2018;154:234–47. Al Alawi AM, Majoni SW, Falhammar H. Magnesium and Human Health: Perspectives and Research Directions. Int J Endocrinol, 2018. 2018: p. 9041694. Lin S, et al. A Magnesium-Enriched 3D Culture System that Mimics the Bone Development Microenvironment for Vascularized Bone Regeneration. Adv Sci (Weinh). 2019;6(12):1900209. Hung CC, et al. The role of magnesium ions in bone regeneration involves the canonical Wnt signaling pathway. Acta Biomater. 2019;98:246–55. Díaz-Tocados JM, et al. Magnesium Chloride promotes Osteogenesis through Notch signaling activation and expansion of Mesenchymal Stem Cells. Sci Rep. 2017;7(1):7839. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5765695","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":398325248,"identity":"2921bfa6-dc9a-4ce5-951c-aab7fb2a7a03","order_by":0,"name":"Linfeng Liu","email":"","orcid":"","institution":"State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology","correspondingAuthor":false,"prefix":"","firstName":"Linfeng","middleName":"","lastName":"Liu","suffix":""},{"id":398325250,"identity":"bad5b623-75ec-4dd1-921e-fc6110f274d5","order_by":1,"name":"Lixuan Huang","email":"","orcid":"","institution":"State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology","correspondingAuthor":false,"prefix":"","firstName":"Lixuan","middleName":"","lastName":"Huang","suffix":""},{"id":398325253,"identity":"5aefbdb5-26a6-43d2-b7b8-754f9738c2c5","order_by":2,"name":"Wei Song","email":"","orcid":"","institution":"State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Song","suffix":""},{"id":398325254,"identity":"a6bf163e-ec48-4fcd-9c06-096e71dc9335","order_by":3,"name":"Danting Zhang","email":"","orcid":"","institution":"State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology","correspondingAuthor":false,"prefix":"","firstName":"Danting","middleName":"","lastName":"Zhang","suffix":""},{"id":398325255,"identity":"e864fb1b-59ea-49aa-aa1f-7c7086c996ec","order_by":4,"name":"Shiwen Zhang","email":"","orcid":"","institution":"State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology","correspondingAuthor":false,"prefix":"","firstName":"Shiwen","middleName":"","lastName":"Zhang","suffix":""},{"id":398325256,"identity":"f05363c6-6777-4684-b833-1523415ef867","order_by":5,"name":"Fan Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYBACfvb+hw8+VLDJ8bM3EKlFsucMs+GMM3zGkj0HiNRicCOHTZq3TS5xw40EYl125uwxyRlnzIwlZz7eeIOhxiaaoA7G9r5kiw8VaXL80mnFFgzH0nIbCGlh5jlgeHPGmWPGkrNzzCQYGw4T1sImkWAA9Mv/xA03zxCphUcixwiohQ3ofR4itUjwHEsGBjIbMJCBfkkgxi/2x5sPQqPy8MYbH2psCGtBBgYSCaQoh2ghVccoGAWjYBSMDAAAD9NEZMquOTYAAAAASUVORK5CYII=","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":true,"prefix":"","firstName":"Fan","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-01-05 02:08:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5765695/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5765695/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73269312,"identity":"3ce46dd4-3c61-4f19-88bb-c3f6fd20c805","added_by":"auto","created_at":"2025-01-08 10:43:07","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":597104,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMg\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e promoted proliferation and osteogenic differentiation of MC3T3-E1 osteoblasts in vitro.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Cell proliferation assay of MC3T3-E1 osteoblasts by CCK-8 test on day 1 and day 3 (n = 3). (B-C) ALP activity quantitation and ALP staining of MC3T3-E1 cells in the medium containing gradient Mg\u003csup\u003e2+\u003c/sup\u003e. (n = 3).\u0026nbsp; (D) qRT-PCR analyses of the expression of \u003cem\u003eCOL1A1, OSX, RUNX2 \u003c/em\u003e(n = 3).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5765695/v1/90e95f7ea04d62373a670391.jpg"},{"id":73269313,"identity":"1ca31e85-50ac-48fe-a79f-468356551036","added_by":"auto","created_at":"2025-01-08 10:43:07","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":931176,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynthesis and characterization of materials.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) SEM scanning images of the implants. (B-C) EDS and XRF showed the elemental composition of the samples. (D) Contact angle and representative images of droplets on different samples (n = 5). (E) Cell proliferation assay of MC3T3-E1 osteoblasts cultured on different samples by CCK-8 test (n = 3). (F) SEM morphology of MC3T3-E1 osteoblasts on different samples (n = 3). (G) Representative images morphology and filopodia counts of MC3T3-E1 osteoblasts on different samples.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5765695/v1/891efc1547e099cab2132095.jpg"},{"id":73271598,"identity":"072b7029-35e6-4ab9-91d9-90bc578e5206","added_by":"auto","created_at":"2025-01-08 10:59:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":779309,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMg\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e promoted osseointegration in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Implant placement and healing 7 days post-surgery. (B) Representative images of μCT reconstruction of the newly formed bone (red) with implants (gray) 7 days post-surgery. Scale bar = 150 μm. (C) Second harmonic generation (SHG) signal (green) around implants. Scale bar = 100 μm. (D) Tissue clearing–based 3-dimensional images around implants showed osteogenesis on day 7 after surgery, with tdTomato signal displaying \u003cem\u003eLepR\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003ecells (red), \u003cem\u003eCol2.3-GFP\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003edisplaying bone (green) and implant (gray). Scale bar = 100 μm. (E) Quantitative analyses of bone-implant contact rate (BIC) and bone volume/tissue volume (BV/TV) (n = 3). (F) Quantitative analyses of SHG, \u003cem\u003eLepR\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003ecells and \u003cem\u003eLepR\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e;Col2.3-GFP\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003ecells around implants (n = 3).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5765695/v1/d87955979e1262869d58ec15.jpg"},{"id":73315071,"identity":"62c92ae5-1ba5-4c22-b5c1-b84a54a077a4","added_by":"auto","created_at":"2025-01-08 19:53:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2844550,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5765695/v1/16093fd6-cb15-40c4-8c3b-e74f56b6103b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Magnesium promotes implant osseointegration by regulating LepR + stem cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSince Professor Branemark proposed the theory of osseointegration in the 1960s, with the continuous development and maturity of implant surgery technology, implant-supported restoration have been more and more clinically applied, and are becoming the preferred treatment option for missing teeth[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In suitable cases, satisfactory osseointegration can be achieved after implant placement. However, in clinical practice, poor osseointegration is often caused by pathological or physiological reasons such as inflammation, systemic diseases, age and hormone levels. While implant failure still occurs due to the particularity and complexity of the local oral environment[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBone reconstruction around the implant exists in the whole functional dynamic cycle. Its occurrence and development require fully mobilizing the skeletal stem cells (SSCs) to function, while the homing and differentiation of SSCs determine the speed and efficiency of osseointegration. When the implant is inserted in the alveolar bone, it is surrounded by cells, cytokines, etc. As healing progresses, SSCs gradually aggregate and differentiate towards osteoblasts which accumulate, secrete osteogenic factors, form new bone tissue, and ultimately promote the formation of osseointegration. In the process of bone healing, various factors such as inflammation, immune response, hormone levels may affect the proliferation and differentiation of SSCs, thereby affecting the effect of osseointegration, and even form fibrous tissue leading to osseointegration failure[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs one of the indispensable mineral elements of the human body, more than half of the magnesium ion (Mg\u003csup\u003e2+\u003c/sup\u003e) exists in the bones, participating in the metabolic processes[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Since Professor Qin first demonstrated in Nature Medicine that Mg\u003csup\u003e2+\u003c/sup\u003e accelerated osteogenic differentiation of stem cells by promoting the secretion of CGRP by dorsal root ganglion in 2016[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], the preferred osteogenic activity of Mg\u003csup\u003e2+\u003c/sup\u003e has attracted widespread attention. In 2021, Professor Kelvin W K Yeung demonstrated in Nature Communication that Mg\u003csup\u003e2+\u003c/sup\u003e contributes to the formation of the osteogenic immune microenvironment, which once again verified that the osteogenic activity of Mg\u003csup\u003e2+\u003c/sup\u003e was closely related to its regulation of the osteoblastic microenvironment[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAt present, the role of Mg\u003csup\u003e2+\u003c/sup\u003e in promoting bone regeneration has been widely confirmed, but almost all in vitro and clinical studies on Mg\u003csup\u003e2+\u003c/sup\u003e to promote SSCs osteogenesis are derived from bone marrow mesenchymal stem cells of long bones, while it remains unknown whether Mg\u003csup\u003e2+\u003c/sup\u003e could affect SSCs in alveolar bone. Alveolar bones and long bones are different in origin and biological characteristics[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In our previous studies, we has proved that LepR\u003csup\u003e+\u003c/sup\u003e cells were osteogenic precursor cells of the alveolar bone and played an important role in the bone healing[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. So we used \u003cem\u003eLepR-Cre;tdTomato\u003c/em\u003e mice to label skeletal stem cells in the alveolar bone. And in order to explore the role of Mg\u003csup\u003e2+\u003c/sup\u003e on SSCs participating in implant osseointegration, we adopted the PEGASOS tissue clearing method[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], which realized the direct three-dimensional observation of soft and hard tissues without sectioning and provided great convenience for the phenotypic study of sketetal stem cells. Since there is no need to remove the implant for traditional tissue sectioning, the separation and destruction of the implant-osseointegration interface is avoided, which allows us to more intuitively and reliably observe the distribution, proliferation and differentiation of skeletal stem cells in tissues.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eMC3T3-E1 osteoblasts were cultured in alpha modified Eagle medium (α-MEM) (Hyclone, Laboratories, Logan, UT, USA) containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 U/mL penicillin (Hyclone, Cytiva, Marlborough, MA, USA) and 100\u0026micro;g/mL streptomycin (Hyclone, Cytiva, Marlborough, MA, USA) and changed every other day. MgSO4 (MACKLIN, Shanghai China) was added to the medium as an additional source of Mg\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell counting kit-8 assay\u003c/h3\u003e\n\u003cp\u003eMC3T3-E1 osteoblasts were seeded in a 96-well plate at a density of 5000 cells per well with medium containing 2.5mM, 5mM, 10mM, or 20mM Mg\u003csup\u003e2+\u003c/sup\u003e. Cell counting kit-8 (CCK-8) assay was conducted after 1day and 3days separately. Optical density (OD) values were measured by a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm.\u003c/p\u003e\n\u003ch3\u003eAlkaline phosphatase staining\u003c/h3\u003e\n\u003cp\u003eMC3T3-E1 osteoblasts were cultured with osteogenic medium for 7 days and fixed with 4% PFA for 20 minutes. Then staining was performed with commercial Alkaline Phosphatase Assay Kit (Beyotime Biotechnology, Shanghai, China). ALP activity quantitation was performed with a commercial kit (Beyotime Biotechnology, Shanghai, China) and detected by spectrophotometer (Thermo Fisher Scientifific, Waltham, MA, USA) at 450 nm.\u003c/p\u003e\n\u003ch3\u003eQuantitative reverse-transcription PCR (qRT-PCR)\u003c/h3\u003e\n\u003cp\u003eMC3T3-E1 osteoblasts were cultured in normal medium and medium containing 5mM Mg\u003csup\u003e2+\u003c/sup\u003e for 72h. Then total RNA of MC3T3-E1 osteoblasts were extracted by Trizol Reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription complementary DNA (cDNA) from 1 \u0026micro;L of RNA using the PrimeScript RT Kit (TaKaRa Bio,Otsu, Japan), and the genes of interest were measured by SYBR Premix Ex Taq II (TaKaRa) in LightCycler 96 (Roche, Basel,Switzerland). Relative microRNA (mRNA) expression was analyzed using a delta delta comparative threshold cycle (2-ΔΔCt) method by normalizing with glyceraldehyde-3-phosphate dehydrogenase (GADPH).\u003c/p\u003e\n\u003ch3\u003eFabrication of Mg-coating Implants\u003c/h3\u003e\n\u003cp\u003eFor hydrothermal synthesis, an aqueous solution of MgO and NaOH (MACKLIN, Shanghai China) was prepared in ddwater. The hydrothermal autoclave was sealed and set up to 180\u0026deg;C for 12 h. Then the plates were washed, respectively, by ddwater and ethanol twice for a period of 10 min. The plates were placed in an oven and heated at 70\u0026deg;C for 1 h for drying.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMaterials Characterization of Samples\u003c/h2\u003e \u003cp\u003eThe surface topography of the samples was scaned by a field-emission SEM (FE-SEM; JSM-7500 F, JEOL, Japan). Elemental analysis was performed by EDS and XRF (X-ray Fluorescence Spectrometer, Japan). Water contact angle test was performed by the WCA apparatus (Zhongchen Digital Technic Apparatus Co., Shanghai, China).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell Morphology and Immunofluorescence Staining\u003c/h3\u003e\n\u003cp\u003eAfter 24h of incubation, cell cytoskeleton was stained by phalloidin (Shanghai Maokang Biotechnology Co., Ltd, China) and the nuclei stained by and DAPI (D8417, Sigma). Laser scanning confocal microscopy (FV3000; Olympus) was used to detect fluorescent imaging. For SEM observation, cells were fixed and dehydrated sequentially.\u003c/p\u003e\n\u003ch3\u003eMice\u003c/h3\u003e\n\u003cp\u003e All protocols for animal care and experiments were reviewed and approved by the Subcommittee on Research and Animal Care of Sichuan University. All mice were C57BL/6 background and bred under a specific pathogen\u0026ndash;free environment with a 12-h light-dark cycle. Rosa26-tdTomato (007905) were from Jackson Laboratory. \u003cem\u003eCol2.3-GFP\u003c/em\u003e and \u003cem\u003eLepR-Cre\u003c/em\u003e mice were kindly provided by Dr. Bo O. Zhou (Institute of Biochemistry and Cell Biology). \u003cem\u003eLepR-Cre; tdTomato\u003c/em\u003e mice were generated by mating \u003cem\u003eLepR-Cre\u003c/em\u003e mice with \u003cem\u003eRosa26-tdTomato\u003c/em\u003e mice. \u003cem\u003eLepR-Cre;tdTomato;Col2.3-GFP\u003c/em\u003e mice were generating by crossing \u003cem\u003eLepRCre;tdTomato\u003c/em\u003e mice with \u003cem\u003eCol2.3-GFP\u003c/em\u003e mice. C57BL/6J wild-type mice were purchased from Chengdu Dossy Experimental Animals CO.LTD. Every effort has been made to minimize the suffering of mice, reduce the number of mice used, and use alternatives to in vivo techniques if possible.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSurgery\u003c/h2\u003e \u003cp\u003eFirst mandibular molars were removed under anesthesia with a 26G syringe needle and forceps. For immediate implant placement, a self-designed sandblasted, large grit and acid etched (SLA) titanium implant (0.6-mmdiameter; WEGO China) was screwed in the socket after extraction under the stereomicroscope. On day 7 after extraction or implantation, mandibles were harvested.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePEGASOS tissue clearing process\u003c/h2\u003e \u003cp\u003ePEGASOS tissue clearing process was performed as reported before [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImage acquisition and Analyses\u003c/h2\u003e \u003cp\u003eImages were acquired through a multiphoton microscope (Lecia SP8 DIVE). IMARIS software (version 9.1.2; Oxford Instruments) was used for data analysis and image processing[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMicro\u0026ndash;computed tomography Image and Analyses\u003c/h2\u003e \u003cp\u003eThe samples were scanned with a \u0026micro;CT-50 system (Scanco Medical) at a medium resolution and a voxel size of 7\u0026micro;m. IMARIS software (version 9.1.2; Oxford Instruments) was used for data analysis and image processing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyses\u003c/h2\u003e \u003cp\u003eAll data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. For each group, at least three independent experiments were conducted with duplicate sample. Statistical analysis was done with a one-way ANOVA followed by Bonferroni\u0026rsquo;s multiple comparison test. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e1. Mg\u003csup\u003e2+\u003c/sup\u003e promoted proliferation and osteogenic differentiation of MC3T3-E1 osteoblasts in vitro\u003c/h2\u003e \u003cp\u003eTo screen the optimal Mg\u003csup\u003e2+\u003c/sup\u003e concentration for cell survival, MC3T3-E1 osteoblasts were cultured in medium containing varying concentrations of Mg\u003csup\u003e2+\u003c/sup\u003e. The cell counting kit 8 (CCK-8) assay was performed to assess the proliferation on day 1 and 3. Notably, the proliferation was significantly promoted on 1 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 3 days (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in the medium containing 2.5mM, 5mM, and 10mM Mg\u003csup\u003e2+\u003c/sup\u003e compared with the control group. However, when the Mg\u003csup\u003e2+\u003c/sup\u003e concentration reached 20mM, cell proliferation was inhibited. This phenomenon was even more pronounced on day 3 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Figure.1A). Based on these results, medium with 20mM Mg\u003csup\u003e2+\u003c/sup\u003e was detrimental to cell proliferation while 2.5mM, 5mM, and 10mM Mg\u003csup\u003e2+\u003c/sup\u003e could promote proliferation, with no obvious difference in three groups.\u003c/p\u003e \u003cp\u003eTo further explore the optimal Mg2\u0026thinsp;+\u0026thinsp;concentration for creating osteoblastic microenvironment, Alkaline phosphatase (ALP) staining was performed to evaluate osteogenic differentiation activity in vitro. ALP staining suggested that compared with the control group, Mg2\u0026thinsp;+\u0026thinsp;evidently exhibited a significant effect on promoting osteogenic differentiation (Figure.1C). While 5mM and 10mM staining was more obvious and more effective in promoting osteogenic differentiation than 2.5mM. But this might be related to promoting cell proliferation. Therefore, we used ALP activity quantification and showed that 5mM and 10mM increased ALP activity without a statistically significant difference between the two groups (Figure.1B). Under the premise that 5mM and 10mM promoted osteogenesis without difference, we selected a low concentration of 5mM Mg2\u0026thinsp;+\u0026thinsp;loaded onto the titanium surface which was optimal for the proliferation and osteogenic differentiation of the osteoblastic precursor cells. While qPCR results also indicated that 5mM Mg2\u0026thinsp;+\u0026thinsp;could promote the expression of osteogenic-related genes like COL1A1 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), OSX (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and RUNX2 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Figure.1D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2. Synthesis and characterization of implants\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eMg\u003csup\u003e2+\u003c/sup\u003e were hydrothermally assembled onto the titanium surface at the optimal concentration. SEM scanning suggested that the coating of the hydrothermal load retains the thread structure of the implant at the magnification of 1000 and 10000 (Figure.2A). While we could clearly observe that the surface of the implant is an accumulation of inorganic salts under 40000 (Figure.2A). To determine the elemental composition of the inorganic salts, elemental analysis was performed by EDS and XRF, which fully corroborated Mg successfully decorated onto the surface (Figure.2B, C). Then, we measured the water contact angle to detect the hydrophilicity of the samples after modification and the results showed that the water contact angle of the coating material was reduced remarkably (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Figure.2D).\u003c/p\u003e \u003cp\u003eTo assess the bio-compatibility, we detected cell proliferation by cck8 experiment and SEM scanning. The results indicated that the Mg-coating group increased the cell proliferation of osteoblast precursor cells compared with the SLA group (Figure.2E), with more cells observed under scanning electron microscopy (Figure.2F). Interestingly, by hochest/pallion cytoskeleton staining, we found Mg-coating group had better surface osteoblast precursor cell stretching and more pseudopodia protrusion than the SLA group, proving that the Mg-coating group enhanced the extension of osteoblast precursor cells (Figure.2G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3. Mg\u003csup\u003e2+\u003c/sup\u003e promoted the aggregation and osteogenic differentiation of LepR\u003csup\u003e+\u003c/sup\u003e cells around implants\u003c/h2\u003e \u003cp\u003eIn order to verify the effects of Mg-coating implants on osseointegration in mice, SLA implants and Mg-coating implants were placed in the the first molar extraction sockets of the mandibles on both sides of the mice and as the Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA showed, the mucosa at the implant site healed well after one week, on which day samples were harvested (Figure.3A). Visually, the CT images showed newly formed bone surrounding the implants postoperatively (Figure.3B). Micro\u0026ndash;computed tomography analysis revealed bone-implant contact rate (BIC) and bone volume/tissue volume (BV/TV) of Mg-coating implants were remarkably higher compared with SLA implants qualitatively and quantitatively (Figure.3E), suggesting that Mg-coating implants truly enhanced osseointegration in mice, which was consistent with increased bone density, shown by the SHG signal around the implants (Figure.3C, F).\u003c/p\u003e \u003cp\u003eIn turn, \u003cem\u003eLepR-Cre; tdTomato\u003c/em\u003e mice were harvested and treated following PEGASOS tissue clearing method to display cell distribution around the implant in three dimensions. The results indicated that more Tomato\u003csup\u003e+\u003c/sup\u003e cells were recruited around the Mg-coating implants compared with the SLA implants(P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), reflecting the ability of Mg\u003csup\u003e2+\u003c/sup\u003e to recruit LepR\u0026thinsp;+\u0026thinsp;cells. While osteoblasts differentiated from osteoblast precursor, (LepR\u003csup\u003e+\u003c/sup\u003e cells), the yellow signal was a overlap of the red (LepR\u003csup\u003e+\u003c/sup\u003e cells) and the green (Col2.3\u003csup\u003e\u0026minus;\u003c/sup\u003eGFP\u003csup\u003e+\u003c/sup\u003e) signal, indicating osteoblast precursor cells differentiated to osteoblasts. As we had seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, the Mg-coating implants appeared to be surrounded by more of the yellow SHG signal, implying that Mg promotes differentiation of osteoblast precursor cells toward osteoblasts (Figure.3D, G).\u003c/p\u003e \u003cp\u003eAltogether, through in vivo model validation, we observed for the first time that Mg promotes the aggregation of LepR\u003csup\u003e+\u003c/sup\u003e cells in vivo. What\u0026rsquo;s more, we demonstrated the ability of Mg-coating implants to promote osseointegration by promoting the aggregation and differentiation towards osteoblasts of osteoblast precursor cells.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs the best way to restore missing teeth, dental implants are becoming more and more widely used in clinical practice. The healing response process after implant placement can be divided into three phases: the inflammatory phase, the stromal formation phase and the remodeling phase. After implant placement, the surrounding blood clot is gradually replaced by granulation tissue rich in skeletal stem cells and matrix components. Skeletal stem cells then increases and the transcription of bone-specific genes occurs under the action of transcription factors, which promotes the differentiation of stem cells into osteoblasts and forms woven bone with the deposited mineralized extracellular matrix. This forms the core of implant success: good osseointegration[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In this process, various factors such as inflammation, immune response, hormone levels may affect the proliferation and differentiation of sketetal stem cells (SSCs), thereby affecting the effect of osseointegration, and even forming fibrous tissue leading to osseointegration failure[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. At the same time, the proliferation and differentiation of stem cells also regulate the host immune response by secreting various signaling molecules, effectively maintaining the osteogenesis-osteoclastic balance around the implant[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMassive research has been carried out on the surface modification of titanium in order to improve the biological activity of implants such as changing the surface morphology and elemental composition. As an essential element for cellular metabolic activities, Mg\u003csup\u003e2+\u003c/sup\u003e participates in a variety of enzymatic reactions in cells[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Evidence has proved that Mg\u003csup\u003e2+\u003c/sup\u003e can directly act on SSCs and promote osteogenic differentiation through multiple signaling pathways. S. Lin et al. found that Mg\u003csup\u003e2+\u003c/sup\u003e significantly enhances stem cell proliferation, migration, and osteogenic differentiation by activating the MAPK/ERK signaling pathway[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. C. C. Hung et al. found that Mg\u003csup\u003e2+\u003c/sup\u003e significantly increased the protein expression of active β-catenin in SSCs and activated the classical Wnt pathway of SSCs to induce osteogenic differentiation[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Juan M. D\u0026Atilde;az-Tocados et al. investigated the role of Mg\u003csup\u003e2+\u003c/sup\u003e in activating Notch1 signaling to promote its proliferation and osteogenic differentiation[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, previous studies have focused on the effect of Mg\u003csup\u003e2+\u003c/sup\u003e on long bones, but its function on the alveolar bone repair remains unknown. In terms of developmental origin and biological characteristics, the alveolar bone mainly originated from neural crest cells with intramembranous osteogenesis; the long bone originated from the mesoderm with endochondral osteogenesis.\u003c/p\u003e \u003cp\u003eIn our study, the addition of Mg\u003csup\u003e2+\u003c/sup\u003e has the obvious ability to hasten proliferation, and osteogenic differentiation of osteoblastic precursor cells. For screening optimal Mg\u003csup\u003e2+\u003c/sup\u003e in vitro, we performed cck8 test and ALP staining. The same proliferation, stretching, and osteogenesis experiments once again validate the biosafety of the Mg-coating and the osteogenesis-promoting phenotype.\u003c/p\u003e \u003cp\u003eFurthermore, the implant-tissue interface is a key issue in implant research. Due to the limitations of traditional technical means, it was difficult to visually observe the changes of the implant-osseointegration interface in three dimensions. Drawing on the tissue clearing technology previously developed by our research group, we visually observed for the first time the change of Mg\u003csup\u003e2+\u003c/sup\u003e on the three-dimensional microenvironment around the implant, which clearly presented more LepR\u003csup\u003e+\u003c/sup\u003e cells recruitment and osteogenic differentiation towards Col2.3\u003csup\u003e\u0026minus;\u003c/sup\u003eGFP\u003csup\u003e+\u003c/sup\u003e cells around Mg-coating implants. In conclusion, through in vitro and in vitro experiments we have demonstrated that Mg\u003csup\u003e2+\u003c/sup\u003e promotes the SSCs rescruitment, osteogenic differentiation and thus promotes peri-implant osseointegration.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Qiang Guo from the State Key Laboratory of Oral Diseases, Sichuan University, for assistance with micro-CT scanning. We thank Dr. Yinghui Wen and Dr. Manlu Wang from the State Key Laboratory of Oral Diseases, Sichuan University, for assistance with the multiphoton microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by ITI Research Grant (No. 1477_2020) and Sichuan Science and Technology Program (No. 2022NSFSC1384).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL. Liu contributed to the conception, design, data acquisition, analysis and interpretation, drafted and critically revised the manuscript; L. Huang, contributed to data interpretation, drafted and critically revised the manuscript; W. Song, contributed to data interpretation, drafted and critically revised the manuscript; D. Zhang, contributed to data interpretation, drafted and critically revised the manuscript; S. Zhang contributed to the conception, design, drafted and critically revised the manuscript. F. Yang contributed to the conception, design, drafted and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Conflicting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declared no potential conflicts of interest concerning the research, authorship, and/or publication of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSadowsky SJ. \u003cem\u003eComprar Evidence-Based Implant Treatment Planning And Clinical Protocols | Steven J. Sadowsky | 9781119080039 | Wiley\u003c/em\u003e. 2017.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Angelis F, et al. Implant survival and success rates in patients with risk factors: results from a long-term retrospective study with a 10 to 18 years follow-up. Eur Rev Med Pharmacol Sci. 2017;21(3):433\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTerheyden H, et al. Osseointegration\u0026ndash;communication of cells. Clin Oral Implants Res. 2012;23(10):1127\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrindade R, Albrektsson T, Wennerberg A. Current concepts for the biological basis of dental implants: foreign body equilibrium and osseointegration dynamics. Oral Maxillofac Surg Clin North Am. 2015;27(2):175\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Baaij JH, Hoenderop JG, Bindels RJ. Magnesium in man: implications for health and disease. Physiol Rev. 2015;95(1):1\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, et al. Implant-derived magnesium induces local neuronal production of CGRP to improve bone-fracture healing in rats. Nat Med. 2016;22(10):1160\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQiao W, et al. TRPM7 kinase-mediated immunomodulation in macrophage plays a central role in magnesium ion-induced bone regeneration. Nat Commun. 2021;12(1):2885.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaruyama T. Stem cells of the suture mesenchyme in craniofacial bone development, repair and regeneration. Keio J Med. 2019;68(2):42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChai Y, Maxson RE Jr.. Recent advances in craniofacial morphogenesis. Dev Dyn. 2006;235(9):2353\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMavropoulos A, Rizzoli R, Ammann P. Different responsiveness of alveolar and tibial bone to bone loss stimuli. J Bone Min Res. 2007;22(3):403\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang D, et al. LepR-Expressing Stem Cells Are Essential for Alveolar Bone Regeneration. J Dent Res. 2020;99(11):1279\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJing D, et al. Tissue clearing of both hard and soft tissue organs with the PEGASOS method. Cell Res. 2018;28(8):803\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYi Y, et al. 3-dimensional visualization of implant-tissue interface with the polyethylene glycol associated solvent system tissue clearing method. Cell Prolif. 2019;52(3):e12578.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlbrektsson T et al. \u003cem\u003eOsseointegration of Implants: A Biological and Clinical Overview\u003c/em\u003e. 2017.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMavrogenis AF, et al. Biology of implant osseointegration. J Musculoskelet Neuronal Interact. 2009;9(2):61\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKikuiri T, et al. Cell-based immunotherapy with mesenchymal stem cells cures bisphosphonate-related osteonecrosis of the jaw-like disease in mice. J Bone Min Res. 2010;25(7):1668\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa QL, et al. Bone mesenchymal stem cell secretion of sRANKL/OPG/M-CSF in response to macrophage-mediated inflammatory response influences osteogenesis on nanostructured Ti surfaces. Biomaterials. 2018;154:234\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl Alawi AM, Majoni SW, Falhammar H. \u003cem\u003eMagnesium and Human Health: Perspectives and Research Directions.\u003c/em\u003e Int J Endocrinol, 2018. 2018: p. 9041694.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin S, et al. A Magnesium-Enriched 3D Culture System that Mimics the Bone Development Microenvironment for Vascularized Bone Regeneration. Adv Sci (Weinh). 2019;6(12):1900209.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHung CC, et al. The role of magnesium ions in bone regeneration involves the canonical Wnt signaling pathway. Acta Biomater. 2019;98:246\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD\u0026iacute;az-Tocados JM, et al. Magnesium Chloride promotes Osteogenesis through Notch signaling activation and expansion of Mesenchymal Stem Cells. Sci Rep. 2017;7(1):7839.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"magnesium, implant, osseointegration, LepR+ stem cells, PEGASOS","lastPublishedDoi":"10.21203/rs.3.rs-5765695/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5765695/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eHoming and differentiation of stem cells is a necessary step in achieving implant osseointegration. However, the specific role and mechanism about stem cells in the osseointegration process were rarely reported. Rencently, LepR\u003csup\u003e+\u003c/sup\u003e cells were identified as crucial skeletal stem cells in the long bone and alveolar bone, which participated homeostasis maintenance and damage repair. As one of the indispensable trace elements in the human body, Mg\u003csup\u003e2+\u003c/sup\u003e were proved to promote vascularized bone regeneration. Here, by combining tissue clearing technique and immunofluorescent mice, we proved that Mg\u003csup\u003e2+\u003c/sup\u003e promoted implant osseointegration by regulating LepR\u003csup\u003e+\u003c/sup\u003e stem cells aggregation and differentiation at the implant-bone interface in vivo and provided a new perspective to promote the study of implant research.\u003c/p\u003e\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eThe aim of the study was to explore the effects of Mg\u003csup\u003e2+\u003c/sup\u003e in promoting implant osseointegration in the alveolar bone.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe cell proliferation and osteogenic differentiation were performed to screening the optimal Mg\u003csup\u003e2+\u003c/sup\u003e concentration for MC3T3-E1 osteoblasts. Then the optimal Mg\u003csup\u003e2+\u003c/sup\u003e was loaded on a self-designed SLA implant hydrothermally. Characterization and bio-compatibility of materials were detected. In vivo, combining the PEGASOS with \u003cem\u003eLepR-Cre;tdTomato;Col2.3-GFP\u003c/em\u003e mice, we traced the \u003cem\u003eLepR\u003c/em\u003e positive \u003cb\u003e(\u003c/b\u003e LepR\u003csup\u003e+\u003c/sup\u003e \u003cb\u003e)\u003c/b\u003e stem cells aggregation and differentiation at the implant-bone interface.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003e5mM Mg\u003csup\u003e2+\u003c/sup\u003e was screened to be the optimal concentration for MC3T3-E1 osteoblasts. EDS and XRF proved that Mg\u003csup\u003e2+\u003c/sup\u003e was successfully corroborated onto the implants. Further detection confirmed the better stretching and proliferatio of Mg-coating implants. 3-D imaging revealed that Mg-coating implants facilitated LepR\u003csup\u003e+\u003c/sup\u003e cells aggregation and differentiation to Col2.3-GFP\u003csup\u003e+\u003c/sup\u003e cells.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eMg\u003csup\u003e2+\u003c/sup\u003e promoted osseointegration around implants by facilitating the LepR\u003csup\u003e+\u003c/sup\u003e cells accumulation and osteogenic differentiation.\u003c/p\u003e","manuscriptTitle":"Magnesium promotes implant osseointegration by regulating LepR + stem cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-08 10:43:03","doi":"10.21203/rs.3.rs-5765695/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d6acf07b-2a31-4e49-9b27-d77ab01507fa","owner":[],"postedDate":"January 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-08T19:53:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-08 10:43:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5765695","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5765695","identity":"rs-5765695","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}