Mg²⁺-doped Carbon Dots Synthesized Based On Lycium Ruthenicum In Cell Imaging And Promoting Osteogenic Differentiation In Vitro

Objective:Large jaw defects are still a difficult medical disease to overcome,and how to repair bone defects and promote bone tissue regeneration is a major challenge in the medical field.The commonly used means are autograft and allograft,but they are limited by their limited source,donor injury,and immune rejection.As a new alternative to transplantation,bone tissue engineering constructs an active osteogenic microenvironment to promote bone tissue regeneration by implanting artificial biomaterials in the area of bone defects.Therefore,the selection of suitable biomaterials as bone graft substitutes is crucial in bone tissue engineering.Considering that the microscopic processes of bone regeneration are difficult to detect,the application of fluorescent carbon dots（CDs）to bone regeneration can enable the visual monitoring of osteogenic effects.Carbon dots are simple to synthesize,have unique fluorescent properties,and are biocompatible,but the osteogenic effect of carbon dots alone is insufficient.In this study,we investigated the feasibility of Mg-CDs as bone repair biomaterials by using a one-step hydrothermal method to prepare Mg-doped fluorescent carbon dots（Mg-CDs）,characterized the physicochemical properties of Mg-CDs,observed the fluorescence imaging of Mg-CDs on cells,and investigated their in vitro cytotoxicity and osteogenic induction.The ability of Mg-CDs to induce osteogenic differentiation in vitro was investigated.Methods:In this study,natural lycium ruthenicum was co-mixed with magnesium chloride hexahydrate to synthesize fluorescent carbon dots（Mg-CDs）by a one-step hydrothermal method to investigate the physicochemical properties,fluorescence imaging of cells,cytotoxicity and osteogenic properties of the materials.The materials’osteogenic characteristics,photoluminescence spectra,Fourier transform infrared spectroscopy（FT-IR）,and ultraviolet-visible spectroscopy（UV-vis）were all examined.Energy Dispersive Spectroscopy（EDS）,high-resolution transmission electron microscope,far-infrared（FT-IR）,X-ray photoelectron spectroscopy（XPS）,and（transmission electron microscope,TEM）X-ray photoelectron spectroscopy（XPS）,Energy dispersive spectroscopy（EDS）,high-resolution transmission electron microscope（TEM）,X-ray diffraction（XRD）,and zeta potential meter were used to examine the macroscopic physicochemical properties of the materials to determine their morphology,surface functional groups,and elemental chemical structure states.In addition,mouse embryonic osteoblast precursor cells（MC3T3-e1）were co-cultured with Mg-CDs,and the co-cultured cells were then examined using laser confocal microscopy（LSCM）to achieve fluorescence imaging of the material on the cells.By using the MTT assay,it was possible to measure the survival and cell growth of Mg-CDs co-cultured cells and evaluate the material’s in vitro cytotoxicity.ALP activity assay,alizarin red S（ARS）staining,and quantitative real-time polymerase chain reaction（q RT-PCR）of osteogenic related genes（ALP,Runx-2,Col-I,and OC）were used to evaluate the osteogenic induction effect of the materials.Finally,osteogenic differentiation was induced by MC3T3-e1 under Mg-CDs culture conditions.Results:1.EDS and XPS results together verified the successful doping of Mg elements,and the content of Mg atoms in the products increased with the increase of the mass of the reactant magnesium chloride hexahydrate,so the sample with the mass ratio of lycium ruthenicum and magnesium chloride hexahydrate of 1:4 was named as Mg-CDs.Mg-CDs emitted bright green fluorescence under UV excitation and exhibited the excitation dependence of carbon dots.According to the TEM findings,the Mg-CDs had a spherical form,an average diameter of 2.0±0.02 nm,and distinct lattice stripes.The type of functional groups on the surface of CDs and the location of the absorption peaks were unaffected by the doping of Mg²⁺,according to FT-IR measurements,preserving the CDs original functional groups.XRD demonstrated that both CDs and Mg-CDs were graphitized materials,and the Mg²⁺doping improved the crystallinity of the materials and enhanced the stability of the materials.2.The in vitro cell studies revealed that when the culture dose of Mg-CDs was as high as 800μg m L^-1,the cell survival rate was maintained above 85%.The cell survival rate was 92%when the culture dose of Mg-CDs was 200μg m L^-1.The cells were clearly outlined under laser confocal microscopy and the fluorescence was located in the cytoplasm.The results of in vitro osteogenesis-induced differentiation culture experiments showed that the ALP activity of MC3T3-e1 increased dose-dependently after 3,7 and 14 days of osteogenesis induction under Mg-CDs culture conditions.After 3 and 7 days of osteogenesis induction,intracellular ALP activity was higher in the Mg-CDs group than in the CDs group.Similarly,higher levels of ALP,Runx2,Col-I and OC were expressed in MC3T3-e1 in the Mg-CDs group compared to the pure CDs group after 7 and 14 days of osteogenic induction culture.The results of alizarin red staining were similar to the results of ALP activity,and the higher the level of Mg-CDs in the culture medium after 21 days of osteogenic induction culture,the more calcified nodules were observed.Conclusion:In summary,we successfully prepared Mg-CDs with green fluorescence using natural lycium ruthenicum and magnesium chloride hexahydrate by a one-step hydrothermal method,and the introduction of Mg²⁺did not change the characteristic functional groups of CD s.With good cytocompatibility and the ability to promote osteogenic differentiation,Mg-CDs can be used as a fluorescent bioimaging reagent for the monitoring of bone regeneration and show great potential in bone tissue regeneration.