Effects Of Zn2+-doped Carbon Dots In Cell Imaging And Inducing Osteoblastic Differentiation In Vitro

Background:Osteoblasts and osteoclasts are the main participants in the dynamic balance of bone tissue renewal and remodeling.In oral clinical application,bone mass loss is mainly caused by periodontitis,tumor and trauma.Alveolar bone metabolism and tissue remodeling are the most active of all bones in the human body.When periodontitis and other reasons invade the alveolar bone,it causes bone resorption and loss of tooth support tissue,and tooth looseness also increases gradually.Finally,teeth are fall off or extracted passively.These not only affect their physiological functions,such as chewing,pronunciation,but also affect their physical and mental health.However,the current treatment of bone defects is accompanied by some drawbacks,such as unavoidable donor site defects,limited sources and immune response.Therefore,it is impendence to find a new bone regeneration material.Fluorescent carbon dots are photoactive nanomaterials.Due to its relatively simple structure,small particle size,small toxicity and strong penetration rate,they have been widely concerned by scholars in recent years.The purpose of this study is to investigate the effects of Zn2+-doped carbon dots(Zn-CDs)in cell imaging and osteogenic differentiation in vitro,and to provide a scientific theoretical basis for further biological application of Zn-CDs.Methods:1.Synthesis of Zn-CDs:The Zn-CDs were synthesized by one-step hydrothermal method taking zinc gluconate as raw materias.2.Characterizations of Zn-CDs:The size and morphology of Zn-CDs were observed by TEM,infrared Spectra of samples were obtained by Fourier transform(FT-IR),Fluorescence spectra were obtained by fluorescence spectrometer.3.Cytotoxicity of Zn-CDs:The mouse preosteoblast cell line(MC3T3-E1 cells)were co-cultured with a gradient concentrations of Zn-CDs(0.01?0.1?1?10?100?1000?g/mL).The cytotoxicity of Zn-CDs was tested by MTT.4.Cell uptake of Zn-CDs:Flow cytometry was applied to quantitatively analyze the Zn-CDs uptake of MC3T3-E1 cells.5.Fluorescence imaging of Zn-CDs:Laser confocal imaging was used to observe the imaging characteristics of MC3T3-E1 cells at optimal concentration.6.Determination of osteoactive activity of gradient concentration Zn-CDs:The MC3T3-E1 cells were co-cultured with gradient concentration Zn-CDs(0?0.01?0.1?1?10?100 ?g/mL)solution for 3,7 and 14 days,respectively.The expression of alkaline phosphatase were detected by alkaline phosphatase kit and mRNA expression of osteoblast related gense were detected by qRT-PCR.After 21 days,the mineralization degree was detected by alizarin red staining.Results:1.Synthesis and characterization of Zn-CDs:The TEM results showed that the Zn-CDs of 5.25 nm were successfully synthesized.FT-IR results showed that the surface of Zn-CDs were mainly composed of hydroxyl groups and carboxyl groups.The fluorescence spectra revealed that the excitation and emission spectrums of Zn-CDs were extensive.2.Toxicity of Zn-CDs:The MTT results showed that the concentration of Zn-CDs was below 100 ?g/mL,and there was no obvious cytotoxicity.3.Uptake rate of Zn-CDs:Flow cytometry showed that after treatment with 100?g/mL Zn-CDs for 6 h,the uptake rate of the cells was up to 95%and maintained stably,indicating that the Zn-CDs could be effectively absorbed by cells.4.Fluorescence imaging results Zn-CDs:The MC3T3-E1 cells could present blue,green and red fluorescence images.5.The related results of osteogenic differentiation activity were as follows:At 3,7 and 14 days,the activity of alkaline phosphatase(ALP)increased with the increase of Zn-CDs concentration,and the expression of runt related transcription factor 2(Runx2),alkaline phosphatase(Alp),Osteocalcin(Ocn)increased with the increase of Zn-CDs dose,too.At 21 days,the results of alizarin red staining showed that the amount of calcium nodules in each concentration were more than that in the control group,which was also dose-dependent.Conclusions:1.The synthesized Zn-CDs are a spherical carbon nanoparticles with an average size of 5.25 nm and an excitation wavelength dependent property.2.Zn-CDs have good biocompatibility,can be effectively ingested by MC3T3-E1 cells,and can present polychromatic fluorescence images.3.Zn-CDs can induce osteoblastic differentiation in MC3T3-E1 cells.