| Objective:Bone regeneration is a highly dynamic process of bone remodeling during various stages of human growth and development.Interactions between osteoblasts produce new bone and osteoclasts absorb old bone,maintaining a dynamic balance.The repair of nonunion defects remains an important clinical challenge.The traditional treatment of bone defects is often limited by the shortage of autologous bone,the immune rejection of allograft and the high medical cost.In recent years,new fluorescent carbon dots with simple structure,small size,high tissue permeability and good biocompatibility have attracted extensive attention from researchers.These unique properties make it widely used in biometrics,gene transfer,drug delivery,biological imaging and tissue regeneration.As a new kind of nanometer material,carbon dots has great potential in biomedicine application.We have synthesized novel Zn2+-modified carbon dots?Zn-CDs?which showed good osteogenic activity in vitro.However,the optimal dose with related factors for promoting osteoblast differentiation and bone regeneration in vivo are not clear.We will further study the osteogenesis of Zn-CDs in vivo and vitro,which is essential before clinical use.It is expected that our research will provide a good theoretical basis for the application of Zn-CDs in bone regeneration and provide a new candidate material for bone forming nanomaterials.The effects of Zn-CDs on the proliferation and differentiation of rat bone marrow mesenchymal stem cells?BMSCs?and the effect of Zn-CDs on the repair of rat skull defects were studied in order to clarify the optimal dose and related factors of Zn-CDs in promoting osteogenic differentiation of stem cells and the ability of Zn-CDs to promote bone regeneration in vivo.And it would provide a good theoretical basis for the further clinical application of Zn-CDs.Methods:Zn2+-modified carbon dots?Zn-CDs?were prepared by one-step pyrolysis of zinc gluconate?Zn-G?.The morphology of Zn-CDs was observed by transmission electron microscopy?TEM?and high-resolution transmission electron microscopy?HRTEM?.The optical properties of Zn-CDs were determined by photoluminescence spectroscopy and ultraviolet-visible absorption spectroscopy.Fourier transform infrared spectroscopy?FT-IR?and X-ray photoelectron spectroscopy?XPS?were used to analyze the chemical structure and surface functional groups of Zn-CDs.Isolated and cultured rat bone marrow mesenchymal stem cells?BMSCs?in vitro.MTS was used to examine the biosecurity of Zn-CDs and its effect on the proliferation of BMSCs.The optimum dose for inducing osteoblasts was evaluated by intracellular reactive oxygen species?ROS?detection,alkaline phosphatase?ALP?activity test and alizarin red staining in vitro.Zn-CDs modified gelatin/hydroxyapatite?GH?nanofiber scaffold was designed for bone regeneration experiment in vivo.GH nanofiber scaffold was prepared by electrostatic spinning technology,and the surface morphology of scaffold was studied by SEM.The successful load of Zn-CDs was verified by inverted fluorescence microscope using the spontaneous fluorescence property of Zn-CDs.Ten SD rats?weight:200g?were selected to establish a skull defect model of rats with a defect diameter of 5mm.They were randomly divided into two groups?GH group and Zn-CDs/GH group?.CBCT and HE staining were used to evaluate the repair of skull bone defects at 8 week after surgery.Results:TEM,FT-IR and XPS confirmed the successful synthesis of Zn-CDs.MTS results indicated that zinc gluconate?Zn-G?and Zn-CDs can promote the proliferation of bone marrow mesenchymal stem cells,when the zinc ion concentration was 10-4mol/L?Zn-G:45.6?g/mL?and 10-55 mol/L?Zn-CDs:300?g/mL?or below respectively.In terms of osteogenesis,Zn-CDs induced ALP activity was significantly higher than Zn-G.Alizarin red staining showed a dose-dependent increase in the area of calcium nodules in Zn-CDs group.At the same concentration of Zn2+?10-55 mol/L?,the number of calcium nodules in the Zn-CDs group was significantly higher than that in the Zn-G group.The PL spectrum of Zn-CDs/GH scaffold and fluorescence excitation under the microscope showed the same characteristics of excitation dependence as that of Zn-CDs,which showed the load of Zn-CDs on GH scaffold.The CBCT imaging results of animal experiments showed that the Zn-CDs/GH group had an obvious bone defect repair effect compared with the control group,and the volume of new bone formation in Zn-CDs/GH group?6.66±1.25 mm3?was twice that of the control group?3.33±0.94 mm3?.Further histological evaluation confirmed the markedly new bone formation at 8 weeks in the Zn-CD/GH group.Conclusion:1.Zinc content is an important factor affecting the biocompatibility and osteogenesis ability of Zn-CDs.Zn-CDs solution containing 10-5mol/l Zn2+is the optimal concentration for BMSCs proliferation and differentiation in vitro.2.Zn-CDs can effectively promote bone regeneration in vivo.Zn-CDs/GH can achieve the ideal effect of bone defect repair.Zn-CDs has good osteogenesis and osteoinductive activity,and has potential application prospects in bone diseases and tissue regeneration. |
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