Synthesis And Application Of Dual-function Ce/Gd Co-doped Hydroxyapatite In Bone Defect Repair

Background:Bone transplantation is one of the most commonly used surgical methods for repairing bone defects in clinical practice.In recent years,the incidence of bone defects caused by traumatic tumor osteomyelitis or congenital diseases has been increasing,increasing the social burden and increasing the demand of clinicians for efficient bone transplantation substitutes.At present,the choice of bone graft is mainly autologous bone and allogeneic bone.However,both of them have their own problems,such as painful fractures at the donor site,limited amount of bone resulting in immune rejection and the risk of virus transmission,etc.Therefore,the research and development of bone graft materials has been the focus of scholars at home and abroad.In recent years,the method of preparing multifunctional bone graft materials by combining two or more elements with basic materials by co-doping has attracted more and more attention.Hydroxyapatite（HA）is the main inorganic component of human and animal bones.As an important bone substitute material,HA has great potential in clinical application of orthopedics and dentistry.But HA itself has obvious shortcomings,such as poor mechanical properties and weak bone inductance.As similar to bone mineral density,density of HA in the X-ray and CT scans in the image is very difficult to distinguish,and the X-ray and CT will also not sure of the radioactive harm to human body MRI imaging and outstanding advantage lies in its not radiation damage to human body,and the qualitative and quantitative aspects of bone structure has good capability of imaging.Therefore,it is particularly important to change the physicochemical properties of HA by chemical means.Rare earth elements with excellent physical and chemical properties,was successfully used in microelectronics mechanical energy and biomedical industries such as category,and abundant rare earth resources in our country,the total reserves accounted for a third of the world’s total rare earth resources.The cerium element is a distribution of induction of anti-inflammatory and anti-tumor ability of rare earth elements,scholars at home and abroad has been widely applied to various clinical studies.The biological safety of cerium,the nano materials can be induced bone marrow mesenchymal stem cells differentiate into osteoblasts,between which promote bone defect repair.At the same time,rare earth element gadolinium has seven parallel 4f unpaired electron spin,with a high magnetic moment,so that the gadolinium and its compounds showed good magnetic,as MRI contrast agent enhancement test contrast of the image.Therefore,if cerium and gadolinium can be chemically synthesized into HA,thus enabling it to have MRI imaging ability and enhanced osteogenic induction ability,it is of great significance for future clinical application.Methods:In this paper,we successfully prepared HA nanomaterials with different Ce and Gd doping ratios by hydrothermal synthesis using Ce and Gd as raw materials,and prepared Ce/Gd@HA/PLGA composite supports using NMP（N-methylpyrrolidone）as solvent by phase conversion method.Firstly,the synthesized nanoparticles were characterized,including phase composition analysis of the nanoparticles,determination of the specific content of Ce³⁺,Gd³⁺,and Ca²⁺,observation of the microstructure and magnetic measurement.Then the microstructure and pore structure of the composite scaffold were observed,and the porosity of the scaffold was calculated,and the MRI imaging effect and hydrophilicity of the composite scaffold were characterized.Secondly,the effects of Ce/Gd@HA/PLGA on in vitro cytotoxicity,proliferation,adhesion,alkaline phosphatase expression,calcium deposition and related protein and gene expression during osteogenic differentiation were studied using MC3T3-E1 cells.Finally,the synthesized Ce/Gd@HA/PLGA composite scaffold was implanted into the bone defect of the proximal tibia of rats,and the morphological and histological changes of the bone defect were observed at4W and 8W,and the in vivo safety of the material was evaluated.Through systematic and abundant experiments,this study confirmed that Ce/Gd@HA/PLGA composite material has clear MRI imaging ability,good biocompatibility and osteogenic induction ability,can effectively promote osteogenic differentiation,and has great potential and wide application prospects in the field of bone tissue engineering.Results:Part one:preparation and characterization of Ce/Gd co-doped HA nanoparticles and PLGA compositesOn the premise of determining the molar ratio of Ca+Ce+Gd/P,we used calcium nitrate,gadolinium nitrate hexahydrate,cerium nitrate hexahydrate and ammonium dihydrogen phosphate as raw materials to prepare hydrothermal solution.Using hydrothermal synthesis method,we finally successfully prepared the accurate composition of the image.Pure HA 0.5Ce@HA,1Ce@HA,0.5Gd@HA,1Gd@HA,0.5Ce/1Gd@HA and 1Ce/1Gd@HA nanoparticles with regular amorphous morphologic changes.The HA nanoparticles in each group had typical HA characteristic peaks,and the groups were mainly hydroxyl and phosphate groups.The HA nanoparticles showed typical short rod structure under scanning electron microscope.Gd³⁺doped sample group showed obvious paramagnetism,among which 1Gd@HA group showed the strongest paramagnetism,which was related to the doping amount of Gd³⁺.Then,the composite scaffolds were prepared by mixing each group of nanoparticles with PLGA at a mass ratio of 20%by solution blending.In the view of high magnification,there are abundant microporous structures in the composite material.Micro-ct can find that there are long circular pores in the inner part of the scaffold,which are connected with each other internally and are conducive to the adhesion and growth of cells on the scaffold.Imaging results showed that Gd³⁺incorporation enhanced the MRI imaging capability of the composite scaffold.With the increase of incorporation concentration,the MRI imaging capability of the scaffold was also significantly improved.Contact Angle measurement experiments showed that with the addition of HA nanoparticles,the surface hydrophilicity of all groups of composite scaffolds was significantly improved compared with pure PLGA scaffolds,which was conducive to cell adhesion and proliferation.Part two:In vitro cell biological evaluation of Ce/Gd co-doped HA nanoparticles and PLGA compositesDMEM medium was used to extract each group of nanoparticles and prepare gradient diluent.MC3T3-E1 cells were used to evaluate toxicity.It was found that the survival rate of all components exceeded 80%when the dilution ratio of the extract reached 1/8.The results of cell proliferation experiment showed that the cell proliferation efficiency of all groups could reach 100%on the 7th day,and the cell proliferation efficiency of the ce-doped component was more obvious.In addition,1Ce@HA/PLGA and 1Ce/1Gd@HA/PLGA had the highest value-added efficiency among all groups,indicating that Ce³⁺had a promoting effect on cell proliferation and a concentration-dependent effect.The result of Calcein/PI fluorescence staining was consistent with that of proliferation.Cell adhesion experiment,ALP and ARS staining results proved that Ce doped HA/PLGA composite could not only improve cell proliferation and adhesion,but also promote calcium deposition and osteogenesis induction.The results showed that 1Ce@HA/PLGA and1Ce/1Gd@HA/PLGA could significantly up-regulate the expression of OCN and COL-1 at 4W and 8W with the highest expression levels,thus inducing osteogenic differentiation of MC3T3-E1 cells and promoting the secretion of extracellular matrix and minerals.Part three:In vivo bone repair with Ce/Gd@HA/PLGA composite scaffoldersIn this chapter,the osteogenesis effect of HA/PLGA,1Ce@HA/PLGA,1Gd@HA/PLGA and 1Ce/1Gd@HA/PLGA groups was studied in vivo.T1 MRI results showed that at 4 weeks,no implanted composite materials were observed in the HA/PLGA group and 1Ce@HA/PLGA group,while the composite materials were clearly visible in the 1Gd@HA/PLGA group and 1Ce/1Gd@HA/PLGA group,indicating significant MRI signal enhancement.These results indicate that the incorporation of Gd³⁺enables the scaffold material to have MRI imaging ability.Micro-ct results showed that the addition of Ce element significantly enhanced the repair effect of bone defects in the 1Ce@HA/PLGA and 1Ce/1Gd@HA/PLGA groups.Three-dimensional reconstruction images showed that the surface of bone defects was completely filled with new bone,and axial scanning showed a large number of bone trabeculae.In the quantitative analysis results of bone volume fraction,bone surface area density,bone trabecular number and bone trabecular thickness,only the bone trabecular thickness showed no significant difference between the experimental groups,while other parameters in the Ce doped group were significantly better than those in the non-Ce doped group.The results of histological staining showed that Ce element had a definite effect on promoting new bone,collagen fiber formation and bone formation related protein secretion.Conclusion:In this study,Ce/Gd co-doped HA nanoparticles with pure image composition and regular particle morphology were successfully synthesized by hydrothermal method,and dual-function Ce/Gd@HA/PLGA composite scaffold materials with osteogenic induction ability and MRI imaging ability were prepared.Gd³⁺doping enables 1Ce/1Gd@HA/PLGA composite scaffold material to have MRI imaging capability,which provides a feasible method for non-radioactive non-invasive observation of bone implants.Ce³⁺doping reduced the effect of excessive Gd³⁺doping on HA crystal structure,but did not affect MRI imaging ability.In vivo and in vitro experiments showed that 1Ce/1Gd@HA/PLGA scaffolds could promote cell adhesion,proliferation and osteogenic differentiation,as well as improve the ability of bone defect repair.These results indicate that 1Ce/1Gd@HA/PLGA scaffold has the ability to promote osteogenic differentiation and MRI imaging,and is a bone tissue engineering material with great application potential.