Preparation And Bone Repairment Experiment Study Of Rare-Earth Doped Carbonated-Hydroxyapatite Materials

Bone tissue defects resulted from oral and maxillofacial tumors,trauma,congenital diseases and tooth loss are common problem in the process of oral diagnosis and treatment,and it is also a difficult problem for dentists to repair bone defects.Jaw defects seriously affect chewing,swallowing and language expression.Ineffective treatment or treatment failure of bone defect repair will both directly affect the living quality of patients.Dental implant restoration is the main treatment for dentition loss and dentition defect.Adequate bone mass is one of the keys to ensure the success of implant.However,under the influence of complex occlusal relationship and different oral environment,patients with tooth loss often have different degrees of alveolar bone atrophy in the edentulous area,resulting in the change of dental arch curvature,which is easy to lead to implant failure.Although autogenous bone is considered as the"gold standard"of bone transplantation,the trauma caused by the opening of the second operation area is more serious,the source is limited,and it can not be shaped at will,and certain complications will occur after autogenous bone transplantation.Allogeneic bone is also limited by the demand for raw materials and rejection reaction,resulting in high price and repair risk.The concept of"bone tissue engineering"is put forward to solve the above problems,which is to replace the traditional bone transplantation with seed cells,cytokines and scaffolds.It is the only way to achieve high-level repair of bone defects by using bone repair materials,seed cells and cytokines.In recent years,with the development of cell engineering and gene editing technology,the research and application of seed cells and cytokines in tissue engineering are constantly promoted.However,there are many kinds of scaffolds available in bone tissue engineering.However,their application is still very limited due to their unavoidable problems.For example,the biological inertia and mechanical mismatch of metal stents,the non degradability and rejection of polymer stents,the brittleness and poor bone conductivity of traditional ceramic stents,etc.It is the key of bone tissue engineering in oral and maxillofacial repair from concept to clinical application to find the suitable material system,design and prepare personalized scaffolds to meet different osteogenic needs.Hydroxyapatite is a natural inorganic mineral in bone and tooth tissue.The scaffold made of hydroxyapatite has been highly praised in the field of bone tissue engineering.However,the commonly used hydroxyapatite in the laboratory is mainly Ca₁₀(PO₄)₆(OH)₂ and other forms of calcium phosphate materials,which is quite different from the actual needs of poor crystallinity of hydroxyapatite in bone tissue,the existence of a variety of ion gradient substitution,and the composition difference in different bone positions.In the applications reported in the literature,undoped pure hydroxyapatite has some problems,such as brittleness,poor degradation effect,and interface with new bone tissue,which is still one of the bottlenecks to limit its application as bone tissue engineering scaffold.How to design and prepare biomimetic hydroxyapatite bone powder materials similar to the minerals in human bone tissue,and customized bone repair materials according to the needs of different parts of bone defects,is the key to promote the clinical application of bone tissue engineering.The hydroxyapatite in bone and teeth is mainly carbonate substituted hydroxyapatite with gradient doping of metal ions.The mass fraction of carbonate is3-8wt%,in which carbonate mainly replaces the crystallographic position of phosphate,which is called B-type hydroxyapatite.Carbonate replacement can make the crystallinity of hydroxyapatite worse and the structure more loose,which is conducive to bone breaking and bone reconstruction.The loose structure is more conducive to the combination with collagen and other proteins,thus reducing the brittleness of the material and making it more plastic.Therefore,it is of great significance to develop carbonate substituted hydroxyapatite materials and realize the controllable concentration of carbonate to promote the application of bone tissue engineering in the treatment of oral bone defects.Due to their spectral properties,rare earth elements can be used as fluorescent markers of biomaterials,especially their ionic radius is close to that of Ca²⁺.Many literatures have applied them to the fluorescent labeling of bone implant materials in vivo.In addition,it has been reported that low dose of rare earth ions doping can not only regulate the crystallinity of hydroxyapatite,but also activate calcium channels through slow-release rare earth ions,thus promoting the differentiation of mesenchymal cells into osteoblasts.Based on the above research status of hydroxyapatite used in bone tissue engineering scaffolds,we propose to prepare a series of B-type hydroxyapatite biomimetic bone powder materials with non carbonate and rare earth ion content by controlled substitution of carbonate and controlled doping of rare earth ions as the source,and prepare them into scaffolds by3D printing,and evaluate their catalytic effect at the cellular and animal levels Bone effect provides a customized scaffold system that is closer to bone tissue for bone tissue engineering research,so as to solve the complex osteogenic environment and specific osteogenic needs of oral and maxillofacial repair.This paper is divided into three parts:(1)material design and controllable preparation;(2)evaluation of osteoblast differentiation,proliferation and key protein expression at the cellular level;(3)evaluation of new bone formation,calcification and new bone growth in scaffolds at the animal level.The main findings are listed as follows:(1)The preparation of carbonated and rare-earth doped hydroxyapatite bionics bone powdersAccording to the thermodynamic equilibrium conditions of calcium ion,phosphate ion and carbonate ion in physiological environment,the mineralization conditions and adjustment parameters of B-type hydroxyapatite were calculated and obtained by Pourbaix.A series of carbonate substituted hydroxyapatite nanomaterials were prepared.By controlling the reaction conditions and initial carbonate concentration,the ratio of carbonate in the product hydroxyapatite can be adjusted Example.On this basis,a series of rare earth ions doped hydroxyapatite and carbonate substituted hydroxyapatite materials were prepared by doping rare earth ions with similar ionic radius but different charges with Ca²⁺ions into the lattice of hydroxyapatite and carbonate substituted hydroxyapatite.Structural analysis shows that the carbonate anion with triangular conformation,when replacing phosphate ion with tetrahedral conformation,is twisted and superimposed on a long P-O bond surface of phosphate tetrahedron in a way of 30 ^o angle relative to c-axis.With the increase of carbonation degree,the aspect ratio of nanorods decreased from 3.6 to 2.9.The type of rare earth ions can affect the morphology of B-type hydroxyapatite nanorods,and carbonation does not affect the coordination environment and luminescent properties of rare earth in hydroxyapatite.The B-type hydroxyapatite material obtained by this method is close to the mineralized composition of human bone tissue,and rare earth elements can be evenly doped in the lattice of the material,which can not only use its characteristic fluorescence spectrum to mark its change process in vivo,but also act as a potential osteoblast differentiation promoter to activate the osteogenic process,so as to achieve better bone promoting effect.(2)MC3T3-E1 cell experiment to determine the biological performance of rare-earth doped and carbonated hydroxyapatite materialsHydroxyapatite(HA),1 mol%Eu doped HA,1 mol%Tb doped HA,3.3 wt%carbonate substituted HA,1 mol%Eu doped CHA and 1 mol%Tb doped CHA were selected to study their effects on mouse embryonic osteoblast precursor cells(MC3T3-E1)Objective:To investigate the effects of different concentrations of calcium on cell morphology,proliferation and osteogenic differentiation.The above materials were printed into scaffolds by three-dimensional printer,and their potential as bone repair materials in bone tissue engineering was evaluated by cell proliferation,differentiation and protein expression test.The results of ALP staining and quantification,alizarin red staining and expression of osteogenic related proteins showed that Tb incorporation could promote the adhesion and proliferation of MC3T3-E1 cells,especially Tb-CHA,which showed the best osteogenic effect,significantly better than TB-HA,HA and CHA;Eu incorporation slowed down the proliferation,adhesion and osteogenic differentiation of MC3T3-E1 cells.(3)Skull defect repair experiment assisted by rare-earth doping and carbonated hydroxyapatite scaffoldAccording to the results of cell experiment,the repair effect of carbonate substituted and rare earth doped hydroxyapatite on bone defect was evaluated at animal level.The results of imaging and he staining showed that the blank control group showed that 8 mm critical bone defect was effective in 6 to 12 weeks;HA group rats did not show obvious healing in the skull defect site,while the blank control group showed only a small amount of new bone formation in the defect site;Tb-HA group showed partial repair in the defect site at 6 weeks,and he staining section showed new bone formation;carbonate substituted CHA scaffold also had a certain osteogenic effect,and a small amount of new bone formation in the bone defect site In addition,Tb may participate in the regulation of some calcium channels,thus promoting the differentiation and proliferation of osteoblasts,showing a stronger ability to repair bone defects.In conclusion,based on the origin of the scaffold used in bone tissue engineering to repair bone defects,this paper designed and prepared a series of carbonate substituted hydroxyapatite materials which are closest to the composition of human bone tissue through in-depth analysis of the mineralization mechanism of hydroxyapatite materials Using 3D printing technology,the model materials were printed into scaffolds,and the proliferation,differentiation ability,protein expression level and new bone growth of pre osteoblasts were tested at cell level and animal level respectively.It was found that carbonate substitution and rare earth Tb³⁺ion doping can promote the bone defect repair effect of hydroxyapatite materials.The research of this paper will guide the hydroxyapatite based bone tissue engineering scaffolds to carbonate substituted hydroxyapatite and heterovalent metal ions doped hydroxyapatite from the source.By using the difference of the osteogenic performance controlled by carbonate and rare earth ions,and with the help of 3D printing equipment,the scaffolds are designed according to the osteogenic needs of different locations,and customized with different osteogenic properties Therefore,it can provide personalized implant materials for the repair of oral and maxillofacial bone defects.