Constructing Artificial Enamel Organ By Mimetic The Physicochemical Properties And Spatial Structure Of Natural Enamel Organ For The Regeneration Of Enamel

Backgrounds: Enamel is the most hard and mineralized tissue in the mammalian mineralized tissue.Once tooth enamel is damaged,the body cannot achieve self-healing repair.The traditional methods to repair were often accompanied by secondary caries and microleakage in adhesion interface of the joint of dental tissue and artificial material.It is difficult to regenerate the enamel-like crystal structure with the remineralized materials used in clinic.Moreover,the regenerated irregular mineralized crystals could not withstand the daily chewing movement.The regeneration of enamel microstructure induced by biomimic mineralization was one of the hot spots.The biomineralization of enamel in vivo is secreted by ameloblastic cells in the enamel organ,and the nucleation growth of crystal is regulated by the enamel matrix protein.However,there were some problems in the selection and design of biomimetic mineralized models.The design of templates cannot fully imitate the microenvironment of gel matrix in the growth of enamel in organisms,especially the lack of design basis in physicochemical properties and spatial structure.Objectives: According to tooth tissues biomineralization process of organic ground substance,organic template control and induced apatite crystal nucleation growth,the physicochemical properties and space structure of natural enamel organ are analyzed,such as morphological,type of inorganic mineral ions and form,etc..Providing scientific theoretical basis of the model of enamel remineralization,and constructing an artificial enamel organ for the regeneration of enamel in vitro.Methods: The tooth germ of the 6-month-old pig was obtained,and the enamel organ was obtained after decellularization.Critical point drying,scanning electron microscopy(SEM)to observe its morphological structure,fiber size,pore distribution characteristics and pore size.After the enamel matrix was dissolved by nitric acid,the element types and contents in the matrix were analyzed by ICP-AES.X-ray diffraction(XRD)and Fourier Transform Infrared Spectroscopy(FTIR)were used to analyze the existence form of inorganic minerals in matrix.Design of modified Leucine Rich Amelogenin Protein(m LRAP)and non-Amelogenin biomimetic Analogues(NAA)simulate the functions of amelogenin and non-amelogenin in the enamel organ.The m LRAP and NAA were crosslinked with EDC on the etched enamel for biomimetic simulation of enamel extracellular matrix.The mineral ions in the mineralized system were determined according to the concentration and content of mineral ions in the natural enamel organ.Artificial enamel organ was constructed by organic matrix and inorganic ions.The biocompatibility of the mimetic enamel matrix was tested by the CCK-8.The morphology of regenerated enamel was observed by SEM.XRD and FTIR test the types of regenerated crystals,the hardness of regenerated crystals were tested by microhardness.Results: SEM images showed that the enamel matrix presented a fibrous reticular structure with uniform distribution,with flaky and lumpy structures between the reticular structures.The pore size is about 10 to 20 microns.ICP-AES results showed that the enamel matrix contained Na,Ca,Mg,P,Zn,Si and other elements.The closer the enamel matrix was to the tooth formation site,the contents of Ca,Mg and P were significantly increased.XRD and FTIR show that there are no obvious crystals in the matrix far away from the mineralization,but hydroxyapatite crystals are formed in the area near the mineralization.The pore size and morphology of the artificial enamel organ constructed with the concentration of 3mg/ml m LRAP/NAA were similar to that of the natural enamel organ.The results of molecular dynamics showed the site of the m LRAP and NAA interactions in the fragment we designed.The biomimetic enamel organ has good biocompatibility.Enamel-like crystals were regenerated on demineralized enamel surface with a hardness similar to that of natural enamel.Conclusion:Based on the analysis of physicochemical properties and spatial structure of natural enamel organ,the artificial enamel organ constructed by m LRAP/NAA successfully induced enamel regeneration in vitro,which is a feasible method for the treatment of enamel defects.