A composition for inducing enamel-like hydroxyapatite formation and its use in a method for hydroxyapatite formation

Abstract

The present application relates to the technical field of dental care, and particularly relates to a composition for guiding the generation of enamel-like hydroxyapatite and application and a method for generating hydroxyapatite. The composition of the present application comprises composition 1 and composition 2; the composition 1 takes Tris-HCl solution as a solvent and comprises the following components with final concentrations: 0.1-0.3 mg / ml of amelogenin, 0.1-0.3 mg / ml of amelotin C-terminal peptide; the composition 2 takes Tris-HCl solution as a solvent and comprises the following components with final concentrations: 0.1-0.3 mg / ml of amelogenin, 0.1-0.3 mg / ml of enamel maturation protein. The composition of the present application can realize the biomimetic remineralization of micron-level demineralized enamel and form new hydroxyapatite which is close to the structure of enamel and has no obvious interface.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of dental care, in particular to a composition for guiding the generation of enamel-like hydroxyapatite and application thereof and a method for generating hydroxyapatite. BACKGROUND

[0002] Enamel is made of hydroxyapatite arranged in parallel as its basic structure-enamel rods, and then the enamel rods are arranged in order in height to form the whole layer of enamel covering the tooth crown, which is difficult to regenerate once damaged. At present, the main treatment method for enamel damage such as caries is to repair with materials (such as inlays, resins, glass ionomers, etc.) that are close to enamel in color, shape and quality, but there is always an adhesive interface between the repair material and the original enamel, which is easy to cause secondary caries due to micro-leakage. In recent years, there have been many studies on biomimetic mineralization templates for inducing enamel regeneration in situ, but the final mineralization product and the effect of enamel regeneration in situ are still not satisfactory.

[0003] Amelogenesis is a complex biomineralization process, including programmed physicochemical events: gene expression, protein secretion, protein folding and assembly, mineral growth and protein degradation. Amelogenesis is dominated by the extracellular matrix secreted by ameloblasts, mainly including enamel matrix proteins and proteases, which work together to complete the basic mineralization process: 1) self-assembly of enamel matrix proteins to generate a mineralization scaffold supermolecular structure; 2) acid / phosphorylated proteins inhibit mineral nucleation and transport mineral ions to the mineralization scaffold to induce the formation of amorphous calcium phosphate (ACP); 3) the self-assembly scaffold binds proteins to perform enzymatic treatment on enamel matrix proteins for spatial and temporal mineral nucleation; 4) guiding the transformation of non-crystalline phase to organic scaffold hydroxyapatite crystals. The organic matrix in dental enamel is different from the permanently preserved organic matter in pearls and dentin, and also different from the collagen fibers that can be constantly remodeled in the bone. In order to provide as much hardness as possible for mastication, proteases will degrade enamel matrix proteins, and the protein matrix is almost completely removed when enamel matures. Therefore, once the dental enamel is demineralized or damaged, it cannot be regenerated.

[0004] In the system research of tooth regeneration, it is difficult to achieve the lattice orientation and hierarchical arrangement of enamel by pure chemical synthesis or tissue engineering method. The chemical method obtains a single structure of primary HA unit, which is far from the multi-structure of the hierarchical assembly of "40 nm HA crystal-5 μm HA nanofiber-20 μm crystal fiber bundle" of tooth enamel. The tissue engineering method reconstitutes tooth germ-like structure, and it is still difficult to solve the problems of timely protein secretion and protease release. The dispersed dentin-like tissue cannot be combined into a complete tooth structure, and it is difficult to rebuild a complete tooth with clinical value in the short term. Although the enamel structure is anisotropic and arranged skillfully, the inorganic crystal components of enamel are completely the same, and the only difference is the crystal orientation. The difference between the enamel rods and the interstitial substance is constructed by the angle of the long axis of the nanofiber, and the essence is that the secretory unit Tomes process of ameloblasts secretes two parts of the same protein at an angle of 60°. The arrangement trend in space is divided into radial and tangential directions, and the tennis racket-like human enamel rod structure is formed by moving along the sinusoidal curve. At present, any physical or biological engineering means cannot assemble such a space-time structure with the same matrix but different orientations.

[0005] In early enamel remineralization research, natural inorganic or organic macromolecules are often selected as mineralization templates, such as Langmuir-Blodgett film attached by sodium dodecyl sulfate / isooctane reverse micelles, monolayer film assembled by HS(CH2)nX(-SO3H, -PO4H2, -COOH, -OH, -CH3), self-assembled oligopeptide, and high molecular polypeptide DMPA-PCL-P. Modified gel systems are also widely used, such as agarose gel, gelatin gel containing F- and PO4 3- Although such template materials are simple and easy to obtain, they can only simulate some specific functions of protein templates at a certain stage of enamel development cycle, and the mineralization products obtained are only similar primary products of enamel structure at a certain stage, and there is a "crack" between the mineralization product and the original enamel surface, and the adhesion is poor.

[0006] Based on this, the present application is proposed. SUMMARY

[0007] The present application aims to provide a composition for guiding the generation of tooth enamel-like hydroxyapatite, and application and a method for generating hydroxyapatite.

[0008] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0009] The present application provides a composition for guiding the generation of tooth enamel-like hydroxyapatite, which comprises composition 1 and composition 2.

[0010] The composition 1 is dissolved in Tris-HCl solution, and further comprises the following components with the following final concentrations: 0.1-0.3 mg / ml of amelogenin, 0.1-0.3 mg / ml of C-terminal peptide of enamel band.

[0011] The composition 2 is dissolved in Tris-HCl solution, and further comprises the following components with the following final concentrations: 0.1-0.3 mg / ml of amelogenin, 0.1-0.3 mg / ml of enamel maturation protein.

[0012] Preferably, the composition comprises the composition 1 and the composition 2.

[0013] The composition 1 is dissolved in Tris-HCl solution, and further comprises the following components with the following final concentrations: 0.15-0.25 mg / ml of amelogenin, 0.15-0.25 mg / ml of C-terminal peptide of enamel band.

[0014] The composition 2 is dissolved in Tris-HCl solution, and further comprises the following components with the following final concentrations: 0.15-0.25 mg / ml of amelogenin, 0.15-0.25 mg / ml of enamel maturation protein.

[0015] Preferably, the composition comprises the composition 1 and the composition 2.

[0016] The composition 1 is dissolved in Tris-HCl solution, and further comprises the following components with the following final concentrations: 0.2 mg / ml of amelogenin, 0.2 mg / ml of C-terminal peptide of enamel band.

[0017] The composition 2 is dissolved in Tris-HCl solution, and further comprises the following components with the following final concentrations: 0.2 mg / ml of amelogenin, 0.2 mg / ml of enamel maturation protein.

[0018] The present application also provides the use of the composition in the preparation of a preparation for guiding the production of enamel-like hydroxyapatite.

[0019] The present application also provides a method for guiding the production of enamel-like hydroxyapatite, comprising the following steps:

[0020] The composition is dropped onto the demineralized tooth;

[0021] The dropping method is to alternately drop the composition 1 and the composition 2 every other day.

[0022] The amount of each drop is 4-5 mg / cm 2 .

[0023] The present application provides a composition for guiding the production of enamel-like hydroxyapatite, and the use thereof and a method for the production of hydroxyapatite.

[0024] The composition 1 formed by the amelogenin and the enamel maturation protein of the present application forms a stable nanosphere structure in the concentration range of the present application, and has a typical self-assembly process. The composition 2 formed by the amelogenin and the amelogenin C-terminal peptide of the present application has a stable nanostructure in the concentration range of the present application. The alternation of the two can induce the generation of a typical classic needle-shaped crystal bundle, and has a synergistic effect of combining calcium and phosphate ions to nucleate and promote the growth of mineralized crystals.

[0025] The composition of the present application can achieve the remineralization of the micrometer-level demineralized tooth enamel, and form new hydroxyapatite close to the enamel structure and without obvious interface. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The self-assembly structure observed by electron microscopy (from left to right, AMEL, AMBN-C, AMTN);

[0027] Figure 2 The in vitro mineralization experiment of single protein and X-ray diffraction pattern (from left to right, the single protein is 0.1 mg / ml AMEL, 0.1 mg / ml AMBN-C, and 0.1 mg / ml AMTN; the first three rows are mineralization results; the fourth row is the X-ray diffraction pattern);

[0028] Figure 3 The in vitro mineralization experiment of combined proteins and X-ray diffraction pattern (from left to right, the combined proteins are 0.1 mg / ml AMEL+0.1 mg / ml AMTN, 0.1 mg / ml AMEL+0.1 mg / ml AMBN-C, and 0.1 mg / ml AMTN+0.1 mg / ml AMBN-C; the first three rows are mineralization results; the fourth row is the X-ray diffraction pattern);

[0029] Figure 4 The in vitro tooth enamel defect in situ mineralization experiment and XRD diffraction pattern (from left to right, the mineralization results guided by AMEL alone, the mineralization results guided by the alternation of composition 1 and composition 2, the crystal layer formed by the alternation of composition 1 and composition 2 observed by electron microscopy, and the XRD diffraction pattern). DETAILED DESCRIPTION

[0030] The present application provides a composition for guiding the generation of tooth enamel-like hydroxyapatite, which comprises composition 1 and composition 2.

[0031] The composition 1 is dissolved in Tris-HCl solution, and further comprises the following components with the final concentration: 0.1-0.3 mg / ml of amelogenin, 0.1-0.3 mg / ml of amelogenin C-terminal peptide; the concentration of the amelogenin is preferably 0.15-0.25 mg / ml, and further preferably 0.2 mg / ml; the concentration of the amelogenin C-terminal peptide is preferably 0.15-0.25 mg / ml, and further preferably 0.2 mg / ml.

[0032] The composition 2 is dissolved in Tris-HCl solution, and further comprises the following components with the final concentration: 0.1-0.3 mg / ml of amelogenin, 0.1-0.3 mg / ml of enamel maturation protein; the concentration of the amelogenin is preferably 0.15-0.25 mg / ml, and further preferably 0.2 mg / ml; the concentration of the enamel maturation protein is preferably 0.15-0.25 mg / ml, and further preferably 0.2 mg / ml.

[0033] In the present application, the preparation method of the composition 1 is mixing the amelogenin and the amelogenin C-terminal peptide with Tris-HCl solution; the molar concentration of the Tris-HCl solution is 50 mM.

[0034] In the present application, the preparation method of the composition 2 is mixing the amelogenin and the enamel maturation protein; the molar concentration of the Tris-HCl solution is 50 mM.

[0035] The present application further provides the use of the composition in the preparation of a preparation for guiding the generation of enamel-like hydroxyapatite.

[0036] The present application further provides a method for guiding the generation of enamel-like hydroxyapatite, comprising the following steps:

[0037] Dropping the composition on the demineralized teeth;

[0038] The dropping method is to alternately drop the composition 1 and the composition 2 every other day;

[0039] The amount of each drop is 4-5 mg / cm 2 , preferably 4.5 mg / cm 2 .

[0040] Dropping the composition on the demineralized teeth with a pipette.

[0041] The enamelogenin (AMEL) of the present application is the most abundant protein in the enamel matrix, accounting for more than 90% of the total amount of enamel matrix protein, and is a structural protein necessary for the mineralization of enamel in the body. The coding gene of enamelogenin is present on both X and Y chromosomes of humans, located at Xp22.3-p22.1 (AMELX) and Yp11 (AMELY) respectively. Both coding genes contain 7 exons, although AMELX and AMELY are both expressed in males, the expression of AMELX dominates. Currently, 16 kinds of AMELX mutations can modify enamelogenin and cause enamel hypoplasia, and the enamel of such enamel hypoplasia has low mineralization degree, which is different from the enamel characteristics of enamel hypoplasia caused by other diseases. The pH value of recombinant pig enamelogenin isoform rp172 is 3.8, and it exists in the form of monomer. The sequence of enamelogenin is usually divided into three significant amino acid structure regions: a hydrophobic N-terminal structure region rich in tyrosine, referred to as tyrosine-rich amelogenin peptide (TRAP); a proline-rich central region, which is hydrophobic, mainly composed of repeated protein sequences of X-Y-proline (where X and Y are usually glutamine); and a hydrophilic C-terminal structure region. The N- and C-terminal structure regions of enamelogenin are highly conserved, and in a mouse model, the loss of conserved terminal gene structure leads to disordered enamel crystal formation. The signal activity of the enamelogenin gene is derived from the leucine-rich amelogenin peptide (LRAP) in the gene sequence of the enamelogenin gene. Natural enamelogenin contains a phosphate group at serine-16, which is involved in the interaction of enamelogenin-calcium phosphate and helps to improve the ability of enamelogenin to stabilize the precursor amorphous calcium phosphate. Although enamelogenin is completely unfolded in structure, residual secondary structures (such as alpha helix, extended beta strand, turn / ring and polyalanine type II (PPII) conformation) can be detected locally, which are potential sites for inducing mineralization, binding calcium and phosphorus ions in the process of enamel formation, and play multiple roles in the extracellular matrix of enamel. For example, the unfolded N-terminal TRAP (self-assembled A domain) provides sufficient molecular contact for protein-protein interaction and assembly; the tri-tyrosine motif (PYPSYGYEPMGGW) in the N-terminal region has lectin-like properties (high affinity for N-acetyl-D-glucosamine), increasing the ability of enamelogenin to interact with other enamel matrix proteins or cell surface proteins; the random loop structure of the C-terminal region can enhance the multiple charged contact between enamelogenin and the mineral surface; the region close to the C-terminal is also identified as participating in the self-assembly of enamelogenin (self-assembly B-region). The expression of enamelogenin gradually increases throughout the secretion period of ameloblasts, peaks in the early stage of maturation, and then rapidly decreases. Enamelogenin disappears when the enamel matures.After the mouse amelogenin coding gene was knocked out, the mouse teeth were grayish white, the enamel had no enamel column structure, the crystals were distributed in disorder, and only a thin layer of enamel was secreted (10-20% of the thickness of normal mouse enamel), which indicated that amelogenin was not essential for mineralization initiation, but was essential for enamel crystal growth and thickness. In the in vitro self-assembly experiment of amelogenin, the protein monomer with a diameter of 2 nm first formed an oligomer with a diameter of 5 nm by extending the hydrophilic C-terminal outside and aggregating the hydrophobic N-terminal, and then further assembled into a nanosphere with a diameter of about 20 nm, which had a hexagonal cell shape and linear arrangement, and was extremely similar to the primary structure of hydroxyapatite crystals. In the in vitro mineralization experiment of amelogenin, amelogenin first combined with calcium and phosphate ions to form an amelogenin-calcium-phosphate ion group, and then continued to assemble into larger nanospheres. Due to the electrostatic interaction between the proteins (a, b axis of the crystal), the nanospheres were connected head to tail to form parallel hydroxyapatite crystals, and finally formed hydroxyapatite crystals growing along the c axis. During enamel formation, amelogenin plays a key role in closing the a, b axis of hydroxyapatite crystals, allowing the crystals to grow along the c axis. In addition, when amelogenin is lacking, the pH value in the developing enamel matrix will decrease, and the function of other proteins will be blocked, leading to enamel development defects, so sufficient amelogenin can protect ameloblasts and other proteins from changes in the pH value of the matrix. Amelogenin also plays a certain role in the control of mandibular morphological development, it participates in the short-distance signal transduction of osteoblasts, guides the morphological development of the mandible, and the miRNA of AMELX exon 4 is directly related to the cell activity of osteoblasts.

[0042] The enamel matrix protein C-terminal peptide (AMBN-C) in the application is the second highest secreted enamel matrix protein in ameloblasts, accounting for about 5% of the total enamel matrix protein. The gap between the enamel rods, also known as the sheath protein, is located in the enamel rod sheath. The ameloblastin gene is located on human chromosome 4q21 and contains 13 exons, and is also one of the members of the secretory calcium binding phosphoprotein (SCPP) gene cluster. In pigs, ameloblastin is composed of 359 amino acids and has a molecular weight of 62 kDa. The N-terminal 86 amino acid structure region is the most conserved region among species, which is highly conserved from amphibians to humans. The N-terminal 129 amino acids (pI=10.6) and the C-terminal 66 amino acids (pI=4.5) are connected by a “non-structure” region which is easily hydrolyzed. Ameloblastin starts to express in ameloblasts, reaches a peak in the maturation stage of ameloblasts, and then gradually disappears by hydrolysis. Once secreted into the matrix, ameloblastin is rapidly hydrolyzed into different products and distributed on the enamel rod sheath. In the outermost layer of the newly formed enamel, ameloblastin is distributed on the enamel rod structure. During the expression of porcine ameloblastin, mRNA is selectively and alternately spliced, thereby determining the glycosylation state of the translation product (different ameloblastin subtypes). The translation product is 380 and 395 amino acid two ameloblastin subtypes respectively except for the signal peptide. The difference between the two subtypes is that the 5' end of the 5th exon of the ameloblastin subtype with a slightly smaller molecular weight lacks 15 amino acids (73-YEYSLPVHPPPLPSQ-87). Although their functions are not clear, they are located in the highly conserved region of the N-terminus and contain the Ser86 O-glycosylation site. In humans, ameloblastin mutations have not been found to cause enamel hypoplasia, but enamel hypoplasia can be seen in a mouse model in which the 5th and 6th exons of the ameloblastin coding gene are knocked out. Initially, this mouse model was identified as a true ameloblastin gene knockout model, but it was later found that although the 5th and 6th exons were missing, the incomplete mRNA could still be translated and expressed. Mutant ameloblastin will cause severe enamel defects, and in the teeth of the mutant mouse model, only a thin layer of irregular mineralized product without enamel rod structure is formed. The self-assembly ability of ameloblastin to induce mineralization is mainly controlled by the 5th exon consisting of 36-72 residues. Through immunolocalization studies, it was found that N-terminal cleavage products of ameloblastin (13 kDa and 17 kDa) appeared in the enamel rod sheath of pig teeth, indicating that ameloblastin may be involved in the control of the enamel rod structure of dental enamel. The C-terminal hydrolysis products of ameloblastin have a high affinity for calcium ions, indicating that they can bind mineral ions and participate in the regulation of mineralization. The identification of the 2347 bp homozygous gene deletion of ameloblastin confirms that ameloblastin mutations can cause enamel matrix protein defects (AI, Amelogenesis imperfecta), thereby causing autosomal recessive human enamel hypoplasia.The mechanism leading to AI is not clear, and can be ameloblastin deficiency, which cannot play a key role in ameloblast attachment, maintenance of ameloblast phenotype and regulation of transcription factors. After the inhibition of the above-mentioned transcription factors, the expression of ameloblastin is down-regulated, causing enamel dysplasia. In the process of enamel mineralization, after the appearance of enamel contour, it is found through immunolabeling that ameloblastin presents a "fishing net-like" structure. Because the VTLG motif in ameloblastin has cell adhesion properties, ameloblastin is considered to be able to control ameloblast differentiation. However, in the currently studied species, the VTLG motif which plays a role in cell adhesion is not conserved.

[0043] The specific role of the enamel maturation protein (AMTN) in the process of enamel development in the present application has not been determined. The mouse and human ameloblastin coding genes are highly homologous, and the full-length cDNA has 1022 bp, encoding 213 amino acids, which are rich in leucine, proline, glutamine and threonine residues, accounting for 52% of the total protein. The mouse and human ameloblastin coding gene sequences are composed of 9 exons and 8 introns, respectively, located on chromosomes 5 and 4q13.3, and the distribution of their sites is very close to the coding genes of ameloblastin and enamel protein. The SSEEL motif contained in the AMTN protein sequence can be a key sequence for binding calcium and phosphorus ions in the process of enamel mineralization. In the enamel maturation protein, there is a completely conserved phosphorylation site of protein kinase CK2, which is highly conserved and replicated, and belongs to the secreted calcium-binding phosphoprotein family. The expression of enamel maturation protein starts in the transition stage of enamel development and is continuously expressed in the mature stage, and the expression gradually decreases in the pre-eruptive stage. Studies have shown that enamel maturation protein also has a small amount of expression in the thymus, but the specific role is not clear. In the mature stage of enamel development, it is expressed in the interface between the ameloblast and the third fibrous reticular layer (atypical basement membrane) of the basement membrane-like structure, which indicates that enamel maturation protein forms a basement layer-like structure and a tight binding layer of enamel by promoting the biomineralization of the third layer, indicating that it plays a role in the mature stage of enamel and the formation of a dense enamel surface layer. In experiments of controlling Amtn gene expression (promoting AMTN expression and AMTN knockout), after controlling the Amel promoter, the enamel of AMTN overexpression (pAmel: Amtn+ / +) and AMTN knockout (Amtn- / -) mouse models is defective. Compared with wild type (WT), the pAmel: Amtn+ / + mouse model has a thin and brittle defective enamel surface layer; in contrast, the Amtn- / - mouse model has a rough and chalky enamel. The enamel of the AMTN knockout mouse is not hypomineralized, but is underdeveloped and disordered, indicating that it plays a role in regulating the morphology of the mineralization product.

[0044] The affinity of AMTN to hydroxyapatite particles was found to be 3.86 x 10 -5 M -1 , which was in the same order of magnitude as that of AMEL (6.38 x 10 -5 M -1 ). There was a preferential hydroxyapatite binding motif on AMTN, and the binding to mineral might cause steric hindrance and / or conformational changes of the protein secondary structure, making the potential cleavage sites inaccessible to proteases (e.g. KLK4) present in the matrix. AMTN could bind to calcium and phosphate ions to form stable cluster crystals, and induce the mineralization of calcium and phosphate ions to hydroxyapatite crystals.

[0045] The technical solutions provided by the present application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0046] Example 1

[0047] Self-assembly and mineralization of different proteins

[0048] AMEL was dissolved in 50 mM Tris-HCl solution to obtain an AMEL preparation with a concentration of 0.1 mg / ml;

[0049] AMBN-C was dissolved in 50 mM Tris-HCl solution to obtain an AMBN-C preparation with a concentration of 0.1 mg / ml;

[0050] AMTN was dissolved in 50 mM Tris-HCl solution to obtain an AMTN preparation with a concentration of 0.1 mg / ml;

[0051] AMEL and AMTN were dissolved in 50 mM Tris-HCl solution to obtain a preparation containing AMEL and AMTN, in which the concentration of AMEL was 0.1 mg / ml and the concentration of AMTN was 0.1 mg / ml.

[0052] AMEL and AMBN-C were dissolved in 50 mM Tris-HCl solution to obtain a preparation containing AMEL and AMBN-C, in which the concentration of AMEL was 0.1 mg / ml and the concentration of AMBN-C was 0.1 mg / ml.

[0053] AMTN and AMBN-C were dissolved in 50 mM Tris-HCl solution to obtain a preparation containing AMTN and AMBN-C, in which the concentration of AMTN was 0.1 mg / ml and the concentration of AMBN-C was 0.1 mg / ml.

[0054] The protein stock solution has a pH of 3.5 when stored, and remains in a monomer state. When the solution of Tris-HCl (pH = 8.0) is used to configure, self-assembly is initiated, and the solution is added dropwise to the tooth piece and then placed in the biomimetic saliva, which is full of Ca and P mineral ions, so that the crystal deposition begins to form. The repeated dropwise addition every day is because the single-layer protein cannot simulate the continuous secretion in the body, and when all the mineralization sites are covered with Ca and P ions, the mineralization crystal is irregular, so the second layer is coated in time, and this is repeated for 5-7 times, so that the hydroxyapatite crystal with the best combination degree, biomimetic degree and hardness can be obtained.

[0055] The structure of the self-assembled monomer protein was observed under cryo-EM. The results are shown in Figure 1 .

[0056] Figure 1 It is shown that the three proteins derived from enamel matrix (0.1 mg / ml) exist in a monomer form at pH 3.5, and as soon as they are placed in the Tris-HCl solution (pH 8.0), self-assembly is initiated, and a nano-monomer develops into a nano-chain, a nano-sphere, and finally a network structure formed by nano-spheres connected together.

[0057] The in vitro mineralization results of the monomer protein and the combined protein observed under the electron microscope are shown in Figures 2-3 .

[0058] The X-ray diffraction (XRD) detects the new mineral pattern of the monomer protein and the combined protein, and the results are shown in Figures 2-3 .

[0059] Figures 2-3 The first three rows are mineralization results, and the fourth row is the X-ray diffraction pattern.

[0060] Figures 2-3 It is shown that the preparation containing amelogenin and ameloblastin forms a stable nanosphere structure, and has a typical self-assembly process. The preparation containing ameloblastin and the C-terminal peptide of amelogenin can also be observed to form a nanosphere structure, and induce a typical needle-shaped crystal bundle. This indicates that the combination of amelogenin and ameloblastin and the combination of ameloblastin and the C-terminal peptide of amelogenin have a synergistic effect of combining calcium and phosphate ions to form nuclei and promoting the growth of mineralized crystals. The newly generated mineral detected by X-ray diffraction is hydroxyapatite.

[0061] Example 2

[0062] Amelogenin and the C-terminal peptide of amelogenin were dissolved in 50 mM Tris-HCl solution to obtain composition 1, and the concentration of amelogenin in composition 1 was 0.1 mg / ml, and the concentration of the C-terminal peptide of amelogenin was 0.1 mg / ml.

[0063] The enamel preproteins and enamel maturation proteins were dissolved in 50 mM Tris-HCl solution to obtain composition 2, the concentration of the enamel preproteins in the composition 2 was 0.1 mg / ml, and the concentration of the enamel maturation proteins was 0.1 mg / ml.

[0064] The extracted healthy teeth were polished into 2 mm thick enamel slices, the enamel defects were caused by phosphoric acid demineralization, then composition 1 was added dropwise to the surface of the enamel slices, the dropwise amount was 5 mg / cm 2 , and after standing for 1 min, the enamel slices were placed in artificial saliva for 24 h. After being taken out, the enamel slices were naturally dried at room temperature for 2 min. Composition 2 was added dropwise to the surface of the enamel slices, and after standing for 1 min, the enamel slices were placed in artificial saliva for 24 h to complete one cycle. The above cycle was repeated for 7 times, the enamel slices were taken out, dried and gold sprayed for scanning electron microscope detection, and the newly formed mineral layer was scraped for XRD detection to determine the crystal composition. The detection results are shown in Figure 4 .

[0065] Artificial saliva: including 0.2 mM MgCl2, 4 mM KH2PO4, 16 mM KCl, 4.5 mM NH4Cl, 1 mM CaCl2·H2O, 20 mM HEPES, 0.1 M HCl and 0.1 M NaOH to adjust the pH of the artificial saliva to 7.6, and 300 ppm NaF was added before use. The artificial saliva needs to be prepared and used immediately, and is replaced once a day.

[0066] The experiment was carried out with monomer enamel preproteins 0.1 mg / ml (AMEL) as a control. The mineralization of the monomer enamel preproteins to the enamel defects was detected after 14 d, and the results are shown in Figure 4 .

[0067] Figure 4 It is shown that the urchin-shaped long columnar crystals formed by AMEL alone, and the single crystal length is about 2 μm. The parallelly arranged crystals guided by the composition 1 and the composition 2 alternately are columnar crystals similar to enamel, and the single crystal length is about 300-500 nm, and the height is close to the natural tooth enamel. The structure of the newly formed crystal layer guided by the composition 1 and the composition 2 alternately is hydroxyapatite crystal. The newly formed crystal detected by XRD is hydroxyapatite.

[0068] It can be known from the above examples that the present application provides a composition for guiding the generation of tooth enamel-like hydroxyapatite, and application and a method for generating hydroxyapatite. The composition of the present application can realize the in-situ remineralization of the micron-level demineralized tooth enamel, and form the newly formed hydroxyapatite which is close to the structure of the enamel and has no obvious interface.

[0069] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, some improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A composition for directing the production of enamel-like hydroxyapatite, characterized in that, The composition comprises composition 1 and composition 2; The composition 1 uses Tris-HCl solution as solvent, and further comprises components with the following final concentrations: 0.1-0.3 mg / ml amelogenin, 0.1-0.3 mg / ml ameloblastin C-terminal peptide; The composition 2 uses Tris-HCl solution as solvent, and further comprises components with the following final concentrations: 0.1-0.3 mg / ml amelogenin, 0.1-0.3 mg / ml ameloblastin; The Tris-HCl solution has a pH of 8.0; The Tris-HCl solution has a concentration of 50 mM.

2. The composition of claim 1, wherein, The composition comprises composition 1 and composition 2; The composition 1 uses Tris-HCl solution as solvent, and further comprises components with the following final concentrations: 0.15-0.25 mg / ml amelogenin, 0.15-0.25 mg / ml ameloblastin C-terminal peptide; The composition 2 uses Tris-HCl solution as solvent, and further comprises components with the following final concentrations: 0.15-0.25 mg / ml amelogenin, 0.15-0.25 mg / ml ameloblastin.

3. The composition of claim 2, wherein, The composition comprises composition 1 and composition 2; The composition 1 uses Tris-HCl solution as solvent, and further comprises components with the following final concentrations: 0.2 mg / ml amelogenin, 0.2 mg / ml ameloblastin C-terminal peptide; The composition 2 uses Tris-HCl solution as solvent, and further comprises components with the following final concentrations: 0.2 mg / ml amelogenin, 0.2 mg / ml ameloblastin.

4. Use of the composition according to any one of claims 1-3 in the preparation of a preparation for guiding the production of enamel-like hydroxyapatite.

5. A non-therapeutic method of directing the production of enamel-like hydroxyapatite, characterized in that, The method comprises the following steps: Dropping the composition according to any one of claims 1-3 on the demineralized tooth; The dropping method is to alternately drop composition 1 and composition 2 every other day; The dropping method is to alternately drop composition 1 and composition 2 every other day; The amount of each drop was 4-5 mg / cm 2 .

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