A method for biomimetic mineralization of enamel with fluorine-coordinated gallic acid-stabilized amorphous calcium phosphate nanoclusters

Abstract

The present invention relates to the technical field of enamel restoration, and particularly to a method for biomineralizing enamel with amorphous calcium phosphate nanoclusters stabilized by fluorine and gallic acid. The reagent combination includes a gallic acid-calcium phosphate-fluoride solution, and the raw materials of the gallic acid-calcium phosphate-fluoride solution include gallic acid, a calcium ion source, a phosphorus ion source, and a fluoride ion source; the molar ratio of calcium element in the calcium ion source to gallic acid is 0.01-0.5, and the molar ratio of calcium element in the calcium ion source to phosphorus element in the phosphorus ion source is 0.1-5. This technical solution can solve the technical problems in the prior art of lacking an effective and non-toxic method and system for promoting the stability of calcium phosphate nanoclusters to achieve enamel biomineralization. By using gallic acid and fluoride ions to co-biomineralize enamel, calcium phosphate nanoclusters for biomineralizing dentin can be prepared to achieve effective mineralization repair. Gallic acid is an inexpensive and edible compound, and this solution has an ideal application and promotion prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of enamel restoration, and particularly relates to a method for biomimetic mineralization of enamel with amorphous calcium phosphate nanoclusters stabilized by fluorine and gallic acid. Background Art

[0002] Enamel is a highly mineralized extracellular matrix and the hardest tissue in the human body. Its main component is 95wt%-97wt% nanorod-shaped hydroxyapatite (HA) crystals. Enamel has good mechanical properties, and its excellent mechanical properties stem from its highly ordered spatial structure, which plays an important role in controlling the mechanical strength of teeth and serves as a buffer to protect teeth. Dental caries, trauma, abrasion, and incorrect brushing methods can all cause enamel defects. Mature enamel is a non-living tissue, and the damaged part can hardly repair itself.

[0003] At present, metals, dental composite resins, and ceramic materials are commonly used clinically to fill and repair defective tooth enamel. However, these materials have weak mechanical strength, poor adhesion to teeth and are prone to falling off, and their physical properties and chemical compositions are different from those of natural materials in the human body. In order to achieve a perfect repair at the defect site with chemical composition and structure similar to natural enamel, biomimetic mineralization for repairing tooth enamel is currently an ideal strategy. Based on in-depth exploration and understanding of biological minerals, it is found that at the growth front during the formation of many biological minerals, amorphous minerals mainly fuse onto crystalline minerals for epitaxial growth. In this process, amorphous calcium phosphate plays a crucial role as a precursor for the entire mineralization process. In nature, constructing a biomimetic mineralization front on the tooth enamel surface for tooth enamel repair is a mainstream strategy. Currently, the strategy for constructing a biomimetic mineralization front is mainly achieved by constructing polymer-stabilized calcium phosphate nanoclusters (PNCs). Calcium phosphate precursors such as PNCs are mainly obtained by the double decomposition of calcium salts and phosphates in a liquid medium. However, due to the large proportion of surface atoms, large specific surface area, and high surface energy, PNCs are very unstable, tend to agglomerate, and will transform into large-sized crystals (such as HA, etc.) within a short time during synthesis. Scientists mainly stabilize PNCs by adding stabilizers. Additives with the ability to stabilize precursors such as PNCs can be divided into two major categories: organic and inorganic. Inorganic stabilizers mainly include magnesium ions, strontium, fluoride ions, pyrophosphate ions, and citrate ions, etc. However, the stabilizing effect of inorganic stabilizers is not good, and their addition will also change the chemical composition of precursors such as ACP nanoparticles, forming calcium phosphate salts doped with magnesium, strontium, etc., and may also reduce their biological safety. Organic stabilizers that have been studied more include organic macromolecule stabilizers (polyethylene glycol, polyvinyl alcohol, and polyacrylic acid) and organic small molecule stabilizers (triethylamine, etc.). Since tooth enamel is basically pure mineral, when using polymer-stabilized calcium phosphate clusters to repair enamel, a large amount of organic matter will be introduced, which will damage the structural integrity of the enamel repair layer and also greatly reduce the mechanical strength of the enamel. Therefore, polymer-stabilized calcium phosphate clusters are not suitable for enamel repair. Organic small molecule stabilizers such as triethylamine can bring excellent mechanical strength and biomimetic structure, but organic small molecules such as triethylamine are toxic and easily react with phosphate groups and deposit and remain on the teeth, making them unsuitable for clinical application. In addition, the most crucial problem is that whether it is macromolecule stabilizers or small molecule stabilizers, they have poor ability to stabilize calcium phosphate clusters and short storage time during application. If made into products, the shelf life is extremely short, which greatly affects their commercial application. Therefore, it is urgent to further study the stabilizers and formulation methods of calcium phosphate nanoclusters to further improve the effect of tooth enamel mineralization repair. Summary of the Invention

[0004] The present invention aims to provide a reagent combination for biomimetic mineralization of enamel by using fluorine and gallic acid to stabilize amorphous calcium phosphate nanoclusters, so as to solve the technical problem that the prior art lacks an effective and non-toxic method and system for promoting the stability of calcium phosphate nanoclusters to achieve biomimetic mineralization of enamel.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] A reagent combination for biomimetic mineralization of tooth enamel, the reagent combination comprising a gallic acid-calcium phosphate-fluoride solution, the raw materials of which comprise gallic acid, a calcium ion source, a phosphorus ion source and a fluoride ion source; the molar ratio of the calcium element in the calcium ion source to the gallic acid is 0.01-0.5, and the molar ratio of the calcium element in the calcium ion source to the phosphorus element in the phosphorus ion source is 0.1-5.

[0007] Furthermore, the gallic acid-calcium phosphate-fluoride solution is prepared by the following method: dissolving gallic acid in water to obtain a gallic acid solution; adding a calcium ion source to the gallic acid solution under stirring to obtain a gallic acid-calcium solution; dissolving a phosphorus ion source and a fluoride ion source in water to obtain a fluorine-containing phosphorus ion source solution, and dropwise adding the fluorine-containing phosphorus ion source solution to the stirring gallic acid-calcium phosphate solution, and adjusting the pH value to obtain a gallic acid-calcium phosphate-fluoride solution.

[0008] Furthermore, the mass fraction of gallic acid in the gallic acid solution is 0.01-2%; in the gallic acid-calcium solution, the concentration of calcium is 0.01-30mM; in the fluorine-containing phosphorus ion source solution, the concentration of phosphorus is 0.01-30mM, and the concentration of fluorine is 10ppm-1500ppm.

[0009] Further, gallic acid is dissolved in water and stirred at 35-50° C. for 10-120 min to obtain a gallic acid solution.

[0010] Further, under the stirring condition of >300 rpm, a calcium ion source is added to the gallic acid solution and stirred for 10-120 min to obtain a gallic acid-calcium solution.

[0011] Further, the fluorine-containing phosphorus ion source solution is added dropwise at a rate of 1-4 drops / second to the gallic acid-calcium solution being stirred at >300 rpm, with a pH value of 5-7, to obtain a gallic acid-calcium phosphate-fluoride solution.

[0012] Furthermore, the reagent combination also includes artificial saliva or saliva.

[0013] Furthermore, the reagent combination also includes a washing solution; the washing solution includes anhydrous ethanol and water.

[0014] The present technical solution also provides an application of a reagent combination for biomimetic mineralization of tooth enamel in the preparation of a drug for promoting tooth enamel mineralization. The drug for promoting tooth enamel mineralization includes a gallic acid-calcium phosphate-fluoride solution and a washing solution; the washing solution includes absolute ethanol and water.

[0015] Further, the gallic acid-calcium phosphate-fluoride solution is used to contact the tooth enamel to be repaired, and then the tooth enamel is washed successively with absolute ethanol and water and then contacted with saliva or artificial saliva.

[0016] The principle and beneficial effects of the present technical solution are as follows:

[0017] The present technical solution provides an actual combination and method for biomimetic mineralization of tooth enamel that can be clinically applied and has great commercial promotion value. By mixing a specific molar ratio of gallic acid and calcium ion solution, and then slowly dripping a fluoride-containing phosphate ion solution into the previous mixture, stable calcium phosphate nanoclusters (PNCs) can be prepared. Using the fluoride-added PNCs to treat demineralized tooth enamel, after natural drying in air, the treated tooth enamel is washed with absolute ethanol, and then immersed in artificial saliva or saliva to achieve the directional transformation growth of HA crystals on the surface of the tooth enamel to be biomimetically mineralized, forming a structure with chemical composition and mechanical properties similar to those of natural tooth enamel, which can well achieve the biomimetic mineralization of tooth enamel. This method has great potential for clinical transformation of the biomimetic mineralization strategy of tooth enamel and is very helpful for treating oral problems such as microdefects of tooth enamel, early caries, and enamel leukoplakia.

[0018] Adopting this solution, in the presence of fluoride, using gallic acid-stabilized calcium phosphate nanoclusters, and then through treatment with absolute ethanol, a relatively pure biomimetic mineralized tooth enamel front is constructed to achieve the biomimetic mineralization of tooth enamel. The general process is as follows: Dissolve a calcium ion source compound and gallic acid in water to obtain a gallic acid-calcium solution; dissolve a phosphate and fluoride ion source compound in water to obtain a fluorine-added phosphorus source solution; slowly drip the fluorine-added phosphorus source solution into the stirring gallic acid-calcium solution to obtain a GA-CaP-F solution (calcium phosphate nanoclusters containing gallic acid and fluoride ions), that is, fluoride-added gallic acid-stabilized calcium phosphate nanoclusters (PNCs); use the GA-CaP+F solution to treat demineralized tooth enamel for a certain period of time; contact the tooth enamel with saliva or artificial saliva for a certain period of time, and the biomimetic mineralization of tooth enamel can be achieved by this method.

[0019] Gallic acid is an inexpensive and edible compound, and the biomimetic mineralization of tooth enamel can be well achieved through the above process. This method has great potential for the clinical transformation of the biomimetic mineralization strategy of tooth enamel and is very helpful for treating oral problems such as enamel microdefects, early caries, and enamel leukoplakia. Currently, there are no research and patent reports on using gallic acid as a stabilizer to construct PNCs, nor are there research and patent reports on using fluorine in combination with gallic acid-stabilized PNCs and then realizing the biomimetic mineralization of tooth enamel through anhydrous ethanol treatment. Description of the Drawings

[0020] Figure 1 Scanning electron micrograph of the surface morphology of natural tooth enamel in Example 1 (scale bar: 10 μm).

[0021] Figure 2 Scanning electron micrograph of the surface morphology of natural tooth enamel in Example 1 (scale bar: 5 μm).

[0022] Figure 3 Scanning electron micrograph of the surface morphology of acid-etched natural tooth enamel in Example 1 (scale bar: 10 μm).

[0023] Figure 4 Scanning electron micrograph of the surface morphology of acid-etched natural tooth enamel in Example 1 (scale bar: 5 μm).

[0024] Figure 5 Scanning electron micrograph of the surface morphology of mineralized tooth enamel in Example 1 (scale bar: 10 μm).

[0025] Figure 6 Scanning electron micrograph of the surface morphology of mineralized tooth enamel in Example 1 (scale bar: 5 μm).

[0026] Figure 7 Scanning electron micrograph of the surface morphology of mineralized tooth enamel in Example 2 (scale bar: 10 μm).

[0027] Figure 8 Scanning electron micrograph of the surface morphology of mineralized tooth enamel in Example 2 (scale bar: 5 μm).

[0028] Figure 9 Scanning electron micrograph of the surface morphology of mineralized tooth enamel in Example 3 (scale bar: 10 μm).

[0029] Figure 10 Scanning electron micrograph of the surface morphology of mineralized tooth enamel in Example 3 (scale bar: 5 μm).

[0030] Figure 11 Scanning electron micrograph of the surface morphology of mineralized tooth enamel in Example 4 (scale bar: 10 μm).

[0031] Figure 12 Scanning electron microscopy image of the surface morphology of mineralized tooth enamel in Example 4 (scale bar: 5 μm).

[0032] Figure 13 Scanning electron microscopy image of the surface morphology of mineralized tooth enamel in Example 5 (scale bar: 10 μm).

[0033] Figure 14 Scanning electron microscopy image of the surface morphology of mineralized tooth enamel in Example 5 (scale bar: 5 μm).

[0034] Figure 15 Statistical chart of the hardness of mineralized tooth enamel obtained by different treatment methods in the examples.

[0035] Figure 16 Statistical chart of the carbon element content on the surface of mineralized tooth enamel obtained by different treatment methods in the examples. Detailed implementation manners

[0036] The present invention will be further described in detail below in conjunction with the examples, but the implementation manners of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can all be obtained from commercial channels.

[0037] The overall technical process of this technical solution is as follows:

[0038] A reagent combination for biomimetic mineralization of tooth enamel includes: gallic acid (GA), calcium ion source, phosphate ion source, and fluoride ion source. Among them, the molar ratio of calcium in the calcium ion source to GA in the gallic acid solution is 0.01 - 0.5, and the molar ratio of calcium in the calcium ion source to phosphorus in the phosphate ion source is 0.1 - 5.

[0039] The calcium ion source is at least one of calcium chloride, calcium nitrate, and calcium acetate; the phosphate ion source is at least one of phosphoric acid, sodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, ammonium phosphate, ammonium hydrogen phosphate, and ammonium dihydrogen phosphate. Deionized water is used as the solvent for preparing various solutions.

[0040] At room temperature, gallic acid is added to water, and the mixture is stirred in a water bath at 35 - 50 °C (preferably 45 °C) for 10 - 120 min (preferably 30 min) to prepare an aqueous GA solution. Then, a calcium ion source is added thereto, and the mixture is stirred with a magnetic stirrer (500 rpm, which needs to be ensured to be above 300 rpm, and the upper limit depends on the equipment used, for example: 20000 rpm) for 10 - 120 min (preferably 60 min) to obtain an aqueous GA-Ca solution. At room temperature, a phosphorus ion source and a fluorine ion source are added to water to obtain a fluorine-added phosphate solution. Under stirring with a magnetic stirrer (500 rpm, which needs to be ensured to be above 300 rpm, and the upper limit depends on the equipment used, for example: 20000 rpm) at room temperature, the fluorine-added phosphate solution is slowly added to the aqueous GA-Ca solution (2 drops / second). The pH value is adjusted to 5 - 7 (preferably 5.5) to obtain a GA-CaP-F solution (a solution containing gallic acid, a calcium ion source, a phosphorus ion source, and a fluorine ion source). The GA-CaP-F solution is applied to the enamel to be repaired, and after the excess liquid is blotted dry with filter paper, it is placed in air to dry naturally, and then rinsed with absolute ethanol for 1 - 5 min (preferably 3 min), followed by washing with water for 1 min. By the above method, in the prepared GA solution, the mass percentage of GA is 0.01 - 2 wt%; in the prepared GA-Ca solution, the calcium concentration is 0.01 - 30 mM; in the prepared fluorine-added phosphate solution, the phosphorus concentration is 0.01 - 30 mM, and the fluorine concentration is 10 ppm - 1500 ppm.

[0041] A reagent combination for biomimetic mineralization of enamel further includes saliva or artificial saliva. Artificial saliva is a conventional reagent in the prior art. Commercial artificial saliva can be purchased, or it can be prepared by oneself. In this technical solution, the specific formula of the artificial saliva used is: 1.5 mM CaCl2·2H2O, 0.9 mM K2HPO4, 15 ppm F - 、130 mM KCL, 1 mM NaN3, which is prepared using 20 mM HEPES buffer solution, and the pH of the solution is adjusted to 7.00 ± 0.03. Artificial saliva is a conventional reagent in the art. In actual use, artificial saliva with other formulas can also be used, mainly to provide calcium and phosphorus elements and simulate the role of saliva. After applying the GA-CaP-F solution to treat the enamel, the enamel needs to be further mineralized in an environment of saliva or artificial saliva to form a sufficient mineralized layer. Since the subsequent experiments are carried out on enamel samples, artificial saliva is specifically used. The enamel after soaking in the GA-CaP-F solution, absolute ethanol, and water washing is placed in saliva or artificial saliva for soaking, and fresh artificial saliva is replaced every 6 h, lasting for 72 - 144 h in total.

[0042] Example 1

[0043] (1) Preparation of enamel specimens

[0044] Cut the enamel of healthy bovine teeth free of soft deposits, calculus, and soft tissues into enamel pieces with a size of 0.5×0.5×0.2 cm. Prepare 5 enamel pieces for each group of samples. Polish them sequentially with 360-mesh, 1200-mesh, 3000-mesh, and 5000-mesh SiC sandpapers until the surface is smooth and flat. Keep the enamel pieces moist throughout the process. 3 Invert the enamel pieces in a dental casting mold and embed them with denture base resin. After solidification, demold, and then grind the surface of the enamel pieces to be on the same surface as the resin. Ultrasonically clean them in water for 20 min. After natural drying in air, cover the surface of the tooth samples with a polyester tape with a window to create a test area with a size of approximately 3×6 mm

[0045] , and measure its hardness with a hardness tester, which is the hardness of natural enamel. The scanning electron microscope images of natural enamel are shown in 2 , and Figure 1 and Figure 2 .

[0046] Soak the prepared enamel pieces in 37% phosphoric acid for 40 s, wash them with water for 1 min, and ultrasonically treat them for 20 min. Measure the hardness after acid etching with a digital display hardness tester, and measure the surface morphology of the enamel after acid etching with a scanning electron microscope. The surface morphology of the enamel after acid etching is shown in Figure 3 and Figure 4 . Store the acid-etched enamel pieces in deionized water at 4℃ and air-dry them before use.

[0047] (2) GA-CaP-F solution

[0048] At room temperature, 29.7 ml of deionized water was taken, and 0.40 g of gallic acid (molecular weight 170.12, about 0.00235 mol) was added thereto. It was stirred for 30 min in a water bath at 45 °C to prepare a 1% GA solution. Then, 0.0173 g of CaCl2·2H2O (molecular weight 147.01, the concentration of calcium element in the GA-Ca solution was about 0.000118 mol / 29.7 ml) was added thereto, and it was stirred with a magnetic stirrer (500 rpm) for 60 minutes. At this time, the molar ratio of calcium ion to GA was 0.05. At room temperature, 10 ml of deionized water was taken, and 0.01 g of Na2HPO4 (molecular weight 141.96, the concentration of phosphorus in the phosphate solution with added fluorine was about 0.00007 mol / 10 ml) and 0.004 g of NaF (molecular weight 41.99, the concentration of F in the phosphate solution with added fluorine was 180 ppm) were added thereto to obtain a phosphate solution with added fluorine. Under stirring with a magnetic stirrer (500 rpm) at room temperature, the phosphate solution with added fluorine was slowly added to the GA-Ca aqueous solution (2 drops / second). The pH value was adjusted to 5.5 with 1 M NaOH. The molar ratio of CaCl2·2H2O / Na2HPO4 in the final solution was about 1.67 to obtain a GA-CaP-F solution.

[0049] A small dental cotton swab was dipped in a small amount of GA-CaP-F and applied to the demineralized (acid-etched) enamel slices for 5 min. More specifically, an ordinary cotton swab was fully wetted with the GA-CaP-F solution (saturated dipping), and then the wetted cotton swab was used to wipe the dentin surface. It was wiped back and forth for 2.5 min. Then, a new cotton swab was dipped in the GA-CaP-F solution and the enamel slices were wiped again, that is, the dentin surface was applied for a total of 5 min. The excess liquid around the dentin was blotted dry with filter paper. After the liquid was fully dried (naturally dried in the air), it was then rinsed with absolute ethanol for 3 min and washed with water for 1 min. Then, it was placed in artificial saliva at 37 °C with the surface of the dentin coated with the GA-CaP-F solution facing down, and the surface of the dentin coated with the GA-CaP solution was kept in full contact with the artificial saliva. The fresh artificial saliva was changed every 6 h. After 72 h, the hardness after mineralization was measured with a digital hardness tester, and the surface morphology of the enamel after mineralization was measured with a scanning electron microscope. See Figure 5 and Figure 6 。

[0050] Example 2

[0051] The preparation and acid etching of enamel specimens and the preparation of artificial saliva are the same as those in Example 1. In this example, we did not add gallic acid solution in the treatment steps of enamel specimens to observe the biomimetic mineralization of enamel after acid etching. That is, take 29.7 ml of deionized water, add 0.0173 g of CaCl2·2H2O to it, stir with a magnetic stirrer (500 rpm) for 60 minutes to obtain a calcium ion source solution, and use the calcium ion source solution to replace the GA-Ca solution for the subsequent steps. The finally obtained CaP-F solution is used to treat tooth enamel (instead of the GA-CaP-F solution, without adding gallic acid), and other operations are the same as those in Example 1. The SEM images of the biomimetic mineralized enamel are shown in detail in Figure 7 and Figure 8 .

[0052] Example 3

[0053] The preparation and acid etching of enamel specimens and the preparation of artificial saliva are the same as those in Example 1. In this example, we did not add fluorine to the phosphate source solution in the treatment steps of enamel specimens to observe the biomimetic mineralization of enamel after acid etching. That is, take 10 ml of deionized water, add 0.01 g of Na2HPO4 to it to obtain a phosphate root solution, which is used to replace the phosphate root solution with added fluorine. The finally obtained GA-CaP solution is used to treat tooth enamel (instead of the GA-CaP-F solution, without adding fluorine element), and other operations are the same as those in Example 1. The SEM images of the biomimetic mineralized enamel are shown in detail in Figure 9 and Figure 10 .

[0054] Example 4

[0055] The preparation and acid etching of enamel specimens and the preparation of artificial saliva are the same as those in Example 1. In this example, we removed the anhydrous ethanol water washing step in the treatment steps of enamel specimens to observe the biomimetic mineralization of enamel after acid etching. That is, after the enamel slices are treated with the GA-CaP-F solution and dried, they are not rinsed with anhydrous ethanol, but directly washed with water for 1 min, and other operations are the same as those in Example 1. The SEM images of the biomimetic mineralized enamel are shown in detail in Figure 11 and Figure 12 .

[0056] Example 5

[0057] The preparation and acid etching of enamel specimens and the preparation of artificial saliva are the same as those in Example 1. In this example, on the basis of Example 1, the calcium source solution was replaced with calcium nitrate, and the phosphate source solution was replaced with sodium phosphate to observe the biomimetic mineralization of enamel after acid etching. Specifically as follows:

[0058] At room temperature, take 29.7 ml of deionized water, add 0.40 g of GA thereto, stir in a 45°C water bath for 30 min to prepare a 1% GA aqueous solution, then add 0.0193 g of Ca(NO3)2 (molecular weight 164.09, the concentration of calcium in the GA-Ca solution is about 0.000118 mol / 29.7 ml), stir with a magnetic stirrer (500 rpm) for 60 minutes, at which time the molar ratio of calcium ions to GA is 0.05; at room temperature, take 10 ml of deionized water, add 0.0115 g of Na3PO4 (molecular weight 164, the concentration of phosphorus in the fluorinated phosphate solution is about 0.00007 mol / 10 ml) and 0.004 g of NaF (molecular weight 41.99, the concentration of F in the fluorinated phosphate solution is 180 ppm) thereto to obtain a fluorinated phosphate solution. At room temperature, under the stirring action of a magnetic stirrer (500 rpm), the fluorinated phosphate solution was slowly added to the GA-Ca aqueous solution (2 drops / second). The pH value was adjusted to 5.5 with 1M NaOH to obtain a GA-CaP-F solution. The Ca(NO3)2 / Na3PO4 in the final solution was 1.67. Other operations were the same as in Example 1. The electron microscope scanning image of the biomimetic mineralized enamel is shown in detail. Figure 13 and Figure 14 .

[0059] Based on the experimental results of Examples 1-5: the surface of the natural enamel flakes has a fuzzy scaly structure, the cross section is neat, and the enamel columns with a prismatic structure of about 5 μm in cross-sectional diameter are highly parallel to each other along the c-axis direction (the long axis of the apatite crystal) ( Figure 1 , 2 After acid etching, the scaly structure of the enamel surface became clear, the enamel interstitium was largely damaged, the enamel prisms became sparse, and the enamel fibers were incomplete and scattered ( Figure 3 , 4 After being treated in Example 1, the mineralized layer grew epitaxially along the enamel column, presenting a dense structure layer perpendicular to the surface. The scale-like structure clearly visible after acid etching was completely covered, and the surface became smooth and flat, which was closer to the surface structure of natural enamel ( Figure 5 , 6 After the treatment of Example 5, similar results as those of Example 1 were obtained ( Figure 13 , 14 ), indicating that changing the anion type of the calcium source and phosphorus source has little effect on the method results. In Example 2, after removing gallic acid, the enamel surface after acid etching is covered with a layer of material, but there is no orderly structure, which is quite different from the natural enamel surface structure ( Figure 7 , 8 In Example 3, without the addition of fluoride, the mineralized layer showed a sparse fibrous structure and was relatively messy ( Figure 9 , 10)。In Example 4, the anhydrous ethanol washing step was removed, and the mineralized layer was thinner and had no effective ordered structure. Figure 11 and 12 )。

[0060] The hardness of the mineralized dentin samples, the dentin samples before and after acid etching obtained in the examples and comparative examples was measured using a hardness tester (the dentin was pretreated with the GA-CaP solution, CaP-F solution or GA-CaP-F solution prepared according to each example and comparative example, and after continuing to be treated with artificial saliva for 72 hours, the dentin samples were taken out for hardness testing). For the initial natural dentin, acid-etched dentin, and mineralized dentin of each dentin sample, a digital display hardness tester was used for hardness measurement. Each time a measurement was taken, 3 points were selected for measurement on the same dentin sample, and the average value of these 3 measurement points was taken as the hardness of the dentin sample. 9 dentin samples were measured for each example or comparative example, and the average hardness values of the 9 dentin samples of each example or comparative example were shown in the table. The experimental results are shown in Figure 15 . In the figure, **** indicates that the two groups of data were analyzed by ordinary one-way ANOVA, p < 0.0001; ns indicates that there was no significant difference between the two groups of data; the hardness was Vickers hardness HV. The hardness of natural tooth enamel was 328.10 ± 10.73; the hardness of natural tooth enamel after acid etching decreased to 131.4 ± 13.65; after the treatment of Example 1, the hardness was also restored to 334.9 ± 18.78, with no significant difference from the hardness of natural enamel. The hardness after acid etching was increased to 233.1 ± 21.60 in Example 2, but there was still a significant difference from the hardness of natural enamel due to the absence of gallic acid. The hardness of the mineralized layer in Example 3 decreased significantly to 218.6 ± 26.92, but there was a significant difference from the hardness of natural enamel due to the absence of fluoride ions. The hardness of tooth enamel in Example 4 was 225.6 ± 19.86; the hardness of tooth enamel in Example 5 was 330.4 ± 22.72. In Example 4, the anhydrous ethanol washing step was removed, and the mineralization effect was not ideal, resulting in the inability to effectively improve the hardness of the biomimetic mineralized tooth enamel.

[0061] Compare the hardness data of the acid-etched enamel in Examples 1, 2, and 3. After treating the enamel with the GA-CaP-F solution (containing gallic acid, fluoride ions, and calcium phosphate nanoclusters), the increase in the hardness of the enamel is 334.9 - 131.4 = 203.5; after treating the enamel with the CaP-F solution (containing fluoride ions and calcium phosphate nanoclusters), the increase in the hardness of the enamel is 233.1 - 131.4 = 102.7; after treating the enamel with the GA-CaP solution (containing gallic acid and calcium phosphate nanoclusters), the increase in the hardness of the enamel is 218.6 - 131.4 = 87.2. It can be seen that the effect of increasing the hardness of the enamel brought about by using the GA-CaP-F solution (using fluoride ions and gallic acid simultaneously) (203.5) is stronger than the sum of the effects of increasing the hardness of the enamel brought about by using fluoride ions and gallic acid separately (102.7 + 87.2), indicating that the combined use of fluoride ions and gallic acid, in the presence of calcium phosphate nanoclusters (CaP), produces a synergistic effect in enhancing the hardness of biomimetic mineralized enamel. In addition, the ethanol washing step is very crucial, otherwise, the synergistic effect of fluoride ions and gallic acid is difficult to exert (Example 4).

[0062] After statistical analysis by EDS scanning equipped with a scanning electron microscope, it was found that the carbon element content in Example 4 reached 13.08 ± 1.99, showing a significant difference compared with the carbon element content of natural enamel, which is 3.27 ± 0.36. In other examples, after ethanol washing, there was no significant difference in the carbon element content compared with natural enamel( Figure 16 ). Therefore, ethanol washing for 1 - 5 minutes is a key technical point for both enhancing the hardness of biomimetic mineralized enamel and ensuring that the carbon element content remains at the natural level.

[0063] The above are only examples of the present invention. Specific technical solutions and / or common knowledge such as characteristics well known in the art are not described in detail herein. It should be noted that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application should be based on the content of its claims, and the specific implementation manners described in the specification can be used to interpret the content of the claims.

Claims

1. A reagent combination for biomimetic mineralization of tooth enamel, characterized in that: The reagent combination includes a gallic acid-calcium phosphate-fluoride solution, and the raw materials of the gallic acid-calcium phosphate-fluoride solution include gallic acid, a calcium ion source, a phosphorus ion source and a fluoride ion source; the molar ratio of the calcium element in the calcium ion source to the gallic acid is 0.05, and the molar ratio of the calcium element in the calcium ion source to the phosphorus element in the phosphorus ion source is 1.67; Also included is a washing solution, the washing solution comprising anhydrous ethanol and water; Also includes saliva or artificial saliva; The gallic acid-calcium phosphate-fluoride solution is used to contact the tooth enamel to be repaired; Absolute ethanol was used to wash tooth enamel after contact with gallic acid-calcium phosphate-fluoride solution; Water is used to wash the enamel after washing with anhydrous ethanol; saliva or artificial saliva is used to contact the enamel after washing with water.

2. The reagent combination for biomimetic mineralization of tooth enamel according to claim 1, characterized in that: The gallic acid-calcium phosphate-fluoride solution is prepared by the following method: dissolving gallic acid in water to obtain a gallic acid solution; adding a calcium ion source into the gallic acid solution under stirring to obtain a gallic acid-calcium solution; dissolving a phosphorus ion source and a fluoride ion source in water to obtain a fluorine-containing phosphorus ion source solution; dropping the fluorine-containing phosphorus ion source solution into the stirred gallic acid-calcium solution, and adjusting the pH value to obtain a gallic acid-calcium phosphate-fluoride solution.

3. A reagent combination for biomimetic mineralization of tooth enamel according to claim 2, characterized in that: The mass fraction of gallic acid in the gallic acid solution is 0.01-2%; in the gallic acid-calcium solution, the concentration of calcium element is 0.01-30mM; in the fluorine-containing phosphorus ion source solution, the concentration of phosphorus element is 0.01-30mM, and the concentration of fluorine element is 10ppm-1500ppm.

4. The reagent combination for biomimetic mineralization of tooth enamel according to claim 3, characterized in that: Dissolve gallic acid in water and stir at 35-50° C. for 10-120 min to obtain a gallic acid solution.

5. The reagent combination for biomimetic mineralization of tooth enamel according to claim 4, characterized in that: Under stirring conditions of >300 rpm, a calcium ion source is added to the gallic acid solution and stirred for 10-120 min to obtain a gallic acid-calcium solution.

6. The reagent combination for biomimetic mineralization of tooth enamel according to claim 5, characterized in that: The fluorine-containing phosphorus ion source solution is added dropwise at a rate of 1-4 drops / second to the gallic acid-calcium solution being stirred at >300 rpm, with a pH value of 5-7, to obtain a gallic acid-calcium phosphate-fluoride solution.

7. Use of a reagent combination for biomimetic mineralization of tooth enamel according to any one of claims 1 to 6 in the preparation of a drug for promoting tooth enamel mineralization, characterized in that: Drugs that promote enamel mineralization include gallic acid-calcium phosphate-fluoride solution and washing solution; washing solution includes anhydrous ethanol and water; The gallic acid-calcium phosphate-fluoride solution is used to contact the tooth enamel to be repaired, and then the tooth enamel is washed with anhydrous ethanol and water in sequence and then contacted with saliva or artificial saliva.

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