A method for constructing calcium phosphate nanoclusters biomimetic mineralized dentin using gallic acid

Abstract

The present invention relates to the technical field of dentin repair, and in particular to a method for constructing a biomimetic mineralized dentin with calcium phosphate nanoclusters using gallic acid. The reagent combination includes a gallic acid-calcium phosphate solution, and the raw materials of the gallic acid-calcium phosphate solution include gallic acid, a calcium ion source, and a phosphorus ion source; the molar ratio of the calcium element in the calcium ion source to the gallic acid is 0.01-0.1, 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. This technical scheme can solve the technical problem that the prior art lacks an effective method for promoting dentin mineralization. By using gallic acid biomimetic mineralized dentin, by mixing gallic acid and calcium ion solution in a specific molar ratio, slowly adding a phosphate ion solution dropwise to the pre-mixed solution, a calcium phosphate nanocluster of biomimetic mineralized dentin can be prepared to achieve effective mineralization repair. Gallic acid is a cheap, edible compound, and this scheme has an ideal application and promotion prospect.

Description

Technical Field

[0001] The invention relates to the technical field of dentin repair, and in particular to a method for constructing calcium phosphate nanocluster biomimetic mineralized dentin by utilizing gallic acid. Background Art

[0002] Caries and dentin hypersensitivity are currently troublesome oral problems worldwide, with a high incidence and a wide range of impact, seriously affecting the quality of human life. Efficient dentin bionic mineralization is considered to be an ideal strategy for treating oral problems such as dentin micro-defects, early caries and dentin hypersensitivity.

[0003] Dentin is composed of dentinal tubules, dentinal cell protrusions and intercellular matrix. It has carbonated hydroxyapatite (HAP) as the main inorganic component, accounting for about 70% by weight (40-45% by volume). The organic matrix is ​​mainly type I collagen, accounting for about 20%, and the remaining 10% is mainly water. Although dentin is not as hard as enamel, it has higher toughness and can buffer and absorb the force from enamel.

[0004] The biomineralization process of dentin is a process in which HAP is deposited and formed in an orderly manner within and between dentin collagen fibers under the regulation of non-collagenous proteins (NCPs) such as dentin matrix protein (DMP) and dentin phosphoprotein (DPP). Compared with enamel, dentin contains more type I collagen fibers. To achieve the biomimetic mineralization of dentin, it is necessary to first achieve mineralization within the collagen fibers, and then achieve the mineralization of the dentin hard tissue, and even the closure of the dentinal tubules, because the support of the collagen scaffold by the minerals within the fibers is the key to the excellent mechanical properties of dentin. The length of the collagen molecule is about 300nm and the width is about 1.5nm. Adjacent collagen microfibrils are arranged in a way of 67nm offset translation to form collagen fibers. Every 67nm offset forms a periodic structure of collagen, called the D period. Among them, there is a gap of about 40nm between the C-terminus of a collagen microfibril and the N-terminus of the next adjacent microfibril, which is called the pore area. If nanoparticles are to enter collagen fibers through the pores, the smaller the particle size, the better. NCPs carry carboxyl-rich amino acids, which can act like capping agents to prevent amorphous calcium phosphate (ACP, which is the precursor of HAP) from agglomerating and enlarging, making it easier for it to enter the interior of type I collagen fibers. At the same time, NCPs can also bind to type I collagen, providing time for the conversion of ACP to HAP. However, it is very difficult to separate and purify natural NCPs, which also leads to its high price, greatly limiting the in vitro research and clinical application of NCPs. Current research mainly focuses on finding NCPs analogs such as poly aspartic acid (PASP), polyacrylic acid (PAA), and polyvinylphosphonic acid (PVPA) to simulate the role of NCPs to achieve biomimetic mineralization of dentin.

[0005] Although NCPs analogs induce orderly remineralization of dentin, this process takes a considerable amount of time, usually several weeks or even months, which is clinically unacceptable and hinders the development of these technologies from the laboratory to the clinic. In addition, since this type of polyelectrolyte contains a large number of carboxyl groups and exhibits strong anionic properties, many free carboxyl groups have certain cytotoxicity. Even if they can be used in vivo, biocompatibility should be taken into account. Based on the above safety and clinical efficiency considerations, it is urgent to develop a new substance that can replace the role of NCPs, thereby achieving the promotion of dentin mineralization. Summary of the invention

[0006] The present invention is intended to provide a reagent combination for constructing calcium phosphate nanoclusters for biomimetic mineralization of dentin using gallic acid, so as to solve the technical problem that the prior art lacks an effective method for promoting dentin mineralization.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme:

[0008] A reagent combination for constructing calcium phosphate nanocluster biomimetic mineralized dentin using gallic acid, the reagent combination comprising gallic acid-calcium phosphate solution, the raw materials of which include gallic acid, a calcium ion source, and a phosphorus 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.

[0009] Furthermore, the gallic acid-calcium phosphate 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 in water to obtain a phosphorus ion source solution, and dropwise adding the phosphorus ion source solution to the stirred gallic acid-calcium solution, and adjusting the pH value to obtain a gallic acid-calcium phosphate solution.

[0010] 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 element is 0.01-30mM; in the phosphorus ion source solution, the concentration of phosphorus element is 0.01-30mM.

[0011] Further, gallic acid is dissolved in water and stirred at 35-50° C. for 10-120 min to obtain a gallic acid solution.

[0012] 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.

[0013] Furthermore, the phosphorus ion source solution is added dropwise at a rate of 1-4 drops / second into the gallic acid-calcium solution being stirred at >300 rpm.

[0014] Furthermore, the pH value of the gallic acid-calcium phosphate solution is 5-7.

[0015] Furthermore, the reagent combination also includes artificial saliva or saliva.

[0016] The technical solution also provides an application of a reagent combination for constructing calcium phosphate nanoclusters for biomimetic mineralization of dentin using gallic acid in the preparation of a drug for promoting dentin mineralization. The drug for promoting dentin mineralization includes gallic acid-calcium phosphate solution.

[0017] Furthermore, the gallic acid-calcium phosphate solution is used to contact with the dentin to be repaired, and the dentin after contacting with the gallic acid-calcium phosphate solution is used to be immersed in saliva or artificial saliva.

[0018] The principle and beneficial effects of this technical solution are:

[0019] The present invention provides a reagent combination and a method for using gallic acid to construct calcium phosphate nanocluster biomimetic mineralization of dentin. A calcium ion source compound is dissolved in a gallic acid solution, and a phosphorus source solution is slowly added dropwise to a stirred mixed solution of gallic acid and calcium to obtain a gallic acid-calcium phosphate solution (GA-CaP solution). The demineralized dentin is treated with the GA-CaP solution for a certain period of time, and the dentin is placed in saliva or artificial saliva. The method can achieve biomimetic mineralization of dentin.

[0020] In this technical solution, gallic acid from a natural plant gallnut extract is used to construct non-collagen PNCs to biomimetic mineralized dentin. Gallic acid is a cheap, edible compound. By mixing a specific molar ratio of gallic acid and a calcium ion solution, and then slowly dropping a phosphate ion solution into the premixed solution, calcium phosphate nanoclusters (PNCs) for biomimetic mineralized dentin can be prepared, and the particle size can achieve an effect similar to that of polyanion electrolytes such as PAA and PASP. The demineralized dentin is treated with such PNCs for 5 minutes, and then immersed in artificial saliva or saliva, which can well achieve the biomimetic mineralization of dentin. This method has a great potential for clinical transformation of dentin biomimetic mineralization strategies, and is very helpful for treating oral problems such as dentin micro-defects, early caries and dentin hypersensitivity. At present, there is no research and patent report on the use of gallic acid as a stabilizer to construct PNCs, nor is there any research and patent report on the use of gallic acid-stabilized PNCs to achieve intra-collagen and inter-collagen biomimetic mineralization of dentin hard tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a scanning electron microscope image of the natural dentin surface morphology of Example 1.

[0022] Figure 2 This is a transmission electron microscopy image of the natural dentin slice of Example 1.

[0023] Figure 3 This is the transmission electron microscopy SAED image of the natural essence slice of Example 1.

[0024] Figure 4 This is a scanning electron microscope image of the intrinsic surface morphology after acid etching in Example 1.

[0025] Figure 5 This is a transmission electron microscope image of the intrinsic section after acid etching in Example 1.

[0026] Figure 6 This is the transmission electron microscopy SAED image of the intrinsic slice after acid etching in Example 1.

[0027] Figure 7 This is a transmission electron microscopic morphology image of the calcium phosphate nanoclusters of Example 1.

[0028] Figure 8 This is a scanning electron micrograph of the surface morphology of the treated and mineralized dentin in Example 1.

[0029] Fig. 9 This is a transmission electron micrograph of the treated and mineralized dentin section of Example 1.

[0030] Fig.10 This is the transmission electron microscopy SAED image of the treated and mineralized dentin section in Example 1.

[0031] Fig.11 This is a transmission electron microscopic morphology image of the calcium phosphate nanoclusters of Example 2.

[0032] Fig.12 This is a scanning electron microscope image of the surface morphology of the treated and mineralized dentin in Example 2.

[0033] Fig.13 This is a transmission electron micrograph of the treated and mineralized dentin section of Example 2.

[0034] Fig.14 This is the transmission electron microscopy SAED image of the treated and mineralized dentin section in Example 2.

[0035] Fig.15 The hardness statistics of the treated and mineralized dentin slices of the embodiments and comparative examples are shown in FIG. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with examples, but the embodiments 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 be obtained from commercial sources.

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

[0038] The reagent combination for constructing calcium phosphate nanocluster biomimetic mineralized dentin using gallic acid includes gallic acid, a calcium ion source, and a phosphorus ion source, wherein the molar ratio of calcium in the calcium ion source to GA in the gallic acid solution is 0.01-0.1, and the molar ratio of calcium in the calcium ion source to phosphorus in the phosphorus ion source is 0.1-5.

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

[0040] At room temperature, deionized water is taken, GA is added thereto, stirred for 10-120min (preferably 30min) in a water bath at 35-50°C (preferably 45°C), a GA solution is prepared, a calcium ion source is added thereto, and a magnetic stirrer (500rpm, it needs to be ensured to be above 300rpm, the upper limit depends on the equipment used, for example: 20000rpm) is stirred for 10-120min (preferably 60min) to obtain a GA-Ca solution (gallic acid-calcium solution). At room temperature, deionized water is taken, a phosphorus ion source is added thereto, and a phosphorus ion source solution is obtained. At room temperature, a magnetic stirrer (500rpm, it needs to be ensured to be above 300rpm, the upper limit depends on the equipment used, for example: 20000rpm) is stirred, and the phosphorus ion source solution is slowly added to the GA-Ca solution (1-4 drops / second, preferably 2 drops / second). The pH value is adjusted to 5-7 (preferably 5.5) to obtain a GA-CaP solution (gallic acid-calcium phosphate solution). The GA-CaP solution is applied to the dentin to be mineralized. In the above method, the mass percentage of GA in the prepared GA solution is 0.01-2wt%; the calcium concentration in the prepared GA-Ca solution is 0.01-30mM; and the phosphorus concentration in the prepared phosphorus ion source solution is 0.01-30mM.

[0041] The reagent combination for constructing calcium phosphate nanocluster biomimetic mineralized dentin using gallic acid also includes artificial saliva / saliva. Artificial saliva is a conventional reagent in the prior art of the art. Commercial artificial saliva can be purchased or prepared by oneself. In the present technical solution, the specific formula of artificial saliva used is: 1.5mM CaCl2·2H2O, 0.9mM K2HPO4, 15ppm F -, 130mM KCL, 1mM NaN3, prepared with 20mM HEPES buffer, and adjusted the solution pH to 7.00±0.03. Artificial saliva is a conventional reagent in this field. Artificial saliva of other formulas can also be used in actual use, mainly to provide calcium and phosphorus elements to simulate the effect of saliva. After the GA-CaP solution is applied to treat the dentin, the dentin needs to be further mineralized in the environment of saliva or artificial saliva. Because the subsequent experiments were carried out in dentin samples, artificial saliva was specifically used. Demineralized collagen and dentin are further mineralized on the basis of the dentin samples treated with GA-CaP solution to form a sufficient amount of mineralized layer. The dentin slices treated with GA-CaP solution can be placed in artificial saliva, and fresh artificial saliva is replaced every 6 hours for a total of 72-144 hours.

[0042] Example 1

[0043] (1) Dentin sample preparation

[0044] Healthy bovine teeth without cracks, caries, white spots or dysplasia were selected, and the teeth were thoroughly cleaned with deionized water to remove soft plaque, hard plaque and soft tissue. Then disinfected in 2% glutaraldehyde for 30 minutes. Bovine dentin was cut into dentin slices of 5mm×5mm×1.5mm in size using a slow cutter under running water. All dentin slices were polished on a grinding and polishing machine using 360, 1200, 3000 and 5000 mesh SiC sandpaper in sequence until the surface was smooth and flat. The dentin slices were kept moist during the whole process. Ultrasonic cleaning was performed in water for 20 minutes (ultrasonic frequency 40KHz, power 400W, continuous ultrasonic treatment) to obtain a dentin sample. The scanning electron microscope morphology of the natural dentin surface is shown in Figure 1 , transmission electron microscopy of natural dentin slices Figure 2 , Figure 3 This is a transmission electron microscopy SAED image of a natural dentin section.

[0045] (2) Acid etching of dentin samples

[0046] The polished dentin samples were soaked in 37% phosphoric acid for 15 seconds, washed with deionized water, treated with 5.25% sodium hypochlorite for 30 seconds, and rinsed with sufficient deionized water. A three-dimensional demineralized dentin sample with almost no mineralization within a few microns near the surface was obtained. The typical scanning electron micrograph of the dentin surface after acid etching is shown in Figure 4 , transmission electron microscopy of the dentin section after acid etching is shown in Figure 5 , Figure 6 This is the transmission electron microscopy SAED image of the dentin section after acid etching.

[0047] (3) Preparation of GA-CaP solution

[0048] At room temperature, 29.7 ml of deionized water was taken, 0.40 g of GA (molecular weight 170.12, about 0.00235 mol) was added thereto, and stirred in a 45°C water bath for 30 min to prepare a 1% GA aqueous solution, to which 0.0173 g of CaCl2·2H2O (molecular weight 147.01, the concentration of calcium in the GA-Ca solution was about 0.000118 mol / 29.7 ml) was added, and a magnetic stirrer (500 rpm) was stirred for 60 minutes to form a GA-Ca solution, at which the molar ratio of calcium ions to GA was 0.05. At room temperature, 10 ml of deionized water was taken, 0.01 g of Na2HPO4 was added thereto, and a phosphorus ion source solution (molecular weight 141.96, the concentration of phosphorus in the phosphorus ion source solution was about 0.00007 mol / 10 ml) was obtained. At room temperature, under the stirring action of a magnetic stirrer (500 rpm), the phosphate solution was slowly added to the GA-Ca solution (2 drops / second). The pH value was adjusted to 5.5 with 1M NaOH. The molar ratio of CaCl2·2H2O / Na2HPO4 in the final solution was about 1.67, and a GA-CaP solution was obtained. Microscopic observation of the GA-CaP solution, transmission electron microscopy morphology of calcium phosphate nanoclusters can be found in Figure 7 .

[0049] (4) Processing of dentin samples

[0050] Use a small dental cotton swab to dip a small amount of GA-CaP solution and apply it to the demineralized dentin sample (dentin sample after acid etching) for 5 minutes. More specifically, use an ordinary cotton swab to fully soak the GA-CaP solution (saturated dipping), and then use the soaked cotton swab to wipe the dentin surface, and wipe back and forth for 2.5 minutes. Then, take a new cotton swab and saturate it with GA-CaP solution, and repeat wiping the dentin surface once, that is, apply the dentin surface for a total of 5 minutes. Use filter paper to absorb excess liquid around the dentin, and after the liquid is fully dried (natural drying), place the dentin with the surface coated with GA-CaP solution facing down in 37°C artificial saliva, keeping the surface of the dentin coated with GA-CaP solution in full contact with the artificial saliva. Replace fresh artificial saliva every 6 hours and continue soaking for 4 days. The dentin slices taken out after treatment were cut into two halves, and dehydrated with 30%, 50%, 70%, 80%, 90%, 95%, and 100% alcohol in turn, each time for 15 minutes. One half was air-dried for SEM analysis, and the other half was treated with acetone solution for 20 minutes. Then, the samples were treated with 1:1 epoxy resin embedding agent / acetone mixture, 3:1 epoxy resin embedding agent / acetone mixture, and pure epoxy resin embedding agent in turn for 1h, 3h, and 8h, and finally cured at 70°C. A transmission electron microscopy sample of about 70-90nm thickness was cut with an ultrathin slicer for transmission electron microscopy observation. The scanning electron microscopy image of the treated dentin in this embodiment is shown in Figure 8 , transmission electron microscopy of dentin sections see Fig. 9 , TEM SAED images of dentin sections are shown in Fig.10 .

[0051] Example 2

[0052] In this example, the preparation and acid etching of the dentin specimen, the preparation of artificial saliva and the treatment of the dentin specimen are the same as in Example 1. In this example, the GA-CaP solution is replaced with a CaP solution of the same concentration to observe the biomimetic mineralization of the dentin after acid etching. That is, the specific difference is: at room temperature, take 29.7 ml of deionized water, add 0.0173 g of CaCl2·2H2O thereto, and stir with a magnetic stirrer (500 rpm) for 60 minutes; at room temperature and with stirring of a magnetic stirrer (500 rpm), slowly add the phosphate ion source solution to the aqueous solution containing only calcium ions (2 drops / second). Use 1M NaOH to adjust the pH value to 5.5. The CaCl2·2H2O / Na2HPO4 in the final solution is 1.67. For transmission electron microscopy morphology of calcium phosphate nanoclusters, see Fig.11 , and Example 1 Figure 7 Compared with Fig.11 The particles are in a state of large agglomeration and have a relatively large particle size. The scanning electron microscopy image of the surface morphology of the dentin after treatment with the GA-free CaP solution and mineralization in this example is shown in Fig.12 , the transmission electron microscopy images of the dentin sections treated with the CaP solution without GA and mineralized in this example are shown in Fig.13 , the transmission electron microscopy SAED image of the dentin slice after treatment and mineralization with CaP solution without GA in this example is shown in Fig.14 .

[0053] Example 3

[0054] This embodiment is basically the same as embodiment 1, except that the amount of CaCl2·2H2O is increased to 0.173 g, the molar ratio of calcium ion to GA is increased, and the amount of Na2HPO4 is increased to 0.1 g, while the other parameters and operating procedures / methods remain unchanged.

[0055] Comparative Example 1

[0056] This comparative example is basically the same as Example 1, except that the phosphate solution is directly added to the GA-Ca solution instead of being slowly added dropwise as in Example 1, and the other parameter conditions and operation process / method remain unchanged.

[0057] Comparative Example 2

[0058] The preparation and acid etching of the dentin specimens, the preparation of artificial saliva and the treatment of the dentin specimens in this comparative example are the same as those in Example 1. In this comparative example, the order of adding calcium ions and phosphate ions is changed, that is, the phosphate ions are first mixed with the GA solution, and then the calcium ions are added to the mixed solution. That is, the specific difference is: at room temperature, 29.7 ml of deionized water is taken, 0.30 g of GA is added thereto, and stirred in a water bath at 45°C for 30 minutes to prepare a 1% GA aqueous solution, and then 0.01 g of Na2HPO4 is added thereto, and stirred for 60 minutes with a magnetic stirrer (500 rpm); at room temperature, 10 ml of deionized water is taken, 0.0173 g of CaCl2·2H2O is added thereto to obtain a calcium ion solution. At room temperature, under the stirring action of a magnetic stirrer (500 rpm), the calcium ion solution is slowly added to the mixed aqueous solution of GA and phosphate (2 drops / second). The remaining parameter conditions and operation process / method are the same as in Example 1.

[0059] Comprehensive analysis of the dentin mineralization of Examples 1-3 and Comparative Examples 1 and 2 shows that the natural dentin structure is dense and flat, and the dentin collagen is embedded in a large number of hydroxyapatite crystals ( Figure 1 , Figure 2 ), SAED detection shows obvious crystallization ( Figure 3 ). Through phosphoric acid etching, a dentin demineralization layer of about 3-5μm was obtained, the dentinal tubules were opened, and the dentin collagen fibers were exposed to the external environment after demineralization. Under transmission electron microscopy, the minerals in the dentin collagen network after demineralization were lost ( Figure 4 , Figure 5 ), with a light grey structure and a clear boundary with natural dentin, and no crystalline phase was detected by SAED ( Figure 6 ), indicating that the demineralization effect is obvious.

[0060] In Example 1, a particle size of about 11.62 nm ( Figure 7 ) calcium phosphate nanoclusters. After being treated with the GA-CaP solution in Example 1 for 5 minutes and then immersed in artificial saliva for 4 days, the electron density of the collagen fiber network in the mineral layer increased significantly, and obvious remineralized crystals in the collagen fibers were observed. These crystals were arranged in parallel and orderly, showing the rope-like fiber structure of the collagen fibers ( Fig. 9 ), achieving better mineralization within the fibers. Scanning electron microscopy revealed that the orifices of the tubules shrank, and the minerals gradually extended from the periphery to the center, and the collagen fibers exposed on the surface were basically covered ( Figure 8 ), the collagen exposed at the tube orifice is covered by a layer of mineralization, and its morphology is similar to that of natural dentin.

[0061] The CaP solution without GA in Example 2 was turbid and had a lot of precipitation. Transmission electron microscopy showed that most of the precipitation was in the form of agglomerates of several microns in size ( Fig.11), after 4 days of mineralization in the pretreatment group, a large number of large mineralized materials were deposited on the surface, but in a disordered accumulation state ( Fig.12 ). Under transmission electron microscopy, the dentin collagen network showed a light grey structure and no mineralized material was observed ( Fig.13 ), indicating that it is only the external deposition of larger particles, without the occurrence of intrafibrous mineralization and dentin fiber mineralization, and no crystalline phase was detected by SAED ( Fig.14 ).

[0062] The phenomena in Example 3, Comparative Example 1 and Comparative Example 2 are similar to those in Example 2, and effective biomimetic mineralization in dentin collagen fibers and hard tissues cannot be achieved. It is suggested that the ratio of calcium ions to gallic acid, the order of adding calcium ion source and phosphorus ion source, and the dripping speed of phosphate ions are all key factors in this method. That is, when using gallic acid to stabilize calcium phosphate nanoclusters, it is best to control the molar ratio of calcium to gallic acid to about 0.05, and first mix the gallic acid and calcium ion source solutions, and then slowly add the phosphorus ion source to the mixed solution of gallic acid and calcium ion source.

[0063] The hardness of the mineralized dentin samples obtained in the examples and comparative examples, and the dentin samples before and after acid etching were measured using a hardness tester (the dentin was pretreated with the CaP solution or GA-CaP solution prepared in each example and comparative example, and artificial saliva was continued to be used for treatment for 4 days, and the dentin samples were taken out for hardness testing). For the initial natural dentin, acid-etched dentin, and mineralized dentin of each dentin sample, the hardness was measured using a digital hardness tester. In each measurement, 5 points were selected for the same dentin sample for measurement, and the average value of the 5 measurement points was taken as the hardness of the dentin sample. Each example or comparative example measured 9 dentin samples, and the table shows the average hardness of the 9 dentin samples of each example or comparative example.

[0064] The experimental results are detailed in Fig.15 In the figure, *** indicates that the two groups of data were analyzed by ordinary one-way ANOVA, p < 0.001; ns indicates that there is no significant difference between the two groups of data; hardness is Vickers hardness HV. From the experimental results, it can be seen that after acid etching, the hardness of natural dentin is significantly reduced, and after 4 days of mineralization in the CaP pretreatment group, the hardness of dentin did not increase significantly; after treatment in Example 1, the hardness was significantly improved, even exceeding the hardness of natural dentin, while there was no significant difference between Example 2, Example 3, Comparative Example 1 and Comparative Example 2 compared with the acid etching baseline.

[0065] It can be seen that the operation method of Example 1 of this scheme can effectively ensure the mineralization of dentin, thereby improving the overall hardness of dentin. Among them, the use of gallic acid is the key to achieving the above effect. When using gallic acid, it is necessary to control the molar ratio of gallic acid and calcium element, control the addition method of the phosphorus ion source, and control the order of adding the calcium ion source and the phosphorus ion source, so as to effectively achieve the mineralization of dentin.

[0066] The inventor analyzed the reason according to the above phenomenon: there are great differences in the composition and structure of dentin and enamel, and dentin contains more type I collagen fibers. To realize the bionic mineralization of dentin, it is necessary to first realize the mineralization in the collagen fibers, and then realize the mineralization of the hard tissue of the essence, and even the closure of the dentin tubules, because the support of the collagen scaffold by the minerals in the fibers is the key to the excellent mechanical properties of dentin. In order to realize the full mineralization of dentin and the effective improvement of its hardness, it is necessary to ensure that the collagen fibers are effectively mineralized inside and outside. Adjacent collagen microfibrils are arranged in a dislocated translation of 67nm to form collagen fibers, and the dislocation of each 67nm forms a periodic structure of collagen. Amorphous calcium phosphate (ACP) is formed in the CaP solution prepared in this scheme, and it is necessary to ensure that it fully enters the gap of the above-mentioned dislocated translation to realize the effective mineralization inside the collagen fibers. The amorphous calcium phosphate (ACP, forming calcium phosphate nanoclusters) located in the gap then forms hydroxyapatite (HAP) to achieve mineralization. The addition of gallic acid can effectively control the particle size of calcium phosphate nanoclusters, avoiding its excessive particle size, which makes it difficult for amorphous calcium phosphate to enter the gaps in the fibers (the particle size of calcium phosphate nanoclusters is preferably controlled at 1-20nm). The average particle size of the calcium phosphate nanoclusters obtained by the method of Example 1 is about 11.62nm, 323.38nm for Example 2, 89.14nm for Example 3, 172.68nm for Comparative Example 1, and 128.94nm for Comparative Example 2. Since gallic acid is not used in Example 2, the average particle size of the calcium phosphate nanoclusters is too large, and the mineralization inside the collagen fibers cannot be effectively achieved, resulting in the overall hardness of the dentin not being effectively improved. The calcium ion to GA molar ratio of Example 3 is too high, resulting in the average particle size of the calcium phosphate nanoclusters being too large. Due to the adjustment of the addition method of the calcium ion source and the phosphorus ion source in Comparative Examples 1 and 2, the average particle size of the calcium phosphate nanoclusters is also affected to a certain extent, which ultimately causes the internal mineralization of the collagen fibers to be negatively affected.

[0067] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out 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 effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A reagent combination for constructing calcium phosphate nanoclusters biomimetic mineralized dentin using gallic acid, characterized in that: The reagent combination includes a gallic acid-calcium phosphate solution, and the raw materials of the gallic acid-calcium phosphate solution include gallic acid, a calcium ion source, and a phosphorus 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 0.1-5; The reagent combination also includes artificial saliva or saliva; The gallic acid-calcium phosphate 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 a stirring condition of >300 rpm, and stirring for 10-120 minutes to obtain a gallic acid-calcium solution; dissolving a phosphorus ion source in water to obtain a phosphorus ion source solution, and adding the phosphorus ion source solution dropwise at a speed of 1-4 drops / second to the gallic acid-calcium solution under a stirring condition of >300 rpm, and adjusting the pH value to 5-7 to obtain a gallic acid-calcium phosphate solution; Calcium phosphate nanoclusters are formed in the gallic acid-calcium phosphate solution, and the average particle size of the calcium phosphate nanoclusters is 11.62 nm.

2. The reagent combination for constructing calcium phosphate nanoclusters biomimetic mineralized dentin using gallic acid according to claim 1, 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 phosphorus ion source solution, the concentration of phosphorus element is 0.01-30mM.

3. The reagent combination for constructing calcium phosphate nanoclusters biomimetic mineralized dentin using gallic acid according to claim 2, characterized in that: Dissolve gallic acid in water and stir at 35-50° C. for 10-120 min to obtain a gallic acid solution.

4. Use of a reagent combination for constructing calcium phosphate nanoclusters biomimetic mineralized dentin using gallic acid according to any one of claims 1 to 3 in the preparation of a drug for promoting dentin mineralization.

5. The use of a reagent combination for constructing calcium phosphate nanoclusters biomimetic mineralization of dentin using gallic acid according to claim 4 in the preparation of a drug for promoting dentin mineralization, characterized in that: The gallic acid-calcium phosphate solution is used to contact with the dentin to be repaired, and the dentin after contacting with the gallic acid-calcium phosphate solution is used to be immersed in saliva or artificial saliva.

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