A composition for dentin biomimetic remineralization and applications thereof

Abstract

The application discloses a composition for dentin biomimetic remineralization and application thereof. The composition comprises a dentin pretreatment agent for providing modification function and infiltration function; and a mixed mineralization solution for providing remineralization and reconstructing an enamel-like layer, which comprises a mineralization solution A and a mineralization solution B; wherein the dentin pretreatment agent comprises citric acid and / or a salt thereof and 10-methacryloyloxydecyl phosphate; the mineralization solution A is a buffer solution containing a soluble calcium salt; the mineralization solution B is a buffer solution containing a soluble fluoride and a soluble phosphate; and the volume ratio of the mineralization solution A to the mineralization solution B is 0.1-10:1. The above composition is used for treating demineralized dentin, so that the collagen fibers and dentin tubules are completely mineralized and protected, and micron-sized enamel-like layers are newly generated on the surface of the collagen fibers, the purpose of quickly and efficiently repairing the demineralized dentin is achieved, and a scientific and feasible research direction for the repair of the dentin is provided.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of dentin biomimetic remineralization. More particularly, it relates to a composition for dentin biomimetic remineralization and its application. BACKGROUND

[0002] The main structure of teeth includes outer enamel and inner dentin and dental pulp. Among them, dentin is a highly complex mineralized tissue, whose core is composed of mineralized collagen fibers, which exhibit a multi-level, ordered arrangement structure. Hydroxyapatite crystals are deposited on the surface and internal interstitial regions of collagen fibers in a precise and ordered manner, which together endow dentin with excellent mechanical properties and biological functions. In addition, dentin contains dentin tubules connected to the dental pulp, which often triggers dentin sensitivity and other discomfort symptoms when external stimuli are transmitted to the dental pulp. When the enamel or cementum is damaged due to acid erosion, wear and other factors, the dentin is exposed and demineralized, and the dentin tubules are directly exposed to the external environment. At this time, cold and hot stimuli can act on the dental pulp through the dentin tubules, causing significant pain, which is called dentin hypersensitivity (Dentin Hypersensitivity, DH). However, due to the high complexity of dentin tissue and the multi-level fine regulation mechanism in the mineralization process, the realization of its biomimetic mineralization has been one of the research difficulties in the fields of material science and biomedical science.

[0003] At present, the research on dentin remineralization has certain potential in dealing with demineralization problems, but the existing technology still faces the following key challenges: first, low mineralization induction efficiency is one of the main bottlenecks, and the existing methods often need a long processing time to generate a functional and stable mineralized layer, which is difficult to meet the demand of rapid repair in clinical practice. Secondly, the existing technology usually relies on precise equipment, difficult-to-obtain experimental consumables and complex experimental conditions, which has a high operation threshold and is not conducive to popularization to routine medical applications; the most critical point is that the sealing rate of the dentin tubules after biomimetic mineralization is limited, and the existing methods for sealing the dentin tubules are only limited to the inner and outer layers of collagen fibers, which cannot effectively isolate the external stimuli from conducting to the dental pulp through the dentin tubules, and cannot form a complete blockage in the dentin tubules and a micron-sized enamel-like layer on the collagen surface.

[0004] CN112336634A discloses a dentin bonding pretreatment composition based on microenvironment-induced nanoparticle redeposition, which can quickly induce the thermodynamically unstable calcium phosphate precursor solution formed after mixing of a calcium salt solution and a phosphate solution, and quickly form nano-ACP particles in a strongly polar demineralized dentin matrix, thereby forming a new organic / inorganic hybrid three-dimensional composite structure. However, there are still the following defects: first, the remineralized dentin repair time is long, up to several days to several weeks, and the clinical operation depends on strict proportion control and water flushing steps, which is highly technical sensitive and cannot meet the needs of convenient use and rapid repair in clinical application scenarios; second, since this remineralization repair is limited to the nanoscale microscopic level inside and outside the dentin collagen fibers, the problem of dentin sensitivity caused by the exposure of dentin tubules is not effectively solved, and the exposure of dentin tubules directly leads to no significant improvement in mechanical strength such as hardness and elastic modulus of the remineralized dentin, and the repaired dentin is also easy to demineralize again. In summary, the ambiguity of dentin remineralization mechanism, low remineralization efficiency, high technical sensitivity in clinical application, limited mechanical performance improvement after repair and other series of problems need to be solved, and there is currently no dentin remineralization technology that can efficiently repair and produce stable repair effect. SUMMARY

[0005] To solve the above problems, the first object of the present application is to provide a composition for bionic remineralization of dentin.

[0006] The second object of the present application is to provide an application of the composition as described above in the preparation of a product for repairing demineralized dentin.

[0007] The third object of the present application is to provide a new remineralization method capable of efficiently inducing mineralization, rebuilding a natural mineral structure and effectively sealing dentin tubules. This method can realize dual remineralization protection of collagen fibers and dentin tubules to generate a core of an enamel-like layer, thereby providing a scientific and feasible technical path for rapid and efficient repair of demineralized dentin.

[0008] To achieve the above first object, the present application adopts the following technical solutions:

[0009] The present application provides a composition for bionic remineralization of dentin, which comprises

[0010] a dentin pretreatment agent for providing modification function and infiltration function; and

[0011] a mixed mineralization liquid for providing remineralization effect and rebuilding an enamel-like layer, which comprises mineralization liquid A and mineralization liquid B;

[0012] The dentin pretreatment agent includes citric acid and / or a salt thereof, and 10-methacryloyloxydecyl phosphate;

[0013] The mineralizing solution A is a buffer solution containing a soluble calcium salt.

[0014] The mineralizing solution B is a buffer solution containing a soluble fluoride and a soluble phosphate.

[0015] In the mixed mineralizing solution, the volume ratio of the mineralizing solution A to the mineralizing solution B is 0.1-10:1.

[0016] Further, the mass concentration of 10-methacryloyloxydecyl phosphate in the dentin pretreatment agent is 10-20%, and the mass concentration of citric acid and / or a salt thereof in the dentin pretreatment agent is 3-10%.

[0017] The citric acid and / or a salt thereof includes but is not limited to one or more of citric acid, citric acid hydrate, sodium citrate, and sodium citrate hydrate.

[0018] In the composition of the present application, the selected dentin pretreatment agent achieves biomimetic mineralization regulation through multi-level synergy. The specific innovation lies in: (1) collagen interface modification and nucleation site construction: the phosphate groups of 10-MDP and the carboxylic acid groups of citric acid (salt) in the pretreatment agent form a multi-point combination with the amino / hydroxyl groups of the collagen fibers through hydrogen bond networks and electrostatic interactions. This process maintains the integrity of the collagen matrix while exposing the nucleation active sites of the collagen molecular chain, providing high-energy binding sites for calcium phosphate heterogeneous nucleation; (2) collagen network structure reconstruction: the pretreatment agent disrupts the intermolecular hydrogen bonds of collagen fibers, uncoils the triple helix of collagen fibers, and changes its topological structure, facilitating the subsequent infiltration of the mixed mineralization liquid. Meanwhile, citric acid and / or its salt can improve the hydrophilicity of the dentin collagen fiber surface and its wetting effect in the early stage of biomineralization, significantly reducing the interfacial energy between dentin collagen and the liquid precursor of calcium phosphate; (3) precursor phase stabilization and crystal orientation regulation: the phosphate groups and carboxylic acid groups in the pretreatment agent stabilize calcium ions through bidentate chelation, maintaining the local free calcium ion concentration in a metastable state. The chelated calcium ions can be gradually released, providing a continuous ion source for the ordered crystallization of hydroxyapatite. This can effectively stabilize the amorphous precursor phase, delay its premature precipitation in solution, inhibit the disordered growth of crystals, promote the directional arrangement along the natural c-axis direction of dentin, and reduce the generation of amorphous and other impurities. This avoids the crystal structure defects caused by disordered crystallization, ensuring the phase purity and mechanical properties of the remineralized material; (4) microenvironment regulation: the two common acidic monomers in the pretreatment agent can regulate the local pH of the microenvironment during dentin remineralization by releasing protons. The initial acidic environment effectively promotes the formation and stabilization of amorphous calcium phosphate precursor phase. As the protons are depleted and the local pH rises, the amorphous precursor phase transforms into a crystalline phase, which is conducive to the formation of an enamel-like layer structure with a columnar structure.

[0019] In the composition of the present application, the calcium salt in mineralization liquid A and the phosphate salt in mineralization liquid B will react after mixing to form a calcium phosphate precursor, providing mineralization material for dentin. The fluoride ions in mineralization liquid B can form a soluble calcium-fluorine-phosphorus complex with the calcium salt in mineralization liquid A and the phosphorus salt in mineralization liquid B, which is conducive to the formation and stabilization of amorphous phase, slows down the premature formation of large crystals in the liquid phase, and thus promotes the penetration of the mineralization liquid system into the deep dentin tubules, promoting uniform and deep mineralization of minerals in the collagen network.

[0020] The study found that if the dentin pretreatment agent only contains 10-methylacryloxydecyl phosphate, the water contact angle of the dentin surface is high, which is not conducive to the infiltration of the mineralization solution. The addition of citric acid and / or its salt can significantly enhance the wettability of the dentin surface. Therefore, under the co-modification of 10-MDP and citric acid and / or its salt, the interface physical and chemical properties change, the water contact angle decreases, the wettability of the dentin surface is significantly improved, the mineralization solution can more easily penetrate into the collagen fibers, and the nucleation energy barrier of the mineralization solution in the collagen protein is reduced.

[0021] In the preparation of the dentin pretreatment agent, 10-methylacryloxydecyl phosphate and citric acid and / or its salt can be dissolved in an aqueous ethanol solution, respectively, and then mixed in proportion. As an example, a preparation method for preparing a dentin pretreatment agent is provided below, which includes the following steps:

[0022] 10-methylacryloxydecyl phosphate with a mass fraction of 20-40% is dissolved in a 50% aqueous ethanol solution, citric acid with a mass fraction of 6-20% is dissolved in a 50% aqueous ethanol solution, and the two solutions obtained above are mixed in a volume ratio of 1:1 to obtain a 50% aqueous ethanol solution of 10-methylacryloxydecyl phosphate (10-MDP) with a mass fraction of 10-20% and citric acid with a mass fraction of 3-10%, which is the dentin pretreatment agent.

[0023] Further, the soluble calcium salt includes but is not limited to one or more of calcium chloride, calcium fluoride, calcium nitrate, and calcium acetate;

[0024] The concentration of calcium ions in the mineralization solution A is 0.1-100 mmol / L. For example, the concentration of calcium ions in the mineralization solution A can be 0.1 mmol / L, 1 mmol / L, 5 mmol / L, 10 mmol / L, 15 mmol / L, 20 mmol / L, 25 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, 50 mmol / L, 55 mmol / L, 60 mmol / L, 65 mmol / L, 70 mmol / L, 75 mmol / L, 80 mmol / L, 85 mmol / L, 90 mmol / L, 95 mmol / L, 100 mmol / L, etc., and preferably 18.2-30 mmol / L.

[0025] The pH of the mineralization solution A is 4.5-7.5, preferably 5.5-6.5.

[0026] Further, the soluble fluoride includes but is not limited to one or more of sodium fluoride, potassium fluoride, potassium monofluorophosphate, stannous fluoride, ammonium fluoride, zinc ammonium fluoride, laurylamine hydrofluoride, and diethylaminoethyl octanoyl amine hydrofluoride.

[0027] The soluble phosphate in the mineralization solution B includes, but is not limited to, one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, tripotassium phosphate, trisodium phosphate, sodium dihydrogen phosphate, triammonium phosphate, diammonium hydrogen phosphate, and dipotassium hydrogen phosphate;

[0028] The pH of the mineralization solution B ranges from 4.5 to 7.5, preferably from 5.5 to 6.5.

[0029] Further, the concentration of fluoride ions in the mineralization solution B is 0.001-1 g / L; for example, the concentration of fluoride ions in the mineralization solution B can be 0.001 g / L, 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, etc., preferably 0.01-0.05 g / L.

[0030] The mineralization solution B is rich in phosphate ions, monohydrogen phosphate ions, and dihydrogen phosphate ions, all of which can react with calcium ions to form calcium phosphate liquid phase precursors. The total concentration of phosphate ions, monohydrogen phosphate ions, and dihydrogen phosphate ions in the mineralization solution B is 0.1-100 mmol / L; for example, the total concentration of phosphate ions, monohydrogen phosphate ions, and dihydrogen phosphate ions in the mineralization solution B can be 0.1 mmol / L, 1 mmol / L, 5 mmol / L, 10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, 100 mmol / L, etc., preferably 5.6-10 mmol / L.

[0031] Further, the mineralization solution A and the mineralization solution B can be mixed in a volume ratio of 0.1:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, preferably 1:1.

[0032] Further, the ratio of the concentration of calcium ions in the mineralization solution A to the total concentration of phosphate ions, monohydrogen phosphate ions, and dihydrogen phosphate ions in the mineralization solution B is 1-10:1; for example, the concentration ratio can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc.

[0033] Further, in the mineralizing solution A and the mineralizing solution B, the buffer is used to stabilize the pH of the mineralizing solution A and the mineralizing solution B in a weakly acidic to neutral environment, including but not limited to one or more of a phosphoric acid or a salt solution thereof, an acetic acid solution, a sodium acetate solution, a carbonic acid solution, a bicarbonate solution, a Tris or a salt solution thereof, a citric acid or a salt solution thereof, a HEPES or a sodium salt solution thereof, a boric acid or a salt solution thereof.

[0034] Further, the mineralizing solution A and the mineralizing solution B can also be added with a sweetener with a mass fraction of 0.1-10%, such as one or more of saccharin, cyclamate, sucrose, glucose, and potassium acesulfame.

[0035] Further, the mineralizing solution A and the mineralizing solution B can also be added with a humectant with a mass fraction of 0.1-10%, such as one or more of polyethylene glycol, propylene glycol, glycerol (glycerin), erythritol, xylitol, sorbitol, and mannitol.

[0036] Further, the dentin pretreatment agent, the mineralizing solution A, and the mineralizing solution B are stored separately after being prepared.

[0037] Further, the dentin pretreatment agent, the mineralizing solution A, and the mineralizing solution B can also be used to prepare a dental care product, including but not limited to a tooth desensitizer for sealing dentin tubules, a drug for preventing or treating dental erosion, a product for dental care, and the like.

[0038] To achieve the second purpose, the present application adopts the following technical solutions:

[0039] The present application provides a use of the composition as described above in the preparation of a product for repairing demineralized dentin.

[0040] To achieve the third purpose, the present application adopts the following technical solutions:

[0041] The present application provides a method for bionic remineralization of dentin, which uses the composition as described above to perform bionic remineralization of dentin, including the following steps:

[0042] 1) Dentine pretreatment:

[0043] The dentin pretreatment agent is applied on the surface of the demineralized dentin, the total application time is 5-10 min, and after the application is completed, the pretreated dentin is obtained by standing for 0-60 min and then washing with deionized water; the surface water contact angle of the pretreated dentin is 30-40°.

[0044] 2) Rebuilding an enamel-like layer:

[0045] The mineralizing solution A and the mineralizing solution B are mixed in proportion to obtain a mixed mineralizing solution.

[0046] The pretreated dentin is immersed in the mixed mineralization solution, the mixed mineralization solution is replaced every 6-24 hours, and the biomimetic remineralization of the demineralized dentin is completed after the constant temperature mineralization at 37 DEG C for 1-14 days and the removal of the loose mineralized layer on the surface.

[0047] The application can be applied once or repeatedly when applying the dentin pretreatment agent, as long as the total application time is reached.

[0048] Further, the constant temperature mineralization time can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, etc., and is preferably 1-2 days.

[0049] The application has the following advantages:

[0050] The application provides a new remineralization method capable of efficiently inducing mineralization, reconstructing a natural mineral structure and effectively sealing dentin tubules.

[0051] 1) In the selection of the dentin pretreatment agent components, unlike the raw materials that are difficult to obtain and prepare in the past, citric acid and / or its salt and 10-MDP are selected, wherein the 10-MDP is a functional monomer commonly used in self-etching adhesive, and the citric acid and / or its salt is an organic small molecule commonly used in bone biological mineralization level. The dentin is treated with the pretreatment agent containing the two functional monomers that are easy to obtain and prepare. The functional groups (carboxyl, carbonyl, decyl and acryloyloxy groups, etc.) of the 10-MDP and the citric acid and / or its salt can be modified on the surface of the collagen fibers, provide nucleation sites for the remineralization of the surface of the dentin collagen fibers, and enhance the penetration of the mineralization solution in the dentin tubules and the collagen fibers. More importantly, with the introduction of the citric acid and / or its salt, the hydrophilicity of the surface of the dentin collagen fibers and the wetting effect in the early stage of biological mineralization are significantly improved, thereby being beneficial to the generation of the dentin-enameloid layer biomimetic mineralization structure.

[0052] 2) By adding fluoride ions in the mineralization solution system, the weak acid environment of stable amorphous calcium phosphate and amorphous calcium fluoride is controlled, the speed of forming hydroxyapatite and fluorapatite crystals is slowed down, thereby being beneficial to the penetration of the mineralization solution system into the deep dentin tubules, the tight sealing of the dentin tubules, and the formation of the main mineral components being hydroxyapatite and fluorapatite, which are extremely similar to the mineral component structure in the natural dentin and have good biocompatibility. In addition, the fluorapatite has a smaller size and lower surface energy, and is easy to deposit in the collagen fibers and the dentin tubules.

[0053] 3) The remineralization method provided by the present application overcomes the problems of high preparation cost and shallow dentin tubule sealing depth in current dentin mineralization technology, can efficiently isolate external stimulation through the dentin tubule to the pulp, and the separated storage of the mineralization solution is conducive to long-term storage, and has a broad market prospect. In addition, due to the efficient functional modification of the pretreatment agent to the demineralized collagen and the reduction of the energy barrier of the heterogeneous nucleation of the amorphous calcium phosphate precursor phase on the collagen matrix, the pretreated dentin can be fully mineralized in the mineralization solution system after 1 day, solving the problems of long remineralization time, incomplete sealing of the dentin tubule and poor effect of the existing dentin remineralization.

[0054] 4) The dentin pretreatment agent and mineralization solution system provided by the present application are not only suitable for the oral field, but also suitable for the field with mineralization demand for collagen fibers, and the synthetic raw materials used are tested for in-vitro and in-vivo biocompatibility, and have good biological safety. BRIEF DESCRIPTION OF DRAWINGS

[0055] The specific embodiments of the present application will be further described in detail below with reference to the accompanying drawings.

[0056] Figure 1 The scanning electron microscope (SEM) images of the demineralized dentin disc and the remineralized dentin prepared by Example 1 and Comparative Examples 1-4 are shown; wherein, Figure 1 a1-a4 are the surface morphology of the demineralized dentin disc, the enlarged view of the surface morphology, the longitudinal section morphology, and the enlarged view of the longitudinal section morphology, b1-b4 are the surface morphology of the remineralized dentin of Example 1, the enlarged view of the surface morphology, the longitudinal section morphology, and the enlarged view of the longitudinal section morphology, c1-c4 are the surface morphology of the remineralized dentin of Comparative Example 1, the enlarged view of the surface morphology, the longitudinal section morphology, and the enlarged view of the longitudinal section morphology, d1-d4 are the surface morphology of the remineralized dentin of Comparative Example 2, the enlarged view of the surface morphology, the longitudinal section morphology, and the enlarged view of the longitudinal section morphology, e1-e4 are the surface morphology of the remineralized dentin of Comparative Example 3, the enlarged view of the surface morphology, the longitudinal section morphology, and the enlarged view of the longitudinal section morphology, and f1-f4 are the surface morphology of the remineralized dentin of Comparative Example 4, the enlarged view of the surface morphology, the longitudinal section morphology, and the enlarged view of the longitudinal section morphology.

[0057] Figure 2 The longitudinal section enlarged element analysis (EDS) image of the remineralized dentin of Example 1 is shown.

[0058] Figure 3 The water contact angles of the dentin pretreated by different pretreatment agents are shown.

[0059] Figure 4 The infiltration of amorphous calcium phosphate (ACP) in the pretreated collagen of Example 1, Comparative Example 1 and Comparative Example 2 is shown.

[0060] Figure 5Transmission electron microscope (TEM) images showing the remineralization of single layer of recombinant type I collagen without pretreatment and after pretreatment; wherein, Figure 5 Fig. 13 shows the remineralization morphology of collagen without pretreatment (a-c, control group) and pretreated collagen in Example 1 (d-f).

[0061] Figure 6 Fig. 14 shows the molecular docking results of the two components in Example 1. DETAILED DESCRIPTION

[0062] In order to more clearly illustrate the present application, the present application will be further described below with reference to the preferred embodiments and accompanying drawings. Like components are denoted by the same reference numerals in the accompanying drawings. It should be understood by those skilled in the art that the specific descriptions below are illustrative rather than limiting, and should not limit the scope of protection of the present application.

[0063] Example 1

[0064] This example provides a composition for dentin biomimetic remineralization, which is prepared as follows:

[0065] Preparation of dentin pretreatment agent: 20wt% 10-MDP in 50% ethanol aqueous solution was mixed with 6wt% citric acid in 50% ethanol aqueous solution at a volume ratio of 1:1, and ultrasonicated for 30 min to ensure complete dispersion, and was ready for use.

[0066] Preparation of mineralization solution A: 2.67325 g of calcium chloride dihydrate, 0.00825 g of sodium chloride, 5.02775 g of Tris, and 0.5 g of sodium benzoate were added to 500 ml of deionized water, and the pH was adjusted to 6.0±0.5, and was ready for use.

[0067] Preparation of mineralization solution B: 0.975 g of potassium phosphate dibasic, 0.015 g of sodium fluoride, 5.02775 g of Tris, and 0.5 g of sodium benzoate were added to 500 ml of deionized water, and the pH was adjusted to 6.0±0.5, and was ready for use.

[0068] Preparation of demineralized dentin disc: a dentin disc with a diameter of about 5 mm and a thickness of 2 mm was prepared, and was etched with 37% phosphoric acid for 10 s, and was rinsed with deionized water for 1 min, and was ready for use.

[0069] This example also provides a method for dentin biomimetic remineralization, and the specific steps are as follows:

[0070] Dentin pretreatment: the above-mentioned dentin pretreatment agent was evenly applied to the surface of the demineralized dentin disc for 5 min, and the excess pretreatment agent was removed by gently rinsing with deionized water for 1 min, to obtain a pretreated dentin disc with a water contact angle of 37.6°;

[0071] Remineralized dentin: Take a test tube to mix 10 mL of mineralization liquid A and 10 mL of mineralization liquid B to obtain a mixed mineralization liquid; immerse the pretreated dentin disc in the mixed mineralization liquid, and replace the mixed mineralization liquid every 12 hours; mineralize at 37°C for 1 day; after the mineralization is completed, ultrasonic cleaning is performed to remove the loose mineralization layer on the surface, and the biomimetic remineralization of the demineralized dentin disc is completed.

[0072] Comparative Example 1

[0073] This example provides a composition for biomimetic remineralization of dentin, which is prepared as follows:

[0074] Preparation of a dentin pretreatment agent: 50% ethanol aqueous solution containing 20 wt% 10-MDP and 50% ethanol aqueous solution containing 2 wt% citric acid are mixed at a volume ratio of 1:1, ultrasonic dispersion is performed for 30 min to ensure uniform dispersion, and the mixture is prepared for use.

[0075] Preparation of mineralization liquid A: 2.67325 g of calcium chloride dihydrate, 0.00825 g of sodium chloride, 5.02775 g of Tris, and 0.5 g of sodium benzoate are added to 500 ml of deionized water, and the pH is adjusted to 6.0±0.5, and the mixture is prepared for use.

[0076] Preparation of mineralization liquid B: 0.975 g of potassium phosphate dibasic, 0.015 g of sodium fluoride, 5.02775 g of Tris, and 0.5 g of sodium benzoate are added to 500 ml of deionized water, and the pH is adjusted to 6.0±0.5, and the mixture is prepared for use.

[0077] Preparation of demineralized dentin disc: a dentin disc with a diameter of about 5 mm and a thickness of 2 mm is prepared, etched with 37% phosphoric acid for 10 s, and washed with deionized water for 1 min, and the mixture is prepared for use.

[0078] This example also provides a method for biomimetic remineralization of dentin, and the specific steps are as follows:

[0079] Dentin pretreatment: the above-mentioned dentin pretreatment agent is uniformly applied to the surface of the demineralized dentin disc for 5 min, and the excess pretreatment agent is removed by gently washing with deionized water for 1 min to obtain a pretreated dentin disc with a water contact angle of 42.8°;

[0080] Remineralized dentin: take a test tube to mix 10 mL of mineralization liquid A and 10 mL of mineralization liquid B to obtain a mixed mineralization liquid; immerse the pretreated dentin disc in the mixed mineralization liquid, and replace the mixed mineralization liquid every 12 hours; mineralize at 37°C for 1 day; after the mineralization is completed, ultrasonic cleaning is performed to remove the loose mineralization layer on the surface, and the biomimetic remineralization of the demineralized dentin disc is completed.

[0081] Comparative Example 2

[0082] The present example provides a composition for dentin biomimetic remineralization, which is prepared as follows:

[0083] Preparation of dentin pretreatment agent: a 50% ethanol aqueous solution containing 10 wt% of 10-MDP was prepared and used as prepared.

[0084] Preparation of mineralization solution A: 2.67325 g of calcium chloride dihydrate, 0.00825 g of sodium chloride, 5.02775 g of Tris, and 0.5 g of sodium benzoate were added to 500 ml of deionized water, and the pH was adjusted to 6.0±0.5, and used as prepared.

[0085] Preparation of mineralization solution B: 0.975 g of potassium phosphate dibasic, 0.015 g of sodium fluoride, 5.02775 g of Tris, and 0.5 g of sodium benzoate were added to 500 ml of deionized water, and the pH was adjusted to 6.0±0.5, and used as prepared.

[0086] Preparation of demineralized dentin disc: a dentin disc with a diameter of about 5 mm and a thickness of 2 mm was prepared, and was etched with 37% phosphoric acid for 10 s, and then rinsed with deionized water for 1 min, and used as prepared.

[0087] The present example also provides a method for dentin biomimetic remineralization, which comprises the following specific steps:

[0088] Dentin pretreatment: the above-mentioned dentin pretreatment agent was evenly applied to the surface of the demineralized dentin disc for 5 min, and then the excess pretreatment agent was removed by gently rinsing with deionized water for 1 min to obtain a pretreated dentin disc with a water contact angle of 69.1°.

[0089] Demineralized dentin: 10 mL of each of the mineralization solution A and the mineralization solution B were mixed to obtain a mixed mineralization solution; the pretreated dentin disc was immersed in the mixed mineralization solution, and the mixed mineralization solution was replaced every 12 hours, and the dentin disc was mineralized at 37°C for 1 day; after the mineralization was completed, the surface loose mineralization layer was removed by ultrasonic cleaning, and the biomimetic remineralization of the demineralized dentin disc was completed.

[0090] Comparative Example 3

[0091] The present example provides a composition for dentin biomimetic remineralization, which is prepared as follows:

[0092] Preparation of dentin pretreatment agent: a 50% ethanol aqueous solution containing 3 wt% of citric acid was prepared and used as prepared.

[0093] Preparation of mineralization solution A: 2.67325 g of calcium chloride dihydrate, 0.00825 g of sodium chloride, 5.02775 g of Tris, and 0.5 g of sodium benzoate were added to 500 ml of deionized water, and the pH was adjusted to 6.0±0.5, and used as prepared.

[0094] Preparation of mineralization solution B: add dipotassium hydrogen phosphate 0.975 g, sodium fluoride 0.015 g, Tris 5.02775 g, sodium benzoate 0.5 g in 500 ml deionized water, adjust pH to 6.0±0.5, and reserve.

[0095] Preparation of demineralized dentin disc: prepare a dentin disc with a diameter of about 5 mm and a thickness of 2 mm, etch with 37% phosphoric acid for 10 s, rinse with deionized water for 1 min, and reserve.

[0096] The example also provides a method for biomimetic remineralization of dentin, and the specific steps are as follows:

[0097] Dentin pretreatment: evenly apply the above-mentioned dentin pretreatment agent on the surface of the demineralized dentin disc for 5 min, and gently rinse with deionized water for 1 min to remove excess pretreatment agent, to obtain a pretreated dentin disc with a water contact angle of 20.0°;

[0098] Remineralized dentin: take a test tube and mix 10 mL of mineralization solution A and 10 mL of mineralization solution B to obtain a mixed mineralization solution; immerse the pretreated dentin disc in the mixed mineralization solution, and replace the mixed mineralization solution every 12 hours; 37°C constant temperature mineralization for 1 day; after mineralization, ultrasonic cleaning is performed to remove the loose mineralized layer on the surface, and the biomimetic remineralization of the demineralized dentin disc is completed.

[0099] Comparative example 4

[0100] The example provides a composition for biomimetic remineralization of dentin, which is prepared as follows:

[0101] Preparation of dentin pretreatment agent: mix 50% ethanol solution containing 20 wt% 10-MDP with 50% ethanol solution of 6 wt% citric acid at a volume ratio of 1:1, ultrasonic for 30 min to ensure uniform dispersion, and reserve.

[0102] Preparation of mineralization solution A: add calcium chloride dihydrate 2.67325 g, sodium chloride 0.00825 g, Tris 5.02775 g, and sodium benzoate 0.5 g in 500 ml deionized water, adjust pH to 6.0±0.5, and reserve.

[0103] Preparation of mineralization solution B without adding fluoride: add dipotassium hydrogen phosphate 0.975 g, Tris 5.02775 g, and sodium benzoate 0.5 g in 500 ml deionized water, adjust pH to 6.0±0.5, and reserve.

[0104] Preparation of demineralized dentin disc: prepare a dentin disc with a diameter of about 5 mm and a thickness of 2 mm, etch with 37% phosphoric acid for 10 s, rinse with deionized water for 1 min, and reserve.

[0105] The example also provides a method for dentin biomimetic remineralization, and the specific steps are as follows:

[0106] Dentin pretreatment: evenly apply the above-mentioned dentin pretreatment agent on the surface of the demineralized dentin disc for 5 minutes, and gently rinse with deionized water for 1 minute to remove the excess pretreatment agent, to obtain a pretreated dentin disc;

[0107] Remineralized dentin: take a test tube, mix 10 mL of mineralization liquid A and 10 mL of mineralization liquid B to obtain a mixed mineralization liquid; immerse the pretreated dentin disc in the mixed mineralization liquid, and replace the mixed mineralization liquid every 12 hours, and mineralize at 37°C for 1 day, then ultrasonic cleaning to remove the loose mineralization layer on the surface, which completes the biomimetic remineralization of the demineralized dentin disc.

[0108] Test 1: SEM electron microscope

[0109] The demineralized dentin disc and the remineralized dentin prepared in Example 1 and Comparative Examples 1-4 are subjected to SEM test.

[0110] Figure 1 The group of a1 to a4 in the middle is the cross-sectional morphology of the demineralized dentin disc, which shows that the collagen fibers of the demineralized dentin disc are completely demineralized and exposed, the dentin tubules are completely exposed, and there is no mineral in the inside of the dentin tubules.

[0111] Figure 1 The group of b1 to b4 in the middle is the cross-sectional morphology of the remineralized dentin of Example 1, and a dense and uniform hydroxyapatite layer can be found on the surface of the dentin, and from the longitudinal section of the dentin (b3, b4), it can also be found that the mixed mineralization liquid of the application can penetrate into the deep part of the dentin tubule, and the remineralization layer on the surface of the dentin tubule is about 3 μm thick. Figure 1 The group of b1 to b4 in the middle is the cross-sectional morphology of the remineralized dentin of Example 1, and a dense and uniform hydroxyapatite layer can be found on the surface of the dentin, and from the longitudinal section of the dentin (b3, b4), it can also be found that the mixed mineralization liquid of the application can penetrate into the deep part of the dentin tubule, and the remineralization layer on the surface of the dentin tubule is about 3 μm thick.

[0112] Figure 1 The group of c1 to c4 in the middle is the cross-sectional morphology of the remineralized dentin of Comparative Example 1, and when the content of citric acid in the pretreatment agent is low, only a small amount of mineral can be found in the dentin tubule, and the mineral shape is irregular and arranged randomly.

[0113] Figure 1 The group of d1 to d4 in the middle is the cross-sectional morphology of the remineralized dentin of Comparative Example 2, and when the pretreatment agent only contains MDP, only part of the mineralization can be formed on the collagen fibers on the surface of the dentin, and cannot penetrate into the dentin tubule, resulting in the emptiness of the dentin tubule.

[0114] Figure 1The middle e1 to e4 group of figures is the remineralized dentin cross-sectional morphology of Comparative Example 3. When the pretreatment agent only contains citric acid, although the mineralizing solution can infiltrate into the dentin tubules to form mineralization, the dentin surface is uneven due to insufficient exposure of nucleation sites, and the surface mineralization morphology is uneven and does not conform to the continuous and ordered structure of the enamel rod.

[0115] Figure 1 The middle f1 to f4 group of figures is the remineralized dentin cross-sectional morphology of Comparative Example 4. When the mineralizing solution does not contain fluorine, the mineralization appears in a sheet shape and is pasted on the dentin surface, and the structure is loose and disordered.

[0116] Test 2: Elemental analysis mapping

[0117] To Figure 1 The middle c1 to c4 group of figures is the elemental analysis mapping after magnification, as shown in Figure 2 , indicating that the dentin tubule lumen is densely closed, and the main elements of the remineralized mineral in the dentin tubule are Ca, P, F and O, indicating that the main components are hydroxyapatite and fluorapatite.

[0118] Test 3: Water contact angle

[0119] After testing, the water contact angle of the pretreated dentin disc of Example 1 is 37.6°, which is lower than that of the dentin disc of Comparative Example 2 treated only with 10-MDP (69.1°) and the natural dentin disc (60.9°) without pretreatment, but higher than that of the dentin disc of Comparative Example 3 treated only with citric acid (20.0°) and the water contact angle of Comparative Example 1 treated with low concentration of citric acid and 10-MDP (42.8°), indicating that the MDP-CA co-modification induces changes in the physical and chemical properties of the interface, significantly improves the wettability of the dentin surface, and potentially reduces the surface energy, making it easier for the mineralizing solution to penetrate into the collagen fibers, which is beneficial for the nucleation of collagen (see Figure 3 ).

[0120] Test 4: Transmission electron microscopy (TEM) and selected area electron diffraction (SEAD)

[0121] Natural dentin is a complex biomineralization system with multi-scale ordered structure, and its basic unit is mineralized collagen fiber. From the nanoscale to the macroscopic scale, this system shows a hierarchical ordered arrangement feature. During the natural mineralization process, the collagen organic matter and the non-collagen protein modified on its surface cooperatively construct a scaffold structure, which in turn guides the formation of mineralized collagen fibers. Therefore, the key of the biomimetic mineralization research lies in the dual biomimetic simulation of the molecular level (primary structure) and the nanometer mineralized fiber level (secondary structure) of the dentin.

[0122] Based on the above structural characteristics, the main component of demineralized dentin, type I collagen fiber, was used as the mineralization matrix. By introducing 10-MDP and citric acid as biomimetic mineralization inducing molecules, a new strategy for efficient mineralization was explored. These two molecules precisely anchor on specific sites on the collagen surface through hydrogen bonding and van der Waals forces, regulating the nucleation and growth of mineral phases, aiming to achieve directional induction of intracellular mineralization. To analyze the mineralization mechanism, the formation process of highly reduced natural dentin was studied, and the re-mineralization mechanism of the pretreatment agent and mineralization solution was studied using a single-layer collagen model system. Transmission electron microscopy (TEM) and selected area electron diffraction (SAED) were used to dynamically characterize the mineralization process, revealing the correlation between molecular anchoring, mineral deposition, and fiber structure evolution at the nanoscale, thus providing a multi-dimensional evidence chain for the analysis of biomimetic mineralization mechanisms.

[0123] Construction of single-layer recombinant type I collagen model: Since collagen can only self-assemble in an alkaline environment, a collagen self-assembly solution was prepared using 50 mM glycine and 200 mM KCl, and the pH was adjusted to 9.2 using 1 M NaOH. 8.33 μL of type I collagen stock solution (3 mg / mL) was added to 0.5 mL of the collagen self-assembly solution to obtain a type I collagen solution of 50 wg / mL, which was then incubated at room temperature for 20 min. Finally, 3 μL of the collagen solution was dropped onto a 300-mesh transmission electron microscope nickel mesh, which was incubated in a constant temperature and humidity environment (humidity 100%, temperature 37°C) for 12 h. To further stabilize the assembled collagen fibers, the assembled collagen fibers were immersed in a 0.1 wt% glutaraldehyde solution for 1 h.

[0124] Transmission electron microscopy (TEM) was used to observe the microstructure of collagen and mineralization, and selected area electron diffraction (SAED) was used to reveal the crystal structure through the diffraction pattern produced by the interaction between the electron beam and the crystal. Amorphous materials produce diffuse diffraction rings, while crystals produce clear spots or sharp diffraction rings. By comparing the experimental diffraction pattern with the standard data card, the phase composition of the sample can be determined.

[0125] Single-layer recombinant collagen fibers pretreated with different concentrations of citric acid: The type I collagen membrane-loaded nickel mesh was floated in the dentin pretreatment agent of Comparative Example 1, Comparative Example 2, and Example 1 for 15 s, and then gently absorbed with filter paper. Then, it was floated in the mineralization solution for 5 min (at the initial stage of collagen mineralization) and immediately taken out. The nickel mesh was dehydrated with deionized water, 50% ethanol aqueous solution, and anhydrous ethanol in gradient, and then characterized by TEM and SAED. With the increase of citric acid concentration in Comparative Example 2, Comparative Example 1, and Example 1, the morphology of the pretreated collagen and the change of ACP contact angle (θ) were observed (see Figure 4 ). Figure 4TEM images show that, in the initial stage of collagen mineralization, calcium and phosphate ions in the mineralization solution first form spherical calcium phosphate precursor phase (indicated by the circular dashed line in the figure) in the solution. The selected area electron diffraction (SAED) of the spherical object indicated by the circular dashed line in the figure proves that it is an amorphous phase, i.e. amorphous calcium phosphate (ACP). The above results show that, in the initial stage of collagen mineralization, spherical amorphous calcium phosphate (ACP) is first formed as a precursor phase of mineralization, and then ACP is deposited on collagen, which is a typical heterogeneous nucleation phenomenon. In this process, the MDP-CA pretreatment agent can regulate the heterogeneous nucleation mineralization behavior by changing the wettability between the collagen matrix and ACP. Figure 4 It is clearly shown that the MDP-CA pretreatment agent combined on the collagen can reduce the contact angle (θ) of ACP on the surface of the collagen fiber, and the effect is enhanced with the increase of the concentration of citric acid in the pretreatment solution. In accordance with the classical nucleation theory, the heterogeneous nucleation energy barrier will be significantly reduced as the contact angle θ decreases.

[0126] Pretreatment and mineralization of single-layer recombinant collagen fibers: the nickel mesh loaded with type I collagen membrane was floated in the dentin pretreatment agent of Example 1 for 15 s, and the type I collagen without pretreatment was used as a blank control group (Example 2) Figure 5 , and then floated in the mineralization solution for 24 hours. After the nickel mesh was dehydrated with deionized water, 50% ethanol aqueous solution and anhydrous ethanol in gradient, it was characterized by TEM and SAED. Figure 5 The re-mineralization morphology of the collagen without pretreatment is shown in a-c, and the re-mineralization morphology of the pretreated collagen is shown in d-f. Figure 5 a-c show that the bare collagen fibers exhibit a characteristic periodic band of 67 nm collagen fibers, and mineralization cannot occur in the mineralization solution or artificial saliva, Figure 5 d-f show that the collagen fibers pretreated in Example 1 are mineralized in the mineralization solution or artificial saliva for 24 h, and obvious mineralization occurs inside and outside the collagen fibers, the collagen fibers are thickened, and the selected area electron diffraction (SAED) pattern shows the (002), (004), (211) characteristic crystal rings of hydroxyapatite, indicating that the re-mineralization crystals inside and outside the collagen are mainly hydroxyapatite.

[0127] Test 5: molecular docking

[0128] The binding poses and interactions of the two small molecules with the protein were obtained using Autodock Vina v.1.2.2, and the binding energy of each interaction was generated, and the docking results were visualized using PyMol (see Figure 6). It is generally accepted that the docking energy value less than -4.25 kcal / mol indicates certain binding activity between the two, less than -5.0 kcal / mol indicates better binding activity, and less than -7.0 kcal / mol indicates strong binding activity. The results show that the collagen and 10-MDP have good binding activity with the binding energy Affinity = -5.2 (kcal / mol). The small molecule forms hydrogen bond interaction with the amino acid residue HIS-457 in the protein. At the same time, the protein and the small molecule have non-bonding contact, forming forces represented by electrostatic potential energy and hydrophobic interaction. The collagen and CA have good binding with the binding energy Affinity = -5.0 (kcal / mol). CA forms hydrogen bond interaction with the amino acid residues ILE-518, ALA-517 and ASN-435 in the protein.

[0129] Test 6: Mechanical properties

[0130] Nanoindentation test: The nanohardness and elastic modulus of dentin were measured on an Agilent G200 nanoindenter, which was equipped with a Berkovich indenter with a tip radius of about 20 nm. The hardness and elastic modulus were measured by force control method, and the maximum force used was 10 mN (1.02 gf) at loading and unloading, the loading time was 10 s, the holding time was 2 s, and the Poisson's ratio was 0.28. At least 25 points were indented for each sample. The mechanical properties of the demineralized dentin discs and the remineralized dentin prepared in Example 1, Comparative Examples 1-3 were tested, and the natural dentin and natural enamel were used as control groups. The results are shown in Table 1.

[0131] Table 1

[0132]

[0133] The elastic modulus and nanohardness of the remineralized dentin prepared in Example 1 are much higher than those of the natural dentin and similar to those of the natural enamel, which is attributed to the fact that the remineralization has penetrated into the dentin tubules and the remineralized dentin has produced a rigid enamel-like surface, thus enhancing the mechanical properties.

[0134] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the implementation modes of the present application. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description, and it is impossible to enumerate all the implementation modes here. Any obvious changes or variations derived from the technical solutions of the present application are still within the protection scope of the present application.

Claims

1. A biomimetic remineralization method for dentin, characterized in that, Dentin biomimetic remineralization using a composition for dentin biomimetic remineralization includes the following steps: 1) Dentin pretreatment: Apply dentin pretreatment agent to the surface of demineralized dentin for a total application time of 5-10 minutes. After application, let stand for 0-60 minutes, then rinse with deionized water to obtain pretreated dentin. The water contact angle of the pretreated dentin surface is 30-40°. 2) Reconstructed enamel layer: Mineralizing solution A and mineralizing solution B are mixed in a certain proportion to obtain a mixed mineralizing solution; The pretreated dentin is immersed in a mixed mineralizing solution, which is replaced every 6-24 hours. The solution is then kept at a constant temperature of 37°C for 1-14 days. After cleaning, the biomimetic remineralization of the demineralized dentin is complete. The composition includes Dentin pretreatment agents; and A mixed mineralizing solution comprising mineralizing solution A and mineralizing solution B; The dentin pretreatment agent includes citric acid and / or its salts, as well as 10-methacryloyloxydecyl phosphate; The mineralizing solution A is a buffer solution containing soluble calcium salts; The mineralizing solution B is a buffer solution containing soluble fluoride and soluble phosphate; In the mixed mineralizing solution, the volume ratio of mineralizing solution A to mineralizing solution B is 0.1-10:1; The dentin pretreatment agent contains 10-20% by mass of 10-methacryloyloxydecyl phosphate, and the dentin pretreatment agent contains 3-10% by mass of citric acid and / or its salts. The soluble calcium salt is selected from one or more of calcium chloride, calcium fluoride, calcium nitrate, and calcium acetate. The soluble fluoride is selected from one or more of sodium fluoride, potassium fluoride, potassium monofluorophosphate, stannous fluoride, ammonium fluoride, and zinc ammonium fluoride. The soluble phosphate is selected from one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, tripotassium phosphate, trisodium phosphate, sodium dihydrogen phosphate, triammonium phosphate, ammonium dihydrogen phosphate, and diammonium hydrogen phosphate. The calcium ion concentration in mineralization solution A is 0.1-100 mmol / L; The pH range of the mineralization solution A is 4.5-7.5; The fluoride ion concentration in mineralization solution B is 0.001-1 g / L; The total concentration of phosphate, monohydrogen phosphate and dihydrogen phosphate in mineralization solution B is 0.1-100 mmol / L; The pH range of the mineralization solution B is 4.5-7.5; The ratio of the calcium ion concentration in mineralization solution A to the total concentration of phosphate, monohydrogen phosphate and dihydrogen phosphate in mineralization solution B is 1-10:

1.

2. The dentin biomimetic remineralization method according to claim 1, characterized in that, The citric acid and / or its salts are selected from one or more of citric acid, citric acid hydrate, sodium citrate, and sodium citrate hydrate.

3. The dentin biomimetic remineralization method according to claim 1, characterized in that, The calcium ion concentration in mineralization solution A is 18.2-30 mmol / L; The pH range of the mineralization solution A is 5.5-6.5; The fluoride ion content in mineralization solution B is 0.01-0.05 g / L; The total concentration of phosphate, monohydrogen phosphate and dihydrogen phosphate in mineralization solution B is 5.6-10 mmol / L; The pH range of the mineralization solution B is 5.5-6.

5.

4. The dentin biomimetic remineralization method according to claim 1, characterized in that, In mineralizing solution A and mineralizing solution B, the buffer solution is selected from one or more of the following: phosphate or its salt solution, acetic acid solution, sodium acetate solution, carbonic acid solution, bicarbonate solution, Tris or its salt solution, citric acid or its salt solution, HEPES or its sodium salt solution, and boric acid or its salt solution.

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