Composition for biomimetic remineralization of dentin and application thereof

Abstract

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

Description

Technical Field

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

[0002] The main structures of teeth include the outer enamel and the inner dentin and pulp. Among them, dentin is a highly complex mineralized tissue, the core of which is composed of mineralized collagen fibers, and these collagen fibers exhibit a multi-level and ordered arrangement structure. Hydroxyapatite crystals are deposited on the surface and internal interstitial regions of collagen fibers in a precise and orderly manner, jointly endowing dentin with excellent mechanical properties and biological functions. In addition, dentinal tubules connected to the pulp are distributed in dentin. When external stimuli are transmitted to the pulp, it often causes discomfort symptoms such as dentin hypersensitivity. When enamel or cementum is damaged due to factors such as acid erosion and abrasion, resulting in the exposure and demineralization of dentin, the dentinal tubules will be directly exposed to the external environment. At this time, thermal and cold stimuli can act on the pulp through the dentinal tubules, resulting in obvious pain, which is called dentin hypersensitivity (DH). However, due to the high complexity of dentin tissue and the multi-level fine regulation mechanism during the mineralization process, the realization of its biomimetic mineralization has always been one of the research difficulties in the fields of materials science and biomedicine.

[0003] Currently, dentin remineralization research has certain potential in dealing with demineralization problems, but the existing technologies still face the following key challenges: First, the low mineralization induction efficiency is one of the main bottlenecks. Existing methods often require a long processing time to generate a functional and stable mineralized layer, which is difficult to meet the requirements of rapid clinical repair. Second, existing technologies usually rely on precision equipment, difficult-to-obtain experimental consumables, and complex experimental conditions, with a high operation threshold, which is not conducive to popularization to conventional medical applications; the most critical point is that the closure rate of dentinal tubules after biomimetic mineralization is limited. Existing methods only limit the closure of dentinal tubules to the inner and outer layers of collagen fibers, and cannot effectively isolate the direct impact of external stimuli transmitted through the dentinal tubules on the pulp, and cannot form a complete plug in the dentinal tubules and a micron-scale enamel-like layer on the collagen surface.

[0004] CN112336634A discloses a dentin bonding pretreatment composition based on microenvironment-induced nanoparticle redeposition. This pretreatment composition can rapidly induce a thermodynamically unstable calcium phosphate precursor solution formed by mixing a calcium salt solution and a phosphate solution, and rapidly form nano-ACP particles within the strongly polar demineralized dentin matrix, thereby forming a brand-new organic / inorganic hybrid three-dimensional composite structure. However, there are still the following defects: First, the remineralization of dentin takes a long time, ranging from several days to several weeks, and clinical operations rely on strict proportion control and water rinsing steps, with high technical sensitivity, unable to meet the requirements of easy use and rapid repair in clinical application scenarios; Second, since this remineralization repair is limited to the nano-scale micro level inside and outside the dentin collagen fibers, the problem of dentin sensitivity that may be caused by widely exposed dentinal tubules has not been effectively solved. The exposure of dentinal tubules directly results in no significant improvement in the mechanical strength such as hardness and elastic modulus of the remineralized dentin, and it is also very easy to demineralize again after repair. In summary, a series of problems such as the ambiguity of the dentin remineralization mechanism, low remineralization efficiency, high technical sensitivity in clinical application, and limited improvement in mechanical properties after repair need to be solved urgently. At present, there is no dentin remineralization technology that can efficiently repair and produce a stable repair effect. Summary of the Invention

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

[0006] The second object of the present invention 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 invention is to provide a new remineralization method that can efficiently induce mineralization, reconstruct the natural mineral-like structure, and effectively seal the dentinal tubules. This method can achieve double remineralization protection of collagen fibers and dentinal tubules, with the formation of an enamel-like layer as the core, providing a scientific and feasible technical path for rapid and efficient repair of demineralized dentin.

[0008] To achieve the above first object, the present invention adopts the following technical solutions: The present invention provides a composition for dentin biomimetic remineralization, and the composition includes a dentin pretreatment agent for providing modification function and infiltration function; and a mixed mineralization solution for providing remineralization effect and reconstructing the enamel-like layer, which contains mineralization solution A and mineralization solution B; wherein, the dentin pretreatment agent includes citric acid and / or its salt, 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 soluble fluoride and soluble phosphate; In the mixed mineralization solution, the volume ratio of the mineralization solution A to the mineralization solution B is 0.1 - 10:1.

[0009] Furthermore, the mass concentration of 10 - methacryloyloxydecyl phosphate in the dentin pretreatment agent is 10 - 20%, and the mass concentration of citric acid and / or its salt in the dentin pretreatment agent is 3 - 10%; The citric acid and / or its salt include, but are not limited to, one or more of citric acid, citric acid hydrate, sodium citrate, and sodium citrate hydrate.

[0010] In the composition of the present invention, the selected dentin pretreatment agent realizes the regulation of biomimetic mineralization through multi - level synergistic effects. The specific innovations are as follows: (1) Collagen interface modification and nucleation site construction: The phosphate group of 10 - MDP and the carboxyl group of citric acid (salt) in the pretreatment agent form multiple - point binding with the amino / hydroxyl groups of dentin collagen fibers through hydrogen - bond networks and electrostatic interactions. This process exposes the nucleation active sites of collagen molecular chains while maintaining the integrity of the collagen matrix, providing high - energy binding sites for the heterogeneous nucleation of calcium phosphate; (2) Collagen network structure reconstruction: The pretreatment agent disrupts the intermolecular hydrogen bonds of collagen fibers, unwinds the triple helix of collagen fibers, and changes its topological structure, facilitating the infiltration of the subsequent mixed mineralization solution. At the same time, citric acid and / or its salt can improve the hydrophilicity of the surface of dentin collagen fibers and their wetting effect in the early stage of biomineralization, significantly reducing the interfacial energy between dentin collagen and the calcium phosphate liquid - phase precursor; (3) Precursory phase stabilization and crystal orientation regulation: The phosphate group and carboxyl group in the pretreatment agent chelate calcium ions through bidentate chelation, maintaining the local free calcium ion concentration in a metastable state. The chelated calcium ions can be gradually released, and this slow - release effect provides a continuous ion source for the orderly crystallization of hydroxyapatite, effectively stabilizing the amorphous precursor phase, delaying its premature precipitation in the solution, inhibiting the disordered growth of crystals, promoting the directional arrangement along the natural c - axis direction of dentin, and reducing the formation of amorphous and other impurity phases, thus avoiding crystal structure defects caused by disordered crystallization and ensuring the phase purity and mechanical properties of the remineralized material; (4) Micro - environment regulation: Two common acidic monomers in the pretreatment agent can regulate the local pH of the micro - environment during the dentin remineralization process through proton release. The initial acidic environment effectively promotes the formation and stabilization of the amorphous calcium phosphate precursor phase. As the protons are consumed, the local pH increases, triggering the transformation of the amorphous precursor phase into the crystalline phase, which is conducive to the formation of an enamel - like layer structure with an enamel rod - like structure.

[0011] In the composition of the present invention, the calcium salt in Mineralizing Solution A and the phosphate in Mineralizing Solution B react after mixing to form a calcium phosphate precursor, providing a mineralizing material for dentin. The fluoride ions in Mineralizing Solution B can form soluble calcium-fluoride-phosphate complexes with the calcium salt in Mineralizing Solution A and the phosphate salt in Mineralizing Solution B, which is beneficial to the formation and stability of the amorphous phase, slowing down the premature formation of large crystals in the liquid phase, thereby promoting the penetration of the mineralizing solution system in the liquid phase into the deep dentinal tubules and promoting the uniform and deep mineralization effect of minerals in the collagen network.

[0012] Research has found that if the dentin pretreatment agent only contains 10-methacryloyloxydecyl phosphate, it will cause a relatively high water contact angle on the dentin surface, which is not conducive to the infiltration of the mineralizing solution. Adding citric acid and / or its salts can significantly enhance the wettability of the dentin surface. Therefore, under the co-modification of 10-MDP and citric acid and / or its salts, the changes in the interfacial physicochemical properties and the decrease in the water contact angle significantly improve the wettability of the dentin surface, making it easier for the mineralizing solution to penetrate into the interior of the collagen fibers and reducing the nucleation energy barrier of the mineralizing solution inside and outside the collagen.

[0013] When preparing the dentin pretreatment agent, 10-methacryloyloxydecyl phosphate and citric acid and / or its salts can be dissolved in an ethanol aqueous solution respectively and then mixed in proportion; as an example, a preparation method for formulating a dentin pretreatment agent is provided below, including the following steps: Dissolve 10-methacryloyloxydecyl phosphate with a mass fraction of 20-40% in a 50% ethanol aqueous solution, dissolve citric acid with a mass fraction of 6-20% in a 50% ethanol aqueous solution, and mix the two solutions obtained above according to a volume ratio of 1:1 to obtain a 50% ethanol aqueous solution containing 10-methacryloyloxydecyl 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.

[0014] Furthermore, the soluble calcium salt includes but is not limited to one or more of calcium chloride, calcium fluoride, calcium nitrate, and calcium acetate; The calcium ion concentration in Mineralization Solution A is 0.1 - 100 mmol / L; for example, the calcium ion concentration in 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; The pH range of the Mineralization Solution A is 4.5 - 7.5, and preferably 5.5 - 6.5.

[0015] Furthermore, the soluble fluoride includes but is not limited to one or more of sodium fluoride, potassium fluoride, potassium monofluorophosphate, stannous fluoride, ammonium fluoride, ammonium zinc fluoride, laurylamine hydrofluoride, and octanamide diethylaminoethyl hydrofluoride; The soluble phosphate in 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 ammonium dihydrogen phosphate; The pH range of the Mineralization Solution B is 4.5 - 7.5, and preferably 5.5 - 6.5.

[0016] Furthermore, the fluoride ion concentration in Mineralization Solution B is 0.001 - 1 g / L; for example, the fluoride ion concentration in 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., and preferably 0.01 - 0.05 g / L; Mineralization solution B is rich in various phosphate ions such as phosphate, monohydrogen phosphate, and dihydrogen phosphate, all of which can react with calcium ions to form a calcium phosphate liquid-phase precursor. Among them, the total concentration of phosphate, monohydrogen phosphate, and dihydrogen phosphate in mineralization solution B is 0.1 - 100 mmol / L. Exemplarily, the total concentration of phosphate, monohydrogen phosphate, and dihydrogen phosphate in 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., and preferably 5.6 - 10 mmol / L.

[0017] Furthermore, the mineralization solution A and the mineralization solution B can also be mixed at 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, and preferably 1:1.

[0018] Furthermore, the ratio of the calcium ion concentration in the mineralization solution A to the total concentration of phosphate, monohydrogen phosphate, and dihydrogen phosphate in the mineralization solution B is 1 - 10:1. Exemplarily, 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.

[0019] Furthermore, in the mineralization solution A and the mineralization solution B, the buffer solution is used to stabilize the pH of the mineralization solution A and the mineralization solution B in a weakly acidic to neutral environment, including but not limited to one or more of phosphoric acid 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, boric acid or its salt solution.

[0020] Furthermore, a sweetener with a mass fraction of 0.1 - 10% can also be added to the mineralization solution A and the mineralization solution B, such as one or more of saccharin, cyclohexylaminosulfonate, sucrose, glucose, and potassium cyclamate.

[0021] Furthermore, a wetting agent with a mass fraction of 0.1 - 10% can also be added to the mineralization solution A and the mineralization solution B, such as one or more of polyethylene glycol, propylene glycol, glycerol (glycerin), erythritol, xylitol, sorbitol, mannitol, and lactitol.

[0022] Furthermore, the dentin pretreatment agent, the mineralization solution A, and the mineralization solution B are stored separately after being prepared.

[0023] Furthermore, the dentin pretreatment agent, mineralization solution A, and mineralization solution B can also be used to prepare dental care products, including but not limited to tooth desensitizers for sealing dentinal tubules, drugs for preventing or treating dental erosion, products for dental care, and the like.

[0024] To achieve the second objective above, the present invention adopts the following technical solution: The present invention provides an application of the composition as described above in the preparation of a product for repairing demineralized dentin.

[0025] To achieve the third objective above, the present invention adopts the following technical solution: The present invention provides a method for biomimetic remineralization of dentin, which uses the composition as described above for biomimetic remineralization of dentin and includes the following steps: 1) Dentin pretreatment: Apply the dentin pretreatment agent to the surface of demineralized dentin, with a total application time of 5 - 10 min. After the application, let it stand for 0 - 60 min, and then rinse with deionized water to obtain pretreated dentin; the water contact angle of the surface of the pretreated dentin is 30 - 40°. 2) Reconstruct the enamel - like layer: Mix mineralization solution A and mineralization solution B in a certain proportion to obtain a mixed mineralization solution; Immerse the pretreated dentin in the mixed mineralization solution, and change the mixed mineralization solution every 6 - 24 hours. Carry out constant - temperature mineralization at 37°C for 1 - 14 days. After washing to remove the loose mineralized layer on the surface, the biomimetic remineralization of demineralized dentin is completed.

[0026] When applying the dentin pretreatment agent in the present invention, it can be applied once or repeatedly, as long as the required total application time is reached, and no specific limitation is made here.

[0027] Furthermore, the time of the constant - temperature mineralization 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 preferably 1 - 2 days.

[0028] The beneficial effects of the present invention are as follows: The present invention provides a novel remineralization method that can efficiently induce mineralization, reconstruct the natural mineral structure, and effectively seal dentinal tubules. Compared with the prior art, the remineralization method provided by the present invention has the following advantages: 1) In the selection of the components of the dentin pretreatment agent, different from the raw materials that were difficult to obtain and prepare in the past, citric acid and / or its salts and 10-MDP are selected. Among them, 10-MDP is a common functional monomer in self-etching adhesives, and citric acid and / or its salts are common organic small molecules in the level of bone biomineralization. Treating dentin with a pretreatment agent containing these two easily obtainable and easily preparable functional monomers can not only modify the functional groups (carboxyl group, carbonyl group, decyl group, acryloyloxy group, etc.) of 10-MDP and citric acid and / or its salts on the surface of collagen fibers, providing nucleation sites for remineralization on the surface of dentin collagen fibers, but also enhance the penetration of the mineralization solution into dentinal tubules and collagen fibers. More importantly, with the introduction of citric acid and / or its salts, the hydrophilicity of the surface of dentin collagen fibers and its wetting effect in the early stage of biomineralization are significantly improved, which is conducive to the generation of a biomimetic mineralized structure of dentin-enamel-like layer.

[0029] 2) By adding fluoride ions to the mineralization solution system, controlling the weakly acidic environment of stable amorphous calcium phosphate and amorphous calcium fluoride, and slowing down the rate of forming hydroxyapatite and fluorapatite crystals, it is thus conducive to the penetration of the mineralization solution system into the deep dentinal tubules, tightly sealing the dentinal tubules, and the main components of the formed minerals are hydroxyapatite and fluorapatite, which are extremely similar to the mineral component structure in natural dentin and have good biocompatibility. In addition, the size of fluorapatite is smaller and its surface energy is lower, making it easy to deposit inside and outside collagen fibers and in dentinal tubules.

[0030] 3) The remineralization method provided by the present invention overcomes the problems of high preparation cost and shallow closure depth of dentinal tubules existing in current dentin mineralization technologies. It can efficiently isolate the stimulation of the outside world to the dental pulp through dentinal tubules, and the separate storage of the mineralization solution is conducive to long-term storage, having a broad market prospect. In addition, thanks to the efficient functional modification of the pretreatment agent on demineralized collagen and the reduction of the energy barrier for heterogeneous nucleation of amorphous calcium phosphate precursor phase on the collagen matrix, the pretreated dentin can achieve full-layer mineralization in the mineralization solution system after 1 day, solving the problems of long dentin remineralization time, incomplete closure of dentinal tubules, and poor effect in the existing technology.

[0031] 4) The dentin pretreatment agent and mineralization solution system provided by the present invention are not only applicable to the oral field, but also applicable to fields with mineralization requirements for collagen fibers. The synthetic raw materials used have passed in vitro and in vivo biocompatibility tests and have good biological safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The following further elaborates on the specific implementation manners of the present invention with reference to the accompanying drawings.

[0033] Figure 1Scanning electron microscope (SEM) images of demineralized dentin discs and remineralized dentin prepared in Example 1 and Comparative Examples 1-4 are shown; wherein, Figure 1 Among them, a1 to a4 are the surface morphology, magnified surface morphology, longitudinal section morphology, and magnified longitudinal section morphology of the demineralized dentin disc, b1 to b4 are the surface morphology, magnified surface morphology, longitudinal section morphology, and magnified longitudinal section morphology of the remineralized dentin in Example 1, c1 to c4 are the surface morphology, magnified surface morphology, longitudinal section morphology, and magnified longitudinal section morphology of the remineralized dentin in Comparative Example 1, d1 to d4 are the surface morphology, magnified surface morphology, longitudinal section morphology, and magnified longitudinal section morphology of the remineralized dentin in Comparative Example 2, e1 to e4 are the surface morphology, magnified surface morphology, longitudinal section morphology, and magnified longitudinal section morphology of the remineralized dentin in Comparative Example 3, and f1 to f4 are the surface morphology, magnified surface morphology, longitudinal section morphology, and magnified longitudinal section morphology of the remineralized dentin in Comparative Example 4.

[0034] Figure 2 An enlarged element analysis of dentin tubules (EDS) map of the longitudinal section of the remineralized dentin in Example 1 is shown.

[0035] Figure 3 The water contact angles before and after pretreatment of dentin with different pretreatment agents are shown.

[0036] Figure 4 The infiltration of amorphous calcium phosphate (ACP) into the pretreated collagen in Example 1, Comparative Example 1, and Comparative Example 2 is shown.

[0037] Figure 5 Transmission electron microscope (TEM) images of remineralization of monolayer recombinant type I collagen before and after pretreatment are shown; wherein, Figure 5 Among them, a-c are the remineralization morphologies of untreated collagen (control group), and d-f are the remineralization morphologies of pretreated collagen in Example 1.

[0038] Figure 6 The molecular docking results of the binary component in Example 1 are shown. Detailed implementation manners

[0039] To illustrate the present invention more clearly, the present invention will be further described below in conjunction with preferred embodiments and drawings. Similar components in the drawings are denoted by the same reference numerals. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0040] Example 1 In this example, a composition for dental bionic remineralization is provided and prepared as follows: Preparation of dentin pretreatment agent: Mix 20wt% 10-MDP in 50% ethanol aqueous solution and 6wt% citric acid in 50% ethanol aqueous solution in a volume ratio of 1:1, ultrasonicate for 30 minutes to ensure full dispersion, and set aside.

[0041] Preparation of mineralization solution A: Add 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 to 500 ml of deionized water, adjust the pH to 6.0±0.5, and set aside.

[0042] Preparation of mineralization solution B: Add 0.975 g of dipotassium hydrogen phosphate, 0.015 g of sodium fluoride, 5.02775 g of Tris, and 0.5 g of sodium benzoate to 500 ml of deionized water, adjust the pH to 6.0±0.5, and set aside.

[0043] Preparation of demineralized dentin disk: Prepare a dentin disk with a diameter of about 5 mm and a thickness of 2 mm, etch it with 37% phosphoric acid for 10 seconds, rinse it with deionized water for 1 minute, and set aside.

[0044] This example also provides a dentin bionic remineralization method, the specific steps are as follows: Dentin pretreatment: The above-mentioned dentin pretreatment agent was evenly applied on the surface of the demineralized dentin disk for 5 minutes, and then gently rinsed with deionized water for 1 minute to remove the excess pretreatment agent, thereby obtaining a pretreated dentin disk with a water contact angle of 37.6°; Remineralized dentin: Take a test tube and take 10 mL of mineralization solution A and 10 mL of mineralization solution B and mix them thoroughly to obtain a mixed mineralization solution; immerse the pretreated dentin disc in the mixed mineralization solution, replace the mixed mineralization solution every 12 hours, and mineralize at a constant temperature of 37°C for 1 day. After the mineralization is completed, ultrasonic cleaning is performed to remove the loose mineralized layer on the surface, thereby completing the bionic remineralization of the demineralized dentin disc.

[0045] Comparative Example 1 This example provides a composition for dentin biomimetic remineralization, which is prepared as follows: Preparation of dentin pretreatment agent: Mix 20wt% 10-MDP in 50% ethanol aqueous solution and 2wt% citric acid in 50% ethanol aqueous solution in a volume ratio of 1:1, ultrasonicate for 30 minutes to ensure full dispersion, and set aside.

[0046] Preparation of mineralization solution A: Add 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 to 500 ml of deionized water, adjust the pH to 6.0±0.5, and set aside.

[0047] Preparation of mineralization solution B: Add 0.975 g of dipotassium hydrogen phosphate, 0.015 g of sodium fluoride, 5.02775 g of Tris, and 0.5 g of sodium benzoate to 500 ml of deionized water, adjust the pH to 6.0±0.5, and set aside.

[0048] Preparation of demineralized dentin disk: Prepare a dentin disk with a diameter of about 5 mm and a thickness of 2 mm, etch it with 37% phosphoric acid for 10 seconds, rinse it with deionized water for 1 minute, and set aside.

[0049] This example also provides a dentin bionic remineralization method, the specific steps are as follows: Dentin pretreatment: The above-mentioned dentin pretreatment agent was evenly applied on the surface of the demineralized dentin disk for 5 minutes, and then gently rinsed with deionized water for 1 minute to remove the excess pretreatment agent, thereby obtaining a pretreated dentin disk with a water contact angle of 42.8°; Remineralized dentin: Take a test tube and take 10 mL of mineralization solution A and 10 mL of mineralization solution B and mix them thoroughly to obtain a mixed mineralization solution; immerse the pretreated dentin disc in the mixed mineralization solution, replace the mixed mineralization solution every 12 hours, and mineralize at a constant temperature of 37°C for 1 day. After the mineralization is completed, ultrasonic cleaning is performed to remove the loose mineralized layer on the surface, thereby completing the bionic remineralization of the demineralized dentin disc.

[0050] Comparative Example 2 This example provides a composition for dentin biomimetic remineralization, which is prepared as follows: Preparation of dentin pretreatment agent: Prepare a 50% ethanol aqueous solution containing 10 wt% of 10-MDP and set aside.

[0051] Preparation of mineralization solution A: Add 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 to 500 ml of deionized water, adjust the pH to 6.0±0.5, and set aside.

[0052] Preparation of mineralization solution B: Add 0.975 g of dipotassium hydrogen phosphate, 0.015 g of sodium fluoride, 5.02775 g of Tris, and 0.5 g of sodium benzoate to 500 ml of deionized water, adjust the pH to 6.0±0.5, and set aside.

[0053] Preparation of demineralized dentin disk: Prepare a dentin disk with a diameter of about 5 mm and a thickness of 2 mm, etch it with 37% phosphoric acid for 10 seconds, rinse it with deionized water for 1 minute, and set aside.

[0054] This example also provides a dentin bionic remineralization method, the specific steps are as follows: Dentin pretreatment: The above-mentioned dentin pretreatment agent was evenly applied on the surface of the demineralized dentin disk for 5 minutes, and then gently rinsed with deionized water for 1 minute to remove the excess pretreatment agent, thereby obtaining a pretreated dentin disk with a water contact angle of 69.1°; Remineralized dentin: Take a test tube and take 10 mL of mineralization solution A and 10 mL of mineralization solution B and mix them thoroughly to obtain a mixed mineralization solution; immerse the pretreated dentin disc in the mixed mineralization solution, replace the mixed mineralization solution every 12 hours, and mineralize at a constant temperature of 37°C for 1 day. After the mineralization is completed, ultrasonic cleaning is performed to remove the loose mineralized layer on the surface, thereby completing the bionic remineralization of the demineralized dentin disc.

[0055] Comparative Example 3 This example provides a composition for dentin biomimetic remineralization, which is prepared as follows: Preparation of dentin pretreatment agent: Prepare a 50% ethanol aqueous solution containing 3 wt% citric acid and set aside.

[0056] Preparation of mineralization solution A: Add 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 to 500 ml of deionized water, adjust the pH to 6.0±0.5, and set aside.

[0057] Preparation of mineralization solution B: Add 0.975 g of dipotassium hydrogen phosphate, 0.015 g of sodium fluoride, 5.02775 g of Tris, and 0.5 g of sodium benzoate to 500 ml of deionized water, adjust the pH to 6.0±0.5, and set aside.

[0058] Preparation of demineralized dentin disk: Prepare a dentin disk with a diameter of about 5 mm and a thickness of 2 mm, etch it with 37% phosphoric acid for 10 seconds, rinse it with deionized water for 1 minute, and set aside.

[0059] This example also provides a dentin bionic remineralization method, the specific steps are as follows: Dentin pretreatment: The above-mentioned dentin pretreatment agent was evenly applied on the surface of the demineralized dentin disk for 5 minutes, and then gently rinsed with deionized water for 1 minute to remove the excess pretreatment agent to obtain a pretreated dentin disk with a water contact angle of 20.0°; Remineralized dentin: Take a test tube and take 10 mL of mineralization solution A and 10 mL of mineralization solution B and mix them thoroughly to obtain a mixed mineralization solution; immerse the pretreated dentin disc in the mixed mineralization solution, replace the mixed mineralization solution every 12 hours, and mineralize at a constant temperature of 37°C for 1 day. After the mineralization is completed, ultrasonic cleaning is performed to remove the loose mineralized layer on the surface, thereby completing the bionic remineralization of the demineralized dentin disc.

[0060] Comparative Example 4 This example provides a composition for dentin biomimetic remineralization, which is prepared as follows: Preparation of dentin pretreatment agent: Mix 20wt% 10-MDP in 50% ethanol aqueous solution and 6wt% citric acid in 50% ethanol aqueous solution in a volume ratio of 1:1, ultrasonicate for 30 minutes to ensure full dispersion, and set aside.

[0061] Preparation of mineralization solution A: Add 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 to 500 ml of deionized water, adjust the pH to 6.0±0.5, and set aside.

[0062] Preparation of mineralization solution B without fluorine addition: add 0.975 g of dipotassium hydrogen phosphate, 5.02775 g of Tris, and 0.5 g of sodium benzoate to 500 ml of deionized water, adjust the pH to 6.0±0.5, and set aside.

[0063] Preparation of demineralized dentin disk: Prepare a dentin disk with a diameter of about 5 mm and a thickness of 2 mm, etch it with 37% phosphoric acid for 10 seconds, rinse it with deionized water for 1 minute, and set aside.

[0064] This example also provides a dentin bionic remineralization method, the specific steps are as follows: 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 excess pretreatment agent to obtain a pretreated dentin disc; Remineralized dentin: Take a test tube and take 10 mL of mineralization solution A and 10 mL of mineralization solution B and mix them thoroughly to obtain a mixed mineralization solution; immerse the pretreated dentin disc in the mixed mineralization solution, replace the mixed mineralization solution every 12 hours, and mineralize at a constant temperature of 37°C for 1 day. After the mineralization is completed, ultrasonic cleaning is performed to remove the loose mineralized layer on the surface, thereby completing the bionic remineralization of the demineralized dentin disc.

[0065] Test 1: SEM The demineralized dentin disks and the remineralized dentin prepared in Example 1 and Comparative Examples 1-4 were subjected to SEM tests.

[0066] Figure 1 Figures a1 to a4 in the middle are the transverse and longitudinal cross-sectional morphologies of the demineralized dentin disk, indicating that after acid etching and demineralization, the dentin collagen fibers are completely demineralized and exposed, the dentinal tubules are completely exposed, and the interior of the dentinal tubules is empty without the presence of minerals.

[0067] Figure 1 Figures b1 to b4 are the cross-sectional and longitudinal morphologies of the remineralized dentin of Example 1. It can be found that a dense and uniform hydroxyapatite layer appears on the dentin surface, and from the longitudinal section of the dentin ( Figure 1It can also be found from (b3 and b4 in the text) that the mixed mineralizing solution of the present invention can penetrate deep into the dentinal tubules, and the remineralized substances biomineralize from about 15 μm deep in the dentin to the dentin surface, and the remineralized layer on the surface of the collagen between the dentinal tubules is about 3 μm thick.

[0068] Figure 1 Figures c1 to c4 in the text are the transverse and longitudinal cross-sectional morphologies of the remineralized dentin of Comparative Example 1. When the content of citric acid in the pretreatment agent is low, only a small amount of minerals can be seen in the dentinal tubules, and the mineral morphology is irregular and arranged randomly.

[0069] Figure 1 Figures d1 to d4 in the text are the transverse and longitudinal cross-sectional morphologies of the remineralized dentin of Comparative Example 2. When the pretreatment agent only contains MDP, only partial mineralization can be formed on the collagen fibers on the dentin surface and cannot penetrate deep into the dentinal tubules, resulting in empty dentinal tubules.

[0070] Figure 1 Figures e1 to e4 in the text are the cross-sectional morphologies of the remineralized dentin of Comparative Example 3. When the pretreatment agent only contains citric acid, although the mineralizing solution can infiltrate into the dentinal tubules to form mineralization, due to insufficient exposure of the nucleation sites on the dentin surface, the morphology of the surface mineralized substances is uneven and does not conform to the continuous and orderly structure imitating enamel rods.

[0071] Figure 1 Figures f1 to f4 in the text are the transverse and longitudinal cross-sectional morphologies of the remineralized dentin of Comparative Example 4. When the mineralizing solution does not contain fluorine, the mineralized substances present in flakes and stick to the dentin surface, with a loose and disordered structure.

[0072] Test 2: Elemental analysis mapping For Figure 1 Figures c1 to c4 in the text are magnified and tested for elemental analysis mapping. See Figure 2 , indicating that the lumen of the dentinal tubules is tightly closed. The main elements of the remineralized minerals in the dentinal tubules are Ca, P, F, and O, indicating that its main components are hydroxyapatite and fluorapatite.

[0073] Test 3: Water contact angle After testing, the water contact angle of the pretreated dentin disc of Example 1 is 37.6°, lower than that of the dentin disc of Comparative Example 2 (69.1°) treated only with 10-MDP and the natural dentin disc without pretreatment (60.9°), but higher than the water contact angle 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 citric acid and 10-MDP (42.8°). This shows that the MDP-CA co-modification induces changes in the interfacial physicochemical properties, significantly improves the wettability of the dentin surface, potentially reduces the surface energy, makes the mineralizing solution easier to penetrate into the collagen fibers, and is beneficial to collagen nucleation (seeFigure 3 ).

[0074] Test 4: Transmission electron microscopy (TEM) and selected area electron diffraction (SEAD) Natural dentin is a complex biomineralization system with a multi-scale ordered structure, and its basic unit is mineralized collagen fibers. From the nanoscale to the macroscale, this system exhibits hierarchical ordered arrangement characteristics. During the natural mineralization process, collagen organic matter and non-collagen proteins modified on its surface synergistically construct a scaffold structure, thereby guiding the formation of mineralized collagen fibers. Therefore, the key to biomimetic mineralization research lies in the dual biomimetic simulation at the molecular level (primary structure) and nano-mineralized fiber level (secondary structure) of dentin.

[0075] Based on the above structural characteristics, this study uses type I collagen fibers, the main component of demineralized dentin, as the mineralization matrix, and explores new strategies for efficient mineralization by introducing 10-MDP and citric acid as biomimetic mineralization inducing molecules. These two molecules are precisely anchored to specific sites on the collagen surface through hydrogen bonds and van der Waals forces, regulating the nucleation and growth of the mineral phase, aiming to achieve the directional induction of mineralization within the fibers. To analyze the mineralization mechanism, the study will highly reproduce the formation process of natural dentin and combine it with a single-layer collagen model system to study the remineralization mechanism of the pretreatment agent and mineralization solution. Transmission electron microscopy (TEM) and selected area electron diffraction (SAED) are used to dynamically characterize the mineralization process, revealing the correlation between molecular anchoring, mineral deposition, and fiber structure evolution at the nanoscale, thereby providing a multi-dimensional evidence chain for the analysis of the biomimetic mineralization mechanism.

[0076] Construction of a single-layer recombinant type I collagen model: Since collagen can only self-assemble in an alkaline environment, a collagen self-assembly solution is prepared with 50 mM glycine and 200 mM KCl, and the pH is adjusted to 9.2 using 1 M NaOH. 8.33 μL of type I procollagen mother liquor (3 mg / mL) is added to 0.5 mL of the collagen self-assembly solution to obtain a 50 μg / mL type I procollagen solution, and then left to stand at room temperature for 20 min. Finally, 3 μL of the collagen solution is dropped onto a 300-mesh transmission electron microscopy nickel grid and incubated at a constant temperature and humidity (humidity 100%, temperature 37 °C) for 12 h. To further stabilize the assembled collagen fibers, the assembled collagen fibers are immersed in a 0.1 wt% glutaraldehyde solution for 1 h.

[0077] Transmission electron microscopy (TEM) is used to observe the microscopic morphology of collagen and mineralized substances. Selected area electron diffraction (SAED) can reveal the crystal structure through the diffraction pattern generated by the interaction between the electron beam and the crystal. Amorphous materials will produce diffuse diffraction rings, while crystals will show clear spots or sharp diffraction rings. By comparing the experimental diffraction pattern with the standard data card, the phase composition in the sample can be determined.

[0078] Single-layer recombinant collagen fibers under pretreatment with different concentrations of citric acid: The nickel mesh loaded with type I collagen membrane was respectively floated in the dentin pretreatment agents in Comparative Example 1, Comparative Example 2, and Example 1 for 15 s, gently blotted with filter paper, and then floated in the mineralization solution for 5 minutes (at the initial stage of collagen mineralization), and immediately taken out. After the nickel mesh was dehydrated with deionized water, 50% ethanol aqueous solution, and absolute ethanol in gradient, it was characterized by TEM and SAED. As the citric acid concentration increased in Comparative Example 2, Comparative Example 1, and Example 1, the morphology of the pretreated collagen and the change of the ACP contact angle (θ) were observed (see Figure 4 ). Figure 4 The transmission electron microscopy (TEM) images of Figure 4 show that at the initial stage of collagen mineralization, calcium and phosphorus ions in the mineralization solution first form spherical calcium phosphate mineralization precursor phases in the solution (indicated by the circular dotted line frame in the figure). Selected area electron diffraction (SAED) of the spherical objects indicated by the circular dotted line frame in the figure confirmed that it is an amorphous phase, that is, amorphous calcium phosphate (ACP). The above results show that at the initial stage of collagen mineralization, spherical amorphous calcium phosphate (ACP) is first formed as the mineralization precursor phase, and then ACP is deposited on the 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 clearly shows that the MDP-CA pretreatment agent bound to the collagen can reduce the contact angle (θ) of ACP on the surface of collagen fibers, and this effect increases with the increase in the concentration of citric acid in the pretreatment solution. In line with the classical nucleation theory, the heterogeneous nucleation energy barrier will be significantly reduced as the contact angle θ decreases.

[0079] 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 untreated type I collagen was used as the blank control group ( Figure 5 a-c in Figure 5 ), gently blotted with filter paper, and then floated in the mineralization solution for 24 hours. After the nickel mesh was dehydrated with deionized water, 50% ethanol aqueous solution, and absolute ethanol in gradient, it was characterized by TEM and SAED. Figure 5 a-c in Figure 5 are the remineralization morphologies of untreated collagen, and d-f are the remineralization morphologies of pretreated collagen. Figure 5 a-c in Figure 5 show that bare collagen fibers exhibit the characteristic periodic banding of 67 nm collagen fibers and cannot be mineralized in the mineralization solution or artificial saliva. Figure 5 d-f in Figure 5 show that the collagen fibers pretreated with Example 1 were mineralized in the mineralization solution or artificial saliva for 24 h, and obvious mineralization occurred both inside and outside the collagen fibers, and the collagen fibers thickened. The selected area electron diffraction (SAED) pattern shows the characteristic diffraction rings of (002), (004), and (211) of hydroxyapatite, indicating that the remineralized crystals inside and outside the collagen are mainly hydroxyapatite.

[0080] Test 5: Molecular Docking The binding poses and interactions between two small molecules and a protein were obtained using Autodock Vina v.1.2.2, and the binding energy of each interaction was generated. The docking results were visualized using PyMol (see Figure 6 ). Generally, a docking energy value less than -4.25 kcal / mol indicates a certain binding activity between the two, less than -5.0 kcal / mol indicates good binding activity, and less than -7.0 kcal / mol indicates strong binding activity. The results showed that the binding energy between collagen and 10-MDP was Affinity = -5.2 (kcal / mol), indicating good binding activity between the two. The small molecule formed a hydrogen bond interaction with the amino acid residue HIS-457 in the protein. At the same time, the protein and the small molecule had non-bonded contacts, forming forces represented by electrostatic potential energy and hydrophobic forces. The binding energy between collagen and CA was Affinity = -5.0 (kcal / mol), indicating good binding between the two. CA formed hydrogen bond interactions with the amino acid residues ILE-518, ALA-517, and ASN-435 in the protein.

[0081] Test 6: Mechanical Properties Nanoindentation test: The measurement of the nano-hardness and elastic modulus of dentin was performed on an Agilent G200 nanoindenter equipped with a Berkovich indenter with a radius of about 20 nm. The hardness and elastic modulus were measured by the force control method. During loading and unloading, the maximum force used was 10 mN (1.02 gf), 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 on each sample. The mechanical properties of the demineralized dentin discs and the remineralized dentin prepared in Example 1 and Comparative Examples 1-3 were tested, with natural dentin and natural enamel as the control groups. The results are shown in Table 1.

[0082] Table 1

[0083] The remineralized dentin prepared in Example 1 was much higher than natural dentin in terms of elastic modulus and nano-hardness, and was similar to natural enamel, which was attributed to the fact that remineralization had penetrated deep into the dentinal tubules, and the remineralized dentin had produced a rigid enamel-like surface, enhancing the mechanical properties.

[0084] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is impossible to enumerate all the implementation manners here. Any obvious changes or variations derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. A composition for dentin biomimetic remineralization, characterized in that: The composition comprises Dentin pretreatment agents for providing modification and infiltration functions; as well as A mixed mineralizing solution for providing remineralization and rebuilding an enamel-like layer, comprising a mineralizing solution A and a mineralizing solution B; Wherein, the dentin pretreatment agent includes citric acid and / or its salt, 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 soluble fluoride and soluble phosphate; In the mixed mineralizing liquid, the volume ratio of the mineralizing liquid A to the mineralizing liquid B is 0.1-10:

1.

2. The composition according to claim 1, characterized in that The mass concentration of 10-methacryloyloxydecyl phosphate in the dentin pretreatment agent is 10-20%, and the mass concentration of citric acid and / or its salt in the dentin pretreatment agent is 3-10%.

3. The composition according to claim 1, characterized in that The citric acid and / or its salt is selected from one or more of citric acid, citric acid hydrate, sodium citrate, and sodium citrate hydrate.

4. The composition according to claim 1, characterized in that 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, zinc ammonium fluoride, lauryl amine hydrofluoride, and diethylaminoethyl caprylamide hydrofluoride; 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.

5. The composition according to claim 4, characterized in that The calcium ion concentration in the mineralization solution A is 0.1-100mmol / L; The pH range of the mineralization liquid A is 4.5-7.5; The fluoride ion concentration in the mineralization solution B is 0.001-1 g / L; The total concentration of phosphate, monohydrogen phosphate and dihydrogen phosphate in the mineralization solution B is 0.1-100 mmol / L; The pH range of the mineralization solution B is 4.5-7.

5.

6. The composition according to claim 5, characterized in that The ratio of the calcium ion concentration in the mineralization solution A to the total concentration of phosphate, monohydrogen phosphate and dihydrogen phosphate in the mineralization solution B is 1-10:

1.

7. The composition according to claim 5, characterized in that The calcium ion concentration in the mineralization solution A is 18.2-30mmol / L; The pH range of the mineralization liquid A is 5.5-6.5; The fluoride ion content in the mineralization solution B is 0.01-0.05 g / L; The total concentration of phosphate, monohydrogen phosphate, and dihydrogen phosphate in mineralization solution B was 5.6-10 mmol / L; The pH range of the mineralization solution B is 5.5-6.

5.

8. The composition according to claim 1, characterized in that In the mineralization liquid A and the mineralization liquid B, the buffer solution is selected from one or more of phosphoric acid 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, boric acid or its salt solution.

9. Use of the composition according to any one of claims 1 to 8 in preparing a product for repairing demineralized dentin.

10. A dentin bionic remineralization method, characterized in that: Using the composition according to any one of claims 1 to 8 to carry out dentin biomimetic remineralization comprises the following steps: 1) Dentin pretreatment: Apply a dentin pretreatment agent on the surface of demineralized dentin for a total application time of 5-10 minutes, let it stand for 0-60 minutes after application, and then rinse with deionized water to obtain pretreated dentin; the water contact angle of the pretreated dentin surface is 30-40°; 2) Reconstruction of enamel-like layer: Mixing the mineralizing liquid A and the mineralizing liquid B in proportion to obtain a mixed mineralizing liquid; The pretreated dentin is immersed in a mixed mineralizing solution, which is replaced every 6-24 hours. The solution is mineralized at a constant temperature of 37°C for 1-14 days, and the bionic remineralization of the demineralized dentin is completed after cleaning.

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