A composition for forming a composite repair layer on a substrate surface and use thereof

Abstract

The application discloses a composition for forming a composite repair layer on a substrate surface and application thereof. The composition comprises a first component and a second component, wherein the first component comprises a solution containing lysozyme, sodium dihydrogen phosphate, a polypeptide and a reducing agent, and the second component comprises a zirconium ion solution. The application aims at the problems of current repair layer structure disorder, poor performance, insufficient durability and the like, and provides a substrate surface in-situ crystal / amorphous phase composite repair layer with good repair effect and durability and a preparation method thereof.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, and in particular relates to a composition for forming a composite repair layer on a substrate surface and its application. Background Technology

[0002] Tooth loss refers to the loss of part or all of the hard tissue of a tooth due to trauma, caries, abrasion, or other causes. Statistics show that the prevalence of tooth loss among adults worldwide is over 90%. The hard tissue of the tooth crown is mainly composed of the inner dentin and the outer enamel. When enamel is lost due to various reasons, the exposed dentin not only causes sensitivity and pain but also accelerates damage to tooth structure and physiological function due to decreased mechanical properties and acid erosion resistance, severely impacting the patient's quality of life. Natural tooth enamel is a composite biomineralized material composed of highly ordered hydroxyapatite and amorphous phases. Its multi-scale hierarchical structure and excellent mechanical properties together form the core barrier against mechanical and chemical damage to the oral cavity. Compared to enamel caries, dentin caries progresses faster and is more difficult to prevent and treat due to its higher organic content. The mechanical properties and stability of dentin itself are also less able to withstand the multiple challenges of acid erosion and abrasion within the oral cavity.

[0003] For tooth defects, current clinical practice primarily relies on macroscopic filling techniques, with widely used restorative materials including resin, metal, and ceramics. However, their drawbacks are obvious. Resin restorations mainly use light-cured resins (such as ordinary filler resins or penetrating resins). The defect site is mechanically or chemically prepared, acid-etched, and then an adhesive is applied before filling with resin. This method requires invasive shaping of the existing defect to provide retention or bonding sites for the resin. Furthermore, resin polymerization shrinkage leads to microleakage (occurrence >60%), aging of the bonding interface can cause secondary caries, and the expansion of microcracks can lead to restoration failure, resulting in poor long-term stability. Metal and ceramic restorative materials typically require extensive removal of healthy tooth tissue, making biointegration difficult. These methods are essentially "replacement restorations" rather than "regenerative restorations," and cannot fundamentally restore the physiological structure and function of tooth tissue.

[0004] Remineralization therapy has the advantages of being minimally invasive and biocompatible. It primarily utilizes disordered minerals such as fluoride or CPP-ACP (casein phosphopeptide-amorphous calcium phosphate). By providing a supersaturated calcium phosphate ion solution, crystal deposition relies on residual hydroxyapatite (HAp) within the dentin-collagen network as seed crystals. However, this remineralization method is relatively slow, and the resulting remineralized crystal structure is typically disordered and loose, with mechanical properties far lower than natural teeth. It is ill-suited to withstand chewing forces and acid erosion, and its application is limited to early enamel caries before substantial defects have formed, resulting in significant limitations. Furthermore, traditional remineralization directly deposits new HAp crystals from a supersaturated calcium phosphate solution, forming a surface mineralization layer or large crystals. This blocks the diffusion channels of mineralized ions into the collagen network, preventing effective internal remineralization. The resulting crystals are disordered, loose, and prone to detachment.

[0005] To overcome the limitations of traditional methods, cutting-edge international research has focused on biomimetic remineralization, aiming to simulate the biomineralization process to achieve in-situ tooth regeneration. While biomimetic remineralization has made significant progress, it still faces the following core challenges: 1) Structural mismatch: mostly disordered mineralized layers with chaotic structure or crystal-amorphous heterogeneous interfaces that cannot reproduce natural enamel, with insufficient biomimicry, poor compactness of the repair layer, corresponding poor mechanical properties, and similarity to the inner demineralized dentin. 2) Low mineralization rate: The growth rate of remineralized crystals induced by traditional regulators such as casein phosphopeptides and amelogenin-derived synthetic peptides is mostly limited to 0-1.5 μm / d, which is difficult to meet the clinical time requirements; while the mineralization rate of recombinant amelogenin and natural amelogenin, which are extremely expensive, is close to 2 μm / d, and they do not show a significant advantage in the context of clinical defects that are often more than 2 mm deep. 3) Insufficient durability: The remineralized repair layer is easily corroded and loses its function in acidic environments, and has poor corrosion resistance. In addition, the remineralized repair layer is prone to peeling off under mechanical stress (such as chewing, abrasion, etc.) and lacks long-term stability.

[0006] In summary, current biomimetic mineralization restorations for full-thickness enamel defects suffer from technical challenges such as structural-performance mismatch, low restoration efficiency, and material instability, hindering their clinical translation. Therefore, there is an urgent need in dental clinics to develop materials suitable for in-situ dentin restoration, restoring a layer with excellent mechanical properties and stability to the dentin surface. Summary of the Invention

[0007] To address at least some of the technical problems in the prior art, the present invention provides a composition for forming a composite repair layer on a substrate surface and its application. Specifically, the present invention includes the following.

[0008] In a first aspect, the present invention provides a composition for forming a composite repair layer on a substrate surface, comprising a first component and a second component, the first component comprising a solution containing lysozyme, sodium dihydrogen phosphate, a polypeptide and a reducing agent, the polypeptide having a sequence as shown in SEQ ID NO.1, and the second component comprising a zirconium ion solution.

[0009] In some embodiments, the composition for forming a composite restorative layer on a substrate surface according to the present invention is used, wherein the substrate comprises at least one of denture, dentin, enamel, silicon wafer, bioceramics, or titanium alloy.

[0010] In some embodiments, the composition for forming a composite repair layer on a substrate surface according to the present invention further comprises phosphorylated serine.

[0011] In some embodiments, the composition for forming a composite repair layer on a substrate surface according to the present invention includes, wherein the reducing agent comprises at least one of thiol reducing agents, amine reducing agents, sulfonate reducing agents, thiourea reducing agents, and sulfite reducing agents.

[0012] In some embodiments, the composition for forming a composite repair layer on a substrate surface according to the present invention further comprises a buffer solution.

[0013] In some embodiments, the composition for forming a composite repair layer on a substrate surface according to the present invention includes a Tris buffer.

[0014] In some embodiments, the composition for forming a composite repair layer on a substrate surface according to the present invention, wherein the zirconium ion solution comprises at least one of zirconium oxychloride solution, zirconium isopropoxide solution, zirconium oxynitrate solution, zirconium sulfate solution, or zirconium acetate solution.

[0015] In some embodiments, the composition for forming a composite repair layer on a substrate surface according to the present invention, wherein in the first component, the concentration of the lysozyme is 0.1-5 mg / mL, the concentration of the sodium dihydrogen phosphate is 1-30 mM, the concentration of the reducing agent is 1-20 mM, and in the second component, the concentration of the zirconium ions is 0.005-2%.

[0016] A second aspect of the present invention provides a method for forming a composite repair layer on a substrate surface using the composition described in the first aspect of the present invention, comprising the following steps: (1) The first component is coated on the surface of the substrate and reacted for 0.5-5 h to obtain a substrate containing an amyloid protein composite film; (2) The matrix containing the amyloid protein composite film is placed in a mineralization solution and mineralized for 2-5 days to obtain the pretreated matrix; (3) Immerse the pretreated matrix in the second component and react for 5-20 h.

[0017] In some embodiments, according to the method of the second aspect of the present invention, step (3) is performed at 4-37°C.

[0018] A third aspect of the present invention provides a dental article comprising a matrix and a composite restorative layer, the composite restorative layer being prepared by the method described in the second aspect of the present invention.

[0019] This invention provides an in-situ crystalline / amorphous phase composite repair layer on a substrate surface with good repair effect and long-lasting stability, and its preparation method, thereby solving the problems of disordered structure, poor performance and insufficient durability of current repair layers. Attached Figure Description

[0020] Figure 1 This is a flowchart illustrating the process of forming a crystalline / amorphous phase composite repair layer on the substrate surface according to the present invention.

[0021] Figure 2 A represents the Zeta potential analysis of the mixture, the unphosphorylated precursor protein complex membrane, and the phosphorylated precursor protein complex membrane in Example 1. B represents the laser confocal microscopy image observed after ThT staining following RPP coating on the dentin surface. Among them, lyso / C-Ame is the control group, the RPP group is the example group, the ultrasound group is the image of the RPP group after ultrasonic treatment, and C represents the quantitative measurement result of fluorescence intensity.

[0022] Figure 3 A is a scanning electron microscope image of the crystal / amorphous phase composite repair layer RPP-HAp@ZrO2 of the present invention; B is a transmission electron microscope image, HRTEM result and SAED diffraction result of the crystal / amorphous phase composite repair layer RPP-HAp@ZrO2 of the present invention, indicating that the generated crystal is HAp crystal; C is a surface morphology image of the crystal / amorphous phase composite repair layer RPP-HAp@ZrO2 under atomic force microscopy; D is a cross-sectional view of the repair layer and a quantitative distribution diagram of the elemental distribution in the corresponding region, indicating that the amorphous zirconium element is uniformly distributed in the cross-sectional layer.

[0023] Figure 4 The results of the nanoindentation test are for the mechanical properties of the composite repair layer of the present invention.

[0024] Figure 5A, B, and C represent the acid erosion resistance, wear resistance, and fatigue resistance results of the repair layers of Example 1 and Comparative Example 2 of the present invention, obtained through lactic acid etching test, brushing test, and fatigue cycle test, respectively. Among them, RPP-HAp@ZrO2 is the composite repair layer of Example 1, RPP-HAp is the repair layer of Comparative Example 2, Enamel is natural enamel, and Dentin is natural dentin.

[0025] Figure 6 This is a schematic diagram illustrating the construction of the animal in vivo mineralization model of the present invention.

[0026] Figure 7 Image A is an electron micrograph of the repair effect of the repair layer of Example 1 and the control group in an animal in vivo mineralization model. Images B and C are comparisons of the mechanical properties of each group after repair (results of nanoindentation test). Among them, RPP-HAp@ZrO2 is the composite repair layer of Example 1.

[0027] Figure 8 The results show the antibacterial adhesion performance of the composite repair layer in Example 1 and Comparative Example 2 of the present invention, wherein RPP-HAp@ZrO2 is the composite repair layer in Example 1.

[0028] Figure 9 These are the results of the biocompatibility evaluation experiments of the repair layers in Example 1 and Comparative Example 2 of the present invention.

[0029] Figure 10 SEM-EDS energy dispersive spectroscopy analysis of amorphous phase embedded in crystals under different temperature conditions. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Composition In one aspect, the present invention provides a composition that can be used to prepare oral restorative materials, such as materials or reagents for repairing tooth defects. In a preferred embodiment, it is used to form a composite restorative layer on a substrate surface. The composition of the present invention comprises a first component and a second component, the first component comprising a solution containing lysozyme, sodium dihydrogen phosphate, a polypeptide, and a reducing agent, and the second component comprising a zirconium ion solution, the polypeptide having the sequence shown in SEQ ID NO.1.

[0034] In this invention, the term "matrix" refers to any material on which a composite restorative layer can be formed, examples of which include, but are not limited to, dentures, dentin, enamel, silicon wafers, bioceramics, titanium alloys, etc. In a preferred embodiment, the matrix is ​​dentin of a natural tooth.

[0035] In this invention, the term "composite repair layer" specifically refers to a crystalline / amorphous phase composite repair layer, wherein the amorphous phase can enter the interphase of the crystalline phase, thereby giving the composite repair layer excellent resistance to acid corrosion, antibacterial properties, wear resistance, fatigue resistance, etc.

[0036] In one preferred embodiment, the composition comprises a first component and a second component, the first component comprising a solution containing lysozyme, sodium dihydrogen phosphate, a polypeptide, a reducing agent, and phosphorylated serine, and the second component comprising a zirconium ion solution. In another preferred embodiment, the composition comprises a first component and a second component, the first component comprising a solution containing lysozyme, sodium dihydrogen phosphate, a polypeptide, and a reducing agent, and the second component comprising a zirconium ion solution and a buffer solution. In yet another preferred embodiment, the composition comprises a first component and a second component, the first component comprising a solution containing lysozyme, sodium dihydrogen phosphate, a polypeptide, a reducing agent, and phosphorylated serine, and the second component comprising a zirconium ion solution and a buffer solution.

[0037] In this invention, the reducing agent is not particularly limited, and examples include, but are not limited to, thiol reducing agents (e.g., but not limited to, tris(2-carboxyethyl)phosphine, 2-mercaptobenzimidazole, 2-mercaptoethanol, etc.), amine reducing agents (e.g., but not limited to, dimethyl-p-toluidine, tert-butyldimethylaniline, dihydroxyethyl-p-toluidine, etc.), sulfonate reducing agents (e.g., but not limited to, benzenesulfinate, p-toluenesulfinate, etc.), and thiourea reducing agents (e.g., but not limited to, 1...). Ethyl 2 Thiourea, tetramethylthiourea, tetraethylthiourea, 1,1 Dibutylthiourea, 1,3 Dibutylthiourea, etc.), sulfites, dithionites, hydrazides, ascorbic acid and its derivatives, metal oxides, coordination metal compounds, etc. In a preferred embodiment, the reducing agent is tris(2-carboxyethyl)phosphine.

[0038] In this invention, the zirconium ion solution is not particularly limited, and examples include, but are not limited to, zirconium oxychloride solution, zirconium isopropoxide solution, zirconium oxynitrate solution, zirconium sulfate solution, and zirconium acetate solution. In a preferred embodiment, the zirconium ion solution is a zirconium oxychloride solution.

[0039] To form a durable and stable in-situ composite repair layer on the repair, the amount or concentration of each substance in the first component can be controlled within a suitable range. In a preferred embodiment, the concentration of the lysozyme in the first component is 0.1-5 mg / mL, preferably 0.2-4.5 mg / mL, even more preferably 0.3-4 mg / mL, further preferably 0.4-3.5 mg / mL, and more preferably 0.5-3 mg / mL, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3 mg / mL; the concentration of the sodium dihydrogen phosphate is 1-30 mM, preferably 2-25 mM, even more preferably 3-20 mM, and more preferably 4-15 mM. The concentration of the polypeptide is 1-10 mg / mL, preferably 1-9 mg / mL, even more preferably 1-8 mg / mL, and more preferably 1-7 mg / mL, for example 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7 mg / mL; the concentration of the reducing agent is 1-20 mM, preferably 1-19 mM, even more preferably 1-18 mM, further preferably 1-17 mM, more preferably 1-16 mM, and more preferably 1-15 mM. mM, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 mM; the concentration of phosphorylated serine is 0.0001-0.5 wt%; preferably 0.0001-0.4 wt%, more preferably 0.0001-0.3 wt%, and even more preferably 0.0001-0.2 wt%, for example 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001 mM. , 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0 .06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2wt%.

[0040] In order to form an in-situ composite repair layer with good repair effect and long-lasting stability on the repair, the amount or concentration of each substance in the second component can be controlled within a suitable range. In the second component, the concentration of zirconium ions is 0.005-2wt%, preferably 0.005-1.8wt%, even more preferably 0.005-1.6wt%, and more preferably 0.005-1.4wt%, for example 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4wt%.

[0041] Based on the above composition, this invention also provides a kit for forming a composite repair layer on a substrate surface, comprising the composition and an instruction manual for forming the composite repair layer on the substrate surface. In this invention, the components of the composition can be placed independently in a container or formed into a premix, and there is no particular limitation thereto.

[0042] Method One aspect of the present invention provides a method for forming a composite repair layer on a substrate surface using the composition described herein, comprising the following steps: (1) The first component is coated on the surface of the substrate and reacted for 0.5-5 h to obtain a substrate containing an amyloid protein composite film; (2) The matrix containing the amyloid protein composite film is placed in a mineralization solution and mineralized for 2-5 days to obtain the pretreated matrix; (3) Immerse the pretreated matrix in the second component.

[0043] In a preferred embodiment, the method of forming a composite repair layer on a substrate surface according to the present invention includes the following steps: (1) Mix 0.5-5 parts by weight, preferably 0.5-4.5 parts by weight, even more preferably 0.5-4 parts by weight, further preferably 0.5-3.5 parts by weight, more preferably 0.5-3 parts by weight, for example 0.5, 1, 1.5, 2, 2.5, or 3 parts by weight of a lysozyme solution (pH 7-8, preferably 7.1-7.9, even more preferably 7.2-7.8, more preferably 7.2-7.7, such as 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7) with 0.5-5 parts by weight. The sodium dihydrogen phosphate solution is mixed in parts by weight, preferably 0.5-4.5 parts by weight, more preferably 0.5-4 parts by weight, further preferably 0.5-3.5 parts by weight, and even more preferably 0.5-3 parts by weight, for example, 0.5, 1, 1.5, 2, 2.5, or 3 parts by weight of 10-30 mM, preferably 11-29 mM, even more preferably 12-28 mM, further preferably 13-27 mM, and even more preferably 14-26 mM, for example, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 mM, for 5-30 min, preferably 5-28 min, even more preferably 5-26 min, and even more preferably 5-24 min, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 mM. Add 5-20 parts by weight, preferably 5-19 parts by weight, even more preferably 5-18 parts by weight, further preferably 5-17 parts by weight, and more preferably 5-16 parts by weight, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 parts by weight of a polypeptide with the sequence shown in SEQ ID NO.1, to obtain a mixture; (2) Add 1-20 mM, preferably 1-19 mM, even more preferably 1-18 mM, further preferably 1-17 mM, more preferably 1-16 mM, even more preferably 1-15 mM, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 mM of TCEP solution to the mixture, adjust the pH to 5.8-6.0, for example 5.8, 5.9, 6.0, and react at 30-45°C, preferably 31-44°C, even more preferably 32-43°C, even more preferably 33-42°C, for example 33, 34, 35, 36, 37, 38, 39, 40, 41, 42°C to obtain the first component; (3) The first component is uniformly coated on the surface of the substrate, and the reaction time is 0.5-5 hours, preferably 0.5-4.5 hours, even more preferably 0.5-4 hours, further preferably 0.5-3.5 hours, more preferably 0.5-3 hours, 0.5, 1, 1.5, 2, 2.5, 3 hours, to obtain a substrate with an amyloid protein complex; (4) Add LO-phosphoserine to the PBS buffer to prepare a concentration of 0.0008-0.5 wt% (preferably 0.0008-0.45 wt%, more preferably 0.0008-0.4 wt%, further preferably 0.0008-0.35 wt%, more preferably 0.0008-0.3 wt%, more preferably 0.0008-0.25 wt%, more preferably 0.0008-0.2 wt%, for example 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.0...). A solution containing 7, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, and 0.2 wt%) is used to immerse the matrix containing the amyloid protein complex in the solution. The reaction is carried out at 30-45°C, preferably 31-44°C, even more preferably 32-43°C, and more preferably 33-42°C, for example, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42°C with shaking for 0.5-5 hours, preferably 0.5-4.5 hours, even more preferably 0.5-4 hours, further preferably 0.5-3.5 hours, and more preferably 0.5-3 hours, for example, 0.5, 1, 1.5, 2, 2.5, and 3 hours. (5) Rinse the matrix obtained in step (4) with deionized water or ultrapure water, mineralize it in a mineralizing solution (e.g., but not limited to artificial saliva) for 2-5 days, for example, 2, 2.5, 3, 3.5, 4, 4.5, 5 days, then place it in a tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution with a pH of 7.0-7.5 (e.g., 7.0, 7.1, 7.2, 7.3, 7.4, 7.5), and cool it to 4-37°C, preferably 4-36°C, and even more preferably 4-35°C. The solution is first heated at a temperature preferably 4-34°C, more preferably 4-33°C, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33°C, and then 5-30 parts by weight, preferably 5-28 parts by weight, even more preferably 5-26 parts by weight, and even more preferably 5-24 parts by weight, are added to the solution. 5-22 parts by weight, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 parts by weight of the second component, zirconium oxychloride octahydrate, at 4-37°C, preferably 4-36°C, even more preferably 4-35°C, further preferably 4-34°C, and more preferably 4-33°C, for example 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18... The reaction is carried out at 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33°C for 5-20 hours, preferably 5-19 hours, even more preferably 5-18 hours, further preferably 5-17 hours, and more preferably 5-16 hours, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 hours. After rinsing with deionized water, the mixture is air-dried at room temperature to obtain a composite repair layer formed on the substrate surface.

[0044] It is understandable that, in order to make the reaction more complete, stirring can be performed during the above steps. The stirring method is not particularly limited and can be any stirring method known in the prior art.

[0045] The present invention also provides a method for controlling the depth of amorphous phase entering the interstitial space of crystalline phases, which includes the following steps: (1) The first component is coated on the surface of the substrate and reacted for 0.5-5 h to obtain a substrate containing an amyloid protein composite film; (2) The matrix containing the amyloid protein composite film is placed in a mineralization solution and mineralized for 2-5 days to obtain the pretreated matrix; (3) Immerse the pretreated matrix in the second component at 4-37°C for 5-20 h.

[0046] In a preferred embodiment, the method of the present invention for controlling the depth of amorphous phase entering the interstitial space of crystalline phases includes the following steps: (1) Mix 0.5-5 parts by weight, preferably 0.5-4.5 parts by weight, even more preferably 0.5-4 parts by weight, further preferably 0.5-3.5 parts by weight, more preferably 0.5-3 parts by weight, for example 0.5, 1, 1.5, 2, 2.5, or 3 parts by weight of a lysozyme solution (pH 7-8, preferably 7.1-7.9, even more preferably 7.2-7.8, more preferably 7.2-7.7, such as 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7) with 0.5-5 parts by weight. The sodium dihydrogen phosphate solution is mixed in parts by weight, preferably 0.5-4.5 parts by weight, more preferably 0.5-4 parts by weight, further preferably 0.5-3.5 parts by weight, and even more preferably 0.5-3 parts by weight, for example, 0.5, 1, 1.5, 2, 2.5, or 3 parts by weight of 10-30 mM, preferably 11-29 mM, even more preferably 12-28 mM, further preferably 13-27 mM, and even more preferably 14-26 mM, for example, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 mM, for 5-30 min, preferably 5-28 min, even more preferably 5-26 min, and even more preferably 5-24 min, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 mM. Add 5-20 parts by weight, preferably 5-19 parts by weight, even more preferably 5-18 parts by weight, further preferably 5-17 parts by weight, and more preferably 5-16 parts by weight, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 parts by weight of a polypeptide with the sequence shown in SEQ ID NO.1, to obtain a mixture; (2) Add 1-20 mM, preferably 1-19 mM, even more preferably 1-18 mM, further preferably 1-17 mM, more preferably 1-16 mM, even more preferably 1-15 mM, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 mM of TCEP solution to the mixture, adjust the pH to 5.8-6.0, for example 5.8, 5.9, 6.0, react at 30-45°C, preferably 31-44°C, even more preferably 32-43°C, even more preferably 33-42°C, for example 33, 34, 35, 36, 37, 38, 39, 40, 41, 42°C to obtain the first component; (3) The first component is uniformly coated on the surface of the substrate, and the reaction time is 0.5-5 hours, preferably 0.5-4.5 hours, even more preferably 0.5-4 hours, further preferably 0.5-3.5 hours, more preferably 0.5-3 hours, 0.5, 1, 1.5, 2, 2.5, 3 hours, to obtain a substrate with an amyloid protein complex; (4) Add LO-phosphoserine to the PBS buffer to prepare a concentration of 0.0008-0.5 wt% (preferably 0.0008-0.45 wt%, more preferably 0.0008-0.4 wt%, further preferably 0.0008-0.35 wt%, more preferably 0.0008-0.3 wt%, more preferably 0.0008-0.25 wt%, more preferably 0.0008-0.2 wt%, for example 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.0...). A solution containing 7, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, and 0.2 wt%) is used to immerse the matrix containing the amyloid protein complex in the solution at 30-45°C, preferably 31-44°C, even more preferably 32-43°C, and more preferably 33-42°C, for example, at 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42°C, with shaking reaction for 0.5-5 hours, preferably 0.5-4.5 hours, even more preferably 0.5-4 hours, further preferably 0.5-3.5 hours, and more preferably 0.5-3 hours, for example, 0.5, 1, 1.5, 2, 2.5, and 3 hours; (5) Rinse the matrix obtained in step (4) with deionized water or ultrapure water, mineralize it in a mineralizing solution (e.g., but not limited to artificial saliva) for 2-5 days, for example, 2, 2.5, 3, 3.5, 4, 4.5, 5 days, then place it in a tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution with a pH of 7.0-7.5 (e.g., 7.0, 7.1, 7.2, 7.3, 7.4, 7.5), and cool it to 4-37°C, preferably 4-36°C, and even more preferably 4-35°C, for further processing. The preferred temperature is 4-34℃, more preferably 4-33℃, for example 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33℃. Then, 5-30 parts by weight, preferably 5-28 parts by weight, even more preferably 5-26 parts by weight, further preferably 5-24 parts by weight, and more preferably 5 parts by weight, are added to the solution. -22 parts by weight, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 parts by weight of the second component, zirconium oxychloride octahydrate, at 4-37°C, preferably 4-36°C, more preferably 4-35°C, further preferably 4-34°C, and more preferably 4-33°C, for example 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1 The reaction is carried out at 9, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33°C for 5-20 hours, preferably 5-19 hours, even more preferably 5-18 hours, further preferably 5-17 hours, and more preferably 5-16 hours, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 hours. After rinsing with deionized water and air-drying at room temperature, the depth of the amorphous phase entering the interstitial space of the crystalline phase is controlled. In a specific embodiment, the control of this invention refers to increasing the interstitial filling rate when zirconia is embedded in HAp crystals.

[0047] The method of the present invention can embed zirconium oxide into the gaps of HAp crystals, achieving seamless integration of HAp crystals and amorphous ZrO2 at the nanoscale, forming a crystal-amorphous heterogeneous interface with a gap filling rate of >90%, thereby achieving excellent antibacterial and mechanical properties of the crystal / amorphous phase composite repair layer.

[0048] This invention utilizes a biomimetic design of a precursor protein composite membrane with dual functional domains inspired by natural amelogenin. This membrane simultaneously achieves multiple functions, including interfacial ion enrichment and strong adhesion, HAp crystal directional growth, and rapid remineralization, enabling the rapid construction of a surface remineralized repair layer. Furthermore, amorphous zirconia fills the intercrystalline gaps, forming a heterogeneous interface that simulates the crystalline / amorphous dual-component structure of natural enamel. The mechanical properties of this repair layer are far superior to similar dentin repair layers, closely resembling those of natural enamel, effectively restoring the physiological function of the hard dental tissues. Figure 1).

[0049] Dental article In one aspect, a dental article is provided, comprising a matrix and a composite restorative layer, said composite restorative layer being prepared by the method described in this invention. Examples of the matrix include, but are not limited to, dentures, dentin, enamel, silicon wafers, bioceramics, titanium alloys, etc.

[0050] Example 1 The following illustrates the method for forming a composite repair layer on the substrate surface and the performance testing of the repair layer.

[0051] 1. Preparation method (1) Artificial polypeptides were synthesized by solid-phase synthesis with a purity of 98.31%, and the sequence of the polypeptides is shown in SEQ ID NO.1.

[0052] (2) Mix 1 mL of lysozyme solution (Sigma-Aldrich, 2 mg / mL, dissolved in 10 mM PBS buffer, pH=7.4) with 1 mL of sodium dihydrogen phosphate solution (20 mM NaH2PO4, dissolved in deionized water) in equal amounts, mix for a period of time (about 15 minutes), and then add 10 mg of synthetic peptide to obtain a mixture.

[0053] (3) Add tris(2-carboxyethyl)phosphine (TCEP) solution to the mixture to a final concentration of 10 mM, adjust the pH to 5.8-6.0 with 5 M NaOH solution, and react at 37°C to obtain the first component.

[0054] (4) The first component is uniformly coated on the surface of dentin (obtained after removing the outer enamel layer), and the reaction time is about 1 hour to obtain dentin with amyloid protein complex.

[0055] (5) Add LO-phosphoserine to PBS buffer to prepare a solution with a concentration of 0.0008%-0.08%, immerse the dentin with the amyloid protein complex in the solution, and shake at 37°C for 1 hour.

[0056] (6) Rinse the dentin obtained in step (5) with deionized water or ultrapure water, mineralize it in artificial saliva for 3 days, then place it in a tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution with a pH of 7.0-7.5, cool it to 4°C in a low-temperature constant temperature cooling bath, then add 14 mg of the second component zirconium oxychloride octahydrate to the solution, and continue to react on a magnetic stirrer at 4°C for 8 hours. Rinse with deionized water, and then air dry at room temperature for 10 minutes to form a composite restoration layer on the substrate surface.

[0057] 2. Performance Testing 2.1 Zeta potential analysis Zeta potential analysis was performed on the solutions obtained in steps (2), (3), and (5) respectively, and the results are as follows: Figure 2 As shown in A, RPP(+P) is the solution group after phosphorylation modification in step (5). The protein membrane of the RPP(+P) group has the strongest negative charge and the strongest affinity for calcium ions.

[0058] In addition, after applying RPP to the dentin surface, ThT staining was performed, followed by observation using a laser confocal microscope. The results are as follows: Figure 2 As shown in Figure B, the RPP group exhibited green fluorescence, indicating that the RPP was successfully integrated into the dentin surface and was rich in β-sheet structures. The ultrasound group showed that the composite membrane remained adhered to the substrate surface after ultrasonic treatment.

[0059] 2.2 Electron Microscopy Analysis Scanning electron microscopy (SEM) analysis was performed on the lower surface structure and longitudinal section structure of the final crystalline / amorphous phase composite repair layer RPP-HAp@ZrO2. The results are as follows: Figure 3 As shown in Figure A, the characteristic tubular structures of the underlying dentin basal layer are visible, and the thickness of the overlying restorative layer is approximately 13 μm. Transmission electron microscopy analysis of the final crystalline / amorphous composite restorative layer RPP-HAp@ZrO2 yielded the following results: Figure 3 As shown in Figure B, ZrO2 is visible intercalated into the gaps of the crystalline HAp layer. Atomic force microscopy analysis of the crystalline / amorphous phase composite repair layer RPP-HAp@ZrO2 is performed, and the surface morphology is shown in the figure below. Figure 3 As shown in C.

[0060] 2.3 Mechanical Performance Testing Nanoindentation testing was performed on the crystalline / amorphous composite restoration layer RPP-HAp@ZrO2. Acid-etched Dentin represented the dentin group, NaF the sodium fluoride group, CPP-ACP the casein phosphopeptide-amorphous calcium phosphate group, and RPP-HAp@ZrO2 the group used in this embodiment. Results are as follows: Figure 4 As shown, the crystalline / amorphous phase composite repair layer obtained in this embodiment has extremely excellent hardness and elastic modulus.

[0061] 2.4 Resistance to acid corrosion / wear / fatigue The acid corrosion resistance, wear resistance, and fatigue resistance of the crystalline / amorphous phase composite repair layer RPP-HAp@ZrO2 were tested. The results are as follows: Figure 5 As shown, the crystalline / amorphous phase composite repair layer obtained in this embodiment has extremely excellent resistance to acid corrosion, wear, and fatigue.

[0062] 3. Security Testing Each group of repair layers was co-cultured with human fibroblasts, and the effects of the materials on cell proliferation and cytotoxicity were evaluated using a live-death assay. The results are as follows: Figure 9 As shown in AC, the repair layer material has virtually no cytotoxicity at in vitro mineralization concentrations, meaning that the crystalline / amorphous phase composite repair layer obtained in this embodiment has good biocompatibility.

[0063] Example 2 The following describes a method for forming a composite repair layer on a substrate surface.

[0064] (1) Artificial polypeptides were synthesized by solid-phase synthesis with a purity of 98.31%, and the sequence of the polypeptides is shown in SEQ ID NO.1.

[0065] (2) Mix 1 mL of lysozyme solution (Sigma-Aldrich, 2 mg / mL, dissolved in 10 mM PBS buffer, pH=7.4) with 1 mL of sodium dihydrogen phosphate solution (20 mM, dissolved in deionized water) in equal amounts, mix for a period of time (about 15 minutes), and then add 10 mg of synthetic peptide to obtain a mixture.

[0066] (3) Add tris(2-carboxyethyl)phosphine (TCEP) solution to the mixture to a final concentration of 10 mM, adjust the pH to 5.8-6.0 with 5M NaOH solution, and react at 37°C to obtain the first component.

[0067] (4) The first component is uniformly coated on the surface of dentin (obtained after removing the outer enamel layer), and the reaction time is about 1 hour to obtain dentin with amyloid protein complex.

[0068] (5) Add LO-phosphoserine to PBS buffer to prepare a solution with a concentration of 0.0008%-0.08%, immerse the dentin with the amyloid protein complex in the solution, and shake at 37°C for 1 hour.

[0069] (6) Rinse the dentin obtained in step (5) with deionized water or ultrapure water, mineralize it in artificial saliva for 3 days, then place it in a tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution with a pH of 7.0-7.5, cool it to 10°C in a low-temperature constant temperature cooling bath, then add 14 mg of the second component zirconium oxychloride octahydrate to the solution, and continue to react on a magnetic stirrer at 4°C for 8 hours. Rinse with deionized water, and then air dry at room temperature for 10 minutes to form a composite restoration layer on the substrate surface.

[0070] Example 3 The following describes a method for forming a composite repair layer on a substrate surface.

[0071] (1) Artificial polypeptides were synthesized by solid-phase synthesis with a purity of 98.31%, and the sequence of the polypeptides is shown in SEQ ID NO.1.

[0072] (2) Mix 1 mL of lysozyme solution (Sigma-Aldrich, 2 mg / mL, dissolved in 10 mM PBS buffer, pH=7.4) with 1 mL of sodium dihydrogen phosphate solution (20 mM, dissolved in deionized water) in equal amounts, mix for a period of time (about 15 minutes), and then add 10 mg of synthetic peptide to obtain a mixture.

[0073] (3) Add tris(2-carboxyethyl)phosphine (TCEP) solution to the mixture to a final concentration of 10 mM, adjust the pH to 5.8-6.0 with 5 M NaOH solution, and react at 37°C to obtain the first component.

[0074] (4) The first component is uniformly coated on the surface of dentin (obtained after removing the outer enamel layer), and the reaction time is about 1 hour to obtain dentin with amyloid protein complex.

[0075] (5) Add LO-phosphoserine to PBS buffer to prepare a solution with a concentration of 0.0008%-0.08%, immerse the dentin with the amyloid protein complex in the solution, and shake at 37°C for 1 hour.

[0076] (6) Rinse the dentin obtained in step (5) with deionized water or ultrapure water, mineralize it in artificial saliva for 3 days, then place it in a tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution with a pH of 7.0-7.5, cool it to 37°C in a low-temperature constant temperature cooling bath, then add 14 mg of the second component zirconium oxychloride octahydrate to the solution, and continue to react on a magnetic stirrer at 37°C for 5 hours, rinse with deionized water, and then air dry at room temperature for 10 minutes to form a composite restoration layer on the substrate surface.

[0077] Example 4 The following describes a method for forming a composite repair layer on a substrate surface.

[0078] (1) Artificial polypeptides were synthesized by solid-phase synthesis with a purity of 98.31%, and the sequence of the polypeptides is shown in SEQ ID NO.1.

[0079] (2) Mix 1 mL of lysozyme solution (Sigma-Aldrich, 2 mg / mL, dissolved in 10 mM PBS buffer, pH=7.4) with 1 mL of sodium dihydrogen phosphate solution (20 mM, dissolved in deionized water) in equal amounts, mix for a period of time (about 15 minutes), and then add 10 mg of synthetic peptide to obtain a mixture.

[0080] (3) Add tris(2-carboxyethyl)phosphine (TCEP) solution to the mixture to a final concentration of 10 mM, adjust the pH to 5.8-6.0 with 5 M NaOH solution, and react at 37°C to obtain the first component.

[0081] (4) The first component is uniformly coated on the surface of the dentin of the extracted human tooth and left for 1 hour.

[0082] (5) Add LO-phosphoserine to PBS buffer to prepare a solution with a concentration of 0.0008%-0.08%, apply the solution to human dentin with a toothbrush, and react at 37°C for 1 hour.

[0083] (6) The growth of the repair layer was induced in the oral saliva environment of rats for 3 days. Then, the human extracted tooth dentin surface was coated with a mixture of tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution and zirconium oxychloride octahydrate three times a day to simulate brushing. Finally, the crystalline / amorphous composite repair layer was synthesized in the oral saliva environment.

[0084] The results of the above embodiments show that in the crystalline / amorphous phase composite repair layer obtained by isothermal stirring at 4°C, the ZrO2 component can achieve full-layer embedding (depth of about 10 μm), encapsulating the HaAp nanorod structure to form a crystalline-amorphous heterostructure interface. The crystalline / amorphous phase composite repair layer obtained by isothermal stirring at 10°C can also achieve full-layer embedding, but the required isothermal stirring time is longer. The amorphous phase embedding depth formed at 37°C is slightly shallower than in Examples 1 and 2, and EDS elemental analysis shows that the surface Zr element distribution content is lower than the first two groups (…). Figure 10 Using the method of this invention, the mineralization rate can reach 3 μm / d.

[0085] Comparative Example 1 The following describes a method for forming a composite repair layer on a substrate surface.

[0086] (1) Artificial polypeptides were synthesized by solid-phase synthesis with a purity of 98.31%, and the sequence of the polypeptides is shown in SEQ ID NO.1.

[0087] (2) Mix 1 mL of lysozyme solution (Sigma-Aldrich, 2 mg / mL, dissolved in 10 mM PBS buffer, pH=7.4) with 1 mL of sodium dihydrogen phosphate solution (20 mM, dissolved in deionized water) in equal amounts, mix for a period of time (about 15 minutes), and then add 10 mg of synthetic peptide to obtain a mixture.

[0088] (3) Add tris(2-carboxyethyl)phosphine (TCEP) solution to the mixture to a final concentration of 10 mM, adjust the pH to 5.8-6.0 with 5 M NaOH solution, and react at 37°C to obtain the first component.

[0089] (4) The first component is uniformly coated on the surface of dentin (obtained after removing the outer enamel layer), and the reaction time is about 1 hour to obtain dentin with amyloid protein complex.

[0090] (5) Rinse the dentin obtained in step (4) with deionized water or ultrapure water, mineralize it in artificial saliva for 3 days, then place it in a tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution with a pH of 7.0-7.5, cool it to 10°C in a low-temperature constant temperature cooling bath, then add 14 mg of the second component zirconium octahydrate to the solution, and continue to react on a magnetic stirrer at 10°C for 8 hours. Rinse with deionized water, and then air dry at room temperature for 10 minutes to form a composite restoration layer on the substrate surface.

[0091] Comparative Example 2 The following describes a method for forming a crystal repair layer on a substrate surface.

[0092] (1) Artificial polypeptides were synthesized by solid-phase synthesis with a purity of 98.31%, and the sequence of the polypeptides is shown in SEQ ID NO.1.

[0093] (2) Mix 1 mL of lysozyme solution (Sigma-Aldrich, 2 mg / mL, dissolved in 10 mM PBS buffer, pH=7.4) with 1 mL of sodium dihydrogen phosphate solution (20 mM, dissolved in deionized water) in equal amounts, mix for a period of time (about 15 minutes), and then add 10 mg of synthetic peptide to obtain a mixture.

[0094] (3) Add tris(2-carboxyethyl)phosphine (TCEP) solution to the mixture to a final concentration of 10 mM, adjust the pH to 5.8-6.0 with 5 M NaOH solution, and react at 37°C to obtain the first component.

[0095] (4) The first component is uniformly coated on the surface of dentin (obtained after removing the outer enamel layer), and the reaction time is about 1 hour to obtain dentin with amyloid protein complex.

[0096] (5) Rinse the dentin obtained in step (4) with deionized water or ultrapure water and mineralize it in artificial saliva for 3 days.

[0097] The performance testing of Comparative Examples 1-2 was conducted as described in the examples. Comparative Example 1 showed weaker orientation of the grown Hap crystals compared to the examples. Comparative Example 2, lacking embedded amorphous components, exhibited mechanical properties far superior to dentin, but inferior to the composite restoration layer and natural enamel. Furthermore, it demonstrated weaker acid resistance under oral acid etching conditions than the Hap crystal / ZrO2 amorphous phase composite restoration layer, and its performance in fatigue cycling and brushing tests was also weaker than the example group with the composite restoration layer.

[0098] The RPP-HAp@ZrO2 composite restorative layer prepared in this invention can be directly used for the rapid construction of enamel-like restorative layers on dentin surfaces. The composite restorative layer of this invention exhibits excellent long-term stability under complex oral conditions, maintaining excellent resistance to severe challenges such as acid etching, wear, and fatigue stress cycles. Furthermore, in vivo experiments (rat model) show that full-thickness dentin coverage is achieved within 7 days. Figure 7 The material exhibits excellent crystal orientation and superior biocompatibility (CCK-8 assay showed cell viability > 95%), while also possessing good antibacterial properties. Figure 8 (AE).

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition for forming a composite repair layer on a substrate surface, characterized in that, It includes a first component and a second component. The first component includes a solution containing lysozyme, sodium dihydrogen phosphate, a polypeptide, and a reducing agent. The polypeptide has the sequence shown in SEQ ID NO.

1. The second component includes a zirconium ion solution.

2. The composition for forming a composite repair layer on a substrate surface according to claim 1, characterized in that, The substrate includes at least one of dentures, dentin, enamel, silicon wafers, bioceramics, or titanium alloys.

3. The composition for forming a composite repair layer on a substrate surface according to claim 1, characterized in that, The first component further includes phosphorylated serine.

4. The composition for forming a composite repair layer on a substrate surface according to claim 1, characterized in that, The reducing agent includes at least one of thiol reducing agents, amine reducing agents, sulfonate reducing agents, thiourea reducing agents, and sulfite reducing agents.

5. The composition for forming a composite repair layer on a substrate surface according to claim 4, characterized in that, The second component further includes a buffer solution; Preferably, the buffer solution comprises Tris buffer.

6. The composition for forming a composite repair layer on a substrate surface according to claim 1, characterized in that, The zirconium ion solution includes at least one of zirconium oxychloride solution, zirconium isopropoxide solution, zirconium oxynitrate solution, zirconium sulfate solution, or zirconium acetate solution.

7. The composition for forming a composite repair layer on a substrate surface according to claim 1, characterized in that, In the first component, the concentration of the lysozyme is 0.1-5 mg / mL, the concentration of the sodium dihydrogen phosphate is 1-30 mM, and the concentration of the reducing agent is 1-20 mM. In the second component, the concentration of the zirconium ion is 0.005-2%.

8. A method for forming a composite repair layer on a substrate surface using the composition according to any one of claims 1-7, characterized in that, Includes the following steps: (1) The first component is coated on the surface of the substrate and reacted for 0.5-5 h to obtain a substrate containing an amyloid protein composite film; (2) The matrix containing the amyloid protein composite film is placed in a mineralization solution and mineralized for 2-5 days to obtain the pretreated matrix; (3) Immerse the pretreated matrix in the second component and react for 5-20 h.

9. The method according to claim 8, characterized in that, Step (3) is performed at 4-37℃.

10. A dental product, characterized in that, It includes a matrix and a composite repair layer, wherein the composite repair layer is prepared by the method of claim 8 or 9.

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