A tooth enamel-like ordered structure hydroxyapatite, and a preparation method and application thereof

Abstract

This invention discloses a tooth enamel-like ordered hydroxyapatite, its preparation method, and its applications. The preparation method includes: mixing an orthophosphate solution, a calcium salt solution, a fluoride salt solution, a magnesium salt solution, and an organic macromolecule solution to obtain a mineralization solution, wherein the organic macromolecules include high molecular weight compounds rich in carboxyl and / or sulfonyl functional groups; immersing acid-etched tooth enamel in the mineralization solution and incubating at a constant temperature. This invention regulates the nucleation and crystallization process of calcium phosphate on the enamel crystal surface through organic macromolecules, constructing a layer of ordered hydroxyapatite mineral with excellent mechanical properties on the tooth enamel surface. This tooth enamel-like ordered hydroxyapatite has a continuous structure with natural enamel crystals, without obvious boundaries, with consistent crystal orientation and similar size. Its mechanical properties are also similar to those of natural enamel, making it suitable for clinical caries restoration. This invention also provides a new approach for preparing other ordered mineral functional materials.

Description

Technical Field

[0001] This invention relates to the preparation of inorganic ordered materials with enamel-like structures, specifically to a hydroxyapatite with an enamel-like ordered structure, its preparation method, and its applications. Background Technology

[0002] Tooth enamel is the only acellular tissue in the body that is secreted and mineralized by epithelial cells, and it cannot repair itself after damage. Composed of hydroxyapatite, enamel covers the surface of the tooth crown and is the first tissue affected by dental caries. If not repaired and treated promptly, it can lead to dentin hypersensitivity, pulpitis, periapical periodontitis, and jawbone inflammation, seriously impacting oral health. Furthermore, infections and inflammatory factors in odontogenic lesions such as dental caries can cause or exacerbate chronic diseases such as cardiovascular disease and diabetes, harming overall health.

[0003] Currently, the main materials used for direct enamel restoration in clinical practice are penetrating resin and enamel composite resin. Their composition, structure, and properties are completely different from natural tooth tissue, making it difficult to meet the clinical need for long-term stable enamel restoration.

[0004] Biomimetic mineralization strategies are a cutting-edge research area in enamel restoration, aiming to regenerate enamel crystals in areas of tooth loss. In research using calcium phosphate as a restorative material, tooth restoration involves placing an acid-etched tooth in a supersaturated calcium phosphate solution, allowing hydroxyapatite to grow on the tooth surface through heterogeneous nucleation. With a deeper understanding of biomineralization, scientists are incorporating organic macromolecules such as amelioproteins, peptides / oligopeptides, polysaccharides, and polymers into this restorative process to regulate the nucleation and growth of calcium phosphate, enhancing its orderliness and restoring its mechanical properties.

[0005] Patent document CN103230342A discloses a fluorohydroxyapatite film grown on the surface of tooth enamel. The preparation steps are as follows: first, the tooth enamel sample is pretreated in 30% phosphoric acid; then, the tooth enamel sample is immersed in a solution containing a certain amount of calcium nitrate, disodium hydrogen phosphate, disodium ethylenediaminetetraacetate, and sodium fluoride; the pH and temperature of the solution are adjusted; after 3-7 days, rod-shaped crystals grow on the surface of the tooth enamel, and the rod-shaped crystals form a cluster structure distributed on the surface of the tooth enamel to form a growth film.

[0006] Patent document CN107343857B discloses a tooth enamel-like hydroxyapatite and its preparation method. Using calcium phosphate polymer ions as a precursor, a tooth enamel-like hydroxyapatite is prepared on the enamel surface. This tooth enamel-like hydroxyapatite, when used as a tooth enamel repair material, can achieve rapid repair of the enamel structure in superficial caries. The repaired structure exhibits complete structural order and orientation consistent with the enamel to be repaired, and the mechanical properties of the repaired enamel are identical to those of natural enamel.

[0007] Although existing technologies have been used to study enamel restoration materials, current research methods still struggle to achieve enamel regeneration and biomimetic preparation of enamel-like materials. Based on biomimetic mineralization strategies and the regulatory role of organic macromolecules in enamel growth, this invention will explore the preparation of ordered enamel-like materials. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a method for preparing hydroxyapatite with an ordered enamel structure. This method regulates the nucleation and crystallization process of calcium phosphate on the surface of enamel crystals by constructing a biomimetic mineralization system, thereby preparing hydroxyapatite materials with certain natural enamel structures and mechanical properties.

[0009] A method for preparing hydroxyapatite with an ordered enamel-like structure includes the following steps:

[0010] (1) A mineralizing solution is obtained by mixing an orthophosphate solution, a calcium salt solution, a fluoride salt solution, a magnesium salt solution and an organic macromolecule solution, wherein the organic macromolecules include high molecular weight compounds rich in carboxyl groups and / or sulfonyl functional groups.

[0011] (2) Immerse the acid-etched enamel in the mineralization solution prepared in step (1) and incubate at a constant temperature to obtain the enamel-like ordered structure hydroxyapatite.

[0012] This invention uses tooth enamel as a base and regulates the nucleation and crystallization process of calcium phosphate on the surface of enamel crystals through organic macromolecules. This results in the formation of an ordered hydroxyapatite mineral layer with excellent mechanical properties on the enamel surface. Furthermore, this invention can continuously deposit hydroxyapatite mineral on the enamel surface through a cyclic mineralization treatment strategy, resulting in a thicker mineral layer. This allows for control over the thickness of the restorative layer and provides a potential strategy for subsequent clinical applications.

[0013] Preferably, the concentration of the orthophosphate solution is 0.5 to 6.0 mM.

[0014] Preferably, the concentration of calcium ions in the calcium salt solution is 0.5–6 mM.

[0015] Preferably, the concentration of fluoride ions in the fluoride salt solution is 0.05–1.00 mM.

[0016] Preferably, the concentration of magnesium ions in the magnesium salt solution is 0.1–6 mM.

[0017] Preferably, the concentration of the organic macromolecule solution is 0.005–10 mg / mL.

[0018] Preferably, the molar ratio of orthophosphate to calcium ions is in the range of 1:1 to 1.5, the molar ratio of fluoride ions to calcium ions is in the range of 1:3 to 10, the molar ratio of magnesium ions to calcium ions is in the range of 1 to 5:1, and the mass ratio of organic macromolecules to calcium ions is 1 to 20:1 to 42.

[0019] Preferably, the polymeric compound rich in carboxyl and / or sulfonyl functional groups includes amphiphilic polypeptides or polysaccharides.

[0020] Preferably, the amphiphilic polypeptide includes C 11 H 23 CO-KDDDDKDDDD(COOH) or C 11 H 23 CO-WPATDKTKREEVD(COOH).

[0021] Preferably, the polysaccharide includes chondroitin sulfate, hyaluronic acid, alginate, or carboxymethyl chitosan.

[0022] The functional groups (carboxyl and / or sulfonyl groups) carried by the organic macromolecules of this invention have certain coordination or electrostatic interactions with calcium, thereby effectively regulating the nucleation and growth of calcium phosphate during the mineralization process and controlling the morphology of the final crystal. By precisely controlling the proportions of each chemical component, the preparation of enamel-like ordered hydroxyapatite can be achieved through the crystallization process mediated by the organic macromolecules.

[0023] Preferably, the constant temperature incubation is 25–40°C, and the incubation time is 1–9 days.

[0024] More preferably, the constant temperature incubation is 37°C for 1 to 7 days.

[0025] Preferably, before step (2), the method further includes adding auxiliary components to the mineralization solution prepared in step (1), wherein the auxiliary components include sodium chloride solution, potassium chloride solution, sodium sulfate solution, and HEPES. This invention adds auxiliary components, sodium chloride, potassium chloride, and sodium sulfate, to the mineralization solution to adjust the ionic strength of the solution and the pH of the HEPES buffer solution, thereby improving the repair effect.

[0026] This invention also provides enamel-like ordered hydroxyapatite prepared by the aforementioned method. The enamel-like ordered hydroxyapatite of this invention forms a continuous structure with natural enamel crystals, without clear boundaries, exhibiting consistent crystal orientation and similar dimensions, and its mechanical properties are also similar to those of natural enamel.

[0027] Preferably, the micro-components of the enamel-like ordered hydroxyapatite are nano-hydroxyapatite rods with a diameter of 25-350 nm, which are arranged in an orderly manner along the c-axis of the natural enamel pillars and have the same orientation as the natural enamel pillar structure.

[0028] This invention also provides the application of the aforementioned enamel-like ordered hydroxyapatite in dental restoration. The enamel-like ordered hydroxyapatite of this invention has the same crystal orientation as natural enamel and similar mechanical properties and dimensions, making it suitable for clinical caries restoration.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) This invention is the first to obtain a highly ordered calcium phosphate material on the surface of enamel by regulating the nucleation and growth process of crystals through organic macromolecules, and can control its growth thickness. The resulting enamel-like material has excellent material properties, providing a new idea for the preparation of other ordered mineral functional materials.

[0031] (2) The hydroxyapatite structure of the tooth enamel ordered structure of the present invention is a continuous structure with natural enamel crystals, without obvious boundaries, with consistent crystal orientation and similar size, and its mechanical properties are also similar to those of natural enamel. It can be applied to tooth restoration and provides a potential method for clinical caries restoration. Attached Figure Description

[0032] Figure 1 The images shown are SEM and AFM images of the enamel-like hydroxyapatite prepared in Example 1. Figure 1 a is a SEM image of hydroxyapatite, a tooth-like structure on the surface of tooth enamel; Figure 1 b is a SEM image of an individual glaze column; Figure 1 c is a SEM image of the cross section of hydroxyapatite with a tooth-like enamel structure; Figure 1 d is the AFM diagram of hydroxyapatite with a tooth-enamel-like structure; Figure 1 e is a SEM image of the cross-section of tooth enamel after cyclic remineralization; Figure 1 f is Figure 1 Enlarged view of the area within the yellow box in image e.

[0033] Figure 2 The image shows the performance test results of the enamel-like hydroxyapatite prepared in Example 1. Figure 2 a is a graph showing the elastic modulus and hardness of the sample surface; Figure 2 b represents the load-displacement curve; Figure 2 c represents the friction coefficient diagram. Figure 2 d is the XRD pattern.

[0034] Figure 3Here is a SEM image of the enamel-like hydroxyapatite prepared in Example 2, in which... Figure 3 Image a is a SEM image of hydroxyapatite, a tooth-like structure on the surface of tooth enamel. Figure 3 b is an SEM image of an individual glaze column.

[0035] Figure 4 This is a schematic diagram illustrating the principle of the formation of hydroxyapatite with an ordered structure mediated by organic macromolecules on the glaze surface according to the present invention. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0037] Example 1

[0038] Cut enamel slices (6mm × 6mm × 0.5mm) perpendicular to the crown using a cutting machine. After progressively polishing with 600, 1200, and 2400 grit sandpaper, ultrasonically clean with deionized water for 5 minutes, then etch with 37% phosphoric acid for 30 seconds, ultrasonically clean with deionized water for 5 minutes, and air-dry for later use. Prepare a mineralization solution containing 10 μg / mL C. 11 H 23 CO-KDDDDKDDDD(COOH), 137.5mM NaCl, 3.0mM KCl, 1.0mM K2HPO4, 1.5mM MgCl2, 2.5mM CaCl2, 0.5mM Na2SO4, 10.0mM HEPES, 15ppm NaF. Acid-etched tooth enamel was immersed in a mineralizing solution and mineralized at 37℃ for 3 days to obtain hydroxyapatite with a tooth-enamel-like structure, designated as the restorative group.

[0039] The mineralization solution was changed, and the obtained enamel-like hydroxyapatite was placed in a new mineralization solution. After mineralization at 37°C for 3 days, the mineralization solution was changed again, and the enamel-like hydroxyapatite was mineralized at 37°C for 3 days. This process was repeated to obtain enamel-like hydroxyapatite after 3 cycles of remineralization.

[0040] The organic macromolecule C used in this embodiment 11 H 23 The structural formula of CO-KDDDDKDDDD(COOH) is as follows:

[0041]

[0042] Example 2

[0043] Cut enamel slices (6mm × 6mm × 0.5mm) perpendicular to the crown using a cutting machine. After progressively polishing with 600, 1200, and 2400 grit sandpaper, ultrasonically clean with deionized water for 5 minutes, then etch with 37% phosphoric acid for 30 seconds, ultrasonically clean with deionized water for 5 minutes, and air-dry for later use. Prepare a mineralization solution containing 10 μg / mL C. 11 H 23 CO-WPATDKTKREEVD-(COOH)(where C 11 H 23 - is a straight-chain alkyl group, WPATDKTKREEVD is a polypeptide), 137.5 mM NaCl, 3.0 mM KCl, 1.0 mM K2HPO4, 1.5 mM MgCl2, 2.5 mM CaCl2, 0.5 mM Na2SO4, 10.0 mM HEPES, 15 ppm NaF. Acid-etched tooth enamel was immersed in a mineralizing solution and mineralized at a constant temperature of 37°C for 3 days to obtain hydroxyapatite with a tooth enamel-like structure.

[0044] Example 3

[0045] The enamel was cut into 6mm × 6mm × 0.5mm slices perpendicular to the crown using a cutting machine. After progressive polishing with 600, 1200, and 2400# sandpaper, it was ultrasonically cleaned with deionized water for 5 minutes, then etched with 37% phosphoric acid for 30 seconds, ultrasonically cleaned with deionized water for 5 minutes, and air-dried for later use. A mineralization solution was prepared containing 5 μg / mL chondroitin sulfate, 150 mM NaCl, 1.0 mM K₂HPO₄, 2 mM MgCl₂, 1.67 mM CaCl₂, 20.0 mM HEPES, and 15 ppm NaF. The etched enamel was immersed in the mineralization solution and mineralized at 37°C for 3 days to obtain hydroxyapatite with an enamel-like structure.

[0046] Comparative Example 1

[0047] Using a cutting machine, cut enamel slices of 6mm×6mm×0.5mm along the direction perpendicular to the crown. After gradually polishing with 600, 1200, and 2400# sandpaper, ultrasonically clean with deionized water for 5 minutes and dry in air for later use, the resulting natural enamel is designated as the natural group.

[0048] Comparative Example 2

[0049] The enamel was cut into 6mm×6mm×0.5mm sections perpendicular to the crown using a cutting machine. After being polished step by step with 600, 1200, and 2400# sandpaper, it was ultrasonically cleaned with deionized water for 5 minutes, then etched with 37% phosphoric acid for 30 seconds, ultrasonically cleaned with deionized water for 5 minutes, and then dried in air for later use. The resulting etched enamel was designated as the etched group.

[0050] Comparative Example 3

[0051] The difference between this comparative example and Example 1 is that the acid-etched enamel is immersed in an environment free of organic macromolecules (C). 11 H 23 The enamel material obtained in the mineralized solution of CO-KDDDDKDDDD(COOH)) was recorded as the control group.

[0052] The morphology and properties of the enamel materials prepared in the examples and comparative examples were observed and characterized.

[0053] Figure 1 SEM and AFM images of the enamel-like hydroxyapatite prepared in Example 1. Figure 1 a indicates that this embodiment constructs an ordered enamel-like hydroxyapatite layer on the enamel surface. (From...) Figure 1 As shown in b, the microscopic constituent units of ordered hydroxyapatite are hydroxyapatite rods with a diameter of 25–350 nm. The nano-hydroxyapatite rods are arranged in an orderly manner along the c-axis of the natural glaze pillars, forming hydroxyapatite minerals with an orientation similar to that of the natural glaze pillars. Figure 1 The cross-section shows that the newly formed mineralized hydroxyapatite is oriented in the same direction as the natural enamel rod. Figure 1 d is the AFM diagram of hydroxyapatite, demonstrating the thickness of the new remineralized layer, approximately 1.5 μm. Figure 1 e is a cross-sectional SEM image of tooth enamel after three rounds of remineralization. After three rounds of remineralization, the thickness of the mineral layer can reach 3.4 μm. Figure 1 f is Figure 1 The high-magnification SEM image of the area within the yellow box in image e shows the transition region from the primary layer to the remineralized layer.

[0054] Depend on Figure 2As shown in Figure a, natural enamel possesses excellent material properties, with a hardness (H) of 4.14 ± 0.64 GPA (mean ± standard deviation) and an elastic modulus (E) of 90.97 ± 12.12 GPa. However, the H and E values ​​of acid-etched enamel were significantly reduced, to 0.15 ± 0.01 GPA and 17.26 ± 2.06 GPA, respectively. The control group showed a slight increase in H (0.74 ± 0.14 GPa) and E (40.91 ± 2.43 GPa), but these values ​​remained significantly lower than those of natural enamel. The H and E values ​​of the restored enamel were completely restored, primarily due to the formation of a structure similar to that of natural enamel, with H and E values ​​of 4.12 ± 0.49 GPA and 88.52 ± 11.00 GPA, respectively, approaching those of natural enamel.

[0055] Depend on Figure 2 As can be seen from b, the load-displacement curve of the enamel-like hydroxyapatite prepared in Example 1 is basically consistent with that of natural enamel. Figure 2 c indicates that the hydroxyapatite with a tooth-enamel-like structure prepared in Example 1 has similar frictional properties to natural enamel. Figure 2 XRD spectroscopy of the repaired enamel showed that the mineral phase of the repair layer consisted of hydroxyapatite crystals, with peak values ​​and intensities similar to those of natural enamel.

[0056] Figure 3 This is a SEM image of the enamel-like hydroxyapatite prepared in Example 2. Figure 3 This indicates that the use of organic macromolecules (C 11 H 23 CO-WPATDKTKREEVD(COOH)) also constructed an ordered enamel-like hydroxyapatite layer on the enamel surface.

[0057] Figure 4 This diagram illustrates the principle of the formation of ordered hydroxyapatite on the enamel surface mediated by organic macromolecules according to the present invention. The present invention successfully reconstructs the complex structure of tooth enamel and fully restores its biological function through a biomimetic strategy. In this strategy, organic macromolecules mediate the assembly and fusion of amorphous calcium phosphate particles, forming a biomimetic mineralization front with a continuous structure, ensuring the directional growth of enamel crystals.

Claims

1. A method for preparing hydroxyapatite with an ordered enamel-like structure, characterized in that, Includes the following steps: (1) A mineralizing solution is obtained by mixing an orthophosphate solution, a calcium salt solution, a fluoride salt solution, a magnesium salt solution, and an organic macromolecule solution, wherein the organic macromolecule is an amphiphilic polypeptide or polysaccharide rich in carboxyl and / or sulfonyl functional groups, and the amphiphilic polypeptide is C 11 H 23 CO-KDDDDKDDDD(COOH) or C 11 H 23 CO-WPATDKTKREEV-D(COOH), wherein the polysaccharide is chondroitin sulfate; The molar ratio of orthophosphate to calcium ions ranges from 1:1 to 1.5, the molar ratio of fluoride ions to calcium ions ranges from 1:3 to 10, the molar ratio of magnesium ions to calcium ions ranges from 1 to 5:1, and the mass ratio of the organic macromolecule to calcium ions is 1 to 20:1 to 42. (2) Immerse the acid-etched enamel in the mineralization solution prepared in step (1) and incubate at a constant temperature to obtain the enamel-like ordered structure hydroxyapatite. The constant temperature incubation is 25~40℃, and the time is 1~9 days.

2. The preparation method according to claim 1, characterized in that, The concentration of the orthophosphate solution is 0.5~6.0 mM; the concentration of calcium ions in the calcium salt solution is 0.5~6 mM; the concentration of fluoride ions in the fluoride salt solution is 0.05~1.00 mM; the concentration of magnesium ions in the magnesium salt solution is 0.1~6 mM; and the concentration of the organic macromolecule solution is 0.005~10 mg / mL.

3. The preparation method according to claim 1, characterized in that, Before step (2), the process also includes adding auxiliary components to the mineralization solution prepared in step (1), wherein the auxiliary components include sodium chloride solution, potassium chloride solution, sodium sulfate solution and HEPES.

4. The enamel-like ordered hydroxyapatite prepared by the preparation method according to any one of claims 1-3.

5. The enamel-like ordered hydroxyapatite according to claim 4, characterized in that, The micro-components of the tooth enamel-like ordered hydroxyapatite are nano-hydroxyapatite rods with a diameter of 25~350 nm. The nano-hydroxyapatite rods are arranged in an orderly manner along the c-axis of the natural enamel pillars, and their orientation is consistent with the structure of the natural enamel pillars.

6. The application of the enamel-like ordered hydroxyapatite according to claim 4 or 5 in the preparation of dental restorative materials.

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