Preparation method and application of carboxylated polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite

Abstract

The present invention provides a preparation method and application of a polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite. The preparation method comprises the following steps: (1) mixing a carboxylated polyamide-amine aqueous solution with a magnesium salt aqueous solution, adding a calcium salt, and then adding anhydrous ethanol to obtain an ethanol mixture containing magnesium, carboxylated polyamide-amine, and amorphous calcium; (2) dissolving methacrylate decyl phosphate in an ethanol solution, and adding a phosphoric acid solution or a phosphate solution to obtain a phosphorus solution; (3) mixing the ethanol mixture containing magnesium, carboxylated polyamide-amine, and amorphous calcium with the phosphorus solution to obtain a polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite. The nanocomposite prepared by the present invention can maintain a stable nanometer size in an ethanol solution for a long time and has good permeability. The present invention also provides the application of the above preparation method in inducing collagen fiber biomimetic mineralization and resin bonding.

Description

Technical Field

[0001] The invention relates to the technical field of remineralization of demineralized dentin collagen fibers at a resin bonding interface, in particular to a preparation method and application of a polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite. Background Art

[0002] Resin fillings and restorations in oral clinics primarily rely on bonding for retention. Dentin bonding involves acid etching to demineralize the dentin surface, forming a network of exposed collagen fibers. Resin monomers in the adhesive then replace the water in the network and cross-link with the collagen fibers to form a resin-collagen mixed layer, creating a strong bonding interface. However, during this bonding process, the resin monomers are insufficient to completely replace the water in the collagen fiber network, exposing some collagen fibers at the bonding interface to the aqueous environment. These fibers are easily degraded by various endogenous enzymes, leading to damage to the bonding interface. Promoting the biomimetic remineralization of these exposed, demineralized collagen fibers, protecting them both internally and externally with minerals and restoring their mechanical properties, can enhance the stability of the resin-dentin bonding interface. This is a method that has emerged in recent years and can fundamentally avoid the aforementioned defects of dentin bonding. Studies have found that carboxylated polyamide-amine has the function of stabilizing amorphous phase mineralization precursor amorphous calcium phosphate. For example, the paper "Study on the Remineralization of Collagen Fibers Induced by Carboxylated Polyamide-amine / Amorphous Calcium Phosphate Magnesium" (Yan Kaiqi, Huang Xiaoman, Huang Jingxian, Xie Fangfang, Journal of Guangxi Medical University, 2023, 40(2): 221-227.) disclosed that: In the early stage, a nano-sized carboxylated polyamide-amine / amorphous calcium phosphate nanocomposite containing magnesium was synthesized, and it was confirmed that it has the ability to induce the remineralization of human dentin collagen fibers. However, the complex is synthesized in an aqueous solution and easily agglomerates over time, losing its nano-size. It can only be stored in a freeze-dried powder state and needs to be re-mixed with water when used, which is inconvenient to use and has a large loss. In addition, the complex is difficult to dissolve in the ethanol or acetone solvents used in resin adhesives, which limits its further application in clinical replacement of existing resin adhesives.

[0003] Therefore, further in-depth research on the magnesium-containing nano-sized carboxylated polyamide-amine / amorphous calcium phosphate nanocomposite so that it can be better preserved for a long time and soluble in the solvent used in the resin adhesive is of great significance for its better clinical replacement of existing resin adhesives. Summary of the Invention

[0004] The present invention provides a preparation method and application of a polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite, which can maintain a stable nanometer size in an ethanol solution for a long time, has good permeability, and can induce the remineralization of demineralized dentin collagen fibers.

[0005] To achieve the above object, the technical solution of the present invention is:

[0006] A method for preparing a carboxylated polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite comprises the following steps:

[0007] (1) mixing a carboxylated polyamide-amine aqueous solution and a soluble magnesium salt aqueous solution, allowing the mixture to stand for 4 to 10 hours, adding a soluble calcium salt, continuing to mix, allowing the mixture to stand for 4 to 10 hours, and then adding anhydrous ethanol, or anhydrous ethanol and distilled water, to obtain an ethanol mixture containing magnesium, carboxylated polyamide-amine, and amorphous calcium;

[0008] (2) dissolving decyl methacrylate phosphate in an ethanol solution, adding a phosphoric acid solution or a soluble phosphate solution, and mixing to obtain a phosphorus solution;

[0009] (3) The ethanol mixture containing magnesium, carboxylated polyamide-amine and amorphous calcium is evenly mixed with the phosphorus solution, with the molar ratio of calcium ion to phosphate ion being 2 to 1:1, and stirred or shaken for 20 to 60 minutes to obtain a polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite.

[0010] Preferably, in the ethanol mixture containing magnesium, carboxylated polyamide-amine and amorphous calcium, the concentration of carboxylated polyamide-amine is 0.10-1.50 mmol / L, Mg 2+ The concentration of Ca is 0.05~0.50mmol / L, 2+ The concentration is 1~20mmol / L.

[0011] Preferably, in the ethanol mixture containing magnesium, carboxylated polyamide-amine and amorphous calcium, carboxylated polyamide-amine, Mg 2+ , Ca 2+ The molar ratio is 0.5-5:1:20-50.

[0012] Preferably, the volume percentage of ethanol in the ethanol mixture containing magnesium, carboxylated polyamide-amine and amorphous calcium and / or in the phosphorus solution is 40-60%.

[0013] Preferably, in the phosphorus solution, PO4 3- The concentration of is 0.5~12.0mmol / L, and the concentration of decyl methacrylate phosphate is 0.5~12.0mmol / L.

[0014] Preferably, in the phosphorus solution, decyl methacrylate phosphate and PO4 3- The molar ratio is 1~10:1~10.

[0015] Preferably, in step (3), the reaction temperature is 8-15°C.

[0016] Preferably, step (1) is specifically as follows: 0.8-12 mmol / L of a carboxylated polyamide-amine aqueous solution and 10-100 mmol / L of a magnesium chloride aqueous solution are vortex-mixed at a volume ratio of 15-35:1, and the mixture is allowed to stand for 4-10 hours. Then, 30-50 times the volume of the magnesium chloride aqueous solution and 10-100 mmol / L of a calcium chloride aqueous solution are added, and the mixture is vortex-mixed. The mixture is allowed to stand for 4-10 hours. Then, 30-50 times the volume of the magnesium chloride aqueous solution and 80-120 times the volume of the magnesium chloride aqueous solution are added, and the mixture is vortex-mixed to obtain an ethanol mixture containing magnesium, carboxylated polyamide-amine and amorphous calcium.

[0017] Preferably, step (2) is specifically as follows: preparing 0.5-13.0 mmol / L of 40-60 vol% ethanol solution of decyl methacrylate phosphate, adding concentrated phosphoric acid to make the concentration of phosphoric acid 0.5-12.0 mmol / L, and vortex mixing to obtain the phosphorus solution.

[0018] The above-mentioned decyl methacrylate phosphate, also known as 10-(2-methacryloyloxy) monodecyl phosphate, has the chemical formula:

[0019]

[0020] The present invention further provides the application of the preparation method of the polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite in inducing collagen fiber biomimetic mineralization and resin bonding.

[0021] The present invention utilizes carboxylated polyamidoamine and magnesium ions to stabilize the mineralization precursor, amorphous calcium phosphate, inhibiting its aggregation and mineralization to form hydroxyapatite before entering collagen fibers, thereby facilitating its induction of remineralization within demineralized dentin collagen fibers. Simultaneously, decyl methacrylate phosphate is introduced to increase the lipid solubility of the composite by utilizing the structural properties of its lipophilic end. Experimental studies have confirmed that the nanocomposite incorporating decyl methacrylate phosphate can maintain a stable nanometer size in ethanol solutions for a long period of time, exhibits good permeability, and is capable of inducing remineralization of demineralized dentin collagen fibers. Application experiments in dentin bonding have also confirmed that the polyamidoamine / amorphous calcium phosphate-decyl methacrylate phosphate nanocomposite prepared by the present invention can be used as an additional primer, effectively improving bonding strength and exhibiting improved anti-aging properties.

[0022] The present invention improves the carboxylated polyamide-amine / amorphous amorphous calcium phosphate material, which not only promotes the biomimetic remineralization of exposed demineralized dentin collagen fibers, so that the dentin is protected both inside and outside by minerals and its mechanical properties are restored, but can also be used for clinical resin bonding of dentin, enhancing the bonding strength and anti-aging properties of the bonding and extending the life of the resin restoration. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A is the general view of PAMAM-COOH / ACMP (freshly prepared) in Example 1. Figure 1 B is a general view of PAMAM-COOH / ACMP-MDP (freshly prepared) of Example 1, Figure 1 C is the general view of PAMAM-COOH / ACMP of Example 1 (after storage for 60 days). Figure 1 D is the general appearance of PAMAM-COOH / ACMP-MDP of Example 1 (after 60 days of storage).

[0024] Figure 2 A is the Fourier transform infrared spectra of PAMAM-COOH / ACMP (freshly prepared) of Comparative Example 1 and PAMAM-COOH / ACMP-MDP (freshly prepared) of Example 1, Figure 2 B is the Fourier transform infrared spectra of PAMAM-COOH / ACMP of Comparative Example 1 (after 60 days of storage) and PAMAM-COOH / ACMP-MDP of Example 1 (after 60 days of storage).

[0025] Figure 3 A is the X-ray diffraction pattern of PAMAM-COOH / ACMP (freshly prepared) of Comparative Example 1 and PAMAM-COOH / ACMP-MDP (freshly prepared) of Example 1, Figure 3 B is the X-ray diffraction pattern of PAMAM-COOH / ACMP of Comparative Example 1 (after 60 days of storage) and PAMAM-COOH / ACMP-MDP of Example 1 (after 60 days of storage).

[0026] Figure 4 1 is a transmission electron micrograph of PAMAM-COOH / ACMP of Comparative Example 1 and PAMAM-COOH / ACMP-MDP of Example 1; Figure 4 A is a transmission electron micrograph of PAMAM-COOH / ACMP (freshly prepared) (scale bars 500 nm, 100 nm). Figure 4 B is the transmission electron micrograph of PAMAM-COOH / ACMP-MDP (freshly prepared) (scale bars 500 nm, 100 nm). Figure 4 C is the transmission electron microscopy image of PAMAM-COOH / ACMP (after 60 days of storage) (scale bars 500 nm, 100 nm), Figure 4 D is a transmission electron micrograph of PAMAM-COOH / ACMP-MDP (after 60 days of storage) (scale bars: 500 nm, 100 nm).

[0027] Figure 51 is a particle size analysis diagram of PAMAM-COOH / ACMP of Comparative Example 1 (freshly prepared and after 60 days of storage) and PAMAM-COOH / ACMP-MDP of Example 1 (freshly prepared and after 60 days of storage).

[0028] Figure 6 The STEM-EDX-mapping test results of PAMAM-COOH / ACMP of Comparative Example 1 and PAMAM-COOH / ACMP-MDP of Example 1 are shown; Figure 6 A is the energy spectrum analysis diagram of PAMAM-COOH / ACMP (freshly prepared), Figure 6 B is the energy spectrum analysis diagram of PAMAM-COOH / ACMP-MDP (freshly prepared), Figure 6 C is the energy spectrum analysis diagram of PAMAM-COOH / ACMP (after storage for 60 days), Figure 6 D is the energy spectrum analysis of PAMAM-COOH / ACMP-MDP (after 60 days of storage). From left to right, the figure shows high-angle annular dark field imaging, the overlay of individual element distributions, the calcium distribution, the phosphorus distribution, the magnesium distribution, and the nitrogen distribution.

[0029] Figure 7 These are the CLSM observation results of the remineralization of the longitudinal sections of dentin after mineralization induction in the blank control group, ACP group, MDP group, PAMAM-COOH / ACMP group, and PAMAM-COOH / ACMP-MDP group in experiment (1). The left column of figures is the low-power field of view (×400), and the right column of figures is the high-power field of view (×1000).

[0030] Figure 8 Figure 1 is the scanning electron microscopy observation result of the longitudinal section of the completely demineralized dentin surface in experiment (2); A1-A5 are the experimental results of the blank control group: Figures A2 and A3 are the white box areas in A1, and Figures A4 and A5 are the black box areas in A1. The magnification of Figures A2 and A4 is 25000, and the magnification of Figures A3 and A5 is 50000 ( Figure 8 B1-5, Figure 8 C1-5, Figure 8 D1-5 Figure 8 E1-5 are arranged in the same manner); B1-B5 are test result diagrams of the ACP group, C1-C5 are test result diagrams of the MDP group, D1-D5 are test result diagrams of the PAMAM-COOH / ACMP group (Comparative Example 1), and E1-E5 are test result diagrams of the PAMAM-COOH / ACMP-MDP group (Example 1).

[0031] Figure 9Figure 1 is the transmission electron microscopy observation result of completely demineralized dentin in experiment (2); Figures A1 and A2 are the test results of the blank control group: A1 is a low-power field of view (×3000), and A2 is an enlarged view of the white box area in Figure A1 ( Figure 9 B1-2, Figure 9 C1-2, Figure 9 D1-2, Figure 9 E1-2 are arranged in the same manner); B1-B2 are test result diagrams of the ACP group, C1-C2 are test result diagrams of the MDP group, D1-D2 are test result diagrams of the PAMAM-COOH / ACMP group (Comparative Example 1), and E1-E2 are test result diagrams of the PAMAM-COOH / ACMP-MDP group (Example 1).

[0032] Figure 10 This is a picture of collagen fibers mineralized by the PAMAM-COOH / ACMP-MDP group (×50000). DETAILED DESCRIPTION

[0033] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the embodiments described are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0034] Example 1

[0035] A method for preparing a carboxylated polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite comprises the following steps:

[0036] (1) 625 μL of 5 mmol / L carboxylated polyamide-amine aqueous solution and 25 μL of 50 mmol / L magnesium chloride aqueous solution were vortexed and allowed to stand for 8 h. Then 975 μL of 50 mmol / L calcium chloride aqueous solution were added and vortexed and allowed to stand for 8 h. Then 875 μL of distilled water and 2.5 mL of anhydrous ethanol were added and vortexed to obtain a solution containing 0.25 mmol / L magnesium. 2+ , 0.625mmol / L carboxylated polyamidoamine and 9.75mmol / L amorphous calcium in ethanol and stored at 4℃.

[0037] (2) Prepare 6mmol / L methacrylate decyl phosphate 50vol% ethanol solution, add 85% concentrated phosphoric acid, and make PO4 3- The concentration of 6mmol / L was vortexed to obtain phosphorus solution, which was stored at 4℃.

[0038] (3) The ethanol mixture containing magnesium, carboxylated polyamide-amine and amorphous calcium was vortex-mixed with the phosphorus solution in a volume ratio of 1:1, and the mixture was placed in a shaker at 10°C and continued to react for 30 minutes to obtain a polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite (PAMAM-COOH / ACMP-MDP).

[0039] Example 2

[0040] A method for preparing a carboxylated polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite comprises the following steps:

[0041] (1) 625 μL of 6 mmol / L carboxylated polyamide-amine aqueous solution and 25 μL of 52 mmol / L magnesium chloride aqueous solution were vortexed and allowed to stand for 10 h. Then 975 μL of 52 mmol / L calcium chloride aqueous solution were added and vortexed and allowed to stand for 8 h. Then 875 μL of distilled water and 2.5 ml of anhydrous ethanol were added and vortexed to obtain a solution containing 0.26 mmol / L magnesium chloride. 2+ , 0.75mmol / L carboxylated polyamidoamine and 10.14mmol / L amorphous calcium in ethanol and stored at 4℃.

[0042] (2) Prepare 6mmol / L methacrylate decyl phosphate 55vol% ethanol solution, add 85% concentrated phosphoric acid, and make PO4 3- The concentration of 6mmol / L was vortexed to obtain phosphorus solution, which was stored at 4℃.

[0043] (3) The ethanol mixture containing magnesium, carboxylated polyamide-amine and amorphous calcium was vortex-mixed with the phosphorus solution in a volume ratio of 1:1, and the mixture was placed in a shaker at 10°C and continued to react for 40 minutes to obtain a polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite.

[0044] Example 3

[0045] A method for preparing a carboxylated polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite comprises the following steps:

[0046] (1) 625 μL of 5 mmol / L carboxylated polyamide-amine aqueous solution and 25 μL of 50 mmol / L magnesium chloride aqueous solution were vortexed and allowed to stand for 8 h. Then 975 μL of 50 mmol / L calcium chloride aqueous solution were added and vortexed and allowed to stand for 8 h. Then 875 μL of distilled water and 2.5 mL of anhydrous ethanol were added and vortexed to obtain a solution containing 0.25 mmol / L magnesium. 2+, 0.625mmol / L carboxylated polyamidoamine and 9.75mmol / L amorphous calcium in ethanol and stored at 4℃.

[0047] (2) Prepare 5mmol / L 50vol% ethanol solution of decyl methacrylate phosphate, add 85% concentrated phosphoric acid, and make PO4 3- The concentration was 5 mmol / L, vortex mixed to obtain phosphorus solution, and stored at 4 °C;

[0048] (3) The ethanol mixture containing magnesium, carboxylated polyamide-amine and amorphous calcium was vortex-mixed with the phosphorus solution in a volume ratio of 1:1, and the mixture was placed in a shaker at 10°C and continued to react for 20 minutes to obtain a polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite.

[0049] Comparative Example 1

[0050] (1) 625 μL of 5 mmol / L carboxylated polyamide-amine aqueous solution and 25 μL of 50 mmol / L magnesium chloride aqueous solution were vortexed and allowed to stand for 8 h. Then 975 μL of 50 mmol / L calcium chloride aqueous solution were added and vortexed and allowed to stand for 8 h. Then 875 μL of distilled water and 2.5 mL of anhydrous ethanol were added and vortexed to obtain a solution containing 0.25 mmol / L magnesium. 2+ , 0.625mmol / L carboxylated polyamidoamine and 9.75mmol / L amorphous calcium in ethanol and stored at 4℃.

[0051] (2) Add 85% concentrated phosphoric acid to a 50 vol% ethanol solution to make the concentration of phosphoric acid 6 mmol / L, vortex mix to obtain phosphorus solution, and store at 4°C.

[0052] (3) The ethanol mixture containing magnesium, carboxylated polyamidoamine, and amorphous calcium phosphate and the phosphorus solution in the above step were vortex-mixed at a volume ratio of 1:1, placed in a shaker at 10°C to continue the reaction, removed after 30 minutes, and stored at 4°C. Thus, a magnesium-containing carboxylated polyamidoamine / amorphous calcium phosphate (PAMAM-COOH / ACMP) ethanol solution was obtained.

[0053] Performance test

[0054] 1. Stability test

[0055] The physical and chemical properties of freshly prepared PAMAM-COOH / ACMP-MDP (Example 1) and PAMAM-COOH / ACMP (Comparative Example 1) after 60 days of storage were tested. Figure 1 A- Figure 1 As shown in D, both can maintain their original colorless clear liquid state. Analyze their chemical bond information, such as Figure 2 A and Figure 2 As shown in B, freshly prepared PAMAM-COOH / ACMP-MDP and PAMAM-COOH / ACMP can both be detected at a wavelength of 1080 cm -1 PO4 observed nearby 3- Characteristic peak, 1560cm -1 and 1651cm -1 The characteristic peaks of amide bond (-NH-CO-) and carbonyl group (-C=O) were observed near the PAMAM-COOH / ACMP-MDP at 1730 cm -1 The characteristic peak of carbonyl group (-C=O) of MDP was observed near the wavelength. After 60 days of storage, the characteristic peaks of PAMAM-COOH / ACMP-MDP and PAMAM-COOH / ACMP were still observed near the corresponding wavelength, indicating that both can maintain good chemical stability. Figure 3 A and Figure 3 As shown in Figure B, whether it is freshly prepared PAMAM-COOH / ACMP-MDP, PAMAM-COOH / ACMP, or PAMAM-COOH / ACMP-MDP, PAMAM-COOH / ACMP after storage for 60 days, an independent large peak unique to amorphous phase materials can be seen at 2θ = 24°, indicating that both can maintain the amorphous phase state and have good stability.

[0056] Transmission electron microscopy was used to observe the microscopic morphology and particle size changes of the two. Figure 4 and Figure 5 As shown, Figure 4 In A, the particles of PAMAM-COOH / ACMP are quasi-spherical and well dispersed, but still have a slight tendency to aggregate and are not uniform in size. Under a high-power microscope, they show a quasi-spherical particle structure with an average particle size of 81.97±19.27 nm. Figure 4 In B, it can be seen that the particles of PAMAM-COOH / ACMP-MDP have better dispersion and more consistent particle size. Under high magnification, they appear as round particles with an average particle size of 49.69±6.47nm. Figure 4 C. Figure 4 D shows that PAMAM-COOH / ACMP and PAMAM-COOH / ACMP-MDP maintain similar round particle structures after 60 days of storage, with high particle dispersion and no agglomeration. No diffraction spots or diffraction rings were found in the SEAD test, indicating that they are all amorphous materials ( Figure 4 A, B, C, D upper right corner). Figure 4 and Figure 5The results show that the particles of PAMAM-COOH / ACMP-MDP have better dispersion and more consistent particle size. Under high magnification, they appear as round-shaped particles with an average particle size of 49.69±6.47nm, which is smaller than the average particle size of PAMAM-COOH / ACMP of 81.97±19.27nm. Moreover, the particles can maintain their original nanoparticle size after 60 days of storage, indicating good stability.

[0057] In addition, we also conducted STEM-EDX-mapping tests, and the results were as follows: Figure 6 As shown, Figure 6 A and Figure 6 Figure B shows that calcium and phosphorus are enriched in the particles of the PAMAM-COOH / ACMP and PAMAM-COOH / ACMP-MDP complexes, confirming that these particles are calcium and phosphorus clusters. A small amount of magnesium and nitrogen enrichment can also be seen in the particles. Figure 6 C and Figure 6 D shows that the composite particles after 60 days of storage are basically consistent with the results of fresh configuration. These results indicate that the new mineralization system has good dispersion and can maintain nanometer size, and has good stability.

[0058] 2. Induced mineralization simulation test

[0059] The experimental groups were set as blank control group (50 vol% ethanol solution), ACP group (amorphous calcium phosphate powder was added to 50 vol% ethanol at a concentration of 3 mg / mL), MDP group (decyl methacrylate was added to 50% anhydrous ethanol at a concentration of 3 mmol / L), PAMAM-COOH / ACMP group (synthesized according to the method of Comparative Example 1), and PAMAM-COOH / ACMP-MDP group (synthesized according to the method of Example 1).

[0060] Experiment (1): The remineralization detection method is to use calcein fluorescence labeling technology to evaluate the remineralization effect of the surface of partially demineralized human dentin slices after mineralization induction. Taking advantage of the characteristic that calcein can bind to calcium ions, the newly formed mineralized products can show a special yellow-green fluorescence under a laser scanning confocal fluorescence microscope, thereby distinguishing the newly formed and the original minerals. According to the grouping, each group has 5 tooth slices, and each tooth is immersed in 0.5 ml of the fluorescent labeling solution of each group. After incubation in a constant temperature box at 37°C, 100% humidity, and light-proof conditions for 7 days, the dentin slices are taken out, rinsed with deionized water for 30 seconds, and then stored in deionized water for testing. The longitudinal section of the upper surface of the dentin slice is observed using a laser scanning confocal fluorescence microscope. Five high-power fields (×1000) of the dentin longitudinal section are randomly selected from the observation interface of each sample group. In each field of view, 8 dentinal tubules are randomly selected to measure the penetration depth of the fluorescent label. Each tubule is measured 3 times and the average value is taken. The statistical results of the penetration depth of the dentinal tubules in each group are expressed as x±S.

[0061] Experimental results Figure 7 As shown in the figure, except for the blank control group and the MDP group, new mineral deposition can be seen on the surface of the other groups. Fluorescently labeled calcium ions can be seen penetrating deep into the dentinal tubules in both the PAMAM-COOH / ACMP group and the PAMAM-COOH / ACMP-MDP group. Compared with the control groups, new mineral deposition can be seen on the surface of the dentin after mineralization induction with the PAMAM-COOH / ACMP-MDP ethanol solution, and fluorescently labeled calcium ions can also penetrate deep into the dentinal tubules, indicating that it can induce deep dentin remineralization. The depth of calcium ion penetration into the dentinal tubules in each group is shown in Table 1.

[0062] Table 1 Depth of calcium ion penetration into dentinal tubules in each group (x±S)

[0063] Group Depth (μm) ACP 17.56±4.17 PAMAM-COOH / ACMP 231.33±46.17 PAMAM-COOH / ACMP-MDP 229.29±63.46

[0064] Because no obvious mineral deposition was observed in the blank and MDP groups, they were not included in the statistical analysis. Pairwise comparisons showed that the average penetration depth of the PAMAM-COOH / ACMP and PAMAM-COOH / ACMP-MDP groups into the dentinal tubules was greater than that in the ACMP group (P < 0.001), indicating a statistically significant difference.

[0065] Experiment (2): Scanning electron microscopy and transmission electron microscopy were used to evaluate the remineralization effect of mineralization induction on completely demineralized human dentin slices. According to the grouping, 5 tooth slices were divided into each group. Each tooth was immersed in 0.5 ml of the solution of each group after filtering with a 0.22 μm needle filter. After incubation in a constant temperature box at 37 ° C and 100% humidity for 7 days, the dentin slices were removed, rinsed with deionized water for 30 seconds, and then stored in deionized water for testing. Scanning electron microscopy and transmission electron microscopy were performed respectively. Scanning electron microscopy: After the dentin was removed from the deionized water, it was gradient dehydrated, critical point dried, and gold sprayed. The surface morphology of human dentin collagen fibers was observed using a scanning electron microscope at an accelerating voltage of 5 kV. Transmission electron microscopy: The dentin slices were immersed in electron microscope fixative, fixed at room temperature in the dark for 2 hours, and then transferred to 4 ° C in the dark. A longitudinal section was cut from the middle to prepare ultrathin sections. The remineralization of human dentin collagen fibers was observed at an accelerating voltage of 80 kV.

[0066] Scanning electron microscopy results Figure 8As shown in Figure 2, the blank control group showed no mineralization of collagen fibers, with thin fibers and large interfibrillar spaces (arrows). In the ACP group, mineralization was observed in the collagen fibers at the dentinal tubule openings, limited to the surface layer of the collagen fiber network (arrows in Figures B2 and B3). The collagen fibers became thicker and the spaces between them became smaller. However, the collagen fibers in the deeper tubules showed no segmental thickening and no obvious mineralization (Figure B4). In the MDP group, a membranous adhesion layer appeared on the surface of the collagen fiber network (arrows), while the collagen fiber network beneath the membranous layer showed no mineralization. In the PAMAM-COOH / ACMP and PAMAM-COOH / ACMP-MDP groups, clear mineralization of collagen fibers was observed both on the surface and within the deeper dentinal tubules. The collagen fibers were thicker and mineralized into sheets than in the blank control group, with smaller interfibrillar spaces (arrows). The interfibrillar spaces in the PAMAM-COOH / ACMP-MDP group were smaller than those in the PAMAM-COOH / ACMP group. Compared with the control groups, after the dentin was mineralized with PAMAM-COOH / ACMP-MDP ethanol solution, obvious characteristics of collagen fiber mineralization could be seen on the surface and deep in the dentinal tubules. The fibers became segmentally thicker and mineralized into sheets, and the gaps between the fibers became smaller. Figure 9 As shown in the figure, no dark-stained mineral particles were observed in the collagen fibers of the blank group, ACP group, and MDP group. In the PAMAM-COOH / ACMP group and PAMAM-COOH / ACMP-MDP group, obvious mineralization characteristics of collagen fibers inside and outside the peritubular dentin were observed. Dark-stained mineral particles were densely arranged along the longitudinal direction of the fibers (arrows), indicating that after mineralization induction with PAMAM-COOH / ACMP-MDP ethanol solution, obvious mineralization characteristics of collagen fibers inside and outside the peritubular dentin were observed. Figure 10 Mineralization with the PAMAM-COOH / ACMP-MDP ethanol solution revealed that the characteristic periodic horizontal striations of collagen fibers disappeared, dark-stained mineral particles were densely arranged longitudinally within the fibers, and mineral deposits were visible outside the fibers. These results demonstrate that the magnesium-carboxylated polyamidoamine / amorphous calcium phosphate-decyl methacrylate phosphate nanocomposite has a strong ability to induce remineralization of demineralized dentin.

[0067] 3. Test on the effect of self-etched dentin bonding interface strength and durability

[0068] The effect of PAMAM-COOH / ACMP-MDP ethanol solution as an additional primer on the bonding interface strength and durability of self-etched dentin was evaluated.

[0069] The experimental groups were blank control group (resin bonding was performed on the ex vivo dentin slices according to clinical routine), ACP group (amorphous calcium phosphate powder was added to 50 vol% ethanol solution to prepare a 3 mg / mL ACP ethanol solution. Before bonding, a small amount of ACP was applied to the dentin bonding surface with a small cotton swab for 20 seconds, and then dried with an air gun. The resin was bonded according to the blank group procedure), and MDP group (MDP solid was added to 50 vol% ethanol solution to prepare a 3 mmol / L MDP ethanol solution. Before bonding, a small amount of ACP was applied to the dentin bonding surface with a small cotton swab for 20 seconds, and then dried with an air gun. The resin was bonded according to the blank group procedure). The blank group (PAMAM-COOH / ACMP group) was prepared by the method of comparative example 1 using a small cotton swab to apply a small amount of the ethanol solution to the dentin bonding surface for 20 seconds before bonding, followed by drying with an air gun and then applying the resin according to the steps of the blank group), and the PAMAM-COOH / ACMP-MDP group (PAMAM-COOH / ACMP-MDP ethanol solution was prepared by the method of example 1 using a small cotton swab to apply a small amount of the ethanol solution to the dentin bonding surface for 20 seconds before bonding, followed by drying with an air gun and then applying the resin according to the steps of the blank group).

[0070] After bonding, the dentin slices in the experimental groups were immersed in deionized water at 37°C for 24 hours. Immediate shear strength testing was then performed. Following this testing, the specimens were placed in a hot and cold cycler with the cold water bath set at 5°C and the hot water bath set at 55°C. Each hot and cold bath cycle lasted 30 seconds, for a total of 10,000 cycles. After the hot and cold cycles, the aged specimens were tested for post-aging shear strength. Shear strength testing was performed using a computer-controlled universal mechanical testing machine. The bonded specimens were secured with a dedicated fixture. The loading head was held against the bonding surface, with the load applied parallel to the bonding surface at a rate of 0.5 mm / min until the resin block fractured and fell off. The peak load required to separate the cylinder from the tooth surface was divided by the bonded surface area and recorded as microshear bond strength using the following formula: bond strength (P) = peak load (Fmax) / bonded area (S). Shear strength is expressed in MPa.

[0071] The experimental results, as shown in Table 2, show that the average shear strength of the PAMAM-COOH / ACMP-MDP group was 6.53±3.91 MPa (immediately after bonding) and 5.24±2.79 MPa (after aging), both significantly higher than those of the other control groups (P<0.05). This demonstrates that the PAMAM-COOH / ACMP-MDP ethanol solution, as an additional primer, can improve the immediate and post-aging bond strength of the resin adhesive, enhancing its anti-aging properties.

[0072] Table 2 Shear strength test results

[0073]

Claims

1. A method for preparing a carboxylated polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite, characterized in that The following steps are involved: (1) Mixing the carboxylated polyamide-amine aqueous solution and the soluble magnesium salt aqueous solution, letting it stand for 4 to 10 hours, adding the soluble calcium salt, continuing to mix, letting it stand for 4 to 10 hours, and then adding anhydrous ethanol, or anhydrous ethanol and distilled water, to obtain an ethanol mixture containing magnesium, carboxylated polyamide-amine and amorphous calcium; (2) Dissolve decyl methacrylate phosphate in ethanol solution, add phosphoric acid solution or soluble phosphate solution, mix well, and obtain phosphorus solution; (3) The ethanol mixture containing magnesium, carboxylated polyamide-amine and amorphous calcium is mixed evenly with the phosphorus solution, with the molar ratio of calcium ion to phosphate ion being 2~1:1, and stirred or shaken for 20~60 minutes to obtain a polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite.

2. The method for preparing the carboxylated polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite according to claim 1, wherein: In the ethanol mixture containing magnesium, carboxylated polyamide-amine and amorphous calcium, the concentration of carboxylated polyamide-amine is 0.10-1.50 mmol / L, Mg 2+ The concentration of Ca is 0.05~0.50 mmol / L, 2+ The concentration is 1~20 mmol / L.

3. The method for preparing the carboxylated polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite according to claim 1, wherein: In the ethanol mixture containing magnesium, carboxylated polyamide-amine and amorphous calcium, carboxylated polyamide-amine, Mg 2+ , Ca 2+ The molar ratio is 0.5~5:1:20~50.

4. The method for preparing the carboxylated polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite according to claim 1, wherein: The volume percentage of ethanol in the ethanol mixed solution containing magnesium, carboxylated polyamide-amine and amorphous calcium and / or in the phosphorus solution is 40-60%.

5. The method for preparing the carboxylated polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite according to claim 1, wherein: In the phosphorus solution, PO4 3- The concentration of decyl methacrylate is 0.5~12.0 mmol / L, and the concentration of decyl methacrylate is 0.5~12.0 mmol / L.

6. The method for preparing the carboxylated polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite according to claim 1, wherein: In the phosphorus solution, decyl methacrylate phosphate and PO4 3- The molar ratio is 1~10:1~10.

7. The method for preparing the carboxylated polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite according to claim 1, characterized in that: In step (3), the reaction temperature is 8-15°C.

8. The method for preparing the carboxylated polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite according to claim 1, wherein: Step (1) is specifically as follows: 0.8-12 mmol / L of a carboxylated polyamide-amine aqueous solution and 10-100 mmol / L of a magnesium chloride aqueous solution are vortex-mixed at a volume ratio of 15-35:1, and allowed to stand for 4-10 hours. Then, 30-50 times the volume of the magnesium chloride aqueous solution of 10-100 mmol / L of a calcium chloride aqueous solution are added, and vortex-mixed, and allowed to stand for 4-10 hours. Then, 30-50 times the volume of the magnesium chloride aqueous solution of distilled water and 80-120 times the volume of the magnesium chloride aqueous solution of anhydrous ethanol are added, and vortex-mixed to obtain an ethanol mixture containing magnesium, carboxylated polyamide-amine and amorphous calcium.

9. The method for preparing the carboxylated polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite according to claim 1, characterized in that: Step (2) is specifically as follows: preparing a 0.5-13.0 mmol / L 40-60 vol% ethanol solution of decyl methacrylate phosphate, adding concentrated phosphoric acid to adjust the concentration of phosphoric acid to 0.5-12.0 mmol / L, and vortex mixing to obtain the phosphorus solution.

10. The carboxylated polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite prepared by the method for preparing the carboxylated polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite according to any one of claims 1 to 9.

11. Use of the carboxylated polyamide-amine / amorphous calcium phosphate-methacrylate decyl phosphate nanocomposite according to claim 10 in inducing biomimetic mineralization of collagen fibers and resin bonding.

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