CN111714385A - Formula and application of composite micro-nano particles - Google Patents

Abstract

The invention provides a compound micro-nano particle formula which is applied to the fields of tooth restoration, toothpaste formulas and the like. The composite micro-nano particle material consists of various particles, including calcium phosphate nanospheres (nano-CaP), calcium phosphate microspheres (micro-CaP) and calcium fluoride microspheres (CaF)₂) And calcium hydroxide particles (Ca (OH)₂). Such a multiparticulate system can accommodate a variety of oral environments caused by acidic beverages. Under the moist tooth surface microenvironment, the composite micro-nano particle material can release calcium, phosphate, fluoride ions and hydroxide ions, thereby promoting the remineralization of teeth. Small exposed dentinThe tube will be sealed and, after remineralization, enamel caries can be repaired by epitaxial crystal growth. Microscopic structure analysis through Focused Ion Beam (FIB) cutting and Transmission Electron Microscope (TEM) examination shows that the treatment can cause seamless growth of the enamel, so that the composite micro-nano granular material provided by the invention can play a good role in mineralization and repair of the enamel.

Description

Formula and application of composite micro-nano particles

Technical Field

The invention relates to a toothpaste formula, in particular to a preparation method and an experimental method of composite micro-nano particle toothpaste.

Background

Dentinal sensitivity is a recognized public health problem worldwide with a large impact on the quality of life of patients. Defects in the enamel or recession of the gums result in exposure of the dentin, which in turn leads to tooth sensitivity. Treatment of dentinal sensitivity can be classified as invasive or non-invasive treatment. Invasive treatments include gum surgery, resin application, extraction or laser treatment. The first non-invasive treatment, which typically uses topical drugs and dentifrices containing desensitizing products (such as potassium nitrate), can block synapses between nerve cells and reduce nerve excitation. Twice daily, it takes at least two weeks to observe relief from dentinal hypersensitivity symptoms, and four to eight weeks to show significant relief. The second non-invasive treatment is occlusion of dentinal tubules. The occlusion of the tubules retards fluid movement within the tubules, slowing down the pain response.

Inorganic materials or particles have been widely used for tooth restoration and remineralization over the last decade. For example, spherical tricalcium phosphate (TCP) particles have been used for regeneration of bone defects, and have also been used as remineralizing agents in dentistry. Amorphous Calcium Phosphate (ACP) has attracted more attention in dental applications than TCP and is considered to be an excellent precursor for hydroxyapatite in dental remineralization processes. During use, supersaturated calcium ions and phosphate ions can be released from the ACP complex, which play an important role in the formation of apatite. In addition, by combining other remineralizing materials (e.g., CaF2 spheres), the remineralization performance of ACP will be enhanced. Fluoride is effective in preventing dental caries. The current source of fluorine in dental sensitivity treatment and care is mainly sodium fluoride, which is readily soluble. Because of the good solubility of sodium fluoride, part of sodium fluoride can enter human bodies during tooth brushing or brushing, and excessive fluorine is not beneficial to human bodies. In particular, children easily swallow toothpaste when brushing their teeth, which is more likely to cause fluorine build-up and even fluorosis. Calcium fluoride/hydroxyfluorapatite biphasic nanocrystals have been synthesized for dental applications. Two-phase powder in leachingSignificant bioactivity was shown within hours after entering the SBF solution. From novel CaF incorporated in dental resins₂Sustained fluoride release was detected in the nanoparticles. In addition to this, antibacterial properties are a key factor in dental applications. Experiments have determined the antibacterial and physical properties of chlorhexidine-containing calcium phosphate and calcium fluoride nanocomposites. Calcium hydroxide has a general antibacterial effect against dental pulp pathogens, the mechanism of which is based on hydroxyl groups, thus facilitating its use in endodontics and dental traumatology.

As a non-invasive treatment, neither desensitization nor remineralization can cure tooth sensitivity. The present study is intended to provide a method for radically treating dental hypersensitivity: complete restoration of enamel. The project aims to develop the composite micro-nano particle toothpaste, which can efficiently promote tooth remineralization and realize enamel epitaxial growth, and achieves the purpose of perfectly repairing enamel.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the composite micro-nano particle toothpaste which has the beneficial effects of efficiently promoting tooth remineralization and realizing enamel epitaxial growth and achieving the purpose of repairing tooth enamel.

In order to achieve the purpose, the invention provides the following technical scheme: a formula of composite micro-nano particles is characterized in that: comprises composite micro-nano particles; the particles are calcium phosphate nanospheres, calcium phosphate microspheres, calcium fluoride microspheres and calcium hydroxide particles.

The invention is further configured to: a toothpaste using a composite micro-nano particle formula is characterized in that: also comprises an abrasive, a humectant, a surfactant, a thickening agent, a sweetening agent, a preservative, a pigment, an essence, arginine and glycerol.

The invention is further configured to: a particle synthesis method of a composite micro-nano particle formula is characterized by comprising the following steps: comprises the following steps;

1: separately prepared contains C1a²⁺，F^-，PO₄ ^3-And OH^-An aqueous solution of (a);

2: adding CaC in the range of 0.1-20mMl₂Mixing the aqueous solution with the aqueous solution in the step 1 to form a transparent solution with the initial pH of 6.0-8.0 at 25 ℃;

3: storing the transparent solution obtained in the step 3 in a closed glass bottle at 100 ℃ for 12 hours, and precipitating;

4: the precipitated precipitate was separated by filter paper (400nm, Track-Etch membrane), washed twice with ethanol and dried at 60 ℃.

The invention is further configured to: an experimental method of a compound micro-nano particle formula is characterized in that:

1: cutting the tooth into dentin and enamel pieces using a steel blade, the thickness being about 1 mm in parallel from the crown of a human molar;

2: grinding the slices with 200 to 1200 grit sandpaper and washing with distilled water;

3: subsequently, the slices were phosphorylated for 30 seconds using 37% phosphorous acid and washed with distilled water to eliminate acidic residues after acidic etching;

4: coating each tooth slice with the prepared composite micro-nano particle toothpaste for 3 minutes, and then storing in simulated saliva solution at 37 ℃ for 3 days;

5: the same procedure as in steps 1-4 above was repeated every three days. One group of dental sections was treated for 3 days and the other group was treated for 6 days.

Drawings

FIG. 1 shows synthesized calcium phosphate nanospheres (nano-CaP) (A), calcium phosphate microspheres (micro-CaP) (B), calcium fluoride microspheres (CaF)₂(C) And calcium hydroxide particles Ca (OH)₂(D) SEM image of (a);

FIG. 2 shows the ion release conditions of the composite micro-nano particles, and the release concentrations of Ca, F and P elements at different time points;

fig. 3 is the morphology of the dentin surface after use of the prepared toothpaste. (A) Reference, (B) particles in the composite toothpaste (C) dentin surface after 3 days of single use of the composite toothpaste (D) high resolution C (e) dentin surface after 6 days of double use of the composite toothpaste (F) dentin surface after 6 days of double use of the composite toothpaste;

FIG. 4 is an AFM 3D image of the dentin surface (A) reference, (B) dentin surface after 3 days of single use of the compounded toothpaste;

figure 5 is a morphology of enamel surface (a) reference, (B) high resolution a, (C) high resolution of surface E after 3 days (D) high resolution C (E) compound toothpaste applied for 6 days (F);

FIG. 6 is transmission electron TEM (left) and FFT (right) diffraction patterns of repaired enamel;

FIG. 7 is a graph relating nano-CaP (black bar), micro-CaP (red bar), CaF₂(blue column), Ca (OH)₂(magenta column) activity of cells after co-culture of particles and their complex toothpaste (green column) with cells for 48 hours;

FIG. 8 is a graph of nano-CaP (black bars), micro-CaP (red bars), CaF₂(blue column), Ca (OH)₂(magenta columns) particles and their complex toothpaste (green columns) antibacterial activity against Streptococcus mutans;

FIG. 9 is a schematic illustration of the mineralization of dentin after use of a compound toothpaste;

FIG. 10 is an XRD of calcium phosphate nanospheres (nano-CaP), calcium phosphate microspheres (micro-CaP), calcium fluoride microspheres (CaF2) and calcium hydroxide particles (Ca (OH) 2);

FIG. 11 is a comparison of calcium ion release from amorphous calcium phosphate (nano-CaP) and crystalline calcium phosphate (micro-CaP) in buffer solution at different pH values: (A) nano-Cap at pH 4 (B) micro-Cap at pH 4 (C) nano-Cap at pH7.4 (D) micro-Cap at pH 7.4;

FIG. 12 is an infrared spectrum of the dentin surface after treatment with the compound toothpaste for 0, 3 and 6 days, respectively;

FIG. 13 is a TEM image of an FIB-SEM cut enamel sample 6 days after use of the compound toothpaste;

FIG. 14 is a photomicrograph of viable and dead bacteria in control group (A), nano-CaP (B), micro-CaP (C), CaF2(D), Ca (OH)2(E) and their complex toothpaste (F) at 72 h.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings.

The embodiment discloses a formula of composite micro-nano particles, which can be used for tooth restoration and treatment, and can be used for oral care and restoration products such as toothpaste, bleaching cream and applying ointment. When the toothpaste is used as a formula of the toothpaste, the toothpaste comprises composite micro-nano particles; the composite micro-nano particles are calcium phosphate nanospheres, calcium phosphate microspheres, calcium fluoride microspheres and calcium hydroxide particles; the toothpaste also contains abrasive, humectant, surfactant, thickener, sweetener, antiseptic, pigment, essence, composite micro/nano particles, arginine and glycerol.

The friction agent is one or any combination of calcium carbonate, calcium hydrophosphate, precipitated silica, aluminum hydroxide, light calcium carbonate and anhydrous calcium hydrophosphate; the humectant is glycerin and water; the surfactant is sodium lauryl sulfate; the thickening agent is antler pectin and xanthan gum; the sweetener is saccharin sodium; the preservative is potassium sorbate.

A particle synthesis method of a composite micro-nano particle formula comprises the following steps;

1: separately prepared contains Ca²⁺，F^-，PO₄ ^3-And OH^-An aqueous solution of (a);

2: mixing an aqueous solution of CaCl2 in the range of 0.1-20mM with the aqueous solution of step 1 to form a clear solution with an initial pH of 6.0-8.0 at 25 ℃;

3: storing the transparent solution obtained in the step 3 in a closed glass bottle at 100 ℃ for 12 hours, and precipitating;

4: the precipitated precipitate was separated by filter paper (400nm, Track-Etch membrane), washed twice with ethanol and dried at 60 ℃.

An experimental method of a compound micro-nano particle formula comprises the following steps:

1: cutting the tooth into dentin and enamel pieces using a steel blade, the thickness being about 1 mm in parallel from the crown of a human molar;

2: grinding the slices with 200 to 1200 grit sandpaper and washing with distilled water;

3: subsequently, the slices were phosphorylated for 30 seconds using 37% phosphorous acid and washed with distilled water to eliminate acidic residues after acidic etching;

4: coating each tooth slice with the prepared composite micro-nano particle toothpaste for 3 minutes, and then storing in simulated saliva solution at 37 ℃ for 3 days;

5: the same procedure as in steps 1-4 above was repeated every three days. One group of dental sections was treated for 3 days and the other group was treated for 6 days.

The specific research process comprises the following steps of;

material

Sodium fluoride, potassium sulfate, sodium hydroxide and calcium chloride used in this study were purchased from Sigma-Aldrich. All chemicals were used as analytically pure reagents and were used without further purification. Bacterial live/dead kits and cell count kit 8(CCK-8) were obtained from Beyotime Biotech (Shanghai, China). All dentin samples were collected from the patients following the procedures routinely used in dental hospitals. The teeth were sterilized and kept dry until the experiment.

Synthesis of composite micro-nano particles

Separately prepared containing different ions (Ca)²⁺，F^-，PO₄ ^3-And OH^-) An aqueous solution of (a). Adding CaCl in the range of 0.1-20mM₂The aqueous solutions were rapidly mixed with different anionic solutions to form clear solutions with an initial pH of 6.0-8.0, respectively, at room temperature. The obtained clear solution was stored in a closed glass bottle at 100 ℃ for 12 hours. The precipitated precipitate was then separated by filter paper (400nm, Track-Etch membrane, Sigma), washed twice with ethanol and dried at 60 ℃ for further use. The obtained particles were characterized by field emission scanning electron microscopy (SEM, LEO 1550, germany), transmission electron microscopy (TEM, Tecnai, usa) and X-ray diffraction (XRD, D8 Advanced, Bruker, usa).

Ion release from particles

A total of 10mg of composite micro-nano particles were mixed with 100ml of buffer (20mM Hepes, 130mM KCl, 1.5mM CaCl2 (with or without 3.5mM sodium phosphate, pH 7.05)) and inserted into the solution with stirring at 200rpm with the fluoride sensitive electrode. The time-dependent ions released from the particle mixture were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES, Kleve, germany).

Preparation of composite micro-nano particle toothpaste

Abrasives (calcium carbonate, calcium hydrogen phosphate), humectants (glycerin and water), surfactants (sodium lauryl sulfate), thickeners (antler pectin and xanthan gum), sweeteners (saccharin sodium), preservatives (potassium sorbate), and pigments and essences as basic raw materials. The composite micro-nano particles are calcium phosphate nanospheres (nano-CaP), calcium phosphate microspheres (micro-CaP) and calcium fluoride microspheres (CaF)₂) And calcium hydroxide particles (Ca (OH)₂) And arginine thereof. Mixing the basic raw material and 2-80 wt% of the composite micro-nano particles, and stirring to a uniform state to obtain the toothpaste.

Dentinal tubule obstruction and tooth mineralization the teeth were cut into dentin and enamel pieces using steel blades. The thickness is about 1 mm from the crown of the human molar in a parallel manner. Then, the slices were sanded with 200 to 1200 grit sandpaper and washed with distilled water. Subsequently, the sections were phosphorylated for 30 seconds using 37% phosphorous acid. The sections were washed with distilled water to eliminate acidic residues after acidic etching. Each tooth slice was treated by hand with the prepared smart toothpaste for 3 minutes and then stored in a simulated saliva solution at 37 ℃ for 3 days. The same procedure was repeated every three days. One group of pf dental sections was treated for only 3 days, while the other group was treated for only 6 days.

Antibacterial Properties Streptococcus mutans was cultured in moist Brain Heart Infusion (BHI) broth overnight at 37 ℃ for 24 hours, and then diluted 100-fold with fresh BHI. The diluted solution (2 mL per sample) was co-incubated with 10mg of material per group: (a) nano CaP, (b) CaF₂(c) micro-CaP, (d) Ca (OH)₂And (e) a mixture. After incubation of the samples at 37 ℃ for 72 hours, bacterial viability was determined with a confocal laser scanning microscope (Leica, DMi8, germany).

Cytotoxicity of particles for CCK-8 assay, human dental pulp cells were seeded at a density of 104 cells per well in 96-well plates and cultured in medium for 24 hours. The medium was made up of 89% Dulbecco Modified Eagle Medium (DMEM), 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin. The sterilized smart material powder was added to the wells at a concentration of 0 to 100. mu.g/mL and co-cultured with the cells for 36 hours. Wells without sample were used as control. Cell viability after co-culture was assessed using the Cell Counting Kit-8 according to the manufacturer's instructions. Each sample was run in duplicate and the absorbance of the reaction solution was measured at 450 nm.

Results

Characterization of the particles

Nano CaP, micron CaP, CaF₂And Ca (OH)₂The particles were obtained by a simple and easy co-precipitation method and their morphology was analyzed by SEM image as shown in fig. 1. SEM image shows that the prepared nano CaP, micron CaP and CaF₂The particles are spherical, have a rough surface and are Ca (OH)₂The particles are hexagonal or irregular. All of these particles had slight agglomeration, indicating that they were formed by the fusion of smaller particles. The XRD pattern of the particles (as shown in fig. 10) showed that all particles were fine crystals except for the nano CaP.

Ion release from particles in buffer

To further investigate the applicability of various particles as element reservoirs, their ion release in physiological buffer was analyzed, as shown in figure 2. After 120 hours, the HEPES buffer reached 75, 90 and 105ppm of fluoride, phosphate and calcium ions (pH 4, respectively). At the same time point, the fluoride ion, phosphate ion and calcium ion in the buffer (pH7.4) reached 23, 40 and 43ppm, respectively. The results show that the acidic environment significantly enhances the release of fluorine, phosphate and calcium. A comparison of the calcium ions released from amorphous calcium phosphate (nano-CaP) and crystalline calcium phosphate (micro-CaP) (as shown in fig. 11) shows that amorphous calcium phosphate releases almost twice as much Ca ions as crystalline calcium phosphate. At pH 4 and 7.4.

Effect on dentin remineralization

To analyze the effect of the tooth surface coating material on remineralization of dentin, open dentin on the dentin surface was obtained by acid etchingThe tubules (fig. 3A). As shown in fig. 3B, the dentinal tubules were clogged after further treatment with toothpaste for 3 days. After another 3 days of the same treatment, the dentinal surface (in fig. 3C) was completely covered with particles, and no bare tubules were observed. High resolution SEM images (fig. 3D) show that particles in the toothpaste have good connectivity at the surface of the dentinal tubules. To further confirm dentinal tubule obstruction and dentin mineralization, Atomic Force Microscopy (AFM) was also performed on the treated dentin. Fig. 4 shows that the exposed tubules are clearly visible on the dentin surface, whereas after treatment with the smart material, the exposed tubules are filled. In addition, ATR-FTIR spectra (FIG. 12) of dentin surfaces treated over different times showed the phosphate intensity (1017 cm) of remineralized dentin^-1) The enhancement is obtained. Specifically, at 1637^cm-1And 1543^cm-1The absorption bands at (a) correspond to amide I and amide II, respectively. The results show that the intensity of these two bands in remineralized dentin is significantly reduced, especially in the 6 day treated band.

Effect on enamel remineralization

The effect of tooth surface coating materials on enamel remineralization has also been carried out. As shown in fig. 5, many crevices were observed in the rough surface of enamel after acid etching. Application of the toothpaste for 3 days resulted in partial coverage of the enamel crevices. High resolution SEM images (fig. 5C) show that the particles have good adhesion on the surface. After 6 days with the smart material, more particles were filled on the rough surface and the gaps were almost completely covered. Cross-sectional analysis was also performed on the newly mineralized enamel interface. First, a cross section is cut on a selected area under SEM observation by a Focused Ion Beam (FIB), as shown in fig. 13. Figure 6 shows HRTEM (left) and FFT diffractogram of the interface plotted with white lines (right). Lattice planes (1100) were present on both sides of the interface, indicating that both sides have the same crystal structure and that the extension is the epitaxial growth of the enamel crystal. Lattice planes (1100) and (2200) are marked (right) in the FFT diffractogram.

Cell viability of the particles

Cytotoxicity is critical to assessing the biocompatibility of a material prior to any biomedical application. In this study, the cytotoxicity of isolated particles and multiple particles was assessed using the CCK-8 assay. The results show that all cell viability was higher than 90% except for the calcium hydroxide groups when the cells were co-cultured with the particles in the range of 0-80 μ g/mL for 36 hours (fig. 7). The mixed toothpaste has little toxicity to A549 cells, which shows that the mixed toothpaste has excellent biocompatibility.

Antibacterial property

For antibacterial analysis, Streptococcus mutans was cultured in moist Brain Heart Infusion (BHI) broth overnight at 37 ℃ and then left for 24 hours, then diluted to co-culture with the smart material. Figure 8 shows the percentage of viable bacteria after co-cultivation with the mixture and calcium hydroxide particles was 38% and 22%, respectively. nano-CaP, micro-CaP and CaF under the same conditions₂The percentage of live bacteria in the group was 70%, 73% and 67%, respectively. The percentage of live bacteria in all groups decreased with increasing time. All groups were visually observed for live bacteria at 72 hours from the micrograph of fig. 14.

Discussion of the related Art

Calcium phosphate nanospheres (nano-CaP), calcium phosphate microspheres (micro-CaP), calcium fluoride microspheres and calcium hydroxide particles have been used to improve mineralization, respectively. In this study, it has been demonstrated that the combined use of the above-mentioned particles allows to optimize their function and to achieve a better remineralization. The mixed toothpaste can be used as a smart tooth surface coating material because it can adapt it to various oral environments. The teeth consist primarily of hydroxyapatite, surrounded by saliva in the mouth. Tooth demineralization and remineralization in the oral environment is a dynamic process that occurs at the salivary interface.

Demineralization:

remineralization:

from the dynamic equivalence we can see the hazard of acidic beverages and the importance of multiple particles providing the required ions in the case of remineralisation of tooth surfaces. The results show that various systems have excellent remineralization of both dentin and enamel of teeth. The dentinal surface was completely covered with particles and no bare tubules were found after two applications a week. Multiple systems have proven to be more effective than previous studies in which one type of calcium phosphate particle was used. Enamel remineralization showed the same effect as shown in figure 5. Furthermore, the FIB and TEM results in fig. 6 indicate that the epitaxial growth of the crystal extends from the enamel to the regrown layer. The mechanism by which dentinal tubules are remineralized by a plurality of particles. Ions in the oral environment can be described as shown in fig. 9. The foreign material(s) may prevent release of ions from the teeth by self-depletion (demineralization) and provide a abundance of ions to reach an ion concentration threshold that leads to tooth remineralization.

Furthermore, many particles have excellent biological activity and antibacterial properties, which are also important for dental use and tooth remineralization. According to many studies, bacteria are one of the major factors associated with dental caries, and reduction of bacteria will benefit dental health. In this study, calcium hydroxide particles were incorporated into smart materials, primarily because of their strong antimicrobial properties against endodontic disease. Considering that the high cytotoxicity of the calcium hydroxide particles limits the application thereof in medicine and dentistry, the composition ratio of the calcium hydroxide particles is optimized, and the resulting smart material has excellent antibacterial properties and acceptable cytotoxicity.

Conclusion

Herein we disclose specific functions of arginine and multiparticulate systems as smart materials in the formation of apatite on teeth. The apatite obtained is an epitaxial extension of the enamel crystals. The excellent apatite formation is due to the intelligent behavior of the multiparticulate system, which can provide a variety of ions, an acid-resistant environment and an antibacterial effect. This study may open up a new pathway for the formation of dental apatite and provide a new indication for tooth remineralisation.

Novel formulations of intelligent dental materials in toothpaste applications were investigated. The intelligent material is composed of arginine and a plurality of particlesThe particle composition comprises calcium phosphate nanospheres (nano-CaP), calcium phosphate microspheres (micro-CaP), calcium fluoride microspheres (CaF)₂) And calcium hydroxide particles (Ca (OH)₂). This intelligent multi-particle system can be adapted to a variety of oral environments caused by acidic beverages. Under the moist tooth surface microenvironment, the smart material can release calcium, phosphate, fluoride and hydroxide ions, thereby promoting remineralization of teeth. The exposed dentinal tubules will be sealed and, after remineralization, enamel caries can be repaired by epitaxial crystal growth. Microstructural analysis by Focused Ion Beam (FIB) cutting and Transmission Electron Microscopy (TEM) examination showed that this treatment resulted in seamless growth of enamel. In addition, smart materials also have excellent antibacterial properties and excellent biocompatibility with human dental pulp cells, which are critical for tooth remineralization. Thus, smart materials would be a good choice for enamel mineralization and repair

The invention is not limited to the preferred embodiment, but should be construed to cover all modifications, equivalents, and improvements that fall within the spirit and scope of the invention.

Claims (4)

1. A formula of composite micro-nano particles is characterized in that: comprises composite micro-nano particles; the composite micro-nano particles are calcium phosphate nanospheres, calcium phosphate microspheres, calcium fluoride microspheres and calcium hydroxide particles.

2. A toothpaste using the composite micro-nano particle formulation of claim 1, wherein: also comprises an abrasive, a humectant, a surfactant, a thickening agent, a sweetening agent, a preservative, a pigment, an essence, arginine and glycerol.

3. A method for synthesizing particles according to the formula of composite micro-nano particles of claim 1, which is characterized in that: comprises the following steps;

1: separately prepared contains Ca²⁺，F^-，PO₄ ^3-And OH^-Is dissolved in waterLiquid;

2: adding CaCl in the range of 0.1-20mM₂Mixing the aqueous solution with the aqueous solution in the step 1 to form a transparent solution with the initial pH of 6.0-8.0 at 25 ℃;

3: storing the transparent solution obtained in the step 3 in a closed glass bottle at 100 ℃ for 12 hours, and precipitating;

4: the precipitated precipitate was separated by filter paper (400nm, Track-Etch membrane), washed twice with ethanol and dried at 60 ℃.

4. An experimental method of the formula of the composite micro-nano particles according to claim 1, characterized in that: comprises the following steps;

1: cutting the tooth into dentin and enamel pieces using a steel blade, the thickness being about 1 mm in parallel from the crown of a human molar;

2: grinding the slices with 200 to 1200 grit sandpaper and washing with distilled water;

3: subsequently, the slices were phosphorylated for 30 seconds using 37% phosphorous acid and washed with distilled water to eliminate acidic residues after acidic etching;

4: coating each tooth slice with the prepared composite micro-nano particle toothpaste for 3 minutes, and then storing in simulated saliva solution at 37 ℃ for 3 days;

5: repeating the steps 1-4 every three days; dental sections were divided into two groups, one of which was coated for 3 days and the other for 6 days.

Images