Protein aggregate mineralization material capable of resisting saliva obtaining layer and used for preventing and repairing decayed teeth

Abstract

The invention discloses a protein aggregate mineralization material for preventing and repairing decayed teeth and resisting a salivary gaining layer, the mineralization material is composed of zwitter-ion modified protein, a disulfide bond reducing agent and a buffer solution, and the mineralization material can quickly form a stable nano-coating with biological activity on the surface of damaged teeth under mild conditions. On one hand, the nano-coating can resist saliva adhesion and block formation of an anti-saliva obtaining layer (ASP) on the tooth surface, so that the ASP cannot inhibit mineralization; on the other hand, rich amino acid residues, hydroxyl, amino, carboxyl and the like on the surface can promote crystallization of calcium and phosphorus on the surface of defective teeth, so that the effect of preventing and repairing decayed teeth is achieved. The mineralization material for enhancing remineralization by blocking ASP, provided by the invention, is mild in use condition, simple in method and good in biocompatibility, provides a new thought and a research direction for the development of a dental restoration material in a real oral saliva environment, and has extremely high clinical application value and economic value.

Description

Technical Field

[0001] The invention belongs to the technical field of oral biomedical materials, and in particular relates to a protein aggregate mineralized material that resists saliva acquisition layer and is used for preventing and repairing dental caries. Background Art

[0002] According to the World Health Organization and the World Dental Federation, untreated dental caries in permanent teeth is the most common of all diseases, affecting over 2 billion people worldwide. Untreated dental caries in primary teeth is the most common single chronic childhood disease, affecting 514 million children worldwide. Untreated dental caries can have numerous negative consequences at different stages of life. Recurring pain, as well as difficulties chewing and sleeping, can reduce quality of life and work productivity. Dental caries is a major cause of decreased work productivity, missed educational opportunities, and poor academic performance. Systemic inflammation caused by severe, untreated dental caries and dental pulp infection is also a contributing factor to underweight and developmental delays in children. In China, a populous country, only 13.8% of the 13,464 residents surveyed had intact teeth. Loss of tooth enamel or gum recession can lead to dental caries, especially in children. Tooth enamel is the hardest outer layer of the tooth and serves as the first line of defense for dental health. Its primary component is hydroxyapatite (HAp).

[0003] In the presence of oral saliva, a dense salivary acquired layer (ASP) forms on the enamel surface. The ASP provides specific binding sites for the initial colonization of cariogenic bacteria, specifically binding to these bacteria in the mouth to develop into a biofilm, ultimately causing dental caries. Furthermore, certain membrane proteins in the ASP, such as statherin, inhibit the deposition of calcium and phosphate salts on the enamel surface, inhibiting the crystallization of HAp on teeth or traditional dental restorative materials. This is one of the main reasons why teeth cannot repair themselves or why traditional restorative materials are clinically unavailable. To date, research on the prevention and treatment of dental caries has focused on physical sealing or remineralization. Physical sealing methods primarily repair larger tooth defects, such as resin infiltration and metal-ceramic materials, can quickly repair tooth defects and exhibit excellent mechanical properties. However, physical sealing materials often lack good biocompatibility and do not adhere tightly to the existing tooth, which can easily lead to microleakage and secondary dental caries. Traditional remineralization materials, such as polydopamine, dendrimers, and triethylamine-stabilized amorphous calcium phosphate (ACP), utilize biomineralization and recrystallization to grow HAp to repair damaged teeth. However, these materials often have color or an unpleasant odor, and their repair effects in real oral saliva often fall short of clinical needs. One of the main reasons for this is that the inhibitory effect of ASP on remineralization is ignored, resulting in the loss of remineralization effectiveness of existing enamel remineralization materials.

[0004] Therefore, in order to achieve the purpose of preventing and treating dental caries, restorative materials must meet the following requirements: (1) safe and non-toxic, simple to prepare, and not affect the normal color of teeth; (2) able to stably adhere to the tooth surface and have good stability; (3) able to resist the formation of ASP on tooth enamel and resist bacterial adhesion; (4) have excellent remineralization activity and can promote the crystallization of new HAp from calcium and phosphorus ions in saliva on the surface of defective teeth. Summary of the Invention

[0005] The purpose of the present invention is to provide a protein aggregate mineralized material that resists the saliva-acquired layer and is used for the prevention and repair of dental caries. The material is safe and non-toxic, simple to prepare, does not affect the normal color of teeth, can stably adhere to the tooth surface, has good stability, can resist the formation of ASP on tooth enamel and resist bacterial adhesion, etc., has excellent remineralization activity, and can promote the crystallization of new HAp from calcium and phosphorus ions in saliva on the surface of defective teeth.

[0006] The protein aggregate mineralization material for preventing and repairing dental caries and resisting saliva acquisition layer provided by the present invention is composed of the following raw materials in percentage by mass: 5% to 90% of zwitterion modified protein, 5% to 90% of disulfide bond reducing agent and 5% to 90% of buffer solution.

[0007] Furthermore, the protein aggregate mineralized material for preventing and repairing dental caries with a saliva-receiving layer of the present invention is preferably composed of the following raw materials in the following mass percentages: 10% to 80% zwitterion-modified protein, 10% to 80% reducing agent, and 10% to 80% buffer.

[0008] Furthermore, the protein aggregate mineralized material for preventing and repairing dental caries with a saliva-receiving layer of the present invention is more preferably composed of the following raw materials in the following mass percentages: 20% to 60% zwitterion-modified protein, 20% to 60% reducing agent, and 20% to 60% buffer.

[0009] Furthermore, the zwitterion modified protein is prepared by chemical coupling or physical blending of a zwitterion compound and a protein; the zwitterion compound is selected from sulfobetaine methacrylate (SBMA), 2-methacryloyloxyethyl phosphorylcholine (MPC), carboxybetaine methacrylate (CBMA), carboxybetaine acrylic acid (CBA) sulfobetaine methacrylamide (SBAA), sulfobetaine acrylate (SBA), thiobetaine methacrylate, thiobetaine acrylate, thiobetaine acrylamide, carboxybetaine methacrylate, carboxybetaine acrylate, carboxybetaine acrylamide, imidazole type zwitterion compounds, amino acid derived zwitterion acrylic acid any one or more of ester, difunctional carboxybetaine methacrylate, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propane-1-sulfonate, 3-[[2-(acryloyloxy)ethyl]dimethylammonium]propane-1-sulfonate, 3-[(3-acrylamidopropyl)dimethylammonium]propane-1-sulfonate, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate, 3-[(3-acrylamidopropyl)dimethylammonium]propionate, 2-hydroxy-3-(1-(4-vinylbenzyl)imidazol-3-yl)propane-1-sulfonate, 3-(4-vinylbenzyl)dimethylammonium)-2-hydroxypropane-1-sulfonate, and a phosphorylcholine-based compound.

[0010] Furthermore, the zwitterionic compound is preferably any one or more of sulfobetaine methacrylate, 2-methacryloyloxyethyl phosphorylcholine, carboxybetaine methacrylate, and sulfobetaine methacrylamide.

[0011] Furthermore, the protein in the zwitterion-modified protein is at least one of the following:

[0012] (1) Enzymes: lysozyme, collagenase, ribonuclease A (RNase A), DNA polymerase, lipase, β-galactosidase, α-amylase, chymosin, cellulase, superoxide dismutase, laccase, transglutaminase, pepsin;

[0013] (2) Precursor enzymes / zymogens: trypsinogen, pepsinogen, chymotrypsinogen;

[0014] (3) Structural proteins: keratin, elastin, collagen, fibronectin, laminin, fibrinogen, histones, tubulin;

[0015] (4) Transport / storage proteins: transferrin, lactoferrin, myoglobin, hemoglobin, ferritin;

[0016] (5) Immune-related proteins: immunoglobulin G, immunoglobulin A, immunoglobulin M, immunoglobulin E, immunoglobulin D, complement C3 protein, complement C4 protein, complement C5 protein, CD4 protein, HIV-1 gp120 protein, perforin, coagulation factor VIII, coagulation factor IX, tissue plasminogen activator (tPA);

[0017] (6) Hormones / cytokines: insulin, human chorionic gonadotropin (hCG), follicle-stimulating hormone (FSH), luteinizing hormone (LH), growth hormone, thyroglobulin, prolactin, thyroid-stimulating hormone (TSH), somatostatin, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), interferon (IFN);

[0018] (7) Milk proteins / nutritional proteins: ovalbumin, α-lactalbumin, whey albumin, lactoprotein, whey protein, casein, casein, β-lactoglobulin;

[0019] (8) Plant-derived proteins: soy protein isolate, gluten, concanavalin, cruciferin, plant defensins, and lectins;

[0020] (9) Serum-related proteins: human serum albumin, bovine serum albumin, mouse serum, goat serum, fetal calf serum, C-reactive protein, complement;

[0021] (10) Other / regulatory proteins: any one or more of BPTI, mucin, transcription factor P53, transcription factor NF-κB, inhibitory protein IκB, bovine submandibular mucin, heat shock protein (HSP), epidermal growth factor (EGF), epidermal growth factor receptor (EGFR), and cytochrome c.

[0022] Furthermore, the protein in the zwitterion-modified protein is preferably any one or more of lysozyme, human serum albumin, bovine serum albumin, lactoferrin, cruciferin, milk protein, whey albumin, hemoglobin, transferrin, soy protein isolate, and goat serum.

[0023] Furthermore, the above-mentioned disulfide bond reducing agent is selected from any one or more of tris(2-carboxyethyl)phosphine, glutathione, cysteine, dithiothreitol, dithiothreitol isomers, β-mercaptoethanol, mercaptopropionic acid, trihydroxypropylphosphine, urea, sodium ferrate, azobisisobutyramidine hydrochloride, hexafluoroisopropanol, sodium bismuthate, hydrogen peroxide, trivalent cobalt salt, guanidine hydrochloride, trifluoroethanol, and sodium borohydride.

[0024] Furthermore, the disulfide bond reducing agent is preferably any one or more of tris(2-carboxyethyl)phosphine, glutathione, and cysteine.

[0025] Furthermore, the buffer is selected from any one or two of sodium citrate buffer, sodium acetate buffer, PBS buffer, MES buffer, PIPES buffer, MOPS buffer, HEPES buffer, Tris buffer, Bicine buffer, and CHES buffer.

[0026] The beneficial effects of the present invention are as follows:

[0027] (1) The protein aggregate mineralization material provided by the present invention, which is resistant to saliva acquisition and is used for the prevention and repair of dental caries, uses zwitterionic modified proteins to achieve amyloid aggregation under the action of disulfide bond reduction reagent and buffer solution, and can quickly achieve stable adhesion to damaged teeth under mild conditions, forming a colorless, transparent, and bioactive protein nanocoating on the tooth surface.

[0028] (2) The present invention provides a protein aggregate mineralized material for preventing and repairing dental caries with a saliva-receiving layer, wherein the zwitterionic modified protein can effectively block ASP and achieve high-efficiency antifouling. Compared with traditional antifouling materials (such as polyethylene glycol (PEG)), zwitterionic materials are becoming an important candidate for the next generation of biocompatible materials due to their excellent superhydrophilicity, low immunogenicity, and stable antifouling ability in complex environments. They are also biodegradable, have low toxicity, and good biocompatibility, making them more suitable for human applications.

[0029] (3) The protein aggregate mineralization material provided by the present invention, which resists saliva acquisition layer and is used for the prevention and repair of dental caries, forms a bioactive protein nanocoating on the surface of damaged teeth. Its surface contains multiple amino acid residues and functional groups, which can promote the crystallization of new HAp from calcium and phosphate ions in saliva on the surface of defective teeth. In addition, the β-pleated structure contained in the amyloid-like protein aggregates provides a template for remineralization, restarting remineralization and enabling precise regulation of remineralization.

[0030] (4) The protein nanocoating formed on the tooth surface by the protein aggregate mineralized material for preventing and repairing caries provided by the present invention is stable in nature and resistant to acid and alkali. It can not only block the formation of ASP on the tooth surface but also has excellent anti-fouling and anti-bacterial adhesion properties. It can induce the formed repair layer to grow epitaxially along the original enamel and is tightly bonded to the original tooth. The mechanical properties are comparable to those of natural enamel, avoiding microleakage. In addition, it is inexpensive, simple to synthesize, non-toxic, and has better biocompatibility.

[0031] (5) The protein aggregate mineralized material for preventing and repairing dental caries provided by the present invention, which is resistant to saliva acquisition, can be prepared into various products related to preventing and repairing dental caries. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Scanning electron micrographs of ASP on tooth enamel after incubation in artificial saliva for 7 days (a) and after incubation with Streptococcus mutans for 24 hours (b).

[0033] Figure 2 These are the optical image (a), transmission electron microscope image (b), laser confocal image (c), and water contact angle image (d) of the mineralized material on the tooth in Example 1.

[0034] Figure 3 The optical transmittance (a) and circular dichroism spectrum image (b) of the mineralized material on the quartz plate in Example 1 are shown.

[0035] Figure 4 is the saliva adsorption capacity of the mineralized materials in Examples 1 to 20 and the materials in Comparative Examples 1 to 6.

[0036] Figure 5 These are scanning electron microscope images of the remineralization of the enamel surface after treatment with the mineralized materials of Examples 1 to 20 and the materials of Comparative Examples 1 to 6.

[0037] Figure 6 Elemental energy spectrum analysis (a) and 3D wear image (b) of the repaired layer.

[0038] Figure 7 The hardness (left) and elastic modulus (right) of the enamel repair layer after being treated with the mineralized materials in Examples 1 to 20 and the materials in Comparative Examples 1 to 6.

[0039] Figure 8 It is the cytotoxicity of the mineralized materials in Examples 1 to 20 and the materials in Comparative Examples 1 to 6.

[0040] Figure 9 The optical photograph of Example 1 mineralized material @ enamel fixed in the rat mouth (a), the scanning electron microscope image (b) and the corresponding cross-sectional image (c) after incubation in the rat mouth for 7 days, and the elemental EDX image (d) of the remineralized tooth surface after incubation of the rat oral enamel for 7 days. DETAILED DESCRIPTION

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only 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 any creative efforts are within the scope of protection of the present invention.

[0042] Example 1

[0043] 1.52 mg of SBMA-modified lysozyme (physical blending), 1.48 mg of cysteine, and 1.4 mg of PBS buffer were mixed evenly to obtain a protein aggregate mineralized material that resists saliva acquisition layer and is used for the prevention and repair of dental caries.

[0044] Example 2

[0045] 1.52 mg of SBMA-modified lysozyme (chemical coupling), 1.48 mg of cysteine, and 1.4 mg of PBS buffer were mixed evenly to obtain a protein aggregate mineralized material that resists saliva acquisition layer and is used for the prevention and repair of dental caries.

[0046] Example 3

[0047] 1.01 mg SBMA-modified human serum albumin (physical blend), 3.01 mg tris(2-carboxyethyl)phosphine, and 1.2 mg PBS buffer were mixed evenly to obtain a protein aggregate mineralized material that resists saliva acquisition layer and is used for the prevention and repair of dental caries.

[0048] Example 4

[0049] 1.25 mg SBMA-modified bovine serum albumin (physical blend), 3.2 mg tris(2-carboxyethyl)phosphine, and 1.2 mg sodium citrate buffer were mixed evenly to obtain a protein aggregate mineralized material that resists saliva acquisition layer and is used for the prevention and repair of dental caries.

[0050] Example 5

[0051] 1.4 mg of SBMA-modified lactoferrin (physical blending), 1.52 mg of glutathione, and 1.4 mg of sodium citrate buffer were mixed evenly to obtain a protein aggregate mineralized material that resists saliva acquisition layer and is used for the prevention and repair of dental caries.

[0052] Example 6

[0053] 1.42 mg of SBMA-modified cruciferin (physical blend), 3.82 mg of tris(2-carboxyethyl)phosphine, and 1.59 mg of HEPES buffer were mixed evenly to obtain a protein aggregate mineralized material that resists saliva acquisition layer and is used for the prevention and repair of dental caries.

[0054] Example 7

[0055] 1.42 mg of SBMA-modified cruciferin (chemical coupling), 3.82 mg of tris(2-carboxyethyl)phosphine, and 1.59 mg of HEPES buffer were mixed evenly to obtain a protein aggregate mineralized material that resists saliva acquisition layer and is used for the prevention and repair of dental caries.

[0056] Example 8

[0057] 1.68 mg of CBMA-modified lysozyme (physical blending), 1.72 mg of glutathione, and 1.49 mg of HEPES buffer were mixed evenly to obtain a protein aggregate mineralized material that resists saliva acquisition layer and is used for the prevention and repair of dental caries.

[0058] Example 9

[0059] 1.68 mg of CBMA-modified lysozyme (chemical coupling), 1.72 mg of glutathione, and 1.49 mg of HEPES buffer were mixed evenly to obtain a protein aggregate mineralized material that resists saliva acquisition layer and is used for the prevention and repair of dental caries.

[0060] Example 10

[0061] 1.25 mg of SBMA-modified milk protein (physical blending), 1.32 mg of glutathione, and 1.29 mg of HEPES buffer were mixed evenly to obtain a protein aggregate mineralized material that resists saliva acquisition layer and is used for the prevention and repair of dental caries.

[0062] Example 11

[0063] 1.25 mg of SBMA-modified milk protein (chemical coupling), 1.32 mg of glutathione, and 1.29 mg of HEPES buffer were mixed evenly to obtain a protein aggregate mineralized material that is resistant to saliva acquisition layer and is used for the prevention and repair of dental caries.

[0064] Example 12

[0065] 1.48 mg SBMA-modified whey albumin (physical blending), 3.82 mg tris(2-carboxyethyl)phosphine, and 1.48 mg Tris buffer were mixed evenly to obtain a protein aggregate mineralized material that resists saliva acquisition layer and is used for the prevention and repair of dental caries.

[0066] Example 13

[0067] 1.48 mg of SBMA-modified whey albumin (chemical coupling), 3.82 mg of tris(2-carboxyethyl)phosphine, and 1.48 mg of Tris buffer were mixed evenly to obtain a protein aggregate mineralized material that resists saliva acquisition layer and is used for the prevention and repair of dental caries.

[0068] Example 14

[0069] 1.62 mg of MPC modified hemoglobin (physical blend), 4.02 mg of tris(2-carboxyethyl)phosphine, and 1.52 mg of Tris buffer were mixed evenly to obtain a protein aggregate mineralized material that resists saliva acquisition layer and is used for the prevention and repair of dental caries.

[0070] Example 15

[0071] 1.62 mg of MPC-modified hemoglobin (chemical coupling), 4.02 mg of tris(2-carboxyethyl)phosphine, and 1.52 mg of Tris buffer were mixed evenly to obtain a protein aggregate mineralized material that resists saliva acquisition layer and is used for the prevention and repair of dental caries.

[0072] Example 16

[0073] 1.58 mg of MPC-modified transferrin (physical blend), 3.82 mg of tris(2-carboxyethyl)phosphine, and 1.48 mg of Tris buffer were mixed evenly to obtain a protein aggregate mineralized material that resists saliva acquisition layer and is used for the prevention and repair of dental caries.

[0074] Example 17

[0075] 1.58 mg of MPC-modified transferrin (chemical coupling), 3.82 mg of tris(2-carboxyethyl)phosphine, and 1.48 mg of Tris buffer were mixed evenly to obtain a protein aggregate mineralized material that resists saliva acquisition layer and is used for the prevention and repair of dental caries.

[0076] Example 18

[0077] 1.48 mg of MPC-modified soy protein isolate (physical blending), 4.02 mg of tris(2-carboxyethyl)phosphine, and 1.61 mg of PBS buffer were mixed evenly to obtain a protein aggregate mineralized material that resists saliva acquisition layer and is used for the prevention and repair of dental caries.

[0078] Example 19

[0079] 1.48 mg of MPC-modified soy protein isolate (chemical coupling), 4.02 mg of tris(2-carboxyethyl)phosphine, and 1.61 mg of PBS buffer were mixed evenly to obtain a protein aggregate mineralized material that resists saliva acquisition layer and is used for the prevention and repair of dental caries.

[0080] Example 20

[0081] 1.42 mg of MPC-modified goat serum (physical blend), 4.02 mg of tris(2-carboxyethyl)phosphine, and 1.56 mg of PBS buffer were mixed evenly to obtain a protein aggregate mineralized material that resists saliva acquisition layer and is used for the prevention and repair of dental caries.

[0082] Comparative Example 1

[0083] 1.52 mg of PEG-modified lysozyme (physical blend), 1.48 mg of cysteine, and 1.4 mg of PBS buffer were mixed evenly.

[0084] Comparative Example 2

[0085] Mix 1.52 mg of PEG-modified lysozyme (chemical coupling), 1.48 mg of cysteine, and 1.4 mg of PBS buffer.

[0086] Comparative Example 3

[0087] 1.01 mg of PEG-modified human serum albumin (physical blend), 3.01 mg of tris(2-carboxyethyl)phosphine, and 1.2 mg of PBS buffer were mixed evenly.

[0088] Comparative Example 4

[0089] 1.25 mg of PEG-modified bovine serum albumin (physical blend), 3.2 mg of tris(2-carboxyethyl)phosphine, and 1.2 mg of sodium citrate buffer were mixed evenly.

[0090] Comparative Example 5

[0091] Mix 1.25 mg of lysozyme, 3.2 mg of tris(2-carboxyethyl)phosphine, and 1.2 mg of PBS buffer.

[0092] Comparative Example 6

[0093] Mix 1.25 mg of natural lysozyme, 1.32 mg of glutathione, and 1.2 mg of PBS buffer.

[0094] It should be noted that in the above embodiments and comparative examples, the physical blending method is: the protein corresponding to each embodiment or comparative example is uniformly mixed with the corresponding zwitterionic polymer in a mass ratio of 1:1 to prepare the corresponding zwitterionic modified protein; the chemical coupling method is: using EDC / NHS reagent (EDC: 1-ethyl-(3-dimethylaminopropyl)carbodiimide, NHS: N-hydroxysuccinimide) to esterify the amino group on the protein with the carboxyl group of the zwitterionic compound to prepare the corresponding zwitterionic modified protein.

[0095] In order to demonstrate the beneficial effects of the present invention, the inventors used the mineralized materials obtained in Examples 1 to 20 and the materials obtained in Comparative Examples 1 to 6 for tooth enamel repair, and conducted various performance tests on the formed enamel repair layers. The specific tests are as follows:

[0096] Test Example 1

[0097] Adult third molars without caries were collected (tooth samples were provided by a dental hospital, and the study was approved by the medical ethics committee of the above-mentioned institution). After cleaning, they were processed into enamel slices of uniform thickness, which were then ground, polished, and etched. The etched enamel was immersed in adult sterile saliva for 30 minutes to prepare ASP@enamel. The ASP@enamel was immersed in artificial saliva at 37°C and incubated for 7 days. After removal, it was dried and its surface morphology was observed using a scanning electron microscope. Figure 1 a. After ASP@tooth enamel was co-cultured with Streptococcus mutans for 48 hours, the bacteria were removed and fixed with paraformaldehyde. After the sample was dried, the adhesion of the bacteria was observed using a scanning electron microscope. Figure 1 b.

[0098] The acid-etched enamel treated as above was immersed in the mineralized material of Example 1 for 2 minutes to obtain a sample of the mineralized material @ enamel of Example 1. The optical photograph is as follows: Figure 2 a. The copper mesh was prepared as described above to obtain a mineralized material @ copper mesh. The microscopic morphology thereof was observed using a transmission electron microscope and found to be a thin film composed of protein nanoparticles. Figure 2 b. By comparing the water contact angle data, it can be judged that the mineralized material has achieved the modification of tooth enamel. Figure 2 c. ThT staining can be used to determine that the enamel is successfully modified and rich in β-sheet structure, such as Figure 2 d.

[0099] Test Example 2

[0100] The quartz plate was immersed in the mineralized material of Example 1 for 2 minutes to prepare the mineralized material @ quartz plate. The transmittance thereof was tested and found to be close to that of the blank quartz plate, proving that the mineralized material was colorless and transparent. Figure 3 a. Then, the circular dichroism spectrum test of the mineralized material loaded on the quartz plate was performed. It can be seen that the mineralized material (formed protein aggregates) causes the protein to transform from an α-helical structure to a β-pleated structure, which provides a template for remineralization, such as Figure 3 b.

[0101] Test Example 3

[0102] The mineralized materials @ gold chips of Examples 1 to 20 and the materials @ gold chips of Comparative Examples 1 to 6 were prepared using the method in Experiment 2. The adhesion of the mineralized materials to saliva was tested by QCM-D instrument to determine their blocking effect on ASP. It was found that the adhesion of the mineralized materials of Examples 1 to 20 to saliva was only 230 ng / cm 2 The amount of saliva adhered to the blank gold chip (control group) and the materials of comparative examples 1 to 6 is about 5 times (control group) and about 3 times (comparative examples 1 to 6) of that of examples 1 to 20 (see Figure 4 ). It is proved that the mineralized materials of Examples 1 to 20 have better anti-saliva adhesion effect than the materials of Comparative Examples 1 to 6, and are more likely to block the formation of ASP on tooth enamel.

[0103] Test Example 4

[0104] The mineralized materials @ enamel of Examples 1 to 20 and the materials @ enamel of Comparative Examples 1 to 6 were prepared using the method of Experiment 2. All samples were incubated in artificial saliva for 7 days, taken out and dried, and their surface morphologies were observed using a scanning electron microscope. It was found that after 7 days of incubation, a dense regeneration and repair layer was formed on the surface of the samples of Examples 1 to 20, while after 7 days of incubation, the surface of the samples of Comparative Examples 1 to 6 still retained a fish-scale morphology (the morphology of the acid-etched enamel is fish-scale) (see Figure 5 ).

[0105] The element analysis of the regenerated repair layer on the sample of Example 1 was performed ( Figure 6 a), we can see that hydroxyapatite is generated on the enamel surface along the epitaxial growth of the enamel, and the calcium-phosphorus ratio is close to 1.67, which is consistent with natural enamel. The generated repair layer was further subjected to a 3D wear test ( Figure 6 b), it can be seen that the wear depth and wear amount are comparable to natural enamel.

[0106] Nanoindentation tests were performed on the regenerated and repaired coatings on the samples of Examples 1 to 20 and Comparative Examples 1 to 6. Figure 7 The results showed that the elastic modulus and hardness of the repair layers treated with the mineralized materials of Examples 1 to 20 were comparable to those of natural tooth enamel.

[0107] The mineralized materials@tooth enamel of Examples 1 to 20 and the materials@tooth enamel of Comparative Examples 1 to 6 were placed in a porous plate, and human fourth-generation gingival fibroblasts (5000 cells / cm 2 ) were co-cultured for 1 day, 4 days, and 7 days respectively. Then the cell activity was detected (such as Figure 8 ), we found that the absorbance of Examples 1 to 20 was consistent with that of the control group, while the absorbance of the comparative examples was lower than that of the control group, indicating that the mineralized materials of Examples 1 to 20 had better cell activity and biocompatibility.

[0108] Test Example 5

[0109] The treated acid-etched enamel was immersed in the mineralized material of Example 1, taken out after 2 minutes, and fixed inside the rat's mouth with a wire to ensure that the rat's saliva could contact the sample (such as Figure 9 a). From Figure 9 It can be seen from bc that the mineralized material of Example 1 forms a remineralized layer on the surface of tooth enamel, and its calcium-phosphorus ratio is close to that of natural tooth enamel ( Figure 9 d) The results demonstrate the effectiveness of the animal experiment of the present invention, which is beneficial to the prevention and treatment of dental caries and its clinical transformation.

[0110] In summary, the mineralized material provided by the present invention has the dual functions of resisting saliva adhesion / anti-fouling and remineralization, and can produce amyloid aggregation on the surface of damaged teeth to form a protein nanocoating with the functions of blocking ASP formation, restarting remineralization, and anti-fouling. The process conditions are mild, and stable adhesion to the teeth can be achieved within a few minutes, and at least 1.5 μm of repair layer regeneration can be achieved every day. The elastic modulus and hardness of the repair layer regenerated by the mineralized material of the present invention, as well as the wear resistance and acid corrosion resistance are comparable to natural enamel, and have good mechanical properties. The mineralized material of the present invention has excellent superhydrophilicity, low immunogenicity and stable anti-fouling ability in complex environments. It can resist ASP and then repair and prevent caries. The material has also been shown to have good biocompatibility, no hemolysis risk, no cytotoxicity, no oral mucosal irritation, and no skin sensitization. Animal experiments have shown that the mineralized material of the present invention can achieve the remineralization of tooth enamel in the oral cavity of rats, and still has excellent remineralization performance in the real oral environment of animals, verifying the effectiveness of the present invention and having excellent clinical application value. The mineralized material of the present invention avoids the repair failure phenomenon caused by the mineralization inhibition of ASP, provides a new idea for the development of subsequent tooth repair materials, and has great guiding significance and economic value for the prevention and repair of clinical caries.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A protein aggregate mineralized material for preventing and repairing dental caries that resists saliva acquisition layer, characterized by: The mineralized material comprises by weight: 5% to 90% zwitterion-modified protein, 5% to 90% disulfide bond reducing agent, and 5% to 90% buffer; The zwitterion modified protein is prepared by chemical coupling or physical blending of a zwitterion compound and a protein; the zwitterion compound is selected from sulfobetaine methacrylate, 2-methacryloyloxyethyl phosphorylcholine, methacrylic acid carboxybetaine, carboxybetaine acrylic acid, sulfobetaine methacrylamide, sulfobetaine acrylate, sulfobetaine methacrylate, sulfobetaine acrylate, sulfobetaine acrylamide, carboxybetaine methacrylate, carboxybetaine acrylate, carboxybetaine acrylamide, imidazole type zwitterion compound, amino acid derived zwitterion acrylate, difunctional carboxybetaine methacrylate, 3 any one or more of -[[2-(methacryloyloxy)ethyl]dimethylammonium]propane-1-sulfonate, 3-[[2-(acryloyloxy)ethyl]dimethylammonium]propane-1-sulfonate, 3-[(3-acrylamidopropyl)dimethylammonium]propane-1-sulfonate, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate, 3-[(3-acrylamidopropyl)dimethylammonium]propionate, 2-hydroxy-3-(1-(4-vinylbenzyl)imidazol-3-yl)propane-1-sulfonate, 3-(4-vinylbenzyl)dimethylammonium)-2-hydroxypropane-1-sulfonate, and a phosphorylcholine-based compound.

2. The protein aggregate mineralized material for preventing and repairing dental caries with a saliva-receiving layer according to claim 1, characterized in that: The mineralized material comprises by weight: 10% to 80% of zwitterion-modified protein, 10% to 80% of reducing agent and 10% to 80% of buffer solution.

3. The protein aggregate mineralized material for preventing and repairing dental caries with a saliva-receiving layer according to claim 1, characterized in that: The mineralized material is composed of 20% to 60% of zwitterion-modified protein, 20% to 60% of reducing agent and 20% to 60% of buffer solution in terms of mass percentage.

4. The protein aggregate mineralized material for preventing and repairing dental caries according to any one of claims 1 to 3, characterized in that: The zwitterionic compound in the zwitterionic modified protein is selected from any one or more of sulfobetaine methacrylate, 2-methacryloyloxyethyl phosphorylcholine, carboxybetaine methacrylate, and sulfobetaine methacrylamide.

5. The protein aggregate mineralized material for preventing and repairing dental caries according to any one of claims 1 to 3, characterized in that: The protein in the zwitterion-modified protein is selected from at least one of the following: (1) Enzymes: lysozyme, collagenase, ribonuclease A, DNA polymerase, lipase, β-galactosidase, α-amylase, chymosin, cellulase, superoxide dismutase, laccase, transglutaminase, pepsin; (2) Precursor enzymes / zymogens: trypsinogen, pepsinogen, chymotrypsinogen; (3) Structural proteins: keratin, elastin, collagen, fibronectin, laminin, fibrinogen, histones, tubulin; (4) Transport / storage proteins: transferrin, lactoferrin, myoglobin, hemoglobin, ferritin; (5) Immune-related proteins: immunoglobulin G, immunoglobulin A, immunoglobulin M, immunoglobulin E, immunoglobulin D, complement C3 protein, complement C4 protein, complement C5 protein, CD4 protein, HIV-1 gp120 protein, perforin, coagulation factor VIII, coagulation factor IX, tissue plasminogen activator; (6) Hormones / cytokines: insulin, human chorionic gonadotropin, follicle-stimulating hormone, luteinizing hormone, growth hormone, thyroglobulin, prolactin, thyroid-stimulating hormone, somatostatin, interleukin-2, interleukin-4, interleukin-6, tumor necrosis factor α, interferon; (7) Milk proteins / nutritional proteins: ovalbumin, α-lactalbumin, whey albumin, lactoprotein, whey protein, casein, casein, β-lactoglobulin; (8) Plant-derived proteins: soy protein isolate, gluten, concanavalin, cruciferin, plant defensins, and lectins; (9) Serum-related proteins: human serum albumin, bovine serum albumin, mouse serum, goat serum, fetal calf serum, C-reactive protein, complement; (10) Other / regulatory proteins: any one or more of trypsin inhibitor, mucin, transcription factor P53, transcription factor NF-κB, inhibitory protein IκB, bovine submandibular mucin, heat shock protein, epidermal growth factor, epidermal growth factor receptor, and cytochrome c.

6. The protein aggregate mineralized material for preventing and repairing dental caries according to any one of claims 1 to 3, characterized in that: The protein in the zwitterion-modified protein is selected from any one or more of lysozyme, human serum albumin, bovine serum albumin, lactoferrin, cruciferin, milk protein, whey albumin, hemoglobin, transferrin, soy protein isolate, and goat serum.

7. The protein aggregate mineralized material for preventing and repairing dental caries according to any one of claims 1 to 3, characterized in that: The disulfide bond reducing agent is selected from any one or more of tris(2-carboxyethyl)phosphine, glutathione, cysteine, dithiothreitol, dithiothreitol isomers, β-mercaptoethanol, mercaptopropionic acid, trihydroxypropylphosphine, urea, sodium ferrate, azobisisobutyramidine hydrochloride, hexafluoroisopropanol, sodium bismuthate, hydrogen peroxide, trivalent cobalt salt, guanidine hydrochloride, trifluoroethanol, and sodium borohydride.

8. The protein aggregate mineralized material for preventing and repairing dental caries according to any one of claims 1 to 3, characterized in that: The disulfide bond reducing agent is selected from any one or more of tris(2-carboxyethyl)phosphine, glutathione, and cysteine.

9. The protein aggregate mineralized material for preventing and repairing dental caries according to any one of claims 1 to 3, characterized in that: The buffer is selected from any one or two of sodium citrate buffer, sodium acetate buffer, PBS buffer, MES buffer, PIPES buffer, MOPS buffer, HEPES buffer, Tris buffer, Bicine buffer, and CHES buffer.

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