A kind of dialdehyde carboxymethyl chitosan and its preparation method and application

Abstract

The present invention discloses a dialdehyde carboxymethyl chitosan, its preparation method, and application, relating to the field of medical materials technology. The present invention provides a dialdehyde carboxymethyl chitosan, which is a compound represented by formula (I). The structural formula of the compound represented by formula (I) is as follows. The present invention provides a dialdehyde carboxymethyl chitosan (DCC), which, by introducing an aldehyde group, retains the structural and functional characteristics of carboxymethyl chitosan (CMC) while also acquiring the ability to anchor to collagen. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, in particular to dialdehyde carboxymethyl chitosan and a preparation method and application thereof. Background Art

[0002] Composite resins are widely used in tooth defect restoration due to their minimally invasive and aesthetically pleasing properties. However, their high rate of filling loss continues to impact their effectiveness. This loss is primarily due to aging of the bonding interface or the development of secondary caries.

[0003] It has been found that many modifiers of dentin adhesives can enhance the stability of the composite resin-dentin bonding interface. The addition of antibacterial ingredients can resist biofilm adhesion and prevent secondary caries. MMPs inhibitors can inhibit the activation of MMPs by chelating metal cations, competitively binding to dentin collagen, etc. The use of collagen cross-linkers can enhance the resistance to enzymatic degradation and mechanical strength of collagen fibers. The use of ethanol wet bonding technology can reduce the use of heavy hydrophilic monomers in the resin component, reduce water leakage and absorption, and prevent hydrolysis of the resin and collagen components in the mixed layer. The addition of biomimetic mineralization inducing components can enable dentin collagen to form a highly biomimetic intrafiber remineralization structure, obtaining good mechanical properties and anti-enzymatic properties.

[0004] In previous studies, carboxymethyl chitosan (CMC), which contains numerous carboxyl groups, has garnered widespread attention due to its excellent biological and chemical activities. It can inhibit amorphous calcium phosphate crystallization by chelating metal ions, thereby inducing collagen biomimetic mineralization. However, because CMC binds to collagen only electrostatically and readily dissociates, its long-term efficacy is uncertain.

[0005] In summary, the following problems exist in the current existing technologies: (1) The total acid etching adhesive system used causes excessive demineralization of dentin collagen, which loses the support of the mineralized components within the fiber and is prone to structural collapse. (2) Most dentin remineralization adhesives add calcium and phosphorus components to the adhesive. In the absence of biomimetic mineralization inducers, calcium and phosphorus crystals are deposited on the collagen surface, forming extra-fiber mineralization, which cannot effectively restore the performance of collagen. (3) Current biomimetic mineralization collagen mainly relies on the induction of polyelectrolytes. Common inducers such as polyacrylic acid, polyaspartic acid, and carboxymethyl chitosan only bind to collagen through electrostatic bonding, are easily hydrolyzed and separated from the dentin collagen surface, and cannot induce dentin biomimetic mineralization for a long time. (4) The large amount of hydrophilic monomers in the pretreatment agent of the current total acid etching adhesive system can cause water to easily penetrate into the mixed layer. The presence of a large amount of water affects the polymerization of resin monomers, causes phase separation between hydrophilic resin and hydrophobic resin, and the resin is easily hydrolyzed due to water absorption, resulting in reduced bonding durability. (5) The current carboxymethyl chitosan has carboxymethyl groups replacing hydroxyl groups at the N and O positions, which results in the lack of suitable sites for oxidation to aldehydes. Summary of the Invention

[0006] Based on this, the purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a dialdehyde carboxymethyl chitosan and a preparation method and application thereof.

[0007] To achieve the above object, the technical solution adopted by the present invention is: a dialdehyde carboxymethyl chitosan, wherein the dialdehyde carboxymethyl chitosan is a compound represented by formula (I), and the structural formula of the compound represented by formula (I) is as follows:

[0008]

[0009] The present invention provides a dialdehyde carboxymethyl chitosan (DCC). By introducing aldehyde groups, it retains the structural and functional characteristics of carboxymethyl chitosan (CMC) while also enabling it to anchor to collagen. Application of DCC in inducing collagen biomimetic mineralization can enhance mineralization efficiency and ensure more stable and uniform mineral deposition. Furthermore, DCC can mimic the functions of non-collagenous proteins such as dentin phosphoproteins, cross-link with collagen fibers, stabilize the collagen structure, and improve the mechanical properties of collagen fiber bundles.

[0010] The specific DCC structure of the present invention, with its abundant carboxyl groups, can induce biomimetic mineralization of collagen. The inventors discovered that compared to CMC-induced biomimetic mineralized collagen fibers, DCC-induced biomimetic mineralized collagen fibers exhibited a more uniform mineralization structure, thicker fibers, and cross-linked fibers into bundles. This structure is more conducive to restoring the mechanical properties of demineralized dentin collagen.

[0011] Preferably, the weight average molecular weight of the dialdehyde carboxymethyl chitosan is >40 kDa (degree of polymerization n>195).

[0012] Preferably, the method for preparing the dialdehyde carboxymethyl chitosan comprises the following steps:

[0013] (1) chitosan, alkali, and deionized water are uniformly mixed and alkalized to obtain a mixed solution A, which is then heated to 40-55° C. after adding isopropyl alcohol to obtain a mixed solution B;

[0014] (2) dissolving chloroacetic acid in isopropyl alcohol and adding the solution dropwise to the mixed solution B to obtain a mixed solution C after the reaction is complete. The mixed solution C is mixed with an alcohol compound to perform an alcohol precipitation reaction to obtain a precipitated product. The precipitated product is freeze-dried to obtain carboxymethyl chitosan;

[0015] (3) The carboxymethyl chitosan, acid, and deionized water are mixed uniformly, heated to (37-44)° C., sodium periodate is added, and the mixture is reacted in the dark. After cooling, a mixed solution D is obtained, and the mixed solution D is mixed with an alcohol compound for alcohol precipitation reaction to obtain a precipitated product. The precipitated product is freeze-dried to obtain dialdehyde carboxymethyl chitosan.

[0016] The present invention provides a method for preparing the dialdehyde carboxymethyl chitosan. By regulating the carboxymethylation reaction conditions, the method prepares carboxymethyl chitosan (O-CMC) using chitosan as a raw material, wherein the carboxymethyl group is substituted only at the O-position. O-CMC has a 1-amino-2-hydroxy structure and can be oxidized with sodium periodate at room temperature to produce dialdehyde, thereby introducing aldehyde groups into CMC.

[0017] Preferably, in step (1), the weight-to-volume ratio of chitosan, alkali, and deionized water is chitosan: alkali: deionized water = (4.0-4.4 g): (3.3-3.6) g: 100 mL; the alkalization and swelling time is 2-5 hours; and the weight ratio of isopropyl alcohol and mixed solution A is isopropyl alcohol: mixed solution A = (1.2-1.4): 1.

[0018] Preferably, in step (2), the weight ratio of chloroacetic acid to isopropanol is chloroacetic acid: isopropanol = (18-24) g: 100 mL; the volume ratio of the isopropanol solution of chloroacetic acid in mixed solution C to mixed solution A is chloroacetic acid isopropanol solution: mixed solution A = (0.10-0.12): 1, the dropwise addition time should be 15-30 minutes; the reaction time is 4-6 hours; the weight ratio of mixed solution C to anhydrous ethanol is mixed solution C: anhydrous ethanol = 1: (1.0-1.3).

[0019] Preferably, in step (3): in the mixed solution D, the concentration of carboxymethyl chitosan in the aqueous solution formed by mixing with water is 0.1-20 mg / mL, the mass ratio of carboxymethyl chitosan to sodium periodate is carboxymethyl chitosan: sodium periodate = 1: (0.8-1), the ratio of carboxymethyl chitosan to acid is carboxymethyl chitosan: acid = 1 g: (0.95-1.1) mL, the acid is preferably (0.6-0.7) mL hydrochloric acid + (0.35-0.4) mL glacial acetic acid, and the time for the light-proof reaction is 4-6 h; the volume ratio of the mixed solution D and anhydrous ethanol is mixed solution D: anhydrous ethanol = 1: (1.5-3).

[0020] Preferably, in step (1), the base is at least one of sodium hydroxide and potassium hydroxide; in step (3), the acid is at least one of glacial acetic acid and hydrochloric acid.

[0021] Preferably, the alcohol compounds described in step (2) and step (3) are independently selected from C 1～6 Small molecule polyols, the C 1～6 The small molecule polyol can specifically be at least one of methanol, ethanol, propanol, ethylene glycol, and glycerol.

[0022] Preferably, the pH of the alcohol precipitation reaction in step (3) is 6.4-7.2.

[0023] Preferably, in step (3), to improve the purity, the precipitated product obtained by the alcohol precipitation reaction can also be dissolved in deionized water and dialyzed, and then the dialyzed precipitated product is freeze-dried.

[0024] In addition, the present invention provides the use of the dialdehyde carboxymethyl chitosan in a biomimetic mineralized dentin bonding system.

[0025] Based on the principles of dentin total acid etching, collagen biomimetic mineralization, and ethanol wet bonding, this invention introduces DCC into a biomimetic mineralized dentin bonding system, endowing it with the functions of resisting bacterial adhesion, inhibiting MMPs activity, cross-linking dentin collagen, and inducing dentin collagen biomimetic remineralization.

[0026] Preferably, the biomimetic mineralized dentin bonding system comprises the following components: a pretreatment agent, a water remover, a primer, and an adhesive.

[0027] The pretreatment agent comprises the following components: dialdehyde carboxymethyl chitosan, calcium chloride dihydrate, and dipotassium hydrogen phosphate; the weight ratio of the dialdehyde carboxymethyl chitosan, calcium chloride dihydrate, and dipotassium hydrogen phosphate to water is dialdehyde carboxymethyl chitosan: calcium chloride dihydrate: dipotassium hydrogen phosphate: water = (5-20): (0.95-1.05): (0.7-0.78): 1000; the dehydrating agent is anhydrous ethanol;

[0028] The primer comprises the following components: anhydrous ethanol, tetraethylene glycol dimethacrylate (TEGDMA), bisphenol A glycerol dimethacrylate (Bis-GMA), CQ, and ethyl benzoate (EDMAB); the weight ratio of anhydrous ethanol, tetraethylene glycol dimethacrylate (TEGDMA), bisphenol A glycerol dimethacrylate (Bis-GMA), camphorquinone (CQ), and ethyl benzoate (EDMAB) in the primer is anhydrous ethanol: tetraethylene glycol dimethacrylate (TEGDMA): bisphenol A glycerol dimethacrylate (Bis-GMA): camphorquinone (CQ): ethyl benzoate (EDMAB) = (3.8-4.2): (2.5-3.5): (0.5-1): (0.07-0.08): (0.07-0.08);

[0029] The adhesive comprises the following components: diurethane dimethacrylate (UDMA), tetraethylene glycol dimethacrylate (TEGDMA), bisphenol A glycerol dimethacrylate (Bis-GMA), camphorquinone (CQ), and ethyl benzoate (EDMAB); the diurethane dimethacrylate (UDMA), tetraethylene glycol dimethacrylate (TEGDMA), bisphenol A glycerol dimethacrylate (Bis-GMA) in the adhesive are ), camphorquinone (CQ), and ethyl benzoate (EDMAB) are in a weight ratio of diurethane dimethacrylate (UDMA): tetraethylene glycol dimethacrylate (TEGDMA): bisphenol A glycerol dimethacrylate (Bis-GMA): camphorquinone (CQ): ethyl benzoate (EDMAB) = (2.0-2.4): (2.0-2.4): (0.6-1): (0.05-0.06): (0.05-0.06).

[0030] The biomimetic mineralized dentin bonding system described in this invention utilizes ethanol wet bonding technology, which promotes dehydration of the system and shifts the equilibrium of the Schiff base dehydration reaction to the right. Furthermore, the resin component eliminates the need for hydrophilic monomers, effectively reducing the risk of microleakage and bonding layer degradation. Consequently, this bonding system achieves a stronger and more durable resin-to-dentin bond.

[0031] In this bonding system, ethanol is innovatively used as an adhesive solvent. Its dehydrating effect can remove the water generated by the Schiff base reaction between DCC and collagen cross-linking, shifting the equilibrium in the positive direction, making the cross-linking reaction more complete, and improving the mechanical properties and anti-aging properties of collagen fibers. At the same time, ethanol wet bonding is also an improved total acid etching bonding method proposed based on the solubility parameter theory. By treating dentin with anhydrous ethanol, the water in the dentin collagen is replaced with ethanol. The ethanol solvent can induce the relatively hydrophobic Bis-GMA monomer to penetrate into the ethanol-saturated dentin collagen matrix, eliminating the need for the addition of hydrophilic monomers that are easily hydrolyzed and aged in the bonding system, avoiding phase separation of the mixed layer, and improving the durability of the bond.

[0032] Preferably, the preparation method of the pretreatment agent comprises the following steps: uniformly mixing dialdehyde carboxymethyl chitosan and calcium chloride solution, and then adding dipotassium hydrogen phosphate to obtain the pretreatment agent;

[0033] The preparation method of the primer comprises the following steps: mixing anhydrous ethanol, tetraethylene glycol dimethacrylate (TEGDMA), bisphenol A glycerol dimethacrylate (Bis-GMA), camphorquinone (CQ), and ethyl benzoate (EDMAB) uniformly and storing the mixture in the dark to obtain the primer;

[0034] The preparation method of the adhesive comprises the following steps: uniformly mixing diurethane dimethacrylate (UDMA), tetraethylene glycol dimethacrylate (TEGDMA), bisphenol A glycerol dimethacrylate (Bis-GMA), camphorquinone (CQ), and ethyl benzoate (EDMAB), and then storing the mixture in the dark to obtain the adhesive.

[0035] Preferably, the method for using the biomimetic mineralized dentin bonding system comprises the following steps:

[0036] (1) Clean tooth cavities;

[0037] (2) Treat the tooth surface with a pretreatment agent for 1-3 minutes, remove water and blow dry, then apply primer, blow dry and light cure, apply adhesive, blow thin and light cure to complete dentin bonding.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows: This study uses a specific DCC+ACP solution as a pretreatment agent to pretreat fully acid-etched dentin, which has the following advantages over the current dentin bonding system: (1) DCC induces collagen biomimetic mineralization, improves the mechanical properties of demineralized dentin collagen, and improves the adhesion and anti-aging properties of the dentin bonding layer. (2) DCC can crosslink collagen fibers, causing the collagen synthesized in vitro to aggregate into bundles. Compared with traditional biomimetic mineralization inducers, it can better restore the natural structural morphology of collagen fibers. (3) DCC undergoes Schiff base crosslinking through the aldehyde groups it contains with the collagen amino groups, eliminating the need for additional crosslinking agents, making it easy to prepare and use, and avoiding the biological toxicity that may be caused by small molecule crosslinkers. (4) The introduction of ethanol wet bonding technology can promote the crosslinking reaction and at the same time avoid the problems of microleakage and aging of the bonding layer caused by hydrophilic monomers. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The synthesis path diagram of DCC in Example 1;

[0040] Figure 2 The infrared spectra of O-CMC and DCC in Example 1 are shown below:

[0041] Figure 3 The hydrogen nuclear magnetic resonance spectra of O-CMC and DCC in Example 1 are shown below:

[0042] Figure 4 The OD value-time line graph of DCC-ACP mineralization solution with different DCC concentrations;

[0043] Figure 5 Transmission electron microscopy and SAED images of DCC stabilizing ACP for 7 days;

[0044] Figure 6 SEM images of collagen fibers after 7 days of traditional mineralization and CMC and DCC-induced biomimetic mineralization treatment;

[0045] Figure 7 FTIR spectra of collagen fibers, DCC and DCC-crosslinked collagen fibers;

[0046] Figure 8 Thermogravimetric (TG) curves and differential thermogravimetric (DTG) curves of traditional mineralization, CMC-induced mineralization scaffolds, and DCC-induced mineralization scaffolds;

[0047] Figure 9 TEM / SAED images of collagen fibers with traditional mineralization (A), CMC-induced (B), and DCC-induced (C) biomimetic mineralization.

[0048] Figure 10Scoring of dentin microleakage test after 1000 hot and cold cycles of five pretreatment methods;*

[0049] (P<0.05)**(P<0.01)***(P<0.001)****(P<0.0001);

[0050] Figure 11 This is the Raman spectra of the mixed layer after bonding with pretreated dentin of DCC, DCC / BR, TR and control group;

[0051] Figure 12 The graph shows the results of micro-tensile test on dentin after five pretreatments. The left column in each group indicates the tensile strength after 24 hours of water storage, and the right column indicates the tensile strength after 1000 hot and cold cycles. *(P<0.05)**(P<0.01)***(P<0.001)****(P<0.0001);

[0052] Figure 13 Scanning electron microscopy images of the dentin surface of each group after pretreatment with five reagents;

[0053] Figure 14 Scanning electron microscopy images of the resin-dentine bonding layer in each group after pretreatment with five reagents;

[0054] Figure 15 Inhibitory rates of rhMMP9 by different concentrations of DCC; *(P<0.05)**(P<0.01)***(P<0.001)****(P<0.0001);

[0055] Figure 16 Results of MTT assay for Streptococcus mutans activity treated with 1-10 mg / ml DCC; *(P<0.05)**(P<0.01)***(P<0.001)****(P<0.0001);

[0056] Figure 17 The results of MTT test on the biofilm activity of the samples in each group; *(P<0.05)**(P<0.01)***(P<0.001)****(P<0.0001);

[0057] Figure 18 Figure 3 shows the results of live / dead bacteria staining and semi-quantitative analysis of biofilm on the surface of samples in each group. The same letters indicate no statistical difference among the groups (P<0.05). DETAILED DESCRIPTION

[0058] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to the accompanying drawings and specific examples. In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials and reagents used are all commercially available unless otherwise specified.

[0059] Example 1

[0060] A dialdehyde carboxymethyl chitosan, the synthesis path is as follows Figure 1 As shown, the preparation method of dialdehyde carboxymethyl chitosan comprises the following steps:

[0061] (1) Chitosan, NaOH, and deionized water were mixed evenly and alkalized and expanded for 2 h to obtain mixed solution A. Isopropyl alcohol was added and the temperature was raised to 50°C to obtain mixed solution B. The weight-to-volume ratio of chitosan, alkali, and deionized water was chitosan: alkali: deionized water = 5 g: 4.2 g: 120 mL; the volume ratio of isopropyl alcohol to mixed solution A was isopropyl alcohol: mixed solution A = 150 mL: 120 mL.

[0062] (2) Dissolve chloroacetic acid in isopropanol and add it dropwise to the mixed solution B. React at 50°C for 5 hours. After the reaction is complete, obtain mixed solution C. Add anhydrous ethanol, let it settle, centrifuge to remove the supernatant, and freeze-dry to obtain carboxymethyl chitosan. The weight-to-volume ratio of chloroacetic acid and isopropanol is chloroacetic acid: isopropanol = 6.5g:30mL; the volume ratio of chloroacetic acid isopropanol solution and mixed solution B is chloroacetic acid isopropanol solution: mixed solution B = 30ml:270mL; the dropwise addition time of chloroacetic acid isopropanol solution should be 15-30 minutes; the volume ratio of mixed solution C and anhydrous ethanol is mixed solution C: anhydrous ethanol = 300ml:370mL.

[0063] (3) The carboxymethyl chitosan, glacial acetic acid, and deionized water were mixed uniformly, heated to 40° C., and sodium periodate was added. The mixture was allowed to react in the dark for 5 hours, cooled, and a mixed solution D was obtained. The pH was adjusted to 7, and anhydrous ethanol was added. After standing and settling, the supernatant was removed and freeze-dried to obtain dialdehyde carboxymethyl chitosan. The weight-to-volume ratio of carboxymethyl chitosan, glacial acetic acid, and deionized water was carboxymethyl chitosan: glacial acetic acid: deionized water: sodium periodate = 3 g: 1.15 ml: 100 ml: 3 g. The volume ratio of mixed solution D to anhydrous ethanol was mixed solution D: anhydrous ethanol = 100 ml: 250 ml.

[0064] Application Example 1

[0065] The dialdehyde carboxymethyl chitosan prepared in Example 1 of the present invention is applied to a biomimetic mineralized dentin bonding system, wherein the biomimetic mineralized dentin bonding system comprises the following components: a pretreatment agent, a water scavenger, a primer, and an adhesive;

[0066] The pretreatment agent comprises the following components: dialdehyde carboxymethyl chitosan, calcium chloride dihydrate, and dipotassium hydrogen phosphate; the weight-to-volume ratio of the dialdehyde carboxymethyl chitosan, calcium chloride dihydrate, and dipotassium hydrogen phosphate is dialdehyde carboxymethyl chitosan: calcium chloride dihydrate: dipotassium hydrogen phosphate: water = 100 mg: 10 mg: 7.4 mg: 10 ml;

[0067] The dehydrating agent is anhydrous ethanol;

[0068] The primer comprises the following components: anhydrous ethanol, tetraethylene glycol dimethacrylate (TEGDMA), bisphenol A glycerol dimethacrylate (Bis-GMA), CQ, and ethyl benzoate (EDMAB); the weight ratio of anhydrous ethanol, tetraethylene glycol dimethacrylate (TEGDMA), bisphenol A glycerol dimethacrylate (Bis-GMA), camphorquinone (CQ), and ethyl benzoate (EDMAB) in the primer is anhydrous ethanol: tetraethylene glycol dimethacrylate (TEGDMA): bisphenol A glycerol dimethacrylate (Bis-GMA): camphorquinone (CQ): ethyl benzoate (EDMAB) = 50:40:8:1:1;

[0069] The adhesive comprises the following components: diurethane dimethacrylate (UDMA), tetraethylene glycol dimethacrylate (TEGDMA), bisphenol A glycerol dimethacrylate (Bis-GMA), camphorquinone (CQ), and ethyl benzoate (EDMAB); the weight ratio of diurethane dimethacrylate (UDMA), tetraethylene glycol dimethacrylate (TEGDMA), bisphenol A glycerol dimethacrylate (Bis-GMA), camphorquinone (CQ), and ethyl benzoate (EDMAB) in the adhesive is: diurethane dimethacrylate (UDMA): tetraethylene glycol dimethacrylate (TEGDMA): bisphenol A glycerol dimethacrylate (Bis-GMA): camphorquinone (CQ): ethyl benzoate (EDMAB) = 40:45:13:1:1.

[0070] Application Example 2

[0071] The dialdehyde carboxymethyl chitosan prepared in Example 1 of the present invention is applied to a biomimetic mineralized dentin bonding system, wherein the biomimetic mineralized dentin bonding system comprises the following components: a pretreatment agent, a water scavenger, a primer, and an adhesive;

[0072] The pretreatment agent comprises the following components: dialdehyde carboxymethyl chitosan, calcium chloride dihydrate, and dipotassium hydrogen phosphate; the weight-to-volume ratio of the dialdehyde carboxymethyl chitosan, calcium chloride dihydrate, and dipotassium hydrogen phosphate is dialdehyde carboxymethyl chitosan: calcium chloride dihydrate: dipotassium hydrogen phosphate: water = 50 mg: 10 mg: 7.4 mg: 10 ml;

[0073] The dehydrating agent is anhydrous ethanol;

[0074] The primer comprises the following components: anhydrous ethanol, tetraethylene glycol dimethacrylate (TEGDMA), bisphenol A glycerol dimethacrylate (Bis-GMA), CQ, and ethyl benzoate (EDMAB); the weight ratio of anhydrous ethanol, tetraethylene glycol dimethacrylate (TEGDMA), bisphenol A glycerol dimethacrylate (Bis-GMA), camphorquinone (CQ), and ethyl benzoate (EDMAB) in the primer is anhydrous ethanol: tetraethylene glycol dimethacrylate (TEGDMA): bisphenol A glycerol dimethacrylate (Bis-GMA): camphorquinone (CQ): ethyl benzoate (EDMAB) = 50:35:13:1:1;

[0075] The adhesive comprises the following components: diurethane dimethacrylate (UDMA), tetraethylene glycol dimethacrylate (TEGDMA), bisphenol A glycerol dimethacrylate (Bis-GMA), camphorquinone (CQ), and ethyl benzoate (EDMAB); the weight ratio of diurethane dimethacrylate (UDMA), tetraethylene glycol dimethacrylate (TEGDMA), bisphenol A glycerol dimethacrylate (Bis-GMA), camphorquinone (CQ), and ethyl benzoate (EDMAB) in the adhesive is: diurethane dimethacrylate (UDMA): tetraethylene glycol dimethacrylate (TEGDMA): bisphenol A glycerol dimethacrylate (Bis-GMA): camphorquinone (CQ): ethyl benzoate (EDMAB) = 40:40:18:1:1.

[0076] Application Example 3

[0077] The dialdehyde carboxymethyl chitosan prepared in Example 1 of the present invention is applied to a biomimetic mineralized dentin bonding system. The biomimetic mineralized dentin bonding system comprises the following components: a pretreatment agent, a water scavenger, a primer, and an adhesive;

[0078] The pretreatment agent comprises the following components: dialdehyde carboxymethyl chitosan, calcium chloride dihydrate, and dipotassium hydrogen phosphate; the weight-to-volume ratio of the dialdehyde carboxymethyl chitosan, calcium chloride dihydrate, and dipotassium hydrogen phosphate is dialdehyde carboxymethyl chitosan: calcium chloride dihydrate: dipotassium hydrogen phosphate: water = 80 mg: 10 mg: 7.4 mg: 10 ml;

[0079] The dehydrating agent is anhydrous ethanol;

[0080] The primer comprises the following components: anhydrous ethanol, tetraethylene glycol dimethacrylate (TEGDMA), bisphenol A glycerol dimethacrylate (Bis-GMA), CQ, and ethyl benzoate (EDMAB); the weight ratio of anhydrous ethanol, tetraethylene glycol dimethacrylate (TEGDMA), bisphenol A glycerol dimethacrylate (Bis-GMA), camphorquinone (CQ), and ethyl benzoate (EDMAB) in the primer is anhydrous ethanol: tetraethylene glycol dimethacrylate (TEGDMA): bisphenol A glycerol dimethacrylate (Bis-GMA): camphorquinone (CQ): ethyl benzoate (EDMAB) = 50:33:15:1:1;

[0081] The adhesive comprises the following components: diurethane dimethacrylate (UDMA), tetraethylene glycol dimethacrylate (TEGDMA), bisphenol A glycerol dimethacrylate (Bis-GMA), camphorquinone (CQ), and ethyl benzoate (EDMAB); the weight ratio of diurethane dimethacrylate (UDMA), tetraethylene glycol dimethacrylate (TEGDMA), bisphenol A glycerol dimethacrylate (Bis-GMA), camphorquinone (CQ), and ethyl benzoate (EDMAB) in the adhesive is: diurethane dimethacrylate (UDMA): tetraethylene glycol dimethacrylate (TEGDMA): bisphenol A glycerol dimethacrylate (Bis-GMA): camphorquinone (CQ): ethyl benzoate (EDMAB) = 35:45:18:1:1.

[0082] The method for using the biomimetic mineralized dentin bonding system of the present invention comprises the following steps:

[0083] (1) Clean the tooth cavity; etch the cavity with 35% phosphoric acid for 20 seconds, rinse for 20 seconds, and blow gently until half dry;

[0084] (2) Apply an appropriate amount of pretreatment agent to the tooth surface with a cotton swab for 20 seconds. Blow gently until semi-dry. Apply anhydrous ethyl acetate to the tooth surface with a cotton swab for 15 seconds. Blow gently until semi-dry. Apply resin primer. Continue applying for 15 seconds and then blow dry. Light cure for 20 seconds, apply adhesive, blow thinly, and light cure for 20 seconds to complete dentin bonding and proceed to the next step of repair.

[0085] Test Example 1 Characterization of dialdehyde carboxymethyl chitosan

[0086] Infrared FTIR detection: O-carboxymethyl chitosan and dialdehyde carboxymethyl chitosan powders were scanned using a Fourier transform infrared spectrometer (Nicolet 6700-Continuous μm, Thermo Scientific, USA). The average of 32 scan signals was set for each measurement, with a resolution of 2 cm-1 , wavelength range is 400-4000cm -1 Graphpad Prism 7 software was used to plot the infrared spectra of each DCC group.

[0087] H NMR spectrum detection: O-carboxymethyl chitosan and dialdehyde carboxymethyl chitosan powders were scanned using a Fourier transform infrared spectrometer (Nicolet 6700-Continuum, Thermo Scientific, USA). The average of 32 scan signals was set for each measurement, with a resolution of 2 cm -1 , wavelength range is 400-4000cm -1 Graphpad Prism 7 software was used to plot the infrared spectra of each DCC group.

[0088] Elemental Analysis: Chitosan, O-carboxymethyl chitosan, and dialdehyde carboxymethyl chitosan powders were separately sampled and their carbon and nitrogen contents were measured using an elemental analyzer (Vario EL, Elementar, Germany). The C / N mass ratios of chitosan, O-carboxymethyl chitosan, and dialdehyde carboxymethyl chitosan were converted to C / N atomic ratios, denoted as k1, k2, and k3, respectively. The degree of substitution (DS) was calculated using the following formula:

[0089] DS -CH2COOH =3(k2-k1) / k1;

[0090] DS -CHO =2(k3-k2) / k3.

[0091] Table 1 Elemental analysis of C / N atomic ratio and degree of substitution of carboxymethyl and aldehyde groups in O-carboxymethyl chitosan and dialdehyde carboxymethyl chitosan

[0092] Chitosan O-Carboxymethyl Chitosan Dialdehyde carboxymethyl chitosan C / N 6.55±0.01 8.40±0.02 10.06±0.43 Degree of substitution - 0.85±0.02 0.33±0.06

[0093] FTIR detection of DCC and intermediate product O-CMC prepared in Example 1 showed the following results: Figure 2 As shown, the O-CMC group is at 1620 cm -1 and 1157cm -1 The carboxyl peak and ether bond peak appeared at 1730 cm -1 A new aldehyde absorption peak appeared at 2940 cm -1 The characteristic weak doublet peak of aldehyde group can be seen at 1360~1020cm -1 The CN absorption peak at 881 cm is lower than that of the O-CMC group, indicating that the number of amino groups in DCC is reduced compared with O-CMC and is oxidized to aldehyde groups. -1, 757cm -1 A new strong CH stretching vibration peak can be seen in the DCC group, which further confirms the aldehyde grafting.

[0094] H 1 -NMR detection of DCC and intermediate product O-CMC prepared in Example 1, the results are as follows Figure 3 As shown, DCC and O-CMC have the same characteristic peaks af. However, a new -CHO resonance signal appeared in DCC at 8.0-8.5ppm (x peak). Since the solvent is heavy water, the carboxyl hydrogen ion is dissociated, so the carboxyl signal peak is not found in the hydrogen spectrum. In addition, in the spectrum of the DCC group, the -OH peak at 5.0-5.2ppm (a peak), the -NH2 signal peak at 3.5-3.7ppm (d peak), and the -CH(O-)- signal peak at 2.0-2.5ppm (e peak) are all reduced compared with the O-CMC group. The results can support the synthesis route of this study.

[0095] The C / N content ratios of DCC and O-CMC prepared in Example 1 were analyzed by elemental analysis. The results and the calculated carboxymethyl and aldehyde substitution degrees are shown in Table 1. The carboxymethyl substitution degree in O-CMC is approximately 0.85, and the aldehyde substitution degree in DCC is approximately 0.33.

[0096] Test Example 2 Analysis of DCC Stabilization ACP Effect

[0097] Test process:

[0098] A calcium phosphate turbidity assay was used to assess the ability of various concentrations of DCC to stabilize ACP. A TBS solution (pH 7.4), 9 mM CaCl2·2H2O, and 4.2 mM K2HPO4 solution were prepared. DCC was added to 5 mL of CaCl2 solution at various concentrations (0.5, 1, 3, 5, 7, 10, 15, and 20 mg / mL), then mixed with 5 mL of K2HPO4 solution and thoroughly mixed. The solution was incubated at 37°C. 100 μL of the solution was transferred to a 96-well plate at 0, 24, 48, 72, 96, 120, 144, and 168 hours, respectively. Ultrapure water was used as a control. The absorbance at 650 nm was measured using an automated microplate reader. Three samples were collected at each time point. Data were analyzed and plotted using GraphPad Prism 7.

[0099] The results are as follows Figure 4 As shown, the supersaturated calcium phosphate solution without DCC began to precipitate minerals on the first day. The solution containing 0.1 mg / mL DCC-ACP began to precipitate on the second day. Groups containing 0.5 mg / mL and above were able to stabilize ACP without precipitation for 7 days.

[0100] Transmission electron microscopy observation of ACP agglomerates stabilized with 5 mg / mL DCC after 7 days. A supersaturated calcium phosphate solution stabilized with 5 mg / mL DCC was prepared and allowed to stand for 7 days. 5 μL was taken and placed on a carbon-containing nickel grid for transmission electron microscopy. After drying, it was observed under a 120 kV TEM. Selected area electron diffraction (SAED) was performed on specific areas to detect particle properties. ImageJ 1.51k was used to measure the diffraction ring radius and clarify the particle morphology. Figure 5 After seven days of DCC-stabilized ACP, the particle diameter was approximately 100 nm. SAED diffraction showed no obvious single or polycrystalline structures, but visible diffraction halos from calcium and phosphorus atoms, indicating that DCC-stabilized ACP can maintain its amorphous state after seven days in solution.

[0101] Experimental Example 3: Analysis of DCC-induced collagen fiber biomimetic effects

[0102] Preparation of mineralized collagen samples in each group:

[0103] Rat tail type I collagen was extracted according to the modified Price method

[83] . Sprague-Dawley (SD) rat tail tendons were extracted, washed sequentially with acetone, isopropanol, ethanol, and PBS, and then dissolved in 0.3 M glacial acetic acid. The collagen solution was centrifuged at 4°C and 3000 rpm for 30 min. The supernatant was placed in a 3000 Da dialysis bag and dialyzed against 0.02 M K2HPO4 solution at room temperature for 5-7 days. The collagen gel in the dialysis bag was removed, rinsed repeatedly with ultrapure water, and freeze-dried. The freeze-dried collagen was redissolved in 0.03 M acetic acid at 2 mg / ml. Phenolphthalein was used as an indicator, and 0.3 M NaOH was added dropwise under magnetic stirring to neutralize the collagen-acetic acid solution until the phenolphthalein just changed color. 600 μl of the neutralized solution was quickly added to a 24-well plate, incubated at 37°C for 24 hours, and freeze-dried to obtain the collagen sample.

[0104] Mineralization induction solutions were prepared using 5 mg / ml DCC and CMC as mineralization inducers, respectively, as described above. A conventional mineralization control group without the addition of mineralization inducers served as a control. The prepared collagen samples were placed in the aforementioned mineralization induction solutions and stored at 37°C. After 7 days of mineralization, the samples were removed, washed with ultrapure water, and freeze-dried to produce DCC- and CMC-induced mineralization, as well as conventionally mineralized collagen samples.

[0105] Sample observation:

[0106] SEM observation of traditional mineralization and DCC, CMC (common commercially available) induced biomimetic mineralized collagen fibers. Each group of biomimetic mineralized collagen samples was fixed on the sample stage, sprayed with gold, and the collagen morphology of each group was observed under a cold field emission scanning electron microscope (JSM-6330F, JEOL, Japan) at a voltage of 10kV. The results are shown in Figure 2. Figure 6As shown, the horizontal striations in all three collagen groups disappeared, indicating that mineralization occurred in all three groups, but the surface morphology differed significantly. The collagen fibers in the traditional mineralization group had smaller diameters, and nodular mineral deposits were visible on the surface. The collagen surface in the CMC-induced biomimetic mineralization group was smoother, but the collagen diameters varied. This may be due to the local expansion of the mineralized collagen, while the diameter of the non-mineralized areas remained unchanged. The collagen diameter in the DCC-induced biomimetic mineralization group increased, but the size was more uniform, and multiple collagen fibers were observed to aggregate in bundles.

[0107] FTIR was used to detect the chemical groups of DCC cross-linked unmineralized collagen fibers. A 5 mg / ml concentration of DCC was added to the rat tail type I collagen described above. After 24 hours of reaction, the DCC solution was aspirated, the collagen was rinsed three times with PBS, and lyophilized. Scanning was performed using a Fourier transform infrared spectrometer (Nicolet 6700-Continuous μm, Thermo Scientific, USA), with the average of 32 scan signals per measurement set to 2 cm⁻¹ and a wavelength range of 400–4000 cm⁻¹. -1 Graphpad Prism 7 software was used to draw the infrared spectra of each group of DCC. Figure 7 As shown, the newly appeared 1693cm -1 -C=N-peak, and 1730cm -1 The -CHO peak at 3200 cm -1 The characteristic double peak of amino groups at 147° disappeared. This result indicates that the aldehyde groups of DCC cross-linked with the amino groups of collagen fibers through Schiff base reaction.

[0108] Thermogravimetric analyzer was used to test three groups of mineralized collagen fibers. Each group of mineralized collagen samples was taken and heated from 30°C to 800°C at a rate of 5°C / min using a thermogravimetric analyzer in a nitrogen atmosphere. Graphpad Prism7 was used to plot the mass-temperature curve of each group of mineralized collagen, and the first-order derivative of the thermogravimetric curve with respect to temperature was calculated to plot the derivative thermogravimetric analysis (DTG) curve. The results are shown in Figure 2. Figure 8As shown. TR: traditional mineralization, CMC: CMC-induced biomimetic mineralization, DCC: DCC-induced biomimetic mineralization. The final mass fractions of the TR, CMC, and DCC groups were 42.97%, 38.39%, and 34.93%, respectively. Mineralized material content: traditional mineralization group > CMC-induced biomimetic mineralization group > DCC-induced biomimetic mineralization group. This is presumably due to the additional DCC cross-linking on the collagen surface, resulting in a relatively high mass of organic components and a decrease in the relative mass of the mineralized material. This inference is supported by the higher residual mass in the DCC group during the plateau phase at 400-600°C in the TG images. DTG images show a higher temperature during the rapid decomposition phase in the DCC group than in the CMC group, presumably due to DCC cross-linking collagen, which increases its thermal stability. Although the traditional mineralization group had the highest mineralized material content, its mass decreased by approximately 25% during the rapid decomposition phase at 30-100°C, indicating that the collagen contained a large amount of bound water and that the mineralized components were primarily calcium phosphate hydrates. The DCC and CMC-induced biomimetic mineralization groups showed a smaller mass decrease in the 30-100°C stage, indicating that HA crystals replaced water in collagen during the biomimetic mineralization process, the degree of hydration of HA crystals in collagen was lower, and the crystal structure was more stable.

[0109] TEM was used to observe the collagen structure of each mineralized scaffold. Each mineralized collagen was ground into a fine powder in liquid nitrogen and fully dispersed in 75% ethanol. 5 μl of each sample was loaded onto a carbon-containing nickel transmission electron microscope grid, stained with 1% uranyl acetate for 1 minute, and rinsed with ultrapure water. After drying, the grid was observed under a 120 kV TEM, and selected area SAED was used to detect the mineral components in the collagen fibers. Figure 9 As shown, the mineralized crystals of collagen fibers in the conventional mineralization group are located on the surface of the collagen fibers, resulting in low mineralization efficiency. The crystals within the biomimetic mineralized collagen fibers induced by CMC are heterogeneous, exhibiting different forms of mineralization in different regions, including extra-fiber mineralization, uneven intra-fiber mineralization, and relatively complete intra-fiber mineralization. This is presumably due to the failure to replace the mineralization solution within seven days. The biomimetic mineralized collagen fibers induced by DCC exhibit relatively homogeneous intra-fiber mineralization, and low-power microscopy reveals that the collagen fibers are aggregated into bundles. This demonstrates that DCC crosslinks collagen fibers and induces efficient biomimetic mineralization.

[0110] Test Example 4: Adhesion Effect Detection of DCC Bionic Mineralized Adhesive

[0111] In vitro tooth bonding microleakage tests and microtensile tests were performed using supersaturated calcium phosphate solution (Traditional remineralization, TR), 5 mg / mL DCC solution (DCC), 5 mg / mL DCC-stabilized supersaturated calcium phosphate solution (Biomimetic remineralization, DCC / BR), 5 mg / mL CMC-stabilized supersaturated calcium phosphate solution (CMC / BR) and deionized water (Control) as pretreatment agents.

[0112] Microleakage experiments were conducted using 24 ex vivo human premolars. Class V cavities with a diameter of 3 mm and a depth of 2 mm were created on the buccal and lingual sides of the crowns using a high-speed turbine. The gingival wall of the cavity was located 1 mm above the cementoenamel junction. The specimens were then randomly divided into four groups (n = 6) based on the combination of maxillary and mandibular premolars.

[0113] Bonding steps: After cleaning the tooth sample with running water, 35% phosphoric acid etchant was used to etch the cavity for 20 seconds, rinsed for 20 seconds, and gently blown until semi-dry. The tooth surface was pretreated with 5mg / ml DCC solution, supersaturated calcium phosphate solution, and biomimetic mineralization induction solution (supersaturated calcium phosphate solution containing 5mg / ml DCC) for 2 minutes respectively. Deionized water was used as a control and gently blown until semi-dry. Anhydrous ethanol was added to the cavity to pretreat the demineralized dentin twice, each for 1 minute, and gently blown until semi-dry. The resin primer was gently applied for 15 seconds, blown until semi-dry, and then light-cured for 20 seconds. The adhesive was applied for 15 seconds, blown thinly with air, and light-cured for 20 seconds. The cavity was filled with composite resin Z350 by the same operator and polished.

[0114] Using a hot and cold cycler, the sample teeth were placed alternately in hot and cold water at (5±2)°C and (55±2)°C, repeating the cycle 1000 times with a 30-second dwell time and a 5-second switching time. The sample teeth were dried, the apical foramen sealed with heated red wax, and the tooth surface within 1 mm of the filling margin was covered with a double layer of nail polish. The sample teeth were placed in 2% methylene blue stain and stored in a 37°C incubator for 24 hours. After staining, the teeth were rinsed and dried, and the crowns were cut longitudinally along the buccal and lingual directions toward the long axis of the tooth using a wheel cutter. The test surface was polished with 1600-grit water-abrasive paper and observed under a stereomicroscope to evaluate the dye penetration in each sample.

[0115] Leakage rating standards

[86] :

[0116] Grade 0: No dye penetration.

[0117] Level I: The dye penetrates into the cave wall, with a depth ≤ 1 / 3 of the cave depth.

[0118] Level II: The dye penetrates into the cave wall, with a depth ≤ 2 / 3 of the cave depth.

[0119] Level III: The dye has penetrated the cave wall and is flush with the cave floor.

[0120] Grade IV: The dye penetrates the cavity wall and involves the medullary cavity.

[0121] Microtensile testing: Forty intact, caries-free human molars were collected from the ex vivo specimens. The teeth were stored in chloramine T at 4°C and used within one month after extraction. A wheel cutter (YWQ-50S, China) was used to cut the teeth horizontally under cooling to expose the dentin in the mid-coronal section. The dentin surface was polished with 600-grit silicon carbide sandpaper at 120 rpm and then set aside.

[0122] The excised teeth were randomly divided into four groups (n = 10). The excised teeth in each group were bonded as described above. Five teeth were stored in deionized water for 24 hours before microtensile testing. The remaining five teeth were placed in a hot-cold cycler with a dwell time of 30 seconds and a transition time of 5 seconds. The cold water tank was set at (5 ± 2)°C and the hot water tank was set at (55 ± 2)°C for 1000 cycles.

[0123] Each group of teeth was cut into strip specimens with a cross-sectional area of approximately 0.9 mm × 0.9 mm along the long axis of the tooth using a tooth wheel cutter under running water cooling. The specimens were fixed to the test mold of a universal mechanical tester (ElectroPuls E3000, UK) using cyanoacrylate adhesive. The tensile test was performed at a speed of 0.7 mm / min in the tensile mode. The tensile test was stopped when the specimen broke. The maximum tensile stress during the tensile process was recorded, and the specimen area was measured with a vernier caliper. The formula for calculating the microtensile strength is as follows:

[0124] Tensile strength (MPa) = (maximum load (N)) / (cross-sectional area (mm^ 2 )).

[0125] The result is as follows:

[0126] Microleakage test results ( Figure 10 ) showed that dentin Class V cavities treated with the four pretreatment agents all had microleakage after 1000 hot and cold cycles. The group pretreated with mineralization-inducing solution containing 5 mg / ml DCC achieved the lowest microleakage score, while the group pretreated with 5 mg / ml DCC solution alone had a higher microleakage score than the other three groups.

[0127] Raman spectroscopy was used to analyze the composition of the dentin bonding layer and the 960 cm -1 The Raman intensity (RI) at 960 cm-1 characterizes the apatite content, and the -1 / 1450cm -1 The RI ratio characterizes the relative content of mineral components and organic components. The results show that ( Figure 11, Table 1). A large amount of apatite deposited in the mixed layer of the DCC-stabilized biomimetic mineralization solution pretreatment group and the traditional mineralization solution pretreatment group. The RI values reached 2.45 and 2.29, respectively, which were much higher than the control group (1.29) and the DCC group (0.96). This indicates that mineralized material was deposited in the mixed layer in both the TR and BR groups. The mixed layer RI (960 cm) of the DCC solution pretreatment group was -1 ) value and RI(960cm -1 The ratio of RI (1450 cm-1) decreased significantly. This is presumably because DCC in the mixed layer inhibits the crystallization of calcium and phosphate ions dissolved in the dentin after acid etching and rinsing, causing the calcium and phosphate ions to deposit deeper.

[0128] Table 2 Raman spectrum RI (960 cm -1 ) and RI(960cm -1 ) / RI(1450cm -1 )Analysis results

[0129] Group <![CDATA[RI(960cm -1 )]]> <![CDATA[RI(960cm -1 ) / RI(1450cm -1 )]]> Control <![CDATA[43446.2±153.06 A ]]> <![CDATA[1.29±0.01 a ]]> TR <![CDATA[59046.76±285.11 B ]]> <![CDATA[2.45±0.01 b ]]> DCC <![CDATA[18113.92±77.92 C ]]> <![CDATA[0.96±0.01 c ]]> DCC / BR <![CDATA[41090.40±238.36 D ]]> <![CDATA[2.29±0.01 d ]]>

[0130] *Different superscript letters in each column indicate statistically significant differences (P<0.05).

[0131] Four sets of dentin cutting models were prepared, acid-etched and pre-treated with the four methods mentioned above, then bonded with the adhesive described in this patent and filled with 3M Z350 resin. The cut samples were then tested for microtensile strength. The experimental results showed that ( Figure 12 ). After 24 hours of water storage and 500 cycles of thermal cycling, the microtensile strength of the dentin group pretreated with DCC was higher than that of the other three groups. The group pretreated with DCC alone exhibited the lowest microtensile strength. The microtensile strength of all groups after 500 cycles of thermal cycling was not statistically different from that before aging. However, the mean microtensile strength of the control group and the traditional mineralization group decreased slightly, while the mean microtensile strength of the DCC-induced biomimetic mineralization group remained the same as before aging.

[0132] Studies have shown that negatively charged NCPs exist on the surface of dentin after acid etching, and the high hydration capacity of their polar groups causes a hydrogel-like structure to form on the dentin collagen fibers. The hydrogel-like interface environment acts as a "molecular sieve", hindering the penetration of adhesive monomers. The water molecules bound to NCPs also form a semi-cage structure, surrounding and hydrating the dentin collagen fibers to form a hydration layer, which in turn leads to a decrease in the durability of dentin bonding. The large amount of Ca in the supersaturated calcium phosphate solution stabilized by DCC 2+ It can bind to the carboxyl groups on the DCC surface and exclude water molecules bound to the DCC charged groups, thus facilitating the dehydration of ethanol and the penetration of resin monomers. This explains the significant decrease in adhesion in the DCC group and the significant increase in adhesion in the BR group in this study.

[0133] Experimental Example 5 Observation of dentin surface morphology after treatment with DCC biomimetic mineralization solution

[0134] Dentin surface morphology observation: Twelve intact, caries-free human premolars were collected and sliced perpendicular to the long axis of the tooth using a linear wheel cutter (YWQ-50S, China) under cooling conditions into approximately 1 mm thick slices. After etching with 37% phosphoric acid for 15 seconds, the dentin was pretreated by immersion in 5 mg / ml DCC solution (DCC), 10 mg / ml, 5 mg / ml, and 1 mg / ml DCC biomimetic mineralization induction solution (DCC / BR), 5 mg / ml CMC biomimetic mineralization induction solution (CMC / BR), and traditional mineralization induction solution (TR). Deionized water served as a control. After 2 days of treatment, the dentin was dehydrated in a gradient ethanol solution, gold was sprayed, and the dentin surface morphology of each group was observed by scanning electron microscopy.

[0135] Observation of the resin-dentine bonding layer: Samples of resin-dentine cut from the bonding experiment before the hot and cold cycles were treated with 50% phosphoric acid for 20 seconds. After cleaning, they were treated with 5% sodium hypochlorite solution for 15 minutes. This process was repeated five times. Ultrapure water ultrasonic cleaning was then performed for 20 minutes. Dehydration was performed sequentially with ethanol, followed by drying and gold coating. The morphology of the spikes in the hybrid layer was observed using a scanning electron microscope.

[0136] The result is as follows:

[0137] In the SEM images of the dentin surface morphology after pretreatment ( Figure 13 ). The peritubular dentin surfaces in the TR, DCC, CMC / BR, and 1 mg / ml DCC / BR groups were relatively flat, a result of the collapse of the acid-etched, fluffy dentin collagen during dehydration and gold spraying. Needle-shaped and spherical calcium-phosphate crystals were observed in the TR group, representing calcium phosphate deposits outside the dentin fibers. A small amount of relatively uniform calcifications was observed in the CMC / BR and 1 mg / ml DCC / BR groups, likely due to mineralization within the collagen fibers, but insufficient mechanical strength to maintain the collagen fiber morphology, resulting in collapse. In the 5 mg / ml and 10 mg / ml DCC / BR groups, significant remineralization of peritubular dentin collagen was observed, and the dentin morphology was well preserved. However, microcracks were observed in the peritubular dentin of the 10 mg / ml DCC group (indicated by white arrows). Therefore, 5 mg / ml DCC biomimetic mineralizing solution was selected as the pretreatment agent for the dentin adhesive. In the DCC and DCC / BR groups, the area of peritubular dentin decreased, and the volume of dentinal tubules increased, which may be due to the denser cross-linking of dentin collagen induced by DCC.

[0138] The SEM results of the cross section of the resin-dentin bonding layer ( Figure 14). In the control group, dense resin spikes were regularly arranged, with an average length of approximately 40-45 μm. In the DCC group, the morphology and length of the spikes were similar to those in the control group, but the number was significantly less than that in the control group. In the TR group, the number of spikes was similar to that in the control group, but the length was significantly shorter, averaging only about 20 μm, and a calcium phosphate deposition layer was visible at the base of the spikes (indicated by the white arrow). In the CMC / BR group, a calcium phosphate deposition layer was observed approximately 5 μm near the base of the spikes, and the length of the spikes was slightly shorter than that in the control group, at approximately 35-40 μm. In the DCC / BR group, the spikes were more densely distributed, with a length of up to 60 μm, and no calcium phosphate deposition was observed at the base.

[0139] Experimental Example 6: Antibacterial and MMP9 Inhibitory Effects of DCC

[0140] rhMMP9 inhibition assay: rhMMP9 was added to the activator in the SensoLyte MMP kit and activated at 37°C for 2 h. DCC was prepared in 2 ml centrifuge tubes at concentrations of 150 mg / ml, 100 mg / ml, 50 mg / ml, 25 mg / ml, 12.5 mg / ml, and 5 mg / ml. The following groups were set up according to the kit instructions: ① Negative control group (G1): rhMMP9 (10 μl), substrate (50 μl), buffer (40 μl); ② Positive control group (G2): rhMMP9 (10 μl), MMP inhibitor (10 μl), substrate (50 μl), buffer (30 μl); ③ Substrate control group (G3): substrate (50 μl), buffer (50 μl); ④ Experimental substrate control group (G4): DCC (10 μl) of each concentration group, substrate (50 μl), buffer (40 μl); ⑤ Experimental group (G5): rhMMP9 (10 μl), DCC (10 μl) of each concentration group, substrate (50 μl), buffer (30 μl).

[0141] Add each reagent to a 96-well plate and incubate at 37°C in the dark for 30 minutes. Measure the absorbance at 412 nm using a microplate reader. Calculate the substrate concentrations of each group using the standard curve and record them as C1, C2, C3, C4, and C5. Calculate the inhibition rate (IR) using the following formula:

[0142] IR control% = (C1-C2) / (C1-C3) x 100%

[0143] IR experimental % = ((C1-C3)-(C5-C4)) / (C1-C3)×100%

[0144] Direct DCC inhibition assay: Dissolve an appropriate amount of DCC in ultrapure water to prepare 20 mg / ml, 10 mg / ml, 6 mg / ml, and 2 mg / ml DCC solutions. Filter sterilize and set aside. Single colonies of frozen UA159 mutans Streptococcus were streaked onto BHI solid medium and incubated anaerobically at 37°C for 24 hours in a humidified atmosphere of 85% N₂, 5% CO₂, and 10% H₂. Single colonies were inoculated into BHI liquid medium and incubated anaerobically for 18 hours under the aforementioned conditions. The solution was diluted to 1×10⁶ CFU / ml and set aside. 50 μl of the above solution was inoculated into a 96-well plate, with five replicates per group. 50 μl of the above DCC solution was added to each group, resulting in final concentrations of 10 mg / ml, 5 mg / ml, 3 mg / ml, and 1 mg / ml. BHI medium alone served as a negative control, and the solution without drug served as a positive control.

[0145] After 24 hours of anaerobic incubation, 10 μl of 5 mg / ml MTT reagent was added to each well and incubated for another 4 hours. 100 μl of Formazan solution was then added to each well, mixed, and incubated until the purple crystals were completely dissolved under the microscope. The liquid in each well was transferred to a new 96-well plate, and a zero well (50 μl BHI medium + 50 μl ultrapure water + 10 μl MTT + 100 μl Formazan solution) was set. The absorbance at a wavelength of 570 nm was measured using a multifunctional microplate reader, and the data were analyzed and plotted using GraphPad Prism 7.

[0146] Dentin biofilm resistance after DCC pretreatment: Twelve excised human premolars were collected, and 1 mm thick dentin slices were cut perpendicular to the long axis of the teeth. The length and width were measured and the area was calculated. Six dentin slices of similar area were paired and assigned to experimental groups. Samples from each group were treated with 37% phosphoric acid for 20 seconds and then placed in biomimetic mineralization induction solution containing 1 mg / ml DCC, 5 mg / ml DCC, 10 mg / ml DCC, 5 mg / ml CMC, 5 mg / ml DCC, or traditional mineralization induction solution. After two days of treatment, the samples were immersed in 75% ethanol for 2 hours and sterilized under ultraviolet light for 4 hours before use. Mutans Streptococcus culture was performed as described previously.

[0147] Mutans Streptococcus biofilm activity assay: Four disinfected specimens from each group were placed in a 24-well plate. 1.5 ml of BHIS solution containing 1 × 106 CFU / ml of mutans Streptococcus was added to each well and incubated anaerobically at 37°C for 24 hours. The plates were then washed three times with PBS to remove floating bacteria. The specimens were transferred to a new 24-well plate. MTT reagent was diluted 1:10 with PBS and 300 μl was added to each well. After incubation at 37°C for 4 hours, Formazan solution was added and incubated until all purple crystals dissolved. After that, 100 μl was transferred to a 96-well plate and the absorbance at 570 nm was measured using a microplate reader. Three zero-adjustment wells were set (100 μl BHI medium + 10 μl MTT + 100 μl Formazan solution). The OD value of each group was subtracted from the mean OD value of the zero-adjustment wells to obtain the final result.

[0148] Live / dead staining and semiquantitative analysis of S. mutans biofilms: Four sterilized specimens from each group were placed in a 24-well plate. 1.5 ml of a BHIS solution containing 1 × 106 CFU / ml of S. mutans was added to each well and incubated anaerobically at 37°C for 24 h. The plates were then washed three times with PBS to remove floating bacteria. The specimens were transferred to a new 24-well plate, and 400 μl of live / dead stain prepared according to the product instructions was added to each well. The plates were incubated in the dark for 15 min, washed three times with PBS to remove excess stain, and air-dried. The specimens were transferred to a confocal culture dish and observed using a laser confocal microscope (FV 3000; Olympus, Japan). Laser parameters were adjusted to appropriate values to ensure no overexposed pixels. Four fields of view were randomly selected from each sample, and the z-axis plane with the highest fluorescence intensity was used to image each field. ImageJ software was used to analyze the red and green fluorescence areas, total intensities, and mean intensities of the images, and the green / red total fluorescence intensity ratio and green / red mean fluorescence intensity ratio were calculated.

[0149] The result is as follows:

[0150] The results of rhMMP9 inhibition experiments showed that ( Figure 15 ), DCC exhibited a significant inhibitory effect on rhMMP9 activity, with the inhibitory effect gradually increasing with increasing concentration. At a concentration of 5 mg / ml, DCC achieved an inhibition rate of 79.39 ± 5.37% on rhMMP9 activity. At a concentration of 10 mg / ml, the inhibition rate reached 98.46 ± 1.51%, comparable to that of the positive control MMP9 inhibitor.

[0151] The results of DCC direct antibacterial experiment showed that ( Figure 16 ), the activity of Streptococcus mutans in the DCC-treated group was lower than that in the control group. And with the increase of DCC concentration, the bacterial activity gradually decreased.

[0152] The results of biofilm activity test showed that ( Figure 17 ). MTT assay results of Streptococcus mutans biofilms on dentin surfaces after pretreatment with each reagent group showed that the activity of Streptococcus mutans on dentin surfaces in the 5mg / ml, 10mg / ml DCC / BR, and DCC groups was the lowest. The activity of Streptococcus mutans on dentin surfaces in the CMC / BR, TR, and 1mg / ml DCC / BR groups was relatively high.

[0153] The results of live-dead bacteria staining observation and semi-quantitative analysis showed that ( Figure 18 ), the dentin surface pretreated with 5mg / ml DCC solution had the lowest levels of green / red fluorescence area and intensity. The CMC / BR group had the highest level of biofilm growth, and the biofilm area in the DCC / BR group was relatively low. In the results of semi-quantitative fluorescence analysis, the average live / dead fluorescence intensity ratio in the TR group was significantly higher than that in the other groups. The total live / dead fluorescence intensity ratio in the CMC / BR group was significantly higher than that in the other four groups. In the DCC / BR group, the total live / dead fluorescence intensity ratio gradually decreased with increasing DCC concentration. These results suggest that DCC-induced biomimetic mineralized dentin collagen has the effect of inhibiting the growth of Streptococcus mutans biofilm.

[0154] In summary, the DCC-modified biomimetic mineralized dentin adhesive synthesized in this patent can induce the biomimetic remineralization of demineralized dentin collagen, significantly improving the bonding strength and anti-aging properties of the adhesive.

[0155] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An application of dialdehyde carboxymethyl chitosan in the preparation of a pretreatment agent in a biomimetic mineralized dentin bonding system, characterized in that: The dialdehyde carboxymethyl chitosan is a compound represented by formula (I), and the structural formula of the compound represented by formula (I) is as follows: Formula (I) The weight average molecular weight of the dialdehyde carboxymethyl chitosan is greater than 40 kDa.

2. The use of the dialdehyde carboxymethyl chitosan according to claim 1 in preparing a pretreatment agent in a biomimetic mineralized dentin bonding system, characterized in that: The preparation method of the dialdehyde carboxymethyl chitosan comprises the following steps: (1) Chitosan, alkali, and deionized water are mixed evenly, and after alkalization and expansion, a mixed solution A is obtained. Isopropyl alcohol is added and the temperature is raised to 40-55°C to obtain a mixed solution B; (2) dissolving chloroacetic acid in isopropanol and adding the solution dropwise to the mixed solution B to obtain a mixed solution C after the reaction is complete. The mixed solution C is mixed with an alcohol compound to perform an alcohol precipitation reaction to obtain a precipitated product. The precipitated product is freeze-dried to obtain carboxymethyl chitosan; (3) The carboxymethyl chitosan, acid, and deionized water are mixed uniformly, heated to 37-44° C., sodium periodate is added, and the mixture is reacted in the dark. After cooling, a mixed solution D is obtained, and the mixed solution D is mixed with an alcohol compound for alcohol precipitation reaction to obtain a precipitated product. The precipitated product is freeze-dried to obtain dialdehyde carboxymethyl chitosan.

3. Use of the dialdehyde carboxymethyl chitosan according to claim 2 in preparing a pretreatment agent in a biomimetic mineralized dentin bonding system, characterized in that: Include at least one of the following features: 1) In step (1), the weight-to-volume ratio of chitosan, alkali, and deionized water is chitosan: alkali: deionized water = (4.0-4.4 g): (3.3-3.6) g: 100 mL; 2) The alkalization and expansion time in step (1) is 2-5 hours; 3) In step (1), the volume ratio of isopropyl alcohol to mixed liquid A is isopropyl alcohol: mixed liquid A = (1.2-1.4): 1; 4) In step (2), the weight ratio of chloroacetic acid to isopropyl alcohol is chloroacetic acid: isopropyl alcohol = (18-24) g: 100 mL; 5) In the mixed solution C of step (2), the volume ratio of the isopropanol solution of chloroacetic acid to the mixed solution B is chloroacetic acid isopropanol solution: mixed solution B = (0.10-0.12): 1; 6) In step (2), the volume ratio of mixed solution C to anhydrous ethanol is mixed solution C: anhydrous ethanol = 1: (1.0-1.3); 7) In the mixed solution D of step (3), the concentration of carboxymethyl chitosan in the aqueous solution formed by mixing with water is 0.1-20 mg / mL; the mass ratio of carboxymethyl chitosan to sodium periodate is carboxymethyl chitosan: sodium periodate = 1: (0.8-1); the ratio of carboxymethyl chitosan to acid is carboxymethyl chitosan: acid = 1 g: (0.95-1.1) mL, and the acid is (0.6-0.7) mL hydrochloric acid + (0.35-0.4) mL glacial acetic acid; 8) In step (3), the volume ratio of the mixed solution D to anhydrous ethanol is mixed solution D: anhydrous ethanol = 1: (1.5-3).

4. Use of the dialdehyde carboxymethyl chitosan according to claim 2 in preparing a pretreatment agent in a biomimetic mineralized dentin bonding system, characterized in that: In the step (1), the base is at least one of sodium hydroxide and potassium hydroxide; in the step (3), the acid is at least one of glacial acetic acid and hydrochloric acid.

5. Use of the dialdehyde carboxymethyl chitosan according to any one of claims 1 to 4 in preparing a pretreatment agent in a biomimetic mineralized dentin bonding system, characterized in that: The biomimetic mineralized dentin bonding system comprises the following components: a pretreatment agent, a water scavenger, a primer, and an adhesive; The pretreatment agent comprises the following components: dialdehyde carboxymethyl chitosan, calcium chloride dihydrate, and dipotassium hydrogen phosphate; the weight ratio of the dialdehyde carboxymethyl chitosan, calcium chloride dihydrate, dipotassium hydrogen phosphate, and water is dialdehyde carboxymethyl chitosan: calcium chloride dihydrate: dipotassium hydrogen phosphate: water = (5-10): (0.95-1.05): (0.7-0.78): 1000; The dehydrating agent is anhydrous ethanol; The primer comprises the following components: Anhydrous ethanol, tetraethylene glycol dimethacrylate, bisphenol A glycerol dimethacrylate, camphorquinone, and ethyl benzoate; the weight ratio of anhydrous ethanol, tetraethylene glycol dimethacrylate, bisphenol A glycerol dimethacrylate, camphorquinone, and ethyl benzoate in the primer is anhydrous ethanol: tetraethylene glycol dimethacrylate: bisphenol A glycerol dimethacrylate: camphorquinone: ethyl benzoate = (3.8-4.2): (2.5-3.5): (0.5-1): (0.07-0.08): (0.07-0.08); The adhesive comprises the following components: diurethane dimethacrylate, tetraethylene glycol dimethacrylate, bisphenol A glycerol dimethacrylate, camphorquinone, and ethyl benzoate; the weight ratio of diurethane dimethacrylate, tetraethylene glycol dimethacrylate, bisphenol A glycerol dimethacrylate, camphorquinone, and ethyl benzoate in the adhesive is diurethane dimethacrylate: tetraethylene glycol dimethacrylate: bisphenol A glycerol dimethacrylate: camphorquinone: ethyl benzoate = (2.0-2.4): (2.0-2.4): (0.6-1): (0.05-0.06): (0.05-0.06).

6. Use of the dialdehyde carboxymethyl chitosan according to claim 5 in preparing a pretreatment agent in a biomimetic mineralized dentin bonding system, characterized in that: The preparation method of the pretreatment agent comprises the following steps: mixing dialdehyde carboxymethyl chitosan and calcium chloride solution uniformly, and then adding dipotassium hydrogen phosphate to obtain the pretreatment agent; The preparation method of the primer comprises the following steps: anhydrous ethanol, tetraethylene glycol dimethacrylate, bisphenol A glycerol dimethacrylate, camphorquinone, and ethyl benzoate are uniformly mixed and then stored in the dark to obtain the primer; the preparation method of the adhesive comprises the following steps: diurethane dimethacrylate, tetraethylene glycol dimethacrylate, bisphenol A glycerol dimethacrylate, camphorquinone, and ethyl benzoate are uniformly mixed and then stored in the dark to obtain the adhesive.

7. Use of the dialdehyde carboxymethyl chitosan according to any one of claims 1 to 4 or 6 in preparing a pretreatment agent in a biomimetic mineralized dentin bonding system, characterized in that: The method for using the biomimetic mineralized dentin bonding system comprises the following steps: (1) Cleaning tooth cavities; (2) Treat the tooth surface with a pretreatment agent for 1-3 minutes, remove water and blow dry, then apply primer, blow dry and light cure, apply adhesive, blow thin and light cure to complete dentin bonding.

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