A mussel bionic monomer modified acid etching agent and its preparation method and application

Abstract

The present invention provides a mussel bionic monomer modified etchant and a preparation method and application thereof, and relates to the technical field of oral tooth restoration materials. The mussel bionic monomer modified etchant provided by the present invention comprises the following components: phosphoric acid, mussel bionic monomer and water, the concentration of mussel bionic monomer in the mussel bionic monomer modified etchant is 1~5mg / mL, and the mass concentration of phosphoric acid is 32~37%; the mussel bionic monomer has the structure shown in formula I. The mussel bionic monomer modified etchant provided in the present application can well impart antibacterial activity to demineralized dentin, inhibit endogenous enzyme activity and promote collagen cross-linking on the basis of ensuring demineralization ability, so as to achieve the purpose of improving the stability of the bonding interface and prolonging the service life of resin-dentin bonding, without adding additional clinical operation steps.

Description

Technical Field

[0001] The invention relates to the technical field of oral tooth restoration materials, and in particular to a mussel bionic monomer modified acid etching agent and a preparation method and application thereof. Background Art

[0002] Resin-based materials have replaced traditional silver-mercury alloy materials and become the first choice for the treatment of caries-related diseases due to their advantages such as aesthetics and minimal invasiveness. However, the long-term durability of resin-dentine bonding restorations still faces severe challenges.

[0003] Secondary caries is one of the main reasons for the loss and replacement of resin materials. During the clinical treatment of caries, some tooth tissue with residual bacteria is often retained at the bottom of the cavity. The anaerobic conditions after filling and restoration can provide a favorable reproduction environment for the residual bacteria. At the same time, the existence of micro-leakage and nano-leakage of filling materials forms a channel for bacteria and their products to enter the bonding interface. The proliferation of bacteria in a dominant environment can lead to the occurrence of secondary caries and pulpitis.

[0004] In addition, the activation of endogenous enzymes (including matrix metalloproteinases, cysteine ​​cathepsins, etc.) is another major cause of failure of resin restoration. In clinical practice, regardless of the bonding strategy (full-etching system or self-etching system), etching provides the necessary structure for bonding while causing excessive exposure of collagen, activating the activity of endogenous proteases in dentin. However, the resin material cannot completely penetrate and encapsulate the demineralized collagen, and the exposed collagen is gradually degraded by the activated endogenous proteases, eventually leading to the fall-off of the bonded restoration.

[0005] In order to extend the service life of resin-dentine restoration bonding, providing a solution that has both antibacterial properties and inhibition of endogenous enzyme activity during the bonding process will be an effective strategy. To this end, various active ingredients have been added to the bonding process, including broad-spectrum antibacterial and matrix metalloproteinase inhibitors—chlorhexidine, quaternary ammonium salts, collagen cross-linking agents—glutaraldehyde, epigallocatechin gallate, and fiber demineralization methods. These strategies provide the possibility of antibacterial, increasing the stability of demineralized collagen matrix, or inhibiting endogenous enzyme activity. However, the introduction of the above strategies requires additional clinical operation steps, prolongs the operation time, or has the potential risk of staining and inhibition. Therefore, the development of new dentin bonding strategies is urgently needed in clinical practice. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a mussel biomimetic monomer modified acid etching agent and its preparation method and application. The mussel biomimetic monomer modified acid etching agent provided by the present invention can impart antibacterial activity to demineralized dentin, inhibit endogenous enzyme activity and promote collagen cross-linking on the basis of ensuring demineralization ability, significantly improve the durability of resin-dentin bonding, and no additional clinical operation steps are required.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The invention provides a mussel bionic monomer modified acid etching agent, comprising the following components: phosphoric acid, mussel bionic monomer and water, wherein the concentration of the mussel bionic monomer in the mussel bionic monomer modified acid etching agent is 1-5 mg / mL, and the mass concentration of the phosphoric acid is 32-37%; the mussel bionic monomer has a structure shown in formula I:

[0009] Formula Ⅰ.

[0010] Preferably, the concentration of the mussel bionic monomer in the mussel bionic monomer modified acid etching agent is 2-4 mg / mL.

[0011] Preferably, the concentration of the mussel bionic monomer in the mussel bionic monomer modified acid etching agent is 3-4 mg / mL.

[0012] Preferably, the mass concentration of phosphoric acid in the mussel bionic monomer modified acid etching agent is 35%.

[0013] The present invention provides a method for preparing the mussel bionic monomer modified acid etching agent described in the above technical solution, comprising the following steps:

[0014] A phosphoric acid aqueous solution, a mussel bionic monomer and water are mixed to obtain the mussel bionic monomer modified acid etching agent.

[0015] Preferably, the mass fraction of the phosphoric acid aqueous solution is 85%.

[0016] The present invention provides the use of the mussel bionic monomer modified acid etching agent described in the above technical solution or the mussel bionic monomer modified acid etching agent prepared by the preparation method described in the above technical solution in the preparation of a dentin bonding pretreatment agent.

[0017] The present invention provides a dentin surface pretreatment method, comprising the following steps:

[0018] After the dentin is demineralized by an acid etchant, an adhesive is applied; the acid etchant is the mussel bionic monomer modified acid etchant described in the above technical solution or the mussel bionic monomer modified acid etchant prepared by the preparation method described in the above technical solution.

[0019] Preferably, the demineralization time is 10 to 15 seconds.

[0020] The present invention provides a mussel bionic monomer modified acid etching agent, comprising the following components: phosphoric acid, mussel bionic monomer and water, wherein the concentration of mussel bionic monomer in the mussel bionic monomer modified acid etching agent is 1-5 mg / mL, and the mass concentration of phosphoric acid is 32-37%; the mussel bionic monomer has a structure shown in Formula I (denoted as CLM). In the present invention, the mussel bionic monomer CLM is a lysine cation and catechol functionalized methacrylate, and the -OH in the catechol structure interacts with the -COOH and -NH2 of the collagen amino acid side chain through hydrogen bonds, so that CLM is cross-linked with dentin collagen, and the CLM monomer is chemically bonded with collagen in a wet environment, and the C=C group of the CLM molecule may be located on the periphery of the collagen, providing more possible binding sites for free radicals and resin monomer polymerization; in addition, the catechol and lysine cation structures have the functions of catalyzing the release of active oxygen for long-distance antibacterial and contact antibacterial. The present invention adds CLM monomer into phosphoric acid for modification. In addition to inhibiting the oxidation of catechol groups, the acidic environment can also play a role in destroying the interfacial hydration layer to promote lysine to expel interfacial hydrated cations, which is beneficial to enhance the interaction between the adjacent catechol groups and the interface. Therefore, the mussel biomimetic monomer modified acid etching agent provided by the present invention can well impart antibacterial activity to demineralized dentin, inhibit endogenous enzyme activity and promote collagen cross-linking on the basis of ensuring demineralization ability, thereby achieving the purpose of improving the stability of the bonding interface and extending the service life of resin-dentin bonding.

[0021] The present invention provides the application of the mussel biomimetic monomer modified acid etchant described in the above technical solution in dentin bonding. The mussel biomimetic monomer (CLM) modified phosphoric acid etchant provided by the present invention, during the phosphoric acid demineralization of dentin, the CLM functional monomer enters the dentin tubules and fiber network with the phosphoric acid liquid to chemically combine with the collagen fibers and provide more possible binding sites for the polymerization of resin monomers. The CLM monomer can promote the cross-linking of demineralized collagen fibers, inhibit the endogenous enzyme activity of dentin and give the demineralized collagen antibacterial activity, thereby facilitating the formation of a better quality mixed layer, significantly improving the durability of resin-dentin bonding, and no additional pretreatment steps are required, reducing the clinical operation time and difficulty. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The Raman spectra of dentin demineralized by different acid etching agents in the examples are shown in Figure 2. Figure 1 The middle left picture is 1000~2000cm -1 Wavelength range spectrum, the right picture is 500~800cm -1 Wavelength range spectrum;

[0023] Figure 2~Figure 10Representative surface optical profiles of dentin after being treated with different acid etching agents (NC, CCL, CPC, BCL, CLM1, CLM2, CLM3, CLM4, CLM5) (left / 2D, right / 3D);

[0024] Fig.11 for Figure 2~Figure 10 The corresponding dentin surface roughness of each group;

[0025] Fig.12 Scanning electron microscopy characterization of the dentin surface after treatment with different acid etching agents (scale bar = 1 μm);

[0026] Fig.13 This is the effect diagram of demineralized dentin collagen stability. Fig.13 A is the in situ enzyme spectrum of demineralized dentin surface (scale = 50 μm), B is the microtensile strength of demineralized dentin, and C is the dry weight loss of dentin strips;

[0027] Fig.14 Live and dead bacteria staining on the dentin surface after acid etching in different acid etching groups (scale bar = 50 μm);

[0028] Fig.15 Scanning electron microscopy observation of plaque biofilm on the dentin surface after acid etching with different acid etching agents (scale bar = 10 μm);

[0029] Fig.16 The micro-tensile bonding strength of dentin after treatment with different acid etching agents;

[0030] Fig.17 The representative in situ enzyme spectra of the resin-dentine bonding interface after treatment with different acid etching agents;

[0031] Fig.18 Observation of the resin-dentine bonding interface after treatment with different acid etching agents (scale = 10 μm). Fig.18 A is a representative picture of nano-leakage at the bonding interface before and after aging, and B is a representative picture of the resin protrusion morphology at the bonding interface before and after aging. DETAILED DESCRIPTION

[0032] The invention provides a mussel bionic monomer modified acid etching agent, comprising the following components: phosphoric acid, mussel bionic monomer and water, wherein the concentration of the mussel bionic monomer in the mussel bionic monomer modified acid etching agent is 1-5 mg / mL, and the mass concentration of the phosphoric acid is 32-37%; the mussel bionic monomer has a structure shown in formula I:

[0033] Formula Ⅰ.

[0034] In the present invention, unless otherwise specified, the raw materials involved are commercially available products well known in the art or are prepared by preparation methods well known in the art.

[0035] In the present invention, the concentration of the mussel biomimetic monomer in the mussel biomimetic monomer modified acid etching agent is 1-5 mg / mL, preferably 2-4 mg / mL, and more preferably 3-4 mg / mL. The mussel biomimetic monomer is a resin functional monomer inspired by mussels, which is lysine cation, catechol and Lysine Functionalized Methacrylate (CLM). In the present invention, the mass concentration of phosphoric acid in the mussel biomimetic monomer modified acid etching agent is preferably 32-37%, and more preferably 35%. In the present invention, the water is preferably deionized water.

[0036] Compared with the existing functionalized commercial etchants (containing quaternary ammonium salt benzalkonium chloride phosphate etchant, Bisco), the mussel bionic monomer modified etchant provided by the present invention has similar antibacterial and matrix metalloproteinase inhibition effects. On this basis, benzalkonium chloride quaternary ammonium salts only bind to collagen by electrostatic adsorption, and its strength is weaker than the chemical binding mode of lysine cations and catechol functionalized methacrylates; and CLM also has the effect of promoting dentin collagen cross-linking, so that it can better improve the durability of resin-dentin bonding. In addition, the mussel bionic monomer modified etchant has excellent antibacterial properties due to the CLM functional monomer. The catechol in the CLM structure can catalyze the generation of active oxygen to achieve long-range antibacterial, and the lysine cation has contact antibacterial function. In particular, compared with the scheme of using CLM monomer as a single component for pretreatment and application in dentin bonding, the present invention adds CLM monomer into phosphoric acid for modification and has the following advantages: when CLM monomer is used alone in a neutral or alkaline environment, it is easy to be oxidized and affect the bonding effect, while when CLM modified acid etchant is used to demineralize dentin, the acidic environment is conducive to maintaining the two hydroxyl groups on the catechol benzene ring in CLM from being oxidized, and the phenolic hydroxyl groups remain in an unoxidized state. The -OH interacts with the -COOH and -NH2 of the collagen amino acid side chain through hydrogen bonds to achieve cross-linking of CLM and collagen; in addition, in addition to inhibiting the oxidation of the catechol group, the acidic environment can also play a role in destroying the interfacial hydration layer to promote lysine to expel the interfacial hydrated cations, which is conducive to enhancing the interaction between the adjacent catechol groups and the interface.

[0037] The mussel bionic monomer modified acid etching agent provided by the invention can significantly improve the durability of resin-dentin bonding while ensuring the demineralization ability.

[0038] The present invention provides a method for preparing the mussel bionic monomer modified acid etching agent described in the above technical solution, comprising the following steps:

[0039] A phosphoric acid aqueous solution, a mussel bionic monomer and water are mixed to obtain the mussel bionic monomer modified acid etching agent.

[0040] In the present invention, the mass fraction of the phosphoric acid aqueous solution is preferably 85%.

[0041] The present invention preferably adds mussel bionic monomer and water in sequence to an aqueous phosphoric acid solution with a mass fraction of 85%; the water in the 85% aqueous phosphoric acid solution and the water constitute all the water in the mussel bionic monomer modified acid etching agent.

[0042] The present invention has no special requirements for the mixing method, and a mixing method well known to those skilled in the art can be used to mix uniformly.

[0043] In the present invention, the mussel biomimetic monomer is easily soluble in solvents such as water and ethanol. Therefore, no additional solubilizing solvent is needed in the process of mixing the mussel biomimetic monomer with phosphoric acid.

[0044] The present invention provides the use of the mussel bionic monomer modified acid etching agent described in the above technical solution or the mussel bionic monomer modified acid etching agent prepared by the preparation method described in the above technical solution in the preparation of a dentin bonding pretreatment agent.

[0045] The present invention provides a dentin surface pretreatment method, comprising the following steps:

[0046] After the dentin is demineralized by an acid etchant, an adhesive is applied; the acid etchant is the mussel bionic monomer modified acid etchant described in the above technical solution or the mussel bionic monomer modified acid etchant prepared by the preparation method described in the above technical solution.

[0047] In the present invention, the demineralization time is preferably 10 to 15 seconds. The present invention has no special requirements for the binder, and a binder well known to those skilled in the art can be used. After the adhesive is applied, the resin is layered and stacked. The present invention has no special requirements for the resin, and a resin well known to those skilled in the art can be used.

[0048] The mussel biomimetic monomer (CLM) modified phosphoric acid etchant provided by the present invention, during the phosphoric acid demineralization of dentin, the CLM functional monomer enters the dentin tubules and fiber network with the phosphoric acid liquid to chemically combine with the collagen fibers and provide more possible binding sites for the polymerization of resin monomers. The CLM monomer can promote the cross-linking of demineralized collagen fibers, inhibit the endogenous enzyme activity of dentin and give the demineralized collagen antibacterial activity, thereby facilitating the formation of a better quality mixed layer and significantly improving the durability of the resin-dentin bonding. In addition, compared with traditional methods for improving the durability of resin-dentin bonding, such as ethanol wet bonding, the use of pretreatment agents, etc., the bonding strategy provided by the present invention can avoid adding additional operating steps, reduce operating time, reduce patient discomfort, and improve the clinical chair turnover rate.

[0049] In order to further illustrate the present invention, the mussel bionic monomer modified acid etching agent provided by the present invention and its preparation method and application are described in detail below in combination with examples, but they should not be construed as limiting the protection scope of the present invention.

[0050] In the embodiment, the mussel biomimetic monomer (CLM) has a structure shown in Formula I, and the preparation method is as follows:

[0051] The synthesis of CLM is divided into 4 steps, and the synthesis route is as follows:

[0052] .

[0053] The specific synthesis process is:

[0054] P1 synthesis: Weigh 5g Fmoc-L-Lys(Boc) (10.67mmol) and dissolve it in 25mL dichloromethane (DCM), then add 1.59g hydroxybenzotriazole (HOBt, 11.74mmol) and 2.25g N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC, 11.74mmol) dropwise at room temperature, and stir in an ice-water bath under nitrogen protection for 30min. Then add 3.04g 2-hydroxyethyl methacrylate (HEMA, 23.34mmol) and 2.18mL N, N-diisopropylethylamine (DIPEA, 12.49mmol) dropwise to the reaction solution, and stir at room temperature for 6h. Wash with 20mL saturated sodium bicarbonate solution and collect the DCM layer, wash with deionized water and saturated sodium chloride solution in turn, and then add anhydrous sodium sulfate to dry. Filter and evaporate to remove the solvent. After the solvent was completely evaporated, the mixture was passed through a column at a volume ratio of petroleum ether to ethyl acetate of 2:1, and then rotary evaporated and dried in vacuum at room temperature overnight to obtain 4.2 g of a white solid product P1.

[0055] Synthesis of P2: 4.2 g of P1 was dissolved in 70 mL of dichloromethane / diethylamine (DCM / DEA, volume ratio 1:1), stirred at room temperature for 6 h, and the solvent was removed by rotary evaporation. Dichloromethane was added to dissolve, and the mixture was rapidly passed through a gradient column (petroleum ether: ethyl acetate (volume ratio) = 1:1, dichloromethane: methanol (volume ratio) = 15:1), rotary evaporation, and vacuum drying at room temperature overnight to obtain 2.4 g of light yellow oily liquid P2;

[0056] Synthesis of P3: 2.4 g P2, 0.518 g 2,3-dihydroxybenzoic acid DHBA (3.36 mmol) and 0.454 g HOBT (3.36 mmol) were dissolved in 30 mL tetrahydrofuran (THF) and stirred for 5 min under nitrogen protection. 1.40 mL triethylamine (TEA) and 1.93 g N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) were added to the solution and stirred for 16 h at 35 °C under nitrogen protection. DCM was added to the product to extract the raw materials, and the resulting solution was washed with 0.1 N hydrochloric acid, deionized water, and saturated sodium chloride solution in turn, dried with anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography (dichloromethane: methanol (volume ratio) = 20: 1) and 1.15 g of light yellow oily P3 was obtained after drying;

[0057] Synthesis of P4 (CLM): 1.15 g of P3 was added to 50 mL of 4 N hydrochloric acid dioxane solution and reacted at room temperature for 1 h. After the reaction was completed, the brown CLM was obtained by vacuum drying.

[0058] Example 1

[0059] A mussel biomimetic monomer (CLM) having a structure shown in formula I was added to an aqueous solution of phosphoric acid with a mass fraction of 85%, and then deionized water was added to prepare a mussel biomimetic monomer modified acid etching agent with a phosphoric acid mass concentration of 35%, wherein the concentration of the mussel biomimetic monomer in the mussel biomimetic monomer modified acid etching agent was 1 mg / mL. The mussel biomimetic monomer modified acid etching agent prepared in Example 1 is recorded as 1 mg / mL CLM+35% phosphoric acid (abbreviated as CLM-1 or CLM1).

[0060] Example 2

[0061] The concentration of the mussel biomimetic monomer in Example 1 was changed to 2 mg / mL to obtain a mussel biomimetic monomer modified acid etching agent, which was recorded as 2 mg / mL CLM+35% phosphoric acid (abbreviated as CLM-2 or CLM2).

[0062] Example 3

[0063] The concentration of the mussel biomimetic monomer in Example 1 was changed to 3 mg / mL to obtain a mussel biomimetic monomer modified acid etching agent, which was recorded as 3 mg / mL CLM+35% phosphoric acid (abbreviated as CLM-3 or CLM3).

[0064] Example 4

[0065] The concentration of the mussel biomimetic monomer in Example 1 was changed to 4 mg / mL to obtain a mussel biomimetic monomer modified acid etching agent, which was recorded as 4 mg / mL CLM+35% phosphoric acid (abbreviated as CLM-4 or CLM4).

[0066] Example 5

[0067] The concentration of the mussel biomimetic monomer in Example 1 was changed to 5 mg / mL to obtain a mussel biomimetic monomer modified acid etching agent, which was recorded as 5 mg / mL CLM+35% phosphoric acid (abbreviated as CLM-5 or CLM5).

[0068] Comparative Example

[0069] 35 wt % phosphoric acid without adding CLM monomer was used as blank control group (BCL);

[0070] Bisco acid etchant with 35 wt% phosphoric acid concentration was used as the commercial control group (CCL);

[0071] Bisco acid etchant containing benzalkonium chloride as the active ingredient was used as a commercial positive control (CPC).

[0072] The components of the acid etching agents of the embodiments and comparative examples are shown in Table 1 (Note: the pH values ​​in Table 1 are expressed in the form of mean value ± standard deviation).

[0073] Table 1 Composition of the acid etching agent in the examples and comparative examples

[0074]

[0075] The performance tests of the acid etching agents of the embodiments and comparative examples are as follows:

[0076] 1. Test Method

[0077] 1.1 Demineralized dentin collagen and CLM reaction

[0078] A slow cutter was used to cut the dentin in the middle of the crown parallel to the occlusal surface under running water, and then a dentin slice with a thickness of 1 mm was cut to prepare a standard thickness dentin specimen. The dentin surface was demineralized with each group of acid etchants for 15 seconds, then washed with deionized water and air-dried, and the interaction between demineralized dentin collagen and CLM functional components was evaluated using Raman spectrometry.

[0079] 1.2 Surface roughness of demineralized dentin

[0080] The surface of 1 mm thick dentin slices was graded and polished with sandpaper (400, 600, 1200, 200, 4000, 5000 mesh) under running water. After demineralization with each group of acid etchants for 15 seconds, the slices were rinsed under running water for 30 seconds. The dentin surface was dried with filter paper to maintain the corresponding wettability. The surface roughness of the demineralized dentin was tested using a three-dimensional non-contact surface profilometer.

[0081] 1.3 Observation of demineralized dentin surface morphology

[0082] The demineralized dentin slices in the above groups were fixed with 2.5% glutaraldehyde for 4 h, dehydrated with gradient ethanol (75%, 85%, 95%, 100% for 10 min each), and the dehydrated dentin surface was sprayed with gold and the surface morphology was observed under a scanning electron microscope.

[0083] 1.4 Effect of dentin collagen stability

[0084] 1.4.1 Ultimate tensile strength of demineralized dentin

[0085] The excised third molars were cut into standard dentin strips (1×1×6 mm) using a slow cutter under running water. The dentin strips were thoroughly demineralized (48 h) using each group of acid etchants, washed with deionized water, and the surface moisture was wiped dry with filter paper. The ultimate tensile strength of dentin collagen was tested using a micro-tensile testing machine with a tensile rate of 0.5 mm / min.

[0086] 1.4.2 Collagen dry weight loss

[0087] Standard dentin strips (1×1×6 mm) were prepared and thoroughly demineralized using 0.5 mol / L EDTA (pH=7.4, 4℃). Each demineralized dentin strip was placed in a polypropylene tube and placed in a desiccator containing anhydrous calcium sulfate. After drying to constant weight, the dry weight of each dentin strip was measured using an analytical balance with an accuracy of 0.1 mg. After the measurement, all dentin strips were rehydrated with deionized water to restore their original size. After that, the dentin strips were treated with each group of acid etchants for 20 seconds. After acid etching, they were immersed in buffer (pH=7.2) and stirred for 5 minutes to neutralize the acid components. The surface water was absorbed by filter paper and placed in an acrylic tube containing calcium zinc culture medium (the culture medium contained 5mmol / L HEPES, 2.5mmol / L CaCl2·H2O, 0.05mM ZnCl2, pH=7.2). In this part of the experiment, only EDTA demineralized dentin strips without acid treatment were added as the control group. The polypropylene tube was sealed and incubated in a 37°C constant temperature shaker (60 cycles / min) for 30 days. After the incubation, the dry weight of the dentin strips was measured again according to the above process. The dry weight loss ratio was calculated based on the change in the dry weight of the dentin before and after incubation.

[0088] 1.4.3 In situ zymography of demineralized dentin surface

[0089] 1mm thick dentin specimens were prepared, and the coronal surface was selected as the test surface. After acid etching and demineralization for 15s in each group, the specimens were rinsed with deionized water for 30s, and the surface moisture was dried with filter paper. The treated specimens were placed on a glass slide, and 50μL collagenase activity display agent was added to soak the demineralized dentin surface, covered with a glass slide, and incubated at 37℃ and 100% humidity for 48h in the dark. The surface dye was washed with deionized water and air-dried, and the endogenous enzyme activity on the dentin surface was scanned under a laser confocal microscope to detect it.

[0090] 1.5 Study on the antibacterial activity of dentin after demineralization with CLM acid etching agent

[0091] 1.5.1 Live and dead bacteria staining

[0092] Dentin specimens with a thickness of 1 mm were prepared as described above, demineralized with each group of acid etchants for 15 s, rinsed with deionized water for 30 s, and placed in a 24-well plate. Each side was irradiated with ultraviolet light for 1 h for sterilization. 20 μL of diluted mutans Streptococcus solution (1×10 6 CFU / mL) and 2mL BHI culture medium were cultured anaerobically at 37℃ for 24h. After completion, the specimens were gently rinsed with sterile PBS. 500μL of live-dead bacteria staining reagent was added to each well to cover the dentin specimens. The specimens were incubated at room temperature and away from light for 15min. The excess dye on the surface was rinsed with PBS again, and the bacteria on the specimen surface were observed using a laser confocal microscope.

[0093] 1.5.2 Observation of plaque biofilm

[0094] The bacterial biofilm on the surface of demineralized dentin was cultured, and the planktonic bacteria on the surface were gently rinsed with sterile PBS. The specimens were immersed in 2.5% glutaraldehyde at 4°C overnight, and dehydrated with gradient ethanol (30%, 50%, 70%, 85%, 95%, and 100% for 15 min each). Finally, the specimens were immersed in anhydrous ethanol until gold was sprayed. The bacteria adhering to the surface of the demineralized dentin specimens were observed using a scanning electron microscope.

[0095] 1.6. Dentin bonding performance study

[0096] 1.6.1 Preparation of bonding specimens

[0097] The third molars were selected and cut parallel to the occlusal surface under running water using a slow cutting machine to expose the dentin in the middle of the crown. Sandpaper (600 mesh) was used for polishing. The crown dentin was demineralized for 15 seconds using each group of acid etchants. The dentin was rinsed with deionized water for 30 seconds. The surface moisture was absorbed by filter paper. The adhesive (Single Bond 2, 3M, USA) was applied for 10 seconds, blown for 5 seconds, and cured for 20 seconds. The resin (Z250, 3M, USA) with a thickness of 4 mm was layered and cured. After preparation, the specimens were immersed in 37℃ deionized water for 24 hours as the immediate group, and the hot and cold cycles (5-55℃, stay for 60 seconds) for 10,000 times were used to simulate the oral aging group for 1 year.

[0098] 1.6.2 Microtensile test

[0099] The specimens of the immediate group and the aging group were cut perpendicularly to the bonding interface using a slow cutting machine to prepare standard resin-dentin strip bonding specimens (1×1×6 mm) with a cross-sectional area of ​​1mm 2, keeping the specimens moist, using a micro-tensile testing machine (tensile rate: 0.5 mm / min) to test and record the load at which the specimens broke (n=10, i.e., 10 specimens in each group).

[0100] 1.6.3 Nanoleakage at the bonding interface

[0101] The specimens of the immediate group and the aging group were cut perpendicularly to the bonding interface by a slow cutting machine to prepare 1 mm thick specimens. The specimens were polished with gradient sandpaper (320, 600, 1200, 2000, 4000, 5000 mesh) under running water, and vibrated with deionized water for 5 min. After drying, two layers of nail polish were completely coated on the upper and lower areas of the bonding interface with an interval of 1 mm. After drying, they were immersed in 50wt% ammonium silver nitrate solution for 24 h in the dark. The dye solution covering the surface was thoroughly washed off with deionized water, immersed in developer solution under fluorescent irradiation for 8 h, immersed in fixer solution for 8 h, and polished with gradient sandpaper (320, 600, 1200, 2000, 4000, 5000 mesh) again, ultrasonically vibrated with deionized water for 5 min, and sprayed with gold after air drying. The nano-leakage of the bonding interface was observed using a scanning electron microscope.

[0102] 1.6.4 In situ zymography of the bonding interface

[0103] The specimens of the immediate group and the aging group were cut perpendicularly to the bonding interface by a slow cutting machine to prepare 1 mm thick specimens. The specimens were graded and polished with sandpaper (320, 600, 1200, 2000, 4000, and 5000 mesh) under running water, shaken with deionized water for 5 min, and the surface moisture was dried with filter paper. The treated specimens were placed on a glass slide, and 50 μL of collagenase activity display agent was added to soak the surface of the specimens. The specimens were covered with a glass slide and incubated at 37°C in the dark and 100% humidity for 48 h. The surface dye was washed off with deionized water and air-dried. The endogenous enzyme activity at the bonding interface was detected by scanning under a laser confocal microscope.

[0104] 1.6.5 Bonding interface morphology

[0105] The specimens of the immediate group and the aging group were cut perpendicularly to the bonding interface between the resin and the dentin using a slow cutting machine to prepare 1 mm thick specimens. The specimens were graded polished with sandpaper (320, 600, 1200, 2000, 4000, and 5000 mesh), shaken with deionized water for 5 min, and the surface moisture was dried with filter paper. The 1 mm area around the bonding interface was treated with 35% phosphoric acid for 30 s, rinsed with deionized water, and immersed in 10% sodium hypochlorite solution for 2 min. After washing, they were ultrasonically shaken for 10 min, dried, sprayed with gold, and the bonding interface morphology was observed using a scanning electron microscope.

[0106] 2. Results

[0107] 2.1 CLM acid etching agent demineralization ability test results

[0108] 2.1.1 Reaction of demineralized dentin collagen with CLM

[0109] Raman spectra of dentin demineralized by each group of etching agents Figure 1 As shown in the figure, amide I bands (1655-1680 cm -1 ), amide II band (1550~1590cm -1 ) and amide III band (1265~1274cm -1 ) and other characteristic peaks. After CLM was used as a functional monomer to modify the acid-etching agent to demineralize dentin, the peak intensities of amide I band, amide II band and amide III band increased compared with other groups, indicating that CLM monomer reacted with collagen; 1363~1384cm -1 and 652~657cm -1 The enhanced vibration peak at 1760-1784 cm indicates the characteristic peak of -OH in catechol. -1 The enhanced vibration peak at 1550-1590 cm-1 represents the characteristic peak of ester group, which proves that CLM binds to collagen. -1 ) proves the existence of -NH. The pyridine ring vibration peak (1061~1084cm -1 ) was enhanced, indicating an increase in the degree of collagen cross-linking.

[0110] 2.1.2 Surface roughness of demineralized dentin

[0111] The results of optical profile and roughness of dentin surface after demineralization by different etching agents are as follows Figure 2~Figure 11 As shown, Figure 2~Figure 10 Representative optical profiles of dentin after treatment with each group of etching agents (left / 2D, right / 3D); Fig.11 is the surface roughness of dentin in each group, and different letters indicate statistically significant differences (P<0.05); Figure 2~Figure 11 NC: negative control group without demineralization, CCL: commercial control group, CPC commercial positive control group, BCL: blank control group. Figure 2~Figure 11 It can be seen that the surface roughness of the undemineralized dentin group was the lowest. After acid etching, the surface roughness of the dentin in each group increased. The surface roughness of the blank control group and the CLM test group was higher than that of the commercial control group and the commercial positive control group. In the test group, with the increase of CLM addition, the surface roughness of the dentin in the CLM-5 group was the highest.

[0112] 2.1.3 Observation of demineralized dentin surface morphology

[0113] The surface morphology of dentin after acid etching in each group Fig.12 As shown, Fig.12Scanning electron microscopy characterization of the dentin surface after treatment with different acid etching agents (scale bar = 1μm). Fig.12 NC: negative control group without demineralization, CCL: commercial control group, CPC commercial positive control group, BCL: blank control group. Fig.12 It can be seen that the surface of undemineralized dentin is mineralized, without dentinal tubules and collagen matrix. In each acid etching group, all dentin samples showed uniform dentinal tubules and demineralized collagen matrix on the surface, proving that the dentin specimens achieved effective demineralization after being treated with each group of acid etching agents.

[0114] 2.2 Effect of dentin collagen stability

[0115] 2.2.1 Ultimate tensile strength of demineralized dentin

[0116] Fig.13 This is the effect diagram of demineralized dentin collagen stability. Fig.13 A is the in situ enzyme spectrum of demineralized dentin surface (scale = 50 μm), B is the microtensile strength of demineralized dentin, and C is the dry weight loss of dentin strips. All data are expressed as mean ± standard deviation, and different letters represent statistically significant differences (P < 0.05). Fig.13 NC: negative control non-demineralized group, CCL: commercial control group, CPC commercial positive control group, BCL: blank control group.

[0117] The ultimate tensile strength results of dentin collagen demineralized by different acid etching agents are as follows Fig.13 As shown in B, the ultimate tensile strength of dentin collagen in the blank control group, commercial control group, commercial positive control group and experimental group (CLM 1-5) was 10.31±1.04, 10.51±1.72, 10.59±1.34, 10.33±1.13, 11.26±1.20, 13.40±0.90, 14.63±1.45 and 16.09±1.16 MPa. When the amount of CLM added was greater than 3 mg / mL, the ultimate tensile strength of collagen increased (P<0.05).

[0118] 2.2.2 Dry weight loss

[0119] The percentage of dentin mass loss after being treated with acid etchants in each group and cultured for 30 days is as follows Fig.13 As shown in C, the dentin strips demineralized with EDTA but not treated with acid lost 23.1% of their dry weight. After acid treatment with the commercial control group and blank control group, the dry weight loss of dentin increased by 38.0% and 37.9%, respectively. The reduction in dry weight loss in the commercial positive control group was similar to that in the CLM-3 group (P>0.05). In CLM, the dry weight loss decreased with increasing concentration, and the CLM-5 group had the lowest dentin dry weight loss of 9.5%.

[0120] 2.2.3 In situ zymography of demineralized dentin surface

[0121] Phosphoric acid etching and demineralization can expose and activate endogenous enzymes encapsulated in hydroxyapatite, such as Fig.13 As shown in A, the green fluorescence intensity represents the strength of endogenous enzyme activity on the dentin surface. The commercial control group and blank control group showed strong green fluorescence, while the green fluorescence intensity of the commercial positive group and CLM group was weaker and showed concentration dependence in the CLM group. The green fluorescence on the dentin surface of the CLM-5 group was the weakest.

[0122] 2.3 Study on the antibacterial activity of dentin after demineralization with CLM acid etching agent

[0123] 2.3.1 Live and dead bacteria staining

[0124] Fig.14 This is a representative image of live and dead staining of Streptococcus mutans on the surface of dentin after acid etching and demineralization (scale bar = 50μm), green fluorescence represents live bacteria, and red fluorescence represents dead bacteria. Fig.14 It can be seen that the surfaces of the commercial control group and the blank control group were mainly large numbers of green live bacteria. Compared with the commercial positive control group and the CLM group, the total number of bacteria decreased, and the number of red dead bacteria increased. The bacterial density and the amount of dead bacteria in the commercial positive control group were similar to those in the CLM-3 group. In the CLM group, as the concentration increased, the bacterial density further decreased, and the number of red dead bacteria increased.

[0125] 2.3.2 Observation of plaque biofilm

[0126] Fig.15 Representative images of Streptococcus mutans biofilm on the dentin surface after being treated with each group of acid etchants (scale bar = 10 μm). Fig.15 It can be seen that the dentin surface of the commercial control group and the blank control group was covered with high-density plaque biofilm, covering the demineralized dentinal tubules; in the commercial positive control group and the CLM group, the density of plaque biofilm was significantly reduced, and at the CLM-5 concentration, only scattered bacteria existed on the dentin surface.

[0127] 2.4 Dentin bonding performance study

[0128] 2.4.1 Microtensile strength

[0129] Microtensile bond strength of dentin demineralized with different etching agents Fig.16 All data are shown as mean ± standard deviation. Before aging treatment, the same capital letters represent no statistically significant difference among the groups (P>0.05). After aging treatment, the same lowercase letters represent no statistically significant difference among the groups (P>0.05). If there is an *, it means that there is no statistically significant difference before and after aging in the same group (P>0.05). Fig.16 It can be seen that there was no significant difference among the groups immediately after bonding (P>0.05). After hot-cold cyclic aging treatment, the micro-tensile strength of the commercial control group, commercial positive control group and blank control group decreased the most, and the micro-tensile strength of the CLM 4~5 group decreased the least.

[0130] 2.4.2 In situ zymography of the bonding interface

[0131] Representative in situ zymogram results of the resin-dentine bonding interface after treatment with different acid etchants are shown in Figure 2. Fig.17 As shown (bar = 50 μm), red fluorescence represents the location of the adhesive stained with rhodamine B, and the green fluorescence intensity represents the intensity of endogenous enzyme activity in dentin, R = resin, HL = mixed layer, and D = dentin. Fig.17 It can be seen that immediately after bonding, the blank control group and the commercial control group showed strong green fluorescence, the commercial positive control group had similar green fluorescence intensities to the CLM-2 and CLM-3 groups, and the CLM-4 and CLM-5 groups had the lowest green fluorescence intensities; after hot and cold cycle aging treatment, the green fluorescence intensities of each group increased, but the green fluorescence of the CLM-4 and CLM-5 groups was still at a weak level. The above results show that the endogenous enzyme activity of the CLM-4 and CLM-5 groups is the lowest. Therefore, CLM as a functional monomer added to phosphoric acid has the effect of inhibiting the endogenous enzyme activity of dentin.

[0132] 2.4.3 Nanoleakage at the bonding interface

[0133] Fig.18 This is the observation picture of the resin-dentin bonding interface (scale bar = 10μm). Fig.18 A is a representative picture of nano-leakage at the bonding interface before and after aging, R=resin, HL=mixed layer, D=dentin, and the white arrows at the mixed layer indicate that the white matter is nano-leakage silver-stained particles; B is a representative picture of the morphology of resin protrusions at the bonding interface before and after aging.

[0134] The nanoleakage results of the bonding interface are as follows Fig.18 As shown in A, there were a small amount of scattered silver particles at the bottom of the bonding interface of each group before the hot and cold cycles. After hot and cold cycle simulated aging, the silver particles at the bottom of the bonding interface of each group increased significantly. Among them, continuous silver particle deposition appeared at the bottom of the mixed layer in the commercial control group, commercial positive control group, blank control group, CLM-1, CLM-2, and CLM-3 groups. The silver particles in the CLM-4 and CLM-5 groups were thinner and scattered.

[0135] 2.4.4 Bonding interface morphology

[0136] The morphology of the mixed layer and resin protrusion formed by resin bonding after each group of acid etching agent treatment is as follows Fig.18As shown in B. All groups formed complete mixed layers and resin protrusions. The resin protrusions formed in the CLM4-5 groups were thicker and denser immediately after bonding. After hot and cold cycles, all groups retained a complete mixed layer structure and did not experience resin protrusion breakage. The resin protrusion breakage phenomenon was more obvious in the commercial control group, commercial positive control group, blank control group, and CLM-1 group. In the CLM group, as the concentration increased, the resin protrusions became more complete.

[0137] It can be seen from the above examples that adding CLM functional components to phosphoric acid can improve the stability of the resin-dentin bonding interface, which is beneficial to prolonging the service life of resin restorations. This strategy can avoid additional clinical operational burdens, endow demineralized dentin with antibacterial activity in a wet environment, inhibit endogenous enzyme activity through chemical bonding, promote collagen cross-linking, and enhance the stability of the bonding interface.

[0138] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A mussel biomimetic monomer modified acid etching agent, characterized in that: The invention is composed of the following components: phosphoric acid, mussel bionic monomer and water. The concentration of the mussel bionic monomer in the mussel bionic monomer modified acid etching agent is 1-5 mg / mL, and the mass concentration of phosphoric acid is 35%. The mussel bionic monomer has a structure shown in Formula I: Formula Ⅰ.

2. The mussel biomimetic monomer modified acid etching agent according to claim 1, characterized in that: The concentration of the mussel bionic monomer in the mussel bionic monomer modified acid etching agent is 2-4 mg / mL.

3. The mussel biomimetic monomer modified acid etching agent according to claim 2, characterized in that: The concentration of the mussel bionic monomer in the mussel bionic monomer modified acid etching agent is 3-4 mg / mL.

4. The method for preparing the mussel biomimetic monomer modified acid etching agent according to any one of claims 1 to 3, characterized in that: The following steps are involved: A phosphoric acid aqueous solution, a mussel bionic monomer and water are mixed to obtain the mussel bionic monomer modified acid etching agent.

5. The preparation method according to claim 4, characterized in that: The mass fraction of the phosphoric acid aqueous solution is 85%.

6. Use of the mussel bionic monomer modified acid etching agent according to any one of claims 1 to 3 or the mussel bionic monomer modified acid etching agent prepared by the preparation method according to claim 4 or 5 in the preparation of a dentin bonding pretreatment agent.

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