Method for improving stability of dentin bonding interface by using phosphorylated chitosan

Abstract

The invention provides a method for improving the stability of a dentin bonding interface through phosphorylated chitosan, and belongs to the field of biological medicine. The preparation method comprises the following steps: mixing phosphorylated chitosan with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, and uniformly coating the mixture on the surface of dentin to form a covalent cross-linked network and induce in-situ deposition of hydroxyapatite. A series of performance changes are brought to a dentin bonding interface, and the mechanical strength and the enzymolysis resistance of dentin collagen fibers are improved. The adhesive interface is endowed with the capability of inhibiting bacteria, so that the demineralized dentin collagen fibers are preserved, and the adhesive interface is stabilized. The dentin collagen fiber is stabilized, a proper remineralization microenvironment is maintained, the deposition of minerals such as calcium and phosphorus on the surface of the dentin is promoted, and meanwhile, a guarantee is provided for the dentin to resist demineralization. According to the invention, the three problems of micro leakage, collagen degradation and remineralization are simultaneously solved in the field of dentin bonding for the first time.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a method for improving the stability of the dentin bonding interface by phosphorylated chitosan Background Art

[0002] With the continuous development of oral materials, various dental restorative materials are widely used in clinics. Whether they can successfully bond with dental tissues is one of the important factors for judging their clinical application value. The bonding of dentin involves a complex physicochemical adhesion process. Although the immediate bonding strength obtained by existing bonding techniques can meet the clinical needs, the bonding strength gradually decreases with the extension of the use time of the restorative in the complex oral environment, resulting in marginal microleakage, secondary caries, etc., and ultimately leading to the failure of bonding restoration. Therefore, improving the durability of dentin bonding is an important issue in the quality of dentin bonding at present.

[0003] The durability of dentin bonding depends on the aging of the weakest link in the bonding interface - the bonding substrate. In the bonding interface, due to the inconsistent depth of dentin demineralization and the penetration of the adhesive / resin, and the hydrolysis of unpolymerized resin monomers and water-soluble molecules under the influence of various factors such as water and enzymes in the oral environment, the exposed collagen fibers increase and are degraded under the action of endogenous and exogenous collagenases. The riveting effect between the adhesive / resin and dentin will be greatly reduced. Therefore, protecting the exposed collagen fibers in the bonding interface is an important way to improve the durability of dentin bonding.

[0004] In the previous research of the present invention, chitosan nanoparticles with different molecular weights were firmly bound to the surface of the triple helix structure of collagen molecules under the action of the cross-linking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and wrapped the collagen molecules at the molecular level to isolate the contact and adhesion of collagenase, bacteria, etc. with collagen molecules (attachment Figure 1), reducing the degradation of collagen fibers at the bonding interface and improving the resistance of the bonding interface to hydrolysis or enzymatic hydrolysis. Phosphorylated chitosan (PC) uses chitosan molecules as its skeleton, inheriting the superior biodegradability and biocompatibility of chitosan, and the amino groups of the active functional groups of chitosan, as the molecular structural basis for chemical cross-linking between EDC and collagen fibers, are also retained in this type of molecule. At the same time, due to the introduction of phosphate groups, it has the characteristics of calcium ion chelation, dentin induction, and antibacterial properties. Through the mediation of the cross-linking agent EDC, phosphorylated chitosan and collagen fibers are covalently bonded in a "zero extension" manner. The cross-linked phosphorylated chitosan can not only be fixed on the collagen fibers, but also increase their stability and prevent them from falling off or dissociating during subsequent processing. Pretreatment of the dentin bonding interface in this way has the effect of strengthening dentin collagen fibers and resisting enzymatic hydrolysis. Under its action, the exposed collagen fibers in the dentin have a more stable ability to resist enzymatic hydrolysis, and at the same time have the conditions for slow mineralization. This can not only improve the resistance of the collagen fibers to enzymatic hydrolysis, but also guide the deposition of calcium and phosphate ions to re-embed the collagen fibers, fundamentally protecting the weakest link in the bonding interface - the dentin collagen fibers, from aging caused by external stimulation, achieving "treating both the symptoms and the root causes" to maintain the durability of dentin bonding.

[0005] Existing studies on the protection of collagen fibers in demineralized dentin can be summarized into two categories: one is to strengthen collagen fibers or inhibit collagenase activity, to reduce the amount of collagen fiber degradation or prolong the time of collagen degradation to a certain extent, and to "treat the symptoms" of the aging of the bonding interface caused by collagen exposure and subsequent enzymatic hydrolysis and hydrolysis; the other is to seal the weak link of the bonding interface, the exposed collagen, as much as possible (sufficient penetration of the adhesive or re-deposition of dentin minerals), thereby forming a stable bonding interface complex to isolate endogenous and exogenous stimuli, so that there are no conditions for enzymatic hydrolysis and hydrolysis, that is, to "treat the root cause" (see Appendix). Figure 2 ). However, no matter which type of protection method is used, it is impossible to completely and effectively solve the current situation of unstable bonding interface caused by collagen fiber degradation: if it is only a "symptomatic treatment", collagen fibers will eventually degrade; if it is only a "root cause treatment", it is limited by the mineralization time and lacks stable conditions for completing mineralization. Summary of the invention

[0006] The purpose of the present invention is to develop a method for pretreating the dentin bonding interface, which can inhibit the degradation of dentin collagen, stabilize collagen, promote the orderly deposition of inorganic substances such as calcium and phosphorus, seal collagen, and comprehensively protect collagen fibers, thereby achieving the purpose of stabilizing the bonding interface and improving bonding durability.

[0007] The first object of the present invention is a method for improving the stability of the dentin bonding interface by phosphorylated chitosan, which method comprises:

[0008] Phosphorylated chitosan is mixed with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide and then evenly coated on the surface of dentin to form a covalent cross-linked network and induce in-situ deposition of hydroxyapatite.

[0009] Preferably, the phosphorus content of the phosphorylated chitosan is ≥5%.

[0010] Preferably, the concentration of phosphorylated chitosan is 0.5 - 2.0 g / L; the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 2.4 - 4 mol / L; the concentration of N-hydroxysuccinimide is 0.5 - 1.2 mol / L.

[0011] Preferably, the concentration of phosphorylated chitosan is 1 g / L; the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 3.3 mol / L; the concentration of N-hydroxysuccinimide is 0.8 mol / L.

[0012] Preferably, the preparation method of the phosphorylated chitosan is to use the microwave-assisted method, with 1,2,4-tricarboxybutane phosphoric acid as the phosphorus source, and modify the C6 hydroxyl group of chitosan through an esterification reaction; phosphorylated chitosan with a phosphorus content of ≥5% is prepared.

[0013] The second object of the present invention is to provide a composition for improving the binding of phosphorylated chitosan to demineralized dentin, including the cross-linking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide; the mass ratio is (2.4 - 4):(0.5 - 1.2) mol / L.

[0014] The third object of the present invention is an oral adhesive repair material, including phosphorylated chitosan and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide; the phosphorus content of the phosphorylated chitosan is ≥5%.

[0015] Preferably, the concentration of phosphorylated chitosan is 0.5 - 2.0 g / L; the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 2.4 - 4 mol / L; the concentration of N-hydroxysuccinimide is 0.5 - 1.2 mol / L.

[0016] The fourth object of the present invention is to provide the application of the above composition for improving the binding of phosphorylated chitosan to demineralized dentin and the oral adhesive repair material in the preparation of dental materials.

[0017] The beneficial technical effects of the present invention are that a series of property changes are brought to the dentin bonding interface:

[0018] 1. The mechanical strength and anti-enzymatic hydrolysis ability of dentin collagen fibers are improved.

[0019] 2. Endow the bonding interface with the ability to inhibit bacteria, thereby preserving the demineralized dentin collagen fibers and stabilizing the bonding interface.

[0020] 3. Stabilize the dentin collagen fibers, maintain a suitable microenvironment for remineralization, promote the deposition of minerals such as calcium and phosphorus on the dentin surface, and at the same time provide protection for the dentin against demineralization.

[0021] 4. Through the synergistic effect of covalent cross-linking (anti-degradation) and phosphate group-guided mineralization (promote regeneration), the present invention achieves "prevention and treatment combination"; for the first time, it solves the three major problems of microleakage, collagen degradation and remineralization in the field of dentin bonding, and extends the lifespan of the restoration by more than 2 times. Brief Description of the Drawings

[0022] The present invention is further described with reference to the accompanying drawings, but the embodiments in the drawings do not constitute any limitation to the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the following drawings.

[0023] Figure 1 Molecular structure analysis of chitosan occupying the collagen-CAN binding site.

[0024] Figure 2 Schematic diagram of phosphorylated chitosan protecting demineralized dentin collagen.

[0025] Figure 3 Schematic diagram of the preparation process of extracted teeth.

[0026] Figure 4 Schematic diagram of EDC-crosslinked chitosan and human type I collagen.

[0027] Figure 5 Characterization of demineralized dentin samples.

[0028] Figure 6 Characterization of remineralized dentin samples. Detailed Description of the Invention

[0029] For a better understanding of the present invention, the present invention is further described below with specific serial numbers. The terms used in the serial numbers are for describing specific embodiments and do not constitute a limitation to the protection scope of the present invention.

[0030] It should be noted that the above-described work process is only illustrative and does not limit the protection scope of the present invention. In actual applications, those skilled in the art can select some or all of them according to actual needs to achieve the purpose of the embodiment scheme, and no limitation is made here.

[0031] The meanings of the abbreviations in the present invention:

[0032] PC: Phosphorylated chitosan

[0033] EDC: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide

[0034] NHS: N-Hydroxysuccinimide

[0035] M: mol / L

[0036] CDDM: Completely demineralized dentin

[0037] SEM: Scanning electron microscope

[0038] EDS: Energy dispersive spectrometer

[0039] Example 1

[0040] 1. Preparation of demineralized dentin

[0041] After approval by the ethics committee and informed consent of the patients, fresh extracted molars without caries, cracks and defects were collected. Immediately after extraction, they were washed with 3% hydrogen peroxide solution and stored in 4°C physiological saline. They were used within 1 month after extraction. When in use, the teeth were embedded and fixed in a mold with embedding resin, and a dentin slice with a thickness of about 0.5 mm was cut perpendicularly to the long axis of the tooth crown in the middle of the tooth with a slow cutter under the condition of running water cooling, and the upper and lower surfaces were ensured to be flat( Figure 3 ). Then it was wet polished with 600-mesh silicon carbide sandpaper and then demineralized with 10% phosphoric acid solution. The degree of demineralization was detected by Fourier transform infrared spectroscopy (FTIR). When the vibration peak at 1004 cm 3- representing the PO4 -1 group weakened, it indicated that the dentin slice had reached partial demineralization. After the dentin collagen slice was ultrasonically cleaned 3 times in deionized water to remove the phosphoric acid solution, it was cut into dentin strips of 3 mm×3 mm×0.5 mm and stored at 4°C for standby.

[0042] 2. Chemical cross-linking reaction

[0043] (1) Cross-linking and fixation of phosphorylated chitosan with collagen fibers on the surface of demineralized dentin

[0044] The demineralized dentin strips were placed in 1 ml of a solution containing 3.3 M cross-linking agent EDC, 0.8 M N-hydroxysuccinimide (NHS) and 1 g / L PC and left at 4°C for 24 h. The carboxyl groups of the collagen fibers and the amino groups of PC were cross-linked under the mediation of EDC.

[0045] For the specific cross-linking principle, please refer to the article "Zhou Z, Yang Y, He L, Wang J, Xiong J. Molecular docking reveals Chitosan nanoparticle protection mechanism for dentin against Collagen-binding bacteria. J Mater Sci Mater Med 2022, 33(5): 43."

[0046] EDC first reacts with the carboxyl group of collagen to form an intermediate containing O-acylisourea, which can further react rapidly with the amino group of PC under the stabilization of NHS to form an amide bond and form a chemical cross-link between collagen and PC, while releasing water-soluble isourea by-products ( Figure 4 ).

[0047] (2) Cross-linking stability between collagen fibers inside demineralized dentin

[0048] While treating demineralized dentin by the above method, the carboxyl groups and amino groups that are spatially close to each other between the collagen fibers inside demineralized dentin cross-link under the mediation of EDC. The cross-linking principle is also similar to the above method ( Figure 4 ).

[0049] The preparation of PC refers to the description in CN 113185622 A "A chitosan derivative with high phosphorus content and its preparation and application in loading nano-zero valent iron"; the phosphorus content in the PC of the present invention is ≥5%.

[0050] Example 2 Cross-linking and influence of phosphorylated chitosan and completely demineralized dentin (CDDM) collagen fibers

[0051] The covalent cross-linking of PC on the surface of demineralized dentin and collagen fibers affects the resin adhesive in dentin, including surface components, microstructure, surface properties, etc.

[0052] Specimen preparation and treatment: After approval by the ethics committee and informed consent of the patients, 20 freshly extracted molars without caries, cracks, or defects (n = 5) were collected. Dentin slices with a thickness of about 0.5 mm were cut perpendicular to the long axis of the tooth crown in the middle of the tooth crown using a slow cutter under running water cooling, and then cut into sheet specimens of 3 mm × 3 mm × 0.5 mm, and ensure that the upper and lower surfaces of the specimens are flat ( Figure 3)。Then, wet-polish the dentin surface to be demineralized with 400-mesh, 800-mesh, and 1200-mesh silicon carbide sandpaper, ultrasonically oscillate and clean for 5 min, and carefully check the enamel on the surface of the dentin slices under a stereomicroscope to ensure complete removal. Then, evenly coat nail polish on the remaining surfaces except the surface to be demineralized, soak in 10% phosphoric acid solution for demineralization for 24 h, then rinse with a large amount of deionized water for 3 min, and dry in an oven for 24 h to obtain dry samples. Treat according to the following experimental groups:

[0053] A.Control (blank control group)

[0054] B.EDC cross-linking group (negative control group)

[0055] C.PC film-forming group

[0056] D.PC cross-linking group

[0057] Among them, for the film-forming group, place the dentin specimens in 100 μl of PC solution, and then place them in a desiccator until the water in the dentin completely evaporates. For the cross-linking group, place the dentin slices in 1 ml of solution containing 3.3 M cross-linking agent EDC and 0.8 M N-hydroxysuccinimide (NHS) (with or without 1 g / L PC) and place at 4 °C for 24 h.

[0058] a. Observe the microscopic morphology of the dentin surface and perform elemental analysis with an energy dispersive spectrometer (EDS) under a scanning electron microscope (SEM)

[0059] I. Take a total of 10 specimens (n = 2) from each group after treatment and rinse with deionized water for 20 s.

[0060] II. Then fix with 3% glutaraldehyde solution and 0.1 mol / L PBS buffer (1:1, pH = 7.2) at room temperature for 4 h, rinse with PBS 3 times, and then dehydrate with gradient ethanol (10%, 30%, 50%, 75%, 90%, 100% ethanol solutions, 3 times each, 30 min each time).

[0061] III. Place the specimens in 100% ethanol and dry them with carbon dioxide in a critical point dryer. Fix the dehydrated specimens on an aluminum base and sputter with gold / platinum to a thickness of 20 nm (60Au:40Pd, Hummer X Sputter Coater, Anatech USA, Union City, CA, USA).

[0062] VI. Place under the SEM and observe the collagen fiber structure and the distribution of PC on the dentin surface of each group at 5 kV and a working distance of 10–12 mm. Take 8 pictures of each specimen.

[0063] The demineralization of the samples was observed at the cross-section of the dentinal tubules under SEM. The dentin sections of the control group were relatively flat, and no obvious collagen fiber structure was observed ( Figure 5 A in Figure 5 ); while in the CDDM group, a large number of collagen fibers could be observed, and at the same time, the diameter of the dentinal tubules increased significantly ( Figure 5 B in

[0064] ), indicating good demineralization effect. The EDS results showed that the P and Ca contents in the CDDM group were significantly lower than those in the control group, indicating that the mineral content of the samples decreased significantly (

[0065] C, D in -1 ). -1

[0066] The FTIR test results showed that in the CDDM group, the infrared spectrum showed the characteristic peaks of collagen. Compared with the undemineralized dentin of the control group, the intensity of the P-O peak at 1035 cm -1 decreased significantly, indicating that the CDDM group had achieved the expected effect of complete demineralization ( Figure 5 E in

[0067] ). Example 3 Collagenase Resistance Test

[0068] Preparation of type I collagenase solution: Purchase DPBS buffer solution with pH = 7.4, and then add 50 mg of type I collagenase with specific activity ≥ 125 CDU / mg to 100 ml of DPBS buffer solution, adjust the pH to 7.4 and stir evenly.

[0069] a. Dry weight loss experiment

[0070] Take out 4 demineralized dentin collagen strips from each group, transfer them to separate polypropylene centrifuge tubes, rinse them 3 times with deionized water, and place them in a vacuum dryer to dry to constant weight. Weigh their mass with an analytical balance and record it as m1, accurate to 0.01 mg. Then, place the dentin collagen strips in 2 mL of type I collagenase solution for enzymatic hydrolysis, and hydrolyze them on a shaker in a 37 °C water bath for 24 h. Take out the remaining dentin collagen strips, dry them to constant weight in a vacuum dryer, and weigh the remaining collagen mass with an analytical balance and record it as m2.

[0071] The calculation formula is:​

[0072] Dry weight loss = (m1 - m2) / m1 × 100%.

[0073] Table 1 Collagenase degradation dry weight test

[0074]

[0075] The demineralized dentin of each group was subjected to a collagenase degradation test, and the mass of the dentin collagen strip loss after the reaction was weighed and calculated. The content of free amino acids detected in the PC cross-linked group was extremely low at both 1 day and 2 days of enzymatic hydrolysis, which were 0.042 mg and 0.040 mg respectively, while the dry weight loss masses of the control PC film-forming group were 0.439 mg and 1.143 mg (Table 1). The results showed that after PC was cross-linked with demineralized dentin, it could inhibit the enzymatic hydrolysis reaction of collagen fibers.

[0076] b. Amino acid release experiment

[0077] Take out 0.5 ml of the supernatant from the polypropylene centrifuge tube containing the dentin collagen strip for amino acid kit detection, and the experimental steps are carried out according to the instructions of the kit. Use an enzyme-labeled instrument to measure the absorbance of the solution at a wavelength of 560 nm. Calculate the release amount of amino acids according to the kit formula to evaluate the degradation of dentin collagen.

[0078] Table 2 Collagenase degradation amino acid release test

[0079]

[0080] The demineralized dentin of each group was subjected to a collagenase degradation test to detect the content of free amino acids after the reaction. The content of free amino acids detected in the PC cross-linked group was extremely low at both 1 day and 2 days of enzymatic hydrolysis, which were 0.01 ± 0.23 μmol / L·mg and -0.03 ± 0.34 μmol / L·mg respectively, while the content of free amino acids detected in the control PC film-forming group was close to 5.57 ± 0.22 μmol / L·mg and 13.02 ± 0.44 μmol / L·mg of the blank control group, with significant statistical differences (p < 0.05, Table 2). The results showed that after PC was cross-linked with demineralized dentin, it could greatly protect its collagen fibers from being degraded.

[0081] Example 4 Phosphorylated chitosan promotes the remineralization of the demineralized dentin surface

[0082] Remineralization solution preparation: An artificial body fluid containing 1.7 mM calcium chloride, 9.5 mM disodium hydrogen phosphate, 125 mM sodium chloride, and 2.7 mg / ml sodium polyaspartate was prepared with Tris buffer solution at pH = 7.4, and adjusted to pH 7.4 with sodium hydroxide.

[0083] PC solution preparation: Prepare it with MES (0.5 M, pH 5.0) as the solvent 24 h before the experiment and store it at -20 °C.

[0084] Remineralization treatment of demineralized dentin specimens: Immerse the specimens in the remineralization artificial fluid at 37 °C (each in 30 mL), and change the fluid once every 24 h. The specimens are stored in the mineralization fluid for 7 d and 14 d. After 7 d and 14 d, rinse them with deionized water for 3 minutes and then conduct the detection.

[0085] a. Observe the surface micro-morphology under a scanning electron microscope (SEM)

[0086] I. Take a total of 8 specimens (n = 2) from each treatment group, and rinse them with deionized water for 20 s.

[0087] II. Then fix them with 3% glutaraldehyde solution and 0.1 mol / L PBS buffer (1:1, PH = 7.2) at room temperature for 4 h, rinse them with PBS 3 times, and then dehydrate them with gradient ethanol (3 times each with 10%, 30%, 50%, 75%, 90%, and 100% ethanol solutions, 30 min each time).

[0088] III. Place the specimens in 100% ethanol in a critical point dryer and dry them with carbon dioxide. Fix the dehydrated specimens on an aluminum base and sputter gold / platinum with a thickness of 20 nm (60Au:40Pd, Hummer X Sputter Coater, Anatech USA, Union City, CA, USA).

[0089] VI. Place them under the SEM and observe the collagen fiber structure and PC distribution on the surface of dentin in each group at 5 kV and a working distance of 10–12 mm. Take 8 pictures of each specimen.

[0090] Observe the remineralization situation from the SEM images of the cross-sections of dentin tubules in each group. Mineralization particles can be clearly observed on the surface of collagen fibers in the PC cross-linking group. At the same time, the wall of the dentin tubule thickens and the diameter becomes smaller (D in Figure 6 ); obvious collagen fiber structures can still be observed in the remaining groups (A, B, and C in Figure 6 ).

[0091] b. Detection with an attenuated total reflection Fourier transform infrared spectrometer (ATR-FTIR)

[0092] The qualitative analysis of the demineralization degree on the surface of dentin is determined by ATR-FTIR.

[0093] I. Take 10 teeth (n = 2) from each treatment group and rinse them with deionized water for 20 s.

[0094] II. ATR-FTIR spectra of dentin specimens were collected using an infrared spectrometer (SHIMADZU, 8400S, Japan) in reflection mode in the range of 400 - 4000 cm -1 band with a resolution of 16 cm -1 , and scanned 10 times. Each specimen was scanned at 3 different positions to analyze the content of mineral elements such as calcium and phosphorus on the surface of the specimen under different treatments.

[0095] In the PC cross-linking group, the intensity of the P-O peak at 1035 cm -1 was significantly increased, indicating a higher degree of remineralization of demineralized dentin in the PC cross-linking group ( Figure 6 E in).

[0096] Expansion test between different concentrations in Example 4

[0097] To further explore the effect on remineralization under different concentrations, the following expansion test was conducted.

[0098] The test was carried out using the method of Example 1, except that the concentrations of the cross-linking agents EDC, NHS, and PC in the chemical cross-linking reaction were as shown in Table 3 below; then the dry weight loss experiment and hydroxyproline release experiment were carried out according to the method of Example 3; the results are shown in Table 4.

[0099] Table 3 Different concentration groups in the expansion test

[0100] Serial number EDC concentration (mol / L) NHS concentration (mol / L) PC concentration (g / L) Scale-up test 1 2.0 0.2 0.2 Scale-up test 2 2.4 0.6 0.5 Scale-up test 3 4.0 1.2 2

[0101] Table 4 Results of the expansion test

[0102]

[0103]

[0104] From the results in Table 4, it can be seen that when the PC concentration is 0.2 g / L, the EDC concentration is 0.2 mol / L, and the NHS concentration is 2.0 mol / L, too low a concentration of phosphorylated chitosan (PC) leads to insufficient mineralization induction ability: PC is the "template" for calcium and phosphorus deposition. When the concentration is too low, PC cannot fully cover the surface of collagen fibers, and the remaining collagen is easily degraded by matrix metalloproteinases (MMPs), resulting in insufficient long-term stability.

[0105] When the NHS concentration is too low, the intermediate is easily hydrolyzed and inactivated, and the cross-linking reaction time window is too short; and EDC may preferentially react with free carboxylic acids in the solution (such as PC or collagen hydrolysis products) rather than the target sites of collagen fibers, resulting in ineffective cross-linking.

[0106] If the EDC concentration is too low, the efficiency of converting carboxyl groups into active esters (O-acylisourea) is low, and the cross-linking density between collagen molecules is insufficient. The collagen with insufficient cross-linking cannot provide a stable anchoring site for PC, resulting in the decoupling of the mineralized layer from the collagen network. Collectively, this leads to a low degree of inhibition of the enzymatic reaction of collagen fibers.

[0107] If the PC concentration is too high, there may be a too high local phosphate concentration, which may form a disordered mineralized structure (such as coarse or unevenly distributed hydroxyapatite crystals), thereby reducing the mechanical strength of dentin; covering the active sites (such as carboxyl and amino groups) on the surface of collagen fibers, hindering the cross-linking reaction mediated by EDC, resulting in a decrease in cross-linking density and a decline in the stability of the bonding interface; in addition, high-concentration PC may cause local pH fluctuations or cytotoxicity, inhibiting the activity of odontoblasts and affecting the health of the pulp tissue.

[0108] If the EDC concentration is too high, it will over-activate the carboxyl groups of collagen fibers, resulting in an excessively high intermolecular cross-linking density, increasing the rigidity and decreasing the toughness of the collagen network, making it prone to brittle fracture under stress. EDC is easily hydrolyzed to form inactive urea derivatives, and the side reactions accelerate at high concentrations, not only wasting reagents but also possibly generating impurities that interfere with the cross-linking process. EDC itself has cytotoxicity, and if the concentration exceeds the standard, it may remain in the dentinal tubules and cause damage to the surrounding tissues (such as the pulp) during long-term release.

[0109] If the NHS concentration is too high, it will overly prolong the intermediate lifetime, resulting in an uncontrollable cross-linking reaction time and possibly triggering non-specific cross-linking (such as self-polymerization between PC molecules). High-concentration NHS will increase the ionic strength of the solution, reducing the penetration ability of PC and EDC into the deep part of the dentinal tubules, resulting in only surface cross-linking and no internal repair.

[0110] Difficulty in removing residues: When NHS is in excess, it may be difficult to completely remove it through subsequent rinsing, and the remaining succinimide groups may trigger an inflammatory reaction.

[0111] Therefore, for safety and mechanical property considerations, the present invention did not conduct experiments with too high concentrations.

[0112] In addition, for the technical details not described in detail in this embodiment, reference can be made to the parameter operation method provided in any embodiment of the present invention, which will not be elaborated here.

[0113] It should be noted that, in this document, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or system including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or further includes elements inherent to such process, method, article or system. Without further limitation, an element defined by the statement "including one..." does not exclude the existence of additional identical elements in the process, method, article or system including such element.

[0114] The serial numbers of the embodiments of the present invention above are only for description and do not represent the superiority or inferiority of the embodiments.

[0115] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent structure or equivalent process transformation made by using the content of the specification and drawings of the present invention, or directly or indirectly applied in other related technical fields, shall be equally included in the patent protection scope of the present invention.

Claims

1. A method for improving the stability of the dentin bonding interface by phosphorylated chitosan, characterized in that: The method comprises: Phosphorylated chitosan was mixed with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide and then evenly coated on the dentin surface to form a covalent cross-linked network and induce in situ deposition of hydroxyapatite.

2. The method for improving the stability of the dentin bonding interface by phosphorylated chitosan according to claim 1, characterized in that: The phosphorus content of the phosphorylated chitosan is ≥5%.

3. The method for improving the stability of the dentin bonding interface by phosphorylated chitosan according to claim 1, characterized in that: The concentration of phosphorylated chitosan is 0.5-2.0 g / L; the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 2.4-4 mol / L; and the concentration of N-hydroxysuccinimide is 0.5-1.2 mol / L.

4. The method for improving the stability of the dentin bonding interface by phosphorylated chitosan according to claim 1, characterized in that: The concentration of phosphorylated chitosan was 1 g / L; the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide was 3.3 mol / L; and the concentration of N-hydroxysuccinimide was 0.8 mol / L.

5. The method for improving the stability of the dentin bonding interface by phosphorylated chitosan according to claim 1, characterized in that: The method for preparing phosphorylated chitosan comprises: adopting a microwave-assisted method, using 1,2,4-tricarboxybutane phosphate as a phosphoric acid source, modifying the C6 hydroxyl group of chitosan by esterification reaction, and preparing phosphorylated chitosan with a phosphorus content of ≥5%.

6. A composition for cross-linking demineralized dentin with phosphorylated chitosan to improve the stability of the dentin bonding interface, characterized in that: The cross-linking agent comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide; the mass ratio thereof is (2.4-4): (0.5-1.2) mol / L.

7. An oral adhesive repair material, characterized in that: The invention comprises phosphorylated chitosan, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide; the phosphorus content of the phosphorylated chitosan is ≥5%.

8. The oral bonding repair material according to claim 7, characterized in that: The concentration of the phosphorylated chitosan is 0.5-2.0 g / L; the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 2.4-4 mol / L; and the concentration of N-hydroxysuccinimide is 0.5-1.2 mol / L.

9. Use of the composition for improving the bonding between phosphorylated chitosan and demineralized dentin as claimed in claim 6 and the oral adhesive restoration material as claimed in claim 7 or 8 in the preparation of dental restoration materials.

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