Dental resin cement and method for preparing the same

Abstract

This invention provides a dental resin adhesive and its preparation method, relating to the field of dental restorative materials. The dental resin adhesive comprises a base adhesive and a mussel biomimetic monomer. The base adhesive comprises the following components in weight percentages: 35-40% bisphenol A dimethacrylate, 35-40% hydroxyethyl methacrylate, 5-10% triethylene glycol dimethacrylate, 10-15% diluent, 0.5-1.0% photoinitiator, 0.5-1.0% co-initiator, and 1.0-2.0% oxygen barrier. The mussel biomimetic monomer has the structure shown in Formula 1. The mass of the mussel biomimetic monomer is 1-6% of the total mass of the base adhesive and the mussel biomimetic monomer. The dental resin adhesive exhibits high permeability, resulting in a resin-dentin bond interface with good density, integrity, and long-term stability, and significantly reduces resin polymerization shrinkage and stress concentration. Formula 1

Description

Technical Field

[0001] This invention relates to the field of dental restorative materials, and in particular to a dental resin adhesive and its preparation method. Background Technology

[0002] In dentin-bonded restorations, the "hybrid layer" formed by the adhesive penetrating into the demineralized dentin collagen network and curing is crucial for achieving stable bonding strength, primarily relying on the micromechanical interlocking structures formed within the collagen scaffold. However, the hybrid layer, formed by the interweaving of the adhesive and dentin, often becomes a weak point for interfacial failure. This interface is exposed to the complex oral environment for extended periods, influenced by multiple factors such as saliva composition, tubular exudate, temperature changes, masticatory stress, and endogenous enzymes, making it susceptible to hydrolysis, degradation, and leakage, leading to adhesive failure and restoration detachment. The structural integrity, density, and penetration depth of the hybrid layer are key factors affecting the long-term performance of the bonding system.

[0003] Traditional adhesives primarily achieve micromechanical fixation. Establishing molecular-level chemical bonding structures on this basis would significantly improve the stability of the adhesive interface. Current mainstream self-etching adhesive systems incorporate phosphate ester functional monomers such as 10-methacryloxydecyl dihydrogen phosphate (10-MDP) and 4-methacryloxytrimelliticacid anhydride (4-META), which can react with hydroxyapatite to form phosphate-based bonds. However, their chemical action is limited to the mineral components and they struggle to form stable covalent bonds with the demineralized collagen network. Furthermore, these acidic monomers are sensitive to aqueous environments, easily undergoing hydrolytic degradation, and exhibit synergistic inhibition when coexisting with hydroxyethyl methacrylate (HEMA), further impacting long-term adhesive performance. Other studies have attempted to combine isocyanate monomers with collagen, but their hydrophobic properties hinder efficient penetration and fixation at moist dentin interfaces, limiting practical applications.

[0004] Furthermore, stress concentration and microleakage at the edges caused by resin polymerization shrinkage have long plagued resin bonding systems. Traditional adhesive monomer systems (such as HEMA, Bis-GMA (bisphenol A dimethacrylate), and unsymmetrical dimethylacrylate (UDMA)) generally suffer from low polymerization conversion rates and insufficient cross-linking, making it difficult to form a high-strength, low-stress mixed-layer structure, resulting in microcracks and leakage paths in the adhesive layer. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a dental resin adhesive and its preparation method. The dental resin adhesive provided by this invention has high permeability, and the resulting resin-dentin bond interface exhibits good density, integrity, and long-term stability, while significantly reducing resin polymerization shrinkage and stress concentration.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a dental resin adhesive, comprising a base adhesive and a mussel biomimetic monomer; the base adhesive comprises the following components in weight percentages: 35-40% bisphenol A dimethacrylate, 35-40% hydroxyethyl methacrylate, 5-10% triethylene glycol dimethacrylate, 10-15% diluent, 0.5-1.0% photoinitiator, 0.5-1.0% co-initiator, and 1.0-2.0% oxygen barrier; The mussel biomimetic monomer has the structure shown in Formula 1: Formula 1; The mass of the mussel biomimetic monomer is 1 to 6% of the total mass of the base binder and the mussel biomimetic monomer.

[0007] Preferably, the base adhesive comprises the following components in weight percentage: 40% bisphenol A dimethacrylate, 35% hydroxyethyl methacrylate, 10% triethylene glycol dimethacrylate, 13% diluent, 0.5% photoinitiator, 0.5% co-initiator, and 1% oxygen barrier.

[0008] Preferably, the diluent is ethanol.

[0009] Preferably, the photoinitiator is camphorquinone.

[0010] Preferably, the co-initiator is ethyl p-dimethylaminobenzoate.

[0011] Preferably, the oxygen inhibitor is cysteine.

[0012] Preferably, the mass of the mussel biomimetic monomer is 2-5% of the total mass of the base binder and the mussel biomimetic monomer.

[0013] This invention provides a method for preparing the dental resin adhesive described in the above technical solution, comprising the following steps: The dental resin adhesive is obtained by mixing bisphenol A dimethacrylate, hydroxyethyl methacrylate, triethylene glycol dimethacrylate, diluent, photoinitiator, co-initiator, oxygen barrier and mussel biomimetic monomer.

[0014] This invention provides a dental resin adhesive, which has the following advantages compared with the prior art: (1) Improve the permeability and interfacial density of dental resin adhesives Current dentin bonding systems are based on wet bonding theory, which requires the retention of some moisture after demineralization to maintain the openness of the collagen network, allowing the adhesive resin to penetrate into the collagen structure and form micromechanical interlocking. However, the presence of interfacial moisture also severely hinders the deep penetration of resin monomers, resulting in a loose and defective interfacial mixed layer, affecting bonding performance and durability. This invention introduces a mussel-inspired monomer (lysine cationic-catechol-methacrylate, abbreviated as CLM) into the adhesive system. The cationic groups in the CLM molecular structure can regulate the collagen surface potential, affecting the binding capacity of interfacial water, making the demineralized collagen matrix surface more conducive to the full penetration of resin components. At the same time, the amphiphilic structure of CLM improves its compatibility with resin monomers, promoting the formation of a more continuous and dense mixed layer structure on the wet dentin surface, improving initial bond strength and inhibiting the generation of interfacial microleakage.

[0015] (2) Reduce polymerization shrinkage and stress concentration of resin adhesives Resin adhesives often experience polymerization shrinkage during photocuring, leading to interfacial stress concentration, edge microleakage, and adhesive failure. The CLM molecular structure of this invention contains polymerizable double bonds, enabling copolymerization with monomers such as hydroxyethyl methacrylate (HEMA) and bisphenol A dimethacrylate glycidyl acrylate (Bis-GMA) in the adhesive. It also possesses a relatively rigid framework structure, allowing for the formation of internal crosslinking points within the polymerization system. This enhances the resin's cohesiveness and degree of crosslinking, effectively suppressing polymerization shrinkage deformation and reducing interfacial stress accumulation. Furthermore, the CLM-enhanced crosslinking structure further improves the mechanical strength and morphological integrity of the adhesive layer, resulting in a hybrid layer with superior functionality and stability.

[0016] (3) Enhance the molecular integration capacity of the resin-dentin interface The CLM monomer in this invention possesses a catechol group, a structure exhibiting extremely strong bioadhesive activity. The catechol structure can covalently react with the amino groups in dentin collagen to form stable chemical bonds, achieving molecular-level integration between collagen and resin through micromechanical intercalation. Simultaneously, the acrylate structure of CLM participates in the polymerization of the resin system, achieving synergistic copolymerization with the adhesive resin network. This chemically integrates the dental collagen matrix and the artificial resin matrix into a unified whole, significantly improving the integrity and long-term stability of the resin-dentin bonding interface.

[0017] (4) Promotes the formation of the mixed layer and enhances the initial sealing and permeability stability. Traditional adhesives suffer from insufficient penetration and incomplete curing within the collagen network, leading to the formation of micropores, leakage channels, and degradation starting points within the hybrid layer. The CLM-introduced adhesive in this invention exhibits superior diffusion within the collagen network, resulting in a hybrid layer with better sealing and structural integrity after curing, effectively reducing the risk of moisture penetration and external bacterial invasion. Furthermore, the increased density of the hybrid layer provides a more stable substrate for subsequent bonding of repair materials. Attached Figure Description Figure 1 The polymerization conversion rate of each group of adhesives in the examples; Figure 2 The contact angle of each group of adhesives on the demineralized dentin surface in the examples; Figure 3 The following are thermogravimetric diagrams of the adhesives in each group in the examples; Figure 4 The polymerization shrinkage rate of each group of adhesives in the examples; Figure 5 The results show the flexural strength of each group of adhesives in the examples; Figure 6 The figures represent the relative cell proliferation rates of the adhesive extracts in each group in the examples. Figure 7 The results of live / dead cell staining in each group are shown in the examples; Figure 8 These are representative images of the acute hemolysis experiments of each group of extracts in the examples; Figure 9 HE staining images of major organs 7 days after oral administration of the adhesive extracts in each group of the examples; Figure 10 This is a graph showing the metabolic activity results of bacteria adhering to the surface of the adhesive specimens in the examples; Figure 11 This is a graph showing the metabolic activity results of airborne bacteria on the surface of the adhesive specimens in the examples; Figure 12 This is an observation image of the biofilm adhering to the surface of the adhesive reagent immediately in the example; Figure 13 This is an observation image of the bacterial biofilm adhering to the surface of the bonded specimen after aging treatment in the example; Figure 14 The images shown are stained images of live / dead bacteria adhering to the surface of the specimens before and after the aging treatment in the examples. Figure 15 The image shown is an RMSD image of CLM on the hydroxyapatite surface in the example. Figure 16 The interaction energy between the CLM and the HAP surface in the embodiment; Figure 17 This is the stable configuration of CLM bonded to the HAP (010) surface in the embodiment; Figure 18 The HSQC spectrum of type I collagen in the examples is shown below; Figure 19 The image shown is the HSQC spectrum after the reaction of CLM with type I collagen in the example. Figure 20 The solid-state NMR spectra of hydroxyapatite before and after the reaction with CLM are shown in the examples. Figure 20 In the image, A represents the solid-state NMR of hydroxyapatite, and B represents the solid-state NMR of hydroxyapatite after its reaction with CLM. Figure 21 The images show the Raman spectra of the bonding interfaces of each adhesive group in the examples. Figure 22 The images show the Raman spectra of the bonding interfaces of each adhesive group in the examples. Figure 23 The dentin bond strength of each group of adhesives in the examples; Figure 24 These are representative in-situ enzyme spectra of the bonding interfaces in each group of examples; Figure 25 This illustrates the nano-leakage at the bonding interface in the embodiment. Figure 26 The morphology of each bonding interface was observed under a scanning electron microscope in the example. Figure 27 The results show the sudden penetration depth of the adhesive resin in each group in the examples; Figure 28 These are representative images showing the sudden penetration of adhesive resin in each group in the examples; Figure 29 This shows the water penetration at the bonding interface of each adhesive group in the examples. Detailed Implementation

[0018] This invention provides a dental resin adhesive, comprising a base adhesive and a mussel biomimetic monomer; the base adhesive comprises the following components in weight percentages: 35-40% bisphenol A dimethacrylate, 35-40% hydroxyethyl methacrylate, 5-10% triethylene glycol dimethacrylate, 10-15% diluent, 0.5-1.0% photoinitiator, 0.5-1.0% co-initiator, and 1.0-2.0% oxygen barrier; The mussel biomimetic monomer has the structure shown in Formula 1: Formula 1; The mass of the mussel biomimetic monomer is 1 to 6% of the total mass of the base binder and the mussel biomimetic monomer.

[0019] Unless otherwise specified, all components involved in this invention are commercially available products or prepared according to methods known to those skilled in the art.

[0020] The dental resin adhesive provided by this invention includes a base adhesive. In embodiments of this invention, the base adhesive is abbreviated as BCL. In this invention, the base adhesive comprises the following components in weight percentage: 35-40% bisphenol A glycidyl methacrylate (Bis-GMA), 35-40% hydroxyethyl methacrylate (HEMA), 5-10% triethylene glycol dimethacrylate (TEGDMA), 10-15% diluent, 0.5-1.0% photoinitiator, 0.5-1.0% co-initiator, and 1.0-2.0% oxygen barrier. In this invention, the mass percentage of bisphenol A dimethacrylate glycidyl ester in the base binder can be 35%, 36%, 37%, 38%, 39%, or 40%; the mass percentage of hydroxyethyl methacrylate can be 35%, 36%, 37%, 38%, 39%, or 40%; the mass percentage of triethylene glycol dimethacrylate can be 5%, 6%, 7%, 8%, 9%, or 10%; the mass percentage of diluent can be 10%, 11%, 12%, 13%, 14%, or 15%; the mass percentage of photoinitiator can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%; the mass percentage of co-initiator can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%; and the mass percentage of oxygen barrier can be 1.0%, 1.5%, or 2.0%. As an embodiment of the present invention, the base adhesive comprises the following components in weight percentage: 40% bisphenol A dimethacrylate, 35% hydroxyethyl methacrylate, 10% triethylene glycol dimethacrylate, 13% diluent, 0.5% photoinitiator, 0.5% co-initiator, and 1% oxygen barrier.

[0021] In this invention, the bisphenol A dimethacrylate glycidyl ester is a hydrophobic base resin component, the hydroxyethyl methacrylate is a hydrophilic resin viscosity-reducing component, and the triethylene glycol dimethacrylate is a plasticizing and viscosity-reducing resin component.

[0022] In this invention, the diluent is preferably ethanol.

[0023] In this invention, the photoinitiator is preferably camphorquinone (CQ), and the co-initiator is preferably ethyl p-dimethylaminobenzoate (EDMAB); the photoinitiator and the co-initiator constitute a resin-based photoinitiation system.

[0024] In this invention, the oxygen barrier is preferably cysteine ​​(L-Cys).

[0025] The dental resin adhesive provided by this invention includes a mussel biomimetic monomer having the structure shown in Formula 1 (lysine cationic-catechol-methacrylate). This invention does not have particular requirements regarding the source of the mussel biomimetic monomer, which is prepared using methods well known to those skilled in the art.

[0026] In this invention, the mass of the mussel biomimetic monomer is preferably 2 to 5% of the total mass of the base binder and the mussel biomimetic monomer, and can be 2%, 3%, 4% or 5%.

[0027] This invention introduces a functional monomer, namely the mussel-inspired monomer (CLM), into the adhesive system. This monomer combines polymerizable acrylate groups, bioadhesive catechol groups, and cationic side chains with charge-regulating capabilities. CLM molecules can participate in the polymerization reaction of the resin network through double bonds, enhancing the cohesive force and cross-linking density of the adhesive, thereby significantly reducing volume shrinkage during polymerization and alleviating stress concentration at the bonding interface. Simultaneously, its molecular structure optimizes the resin's flowability and wetting properties, improving penetration depth into the dentin matrix and interfacial adaptability. The catechol structure of this monomer possesses the high adhesion properties of mussel protein, reacting with amino groups in collagen to form stable covalent bonds, achieving molecular integration of the adhesive molecules with dental collagen. This mechanism not only strengthens the chemical bonding of the mixed layer but also significantly improves the overall density and sealing of the mixed layer, inhibiting the degradation risk caused by moisture and microbial infiltration. The dental resin adhesive provided by this invention can also form longer and denser resin protrusion structures, enhancing the sealing of the mixed layer and significantly reducing interfacial microleakage. This invention not only optimizes the initial bonding effect of the adhesive but also provides a solid foundation for long-term stability. Starting from molecular structure regulation, this invention comprehensively improves the adhesive's permeability, polymerization performance, and collagen binding ability, developing a multifunctional dental resin adhesive that offers a new approach to constructing dense, high-strength, and aging-resistant hybrid layer structures.

[0028] This invention provides a method for preparing the dental resin adhesive described in the above technical solution, comprising the following steps: The dental resin adhesive is obtained by mixing bisphenol A dimethacrylate, hydroxyethyl methacrylate, triethylene glycol dimethacrylate, diluent, photoinitiator, co-initiator, oxygen barrier and mussel biomimetic monomer.

[0029] In this embodiment of the invention, the mixing is preferably: bisphenol A dimethacrylate, hydroxyethyl methacrylate, triethylene glycol dimethacrylate, diluent, photoinitiator, co-initiator and oxygen barrier are first mixed to obtain a basic binder; The base adhesive is mixed with the second mussel biomimetic monomer to obtain the dental resin adhesive.

[0030] This invention does not have any special requirements for the specific mixing method, as long as the components are mixed evenly.

[0031] In this invention, the dental resin adhesive is preferably stored in a nitrogen-sealed brown plastic bottle at 4°C.

[0032] To further illustrate the present invention, the dental resin adhesive and its preparation method provided by the present invention are described in detail below with reference to examples, but these should not be construed as limiting the scope of protection of the present invention.

[0033] Example (a) Preparation of dental resin adhesive Base adhesive: 40% bisphenol A dimethacrylate (Bis-GMA), 35% hydroxyethyl methacrylate (HEMA), 10% triethylene glycol dimethacrylate (TEGDMA), 13% ethanol, 0.5% camphorquinone (CQ), 0.5% ethyl p-dimethylaminobenzoate (EDMAB), and 1% cysteine ​​(L-Cys) are mixed to obtain the base adhesive, denoted as BCL.

[0034] Experimental adhesive (i.e., the dental resin adhesive): Different concentrations of mussel biomimetic monomer (abbreviated as CLM) were added to BCL and mixed to obtain the experimental adhesive. The mass of CLM was 1%, 2%, 3%, 4%, 5%, and 6% of the total mass of BCL and CLM, respectively. The resulting experimental adhesives were named CLM1, CLM2, CLM3, CLM4, CLM5, and CLM6, respectively.

[0035] CLM structure: .

[0036] The base adhesive served as a blank control group (BCL); the experimental adhesives (CLM1, CLM2, CLM3, CLM4, CLM5, CLM6) were stored in nitrogen-sealed brown plastic bottles at 4°C; Single bond2, an adhesive with similar base components, served as a commercial control group (CCL). The formulation composition of each adhesive group is shown in Table 1.

[0037] Table 1 Composition of each adhesive formulation

[0038] (II) Performance tests were conducted on each adhesive. The test methods are as follows: (1) Adhesive polymerization conversion rate Potassium bromide wafers were used as salt windows. Before testing, the salt windows and sample holder were wiped with anhydrous ethanol. Fourier transform infrared spectroscopy was used to scan the salt windows as a background reference. Thin layers of each adhesive were coated onto the potassium bromide wafers, and infrared spectra were obtained before curing. The wafer surface was covered with a celluloid film, and a 10 mm light guide rod and an LED curing lamp were used at a distance of approximately 1 mm and a speed of 1470 mW / cm². 2 After curing under light intensity for 20 seconds, the infrared spectrum after curing was obtained again by scanning. The infrared spectra before and after curing were measured at 1638 cm⁻¹. -1 The intensity of the aliphatic C=C absorption peak at 1608 cm⁻¹ -1 The polymerization conversion rate (DC) (%) was calculated by using the intensity of the aromatic CC reference peak according to the formula. The infrared scanning parameters were: resolution 4 cm-1. -1 The number of scans was 24. The formula for calculating DC (%) is as follows: .

[0039] (2) Adhesive wettability A 1 mm thick dentin slice was cut from the mid-crown of the third molar using a slow-speed cutting machine under running water for testing. The dentin slice was then polished with 600-grit sandpaper under running water to simulate a standard smear layer. The crown surface of the dentin was selected for the experiment. The demineralized dentin surface was treated with 35% phosphate for 15 seconds, rinsed with deionized water for 30 seconds, and excess moisture was absorbed with filter paper to maintain a slightly moist state. The treated demineralized dentin slice was placed 5 mm below the needle tip at the center of the contact angle measuring platform, with the sample plane perpendicular to the measuring micro-injector. One drop of adhesive was added to the micro-injector for each group, and after standing for 10 seconds, an image of the adhesive on the demineralized dentin surface was taken, and the contact angle was calculated. The dentin sample was then moved, and the adhesive was added again for calculation. The average of the three measurements was taken as the contact angle of the adhesive on the demineralized dentin surface. The contact angle of the adhesive on the demineralized dentin surface was used to characterize the wettability of the adhesive.

[0040] (3) Thermal stability of adhesive The thermal stability of the adhesives was tested using a TGA Q500 thermogravimetric analyzer. After drying, each adhesive sample was tested under nitrogen protection at a heating rate of 10℃ / min, from room temperature to 600℃. The temperature at which each adhesive lost 5 wt% of its weight was calculated to assess its thermal decomposition temperature.

[0041] (4) Adhesive polymerization shrinkage rate The polymerization shrinkage rate of the resin adhesive was determined using the volume displacement method based on Archimedes' principle. First, the uncured resin adhesive was slowly injected into a polytetrafluoroethylene mold (5.0 mm in diameter and 3.0 mm in height), avoiding the introduction of air bubbles. The mass of the sample in air (m1) and the buoyant mass suspended in distilled water (m2) were measured using a precision electronic balance. The initial volume (V0) of the uncured sample was calculated using the following formula: V0 = (m1 / m2) * (m2 ... 1- m2) / ρ, where ρ is the density of the displacing liquid (the density of distilled water at 25℃ is 1.00 g / cm³). 3 Anhydrous ethanol has a density of 0.789 g / cm³. 3 ).

[0042] Subsequently, the sample was irradiated from above for 40 seconds using an LED curing device (at a distance of approximately 1 mm and a curing rate of 1470 mW / cm²). 2 (Light intensity curing) to ensure uniform curing. After complete curing and cooling to room temperature, the mass of the sample in air and liquid was measured again, and the cured volume (V1) was calculated. The final polymerization shrinkage (PS%) was calculated using the following formula: PS% = ((V0-V1) / V0) × 100%. Each adhesive formulation was measured 5 times (n = 5), and all samples were prepared and cured in an environment of 23 ± 1℃ and 50 ± 5% relative humidity. Experimental data were analyzed using one-way ANOVA with post-hoc multiple comparisons, and the statistical significance level was set at α = 0.05.

[0043] (5) Flexural strength test Each group of adhesives was dripped into a mold, and the surface was covered with a polypropylene film. After curing for 20 seconds, rectangular specimens with a length of 25 mm and a width and height of 2 mm were prepared. The specimens were then immersed in deionized water at 37°C and stored in the dark for 24 hours before testing. Each group of specimens was placed on the loading stage of a multimeter with a span of 10 mm. The specimens were loaded at a loading rate of 1 mm / min until they fractured, and the maximum load (n=6) was recorded. The load was calculated according to the formula FS=3LF. M / 2bh 2 Calculate the flexural strength, where FS is the flexural strength test value, and F... M Where L is the maximum load, b is the span, and h is the specimen width.

[0044] (6) Relative cell proliferation rate Adhesive specimen preparation and extract preparation: Each group of adhesives was cured to prepare standard cylindrical specimens (10 mm in diameter, 3.5 mm in height). After curing, the specimens were immersed in deionized water at 37℃ for 24 h. After complete curing, both sides were sterilized by ultraviolet light irradiation (1 h per side). The sterilized specimens were placed in sterile containers, and 10% fetal bovine serum culture medium was added to prepare the experimental extract. The extract preparation was based on the following formula: specimen surface area / fetal bovine serum culture medium = 1.25 cm². 2 / mL, soaked at 37℃ for 24 h, then filtered using a 0.22 μm filter for later use.

[0045] Human dental pulp fibroblasts were resuscitated, cultured, and passaged in an incubator at 37°C, 5% CO2, and >95% relative humidity using 10% fetal bovine serum (FBS) medium. Experiments were conducted after 4-6 passages. Single-cell suspensions were prepared by digesting cells with 0.25% trypsin and seeded into 96-well plates (5000 cells per well). After 24 h of culture until adherence, the original culture medium was discarded, and 200 μL of each group's extract was added. The blank control group was treated with 200 μL of 10% FBS medium. The plates were then incubated for another 24 h. 20 μL of CCK-8 reagent was added to each well. 200 μL of 10% FBS medium and 20 μL of CCK-8 reagent were used as control wells. After 4 h of further culture, the OD value of each group was measured at 450 nm using a microplate reader.

[0046] (7) Live / dead cell staining Human dental pulp fibroblasts in the logarithmic phase were used at a concentration of 5 × 10⁻⁶ 4 Cells were seeded at a density of [cells / mL] in 6-well plates and cultured for 24 h until adherence. The original culture medium was discarded, and 200 μL of each group's extract was added. For the blank control group, 200 μL of 10% fetal bovine serum was added. The plates were then incubated for 24 h. After discarding the culture medium, live / dead cell staining solution was added for staining, and images were taken using a fluorescence microscope for recording.

[0047] (8) Acute hemolysis test Anticoagulated rabbit whole blood was prepared by extracting blood from rabbit hearts and adding it to 20 g / L potassium oxalate saline solution at a volume ratio of 20 / 1. Then, diluted anticoagulated rabbit whole blood was prepared by adding 0.2 mL of the diluted anticoagulated rabbit whole blood to 10 mL of deionized water. After incubation at 37°C for 1 h, the mixture was centrifuged at 750 times gravity for 5 min. The supernatant was then measured at 545 nm. If the result was within the range of 0.8 ± 0.3, it met the standard. The prepared diluted anticoagulated rabbit whole blood was stored at 4°C for later use.

[0048] Each adhesive experimental group was mixed at a volume ratio of 1 / 50 for anticoagulated rabbit whole blood / extract, incubated at 37℃ for 1 hour, centrifuged at 750 times gravity for 5 minutes, and 200 μL of supernatant from each centrifuge tube was added to a 96-well plate to detect absorbance at 545 nm. The average absorbance of three samples from each group was taken. Blood treated with physiological saline was used as the negative control group, and blood treated with distilled water was used as the positive control group. The acute hemolysis rate was calculated by dividing the difference between the absorbance of the sample group and the absorbance of the negative control group by the difference between the absorbance of the positive control group and the negative control group.

[0049] (9) Short-term systemic toxicity test Eight-week-old SD rats, half male and half female, were used in the experiment, with 10 rats in each group. The experimental group was given the extract by gavage at a ratio of 5 μg / g, while the control group was given physiological saline. The rats were fasted for 8 hours before gavage and were sacrificed after 7 consecutive days of gavage. The vital signs and weight changes of the rats were recorded daily after gavage. After sacrifice, the main organs such as heart, liver, spleen, lungs, and kidneys were dissected and observed by hematoxylin and eosin (HE) staining.

[0050] (10) Effect of adhesive specimens on the growth of Streptococcus mutans Each group of adhesives was dropped into a cylindrical mold (10 mm high, 15 mm in diameter) under light-protected conditions. The mold was covered with a transparent polyethylene film and a glass slide on the top and bottom sides. After curing from both sides for 20 seconds, the mold was removed. The cured adhesive specimens were immersed in deionized water at 37°C for 24 hours to form the immediate group. After drying, the specimens were sterilized with ethylene oxide and then used for later use.

[0051] To investigate the antibacterial durability of each adhesive group, adhesive samples were stored separately in light-proof brown bottles, with 10 mL of artificial saliva added. The artificial saliva was replaced every 48 hours, and the samples were aged for one year. After aging, the samples were dried and sterilized for later use.

[0052] Place the adhesive sample in a 24-well culture plate and add 20 μL of Streptococcus mutans bacterial suspension (1×10⁻⁶). 6 The bacterial culture medium (CFU / mL) was mixed with 2 mL of sterile BHI medium and anaerobic incubated at 37°C for 24 h. The specimens were then removed and gently rinsed three times with sterile PBS to remove airborne bacteria. The rinsed specimens were placed in a 15 mL sterile centrifuge tube, and 2 mL of sterile BHI medium was added. The tubes were vigorously shaken for 2 min to elute any bacteria adhering to the specimen surface. The eluted bacterial solution and the bacterial culture medium in the 24-well plate were serially diluted. 100 μL of each diluted solution was placed on a sterile BHI agar plate, spread evenly with sterile glass beads, and anaerobic incubated at 37°C for 24 h before colony counting (n=6).

[0053] (11) Effect of adhesive specimens on the metabolic activity of Streptococcus mutans Collect the eluted bacterial solution and bacterial culture medium from the 24-well plate as described above. Add 200 μL of each solution to a 96-well plate, and then add 20 μL of CCK-8 reagent to each well. Mix well and incubate anaerobically at 37°C for 2 hours. A control group consists of BHI culture medium (without bacterial solution) and CCK-8 reagent mixed in equal proportions. After incubation, measure the absorbance at 450 nm using a microplate reader (n=6).

[0054] (12) Observation of bacterial plaque biofilm adhering to the surface of the specimen Place the adhesive sample in a 24-well culture plate and add 20 μL of Streptococcus mutans bacterial suspension (1×10⁻⁶). 6 The sample was mixed with 2 mL of sterile BHI culture medium (CFU / mL) and anaerobic incubated at 37°C for 24 h. The sample was then removed and gently rinsed three times with sterile PBS to remove airborne bacteria. The rinsed sample was then placed back into a new sterile 24-well plate and fixed with 5% glutaraldehyde at 4°C for 12 h. After gradient dehydration with anhydrous ethanol, vacuum drying, and gold sputtering, the biofilm adhering to the sample surface was observed under a field emission scanning electron microscope.

[0055] (13) Live / dead staining of bacterial plaque biofilm adhering to the specimen surface The obtained and rinsed adhering specimens were placed in a sterile 24-well culture plate, and 500 μL of bacterial staining solution was added to completely cover the surface of the specimens. The specimens were then wrapped in aluminum foil and incubated in the dark for 15 min at room temperature. The specimens were rinsed again with sterile PBS to remove excess dye, and absorbent paper was used to remove excess moisture from the surface of the specimens from the edge areas. The specimens were then placed on a glass slide and the live / dead state of the bacteria adhering to the surface of the specimens was observed and analyzed using a laser confocal microscope.

[0056] (14) Experiment on the binding of CLM with collagen or hydroxyapatite CLM was dissolved in a buffer solution of 50 mM acetic acid, 150 mM NaCl, 5 mM CaCl2, and 0.02% NaN3 (pH 4.0, adjusted with NaOH) to prepare a 5 wt% CLM acidic solution. With the approval of the Ethics Committee of the 920th Hospital of the Joint Logistics Support Force of the Chinese People's Liberation Army (2019-022-01), freshly extracted third molars were collected with informed consent from the patients. The teeth were required to be caries-free and fully developed. After manually removing the enamel, the dentin crown was preserved. Under liquid nitrogen protection, the dentin was ground into particles with a diameter <75 μm using a high-throughput tissue homogenizer (TL2020, DHS, China). The dentin powder was demineralized with 0.5 M EDTA (pH 7.4) at 4℃ for 10 days, rinsed with deionized water by centrifugation, and then freeze-dried for later use. 0.05 g of demineralized dentin collagen powder or 0.2 g of hydroxyapatite powder (n=6) were added to 1 mL of CLM solution. The absorbance at 280 nm was measured immediately using a spectrophotometer. Each group of solutions was incubated at 37 °C in a shaker (10 cycles / min) for 1 h. After incubation, the solution was centrifuged at 3000 rpm for 5 min, and the CLM concentration in the supernatant was measured again.

[0057] CLM was dissolved in buffer solution at concentration gradients of 0, 0.156, 0.313, 0.625, 1.25, 2.5, and 5 mg / mL. The absorbance at 280 nm was measured using a spectrophotometer, and the standard curve was calculated.

[0058] (15) Molecular simulation Molecular simulations were performed using GROMACS. The hydroxyapatite surface material and the CLM were simulated using the INTERFACE force field and the General AMBER Force Field (GAFF) force field, respectively. The RESP charge of the CLM was obtained by optimizing the CLM using Gaussian at the B3LYP / 6-31G(d) level. The CLM material was placed at a certain distance above the hydroxyapatite surface, and the system was filled with TIP3P water model molecules in a 6.1875 nm × 5.6502 nm × 6.7000 nm box to form the simulation system.

[0059] The system employed the Particle Mesh Ewald (PME) method to handle long-range electrostatic interactions, with a van der Waals interaction cutoff of 10 Å. The system was then minimized using the conjugate gradient method to simply eliminate unreasonable contact between atoms. Next, hydroxyapatite and the CLM were confined, and a brief equilibration of 100 ps with a 1 fs time step was performed to allow water molecules to fully relax, during which the temperature was controlled using the Berendsen thermobath algorithm with a coupling time constant of 0.2 ps. Finally, long-term molecular dynamics simulations were performed with a 1 fs time step, and the temperature of the simulation system was controlled and maintained constant using the V-rescale algorithm; the simulation temperature was set to 300 K over a 100 ns time range, and the simulation was conducted using an isothermal isovolume ensemble (NVT). The results were analyzed using Visual Molecular Dynamics v1.9.3 software for three-dimensional molecular visualization.

[0060] (16) CLM and type I collagen liquid nuclear magnetic resonance Type I collagen powder (CLP-01, Koken Co. Ltd., Japan) was dissolved in a D2O buffer solution containing 50 mM d4-acetic acid, 150 mM NaCl, 5 mM CaCl2, and 0.02% NaN3 (pD 4.0, adjusted with NaOD). The saturated collagen solution was diluted 4-fold with the buffer solution to reduce viscosity. CLM monomer was added to the dilution buffer to adjust the final concentration to 5 wt%. Nuclear magnetic resonance (NMR) experiments were performed on a 600 MHz spectrometer equipped with an ultra-low temperature probe (Bruker BioSpin Corporation, Billerica, USA). 1H and 1H-13C heteronuclear single quantum correlation (HSQC) spectra of the collagen solution and the CLM-treated collagen solution were measured. Watergate W5 was used for water peak suppression during sampling, and proton chemical shifts were measured using tetramethylsilane (TMS) as the reference.

[0061] (17) CLM and solid-state nuclear magnetic resonance of hydroxyapatite 0.2 g of hydroxyapatite powder was mixed with CLM buffer solution, washed three times with acetone, centrifuged, and air-dried at room temperature for 48 h. The 31p nuclear magnetic resonance spectra of hydroxyapatite and CLM-treated hydroxyapatite were measured, and the chemical shifts were recorded in ppm. Crystalline hydroxyapatite powder was used as an external reference (0.0 ppm).

[0062] (18) Raman spectrum of the bonding interface Extracted third molars were selected, and the dentin in the mid-crown was exposed by cutting parallel to the occlusal surface using a slow-speed cutter under running water. The area was then polished with 600-grit sandpaper, etched with 35% phosphoric acid for 15 seconds, rinsed with deionized water for 30 seconds, and the surface moisture was absorbed with filter paper. Each adhesive was applied for 10 seconds, gently blown on for 5 seconds, and cured for 20 seconds. A 4 mm thick layer of resin (Z250, 3M, USA) was then deposited and cured. For cured specimens, a 1 mm thick specimen was prepared by cutting the resin and dentin perpendicular to the bonding interface using a slow-speed cutter (SYJ-150, Shenyang Kejing, China). A micro-Raman spectrometer (Invia Qontor, Renishaw, UK) was used to locate and record the data at the bottom of the bonding interface under the objective lens.

[0063] (19) Polyacrylamide gel electrophoresis detection of collagen crosslinking Cross-linking solution preparation: Experimental solutions of different concentrations: 3.52 mg / mL collagen solution = 1:1 (volume ratio).

[0064] Non-crosslinked solution control group: Deionized water: 3.52 mg / mL collagen solution = 1:1 (volume ratio).

[0065] Collagen crosslinking was detected using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All solutions were stored at room temperature for 24 h to obtain modified crosslinking solutions. The groups were: marker group, control group (non-crosslinked solution), positive control group (5% glutaric dialdehyde, GD), and experimental groups (CLM1-5). 50 μL of each group's crosslinking solution was mixed with 10 μL of SDS protein loading buffer and heated at 98 °C for 10 min. 10 μL of each group was loaded for electrophoresis. Initially, a constant voltage of 80 V was used for approximately 30 min. After the marker entered the gel, the voltage was increased to 100 V until the gel reached the bottom. After electrophoresis, Coomassie blue staining was performed for 2 h, followed by destaining for 1 h, and the data was recorded and analyzed.

[0066] (20) Micro-stretching test Both the immediate and aged groups of specimens were prepared by cutting resin and dentin perpendicularly to the bonding interface using a slow-speed cutter to create standard resin-dentin strip bonded specimens for micro-tensile testing. Vernier calipers were used to ensure that the cross-sectional area was 1 mm². 2 Keep the specimens moist and use a micro tensile testing machine (tensile rate: 0.5 mm / min) to test and record the load at which the specimen breaks (n=10).

[0067] (21) In-situ enzyme profile at the bonding interface For both the immediate and aged groups, 1 mm thick specimens were prepared by cutting resin and dentin perpendicularly to the bonding interface using a slow-speed cutter. These specimens were then used for in-situ enzyme profiling at the bonding interface. The specimens were subjected to gradient sanding with sandpaper (320, 600, 1200, 2000, 4000, and 5000 mesh) under running water, shaken with deionized water for 5 min, and dried with filter paper. The treated specimens were then placed on glass slides, and 50 μL of collagenase activity indicator was added to immerse the surface of the specimens. The slides were then covered, and the specimens were incubated at 37°C in the dark and at 100% humidity for 48 h. The surface dye was washed off with deionized water and the specimens were air-dried. The endogenous enzyme activity at the bonding interface was then detected by scanning under a laser confocal microscope.

[0068] (22) Nano-permeability at the bonding interface For the immediate and aged groups, 1 mm thick specimens were prepared by cutting resin and dentin perpendicular to the bonding interface using a slow-speed cutter. The specimens were then subjected to gradient sanding (320, 600, 1200, 2000, 4000, 5000 grit) under running water, followed by agitation in deionized water for 5 min to remove surface debris. After drying, two layers of hydrophobic nail polish were applied completely to the area 1 mm above and below the bonding interface. After drying, the specimens were immersed in a 50 wt% silver ammonium nitrate solution for 24 h in the dark. The surface was thoroughly rinsed with deionized water to remove the staining solution, then immersed in fluorescent developer for 8 h, followed by immersion in fixer for 8 h. The specimens were then subjected to gradient sanding again (320, 600, 1200, 2000, 4000, 5000 grit), followed by ultrasonic agitation in deionized water for 5 min, air-dried, and then sprayed with gold. The nano-leakage at the bonding interface was observed using FE-SEM.

[0069] (23) Morphology of the bonding interface For both the immediate and aged groups, 1 mm thick specimens were prepared by cutting resin and dentin perpendicular to the bonding interface using a slow-speed cutter. The specimens were then sanded with sandpaper at gradient grits (320, 600, 1200, 2000, 4000, and 5000 mesh), agitated with deionized water for 5 min, dried with filter paper, and treated with 35% phosphoric acid for 30 s in a 1 mm area around the bonding interface. After rinsing with deionized water, the specimens were immersed in a 10% sodium hypochlorite solution for 2 min, washed, and then ultrasonically agitated for 10 min. After drying and gold sputtering, the morphology of the bonding interface was observed using a scanning electron microscope.

[0070] (24) Water penetration at the bonding interface The extracted third molar was cut 2.5 mm coronally at the cementoenamel junction perpendicular to its long axis to remove enamel and superficial dentin. The root and pulp tissue were then removed 3 mm apically at the cementoenamel junction. The root was connected to a polyethylene tube using isocyanate adhesive and fixed below a 20 cm plexiglass tube. Adhesion was performed using 20 cm of water pressure within the glass tube to simulate the pressure of a non-inflammatory pulp state. To distinguish the fluorescence color from other experimental sections, green fluorescent dye (0.1% sodium fluorescein) was added to the adhesive, and orange fluorescent dye (0.1% Alexa Fluor™ 546) was added to water for bonding. The mixture was maintained in a dark environment for 4 hours. Subsequently, the bonded specimen was cut into approximately 1 mm thick slices perpendicular to the bonding interface. After gradient sanding (320–2500 grit), the water permeability at the bonding interface was observed under a laser confocal microscope.

[0071] The test results are as follows: (1) Results of polymer conversion rate of adhesive The polymerization conversion results of each group of adhesives are as follows: Figure 1 As shown ( ), Figure 1 In the diagram, CCL represents the commercialized control group, BCL represents the blank control group, and CLM 1-6 represents the groups supplemented with 1-6 wt% CLM; the same letter indicates that there is no statistically significant difference between the groups. P >0.05.

[0072] Depend on Figure 1 It can be seen that the CCL group (44.4±2.2%) had the highest polymerization conversion rate. P >0.05), the polymerization conversion rate of the BCL group (39.2±2.0%) was slightly lower than that of the CCL group. P <0.05), there was no statistically significant difference between the CLM 1-6 groups (CLM 1 39.0±1.9%, CLM 2 38.4±1.5%, CLM 3 37.5±2.0%, CLM 4 37.6±1.7%, CLM 5 37.1±1.5%, CLM 6 30.5±1.4%) and the CCL group. P >0.05), when the CLM concentration reaches 6 wt%, the polymer conversion rate of the adhesive decreases ( P <0.05).

[0073] (2) Results of adhesive wettability The wettability results of each group of adhesives are as follows: Figure 2 As shown (θ, ), Figure 2 In the diagram, CCL represents the commercialized control group, BCL represents the blank control group, and CLM1-6 represent groups supplemented with 1-6 wt% CLM; the same letter indicates that there is no statistically significant difference between the groups.P >0.05. The contact angle of the adhesive on the demineralized dentin surface is used to express the wettability of the adhesive; the smaller the contact angle, the stronger the wettability. The contact angles of the CCL group (39.1±3.3) and the BCL group (38.5±3.3) are similar. P >0.05), in the CLM group, the contact angle decreased when the added concentration exceeded 3wt%. P <0.05), with the increase of CLM concentration, the contact angle further decreased, CLM 1 group (37.8±2.7), CLM 2 group (38.4±1.3), CLM 3 group (36.1±3.4), CLM 4 group (34.1±3.4), CLM 5 group (29.1±1.7), CLM 6 group (27.3±2.9).

[0074] (3) Thermal stability of adhesive The thermal stability results of each group of adhesives are as follows: Figure 3 As shown, the stability of adhesive components is crucial during fluctuating temperature changes in the oral cavity. Thermogravimetric analysis (TGA) was used to test the stability of each adhesive component. Within 180°C, the weight loss of each adhesive group did not show a specific decrease with increasing temperature.

[0075] (4) Adhesive polymerization shrinkage rate The polymerization shrinkage rate of each group of adhesives is as follows: Figure 4 As shown, Figure 4 In the diagram, CCL represents the commercially available control group, BCL represents the blank control group, and CLM1-6 represent groups supplemented with 1-6 wt% CLM. The same letter indicates no statistically significant difference between groups. P >0.05. (By) Figure 4 It can be seen that the CCL group (3.7±0.4%) and the BCL group (4.5±0.6%) had the highest polymerization shrinkage rates. P > 0.05), the polymerization shrinkage rate of the CLM group was lower than that of the CCL and BCL groups ( P <0.05. Within the CLM groups, there were no significant differences among CLM 1 (2.4±0.6%), CLM 2 (1.9±0.7%), CLM 3 (1.8±1.0%), and CLM 4 (1.4±0.1%). P > 0.05), with increasing CLM concentration, the polymerization shrinkage rates of CLM group 5 (0.6±0.3%) and CLM group 6 (0.5±0.2%) were the lowest. P <0.05).

[0076] (5) Flexural strength test results The flexural strength test results of each group of adhesives are as follows: Figure 5 As shown, Figure 5In this context, CCL represents the commercially available control group, BCL represents the blank control group, and CLM1-6 represent groups supplemented with 1-6 wt% CLM. The same letter indicates no statistically significant difference between groups. P >0.05. (By) Figure 5 It can be seen that the CCL group (55.4±2.9 MPa), BCL group (52.4±4.3 MPa), and CLM group (CLM 1 group 59.0±5.7 MPa, CLM 2 group 62.5±4.3 MPa, CLM 3 group 63.1±2.0 MPa, CLM 4 group 66.3±3.2 MPa, CLM 5 group 70.2±1.8 MPa, CLM 6 group 70.9±1.7 MPa) had the lowest values. P <0.05), the flexural strength of the specimens increased with increasing CLM concentration, with the highest flexural strength observed in groups CLM5 and CLM6. P <0.05).

[0077] Based on the above physicochemical properties, it is recommended that the concentration of CLM added be ≤5 wt% for subsequent applications, so as to reduce polymerization shrinkage and improve wettability and flexural strength while ensuring the polymerization performance of the material.

[0078] (6) Results of relative cell proliferation rate The effects of each adhesive extract on the relative cell proliferation rate are as follows: Figure 6 As shown ( ), Figure 6 In the table, CL represents the negative control group (10% fetal bovine serum culture medium), CCL represents the commercial control group, BCL represents the blank control group, and CLM1-5 represents the groups supplemented with 1-5 wt% CLM. The same letter indicates that there is no statistically significant difference between the groups. P >0.05. The relative cell proliferation rate directly reflects the cytotoxicity of the material. The blank control group was set as 100% as a reference. All adhesive extracts in each group affected the cell proliferation rate, but there was no statistically significant difference in the effect of adhesive extracts on cell proliferation among the groups. P >0.05).

[0079] (7) Results of staining of live and dead cells The cell staining results of each group are as follows Figure 7 As shown (scale bar 500 μm), Figure 7In the diagram, CL represents the negative control group (10% fetal bovine serum culture medium), CCL represents the commercial control group, BCL represents the blank control group, and CLM1-5 represent groups supplemented with 1-5 wt% CLM. Green represents live cells, and red represents dead cells. Under a fluorescence microscope, the cell density in each group was basically the same. Except for the negative control group, a small number of red dead cells appeared in each group. The green live cells were elongated and spindle-shaped, consistent with the morphology of fibroblasts, and were evenly distributed overall.

[0080] (8) Results of acute hemolysis test Figure 8 Representative images of the acute hemolysis experiments for each group of adhesive extracts are shown in Table 2. The results of the acute hemolysis rates for each group of adhesive extracts are also shown in Table 2. In the positive control group, deionized water caused red blood cell rupture and the release of hemoglobin; therefore, no red blood cells settled at the bottom after centrifugation, and the liquid appeared red with surface hypotonic hemolysis due to deionized water. In the negative control group, physiological saline was used; after centrifugation, red blood cells settled at the bottom, the supernatant was clear, and no hemolysis was observed. In each experimental group, red blood cells settled at the bottom, and the supernatant was clear. Microplate reader analysis showed that the acute hemolysis rate of the adhesive extracts in each group was less than 5%, which meets the requirements for hemolysis rate in the national pharmaceutical industry standard YY / T0127.1 1993.

[0081] Table 2. Acute hemolysis rate of adhesive extracts in each group

[0082] (9) Results of short-term systemic toxicity test Seven days after oral administration of the adhesive extract to each group, the major organs of the SD rats were dissected and HE-stained sections were prepared. The results are as follows: Figure 9 As shown (200×, scale bar 200 μm). After dissection, the color, shape, and size of the major organs (heart, liver, spleen, lungs, kidneys, etc.) of the rats in each group showed no abnormalities. HE staining revealed that, compared with the commercial group, the myocardial cells were arranged regularly, without necrosis or inflammatory cell infiltration. The liver lobule structure was clear, without fatty degeneration, inflammatory reaction, hepatocyte necrosis, or other signs of damage. The spleen corpuscles were intact, and the tissue structure of the spleen red pulp and spleen white pulp was normal, without lymphocyte infiltration. The alveolar structure was normal, without interstitial edema, alveolar wall thickening, or inflammatory cell infiltration. The renal tubules and glomeruli were clear, without renal tubular degeneration, interstitial edema, or inflammatory cell infiltration. The above results suggest that the adhesive extract in each group did not cause significant toxic damage to the major organs of SD rats and had virtually no short-term toxicity to the experimental animals.

[0083] (10) Effect of adhesive specimens on the growth of Streptococcus mutans The CFU counts of bacteria adhering to the surface of the adhesive specimens in each group are shown in Table 3 (the same letter in Table 3 indicates that there is no statistically significant difference between the groups before aging treatment). P>0.05). Before aging treatment, there was no statistically significant difference in CFU count between the CCL group and the BCL group. P >0.05), the CFU count in the CLM group was lower than that in the CCL and BCL groups ( P <0.05), in the CLM group, the count of bacteria adhering to the specimen surface decreased with increasing concentration, and the CLM4 and CLM5 groups had the lowest bacterial counts. P <0.05). The adhesion inhibition effect of the bonded specimens after aging treatment was similar to that before aging treatment; the CLM group still effectively reduced the number of colonies. This indicates that the CLM adhesive can inhibit the growth of surface-adhering bacteria.

[0084] Table 3. Effect of adhesive on the growth of surface-adhesive bacteria in the specimens ( )

[0085] The effects of each adhesive group on the growth of planktonic bacteria in the culture medium are shown in Table 4 (the same letter in Table 4 indicates that there was no statistically significant difference between the groups before aging treatment). P >0.05). Before aging treatment, the CCL and BCL groups had the highest colony counts ( P >0.05), in the CLM group, the number of colonies in the culture medium decreased with increasing addition, and the CLM4 and CLM5 groups had the lowest bacterial counts. P <0.05). After aging treatment, the number of colonies in all groups increased significantly, with no statistically significant difference between the CCL group, BCL group, and CLM groups 1-4. P >0.05), the colony count decreased only when the concentration reached 5 wt%. P <0.05). The results indicate that the inhibitory effect of CLM adhesive on airborne bacteria weakened with immersion and aging.

[0086] Table 4. Effects of adhesive specimens on the growth of planktonic bacteria ( )

[0087] (11) Effect of bonded specimens on bacterial metabolic activity The results of bacterial metabolic activity on the surface of each adhesive specimen are as follows: Figure 10 As shown ( ), Figure 10 In the diagram, the same letter indicates that there is no statistically significant difference between the groups. P >0.05). A higher OD value indicates stronger bacterial metabolic activity. The metabolic activity of bacteria adhering to the surface of the specimens in both the CCL and BCL groups was highest before and after aging treatment, and there was no statistically significant difference before and after aging. P>0.05), exhibiting concentration-dependent inhibition of bacterial metabolic activity in the CLM group, with the CLM group showing the strongest inhibitory effect ( P <0.05%. The inhibitory effect of the CLM group on the metabolic activity of bacteria adhering to the specimen surface was weakened after aging treatment. P <0.05).

[0088] The results of the metabolic activity of each group of adhesive specimens on planktonic bacteria in the culture medium are as follows: Figure 11 As shown ( Before aging treatment, the planktonic bacteria in the CCL and BCL groups exhibited the strongest metabolic activity. P >0.05), in the CLM group, the metabolic activity of planktonic bacteria was effectively inhibited with increasing concentration, and the metabolic activity of CLM4-5 group was the weakest. P <0.05). After aging treatment, the inhibitory effect on the metabolic activity of planktonic bacteria was significantly weakened in all groups, with metabolic activity inhibition only observed in groups CLM4-5. P <0.05).

[0089] (12) Observation of the morphology of bacteria adhering to the surface of the bonded specimen Bacteria adhering to the surface of each group of bonded specimens, such as Figures 12-13 As shown, Figure 12 This is an observation image of the biofilm adhering to the surface of the immediate bonding reagent (×1500, scale bar 20 μm). Figure 13 The image shows an observation of bacterial plaque biofilm adhering to the surface of the bonded specimens after aging treatment (×1500, scale bar 20 μm). Under low magnification, both the CCL and BCL groups were covered with a large amount of Streptococcus mutans bacterial plaque biofilm before and after aging. The high-density accumulation of the bacterial plaque biofilm obscured the surface of the underlying material. In the CLM group, the amount of bacteria adhering to the specimen surface gradually decreased with the increase of CLM concentration, exposing the material surface. In the CLM5 group, only scattered bacteria were found on the specimen surface. After aging, the amount of bacteria adhering to the specimen surface increased compared to before aging, but the CLM group could still effectively inhibit bacterial adhesion and reduce the amount of bacteria on the specimen surface.

[0090] (13) Staining of live / dead bacteria on the surface of adhesive specimens The staining results of live / dead bacteria on the surface of each group of adhesive specimens before and after aging are as follows: Figure 14 As shown, the scale bar is 200 μm. Before aging treatment, the surfaces of the CCL and BCL group specimens mainly contained green viable bacteria with a small amount of red dead bacteria. In the CLM group, the amount of green viable bacteria gradually decreased with increasing concentration, while the amount of red dead bacteria increased. After aging treatment, the antibacterial trend was similar to that before aging treatment. The surfaces of the CCL and BCL group specimens mainly contained green viable bacteria. In the CLM group, the number of green viable bacteria decreased in a concentration-dependent manner while the number of red viable bacteria increased, but compared with before aging treatment, the number of green viable bacteria increased.

[0091] (14) Simulation results of CLM and hydroxyapatite molecular docking The simulation results of CLM-hydroxyapatite molecular docking are as follows: Figures 15-17 As shown, Figure 17 In the diagram, green represents Ca; red represents O; pink represents P; yellow represents C; and blue represents N. Previous studies have simulated the molecular docking between CLM and type I collagen; this experiment further refines the simulation of the molecular docking between CLM and hydroxyapatite. Figure 15 As shown, after a simulation time of 100 ns, the RMSD of the CLM basically stabilized, fluctuating within a certain range, indicating that the system had reached equilibrium. Figure 16 As shown, analyzing the interaction energies of the system, the electrostatic interaction energy is much greater than the van der Waals interaction energy, and hydrogen bonds can also be considered as electrostatic interactions. Hydrogen bonds play a dominant role in maintaining the system's binding stability. For example... Figure 17 As shown, the bonding mode between CLM and hydroxyapatite indicates that hydrogen bonding plays a dominant role in the bonding between CLM and hydroxyapatite. Figure 16 The energy analysis is consistent with this. The interaction mode shows that the strong binding mode between CLM and hydroxyapatite involves the amino group of CLM being anchored to hydroxyapatite via multiple pairs of strong hydrogen bonds, and all three hydrogen atoms forming strong hydrogen bonds with the oxygen atoms of the subsurface phosphate groups (each hydrogen bond length is approximately 2.0 Å). In addition, stable hydrogen bonds can also be formed between the methylene group near the amino group of CLM, the hydroxyl group on the benzene ring, and the surface phosphate groups of hydroxyapatite.

[0092] (15) Results of CLM and Type I Collagen Liquid NMR Previous molecular docking simulations have demonstrated that hydrogen bonding and covalent bonding can occur between CLM and type I collagen. In this experiment, liquid two-dimensional NMR HSQC spectra are used to further verify the results of molecular docking simulations. Figure 18 HSQC spectrum of type I collagen ( Figure 18 In this context, alanine (Ala), hydroxyproline (HYP), lysine (Lys), glycine (Gly), and proline (Pro); α, ε, β, and γ represent the C atoms at the corresponding positions. Figure 19The table shows the HSQC spectra after the reaction of CLM with type I collagen. Table 5 shows the chemical shift analysis of amino acids before and after the interaction between type I collagen and CLM. After CLM treatment, the C-CH relationships of all amino acids in type I collagen underwent certain shifts, with Hyp α and Lys δ showing the largest chemical shifts, at (-0.24 ppm, +1.15 ppm) and (+0.03 ppm, +0.71 ppm), respectively. The chemical shifts of C atoms and C-CH relationships in the amino acid skeleton of type I collagen prove that CLM and type I collagen have undergone chemical interaction.

[0093] Table 5 Amino acid chemical shifts before and after the interaction between type I collagen and CLM.

[0094] (16) Solid-state NMR results of CLM and hydroxyapatite Solid-state NMR spectra of hydroxyapatite before and after the reaction with CLM are as follows: Figure 20 As shown, Figure 20 In the image, A is the solid-state NMR spectrum of hydroxyapatite, and B is the solid-state NMR spectrum of hydroxyapatite after the reaction with CLM. Before the reaction, both images show a characteristic signal peak of hydroxyapatite at approximately 2.01 ppm, with no other peaks, indicating a high-intensity chemical interaction between hydroxyapatite and CLM.

[0095] (17) Raman spectrum of the bonding interface Raman spectral results of the bonding interface of each group of adhesives are as follows: Figures 21-22 As shown, an amide I band (1670 cm⁻¹) appeared at the bonding interface. -1 ), Amide III band (1253 cm) -1 Characteristic peaks such as 650 cm⁻¹; -1 The enhanced vibrational peak at 1760 cm⁻¹ indicates the characteristic peak of the -OH group in catechol. -1 The enhanced vibrational peaks at 1638-1646 cm⁻¹, representing characteristic peaks of the ester group, confirm that the -C=O group on the CLM benzene ring is grafted onto the collagen surface, indicating the bonding of CLM to the collagen surface. The increased peak vibrations and low-frequency shifts in the amide I, amide II, and amide III bands of the CLM group indicate that the CLM monomers have undergone Michael addition, hydrogen bonding, and electrostatic interactions with collagen. -1 The emergence of a new peak and the amide I band (1655~1680 cm⁻¹) -1 The increased width and vibration of the pyridine ring indicate the formation of C=N, further illustrating the occurrence of covalent interaction; the vibrational peaks of the pyridine ring (1061~1084 cm⁻¹) -1 The enhanced cross-linking indicates an increase in the degree of collagen cross-linking.

[0096] (18) Results of micro-tensile strength of adhesive The results of dentin bond strength before and after aging of each group of adhesives are as follows: Figure 23 As shown ( ), Figure 23 In the diagram, the same letter indicates that there is no statistically significant difference between the groups. P >0.05). In the immediate BCL bonding group (25.6±3.5 MPa), the dentin bond strength was slightly lower than that of other groups. P <0.05), the CCL group (29.2±3.6 MPa) was similar to that of CLM groups 1-3 (CLM group 1 28.3±2.1 MPa, CLM group 28.8±2.4 MPa, CLM group 3 29.8±2.1 MPa). P >0.05), in the CLM group, the dentin bonding strength increased with increasing concentration (CLM 4 group 33.8±2.1 MPa, CLM 5 group 38.0±1.9 MPa), with CLM 5 group having the highest dentin bonding strength. P <0.05%. After aging treatment, the bonding strength of all groups showed a decreasing trend, with the CCL group (14.5±3.3 MPa), BCL group (13.7±2.0 MPa), and CLM1 group (16.8±3.5 MPa) showing the largest decreases. The decrease in dentin bonding strength decreased with increasing CLM concentration (CLM2 group 21.2±3.0 MPa, CLM3 group 22.1±2.9 MPa, CLM4 group 27.2±3.6 MPa, CLM5 group 31.5±4.4 MPa). There was no statistically significant difference in CLM5 group before and after aging. P >0.05).

[0097] (19) In-situ enzyme profile at the bonding interface Results of endogenous enzyme activity at the dentin bonding interface of each adhesive group are as follows: Figure 24 As shown (scale bar 150 μm) Figure 24 In the diagram, green fluorescence represents the intensity of endogenous enzyme activity, and red fluorescence represents the area where the adhesive is located. R: resin, HL: mixed layer, D: dentin. Strong green fluorescence was observed at the bonding interface in the CCL and BCL groups, indicating strong enzyme activity. In the CLM group, the intensity of green fluorescence at the bonding interface decreased with increasing concentration, indicating decreased enzyme activity. Only weak fluorescence was observed in the CLM 4 and CLM 5 groups. After aging treatment, enzyme activity at the bonding interface increased in all groups, but the enzyme activity remained the weakest in the CLM 4 and CLM 5 groups.

[0098] (20) Nano-leakage at the bonding interface Nano-leakage results of each adhesive group are as follows Figure 25 As shown (scale bar 20 μm). Figure 25In the diagram, white particles or bands at the bottom of the bonding interface represent the deposition of silver nanoparticles. R: resin, HL: mixed layer, D: dentin. Before aging treatment, only a small amount of silver nanoparticles were deposited at the bottom of the bonding interface in each group, with almost no silver particles appearing at the bottom of the bonding interface in CLM 4 and CLM 5 groups. After aging treatment, continuous silver deposition bands appeared at the bottom of the bonding interface in CCL, BCL, and CLM 1 groups. In the CLM group, the number of silver particles at the bottom of the interface decreased with increasing concentration, while only a small number of particles were scattered in CLM4 and CLM5 groups. The results indicate that CLM can enhance the sealing of the bonding interface and reduce nano-leakage.

[0099] (21) Morphology of the bonding interface The bonding interface morphology of each adhesive group is as follows: Figure 26 As shown (scale bar 40 μm). Immediately after bonding, all groups formed complete mixed layer and resin protuberance structures. The resin protuberances in the CCL and BCL groups were mostly smooth and uniform. In the CLM group, lateral structures appeared around the resin protuberances, and the density and length of the resin protuberances increased significantly. In the CLM 4 group, the resin protuberance length even reached more than twice that of the control group. In the CLM 5 group, in addition to the increased resin protuberance length, abundant resin protuberance lateral structures appeared, with the lateral branches intertwined and the structure intact. After aging treatment, all groups still maintained a complete mixed layer structure, but resin protuberance breakage occurred in all groups. However, the resin protuberance density and length in the CLM 3-5 groups were still better than those in the control group. The above results indicate that CLM facilitates deeper penetration of the resin bonding monomer into the dentin, and the resulting resin protuberance structure and quality are more intact.

[0100] (22) Resin penetration depth The results of the adhesive penetration depth for each group are as follows: Figure 27 ( )and Figure 28 As shown, Figure 27 In the diagram, the same letter indicates that there is no statistically significant difference between the groups. P >0.05), Figure 28 Scale bar 200 μm. There was no statistically significant difference in resin penetration depth among the CCL group (39.9±14.7 μm), BCL group (34.4±15.0 μm), and CLM 1 group (32.6±10.2 μm). P >0.05), in the CLM group, when the concentration exceeded 2 wt%, the resin sudden penetration depth increased significantly (CLM 2 group 82.9±23.6 μm, CLM 3 group 94.0±26.2 μm, CLM 4 group 121.5±37.0 μm, CLM 5 group 111.3±23.7 μm), with CLM 4 and CLM 5 groups having the longest resin sudden penetration depth ( P <0.05).

[0101] (23) Water permeation at the bonding interface Dentin bonding was performed using various adhesives under simulated pulp pressure. Dual-fluorescence technology was used to trace the water content in the adhesives and dentinal tubules. The water permeability test results for each adhesive group are as follows: Figure 29 As shown, the scale bar is 500 μm. In the CCL, BCL, and CLM 1 groups, the adhesive area was completely permeated by water. In the CLM 2 group, water permeated about 3 / 4 of the adhesive area. In the CLM 3 group, water was limited to the lower 1 / 3 of the adhesive area, but some areas still showed blister-like permeation. In the CLM 4 group, although the water did not exceed the lower 1 / 3 of the adhesive area, there were still areas of water retention at the bottom of the adhesive interface. In the CLM 5 group, the water was mainly concentrated in the bottom area of ​​the adhesive interface. Although it did not completely inhibit water permeation, it still effectively isolated some water leakage compared to the control group.

[0102] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A dental resin adhesive, characterized in that, It includes a base binder and a mussel biomimetic monomer; the base binder comprises the following components in weight percentage: 35-40% bisphenol A dimethacrylate, 35-40% hydroxyethyl methacrylate, 5-10% triethylene glycol dimethacrylate, 10-15% diluent, 0.5-1.0% photoinitiator, 0.5-1.0% co-initiator, and 1.0-2.0% oxygen barrier; The mussel biomimetic monomer has the structure shown in Formula 1: Formula 1; The mass of the mussel biomimetic monomer is 1 to 6% of the total mass of the base binder and the mussel biomimetic monomer.

2. The dental resin adhesive according to claim 1, characterized in that, The base binder comprises the following components in weight percentage: 40% bisphenol A dimethacrylate, 35% hydroxyethyl methacrylate, 10% triethylene glycol dimethacrylate, 13% diluent, 0.5% photoinitiator, 0.5% co-initiator, and 1% oxygen barrier.

3. The dental resin adhesive according to claim 1 or 2, characterized in that, The diluent is ethanol.

4. The dental resin adhesive according to claim 1 or 2, characterized in that, The photoinitiator is camphorquinone.

5. The dental resin adhesive according to claim 1 or 2, characterized in that, The co-initiator is ethyl p-dimethylaminobenzoate.

6. The dental resin adhesive according to claim 1 or 2, characterized in that, The oxygen inhibitor is cysteine.

7. The dental resin adhesive according to claim 1, characterized in that, The mass of the mussel biomimetic monomer is 2-5% of the total mass of the base binder and the mussel biomimetic monomer.

8. A method for preparing the dental resin adhesive according to any one of claims 1 to 7, characterized in that, Includes the following steps: The dental resin adhesive is obtained by mixing bisphenol A dimethacrylate, hydroxyethyl methacrylate, triethylene glycol dimethacrylate, diluent, photoinitiator, co-initiator, oxygen barrier and mussel biomimetic monomer.

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