Multifunctional sACP-PEP / TMC film, preparation method and application thereof

Abstract

The application discloses a multifunctional SACP-PEP / TMC film and a preparation method and application thereof, and belongs to the technical field of biomaterials. The preparation method comprises the following steps: reacting a phosphate and polyacrylic acid in the presence of a calcium salt to prepare ACP nanoparticles; dispersing the ACP nanoparticles in a solvent, and reacting the ACP nanoparticles with a silane coupling agent to obtain surface-modified SACP nanoparticles; dissolving N,N,N-trimethyl chitosan in a solvent, adding the SACP nanoparticles and a functional polypeptide, uniformly mixing, and then placing the mixture in a mold, removing the solvent, shaping, and obtaining the SACP-PEP / TMC film. The application further discloses the multifunctional SACP-PEP / TMC film prepared by the above method and application of the film. The application constructs an antibacterial-antienzyme-remineralization multi-target intervention system, protects the collagen matrix, induces ordered remineralization of the collagen matrix, realizes all-round reinforcement of a resin-dentin bonding interface, and thus provides an effective technical scheme for improving clinical bonding durability.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a multifunctional SACP-PEP / TMC thin film, its preparation method, and its applications. Background Technology

[0002] Dental caries, as one of the most prevalent chronic infectious oral diseases worldwide, poses a significant burden on public health. When caries progresses to the dentin, it is primarily caused by Streptococcus mutans (S. mutans). Streptococcus mutans Cariogenic bacteria, primarily composed of cariogenic bacteria, continuously produce acid, dissolving the inorganic mineral phase in dentin. Simultaneously, the acidic microenvironment activates endogenous collagen-degrading enzymes, especially matrix metalloproteinases (MMPs), leading to the degradation of the dentin collagen fiber network. The synergistic effect of mineral loss, enhanced enzyme activity, and bacterial metabolic activity severely damages the structural integrity of dentin, posing a significant challenge to dentin restoration treatment.

[0003] Currently, composite resin fillings have become the mainstream method for treating dentin caries in clinical practice due to their excellent aesthetics, mechanical properties, and retention. The stability and durability of the resin-dentin bonding interface are crucial to the long-term success rate of the restoration. This interface achieves bonding through the formation of a unique hybrid layer, composed of light-cured resin adhesive that penetrates into the demineralized collagen fiber network, providing the necessary micromechanical interlocking between the resin and the dentin matrix. However, in the deeper regions of this hybrid layer, demineralized collagen fibers often remain unencapsulated and uninfiltrated by the resin monomers. These exposed fibers are highly susceptible to slow degradation by endogenous collagenases. Defects in the interface structure can lead to microleakage, providing pathways for the invasion of cariogenic bacteria or the proliferation of residual bacteria, ultimately resulting in secondary caries and restoration failure. Therefore, effective strategies to improve the durability of resin-dentin bonding require a synergistic approach to address multiple interfacial challenges, including: promoting biomimetic remineralization of exposed collagen fibers to restore structural protection, inhibiting endogenous collagenase activity to prevent collagen degradation, and endowing the bonding interface with preventative antibacterial capabilities to resist bacterial invasion and secondary caries.

[0004] Compared to liquid or powder formulations, film formulations offer significant advantages, including ease of use, controllable release, close adhesion to the dentin surface, structural stability, and no introduction of excess moisture into the interface. These properties are particularly beneficial for creating localized biomimetic mineralization microenvironments on dentin surfaces where collagen is exposed after acid etching. According to biomimetic mineralization theory, amorphous calcium phosphate (ACP), as a precursor to dentin mineralization, can effectively maintain its mineralization potential when stabilized by non-collagenous proteins (NCPs) or their analogues, or when present in the form of dry nanoclusters. Therefore, integrating ACP into film-based materials is a feasible strategy to promote dentin remineralization. However, existing research is mostly limited to simple remineralization models and has not yet been extended to resin-dentin bonding systems. Under complex conditions approaching clinical settings, the interaction mechanisms between remineralization films and adhesives in terms of penetration, polymerization processes, and interfacial stability still require further investigation.

[0005] Currently, researchers have proposed various activity regulation strategies to achieve biomimetic remineralization of dentin collagen fibers, including bioactive glass, functional peptides, polysaccharides, dendritic macromolecules, and lipid carriers. Among these, bioactive glass can promote mineral deposition by releasing calcium and phosphate ions; polysaccharides and their derivatives can mimic the natural extracellular matrix environment and stabilize ACP precursors; dendritic macromolecules such as polyamidoamine (PAMAM) can serve as mineralization templates to induce hydroxyapatite formation; and lipid carriers facilitate the delivery of active ions and the construction of local mineralization microenvironments. However, these strategies generally suffer from insufficient precision in mineralization regulation, limited specific binding ability to collagen, or complex systems. In contrast, functional peptides have attracted widespread attention in recent years due to their good biocompatibility, well-defined sequence designability, and precise regulation of the mineralization process. Previous studies have shown that various functional peptides derived from amelogenin, dentin sialophospholipids, and other mineralization-related proteins can mimic the active domains of NCPs, achieving biomimetic remineralization of dentin by stabilizing ACP precursors, promoting intracellular mineralization of collagen fibers, and inducing directional deposition of hydroxyapatite. Furthermore, some functional peptides also possess antibacterial and interfacial regulatory activities. Compared to traditional inorganic mineralization materials or complex nanodelivery systems, functional peptides offer advantages such as designable structures, ease of functionalization modification, higher biocompatibility, and greater compatibility with existing dental bonding systems. Therefore, they are considered ideal media for promoting dentin mineralization and regulating interfacial function.

[0006] Chitosan and its derivatives, due to their excellent biocompatibility, antibacterial properties, and good film-forming ability, have been widely used as carriers for drugs and bioactive molecules, and applied in the fields of dentin biomimetic remineralization and adhesive restoration. Trimethylchitosan (TMC), as a quaternized derivative of chitosan, has higher solubility and stronger cationic properties under neutral conditions, thus exhibiting superior processing performance and bioactivity. Studies have reported that TMC-treated dentin not only has a denser collagen arrangement but also shows significantly enhanced antibacterial properties and inhibition of MMP activity. These characteristics indicate that TMC-based functional membranes have broad application prospects in the field of dentin restoration.

[0007] However, current research largely focuses on developing single-function or fixed-combination active molecules, lacking a platform material that combines good film-forming properties, compatibility with resin bonding systems, and the ability to flexibly load different functional peptides as a universal carrier. Specifically, although various functional peptides, such as biomimetic remineralizing peptides, collagenase inhibitory peptides, and antimicrobial peptides, have been reported, effectively loading these peptides onto a thin-film carrier that is easy to handle clinically, structurally stable, and adheres tightly to the etched dentin surface, enabling controlled loading and release of functional peptides according to clinical needs, thereby specifically addressing multiple problems such as mineral loss, collagen degradation, and bacterial invasion at the resin-dentin interface, while maintaining good compatibility with existing resin bonding systems, remains a key technical challenge that urgently needs to be overcome. Summary of the Invention

[0008] One of the objectives of this invention is to provide a method for preparing a multifunctional SACP-PEP / TMC film, in order to solve the problems that the single-function strategy in the prior art is difficult to comprehensively address the multi-factor failure of the resin-dentin bonding interface, as well as the insufficient clinical operability and long-term stability of existing composite functional materials.

[0009] A second objective of this invention is to provide a multifunctional SACP-PEP / TMC thin film prepared by the above method.

[0010] A third objective of this invention is to provide applications of this multifunctional SACP-PEP / TMC film.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention discloses a method for preparing a multifunctional SACP-PEP / TMC thin film, which includes the following steps: S1. Preparation of nanoparticles: ACP nanoparticles were prepared by reacting phosphate and polyacrylic acid in the presence of calcium salt; S2. Surface modification: ACP nanoparticles are dispersed in a solvent and reacted with a silane coupling agent to obtain surface-modified SACP nanoparticles; S3. Preparation of composite slurry: Dissolve N,N,N-trimethyl chitosan in a solvent, add SACP nanoparticles and functional peptides, mix evenly to obtain film-forming slurry; S4. Film forming step: Place the film forming slurry in a mold, remove the solvent, and form to obtain the SACP-PEP / TMC film.

[0012] In some embodiments of the present invention, in step S1, phosphate and polyacrylic acid are dissolved in water to prepare an aqueous phosphate solution; then the pH value is adjusted to 9.5±0.1, and then reacted with calcium salt solution at room temperature. After solid-liquid separation, washing, and drying, ACP nanoparticles are obtained.

[0013] In some embodiments of the present invention, the phosphate aqueous solution contains phosphate at a concentration of 3-12 mM, polyacrylic acid at a concentration of 50-200 mg / L, and the molar ratio of phosphate to calcium salt is 1.2:1. The phosphate includes at least one of Na2HPO4 and K2HPO4; The calcium salt includes CaCl2.

[0014] In some embodiments of the present invention, in step S2, ACP nanoparticles are dispersed in an aqueous ethanol solution with a concentration of 30-90% (v / v), and silane coupling agent 3-methacryloyloxypropyltrimethoxysilane is added. The mixture is then reacted under low temperature conditions to obtain surface-modified SACP nanoparticles.

[0015] In some embodiments of the present invention, in step S2, 100-400 μL of the silane coupling agent is added for every 100 mg of ACP nanoparticles.

[0016] In some embodiments of the present invention, the reaction temperature of step S2 is 2~6°C.

[0017] In this invention, the functional polypeptide is a bioactive polypeptide, such as a polypeptide possessing mineralization-promoting and / or antibacterial and / or anti-inflammatory functions. As an example, this invention uses the polypeptide PEP-QP5, which possesses mineralization-promoting function, for illustration. It should be noted that there are many types of polypeptides with mineralization functions, and PEP-QP5 is only one example; similarly, many other known functional polypeptides exist in other functional categories. The thin film material of this invention can serve as a carrier for loading various functional polypeptides.

[0018] In some embodiments of the present invention, N,N,N-trimethylchitosan is dissolved in water to prepare a mother liquor of 2.5~10 mg / mL; then SACP nanoparticles and peptides are added to the mother liquor and stirred and dispersed at room temperature to form a uniform and stable mixed suspension, which is used as a film-forming slurry.

[0019] In some embodiments of the present invention, the mass ratio of N,N,N-trimethyl chitosan to SACP nanoparticles is 0.5~2:1; the mass ratio of N,N,N-trimethyl chitosan to functional peptides is 5~20:1.

[0020] The second aspect of this invention discloses a multifunctional SACP-PEP / TMC thin film prepared by the above method.

[0021] The third aspect of this invention discloses the application of the above-mentioned multifunctional SACP-PEP / TMC film in the preparation of at least one of the following dental restorative materials: (1) Materials that promote the remineralization of dentin collagen fibers; (2) Materials that enhance the resin-dentin bonding interface; (3) Materials that inhibit bacterial growth; (4) Materials that inhibit collagenase activity.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention, through material structure design and integration of multifunctional components, has significant advantages in terms of interface compatibility, active component delivery mode and multi-target synergistic effect, and solves the problems of interface degradation, low remineralization efficiency and bacterial colonization and invasion in existing oral resin-dentin bonding systems.

[0023] In terms of structural design, this invention employs a silanization modification method to prepare silanized amorphous calcium phosphate, improving upon the problems of traditional bioactive nano-mineral particles easily agglomerating at the bonding interface and having poor compatibility with hydrophobic resin matrices. Using γ-methacryloyloxypropyltrimethoxysilane as a silane coupling agent, polymerizable methacrylate groups are introduced onto the surface of the amorphous calcium phosphate, allowing it to participate in the photopolymerization reaction of the resin matrix through covalent bonds. This stably disperses the mineral precursor within the resin-dentin mixture, avoiding the negative impact of additional additives on the integrity of the interfacial structure and maintaining the stability and density of the interfacial bonding.

[0024] Regarding the delivery mode of active components, this invention abandons the traditional nanoparticle dispersion system and uses a solid film as a functional carrier to construct a novel interface delivery system. This carrier form has the following application characteristics: First, it enables local delivery and high-concentration retention of functional components on the dentin surface, forming a continuous ion-release system; second, after the film swells in water, it can closely adhere to the demineralized collagen matrix without introducing an additional liquid phase into the bonding interface, reducing operational sensitivity and minimizing defects such as interface porosity and microleakage; third, it can be adapted to routine clinical resin bonding procedures, improving the convenience and feasibility of clinical applications.

[0025] In terms of functional synergy, this film constructs a multi-target synergistic system of antibacterial, anti-enzymatic, and remineralization effects, with each component having complementary functions and working synergistically. Trimethyl chitosan, as the film matrix, plays a dual role due to its cationic properties: firstly, it binds to bacterial cell membranes through electrostatic interactions, inhibiting the adhesion of cariogenic bacteria and biofilm formation in the oral cavity; secondly, it binds to the negatively charged regions of endogenous matrix metalloproteinases in dentin, inhibiting collagenase activity, reducing enzymatic degradation of collagen fibers, and maintaining the integrity of the collagen scaffold structure. Silanized amorphous calcium phosphate, as a stable mineral precursor, continuously releases calcium and phosphorus ions, providing an ion source for remineralization within collagen fibers; its amorphous structure can penetrate into the interfibrillary spaces of collagen fibers, subsequently transforming into hydroxyapatite, achieving structural repair of demineralized areas of dentin. Functional peptides can regulate the crystal transformation process of amorphous calcium phosphate, guiding hydroxyapatite crystals along collagen fibers. c Axial-directed deposition promotes ordered intrafiber mineralization and restores the mechanical properties of demineralized dentin.

[0026] In vitro performance tests and in vivo experiments showed that the composite film significantly improved the immediate bond strength between resin and dentin, maintained stable bonding performance even after artificial aging treatment, reduced interfacial nanoleakage, inhibited collagenase activity, and promoted dentinal tubule closure and collagen remineralization. Systemic toxicity and local biocompatibility evaluations indicated that the material had no significant toxic side effects, and its biocompatibility met the requirements for oral biomedical materials.

[0027] In summary, compared with existing single-function interface modification technologies, this invention constructs a multi-target intervention system with antibacterial, anti-enzyme, and remineralization properties. This system protects the collagen matrix while inducing its orderly remineralization, thereby achieving comprehensive strengthening of the resin-dentin bonding interface and providing a feasible technical solution for improving the durability of clinically bonded restorations. Attached Figure Description

[0028] Figure 1 This is a process flow diagram of the SACP-PEP / TMC thin film preparation method of the present invention.

[0029] Figure 2 Typical TEM images, EDS elemental distribution maps, and selected area electron diffraction (SAED) patterns of ACP and SACP nanoparticles are shown. Figure 2 (a) shows the relevant results for ACP nanoparticles: a1 and a2 are low-magnification and high-magnification TEM bright-field images, respectively; a3 is a multi-element EDS surface scan overlay; a4 is a high-angle annular dark-field image (HAADF-STEM); a5~a9 are EDS surface scan images of Ca, P, O, C and Si, respectively; a10 is the corresponding SAED image; Figure 2(b) shows the relevant results of SACP nanoparticles: b1 and b2 are low-magnification and high-magnification TEM bright-field images, respectively; b3 is a multi-element EDS surface scan overlay; b4 is a HAADF-STEM image; b5 to b9 are EDS surface scan images of Ca, P, O, C and Si, respectively; b10 is the corresponding SAED image.

[0030] Figure 3 X-ray diffraction patterns of ACP nanoparticles, SACP nanoparticles, and SACP-PEP / TMC films.

[0031] Figure 4 The images show the Fourier Transform Infrared (FTIR) spectra of ACP nanoparticles, SACP nanoparticles, pure TMC films, and SACP-PEP / TMC films.

[0032] Figure 5 XPS full spectrum and high-resolution spectrum of ACP nanoparticles and SACP nanoparticles; among which Figure 5 (a) shows the XPS full spectrum of ACP nanoparticles and SACP nanoparticles. Figure 5 (b) is the high-resolution XPS spectrum of Si2p in SACP nanoparticles. Figure 5 (c) is the high-resolution XPS spectrum of P2p of SACP nanoparticles. Figure 5 (d) is the high-resolution XPS spectrum of Ca2p in SACP nanoparticles.

[0033] Figure 6 The image shows the morphology of the SACP-PEP / TMC thin film. Figure 6 (a) is a macroscopic diagram. Figure 6 (b) to (d) are micrographs at different magnifications.

[0034] Figure 7 TGA and DTG curves of SACP-PEP / TMC membrane.

[0035] Figure 8 The XRD and FTIR spectra of the SACP-PEP / TMC film in SBF over 48 h are shown; Figure 8 A is the XRD pattern. Figure 8 B is the FTIR spectrum. Figure 8 JCPDS 09-0432 in the code represents the number of hydroxyapatite.

[0036] Figure 9 The graph shows the swelling behavior of SACP-PEP / TMC film in the adhesive and the release curves of Ca, P and PEP-QP5 of the cured film in simulated body fluid for 84 days. Figure 9 A is the swelling behavior diagram. Figure 9 B represents the release curve.

[0037] Figure 10 The image shows the cytotoxicity test results of the SACP-PEP / TMC film. Figure 10 A is the live / dead staining diagram. Figure 10 B is the CCK-8 result graph.

[0038] Figure 11 The image shows the results of the antibacterial effect evaluation of the SACP-PEP / TMC film. Figure 11 A shows the fluorescent staining and SEM characterization of live / dead bacteria; Figure 11 B is a quantitative analysis chart of antibacterial activity based on OD570; Figure 11 C represents the statistical graph of the live / dead bacteria ratio within the biofilm; where a1~a4 are, in order, the live bacteria staining image, dead bacteria staining image, live / dead bacteria fluorescence overlay image, and bacterial morphology SEM image of the control group; b1~b4 are, in order, the live bacteria staining image, dead bacteria staining image, live / dead bacteria fluorescence overlay image, and bacterial morphology SEM image of the TMC group; c1~c4 are, in order, the live bacteria staining image, dead bacteria staining image, live / dead bacteria fluorescence overlay image, and bacterial morphology SEM image of the SACP-PEP / TMC film group.

[0039] Figure 12 The figure shows the elemental distribution characterization results of SACP-PEP / TMC thin films at different mineralization times using transmission electron microscopy (TEM), selected area electron diffraction (SAED), and energy dispersive spectroscopy (EDS). Figure 12 A1-A7 are the results of the control group after 5 days of mineralization, and are, in order: TEM bright-field image of the thin film fiber, high-magnification TEM bright-field image (including SAED inset), STEM image and EDS surface distribution map of N, O, P and Ca elements; Figure 12 Figures b1-b7, c1-c7, and d1-d7 show the corresponding mineralization results of the SACP-PEP / TMC thin film group after 1 day, 3 days, and 5 days, respectively.

[0040] Figure 13 The images show the SEM images of the in vitro remineralization effect of SACP-PEP / TMC film on demineralized dentin samples. Among them, a1~a3 are SEM images of healthy dentin (Sound) at different magnifications, b1~b3 are SEM images of demineralized dentin (Etched) at different magnifications, c1~c3, e1~e3, g1~g3, and i1~i3 are SEM images of demineralized dentin samples after 3 days of different treatments, and d1~d3, f1~f3, h1~h3, and j1~j3 are SEM images after 7 days of corresponding treatments.

[0041] Figure 14 The graph shows the quantitative results of surface roughness (Ra) of healthy dentin, demineralized dentin, and remineralized dentin in each treatment group.

[0042] Figure 15 The graphs show the nanoindentation load-displacement curves, elastic modulus, and hardness results for healthy dentin, demineralized dentin, and each treatment group; among them... Figure 15 A represents the nanoindentation load-displacement curve. Figure 15 B is a graph showing the results of elastic modulus and hardness.

[0043] Figure 16 Infrared spectra and X-ray diffraction patterns of tooth samples from different treatment groups are shown. Figure 16 A is the infrared spectrum. Figure 16 B is the X-ray diffraction pattern. Figure 16 JCPDS 09-0432 in the code represents the number of hydroxyapatite.

[0044] Figure 17 The figure shows the results of the in vitro bonding performance characterization of resin-dentin bonding, in which... Figure 17 Figure A shows the micro-shear bond strength results for each group. Figure 17 B is a diagram showing the distribution of fracture modes at the adhesive interface. Figure 17 In this context, NS indicates no statistically significant difference.

[0045] Figure 18 Representative confocal laser scanning microscopy findings of resin-dentin interface microleakage in different groups after 24 hours and 3 months of preservation. (CLSM) diagram and CLSM in situ enzyme spectrum; among which Figure 18 A is the CLSM diagram. Figure 18 B is the CLSM in situ enzyme spectrum. Figure 18 In this context, R represents resin, HL represents mixed layer, and D represents dentin.

[0046] Figure 19 The graph shows the quantitative calculation results of nanoleakage over 24 hours and 3 months. Figure 19 Figure A shows the quantitative calculation results of nano-leakage over 24 hours. Figure 19 Figure B shows the quantitative calculation results of nano-leakage over 3 months. Figure 19 In this context, NS indicates no statistically significant difference.

[0047] Figure 20 The graph shows the results of in-situ enzyme profiling quantification calculations over 24 hours and 3 months. Figure 20 Figure A shows the results of 24-hour in-situ enzyme profiling for quantitative analysis. Figure 20 B is the result of in-situ enzyme spectrum quantitative calculation over 3 months. Figure 20 In this context, NS indicates no statistically significant difference.

[0048] Figure 21 This is a schematic diagram of the clinical procedure for applying SACP-PEP / TMC film to the adhesive restoration of the maxillary first molar in SD rats. Figure 21Image a shows the original tooth structure of the maxillary first molar in an SD rat. Figure 21 b~c: Hole preparation stage; Figure 21 d: Acid etching stage; Figure 21 e–f: SACP-PEP / TMC thin placement stage; Figure 21 g: Adhesive coating stage; Figure 21 h–i: Adhesive photocuring stage; Figure 21 j: Resin filling stage; Figure 21 k: Resin photocuring stage; Figure 21 l: Final repair completed status.

[0049] Figure 22 The images represent the resin-dentin interface. The images for each group are as follows: 1: Micro-CT image; 2: Pseudo-color micro-CT image; 3: Low-magnification SEM image; 4: High-magnification SEM image; R represents resin; HL represents the mixed layer; D represents dentin. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. The described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0051] All raw materials used in the examples are commercially available; unless otherwise specified, the reagents, methods and equipment used in this invention are conventional reagents, methods and equipment in this technical field.

[0052] The amino acid sequence of the functional polypeptide PEP-QP5 described in this embodiment of the invention is: QPYQPVQPHQPMQPQTKREEV-D.

[0053] The amelogenin-derived peptide QP5 (PEP-QP5) of this invention was prepared by Shanghai GL Biochem Co., Ltd. using the standard Fmoc solid-phase peptide synthesis technology, referring to the method reported by Wang et al. (J Mater Chem B, 2020, 8(45), 10373-10383; Unraveling the mechanism for an amelogenin-derived peptide regulated hydroxyapatitemineralization via specific functional domain identification).

[0054] Example 1 This embodiment discloses a method for preparing the SACP-PEP / TMC thin film of the present invention, the process flow of which is shown in the attached figure. Figure 1 As shown, the details are as follows: S1. Preparation of ACP nanoparticles Weigh appropriate amounts of disodium hydrogen phosphate (Na₂HPO₄) and polyacrylic acid (PAA, weight average molecular weight 450 kDa), dissolve in water to prepare a phosphate aqueous solution containing 6 mM Na₂HPO₄ and 100 mg / L PAA. Stir the solution magnetically for 30 min at room temperature to ensure thorough mixing; then adjust the pH of the system to 9.5 ± 0.1 using sodium hydroxide solution. Rapidly mix the pH-adjusted phosphate solution with an equal volume of 10 mM calcium chloride solution to initiate the precipitation reaction, continuing the reaction at room temperature for 10 min until complete. After the reaction, centrifuge the resulting suspension at 18000 rpm for 5 min at 4°C, discard the supernatant, wash the precipitate three times with anhydrous ethanol, and then freeze-dry to obtain solid ACP nanoparticle powder.

[0055] S2. Preparation of SACP nanoparticles Accurately weigh 100 mg of ACP nanoparticle powder obtained in step S1, add it to 30 mL of 70% (v / v) ethanol aqueous solution, and disperse it ultrasonically. Add 200 μL of 3-methacryloyloxypropyltrimethoxysilane (MPS) to the uniformly dispersed nanoparticle suspension, and then stir the reaction system at 4 °C for 2 h to ensure that the silane coupling agent and the nanoparticle surface undergo sufficient silanization reaction. After the reaction is terminated, the mixture is centrifuged at 4 °C and 18000 rpm for 5 min, and the precipitate is collected. Wash the precipitate three times with anhydrous ethanol to remove unreacted silane, and then freeze-dry to obtain SACP nanoparticle powder.

[0056] S3. Preparation of SACP-PEP / TMC thin films Weigh 50 mg of N,N,N-trimethyl chitosan (TMC, molecular weight approximately 100 kDa, degree of deacetylation 87%, degree of quaternization 92%; purchased from Qingdao Honghai Biotechnology Co., Ltd., China), and magnetically stir at room temperature for 30 min to completely dissolve it in 10 mL of deionized water to obtain a homogeneous TMC stock solution. Subsequently, add 5 mg of PEP-QP5 and 50 mg of SACP nanoparticles prepared in step S2 to the above solution sequentially, and continue to magnetically stir and disperse for 5 min until a homogeneous and stable mixed suspension is formed.

[0057] The prepared mixed suspension was slowly poured into a mold with dimensions of 6cm×6cm×1cm (length×width×height), and vacuum dried overnight at 25℃. After the solvent had completely evaporated, the film was formed and demolded to obtain the SACP-PEP / TMC film.

[0058] The resulting film was then cut into 5mm×5mm standard samples, placed in a desiccator, sealed, and stored for later use.

[0059] Example 2 This embodiment discloses a method for preparing the SACP-PEP / TMC thin film of the present invention, the process flow of which is shown in the attached figure. Figure 1 As shown, the details are as follows: S1. Preparation of ACP nanoparticles Weigh appropriate amounts of disodium hydrogen phosphate (Na₂HPO₄) and polyacrylic acid (PAA, weight average molecular weight 450 kDa), dissolve in water to prepare a phosphate aqueous solution containing 3 mM Na₂HPO₄ and 50 mg / L PAA. Stir the solution magnetically for 30 min at room temperature to ensure thorough mixing; then adjust the pH of the system to 9.5 ± 0.1 using sodium hydroxide solution. Rapidly mix the pH-adjusted phosphate solution with an equal volume of 5 mM calcium chloride solution to initiate the precipitation reaction, continuing the reaction at room temperature for 10 min until complete. After the reaction, centrifuge the resulting suspension at 18000 rpm for 5 min at 4°C, discard the supernatant, wash the precipitate three times with anhydrous ethanol, and then freeze-dry to obtain solid ACP nanoparticle powder.

[0060] S2. Preparation of SACP nanoparticles Accurately weigh 100 mg of ACP nanoparticle powder obtained in step S1, add it to 50 mL of 30% (v / v) ethanol aqueous solution, and disperse it ultrasonically. Add 100 μL of 3-methacryloyloxypropyltrimethoxysilane (MPS) to the uniformly dispersed nanoparticle suspension, and then stir the reaction system at 4 °C for 2 h to ensure that the silane coupling agent and the nanoparticle surface undergo sufficient silanization reaction. After the reaction is terminated, the mixture is centrifuged at 4 °C and 18000 rpm for 5 min, and the precipitate is collected. Wash the precipitate three times with anhydrous ethanol to remove unreacted silane, and then freeze-dry to obtain SACP nanoparticle powder.

[0061] S3. Preparation of SACP-PEP / TMC thin films 25 mg of N,N,N-trimethyl chitosan (TMC, molecular weight approximately 100 kDa, degree of deacetylation 87%, degree of quaternization 92%; purchased from Qingdao Honghai Biotechnology Co., Ltd., China) was weighed and magnetically stirred at room temperature for 30 min to completely dissolve it in 10 mL of deionized water, thus obtaining a homogeneous TMC stock solution. Subsequently, 5 mg of PEP-QP5 and 50 mg of SACP nanoparticles prepared in step S2 were added sequentially to the above solution, and magnetic stirring was continued for 5 min until a uniform and stable mixed suspension was formed.

[0062] The prepared mixed suspension was slowly poured into a mold with dimensions of 6cm×6cm×1cm (length×width×height), and vacuum dried overnight at 25℃. After the solvent had completely evaporated, the film was formed and demolded to obtain the SACP-PEP / TMC film.

[0063] The resulting film was then cut into 5mm×5mm standard samples, placed in a desiccator, sealed, and stored for later use.

[0064] Example 3 This embodiment discloses a method for preparing the SACP-PEP / TMC thin film of the present invention, the process flow of which is shown in the attached figure. Figure 1 As shown, the details are as follows: S1. Preparation of ACP nanoparticles Weigh appropriate amounts of disodium hydrogen phosphate (Na₂HPO₄) and polyacrylic acid (PAA, weight average molecular weight 450 kDa), dissolve in water to prepare a phosphate aqueous solution containing 12 mM Na₂HPO₄ and 150 mg / L PAA. Stir the solution magnetically for 30 min at room temperature to ensure thorough mixing; then adjust the pH of the system to 9.5 ± 0.1 using sodium hydroxide solution. Rapidly mix the pH-adjusted phosphate solution with an equal volume of 20 mM calcium chloride solution to initiate the precipitation reaction, continuing the reaction at room temperature for 15 min until complete. After the reaction, centrifuge the resulting suspension at 18000 rpm for 5 min at 4°C, discard the supernatant, wash the precipitate three times with anhydrous ethanol, and then freeze-dry to obtain solid ACP nanoparticle powder.

[0065] S2. Preparation of SACP nanoparticles Accurately weigh 100 mg of ACP nanoparticle powder obtained in step S1, add it to 20 mL of 90% (v / v) ethanol aqueous solution, and disperse it ultrasonically. Add 400 μL of 3-methacryloyloxypropyltrimethoxysilane (MPS) to the uniformly dispersed nanoparticle suspension, and then stir the reaction system at 2 °C for 2 h to ensure that the silane coupling agent and the nanoparticle surface undergo sufficient silanization reaction. After the reaction is terminated, the mixture is centrifuged at 4 °C and 18000 rpm for 5 min, and the precipitate is collected. Wash the precipitate three times with anhydrous ethanol to remove unreacted silane, and then freeze-dry to obtain SACP nanoparticle powder.

[0066] S3. Preparation of SACP-PEP / TMC thin films 100 mg of N,N,N-trimethyl chitosan (TMC, molecular weight approximately 100 kDa, degree of deacetylation 87%, degree of quaternization 92%; purchased from Qingdao Honghai Biotechnology Co., Ltd., China) was weighed and magnetically stirred at room temperature for 30 min to completely dissolve it in 10 mL of deionized water, thus obtaining a homogeneous TMC stock solution. Subsequently, 5 mg of PEP-QP5 and 50 mg of SACP nanoparticles prepared in step S2 were added sequentially to the above solution, and magnetic stirring was continued for 5 min until a homogeneous and stable mixed suspension was formed.

[0067] The prepared mixed suspension was slowly poured into a mold with dimensions of 6cm×6cm×1cm (length×width×height), and vacuum dried overnight at 25℃. After the solvent had completely evaporated, the film was formed and demolded to obtain the SACP-PEP / TMC film.

[0068] The resulting film was then cut into 5mm×5mm standard samples, placed in a desiccator, sealed, and stored for later use.

[0069] Example 4 This embodiment discloses a method for preparing the SACP-PEP / TMC thin film of the present invention, the process flow of which is shown in the attached figure. Figure 1 As shown, the details are as follows: S1. Preparation of ACP nanoparticles Weigh appropriate amounts of disodium hydrogen phosphate (Na₂HPO₄) and polyacrylic acid (PAA, weight average molecular weight 450 kDa), dissolve in water to prepare a phosphate aqueous solution containing 6 mM Na₂HPO₄ and 200 mg / L PAA. Stir the solution magnetically for 30 min at room temperature to ensure thorough mixing; then adjust the pH of the system to 9.5 ± 0.1 using sodium hydroxide solution. Rapidly mix the pH-adjusted phosphate solution with an equal volume of 10 mM calcium chloride solution to initiate the precipitation reaction, continuing the reaction at room temperature for 10 min until complete. After the reaction, centrifuge the resulting suspension at 18000 rpm for 5 min at 4°C, discard the supernatant, wash the precipitate three times with anhydrous ethanol, and then freeze-dry to obtain solid ACP nanoparticle powder.

[0070] S2. Preparation of SACP nanoparticles Accurately weigh 100 mg of ACP nanoparticle powder obtained in step S1, add it to 30 mL of 60% (v / v) ethanol aqueous solution, and disperse it ultrasonically. Add 300 μL of 3-methacryloyloxypropyltrimethoxysilane (MPS) to the uniformly dispersed nanoparticle suspension, and then stir the reaction system at 6 °C for 2 h to ensure that the silane coupling agent and the nanoparticle surface undergo sufficient silanization reaction. After the reaction is terminated, the mixture is centrifuged at 4 °C and 18000 rpm for 5 min, and the precipitate is collected. Wash the precipitate three times with anhydrous ethanol to remove unreacted silane, and then freeze-dry to obtain SACP nanoparticle powder.

[0071] S3. Preparation of SACP-PEP / TMC thin films 70 mg of N,N,N-trimethyl chitosan (TMC, molecular weight approximately 100 kDa, degree of deacetylation 87%, degree of quaternization 92%; purchased from Qingdao Honghai Biotechnology Co., Ltd., China) was weighed and magnetically stirred at room temperature for 30 min to completely dissolve it in 10 mL of deionized water, thus obtaining a homogeneous TMC stock solution. Subsequently, 5 mg of PEP-QP5 and 50 mg of SACP nanoparticles prepared in step S2 were added sequentially to the above solution, and magnetic stirring was continued for 5 min until a uniform and stable mixed suspension was formed.

[0072] The prepared mixed suspension was slowly poured into a mold with dimensions of 6cm×6cm×1cm (length×width×height), and vacuum dried overnight at 25℃. After the solvent had completely evaporated, the film was formed and demolded to obtain the SACP-PEP / TMC film.

[0073] The resulting film was then cut into 5mm×5mm standard samples, placed in a desiccator, sealed, and stored for later use.

[0074] Experimental Example 1 This experimental example characterizes the SACP-PEP / TMC film, intermediate product ACP nanoparticles, and SACP nanoparticles prepared in Example 1.

[0075] 1. Testing Method To systematically characterize the microstructure, phase structure, elemental composition, and functional group characteristics of the above samples, various large-scale precision instruments were used for testing. All characterization experiments were performed in triplicate (n=3). Details are as follows: 1.1 Characterization of microstructure and elemental distribution Transmission electron microscopy (TEM; Tecnai G2 F20, FEI, USA), combined with selected area electron diffraction (SAED) and a matching elemental surface distribution testing module, was used to analyze the microstructure, particle size distribution, crystallinity, and elemental distribution uniformity of ACP nanoparticles and SACP nanoparticles.

[0076] 1.2 Phase Composition Analysis X-ray diffraction (XRD; Empyrean, PANalytical, Netherlands) was used to determine the phase composition and crystallization state of the SACP-PEP / TMC thin film and the two nanoparticles. Test conditions: Cu Kα target radiation, X-ray wavelength λ = 1.5406 Å, 2θ scan range 10°–50°.

[0077] 1.3 Characterization of Feature Functional Groups Fourier transform infrared spectroscopy (FTIR; Nicolet 670, Thermo, USA) was used to analyze the characteristic functional groups and chemical bonding states of ACP nanoparticles, SACP nanoparticles, and pure TMC films in SACP-PEP / TMC films. The wavenumber range was 500–2500 cm⁻¹. -1 Comparative characterization was carried out in conjunction with XRD results.

[0078] The pure TMC film was prepared according to the method in step S3 of Example 1, except that PEP-QP5 and SACP nanoparticles were not added.

[0079] 1.4 Surface elemental and chemical state analysis X-ray photoelectron spectroscopy (XPS; Thermo Fisher Scientific K-ALPHA, USA) was used to characterize the surface elemental composition, elemental content and chemical bonding state of the two nanoparticles. The binding energy was calibrated using the C 1s characteristic peak at 284.8 eV as the standard.

[0080] 2. Test Results and Analysis 2.1 TEM, SAED and elemental surface distribution analysis Typical TEM images, elemental distribution maps, and selected area electron diffraction (SAED) patterns of ACP and SACP nanoparticles are detailed in the appendix. Figure 2 TEM observations showed that the unmodified ACP nanoparticles were spherical with a diameter of 50-100 nm and exhibited slight aggregation. The SACP nanoparticles prepared by silanization modification showed no significant difference in microstructure and particle size from the original ACP nanoparticles, and no obvious secondary agglomeration or particle adhesion. This indicates that the silanization modification did not destroy the original structure of the nanoparticles and maintained good dispersibility.

[0081] Elemental distribution pattern analysis showed that the four core elements, Ca, P, O, and C, were uniformly distributed in both ACP and SACP nanoparticles, with no obvious elemental enrichment or segregation, proving that the internal composition of the nanoparticles was homogeneous. Compared to ACP nanoparticles, the elemental spectrum of SACP nanoparticles showed a significantly enhanced and uniformly distributed Si signal, directly confirming that the silanization modifier was successfully loaded onto the nanoparticle surface and the modification process was effectively completed.

[0082] SAED results showed that the diffraction patterns of both nanoparticles only showed diffuse halos, without clear, sharp diffraction rings or spots, indicating that both ACP and SACP nanoparticles are amorphous structures and have not formed crystalline calcium phosphate-related phases.

[0083] 2.2 XRD Phase Analysis XRD (X-ray diffraction) patterns of ACP nanoparticles, SACP nanoparticles, and SACP-PEP / TMC films are attached. Figure 3 The spectral characteristics show that the SACP-PEP / TMC film exhibits a broad diffraction halo in the 20°~30° range, without the sharp crystalline peaks corresponding to hydroxyapatite. The diffraction curves of the two nanoparticles show a highly consistent trend, with typical broad diffuse diffraction peaks at approximately 20° and 35° of 2θ, consistent with the XRD characteristics of amorphous materials. No characteristic crystalline diffraction peaks of hydroxyapatite at positions such as 25.9°, 31.7°, 32.9°, and 34.0° were detected, further verifying the conclusions of TEM and SAED, confirming that both ACP and SACP nanoparticles maintain a stable amorphous structure, and that silanization modification did not induce a crystal transformation in amorphous calcium phosphate.

[0084] 2.3 FTIR Analysis The FTIR (infrared spectrum) of each test sample is attached. Figure 4 The typical characteristic absorption peaks of calcium phosphate can be clearly observed in the spectrum: 1030-1100 cm⁻¹. -1 This corresponds to ν3 PO4 3- The antisymmetric stretching vibration peak, 560-600 cm⁻¹ -1 The location corresponds to ν4PO4 3-The bending vibration peaks confirmed that both nanoparticles contain a calcium phosphate core structure.

[0085] Comparison of the spectra revealed that SACP nanoparticles at 1635 cm⁻¹ -1 A new characteristic absorption peak appeared at the position, which is attributed to the C=C double bond stretching vibration peak of the methacrylate group in the silanization modifier 3-methacryloyloxypropyltrimethoxysilane (MPS). This further proves at the functional group level that the silanization modifier was successfully grafted / loaded on the surface of SACP nanoparticles, which is consistent with the elemental distribution test results.

[0086] Furthermore, TMC can be clearly observed at 1635 cm⁻¹ in the SACP-PEP / TMC thin film spectrum. -1 The characteristic peaks of amide I bands and the broad absorption bands of amorphous calcium phosphate are also evident in the nearby area.

[0087] 2.4 XPS photoelectron spectroscopy analysis XPS full-scan and high-resolution narrow-scan spectra of ACP and SACP nanoparticles are attached. Figure 5 ,in Figure 5 (a) is the full scan spectrum. Figure 5 (b) is the high-resolution spectrum of Si 2p in SACP nanoparticles. Figure 5 (c) is the high-resolution P 2p spectrum of SACP nanoparticles. Figure 5 (d) is the high-resolution spectrum of Ca 2p in SACP nanoparticles.

[0088] Depend on Figure 5 It can be seen that both types of nanoparticles showed characteristic element peaks of Ca, P, O, and C. Among them, the Ca 2p orbital exhibited a typical double-peak structure with binding energies of approximately 347.0 eV (Ca 2p3 / 2) and 350.5 eV (Ca 2p1 / 2), respectively. The binding energy of the P 2p characteristic peak was approximately 133.2 eV. These binding energy values ​​are in perfect agreement with the standard spectrum of amorphous calcium phosphate, confirming that the core component of the sample is a calcium phosphate system.

[0089] A distinct Si 2p characteristic peak was detected in the full spectrum of SACP nanoparticles, with a binding energy range of 102-103 eV. The high-resolution Si 2p spectrum showed that the peak center was located at 102.5 eV, corresponding to two bonding environments: Si-O-Ca / P and Si-O-Si. This fully demonstrates that the silanization modifier forms a stable chemical bond with the nanoparticle surface, rather than a simple physical adsorption, and the modification effect is stable and robust.

[0090] In summary, characterization and testing confirmed that the ACP nanoparticles and SACP nanoparticles prepared by the method of this invention are both spherical nanoparticles with regular morphology and good dispersibility, uniform particle size distribution, and stable amorphous structure. The silanization modification step did not induce a calcium phosphate crystal transformation. The SACP nanoparticles successfully loaded with silanization modifiers, with uniform Si element distribution. The modifiers formed stable chemical bonds with the nanoparticle surface, and the modification was sufficient and effective.

[0091] Experimental Example 2 In this experiment, the surface microstructure and cross-sectional morphology of the SACP-PEP / TMC film prepared in Example 1 were observed using a scanning electron microscope (SEM; Thermo Fisher Scientific Apreo 2C, USA) to analyze the uniformity and internal compactness of the film structure.

[0092] Morphology images of the SACP-PEP / TMC thin films are attached. Figure 6 As shown in the figure. The macroscopic image shows that the prepared SACP-PEP / TMC film has a uniform appearance and a complete structure; the SEM surface morphology image shows that the film has a uniform granular microstructure and a uniform distribution of nanoscale mineral domains; the SEM cross-section shows that the internal structure of the film is dense and continuous, with no obvious interfacial gaps, and the thickness is about 4~5μm.

[0093] Experimental Example 3 This experiment evaluated the thermal properties of the SACP-PEP / TMC thin film prepared in Example 1 of this invention using a thermogravimetric analyzer (TGA; STA 449 F3, Netzsch, Germany). The thermogravimetric curves (TGA) and derivative thermogravimetric curves (DTG) are attached. Figure 7 As shown, the composition of the SACP-PEP / TMC film is approximately 30 wt% minerals, 55 wt% organic matter, and 15 wt% water.

[0094] Test Example 4 This experimental example investigated the release behavior and phase transition of the SACP-PEP / TMC thin film prepared in Example 1 of the present invention.

[0095] 1. Ca 2+ PO4 3- and PEP-QP5 release from SACP-PEP / TMC film in vitro The SACP-PEP / TMC membrane (6cm × 6cm) prepared in Example 1 was immersed in 50mL of simulated body fluid (SBF; Beijing Regen Biosciences) and incubated at room temperature for 24 hours. At preset time points (0.5, 1, 2, 4, 8, 12, and 24 hours), 0.5mL of supernatant was collected, and an equal volume of fresh SBF was added to maintain an ideal release environment. The calcium content in the supernatant was detected using a commercially available calcium assay kit (Nanjing Jiancheng, C004-2), a phosphate assay kit (Nanjing Jiancheng, C006-1), and a BCA protein quantification kit (Shanghai Beyotime, P0010), respectively. 2+ PO4 3- And the concentration of PEP-QP5. Three independent samples were set up for each experiment, and results are expressed as mean. The cumulative release rate of PEP-QP5 was calculated using the following formula: Cumulative release rate (%) = (Total PEP-QP5 released / Total PEP-QP5 in the membrane) × 100%.

[0096] 2. SACP-PEP / TMC membrane phase transition analysis To simulate a physiological oral environment and evaluate the phase transition behavior of the membrane under clinically relevant conditions, the SACP-PEP / TMC film sample (6cm × 6cm) prepared in Example 1 was immersed in 15mL SBF and incubated for 48 hours at 100% relative humidity and 37°C. 1 mL of suspension was collected at 4, 8, 12, 24, 36, and 48 hours. The solid phase was separated by centrifugation, washed three times with anhydrous ethanol, and then freeze-dried. Fourier transform infrared spectroscopy and X-ray diffraction were used to analyze the phase transition behavior of the dried sample.

[0097] 3. In vitro Ca2+ of adhesive-cured SACP-PEP / TMC membranes 2+ PO4 3- and PEP-QP5 release characteristics To simulate clinical bonding procedures, the mineralized membrane needs to undergo bonding and curing treatment before the release experiment to evaluate the release characteristics of the functional components after bonding and polymerization. The specific procedures are as follows: One drop of general-purpose single-component adhesive (SBU; 3M ESPE, USA) was applied to the surface of the SACP-PEP / TMC membrane. The dynamic swelling behavior of the membrane in the adhesive was monitored and recorded. After the membrane swelled rapidly, the adhesive was brushed onto the membrane surface for 20 seconds, then gently blown dry for 5 seconds, and then light-cured for 20 seconds to obtain the bonded and cured mineralized membrane.

[0098] Forty sheets of bonded and cured mineralized membranes were immersed in 50 mL of SBF and incubated at room temperature. At designated time points (3, 7, 14, 28, 42, 56, and 84 days), 0.5 mL of supernatant was collected and immediately replenished with an equal volume of fresh SBF. The Ca content in the supernatant collected at each time point was determined. 2+ PO4 3- The concentrations of PEP-QP5 were also measured. All experiments were performed in triplicate, and results are presented as mean values.

[0099] 4. Results and Analysis The release curves of Ca, P, and PEP-QP5 from the SACP-PEP / TMC membrane in SBF over 24 hours showed that PEP-QP5 experienced a rapid initial burst release in the first 30 minutes, with a cumulative release of approximately 70%, followed by a slower, sustained release phase, ultimately reaching approximately 85% by 24 hours. Simultaneously, Ca and P also exhibited burst release within 30 minutes and continued to be released over 24 hours, with Ca concentrations increasing to approximately 0.9–1.1 mmol / L and P concentrations reaching approximately 0.6–0.8 mmol / L.

[0100] The XRD and FTIR spectra of the SACP-PEP / TMC membrane in SBF within 48 hours are attached. Figure 8 As shown. The XRD pattern shows that within 0-8 hours of immersion, the XRD pattern exhibits a broad amorphous halo centered at 20°-35°; after 12 hours of immersion, the characteristic diffraction peaks corresponding to the (002) and (211) crystal planes of HAP (JCPDS 09-0432) become more pronounced. The FTIR pattern shows that within 0-8 hours, the peaks at 570-580 cm⁻¹... -1 A wideband appeared nearby; from 12h to 48h, approximately 600cm -1 Approximately 560cm -1 The phosphate vibration band at that location gradually sharpens.

[0101] The appearance of the SACP-PEP / TMC film after adding the adhesive is shown in the attached figure. Figure 9 As shown in Figure A: The SACP-PEP / TMC film expands rapidly after contact with the adhesive: the film remains dense at 0s, begins to expand at 5s, becomes noticeably loose and partially dispersed at 10s, and shows obvious swelling and diffusion at 20s.

[0102] The cumulative release curves of Ca, P, and PEP-QP5 of the SACP-PEP / TMC film after photocuring are attached. Figure 9As shown in Figure B: After photocuring, the cured adhesive film continuously released Ca, P, and PEP-QP5 during the 84-day observation period, without significant bursting release. By day 84, the cumulative Ca and P concentrations were approximately 0.4 mmol / L and 0.25 mmol / L, respectively, and the cumulative release of PEP-QP5 gradually increased, eventually reaching approximately 70%.

[0103] Experimental Example 5 This experimental example tested the cytotoxicity of the SACP-PEP / TMC membrane prepared in Example 1 of this invention. Details are as follows: 10 mg of SACP-PEP / TMC membrane was placed in 10 mL of DMEM medium and incubated with gentle shaking at 37 °C for 12 h. The suspension was centrifuged at 1000 rpm for 4 min, and the supernatant was collected and sterilized by filtration through a 0.22 μm membrane to obtain the membrane extract. This sterile membrane extract was diluted with intact DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin to achieve final SACP-PEP / TMC concentrations of 0, 0.05, 0.1, and 0.2 mg / mL. Third-generation human dental pulp cells (hDPCs) were cultured at 5 × 10⁻⁶ m³ / mL. 3 Cells were seeded at a density of cells / well in 96-well plates. After the cells had fully adhered, 200 μL of DMEM membrane extract culture medium at different concentrations was added to each well. The cells were then cultured at 37°C in a 5% CO2 incubator for 1, 3, and 7 days, respectively.

[0104] Cell viability was detected using a CCK-8 assay kit (APE-BIO, USA), and absorbance at 450 nm was measured using a multi-functional microplate reader (ThermoScientific, Waltham, MA, USA).

[0105] Cell viability is calculated using the following formula:

[0106] Where As is the absorbance value of the experimental group, Ac is the absorbance value of the control group, and Ab is the absorbance value of the blank well containing only culture medium and no cells.

[0107] Meanwhile, cells cultured for 24 h were stained at 37°C for 30 min using the Calcein AM / PI (live / dead) cell staining kit and observed using an inverted fluorescence microscope.

[0108] The results of live / dead staining are attached. Figure 10As shown in Figure A: On day 1 of culture, the cells in the control group and the 0.05 and 0.1 mg / mL concentration groups mainly showed green fluorescence (live cells), while the red fluorescence (dead cells) signal was weak; the number of PI-positive dead cells in the 0.2 mg / mL group was slightly increased.

[0109] The CCK-8 test results are attached. Figure 10 As shown in B: Except for the 0.2 mg / mL concentration group, which showed a significant decrease in cell viability on day 3 of culture, the cell viability of all other concentration groups remained above 80% on days 1 and 3 (p<0.05). By day 7 of culture, the cell viability of the 0.05 mg / mL and 0.1 mg / mL groups was still above 80%, while the viability of the 0.2 mg / mL group was below 80%.

[0110] Experimental Example 6 This experimental example examines the antibacterial effect of the SACP-PEP / TMC film of the present invention, as detailed below: Streptococcus mutans ( S. mutans The bacterial culture (ATCC 25175) was inoculated into BHI medium and incubated at 37°C for 24 hours. The bacterial concentration was then adjusted to 0.5 McFarland turbidity standard for later use. Dentin samples were taken, etched with 37% phosphoric acid for 30 seconds, rinsed with sterile water for 30 seconds, and then air-dried. Subsequently, the specimens and TMC or SACP-PEP / TMC membranes were sterilized with ethylene oxide (55°C, 6 hours).

[0111] The sterilized dentin samples were divided into three groups (n=6): a blank control group, a TMC group, and an SACP-PEP / TMC film group. The pure TMC film used in the TMC group was prepared according to the method in Example 1, and the SACP-PEP / TMC film used in the SACP-PEP / TMC film group was prepared according to the method in Example 1.

[0112] TMC film and SACP-PEP / TMC film were respectively applied to the surface of the corresponding samples. Each dentin disc was inoculated with 2 μL of bacterial suspension and 500 μL of BHI medium and incubated at 37℃ in an anaerobic environment of 80% N2 / 10% H2 / 10% CO2 for 24 h.

[0113] The absorbance (OD) of bacteria at 570 nm was measured using a microplate reader (Tecan, Switzerland). 570The bacterial growth was assessed. After washing with PBS, three samples from each group were stained using a live / dead bacteria viability staining kit (APE×BIO, USA) and observed using a confocal laser scanning microscope (CLSM; DM-IRE2 CISM, Leica, Heidelberg, Germany). The ratio of live to dead bacteria was quantified. The remaining samples were prepared for scanning electron microscopy (SEM) to observe biofilm morphology.

[0114] For detailed test results, please refer to the appendix. Figure 11 .Depend on Figure 11 As shown in Figure A, in the live / dead staining images, the control group exhibited predominantly green fluorescence (live bacteria) and weak red fluorescence (dead bacteria). Compared to the control group, the TMC group showed enhanced red fluorescence and weakened green fluorescence, while the SACP-PEP / TMC group showed the strongest red fluorescence signal. Combined images revealed that the proportion of live bacteria in both the TMC and SACP-PEP / TMC groups was lower than that in the control group. Further SEM images showed that bacteria were densely packed and morphologically intact on the dentin surface in the control group; bacterial coverage was reduced in the TMC group; and bacterial adhesion was minimal in the SACP-PEP / TMC group, with a significant decrease in intact bacterial cells on the surface.

[0115] Depend on Figure 11 As shown in Figure B, compared with the control group, the OD570 values ​​of both the TMC group and the SACP-PEP / TMC group were significantly reduced (p<0.001).

[0116] Depend on Figure 11 As can be seen from C, compared with the control group, the live bacteria / dead bacteria ratio of both the TMC group and the SACP-PEP / TMC group was significantly reduced (p<0.001).

[0117] The above results indicate that the SACP-PEP / TMC film of the present invention has a significant antibacterial effect.

[0118] Experimental Example 7 This experimental example investigated the inducing effect of the SACP-PEP / TMC film of the present invention on collagen biomimetic mineralization. The specific steps are as follows: 1. Construction of a biomimetic mineralization model for collagen fibrils A 3 mg / mL collagen stock solution (Roche Diagnostics GmbH, Mannheim, Germany) was diluted to 50 μg / mL with an assembly solution containing 200 mM KCl and 50 mM glycine (pH 9.2) and incubated at room temperature for 20 minutes to promote collagen assembly. 14 μL of the diluted collagen solution was drop-coated onto a 300-mesh nickel transmission electron microscope (TEM) grid covered with a carbon film support membrane and incubated overnight in a humidification chamber at 37°C and 100% relative humidity. Subsequently, the collagen-coated grid was cross-linked and fixed with 0.05 wt% glutaraldehyde solution for 2 hours, gently rinsed with deionized water, and then air-dried.

[0119] 2. Mineralization Induction Experiment Nickel meshes covered with SACP-PEP / TMC films were suspended upside down in 5 mL of SBF and incubated at 37°C for 1, 3, and 5 days, with three replicates (n=3) in each group. The control group consisted of collagen-assembled meshes without SACP-PEP / TMC film coverage, incubated in SBF under the same conditions for 5 days. The SACP-PEP / TMC films in this experiment were prepared according to the method in Example 1.

[0120] 3. Mineralization Characterization and Analysis After collecting samples, the mineral deposition morphology inside collagen fibrils was observed by TEM, the distribution characteristics of Ca and P elements were analyzed by elemental mapping, and the crystal phase structure of the minerals was identified by selected area electron diffraction (SAED).

[0121] 4. Experimental Results The biomimetic mineralization characterization results of collagen fibers are attached. Figure 12 Among them, a1-a7 are the control group, and b1-b7, c1-c7, and d1-d7 are representative TEM images, SAED spectra, and elemental mapping maps of the SACP-PEP / TMC thin film treatment group after 1 day, 3 days, and 5 days of culture, respectively.

[0122] The results showed that after 5 days of incubation, the collagen fibers in the control group were sparsely arranged with no obvious mineralization deposition; no clear diffraction rings were detected in SAED, and the Ca and P signals in the elemental mapping were extremely weak. In contrast, the SACP-PEP / TMC thin film treatment group showed initial mineralization nodules distributed along the collagen fibril axis after 1 day of incubation, and obvious Ca and P element signals were detected. After 3 days of incubation, the electron density of collagen fibers increased significantly, and the SAED spectrum showed apatite characteristic diffraction peaks, corresponding to the reflections of the (002), (211), and (004) crystal planes; among them, the (002) and (004) diffraction arcs were distributed along the longitudinal axis of the fibrils, indicating that the c-axis of the apatite crystal was preferentially aligned with the collagen fibril axis. After 5 days of incubation, extensive mineralization occurred in the collagen fibrils, the Ca and P elements were evenly distributed and the signal intensity was significantly enhanced, and the SAED diffraction characteristics were clearer.

[0123] The above results indicate that SACP-PEP / TMC films can effectively induce biomimetic mineralization of collagen fibers and promote the ordered arrangement of apatite crystals.

[0124] Experimental Example 8 This experimental example investigated the effect of the SACP-PEP / TMC film of the present invention on regulating dentin biomimetic remineralization. Details are as follows: 1. Preparation of demineralized dentin specimens Bovine teeth were selected as a substitute for human teeth due to their ease of acquisition and similar chemical composition, and were used to evaluate dentin remineralization and resin-dentin bonding properties. Healthy bovine incisors without cracks, caries, or leukoplakia were collected from local slaughterhouses. The crowns were sectioned using a diamond-coated band saw (Struers Minitom, Denmark) under continuous water cooling, followed by ultrasonic cleaning for 30 minutes. The crown blocks were embedded in polymethyl methacrylate and polished sequentially with 800, 1500, and 3000 grit (Struers, Denmark) silicon carbide sandpaper under water cooling to expose a 4mm × 4mm dentin window. The exposed dentin surface was then smoothed and polished sequentially with 1000 to 5000 grit silicon carbide sandpaper. The polished samples were ultrasonically cleaned in deionized water for 30 minutes to remove residual debris. Except for the exposed dentin window, the remaining surfaces of the samples were sealed with two coats of acid-resistant nail polish. A microhardness tester (Duramin-1 / -2, Struers, Denmark) with a Knoop indenter was used to measure the surface microhardness (SMH) at five randomly selected locations 100 μm apart on the sample surface under a 15 g load. Samples with average SMH values ​​in the range of 100 to 125 Knoop hardness numbers (KHN) were selected for subsequent experiments.

[0125] Demineralized dentin specimens were prepared by etching with 37% phosphoric acid for 30 seconds, followed by rinsing with deionized water for 1 minute. The SMH of the demineralized dentin was determined using the same method, and specimens with SMH values ​​between 40 and 60 kHN were selected. The prepared demineralized dentin samples were stored in phosphate-buffered saline (PBS) at 4°C and used within 24 hours.

[0126] 2. In-situ biomimetic remineralization of demineralized dentin The prepared demineralized dentin discs were randomly divided into four groups (n=15 per group): (1) Control group: incubated only in SBF; (2) TMC membrane treatment group; the pure TMC membrane used was prepared according to the method of Experimental Example 1.

[0127] (3) SACP-PEP / TMC film treatment group: The SACP-PEP / TMC film used was prepared according to the method in Example 1.

[0128] (4) NaF group (positive control), treated with 1000ppm sodium fluoride solution.

[0129] For the membrane-treated group, the corresponding membrane was applied to the dentin surface, followed by immersion in 5 mL of SBF and incubation at 37°C for 7 days. Intact dentin slides incubated under the same conditions served as healthy controls. Fresh SBF and the corresponding membrane were applied daily. After treatment, the dentin specimens were gently rinsed with deionized water to remove loosely bound minerals. The specimens were then dehydrated with a gradient of ethanol concentrations (30%, 50%, 70%, 90%, 100%) for 15 minutes each, and fixed with hexamethyldisilane for 1 hour for subsequent characterization.

[0130] 3. Characterization of dentin specimens The surface and cross-sectional morphology of demineralized and remineralized dentin specimens were observed using scanning electron microscopy. Atomic force microscopy was used to analyze the three-dimensional surface morphology of dentin before, after, and after demineralization. The average surface roughness (Ra) was calculated from five samples in each group. Nanomechanical properties of the dentin specimens were tested using a nanoindenter under load-controlled conditions. The maximum load was 10000 μN, the peak hold time was 10 seconds, and the Poisson's ratio was set to 0.28. The indentation depth was recorded based on the obtained force-displacement curves. Five locations were randomly selected on each dentin disc for testing. X-ray diffraction was used to identify the crystal phase of the dentin samples within a 2θ angle range of 10° to 80°. Attenuated total reflection Fourier transform infrared spectroscopy was used to analyze the chemical composition of the dentin surface, with a wavenumber range of 400 to 2000 cm⁻¹. - ¹, resolution 4cm -¹. All spectra were normalized to the intensity of the amide I band for comparison.

[0131] 4. Results The results of the in vitro remineralization of demineralized dentin samples by SACP-PEP / TMC film are shown in the attached figure. Figures 13-16 As shown.

[0132] Depend on Figure 13 It can be seen that the dentinal tubules were completely open after acid etching, exposing collagen fibers. Three days after remineralization, significant mineral deposition was observed on the dentin surface in both the SACP-PEP / TMC and NaF groups. Seven days after remineralization, the control and TMC groups showed only sparse mineral deposition, with most tubules remaining open; while the SACP-PEP / TMC group showed abundant mineral deposition, extensive occlusion of the dentinal tubules, and dense crystals attached to the collagen fibrils.

[0133] Depend on Figure 14 It can be seen that the surface roughness (Ra) of dentin after acid etching significantly increased from 31.415±3.499 nm in healthy dentin to 324.228±15.177 nm (p<0.05). After remineralization, the Ra value of the SACP-PEP / TMC group significantly decreased to 103.083±9.941 nm (p<0.05 compared with the control group), which was comparable to that of the NaF group (77.238±4.320 nm) (p>0.05).

[0134] Depend on Figure 15 As can be seen from A, under the same maximum load, the indentation depth of healthy dentin is the smallest; after acid etching, the curve shifts significantly to the right, and the indentation depth increases significantly; the curves of the control group and the TMC group are similar to those of the demineralized group, with large indentation depths and limited recovery.

[0135] Depend on Figure 15 As shown in Figure B, the elastic modulus and hardness of dentin after acid etching decreased significantly from 14.379±0.500 GPa and 0.651±0.025 GPa for healthy dentin to 3.361±0.148 GPa and 0.129±0.009 GPa (p<0.05). After treatment with SACP-PEP / TMC membrane, the elastic modulus and hardness significantly recovered to 11.442±0.509 GPa and 0.509±0.007 GPa (p<0.05 compared with the control group).

[0136] Depend on Figure 16 As can be seen from A, all groups showed the characteristic amide II (≈1550 cm) of type I collagen. - ¹) and amide III (≈1450 cm) - ¹) Bands. After acid etching, PO4 3-Spectral bands (approximately 555, 598, and 1020 cm) - The relative strength of PO4 in the SACP-PEP / TMC group and the NaF group was significantly weaker than that in healthy dentin. Seven days after remineralization, the PO4 content in both groups was significantly lower. 3- The intensity of the bands was significantly restored, and was significantly higher than that of the control group and the TMC group.

[0137] Depend on Figure 16 As can be seen from B, healthy dentin exhibits characteristic hydroxyapatite (HAP) diffraction peaks at (002), (211), (112), (202), (213), and (004). After demineralization, the intensity of these diffraction peaks decreases and their shapes broaden. After remineralization, both the SACP-PEP / TMC and NaF groups show enhanced and sharper HAP diffraction peaks, especially on the (211) and (002) crystal planes, with comparable peak intensities in both groups; while the control group and TMC group only show a slight recovery in peak intensity.

[0138] Experimental Example 9 This experimental example investigated the effect of the SACP-PEP / TMC film described in this invention on the in vitro resin-dentin bonding performance, as detailed below: 1. Preparation of resin-dentin bonding specimens Demineralized dentin specimens were prepared according to the method in Example 8. The treated dentin specimens were randomly divided into three groups: control group, TMC group, and SACP-PEP / TMC film group. The pure TMC film used in the TMC group was prepared according to the method in Example 1, and the SACP-PEP / TMC film used in the SACP-PEP / TMC film group was prepared according to the method in Example 1.

[0139] The control group was treated with conventional bonding processes without any coating. The TMC group and the SACP-PEP / TMC film group had their respective films coated on the demineralized dentin surface. Then, Single Bond Universal adhesive was used for bonding. After the film rapidly swelled, the adhesive was continuously applied and allowed to fully penetrate for 10 seconds. The samples were gently dried for 5 seconds, then light-cured for 20 seconds to obtain a mineralized film after adhesive curing. Subsequently, resin composite material (Filtek Z350 XT; 3M, USA) was deposited layer by layer at a thickness of 1-2 mm on the bonding surface, with each layer light-cured for 20 seconds. All the prepared resin-dentin bonded specimens were stored in deionized water at 37°C for 24 hours and 3 months, respectively, before subsequent testing.

[0140] 2. Micro-shear bond strength test Resin-dentin bond specimens were sliced ​​perpendicularly to the bonding interface to obtain beam-shaped specimens with a cross-sectional area of ​​approximately 1 mm × 1 mm, 15 specimens per group. The microshear bond strength of each specimen was measured using a universal testing machine (INSTRON, USA). The loading blade was brought as close as possible to the bonding interface, and a load was applied parallel to the interface at a crosshead speed of 0.5 mm / min until the specimen failed. The maximum load (Newtons, N) at failure was recorded, and the μSBS value was calculated. After testing, all fractured specimens were collected, and their failure modes were observed under a stereomicroscope, and the results were analyzed according to the references (Huang, L., et al.). The Remineralization Potential of Resveratrol and Cucurbit[n]uril. According to the standard of the Journal of Dental Research (2025), the failure modes are classified into adhesive failure, cohesive failure, or mixed failure.

[0141] 3. Interfacial Microleakage Assessment Resin-dentin bonded specimens were immersed in Rhodamine B solution (1 g / L; Solarbio, Beijing, China) for 12 hours to allow fluorescent dye penetration. The specimens were sliced ​​perpendicular to the bonding interface to obtain slices approximately 1 mm thick and 4 mm high, which were then ultrasonically cleaned and air-dried. Each slice was examined using a confocal laser scanning microscope (CLSM) to assess dye penetration along the bonding interface; the excitation and emission wavelengths were set to 543 nm and 590 nm, respectively. Three slices were randomly selected from each group, and semi-quantitative analysis of fluorescence area, total fluorescence intensity, and average fluorescence intensity was performed using ImageJ software.

[0142] 4. In situ enzyme profile analysis Using fluorescein-labeled gelatin as the enzyme substrate, in situ zymography (using the EnzChek gelatinase / collagenase assay kit; Thermo Fisher Scientific, Brazil) was employed to assess the matrix metalloproteinase (MMP) activity at the resin-dentin interface of cross-sectional specimens. Before use, the gelatin stock solution was diluted 1:10 with the provided reaction buffer. Resin-dentin bonded specimens were fixed onto microscope slides using cyanoacrylate adhesive. After polymerization, the specimens were sequentially sanded with 600, 800, 1000, 1200, 1500, 2000, and 3000 grit silicon carbide sandpaper until a thickness of 500 μm was achieved. 200 μL of freshly prepared fluorescein-labeled gelatin solution was added dropwise to each specimen, covered with a coverslip, and incubated in a dark, humid environment at 37°C for 24 hours to allow endogenous MMPs to degrade the gelatin matrix at the resin-dentin interface. After incubation, fluorescence images were acquired using CLSM, and quantitative fluorescence analysis was performed using ImageJ software.

[0143] 5. Results Figures 17-20 The figure shows the effect of the SACP-PEP / TMC film of the present invention on the bonding performance of resin-dentin.

[0144] Depend on Figure 17 As shown in Figure A, at 24 hours, the μSBS values ​​of the control group, TMC group, and SACP-PEP / TMC film were 21.207±1.224 MPa, 21.120±0.494 MPa, and 22.373±1.173 MPa, respectively. There was no significant difference between the control group and the TMC group, while the μSBS value of the SACP-PEP / TMC film group was significantly higher than that of the control group and the TMC group (p<0.001). At 3 months, the μSBS values ​​of the control group, TMC group, and SACP-PEP / TMC film group were 14.967±0.660 MPa, 13.887±0.567 MPa, and 19.953±0.653 MPa, respectively. Compared with their respective 24-hour values, the μSBS values ​​of the control group and the TMC group were significantly lower (p<0.001), while the μSBS value of the SACP-PEP / TMC film group at 3 months was still significantly higher than that of the other two groups (p<0.001).

[0145] Depend on Figure 17 As shown in Figure B, at 24 hours, adhesive, mixed, and cohesive failures occurred in all groups. The control and TMC groups primarily exhibited adhesive and mixed failures, while the SACP-PEP / TMC film group showed fewer adhesive failures and more mixed / cohesive failures. After 3 months, adhesive failures dominated in the control and TMC groups, while the SACP-PEP / TMC film group maintained a lower proportion of adhesive failures and a higher proportion of mixed / cohesive failures.

[0146] Depend on Figure 18 As shown in Figure A, at 24 hours, a continuous strong fluorescent band was visible along the mixed layer in the control group, and significant dye penetration into the dentinal tubules was observed. The fluorescent bands in the TMC group and the SACP-PEP / TMC film group were narrower, indicating less fluorescent labeling. After 3 months, the fluorescence signal along the interface in the control group was stronger and more continuous, while the fluorescence signal in the SACP-PEP / TMC film group was relatively weaker and discontinuous.

[0147] Depend on Figure 18 As shown in Figure B, at 24 hours, the control group exhibited strong green fluorescence within the mixed layer, indicating high enzyme activity. The TMC group and the SACP-PEP / TMC film group showed lower fluorescence intensity. After 3 months, the fluorescence intensity of the control group increased, while the fluorescence level of the SACP-PEP / TMC film group remained low.

[0148] Depend on Figure 19As can be seen from A, the fluorescence intensity of the SACP-PEP / TMC film group was significantly lower than that of the control group at 24 hours (p<0.001).

[0149] Depend on Figure 19 B shows that the fluorescence intensity of the SACP-PEP / TMC film group was still significantly lower than that of the control group after 3 months (p<0.001).

[0150] Depend on Figure 20 As can be seen from A, the integrated fluorescence intensity of the SACP-PEP / TMC film group was significantly lower than that of the control group at 24 hours (p<0.001).

[0151] Depend on Figure 20 B shows that the integrated fluorescence intensity of the SACP-PEP / TMC film group was also significantly lower than that of the control group at 3 months (p<0.001).

[0152] The above results show that the SACP-PEP / TMC film of the present invention can significantly improve the immediate and aged microshear bond strength of resin-dentin bonded specimens, effectively reduce interfacial microleakage and inhibit matrix metalloproteinase activity, exhibiting excellent interfacial stability and durability.

[0153] Experimental Example 10 This experimental example discloses animal experiments conducted using the SACP-PEP / TMC film of the present invention.

[0154] All animal protocols used in this experiment were approved by the Institutional Animal Protection and Use Committee of West China Hospital of Stomatology, Sichuan University (Approval No.: WCHSIRB-D-2024-114). Male Sprague-Dawley rats (3-4 weeks old) were randomly divided into three groups (n=6): control group, TMC group, and SACP-PEP / TMC film group. The pure TMC film used in the TMC group was prepared according to the method in Example 1, and the SACP-PEP / TMC film used in the SACP-PEP / TMC film group was prepared according to the method in Example 1.

[0155] All procedures were performed under inhaled isoflurane anesthesia. Standardized Class I cavities were prepared on the occlusal surface of the maxillary first molars of rats under water-cooled conditions using a 0.6 mm diameter round bur. The cavity diameter was 0.6 mm, and the depth was controlled between 0.3 and 0.6 mm. After cavity preparation, the tooth surface was treated with 37% phosphate gel (Gluma Etch 35, Heraeus, Germany) for 30 seconds, rinsed, and gently dried. During the procedure, cotton rolls were used to isolate saliva, and the cavities were disinfected with 75% ethanol before material placement. Following the in vitro pre-experimental protocol, a TMC membrane or SACP-PEP / TMC membrane was first placed on the demineralized dentin surface, followed by the application of a self-etching adhesive, which was evenly spread, gently dried, and light-cured. Subsequently, composite resin was used for layered filling, each layer light-cured, and occlusion adjusted using a high-speed handpiece. The control group received the same adhesive and composite resin restoration, but without any membrane material. All procedures were performed by licensed dentists; the specific procedures are attached. Figure 21 As shown.

[0156] Three months post-surgery, rats in each group were re-anesthetized, and euthanized after blood collection via the femoral artery. Blood samples were collected for hematological and serum biochemical tests, and major organs (heart, liver, spleen, lung, and kidney) were dissected and fixed in 4% paraformaldehyde for histopathological observation. Maxillary bone specimens were scanned using a micro-computed tomography system (μCT 50, SCANCOMedical AG, Switzerland) in medium resolution mode (500 projections per 180°) with isotropic voxel size of 7 μm. Reconstructed images were analyzed using SCANCO evaluation software (v1.1.11.0, SCANCO Medical AG, Switzerland). After μCT scanning, the samples were sectioned, and the microstructure of the resin-dentin interface was observed using scanning electron microscopy.

[0157] Results showed that 3 months post-surgery, all prostheses remained intact, with no defects or detachment observed. Histopathological examination of major organs revealed no significant abnormalities, and hematological and serum biochemical indicators were within normal ranges, suggesting that the SACP-PEP / TMC membrane has good in vivo compatibility.

[0158] The in vivo evaluation results of SACP-PEP / TMC film in adhesion repair and interface characterization are attached. Figure 22 As shown. By Figure 22As can be seen, the micro-CT images showed that the restorations in each group were morphologically intact. Although varying degrees of radiolucency were visible beneath the restorations, there were no significant differences in the shape and marginal contours of the restorations among the groups, and no obvious structural damage or secondary defects were observed. Scanning electron microscopy images showed that a distinct mixed layer and resin protrusions extending into the dentinal tubules were visible at the resin-dentin interface of each group of restorations, and the overall interface showed good adhesion. Among them, the TMC group showed disordered mixed layer structure in some areas, and partial separation between the resin matrix and dentin; while the control group and the SACP-PEP / TMC film group showed a more continuous and uniform mixed layer, good resin protrusion formation, and fewer interfacial gaps, suggesting that the SACP-PEP / TMC film helps maintain the integrity of the interfacial structure.

[0159] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the embodiments of the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing a multifunctional SACP-PEP / TMC thin film, characterized in that, Includes the following steps: S1. Preparation of nanoparticles: ACP nanoparticles were prepared by reacting phosphate and polyacrylic acid in the presence of calcium salt; S2. Surface modification: ACP nanoparticles are dispersed in a solvent and reacted with the silane coupling agent 3-methacryloyloxypropyltrimethoxysilane to obtain surface-modified SACP nanoparticles; S3. Preparation of composite slurry: N,N,N-trimethyl chitosan is dissolved in a solvent, SACP nanoparticles and functional peptides are added, and the mixture is stirred evenly to obtain a film-forming slurry; the functional peptide is PEP-QP5, and its amino acid sequence is: QPYQPVQPHQPMQPQTKREEV-D. S4. Film forming step: Place the film forming slurry in a mold, remove the solvent, and form to obtain the SACP-PEP / TMC film.

2. The method for preparing the multifunctional SACP-PEP / TMC thin film according to claim 1, characterized in that, In step S1, phosphate and polyacrylic acid are dissolved in water to prepare an aqueous phosphate solution; then the pH value is adjusted to 9.5±0.1, and then reacted with calcium salt solution at room temperature. After solid-liquid separation, washing and drying, ACP nanoparticles are obtained.

3. The method for preparing the multifunctional SACP-PEP / TMC thin film according to claim 2, characterized in that, In the phosphate aqueous solution, the concentration of phosphate is 3~12mM, the concentration of polyacrylic acid is 50~200mg / L, and the molar ratio of phosphate to calcium salt is 1.2:1; The phosphate includes at least one of Na2HPO4 and K2HPO4; The calcium salt includes CaCl2.

4. The method for preparing the multifunctional SACP-PEP / TMC thin film according to claim 1, characterized in that, In step S2, ACP nanoparticles are dispersed in an aqueous ethanol solution with a concentration of 30-90% (v / v), and silane coupling agent 3-methacryloyloxypropyltrimethoxysilane is added. The mixture is then reacted under low temperature conditions to obtain surface-modified SACP nanoparticles.

5. The method for preparing the multifunctional SACP-PEP / TMC thin film according to claim 4, characterized in that, In step S2, 100–400 μL of the silane coupling agent is added for every 100 mg of ACP nanoparticles; the reaction temperature in step S2 is 2–6 °C.

6. The method for preparing the multifunctional SACP-PEP / TMC thin film according to claim 1, characterized in that, N,N,N-trimethyl chitosan was dissolved in water to prepare a stock solution of 2.5~10 mg / mL; SACP nanoparticles and functional peptides were then added to the stock solution and stirred and dispersed at room temperature to form a uniform and stable mixed suspension, which was used as a film-forming slurry.

7. The method for preparing the multifunctional SACP-PEP / TMC thin film according to claim 1, characterized in that, The mass ratio of N,N,N-trimethyl chitosan to SACP nanoparticles is 0.5~2:1; the mass ratio of N,N,N-trimethyl chitosan to functional peptides is 5~20:

1.

8. A multifunctional SACP-PEP / TMC film, characterized in that, Prepared using the method described in any one of claims 1-7.

9. The application of the multifunctional SACP-PEP / TMC film according to claim 8, characterized in that, Used to prepare at least one of the following dental restorative materials: (1) Materials that promote the remineralization of dentin collagen fibers; (2) Materials that enhance the resin-dentin bonding interface; (3) Materials that inhibit bacterial proliferation; (4) Materials that inhibit collagenase activity.

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