Use of a hydrolysate of andrias slime extract in the preparation of an anti-dentine sensitivity agent

Abstract

The application relates to the field of biomaterials, and particularly relates to application of a giant salamander mucus extract hydrolysate in preparation of an anti-dentin sensitivity agent. The anti-dentin sensitivity agent comprises a hydrolysate retaining the biological activity of SSAD, the hydrolysate being a product of reduction of the giant salamander mucus extract by an acid solution with a pH value of 1-5, and the product comprising five active proteins, namely MHC class I molecule alpha chain, homeobox protein Hox-D13, abnormal heavy chain 3 of axonemal dynein, ATP synthase protein and cytochrome b; the particle size of the giant salamander mucus extract is 1-50 mu m; and the pH value of the anti-dentin sensitivity agent is 5.5-7.5. The giant salamander mucus extract is treated by the acid solution to obtain the hydrolysate containing the multiple active proteins, the hydrolysate can effectively promote deep closure of dentin tubules, and has long-term stable biocompatibility; the pH value is controlled to ensure that the biological activity is completely retained, and the curative effect of the anti-dentin sensitivity agent is remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of biomaterials, specifically to the application of a hydrolysate of giant salamander mucus extract in the preparation of dentin hypersensitivity agents. Background Technology

[0002] Dentin hypersensitivity is a common and frequently occurring oral disease with a high incidence rate. More than 80% of people over 35 years old worldwide experience dentin hypersensitivity. The main mechanism of dentin hypersensitivity is that damage to the enamel and gingival recession lead to the exposure of dentin. When exposed dentin is stimulated by external factors such as cold, heat, acid, sweetness, and pressure, the biohydraulic pressure in the dentinal tubules changes. This pressure change is transmitted to the dental nerve connected to the dentinal tubules, causing people to experience varying degrees of tooth discomfort such as pain and soreness.

[0003] Chronic pain caused by dentin hypersensitivity reduces quality of life, affects oral hygiene practices, and plaque buildup harms periodontal health. Untreated, it can lead to pulpitis, maxillofacial cellulitis, and even systemic diseases.

[0004] The most widely accepted mechanism of dentin hypersensitivity is the hydrodynamic theory, proposed by Brännström. The core principle is that dentinal tubules penetrate the entire dentin layer, are filled with dentinal tubular fluid, and contain nerve endings and odontoblast processes. When enamel wear, wedge-shaped defects at the cervical margin, or gingival recession expose the dentin, external stimuli (cold, heat, mechanical friction, and acid / sweet osmotic pressure stimulation) act on the exposed dentin surface. These stimuli trigger rapid flow of dentinal tubular fluid. Cold stimulation, dryness, and mechanical scraping cause the fluid to flow outwards, while heat stimulation and hypertonic acid / sweet stimuli cause it to flow inwards. This rapid flow of fluid mechanically pulls and stimulates the Aδ sensory nerve fibers at the ends of the dentinal tubules and in the superficial pulp layer, triggering nerve impulses that are transmitted to the central nervous system, producing transient, sharp, and sensitive pain symptoms. Clinical treatment primarily involves blocking the dentinal tubules to inhibit the flow of fluid within them. For example, sealing dentinal tubules (applying desensitizing agents, sealing with resin, or depositing fluoride) reduces tubular fluid flow; or nerve depolarization (containing potassium ion desensitizing agents) reduces nerve excitability and blocks pain transmission.

[0005] Currently, commonly used materials for sealing dentinal tubules include fluorides, oxalates, potassium salts, arginine, glutaraldehyde, and adhesives, or laser-assisted sealing. Fluorides, represented by sodium fluoride, stannous fluoride, and sodium monofluorophosphate, allow fluoride ions to deposit within the dentinal tubules, forming fluorapatite crystals that shrink and block the tubules, reducing tubular fluid flow and providing gentle desensitization. They are widely used for daily prevention and mild dentin hypersensitivity. Potassium salts, mainly potassium nitrate and potassium chloride, increase the potassium concentration around the dental pulp nerves by releasing potassium ions, blocking pain transmission through Aδ nerve fibers and reducing nerve excitability. They have a gentle effect and are often used in anti-sensitivity toothpastes and for long-term home desensitization. Among oxalates, potassium oxalate and ferric oxalate are commonly used. They react with calcium ions within the dentin to form insoluble calcium oxalate crystals, mechanically blocking open dentinal tubules and providing strong immediate desensitization. They are often used in professional desensitization treatments in clinics. The adhesive contains dentin bonding agent and resin desensitizing coating. The resin component directly covers and seals the openings of dentinal tubules, isolating them from various external stimuli. The sealing effect is long-lasting and suitable for severe sensitivity, wedge-shaped defects, and stubborn sensitivity caused by gingival recession.

[0006] However, commonly used sealants all have drawbacks, such as shallow sealing of dentinal tubules, short-lasting sealing effect, and corrosiveness to tissues. Ideal dentin desensitizing agents, on the other hand, can achieve deep sealing of dentinal tubules, and possess long-term stable sealing effects and good biocompatibility.

[0007] Dentin mineralization uses collagen fibers as a structural template, while non-collagen proteins are the key regulatory molecules. On the one hand, they can bind tightly to collagen fibers, building stable interfacial connections between collagen and minerals; on the other hand, thanks to the high-density negatively charged surface formed by acidic amino acids such as aspartic acid, glutamic acid, and serine, they chelate calcium ions, stabilize amorphous calcium phosphate, and further promote its conversion to hydroxyapatite, thereby guiding the orderly mineralization process of dentin.

[0008] The skin glands of the giant salamander secrete a large amount of white mucus when stimulated mechanically or electrically. Its main components are proteins, amino acids, mucopolysaccharides, and antimicrobial peptides. Among these, the non-collagenous amino acid composition, such as threonine, aspartic acid, and glutamic acid, is present in high amounts. Therefore, the skin secretion of andriasdavidianus (SSAD) has the potential to promote dentin remineralization and can be used to seal dentinal tubules. However, it also has the following drawbacks: the material gels upon contact with water during liquefaction, leading to uneven distribution of the agent on the root surface; the inconvenience of mixing powder and liquid increases operational complexity; the prepared solution is unstable, with large fluctuations in the concentration of the active ingredient, making it difficult to maintain stable activity; it is also difficult to preserve for long periods, requiring frequent preparation of new solutions, increasing production time and costs; and the amount of dissolved active protein is low.

[0009] Therefore, it is necessary to develop a hydrolysate of the giant salamander mucus extract for use in the preparation of dentin hypersensitivity agents, thereby obtaining a more convenient and efficient dentin hypersensitivity agent to overcome the bottlenecks of existing technologies. Summary of the Invention

[0010] The purpose of this invention is to provide a hydrolysate of giant salamander mucus extract that overcomes the aforementioned problems and its application in the preparation of anti-dentin hypersensitivity agents. By treating the giant salamander mucus extract with a specific acidic solution, a hydrolysate containing various active proteins is obtained. These active proteins can effectively promote the deep sealing of dentinal tubules and possess long-term stable biocompatibility. Furthermore, the hydrolysate's biological activity is fully preserved through precise control of pH and reaction conditions during preparation, thereby significantly improving the efficacy of anti-dentin hypersensitivity agents.

[0011] To address the aforementioned technical problems, this invention provides the application of a hydrolysate of giant salamander mucus extract in the preparation of an anti-dentin hypersensitivity agent. This anti-dentin hypersensitivity agent comprises a hydrolysate that retains the biological activity of the giant salamander mucus extract. The hydrolysate is a product obtained by reducing the giant salamander mucus extract with an acidic solution at a pH of 1-5. The giant salamander mucus extract is a freeze-dried powder obtained by freeze-drying collected giant salamander skin mucus at -80℃ for 72 hours; the particle size of the giant salamander mucus extract is 1~50μm. The acidic solution includes an aqueous solution of citric acid and / or an aqueous solution of vitamin C and / or a solution of tris(2-carboxyethyl)phosphine hydrochloride; The preparation method of the hydrolysate includes the following specific steps: S1: Use the first solvent to prepare an acidic solution with a pH value of 1~5 as a reducing agent; S2: Add the freeze-dried powder of giant salamander mucus extract and acidic solution to the second solvent, mix well to obtain the first mixture, then add alkaline solution dropwise to the first mixture to adjust the pH value of the first mixture to 5.5~7.5, and let the first mixture stand at 0~8℃ for 12~100h to obtain the second mixture; S3: Centrifuge the second mixture and collect the supernatant, which is the hydrolysate; thus, the pH value of the anti-dentin hypersensitivity agent is 5.5~7.5.

[0012] Preferably, the hydrolysate includes five active proteins: MHC class IA α chain, homeobox protein Hox-D13, axonoderm dynamin abnormal heavy chain 3, ATP synthase protein, and cytochrome b; the particle size of the giant salamander mucus extract is 1-50 μm; and the pH value of the anti-dentin hypersensitivity agent is 5.5-7.5.

[0013] Preferably, the pH value of the anti-dentin hypersensitivity agent obtained after adjustment with an alkaline solution is 6.

[0014] Using the above technical solution, the giant salamander mucus extract was reduced in a strongly acidic solution, and then the pH was adjusted to a weakly acidic level to obtain a hydrolysate that retains the biological activity of SSAD. This hydrolysate, formulated as an anti-dentin hypersensitivity agent, fully preserves the regenerative efficacy, antibacterial, antioxidant, and anti-inflammatory effects of the giant salamander skin mucus extract (SSAD). The weakly acidic pH value is gentle and will not irritate or corrode the dentin or surrounding tissues, while ensuring the stability and biocompatibility of the active proteins. Experimental results show that this hydrolysate can significantly promote the deep closure of dentinal tubules and reduce fluid flow caused by external stimuli, thereby effectively relieving dentin hypersensitivity symptoms. It also solves the problem of the inconvenience of using SSAD powder and, through precise pH control, avoids protein inactivation caused by acid-base fluctuations, thus ensuring the long-term effectiveness of the agent. Furthermore, the preparation method is simple, yields high output, and its good biocompatibility reduces the need for processes such as dialysis to remove toxic solubilizing agents. The mild acidic environment helps maintain the natural structure and bioactivity of the giant salamander's skin mucus proteins, avoiding protein denaturation or inactivation caused by strong acid or alkali treatment. Rich in three carboxyl groups (-COOH), it can promote the formation of numerous H bonds with aqueous solutions and proteins through deprotonation and protonation, thus enhancing protein stability and solubility. In addition, by optimizing reaction conditions such as temperature, time, and solvent selection during the preparation process, the efficacy of this hydrolysate in anti-dentin hypersensitivity applications has been further improved.

[0015] Preferably, the acidic solution is an aqueous solution of citric acid with a concentration of 0.2~0.3 mol / L and a pH value of 2~3; the mass ratio of the giant salamander mucus extract to the acidic solution is (1~3):1.

[0016] Preferably, the dentin hypersensitivity agent further includes excipients; the excipients include gel excipients, thickening / thixotropic agents, and medical matrix excipients, wherein the gel excipients include one or more of sodium carboxymethyl cellulose (CMC-Na), hydroxyethyl cellulose (HEC), hydroxypropyl methyl cellulose (HPMC), carbomer, xanthan gum, and sodium alginate; The thickening / thixotropic agent includes one or more of magnesium aluminum silicate, bentonite, polyethylene glycol (PEG), and propylene glycol / glycerin; The medical matrix excipient comprises one or more of polyvinyl carboxylate, polyvinyl alcohol (PVA), and chitosan. To address the problems of uneven distribution and poor stability in traditional formulations, this invention also introduces specific excipients to optimize the physical properties of the agent, making it easier to apply evenly to the root surface, extending shelf life, and reducing usage costs.

[0017] Excipients are mainly divided into three groups, among which the most mainstream gel excipients (for oral mucosa / tooth surface) include sodium carboxymethyl cellulose (CMC-Na), hydroxyethyl cellulose (HEC), hydroxypropyl methyl cellulose (HPMC), carbomer 940 / 980 / Ultrez 10, xanthan gum, and sodium alginate. Among them, sodium carboxymethyl cellulose (CMC-Na) is the most classic and commonly used, being a transparent gel that is easy to apply and stable. Hydroxyethyl cellulose (HEC) has mild properties and high transparency, making it a common substrate for many desensitizing gels. Hydroxypropyl methyl cellulose (HPMC) has good film-forming properties, making it suitable for root surface treatments requiring a "retention" setting. Carbomer 940 / 980 / Ultrez 10 has high transparency, high viscoelasticity, and good thixotropy, making it the preferred choice for modern desensitizing gels. Xanthan gum has good ion and acid resistance, making it suitable for fluoride / potassium nitrate / strontium-containing preparations. Sodium alginate forms a mild film and was commonly used in the early days as an anti-dentin hypersensitivity agent.

[0018] The second category consists of auxiliary thickeners / thixotropic agents, which are often used in combination, including magnesium aluminum silicate, bentonite (thixotropic, anti-sagging), polyethylene glycol (PEG) (lubricating, moisturizing), and propylene glycol / glycerin (moisturizing, solubilizing). The third category consists of special substrates that are more "medical / professional root surface treatments", including polyvinyl carboxylate (similar to carbomer), polyvinyl alcohol PVA (film-forming type), and chitosan (bioadhesion and retention promotion).

[0019] In modern desensitizing gels / anti-dentin hypersensitivity agents, the most common excipients are carbomer and CMC / HEC in combination. Traditional formulations primarily use CMC, HEC, and sodium alginate.

[0020] In some specific embodiments, the most commonly used general-purpose transparent gel matrix excipients have the following specific formulations and dosages: The carbomer system (modern mainstream, with good thixotropy and transparency) includes Carbomer 940 / 980 / Ultrez 10: 0.5%~1.5%; triethanolamine (neutralizing agent): 0.5%~1.0% (adjusting pH to 6.0~7.0); glycerol: 3%~5%; purified water: balance.

[0021] The formulation of the cellulose system (traditional, stable, and inexpensive) includes sodium carboxymethyl cellulose (CMC-Na) (high viscosity): 1.5%~3.0%; hydroxyethyl cellulose (HEC): 1.0%~2.5%; hydroxypropyl methyl cellulose (HPMC): 1.0%~2.0%; glycerol / propylene glycol: 2%~5%; purified water: balance.

[0022] Preferably, both the first solvent and the second solvent are one of ultrapure water, double-distilled water, deionized water, water for injection, and pure water.

[0023] Preferably, in step S3, an excipient is added to the collected supernatant and mixed evenly to obtain an anti-dentin hypersensitivity agent containing the excipient.

[0024] Preferably, both the first solvent and the second solvent are double-distilled water; when the acidic solution is a citric acid aqueous solution with a concentration of 0.2~0.3 mol / L and a pH of 2~3, the ratio of the lyophilized powder of the giant salamander mucus extract in step S2 to the second solvent is 1 g: 10 mL. The introduction of citric acid aqueous solution (CA) alters the hydrogen bond network and carbonyl environment of SSAD, indicating a significant intermolecular interaction between the two, possibly forming a complex or changing the aggregated structure.

[0025] Preferably, the mass ratio of the freeze-dried powder of the giant salamander mucus extract to the citric acid is (1~3):1.

[0026] Preferably, the alkaline solution in step S2 is sodium hydroxide or potassium hydroxide, and the pH value of the first mixture is adjusted to 6.

[0027] Preferably, in step S3, the centrifugation is performed at a speed of 1000~8000 rpm for 10 minutes.

[0028] Preferably, in step S3, the centrifugation is performed at 4000 rpm for 10 minutes.

[0029] Beneficial technical effects: (1) Anti-dentin hypersensitivity agents were prepared using hydrolysates that retain the activity of SSAD. The hydrolysates formed by the acidic solution and SSAD (such as CA-SSAD) have multiple effects, enabling deep sealing of dentinal tubules. Furthermore, they exhibit long-term stable sealing effects and good biocompatibility, providing new possibilities for the treatment of dentin hypersensitivity. In vitro experiments showed that the hydrolysate could form a uniform and dense mineralization layer on the dentin surface, significantly reducing the permeability of dentinal tubules. Animal experiments further confirmed that after using the anti-dentin hypersensitivity agent prepared from this hydrolysate, the dentin hypersensitivity symptoms in experimental animals were significantly reduced, and no adverse reactions or tissue damage were observed.

[0030] (2) By adding appropriate excipients, such as gelling agents, thickeners and medical matrix excipients, not only are the physical properties of the agent improved, but its distribution uniformity on the root surface is also enhanced. This makes the anti-dentin hypersensitivity agent more efficient and convenient in practical applications, while also extending the shelf life of the formulation and reducing the cost and time consumption of frequent preparation.

[0031] (3) The preparation method is simple and easy to operate. By optimizing the reaction conditions, such as temperature, time and solvent selection, its effect in the application of dentin hypersensitivity is further improved. Experimental data show that the optimized preparation process can significantly improve the recovery rate and stability of active protein, ensuring the applicability of the agent in different environments.

[0032] (4) Remineralized dentin treated with the CA-SSAD anti-dentin hypersensitivity agent of the present invention can better achieve dentinal tubule closure and reduce dentinal tubule permeability. The contact angle of the dentin surface increases and the hydrophilicity decreases, which is conducive to the formation of a surface structure that resists bacterial adhesion. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0034] Figure 1 This is a flowchart of the preparation method of the CA-SSAD anti-dentin hypersensitivity agent of the present invention; Figure 2 The image shows the FTIR spectrum of the CA-SSAD anti-dentin hypersensitivity agent of the present invention. Figure 3 The image shows the viscosity test results of the CA-SSAD anti-dentin hypersensitivity agent of the present invention. Figure 4A The protein concentration detection results of CA-SSAD in this invention; Figure 4B The mass spectrometry detection results of CA-SSAD in this invention; Figure 5 Peptide coverage analysis was performed on CA-SSAD, SSAD-S, SSAD-P, and one protein related to regeneration, one related to antibacterial activity, four related to antioxidant activity, and four related to anti-inflammatory activity of the present invention. Figure 6 This is a diagram illustrating the construction of the in vitro dentin-sensitive model of the present invention; Figure 7 This is a scanning electron microscope image of the surface of a dentin section after EDTA treatment according to the present invention; Figure 8 This is a side scanning electron microscope image of a dentin section after EDTA treatment according to the present invention; Figure 9This is a flowchart of the remineralization experiment of the present invention; Figure 10A The images show the surface morphology of the Control group at 1 week, 2 weeks and 4 weeks in the remineralization experiment of this invention. Figure 10B The images show the surface morphology of the NaF group at 1 week, 2 weeks and 4 weeks in the remineralization experiment of this invention. Figure 10C The images show the surface morphology of the CA-SSAD group at 1 week, 2 weeks and 4 weeks in the remineralization experiment of this invention. Figure 11A This is a statistical graph showing the diameter of dentinal tubules in three groups of samples at 1 week, 2 weeks and 4 weeks in the remineralization experiment of this invention; Figure 11B This is a statistical graph showing the unsealed area of ​​three groups of samples at 1 week, 2 weeks and 4 weeks in the remineralization experiment of the present invention. Figure 11C The sealing rates of the three groups of samples in the remineralization experiment of this invention are shown at 1 week, 2 weeks and 4 weeks, respectively. Figure 12A The images show the morphology of the Control group at 1 week, 2 weeks and 4 weeks in the remineralization experiment of this invention, with the right side being a magnified view of the area selected in the middle box on the left side. Figure 12B The images show the morphology of the NaF group at 1, 2 and 4 weeks in the remineralization experiment of this invention, with the right side being a magnified view of the area selected in the middle box on the left. Figure 12C The images show the morphology of the CA-SSAD group at 1, 2 and 4 weeks in the remineralization experiment of this invention, with the right side being a magnified view of the area selected in the middle box on the left. Figure 13A This is a mineralization depth map of the CA-SSAD group observed by SEM after 4 weeks in the remineralization experiment of this invention. Figure 13B This is a diagram showing the results of FITC-labeled SSAD on calcified dentin slices of the CA-SSAD group in the remineralization experiment of this invention; Figure 14 for Figure 10C EDS analysis images of calcified dentin slices from the CA-SSAD group; where (a) is the energy spectrum and (b) is the pseudo-color distribution map of each target element; Figure 15 XRD comparison images of dentin etched with EDTA, native dentin, and dentin treated with CA-SSAD anti-dentin hypersensitivity agent; Figure 16 This is a flowchart of the acid resistance experiment of the present invention; Figure 17The images show the morphology of the Control group after acid treatment, where (a) is the surface morphology and (b) is the side morphology. Figure 18 The images show the morphology of the NaF group after acid treatment, where (a) is the surface morphology and (b) is the side morphology. Figure 19 The images show the morphology of the CA-SSAD group after acid treatment, where (a) is the surface morphology and (b) is the side morphology. Figure 20A The graph shows the statistical data of dentinal tubule diameters of the three groups of samples in this invention before and after acid treatment. Figure 20B This is a statistical graph showing the unsealed area of ​​the three groups of samples in this invention before and after acid treatment. Figure 20C The blocking rates of the three groups of samples in this invention before and after acid treatment are shown. Figure 21 This is a diagram of the permeability experimental apparatus of the present invention; Figure 22 The graph shows the permeability test results of the three sets of samples in this invention. Figure 23A Photographs showing the contact angles of the three sets of samples from this invention; Figure 23B for Figure 23A Statistical results of contact angle in the figure; Figure 24 The relative cell viability of hDPSCs prepared at different concentrations of CA-SSAD dentin hypersensitivity agent according to this invention after culturing for 24 h, 48 h, and 72 h, n=3, * P <0.05,** P <0.01; Figure 25 These are morphological images of the Control group and the CA-SSAD group at 0h in the cell scratch assay of the present invention; where (a) is the Control group; and (b) is the CA-SSAD group. Figure 26 These are morphological images of the Control group and the CA-SSAD group at 24 h in the cell scratch assay of this invention; where (a) is the Control group; and (b) is the CA-SSAD group. Figure 27 This is a statistical graph showing the migration area of ​​cells in the Control group and CA-SSAD group in the cell scratch assay of this invention. Figure 28A These are microscope images of the Control group and the CA-SSAD group in the alkaline phosphatase (ALP) staining experiment of this invention. Figure 28B To and Figure 28A ALP positive expression level graphs for each group in the qualitative analysis; Figure 29A These are microscope images of the Control group and the CA-SSAD group in the Alizarin Red (ARS) staining experiment of this invention; Figure 29B To and Figure 29A Corresponding quantitative analysis data chart; Figure 30 Bar chart showing the semi-quantitative (CPC elution) staining results of alizarin red S (ARS) on dentin slices treated with the two acidic hydrolysis systems of the present invention; Figure 31 Alizarin Red S (ARS) staining results of dentin slices treated with the three acid hydrolysis systems of the present invention to promote the mineralization of PDLSCs; Figure 32 This is a differentially expressed gene volcano diagram of the present invention; Figure 33A This represents the relative expression level of mRNA in the WNT4 pathway of this invention. Figure 33B This represents the relative expression level of mRNA in the BDNF pathway of this invention. Figure 33C This represents the relative expression level of mRNA in the DLX5 pathway of this invention. Figure 33D This represents the relative expression level of mRNA in the STAT3 pathway of this invention. Figure 33E This represents the relative expression level of mRNA in the LEF1 pathway of this invention. Figure 34 This is a diagram illustrating the in vivo animal modeling process of the present invention. Figure 35 The images show the SEM surface morphology of dentin slices from animals in the Control group, NaF group, and CA-SSAD group at 1 week of age; where (a) is the Control group; (b) is the NaF group; and (c) is the CA-SSAD group. Figure 36 The images show the SEM lateral morphology of dentin slices from animals at 1 week of age for the three groups of samples of this invention; where (a) is the Control group; (b) is the NaF group; and (c) is the CA-SSAD group. Figure 37 The images show the SEM surface morphology of dentin slices from animals at 2 weeks of age for the three groups of samples of the present invention; (a) is the Control group; (b) is the NaF group; and (c) is the CA-SSAD group. Figure 38The images show the SEM lateral morphology of dentin slices from animals at 2 weeks of age for the three groups of samples of the present invention; where (a) is the Control group; (b) is the NaF group; and (c) is the CA-SSAD group. Figure 39 The images show the SEM surface morphology of dentin slices from animals at 4 weeks of age for the three groups of samples of the present invention; (a) is the Control group; (b) is the NaF group; and (c) is the CA-SSAD group. Figure 40 The images show the SEM lateral morphology of dentin slices from animals at 4 weeks of age for the three groups of samples of the present invention; where (a) is the Control group; (b) is the NaF group; and (c) is the CA-SSAD group. Figure 41 This is a graph showing the in vivo biocompatibility test results of the CA-SSAD anti-dentin hypersensitivity agent of the present invention; Figure 42 The results of cell proliferation experiments (cck-8) of the three acidic hydrolysis systems of this invention are shown in the figure. Figure 43 The results show the comparison of cell viability at different concentrations and time periods in the VC-SSAD group using specific Example 1; Figure 44 The cell survival rate of the TCEP-SSAD group using Specific Example 1 at different concentrations and time periods; Figure 45 The results show the comparison of cell viability at different times in three acidic hydrolysis systems at a concentration of 0.2 mg / mL; Figure 46 The figure shows the results of the CCK-8 assay used in this invention to detect the cytotoxicity of three hydrolysis systems on BMSCs. Figure 47 The image shows the results of cell scratch experiments on dentin slices treated with the three acidic hydrolysis systems of this invention. Figure 48 The image shows the results of Alizarin Red S (ARS) staining of dentin slices after treatment with the three acidic hydrolysis systems of this invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0036] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0037] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0038] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4%, more typically + / -3%, more typically + / -2%, even more typically + / -1%, even more typically + / -0.5%.

[0039] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0040] Definition of noun: PBS: Phosphate-buffered saline; the standard 1×PBS formula is: 8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4, 0.24g KH2PO4, pH adjusted to 7.2~7.4; autoclaved / filtered sterilized; HEPES: Full name: 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid; Chinese name: 4-hydroxyethylpiperazine ethanesulfonic acid. Trypsin buffer: a buffer solution containing trypsin and a buffer system, used for cell digestion and passage of adherent cells.

[0041] hDPSCs: short for Human Dental Pulp Stem Cells.

[0042] In this invention, SSAD refers to the extract of giant salamander mucus or the extract of giant salamander skin mucus. The anti-dentin hypersensitivity agent of this invention refers to the product of SSAD hydrolyzed and reduced by three acidic solutions, wherein CA-SSAD and SSAD-CA both refer to the product of SSAD hydrolyzed by citric acid.

[0043] Example: Application of the hydrolysate of the giant salamander mucus extract in the preparation of an anti-dentin hypersensitivity agent. The anti-dentin hypersensitivity agent comprises a hydrolysate that retains the biological activity of SSAD. The hydrolysate is a product obtained by reducing the giant salamander mucus extract with an acidic solution at a pH of 1-5. The hydrolysate contains five active proteins: MHC class IA α chain, homeobox protein Hox-D13, axonoderm dynamin abnormal heavy chain 3, ATP synthase protein, and cytochrome b. The particle size of the giant salamander mucus extract is 1-50 μm. The pH of the anti-dentin hypersensitivity agent is 5.5-7.5.

[0044] In some specific embodiments, the pH value of the anti-dentin hypersensitivity agent obtained after adjustment with an alkaline solution is 6.

[0045] In some specific embodiments, the acidic solution includes an aqueous solution of citric acid and / or an aqueous solution of vitamin C and / or a solution of tris(2-carboxyethyl)phosphine hydrochloride.

[0046] In some specific embodiments, the acidic solution is an aqueous solution of citric acid with a concentration of 0.2~0.3 mol / L and a pH value of 2~3; the mass ratio of the giant salamander mucus extract to the acidic solution is (1~3):1.

[0047] In some specific embodiments, the dentin hypersensitivity agent further includes excipients; the excipients include gel excipients, thickening / thixotropic agents, and medical matrix excipients, wherein the gel excipients include one or more of sodium carboxymethyl cellulose (CMC-Na), hydroxyethyl cellulose (HEC), hydroxypropyl methyl cellulose (HPMC), carbomer, xanthan gum, and sodium alginate; The thickening / thixotropic agent includes one or more of magnesium aluminum silicate, bentonite, polyethylene glycol (PEG), and propylene glycol / glycerin; The medical matrix excipients include one or more of polyvinyl carboxylate, polyvinyl alcohol (PVA), and chitosan.

[0048] In some specific embodiments, the preparation method of the dentin hypersensitivity agent includes the following steps: S1: Use the first solvent to prepare an acidic solution with a pH value of 1~5 as a reducing agent; S2: Add the freeze-dried powder of giant salamander mucus extract and acidic solution to a second solvent, wherein the mass ratio of giant salamander mucus extract to citric acid is 2:1; after mixing, a first mixture is obtained; then, an alkaline solution is added dropwise to the first mixture to adjust its pH value to 5.5~7.5; the first mixture is then allowed to stand at 0~8℃ for 12~100h to obtain a second mixture. S3: Centrifuge the second mixture and collect the supernatant, which is the dentin hypersensitivity agent; Both the first solvent and the second solvent are one of ultrapure water, double-distilled water, deionized water, water for injection, and pure water.

[0049] In some specific embodiments, in step S3, an excipient is added to the collected supernatant and mixed evenly to obtain an anti-dentin hypersensitivity agent containing the excipient.

[0050] In some specific embodiments, both the first solvent and the second solvent are double-distilled water (DD water); when the acidic solution is an aqueous solution of citric acid with a concentration of 0.2~0.3mol / L and a pH of 2~3, the ratio of the freeze-dried powder of the giant salamander mucus extract in step S2 to the second solvent is 1g:10mL.

[0051] In some specific embodiments, the alkaline solution in step S2 is sodium hydroxide or potassium hydroxide, and the pH value of the first mixture is adjusted to 6.

[0052] In some specific embodiments, in step S3, centrifugation is performed at a speed of 1000~8000 rpm for 10 minutes.

[0053] In some specific embodiments, step S3 involves centrifugation at 4000 rpm for 10 minutes. Specific Implementation Example 1: This embodiment provides a method for preparing CA-SSAD dentin hypersensitivity agent, the specific preparation process is as follows: Figure 1 As shown.

[0055] 1) Obtaining the skin mucus of the giant salamander Healthy adult Chinese giant salamanders aged 5-7 years were selected according to standard procedures and cleaned with sterile saline. After that, the skin on their backs was repeatedly stimulated with gentle mechanical stimulation methods (such as electric stimulation and skin scraping) to induce the secretion of mucus. No harm was caused to the animals during the whole process. The secreted white mucus was collected in 50mL clean centrifuge tubes and stored in a -20℃ freezer. The healthy adult Chinese giant salamanders selected were from the China Giant Salamander Breeding Base in Chongqing, China. All of them were artificially bred individuals. All experiments were strictly carried out in accordance with the "Wildlife Protection Law of the People's Republic of China". No Chinese giant salamanders were killed throughout the process. Only the mucus secreted by their back skin was collected. The protocol of this invention was approved by the Experimental Animal Ethics Committee of the Affiliated Stomatological Hospital of Chongqing Medical University.

[0056] 2) Preparation of SSAD lyophilized powder The collected mucus was placed in a vacuum dryer at -80°C (vacuum degree 20 Pa) for 72 hours for freeze-drying. After freezing, the freeze-dried giant salamander mucus was ground and then subjected to a two-stage sieving process to obtain a relatively uniform powder. The ground product was then sieved through a 300-mesh sieve to obtain 300-mesh SSAD powder. This powder was then properly stored in a sealed container at -20°C for later use.

[0057] 3) The specific preparation steps for CA-SSAD dentin hypersensitivity agent (CA-SSAD group) are as follows: To prepare a citric acid (CA) solution, 1.25g CA is required for 25mL. Add 2500mg SSAD powder and 1.25g CA to 25mL of DD water. Adjust the pH of the supernatant to 6 with NaOH. Mix thoroughly and let stand at 4℃ for 48h. Centrifuge at 4000rpm for 10min and aspirate the supernatant to obtain liquid CA-SSAD dentin desensitizing agent (i.e., SSAD desensitizing agent, where the concentration of CA-SSAD is 0.15mg / mL).

[0058] I. The CA-SSAD prepared in Specific Example 1 was characterized by material characterization.

[0059] All data in this invention are presented as mean ± standard deviation. Statistical analysis and graphing were performed using GraphPad Prism 10 and Origin 2024 software. Independent samples t-tests were used to compare two groups of samples, while differences among multiple groups were assessed using one-way ANOVA. P A value <0.05 indicates that the difference is statistically significant.

[0060] (1) FTIR detection of protein structure: Sample preparation: The first group was the traditional SSAD lyophilized powder (SSAD group), and the second group was the CA-SSAD anti-dentin hypersensitivity agent prepared in Specific Example 1, which was lyophilized (CA-SSAD group). 10 mg of powder from each of the two groups of samples was taken.

[0061] Adjust test parameters: Set the FTIR spectrometer (Fourier Transform Infrared Spectrometer, Nicolet 6700, Nicolet USA) to transmission recording mode to accurately acquire the spectral information of the target substance. During data recording, set the spectral data to be recorded in absorbance units, serving as the basis for quantitative analysis. The wavenumber range was set to 500–4000 cm⁻¹. -1This range covers most infrared absorption peaks generated by chemical bond vibrations, providing ample information for a comprehensive analysis of the sample's molecular structure. Simultaneously, the instrument's resolution was set to 4 cm⁻¹. -1 This resolution can effectively improve experimental efficiency while ensuring data accuracy and clearly present spectral details. The number of scans is set to 16.

[0062] Collect and analyze spectra: Under room temperature conditions, collect all FTIR spectra, i.e., first collect the background, then collect the FTIR spectra of the sample, and then analyze them using Origin 2010 software.

[0063] The results are as follows Figure 2 As shown, from Figure 2 It can be seen from this that at 600cm -1 The enhanced disulfide bond characteristic peaks of the nearby CA-SSAD anti-dentin hypersensitivity agent indicate increased intermolecular or intramolecular disulfide bond formation, thus increasing protein stability. (1650cm) -1 The nearby absorption peak (amide I band) is of the C=O stretching vibration type, 1550 cm⁻¹. -1 The nearby absorption peaks (amide II band) are mainly related to the type of NH bending vibration and are the main peaks of SSAD powder and CA-SSAD anti-dentin hypersensitivity agent. CA-SSAD anti-dentin hypersensitivity agent shows a peak at 3400 cm⁻¹. - The broadening of the peak near ¹ indicates that the hydroxyl (OH) groups in the system participate in the formation of a more extensive or complex hydrogen bond network. The introduction of citric acid provides an additional carboxyl group (-COOH), whose hydroxyl group can form hydrogen bonds with groups such as OH, NH, or C=O in SSAD, leading to a broadening of the OH stretching vibration band. Specifically, the introduction of CA causes a blue shift in C=O and a red shift in OH, indicating that the introduction of CA alters the hydrogen bond network and carbonyl environment of SSAD, suggesting a significant intermolecular interaction between the two, possibly forming a complex or changing the aggregated structure. This spectrum shows that the sample simultaneously possesses hydroxyl and carbonyl groups and may contain aromatic rings and ether / ester structures.

[0064] The retention of the overall peak shape indicates that the SSAD protein-polysaccharide backbone was not destroyed during the preparation of the desensitizing agent. Intermolecular interactions (mainly hydrogen bonds) occurred between citric acid and the SSAD matrix. These interactions altered the chemical environment around the relevant functional groups (OH), successfully achieving the binding of citric acid to SSAD.

[0065] (2) Viscosity was measured using a rheometer: Sample preparation: Prepare an equal amount of CA-SSAD dentin hypersensitivity agent to be tested, ensuring that the sample is uniform and free of air bubbles.

[0066] Instrument Inspection: The viscosity of CA-SSAD dentin hypersensitivity agent was determined using a rheometer (Anton Paar, Austria). Ensure the instrument is in good working order, and check the power supply, cables, and measuring components. Clean the measuring components to ensure no residue remains. The rotor diameter used for measurement is 25 mm, and the spacing during measurement is set to 1 mm. Place the CA-SSAD dentin hypersensitivity agent between plates, and set the shear rate to 0.1 s. -1 ~100s -1 The viscosity was tested as a function of shear rate.

[0067] Test: Select the test template "Viscosity Curve", set the default parameters, and perform the test. The test results are as follows: Figure 3 As shown. From Figure 3 As can be seen from this, as the shear rate increases from 0.1 s⁻¹... -1 Increase to 100s -1 The viscosity of CA-SSAD dentin hypersensitivity agent exhibits a non-linear decreasing trend with increasing shear rate: in the low shear rate range, the viscosity remains at a high level, while as the shear rate continues to increase, the viscosity decreases rapidly. These results indicate that CA-SSAD dentin hypersensitivity agent is a typical non-Newtonian fluid with significant shear-thinning characteristics. This characteristic allows it to maintain high viscosity under low shear conditions, thus preserving its stability, while its viscosity decreases rapidly under high shear conditions. This feature is highly beneficial for material flow and application in clinical procedures.

[0068] (3) Amino acid analysis in CA-SSAD dentin hypersensitivity agent To investigate the effects of citric acid treatment on the protein components of SSAD, particularly the differences in the content of acidic amino acids that make up non-collagenous proteins, the amino acid composition of pure SSAD powder and CA-SSAD dentin hypersensitivity agent was compared and analyzed. The specific process was as follows: 1) Accurately weigh 100 mg of the lyophilized CA-SSAD dentin hypersensitivity agent sample and place it in a hydrolysis tube, avoiding sample adhesion to the tube walls as much as possible. Slowly add 4 mL of 1:1 analytical grade hydrochloric acid (6 mol / L concentration) to the tube, then purge with nitrogen gas for 15 min using a nitrogen blower. After completion, seal the hydrolysis tube.

[0069] 2) Place the sealed sample tube into an oven and hydrolyze it at 110℃ for 24 hours. After hydrolysis, take it out and, once cooled to room temperature, open the sample tube and bring the volume up to 100mL.

[0070] 3) Place 2 mL of sample on a nitrogen blower and deacidify at 60 °C until completely dry. Add an appropriate amount of 0.02 mol / L hydrochloric acid to the dried sample, mix well using a vortex mixer, filter through a 0.22 µm filter column, collect the filtrate, and analyze it using an amino acid analyzer (Biochrom30+, UK).

[0071] The results are shown in Table 1. Compared with pure SSAD, the CA-SSAD anti-dentin hypersensitivity agent formed by SSAD treatment with citric acid has higher contents of glutamic acid, aspartic acid, threonine and lysine.

[0072] Table 1. Amino acid analysis results in CA-SSAD dentin hypersensitivity agent and pure SSAD. Note: "-" in the table indicates that the amino acid was not detected or was not counted in the data set.

[0073] (4) Protein spectrum analysis: Sample preparation was the same as for FTIR protein structure detection, with 10 mg of powder from each of the three sample groups.

[0074] Peptide digestion: Add 40 μL of trypsin buffer (where trypsin is Trypsin, EC 3.4.21.4) to the sample and incubate at 37°C for 16-18 h.

[0075] 1) Chromatographic separation: Solution A in the liquid chromatography system is an aqueous solution containing 0.1% formic acid, and solution B is an aqueous solution of acetonitrile composed of 0.1% formic acid and acetonitrile (84%). A 0.15mm × 150mm liquid chromatography column was used in this experiment.

[0076] Before the experiment, the system was equilibrated with 95% solution A to ensure stability. After equilibration, the three sets of samples were injected into the peptide trap column (Zorbax 300SB-C18 peptide traps) via an autosampler, and then the samples were separated by liquid chromatography. The liquid chromatography gradient was dynamically adjusted according to the set parameters: during the 0-50 min period, the proportion of solution B gradually increased from 4% to 50% in a linear gradient. This gradient change was designed to achieve preliminary separation based on the differences in the interactions between the components in the sample and the stationary and mobile phases, allowing components with different properties to initially exhibit different migration rates in the column. Then, during the 50-54 min period, the linear gradient of solution B rapidly increased from 50% to 100%. This rapid gradient change facilitated further separation of components that had initially separated but still had some overlap, increasing the difference in migration distance between the components and improving the separation effect. Finally, solution B is maintained at 100% concentration for 54–60 minutes to ensure that all separated components can be completely eluted from the column, completing the entire chromatographic separation process and obtaining clear and accurate chromatograms, providing high-quality data support for subsequent analysis and identification of sample components.

[0077] 2) Mass Spectrometry Identification: The enzymatic digestion products were first separated by high-performance liquid chromatography (HPLC) using a capillary tube, and then analyzed by a Q Exactive mass spectrometer. The entire analysis process was set to last 60 minutes. Positive ion detection was used in this experiment to ensure effective detection of the target substances. During data acquisition, based on the mass-to-charge ratio of the peptide and peptide fragments, 10 fragment spectra (MS2 scans) were acquired immediately after each full scan. The full scan obtains the mass-to-charge ratio information of various ions in the sample, comprehensively reflecting the overall composition of the sample. The subsequent acquisition of 10 fragment spectra involves further fragmentation of selected ions to obtain the mass-to-charge ratio information of their fragment ions. The characteristics of these fragment ions help to further elucidate the structure of the peptide.

[0078] 3) Data Analysis: After the mass spectrometry tests were completed, the raw files were searched in the database using the professional software MaxQuant 1.5.5.1. Through in-depth analysis of the mass spectrometry data using this software, the results of protein identification and quantification were finally generated. The relevant parameters and detailed explanations involved in the database search process are shown in Table 2. In this study, a Caudata database was selected as the comparison reference for the data of the giant salamander samples. This selection is based on the taxonomic characteristic that the giant salamander belongs to the order Caudata.

[0079] Table 2 shows the relevant parameters involved in the database search. 3) Results Analysis: The protein concentration of CA-SSAD prepared in Specific Example 1 was detected and analyzed by mass spectrometry. The results are as follows: Figure 4B As shown, the concentration of dissolved proteins increased more than twice in citric acid-treated solutions compared to untreated solutions. SSAD-P (SSAD lyophilized powder) detected 57 proteins, SSAD-S (SSAD lyophilized powder directly dissolved in water) detected 45 proteins, and the citric acid-treated SSAD-CA solution detected 49 proteins, representing an increase in detectable proteins compared to the directly dissolved SSAD-S solution. Figure 4A As shown, five proteins were uniquely detected in the four SSAD-CA assays. These five proteins are: MHC class IA alpha chain, homecobox protein Hox-D13, dynein anonemal heavy chain 3, ATP synthase protein, and cytochrome b. The results are as follows: Figure 5 As shown, based on the summary analysis of protein functions in organisms, peptide coverage analysis was performed on 5 proteins related to regeneration, 1 related to antibacterial activity, 4 related to antioxidation, and 4 related to anti-inflammation detected in different groups. The proportion of the identified amino acids to the total number of amino acids in the protein was evaluated. Table 3 shows that the proportions of fibroblast growth factor receptor 2, platelet-reactive protein-1, and n-cadherin in the regeneration-related protein peptides dissolved by SSAD-CA increased, the amount of dissolved lysozyme G (EC 3.2.1.17) increased, the amount of dissolved heat shock protein 70 in the antioxidant protein increased, and the amount of interferon-induced GTP-binding protein Mx3 in the anti-inflammatory protein increased.

[0080] Table 3. Coverage of relevant protein peptides (%) Note: Underlined lines indicate that the relevant protein has the highest peptide coverage in SSAD-CA.

[0081] Specific Implementation Example 2: Construction and Characterization of an In Vitro Dentin Sensitive Model 1. The construction process is as follows Figure 6 As shown, the specific process is as follows: 1) All experimental procedures were performed in accordance with the experimental protocol approved by the Medical Ethics Committee of Chongqing Medical University (ethics number: 2025 Lun Shen (065)). Complete and caries-free extracted tooth samples were collected. First, the calculus, plaque, pigment and soft tissue on the sample surface were removed. Then, the samples were thoroughly cleaned with ultrapure water. After cleaning, the samples were stored in 0.1% thymol solution for subsequent experimental use.

[0082] 2) Using a high-speed diamond bur, a dentin sheet with a thickness of about 1.0 mm is prepared at a position about 1.0 mm above the cementoenamel junction, perpendicular to the long axis of the tooth, under cold water cooling conditions. The prepared dentin sheet is polished with silicon carbide sandpaper (600 grit, 800 grit, 1000 grit, 1500 grit and 2000 grit) for 60 seconds under running water rinsing. Finally, the dentin sheet is ultrasonically cleaned with ultrapure water for 10 minutes.

[0083] 3) Prepare 0.5 mol / L EDTA demineralization solution: Accurately weigh 18.6 g of EDTA powder, dissolve it in 80 mL of pure water, then add an appropriate amount of NaOH, and stir continuously with a glass rod until the reagent is completely dissolved; make up the volume of the above solution to 100 mL, adjust the pH of the system to 8, and set aside for later use.

[0084] 4) Dentin slices were placed in 0.5 mol / L EDTA solution for demineralization, and the treatment time was controlled to be 30 min to construct a dentin model with exposed tubules. The samples were then ultrasonically cleaned with ultrapure water for 10 min and dried.

[0085] 2. Remineralization Experiment and Characterization The materials used in the characterization are shown in Table 4.

[0086] Table 4. Materials used in characterization For comparison, three sets of samples were prepared: the first set was the blank experiment (i.e., treated with physiological saline, referred to as the Control group), the second set was the commonly used sodium fluoride (NaF group), and the third set was the CA-SSAD anti-dentin hypersensitivity agent prepared in Specific Example 1 (referred to as the CA-SSAD group).

[0087] The specific procedure for remineralizing demineralized dentin is as follows: 1) Preparation of CA-SSAD anti-dentin hypersensitivity agent coating on demineralized dentin slices: The prepared CA-SSAD anti-dentin hypersensitivity agent was applied to the surface of the dried demineralized dentin slices with a small brush and dried with an ear syringe to obtain a CA-SSAD anti-dentin hypersensitivity agent coating on the dentin slices. The Control group and NaF group were treated using the same method.

[0088] 2) The solution system used for mineralization induction contains the following components: 20 mM HEPES, 1.5 mM anhydrous calcium chloride, 130 mM potassium chloride, 0.9 mM potassium dihydrogen phosphate, and the pH is adjusted to 7 with NaOH.

[0089] 3) Place dentin slices from the CA-SSAD group, NaF group, and Control group into six-well plates containing 5 mL of mineralization solution, with 20 samples in each group.

[0090] 4) Apply CA-SSAD anti-dentin hypersensitivity agent daily for one week. After one week, brush the dentin surface with a toothbrush for 2 minutes daily (applying a constant force of about 150g perpendicular to the plane) to conduct an abrasion resistance test. The Control group and NaF group were treated in the same way.

[0091] 5) Place the six-well plate in a 37°C shaker and oscillate it at a speed of 100 revolutions per minute, while changing the prepared mineralization solution once a day.

[0092] 6) Dentin slices were taken out after 1 week, 2 weeks and 4 weeks. The samples of each group were characterized and analyzed by scanning electron microscopy (SEM). The samples after 4 weeks of mineralization were subjected to X-ray diffraction (XRD), X-ray energy dispersive spectroscopy (EDS), acid resistance test and permeability measurement experiment using a permeability device.

[0093] In vitro remineralization of dentin, such as Figure 9 As shown, CA-SSAD anti-dentin hypersensitivity agent and NaF were evenly applied to the demineralized dentin area. The dentin tablets of the CA-SSAD group, NaF group and Control group were immersed in mineralization solution. The entire process was carried out in a shaker at 37°C to simulate the temperature environment and saliva flow of the human oral cavity. Fresh mineralization solution was required to be replaced daily to ensure the stable progress of the mineralization process.

[0094] The surface scanning electron microscope image of the dentin section after EDTA treatment is shown below. Figure 7 As shown, the scanning electron microscope image of the side is as follows. Figure 8 As shown. From Figure 7 and Figure 8 As can be seen, after demineralization treatment with 0.5 mol / L EDTA for 30 min, a large number of regularly shaped and evenly arranged circular open tubules were distributed on the dentin surface. Figure 7 The results showed that demineralization treatment effectively exposed dentinal tubules, thus successfully constructing an exposed dentin model. Observation of the cross-section revealed (…). Figure 8 The dentinal tubules exhibit a nearly parallel tubular shape with an open lumen.

[0095] like Figure 10A , Figure 10B and Figure 10C As shown, where Figure 10A The surface morphology of the Control group at 1 week, 2 weeks and 4 weeks are shown. Figure 10B The surface morphology of the NaF group at 1 week, 2 weeks and 4 weeks are shown. Figure 10CThe surface morphology of the CA-SSAD group at 1 week, 2 weeks and 4 weeks are shown. Figure 12A The images show the side profile of the Control group at 1, 2, and 4 weeks, with the right side being a magnified view of the area selected in the middle box on the left. Figure 12B The images show the lateral morphology of the NaF group at 1, 2 and 4 weeks, with the right side being a magnified view of the area selected in the middle box on the left. Figure 12C The images show the lateral morphology of the CA-SSAD group at 1, 2, and 4 weeks, with the right side being a magnified view of the area selected in the middle frame on the left. Figures 10A-10C and Figures 12A-12C The comparison shows that after one week of mineralization, surface SEM in the Control group revealed clear openings of dentinal tubules, which were circular or elliptical in shape, with larger diameters and exposed dentin between the tubules. Cross-sectional SEM showed clear dentinal tubule structures, open lumens, and no mineral deposits. In the NaF group, the openings of dentinal tubules were slightly smaller than those in the control group, and mineral deposits were visible in some areas. A small amount of mineral deposits were also visible within the dentinal tubule lumens, but most of the lumens remained open. In the CA-SSAD group, the openings of dentinal tubules were smaller, and the surface was covered with mineral deposits. Some dentinal tubules appeared to be closed. Cross-sectional SEM showed obvious mineral deposits filling the lumens, and some areas of the lumens were partially closed. Two weeks after mineralization, the openings of the dentinal tubules in the Control group were still clearly visible, with a morphology similar to that in the first week. The demineralization state continued, and the cross-section showed that the dentinal tubule structure remained open, with obvious demineralization characteristics and no signs of spontaneous remineralization. In the NaF group, the openings of the dentinal tubules further narrowed, and the mineral deposition increased, but there were still identifiable openings. More minerals were deposited inside the tubules, but the deposition layer was relatively loose and did not form a tight seal. In the CA-SSAD group, the openings of the dentinal tubules were significantly reduced, the surface mineral deposition layer was denser, most tubules were closed, the mineral deposition inside the lumen was denser, crystal growth filled the lumen, forming a better sealing effect, and the sealing layer remained intact after the wear resistance test. Four weeks after mineralization, the dentinal tubule openings in the Control group remained clearly visible and open, with a rough dentin surface and persistent demineralization characteristics. In the NaF group, the dentinal tubule openings decreased, and mineral deposition increased but was uneven, with some areas still showing open lumens. In the CA-SSAD group, the dentinal tubules were almost completely closed, with a continuous mineral deposition layer forming on the surface. The structure was dense, and the lumen was filled with dense minerals. The closed structure remained stable after the wear resistance test, demonstrating excellent mechanical stability.

[0096] Figure 11A Statistical graphs of dentinal tubule diameter in three groups of samples at 1 week, 2 weeks, and 4 weeks (n=5, **) P <0.01, *** P <0.001, **** P <0.0001); Figure 11B This is a statistical graph showing the unsealed area of ​​three groups of samples at 1 week, 2 weeks, and 4 weeks (n=5, **). P <0.01, *** P <0.001, **** P <0.0001); Figure 11C The sealing rates of the three groups of samples at 1 week, 2 weeks, and 4 weeks, respectively; from Figure 11A Statistical results show that the diameter of dentinal tubules in all three groups decreased with prolonged mineralization time. At week 1, the diameters in the Control group, NaF group, and CA-SSAD group were 4.04±0.23 μm, 3.11±0.38 μm, and 1.02±0.55 μm, respectively; by week 2, the diameters decreased to 3.28±0.17 μm, 2.64±0.24 μm, and 0.49±0.45 μm, respectively; and by week 4, the diameters decreased to 2.94±0.12 μm, 1.37±0.84 μm, and 0.33±0.43 μm, respectively. Figure 11B Statistical results showed that the unsealed area of ​​dentinal tubules in all groups decreased over time, but the rate of decrease varied among groups. In the Control group, the unsealed area of ​​dentinal tubules decreased from 10.90 ± 1.39 μm at week 1. 2 It gradually decreased to 7.39±0.52μm in the second week. 2 And 6.44±0.63μm in week 4. 2 The area of ​​unsealed dentinal tubules in the NaF group decreased significantly, from 7.87±2.14 μm in week 1. 2 It decreased to 3.77±1.08 μm in week 2. 2 The value further decreased to 1.69±0.99μm in week 4. 2 The CA-SSAD group showed the best sealing effect, with its unsealed area decreasing from 1.01 ± 0.59 μm in week 1. 2 The value decreased to 0.51 ± 0.45 μm in week 2. 2 And it reached its lowest value of 0.16±0.25μm in the 4th week. 2 .from Figure 11C It can be seen that the closure rate of the CA-SSAD group reached over 90% in week 4, which was significantly higher than that of the NaF group.

[0097] like Figure 13A The image shows the mineralization depth map of the CA-SSAD group observed by SEM after 4 weeks. Figure 13AThe deepest mineralization depth is 230 μm. To further analyze the penetration of CA-SSAD dentin hypersensitivity agent into the dentinal tubules, fluorescein isothiocyanate (FITC)-labeled SSAD was used to trace the distribution of CA-SSAD dentin hypersensitivity agent within the dentinal tubules. Fluorescence images obtained by laser confocal scanning microscopy (CLSM) under 488 nm laser excitation show the distribution of FITC-SSAD in EDTA-etched dentin. Figure 13B The dentin surface exhibited a continuous and uniform green fluorescent band, indicating that FITC-SSAD successfully bound to and penetrated into the dentinal tubules. The fluorescence signal gradually weakened from the dentin surface towards the interior of the tubules, with a maximum penetration depth of 60 μm after 1 hour. These results demonstrate that CA-SSAD, an anti-dentin hypersensitivity agent, possesses excellent flowability and can penetrate into the interior of the dentinal tubules, providing a delivery basis for intratubular mineralization.

[0098] Four weeks after treatment with CA-SSAD dentin hypersensitivity agent, the results of energy dispersive X-ray spectroscopy (EDS) analysis of the dentin surface are as follows: Figure 14 As shown, Figure 14 (a) is an energy spectrum, which shows that it mainly contains five elements: carbon (C) (30.57 At%), oxygen (O) (39.89 At%), calcium (Ca) (14.49 At%), phosphorus (P) (8.61 At%), and nitrogen (N) (6.44 At%). Figure 14 The distribution diagram in (b) shows that the above elements are uniformly distributed on the dentin surface without local heterogeneity. A large amount of Ca and P elements co-located on the surface of the dentin slices from the CA-SSAD group, with significantly enhanced signal intensity. Furthermore, atomic percentage calculations showed that the calcium-to-phosphorus ratio (Ca / P) of the remineralized dentin surface treated with SSAD anti-dentin hypersensitivity agent was 1.68, close to the Ca / P ratio of natural hydroxyapatite (1.67). This suggests that CA-SSAD anti-dentin hypersensitivity agent treatment did not significantly alter the calcium-to-phosphorus mineralization structure of the dentin surface, and that CA-SSAD anti-dentin hypersensitivity agent induced the formation of a hydroxyapatite deposition layer.

[0099] XRD was used to characterize dentin after EDTA demineralization, natural dentin, and dentin remineralized for 4 weeks after CA-SSAD treatment. Figure 15 As shown, hydroxyapatite crystals were formed on the remineralized dentin surface after CA-SSAD treatment, and the crystallinity was higher.

[0100] 3. Acid resistance test and characterization For comparison, three sets of samples were prepared: the first set was the blank experiment (i.e., treated with physiological saline, referred to as the Control group), the second set was the commonly used sodium fluoride (NaF group), and the third set was the SSAD anti-dentin hypersensitivity agent prepared in Specific Example 1 (referred to as the CA-SSAD group).

[0101] like Figure 16 As shown, three identical dentin slices were processed into three groups of samples. The processed dentin slices were then immersed in a 6% citric acid solution (pH 1) for 1 minute, followed by ultrasonic rinsing for 5 minutes. The acid resistance was then observed using SEM. Figures 17-19 As shown.

[0102] Figure 17 (a) in the diagram is the surface morphology of the Control group after acid treatment; Figure 17 (b) in the figure is a side view of the Control group after acid treatment; Figure 18 (a) in the figure shows the surface morphology of the NaF group after acid treatment; Figure 18 (b) in the figure is a side view of the NaF group after acid treatment; Figure 19 (a) in the figure is the surface morphology of the CA-SSAD group after acid treatment; Figure 19 Image (b) shows the side morphology of the CA-SSAD group after acid treatment. Figures 17-19 It can be seen that in the Control group, dentin demineralization was aggravated, the dentinal tubules were enlarged and the edges were rounded, and no deposits were observed in the tubules on the cross-section. In the NaF group, most of the deposits on the dentin surface dissolved after acid etching, the tubules reopened, and a small number of particles remained. In the CA-SSAD group, the dentin surface remained almost intact after acid etching, with only slight dissolution. The cross-section showed that the sealant was retained and there was no obvious detachment. The results indicate that the CA-SSAD anti-dentin hypersensitivity agent can effectively seal the dentinal tubules and significantly improve the acid resistance of dentin. Its protective effect is better than that of the NaF group and the Control group.

[0103] Figure 20A Statistical graphs of dentinal tubule diameters in three groups of samples before and after acid treatment; Figure 20B The graph shows the statistical data of the unsealed area of ​​the three groups of samples before and after acid treatment. Figure 20C The blocking rates of the three groups of samples before and after acid treatment are shown; from Figure 20A , Figure 20B and Figure 20C Statistical results showed that the CA-SSAD group had the smallest dentinal tubule diameter after remineralization, and maintained the lowest degree of tubule enlargement (average 0.38 μm) and the smallest unsealed area (average 0.39 μm) even after acid etching. 2In contrast, both the NaF group and the Control group showed an increase in dentinal tubule diameter and unsealed area after acid etching. In conclusion, the CA-SSAD anti-dentin hypersensitivity agent remineralized layer exhibits excellent acid resistance. 4. Permeability Test Three groups of remineralized dentin slices (three parallel samples per group) were subjected to a permeability test device (e.g., Figure 21 (As shown) The device was used to test the sealing effect of dentinal tubules in remineralized dentin. The device mainly consists of the following parts: a storage bottle for the permeate, tubing connecting the various parts, a clamping device for fixing the dentin sheet, a syringe, and a timer. When mounting the dentin sheet sample, it is fixed between two "O"-ring rubber seals, each with an inner diameter of 0.6 cm, thus limiting the effective contact area of ​​the permeate through the dentin sheet during the experiment. In this experiment, 0.9% physiological saline was used as the test medium, and the vertical distance between the liquid level and the measuring platform was strictly controlled at 100 cm. This height remained constant throughout the test to ensure that the hydraulic pressure acting on the dentin sheet surface remained stable throughout the experiment. The 0.9% physiological saline was introduced through a connecting tube, allowing it to flow slowly through the dentin sheet to complete the permeation process, and then guided through a three-way tube into a dedicated 15 cm glass tube. During the procedure, first, inject 0.9% saline solution into the three-way connector using a syringe to thoroughly remove any air bubbles that may be present inside the glass tube, ensuring no gas residue remains. After all air bubbles have been expelled, introduce a single air bubble into the system. Then, begin the formal measurement and recording: precisely determine the total time required for the small air bubble to pass through five pre-marked intervals on the glass tube, recorded in minutes (min). This time serves as the core data for permeability calculation. The formula for calculating dentin permeability (Lp) is: Lp = V / PS, where V, P, and S represent the volumetric flow rate (L / min), water pressure (cmH2O), and exposed dentin surface area (cm²). 2 The water conductivity of the EDTA-demineralized dentin sample was taken as the maximum permeability (100%), and the permeability of the remineralized dentin sample was expressed as a percentage of this water conductivity.

[0104] Test results as follows Figure 22As shown, after 4 weeks of remineralization treatment, the permeability of all three groups of samples decreased to varying degrees. The permeability of the CA-SSAD group was significantly lower than that of the NaF and Control groups. After the acid resistance test, the permeability of the Control and NaF groups rebounded to approximately 90% and 75%, respectively, while the CA-SSAD group only rebounded slightly to approximately 30%. The remineralized dentin in the CA-SSAD group had a lower permeability than the Control and NaF groups, indicating that CA-SSAD treatment can better seal dentinal tubules and reduce their permeability. Simultaneously, the experimental results show that the CA-SSAD anti-dentin hypersensitivity agent has a significantly better sealing effect on dentinal tubules than the NaF and Control groups. The mineralized sealing layer it induces is more stable in an acidic environment, effectively maintaining dentin permeability at a low level and exerting a stable desensitizing effect.

[0105] 5. Contact Angle Test The three sets of dentin sheets were subjected to contact angle testing. The water contact angle reflects the surface wettability and hydrophilicity of the material. A water contact angle <90° generally indicates good hydrophilicity of the material surface, which is conducive to cell attachment and growth, thereby promoting periodontal regeneration. Conversely, a water contact angle >90° may indicate a more hydrophobic material surface, potentially leading to poor cell attachment and affecting the restorative effect. Results are as follows... Figure 23A and Figure 23B As shown, from Figure 23A It can be clearly seen that the water contact angle of the CA-SSAD group is larger than that of the Control group and the NaF group; from Figure 23B (n=3, * P <0.05, **** P The results (<0.0001) showed that the static water contact angle of the Control group was 44.95±1.82°, and that of the NaF group was 52.51±3.65°. After treatment with CA-SSAD dentin hypersensitivity agent, the contact angle of the dentin surface increased, but it remained hydrophilic (<90°), with a static water contact angle of 67.17±1.91°. The test results indicate that after CA-SSAD treatment, the contact angle of the dentin surface increased, and the hydrophilicity decreased, which is beneficial for forming a surface structure that resists bacterial adhesion. CA-SSAD dentin hypersensitivity agent can significantly change the physicochemical properties of the dentin surface, reducing its hydrophilicity and increasing its hydrophobicity, thereby helping to reduce the adhesion and penetration of external liquids and enhancing the desensitization and acid-resistant stabilization effects.

[0106] 6. Cytotoxicity test To investigate the biocompatibility of CA-SSAD anti-dentin hypersensitivity agent (specific example 1), a cytotoxicity test (CCK-8) was performed. After pasteurization, CA-SSAD anti-dentin hypersensitivity agent was added to α-MEM complete medium. Third-generation human dental pulp stem cells (hDPSCs) were seeded into 96-well plates at a density of 5 × 10³ cells / well, with three replicates per group to ensure experimental reproducibility. The 96-well plates were then placed in a cell culture incubator. After 24 hours, the original medium in the plates was discarded, and the cells were washed with PBS. The corresponding concentration of CA-SSAD anti-dentin hypersensitivity agent was added to the experimental groups, while the control group received complete medium. In the blank control group, an equal volume of PBS solution was added to each well. Three replicates were set up for each group to control experimental error. The cells were then cultured under suitable conditions for 24–72 hours.

[0107] CCK-8 assay: After cell intervention, discard the liquid in each well, then wash the cells with PBS solution to thoroughly remove any residual impurities. Add fresh α-MEM complete medium containing 10% CCK-8 reagent to each well, and incubate in the dark for 1 hour. OD value determination: Place the 96-well plate in a microplate reader and measure the absorbance (OD value) of each well at 450 nm. Calculate the relative cell viability based on the OD value: (Experimental well absorbance - Blank well absorbance) / (Control well absorbance - Blank well absorbance) × 100%.

[0108] The assay involved culturing different concentrations of SSAD (suppressant for dentin hypersensitivity) with dental pulp stem cells for 24 h, 48 h, and 72 h, respectively. Figure 24 As shown, the results indicated that the relative cell viability in each group was greater than 90%, demonstrating that CA-SSAD anti-dentin hypersensitivity agent has good biocompatibility with cells. The 100 μg / mL concentration of CA-SSAD anti-dentin hypersensitivity agent showed a statistically significant difference in promoting cell proliferation. Therefore, CA-SSAD anti-dentin hypersensitivity agent did not inhibit the growth of hDPSCs and had no cytotoxic effect.

[0109] 7. Cell scratch test To investigate the cell migration-promoting ability of CA-SSAD, an anti-dentin hypersensitivity agent, a cell scratch assay was performed. Specifically: 1) Experimental grouping: This experiment was divided into a control group and an experimental group. The control group was in α-MEM complete culture medium, and the experimental group was in CA-SSAD anti-dentin hypersensitivity agent at 100 μg / mL. This concentration was determined by the CCK-8 experiment and was the optimal proliferation concentration. The ability of this concentration to promote hDPSC migration was evaluated.

[0110] 2) Constructing a cell scratch model: First, draw positioning lines on the bottom of a 6-well plate, then add hDPSCs at 2×10⁻⁶ intervals. 5 Cells were evenly seeded at a density of 10 cells / well in a 6-well plate and allowed to adhere and grow for 24 hours.

[0111] 3) Cells from both groups (Control group and CA-SSAD group) were seeded into culture plates and cultured until complete confluence. A uniform straight line was drawn in the center of each culture plate using a 200 μL sterile pipette tip. After washing with PBS to remove free cells, the CA-SSAD group was added to α-MEM complete medium containing CA-SSAD dentin hypersensitivity agent, while the Control group was added to α-MEM complete medium. Each group was prepared in triplicate. Observations and photographs were taken under a microscope at 0 h and 24 h, and the scratch width was measured. The scratch healing rate was calculated to assess cell migration levels. The relative migration area of ​​hDPSCs in each group was analyzed using ImageJ, calculated using the following formula: Relative migration area percentage (%) = [(Area at 0h) - (Area after 24h)] / (Area at 0h) × 100%.

[0112] The results are as follows Figure 25 , Figure 26 As shown, where Figure 25 (a) in the figure is the topography of the Control group at 0h. Figure 25 (b) in the figure shows the morphology of the CA-SSAD group after 0 hours of culture. Figure 26 (a) in the figure shows the morphology of the Control group at 24 hours. Figure 26 Image (b) shows the morphology of the CA-SSAD group after 24 hours of culture; from Figure 25 and Figure 26 As can be seen, the scratch area in the CA-SSAD group was significantly narrower than that in the control group, and the migration distance of cells to the scratch area was significantly increased. Figure 27 Quantitative analysis showed that the relative migration area percentage of hDPSCs in the control group was approximately 50%, while that in the CA-SSAD group was approximately 70%. Statistical analysis indicated that the migration area in the CA-SSAD group was significantly higher than that in the control group, reflecting the ability of CA-SSAD anti-dentin hypersensitivity agent to promote hDPSC migration.

[0113] 8. Alkaline phosphatase (ALP) and Alizarin Red (ARS) staining experiments To evaluate the mineralization ability of CA-SSAD anti-dentin hypersensitivity agent, alkaline phosphatase (ALP) and alizarin red (ARS) staining experiments were performed on two groups of samples (Control group and CA-SSAD group, each with 3 duplicate wells).

[0114] (1) The specific steps of the alkaline phosphatase (ALP) experiment are as follows: 1) Prepare mineralization induction medium based on α-MEM medium, supplemented with the following components: 10% fetal bovine serum (FBS), 1% penicillin-streptomycin solution, 10mM sodium β-glycerophosphate, 10nM dexamethasone, and 50μg / mL vitamin C.

[0115] 2) Select P3 generation hDPSCs, wash the cells with PBS buffer, then add trypsin for digestion, then add complete culture medium to neutralize the trypsin activity, and resuspend the cells to obtain a cell suspension with good homogeneity.

[0116] 3) Accurately count the cells in the suspension using a hemocytometer, and calculate the cell density based on the count results. Based on this data, moderately dilute the original cell suspension with complete culture medium to a concentration of 2 × 10⁶ cells per well. 5 The cells were seeded at a density of 1,000 cells into a six-well culture plate.

[0117] 4) When the cells in the wells reach 80% confluence, discard the original culture medium and replace it with pre-prepared mineralization induction medium. At the same time, wrap the culture plate with aluminum foil and culture it in the dark. Thereafter, change the osteogenic induction medium every two days and continue culturing for 7 days.

[0118] 5) On day 7 of culture, the osteogenic induction effect of CA-SSAD anti-dentin hypersensitivity agent was detected: the cells were washed with PBS solution 2-3 times, and then stained with BCIP / NBT alkaline phosphatase chromogenic kit; after staining, the cells were washed with PBS twice, and the stained images were taken. Finally, the staining results were semi-quantitatively analyzed with image analysis software (Image J).

[0119] Alkaline phosphatase (ALP) activity reflects the ability of dental pulp stem cells (hDPSCs) to differentiate into odontoblasts. ALP staining after 7 days was used to assess the odontogenic differentiation of hDPSCs under CA-SSAD anti-dentin hypersensitivity agent stimulation. Figure 28A As shown, the bottom row is a magnified view of a portion of the image. Cells in the CA-SSAD group are stained a deep blue, significantly darker than the lighter staining in the Control group. Furthermore, semi-quantitative analysis reveals... Figure 28B As shown, compared with the Control group, the CA-SSAD group showed significantly enhanced ALP activity, and the difference between the groups was statistically significant. This indicates that the CA-SSAD anti-dentin hypersensitivity agent can significantly promote ALP expression in hDPSCs, suggesting that this desensitizing agent has tooth-derived induction properties and provides cellular support for dentin remineralization.

[0120] (2) Alizarin Red S can react with Ca²⁺ in calcium salts under weakly acidic conditions.+ It binds specifically to form an orange-red complex, which is used to visually display mineralized calcium nodules formed in cell culture. It is the gold standard test for evaluating the degree of late osteogenic differentiation / calcification.

[0121] The specific steps of the Alizarin Red (ARS) staining experiment are as follows: 1) Reagent preparation: Alizarin Red S staining solution (pH 4.2) is prepared by weighing 1g of Alizarin Red S and dissolving it in 100mL of deionized water to make a 1% stock solution; adjusting the pH to 4.1-4.3 with 10% NH4OH or HCl; filtering through a 0.22μm filter membrane for sterilization and storing at 4℃ protected from light.

[0122] Other reagents: PBS buffer, 4% paraformaldehyde fixative, 10% cetylpyridine chloride (CPC, for semi-quantitative purposes).

[0123] 2) hDPSCs cells are cultured in the same way as ALP cells, with continuous culture for 21 days to provide sufficient time for mineralized nodule formation.

[0124] 3) The formation of mineralized nodules was observed under a microscope, and then the calcium nodules were further detected by alizarin red staining: the original culture medium in the well was first aspirated and the cells were washed with PBS solution; 4% paraformaldehyde was added to fix the cells for 10 min, and after fixation, they were washed with PBS; the staining solution in the alizarin red S staining quantitative detection kit was added to each well, and the well plate was placed on a shaker at 37℃ for 5 min; after staining, the staining solution was discarded and the wells were washed with distilled water to completely remove the residual staining solution in the wells.

[0125] 4) Place the treated cells under a microscope to identify the mineralized nodules (i.e., calcium nodules) stained orange-red, and then capture images using an image acquisition system for subsequent analysis. 5) Incubate at room temperature for 15 min using the elution buffer from the Alizarin Red S staining quantitative detection kit until Alizarin Red S staining is completely eluted.

[0126] 6) Collect the eluted Alizarin Red S solution in a centrifuge tube and shake well. Place it in a 96-well plate, add 100 μL to each well, and repeat for three wells. Measure the OD value at 560 nm using a microplate reader.

[0127] To assess the odontoblastic differentiation potential of hDPSCs, mineralization induction treatment was performed on adherent hDPSCs using a mineralization-inducing medium. Under induction, hDPSCs exhibiting stem cell characteristics secreted extracellular matrix, and calcium salt deposition eventually formed calcium nodules. Alizarin Red dye stained these calcium nodules reddish-brown. The experimental results are as follows: Figure 29AAs shown in the image, the bottom row is a magnified view of a portion of the cells. After 21 days of osteogenic induction, numerous orange-red mineralized nodules were visible on the surface of cells in the CA-SSAD group, with both the number and staining depth significantly superior to those in the Control group. The results of ELISA reader quantitative detection are also shown. Figure 29B The results showed that the OD value of the CA-SSAD group was significantly higher than that of the Control group, and the difference between the groups was statistically significant, indicating that CA-SSAD anti-dentin hypersensitivity agent can significantly promote the formation of mineralized nodules in hDPSCs and enhance the cell mineralization differentiation capacity.

[0128] Specific Implementation Example 3: Exploring the Mechanism of Action 1. Transcriptome sequencing Total RNA was extracted from cells / tissues, and high-throughput sequencing was performed to detect the expression levels of all mRNAs. Differentially expressed genes were screened, pathways were enriched, and core mechanisms were identified. Specifically, hDPSCs were cultured in a-MEM medium with or without CA-SSAD dentin hypersensitivity agent for 7 days and then transported to Shanghai for testing on dry ice. The transcriptomic sequencing of hDPSCs was performed by Shanghai Meiji Biopharmaceutical Technology Co., Ltd.

[0129] Library construction and sequencing: Eukaryotic mRNA sequencing was performed using the Nova Seq X Plus platform, analyzing all mRNAs generated during cellular transcription. Library construction was completed using the Inmena NovaSeq sequencing kit. The specific methods used were standard procedures familiar to those skilled in the art. RNA was extracted from hDPSCs, and its concentration and purity were measured using a Nanodrop 2000 ultra-micro nucleic acid and protein analyzer. Nova sequencing technology belongs to the category of second-generation sequencing, and a single run can generate billions of sequencing fragments, clearly reflecting the sequencing effect and library construction level from a macroscopic perspective. Simultaneously, for each sample's raw sequencing data, three assessments are required: base content, quality distribution, and error rate distribution.

[0130] (1) Expression level and differential expression analysis: Transcript abundance is a direct reflection of gene expression level, and the two are positively correlated; that is, the higher the abundance, the stronger the expression activity of the corresponding gene. The criteria for determining differentially expressed genes were set as follows: P A gene can be defined as a differentially expressed gene (DEG) if it meets both of these conditions: <0.05 and |log2FC|≥1.

[0131] Using the blank control group as a reference, and comparing it with the CA-SSAD group, this study screened and identified a total of 3959 differentially expressed genes. Among these differentially expressed genes, 2177 genes showed upregulated expression levels, while 1782 genes showed downregulated expression levels. To visually demonstrate the expression changes of all genes, a differential gene volcano plot was created. For detailed results, please refer to [link to relevant documentation]. Figure 32 As shown in the volcano diagram, different colors are used to distinguish the differential expression status of genes: red data points represent genes with increased expression levels, blue data points represent genes with decreased expression levels, and genes whose expression levels did not change statistically significantly are marked with gray data points. Among these, odontoblast-related genes, such as WNT4, BDNF, DLX5, STAT3, and LEF1, show upregulated expression levels. The core function of WNT4 is to promote differentiation through the non-classical Wnt pathway, as manifested in: 1) WNT4 is involved in fluoride-mediated proliferation and mineralization of dental pulp cells.

[0132] 2) Upregulation of WNT4 can promote the recovery of the differentiation of dental pulp stem cells into dentin cells in pulpitis.

[0133] 3) WNT4 can inhibit lipopolysaccharide-induced apoptosis and inflammation of dental pulp cells.

[0134] (2) GO and KEGG annotation and enrichment analysis of differentially expressed genes: GO enrichment analysis was conducted using Goatools software. To effectively control the false positive rate, four multiple test methods were used: Bonferroni method, Holm method, Westdak method, and false discovery rate. P The value is corrected, where the corrected value is... P A GO function value <0.05 was considered significantly enriched. KEGG pathway enrichment analysis was performed using KOBAS software, with Fisher's exact test as the basis for the analysis. Multiple tests were performed using the BH (FDR) method to effectively control the false positive rate. After correction... P KEGG pathways with a value <0.05 are defined as pathways with significant enrichment in differentially expressed genes.

[0135] Gene Ontology (GO) refers to differential signaling pathways and is widely used to systematically describe the properties of genes and their encoded products, covering functional annotation in three aspects: biological processes, molecular functions, and cellular components.

[0136] GO analysis of upregulated genes showed that, specifically in terms of biological processes, the gene was enriched in the regulation of chemical synaptic transmission, regulation of transsynaptic signal transduction, adipocyte differentiation, reproductive system development, regulation of the Wnt signaling pathway, reproductive structure development, and synaptic tissue. In terms of cellular component function, the gene was enriched in dendritic spines, neuronal spines, interneuronal synapses, asymmetric synapses, exocytic vesicles, postsynaptic density, postsynaptic specialized structures, endoplasmic reticulum lumen, synaptic vesicles, and neuronal cell bodies. In terms of molecular function, the gene was enriched in receptor ligand activity, signal receptor activator activity, glycosaminoglycan binding, hormone activity, protein tyrosine kinase activity, G protein-coupled receptor binding, heparin binding, transmembrane receptor protein tyrosine phosphatase activity, transmembrane receptor protein phosphatase activity, and insulin-like growth factor I binding.

[0137] KEGG (Kyoto Genetics & Genome) focuses on the analysis of biological and metabolic pathways. Through comparative analysis with the KEGG database, KEGG pathway enrichment maps corresponding to upregulated and downregulated differentially expressed genes were obtained. Upregulated differentially expressed genes are mainly concentrated in the following pathways: motor proteins, neuroactive ligand-receptor interactions, PI3K-Akt signaling pathway, cytokine-cytokine receptor interactions, signaling pathways, Ras signaling pathway, cAMP signaling pathway, calcium signaling pathway, Wnt signaling pathway, Rap1 signaling pathway, TNF signaling pathway, FoxO signaling pathway, human papillomavirus infection, transcriptional dysregulation in cancer, lipids and atherosclerosis, human T-cell leukemia virus type 1 infection, and chemokine signaling pathways.

[0138] In summary, the results indicate that the Wnt signaling pathway comprises 19 Wnt ligand members, divided into two main families (β-catenin-dependent / canonical and β-catenin-independent / atypical). The Wnt signaling pathway plays a crucial role in dentin formation and regeneration, primarily influencing dentin formation by regulating cell proliferation, differentiation, and mineralization. The CA-SSAD group showed significant enrichment of the Wnt signaling pathway, suggesting its key role in the mineralization process induced by CA-SSAD anti-dentin hypersensitivity agents. Simultaneously, cell adhesion and skeletal-related pathways, as well as the calcium signaling pathway, were also significantly upregulated, indicating that cellular remodeling and ion transport are involved in mineralization regulation. CA-SSAD anti-dentin hypersensitivity agents may promote odontoblastic differentiation and mineralization in hDPSCs through the synergistic regulation of multiple pathways, including Wnt signaling, cell adhesion, and calcium ion metabolism.

[0139] (3) qPCR detection of odontoblast-related gene expression: Two groups were set up: CA-SSAD group and Control group. The cell seeding and culture procedures were as follows: 1) Prepare α-MEM culture medium containing 1% penicillin and no serum in 15mL centrifuge tubes and place them in an ice box.

[0140] 2) All experimental procedures were performed in accordance with the experimental protocol approved by the Medical Ethics Committee of Chongqing Medical University (ethics number: 2025 Lun Shen (065)). Young third molars or orthodontic extraction teeth under the age of 25 with no caries, no filling treatment and intact tooth structure were collected and placed in 15mL centrifuge tubes.

[0141] 3) Remove the dental pulp in a sterile laminar flow hood and rinse it in a sterile dish with PBS containing 1% double antibiotics.

[0142] 4) Prepare fresh α-MEM complete medium: Add 5 mL fetal bovine serum and 500 μL penicillin-streptomycin solution to a 50 mL centrifuge tube, and then add α-MEM medium to bring the total volume to the 50 mL mark.

[0143] 5) Prepare a 1.5 mL EP tube, add 1.2 mL of α-MEM complete culture medium containing 1% double antibiotics to the tube, and transfer the dental pulp tissue into the 1.5 mL EP tube.

[0144] 6) Centrifuge the EP tube at 1000 rpm for 5 min, discard the upper culture medium, add 70 μL of 2% type I collagenase to the EP tube, mix well and place in a 37℃ incubator for 15 min for digestion.

[0145] 7) After 5 min, add 100 μL of α-MEM complete medium containing 1% penicillin and antibiotics to a 1.5 mL EP tube to stop digestion. Centrifuge at 1000 rpm for 5 min, discard the upper medium, add 800 μL of complete medium containing 1% penicillin / streptomycin mixture and 40% FBS, mix well by pipetting and place in a T25 culture flask, making sure to wet the entire bottom of the culture flask. You can use a long pipette tip to move the tissue block to make the tissue block relatively concentrated and evenly distributed.

[0146] 8) Invert the culture flask into the cell culture incubator. Set the incubator parameters as follows: temperature 37℃, relative humidity 95%, 5% CO2. After 5 hours, invert the culture flask. After 12 hours, add liquid to 2.5 mL. Once the cells adhere and grow, these are primary cells.

[0147] When the cells reach approximately 80% growth, replace the medium with fresh mineralization induction medium to induce odontogenic differentiation in hDPSCs. Change the induction medium every two days to maintain a stable induction environment.

[0148] On day 7 of culture, qPCR was used to detect changes in the expression levels of genes related to odontoblast differentiation in hDPSCs, including WNT4, BDNF, DLX5, STAT3, and LEF1.

[0149] The expression of dentin-related genes was verified by qRT-PCR, and the results are as Figures 33A-33E shown, where Figure 33A is the relative expression of mRNA in the WNT4 pathway, Figure 33B is the relative expression of mRNA in the BDNF pathway; Figure 33C is the relative expression of mRNA in the DLX5 pathway; Figure 33D is the relative expression of mRNA in the STAT3 pathway; Figure 33E is the relative expression of mRNA in the LEF1 pathway; It can be seen from the results that on the 7th day of mineralization induction culture, compared with the Control group, the expression of dentinogenic differentiation-related genes in hDPSCs in the CA-SSAD group was significantly up-regulated, that is, the up-regulation of the expression levels of WNT4, BDNF, DLX5, STAT3, and LEF1 was verified at the mRNA level. This result indicates that the CA-SSAD dentin hypersensitivity inhibitor can promote the differentiation of hDPSCs into odontoblasts by up-regulating the expression of dentinogenic differentiation-related genes, providing a basis for its role in dentin regeneration.

[0150] Specific Example 4: Animal experiment in vivo I. Animal experiment modeling in vivo Six-week-old male SD rats (180±20 g), a total of 15, were selected, all from Hunan Slack Jingda Experimental Animal Co., Ltd., and were housed in the animal house of the Key Laboratory of the Affiliated Stomatological Hospital of Chongqing Medical University. All experimental operations were carried out in accordance with the experimental protocol approved by the Medical Ethics Committee of Chongqing Medical University (Ethical number: Approval No. (065) in 2025).

[0151] As Figure 34 shown, the model construction steps are as follows: ① The SD rats were anesthetized by isoflurane inhalation. After successful anesthesia, they were fixed on the animal experimental table.

[0152] ② When performing oral operations on the rats, the mouth was opened and firmly fixed with an oral speculum. Then, a cotton ball soaked with physiological saline was taken, and the oral cavity of the rats was carefully wiped and cleaned gently to remove impurities in the oral cavity.

[0153] ③ The demineralized dentin slices were fixed with ligature wires in advance, and the dentin slices were fixed between the incisors and the first molars of the rats. The appropriate tension at the ligature site was maintained and the knot was fixed. Then, the excess ligature wire was cut off to ensure that the edge of the ligature wire end was smooth, so as to avoid scratching the oral mucosa of the rats.

[0154] ④ After the model preparation is completed, observe and confirm that the demineralized dentin sheet is firmly fixed and there is no damage to the oral mucosa of the rat, thus completing the in vivo construction of the SD rat demineralized dentin model.

[0155] II. Animal in vivo remineralization experiments For comparison, three sets of samples were prepared: the first set was a blank experiment (treated with physiological saline, referred to as the Control group); the second set was commonly used sodium fluoride (NaF group); and the third set was the CA-SSAD anti-dentin hypersensitivity agent prepared in Specific Example 1 (referred to as the CA-SSAD group). The specific process is as follows: ① Dentin slices were randomly selected from the oral cavity of SD rats. The demineralized dentin slices were first dried using an ear syringe. Then, the prepared CA-SSAD anti-dentin hypersensitivity agent was evenly applied to the surface using a small brush. After application, the slices were dried again to form a stable CA-SSAD anti-dentin hypersensitivity agent coating on the surface. The same procedure was followed for both the NaF group and the Control group, with 10 experimental samples in each group.

[0156] ② Soak the feed in sterile water until it is soft, and use it as the food for SD rats. Observe and check the fixation status of the dentin slices in the oral cavity of the SD rats every day.

[0157] ③ After the mineralization culture cycle ends at 1 week, 2 weeks and 4 weeks respectively, SD rats are first subjected to inhalation anesthesia. After the anesthesia takes effect, dentin slices are taken out from their oral cavity. The taken dentin slices are then dried for subsequent testing.

[0158] ④ After 4 weeks of mineralization culture and all experimental samples have been collected, the SD rats used in the experiment were disposed of using the CO2 euthanasia method to ensure that the experimental procedures comply with animal ethics standards.

[0159] Scanning electron microscopy was used to observe dentin slices from SD rats at 1, 2, and 4 weeks of intraoral mineralization. The results at 1 week are as follows: Figures 35-36 As shown, Figure 35 (a) in the figure is the surface SEM characterization image of the Control group at 1 week; Figure 35 (b) shows the surface SEM characterization of the NaF group at 1 week; Figure 35 (c) in the figure shows the surface SEM characterization of the CA-SSAD group at 1 week; Figure 36 (a) in the figure is a lateral SEM image of the Control group at 1 week; Figure 36 (b) is a side SEM image of the NaF group at 1 week; Figure 36(c) shows the lateral SEM image of the CA-SSAD group at week 1. The results indicate that at week 1, a large number of open dentinal tubules with clear circular openings were visible on the dentin surface of the Control group; open tubular structures were visible in the cross-section, with no obvious mineral deposition; the openings of the tubules on the dentin surface of the NaF group were slightly narrowed, and a small amount of scattered minerals were visible; only trace amounts of minerals were visible on the tubule edges in the cross-section; some tubule openings on the dentin surface of the CA-SSAD group were blocked by minerals, and minerals began to deposit on the inner wall of the tubules in the cross-section, forming a thin mineralized layer.

[0160] The results at 2 weeks were as follows Figures 37-38 As shown, Figure 37 SEM images of the surface of the Control group, NaF group and CA-SSAD group at 2 weeks; Figure 37 (a) shows the surface SEM characterization of the Control group at 2 weeks; Figure 37 (b) shows the surface SEM characterization of the NaF group at 2 weeks; Figure 38 (c) in the figure shows the surface SEM characterization of the CA-SSAD group at 2 weeks; Figure 38 (a) is a lateral SEM image of the Control group at 2 weeks; Figure 38 (b) is a side SEM image of the NaF group at 2 weeks; Figure 38 (c) shows the lateral SEM image of the CA-SSAD group at 2 weeks. The results indicate that at 2 weeks, the dentinal tubules in the Control group remained open with no significant mineralization progress; the openings of the surface tubules in the NaF group further narrowed, the amount of minerals increased, and local fusion occurred. The cross-section showed a slight increase in the thickness of the mineral deposits, but it was still discontinuous; in the CA-SSAD group, most of the tubule openings on the dentin surface were closed by minerals, and crystal fusion formed plate-like mineralized areas. The cross-section showed that the minerals had formed a continuous mineralized layer inside the tubules and extended towards the center of the lumen.

[0161] The results at 4 weeks were as follows Figures 39-40 As shown, Figure 39 SEM images of the surface of the Control group, NaF group and CA-SSAD group at 4 weeks; Figure 39 (a) shows the surface SEM characterization of the Control group at 4 weeks; Figure 39 (b) shows the surface SEM characterization of the NaF group at 4 weeks; Figure 39 (c) in the figure shows the surface SEM characterization of the CA-SSAD group at 2 weeks; Figure 40 (a) is a lateral SEM image of the Control group at 4 weeks; Figure 40 (b) is a side SEM image of the NaF group at 4 weeks; Figure 40Image (c) shows the lateral SEM image of the CA-SSAD group at 2 weeks. The results indicate that at week 4 ( Figure 39 and Figure 40 In the Control group, the dentinal tubules remained open, and no effective mineralization repair was observed. In the NaF group, most of the tubule openings on the dentin surface were covered by mineralized crystals, but some tubules that were not completely closed were still visible. The thickness of the mineralized layer on the cross-section increased compared to 2 weeks, but the density was insufficient. In the CA-SSAD group, a dense and continuous mineralized repair layer was formed on the dentin surface, and the tubule openings were almost completely closed. The mineralized layer on the cross-section was uniform and significantly thicker, filling the dentinal tubules and forming a complete mineralized repair structure.

[0162] The above results indicate that CA-SSAD dentin hypersensitivity agent can effectively and stably seal dentinal tubules in the oral environment, and its sealing effect is superior to that of traditional NaF treatment.

[0163] III. Biocompatibility Testing of CA-SSAD Dentin Sensitizer in Vivo: Tissue HE Staining 1. Sampling: After 4 weeks of animal experiments, SD rats were dissected, and vital organs—heart, liver, spleen, lungs, and kidneys—were removed. These organs were immersed in 4% paraformaldehyde fixative and fixed at room temperature for 24 hours to ensure stable preservation of tissue morphology and structure. After fixation, the specimens were rinsed under running tap water for 24 hours to thoroughly remove any residual fixative and avoid interference with subsequent staining. After rinsing, subsequent processing steps were performed, including tissue dehydration, clearing, paraffin embedding, embedding, sectioning, and HE staining.

[0164] ① Tissue dehydration: 75% ethanol for 1 hour, 85% ethanol for 1 hour, 90% ethanol for 1 hour, 95% ethanol for 1 hour, 100% ethanol (I) for 1 hour, 100% ethanol (II) for 1 hour; ② Transparent: Xylene (I) 15 min, Xylene (II) 15 min; ③ Wax impregnation: Paraffin (I) for 1 hour, paraffin (II) for 1 hour; ④ Embedding: After the tissue has undergone paraffin impregnation, it is placed in a paraffin embedding machine containing liquid paraffin for embedding. Care must be taken to place the tissue block in the appropriate position within the mold. Once the liquid embedding paraffin has completely filled the mold, the embedding cap is placed on the mold and the tissue is placed on a -20℃ freezing platform to cool. After the paraffin block has completely solidified, it is demolded and then stored in a -20℃ freezer. ⑤ Sectioning: First, remove the paraffin tissue block from the refrigerator and place it in the slot of the paraffin microtome, clamping it securely. Adjust the section thickness to 4μm and begin slicing. Place the cut tissue sections in a 38℃ water bath to allow them to expand naturally. Then, place a glass slide on top of the slide and lay it flat. Shake off any water droplets on the surface of the slide to prevent cracking. Bake the slide at 55℃ for 30 minutes, then store it at room temperature.

[0165] 2. HE staining ① Baking: Arrange the paraffin slices in an orderly manner on the staining rack, and then place the staining rack in a 65℃ oven for baking for 2 hours.

[0166] ②Dewaxing: After baking, the slices are immediately transferred to xylene for dewaxing. The specific process and corresponding time are: xylene (I) 8 min, xylene (II) 8 min.

[0167] ③ Hydration: After dewaxing, the sections need to be placed in alcohol of varying concentrations to remove residual xylene. The order of operations and the duration of each step are as follows: 100% ethanol (I) 5 min, 100% ethanol (II) 5 min, 95% ethanol 2 min, 90% ethanol 2 min, 85% ethanol 2 min, 80% ethanol 2 min, 75% ethanol 2 min, and water 2 min. ④ Hematoxylin staining: Immerse the tissue sections in hematoxylin staining solution for 5 minutes. After staining, rinse the sections with running water for 1 minute to remove excess staining solution.

[0168] ⑤ Eosin staining: Immerse the slides in eosin staining solution for 2 minutes. After staining, rinse with running water for 1 minute to ensure that the slides are stained evenly and without any residual staining solution.

[0169] ⑥ Dehydration: The stained sections need to be dehydrated by sequentially passing them through ethanol solutions of increasing concentration. First, immerse the sections in 80% ethanol for 10 seconds; then transfer them to 90% ethanol and immerse them for 10 seconds; finally, place them in anhydrous ethanol (100%) and immerse them for 10 seconds to completely remove the water from the tissue.

[0170] ⑦ Clearing: After the dehydration step, the sections are transferred to a xylene solution for clearing. The clearing process is completed in two steps: first, the sections are immersed in xylene (I) for 2 minutes, and then transferred to fresh xylene (II) for another 2 minutes to make the sections clear.

[0171] ⑧ Drying: Remove the cleared sections from the xylene, arrange them neatly on a section rack, and transfer them to a fume hood. Allow them to stand overnight at room temperature to allow the xylene adhering to the surface of the sections to evaporate completely, ensuring that the sections are completely dry.

[0172] 9. Mounting: Place a suitable amount of neutral resin in the center of the dried tissue section, then gently cover it with a clean coverslip. Press the coverslip lightly to spread the resin evenly and remove any remaining air bubbles, especially ensuring that no air bubbles obstruct the tissue area. After mounting, store the section flat at room temperature.

[0173] ⑩ Scanning: After mounting, the slides need to stand for 5-7 days until the resin has completely solidified. Then, place the slides on a slide scanner for scanning. The scanned images will be analyzed using Olympus image software.

[0174] 3. Results Analysis: like Figure 41 As shown, Figure 41 The first row in the diagram is a representation of the heart, liver, spleen, lungs, and kidneys in the Control group; Figure 41 The second row shows the representation of NaF in the heart, liver, spleen, lungs, and kidneys; Figure 41 The third row shows the characterization of the heart, liver, spleen, lungs, and kidneys in the CA-SSAD group. The results indicate that, compared with the normal control group, the tissue structure of important organs in the CA-SSAD group was not damaged. The morphology and structure of myocardial fibers, liver lobules, spleen lymphoid follicles, alveoli, and nephrons remained intact, without inflammatory infiltration, cell necrosis, fibrosis, structural abnormalities, etc., confirming that CA-SSAD anti-dentin hypersensitivity agent did not produce toxicity to important organs.

[0175] Specific Example 5: Comparative Analysis of Three Hydrolysis Systems The first type: VC-SSAD group: Vitamin C (VC) hydrolyzes SSAD lyophilized powder. The preparation method is the same as in specific example 1, except that citric acid is replaced with vitamin C. The ratio is: 1g of vitamin C and 1g of SSAD powder are dissolved in 10mL of DD water, and then diluted with DD water to obtain five different concentrations of VC-SSAD hydrolysate: 0.05mg / mL, 0.1mg / mL, 0.15mg / mL, 0.2mg / mL and 0.25mg / mL.

[0176] The second type: TCEP-SSAD group: adopting TCEP (three (2) The preparation method of the carboxyethyl phosphonic acid hydrochloride hydrolyzed SSAD lyophilized powder is as described in Specific Example 1, except that citric acid is replaced with TCEP. The ratio is: 1g TCEP and 1g SSAD powder are dissolved in 10mL DD water, and then diluted with DD water to obtain five different concentrations of TCEP-SSAD hydrolysate: 0.05mg / mL, 0.1mg / mL, 0.15mg / mL, 0.2mg / mL and 0.25mg / mL.

[0177] The third type: CA-SSAD group: SSAD lyophilized powder was hydrolyzed with citric acid (CA). The preparation method was the same as in specific example 1. The ratio was: 1g of citric acid and 1g of SSAD powder were dissolved in 10mL of DD water, and then diluted with DD water to obtain five different concentrations of CA-SSAD hydrolysate: 0.05mg / mL, 0.1mg / mL, 0.15mg / mL, 0.2mg / mL and 0.25mg / mL.

[0178] 1. Cell proliferation experiment (CCK-8) The effects of the VC-SSAD group and the TCEP-SSAD group were verified by cell proliferation assay (CCK-8), and the results are as follows: Figures 42-45 As shown, where, Figure 42 The results show the comparison of cell survival rates at different concentrations and time points in the CA-SSAD group using specific Example 1. The results indicate that the CA-SSAD group with a concentration of 0.15 mg / mL showed a stronger tendency to promote the proliferation of BMSCs (bone marrow mesenchymal stem cells) after three days. Figure 43 The results show the comparison of cell survival rates at different concentrations and time points in the VC-SSAD group using specific Example 1; in the VC-SSAD group, the concentration of 0.2 mg / mL showed a stronger trend of promoting the proliferation of BMSCs (bone marrow mesenchymal stem cells) after three days. Figure 44 The cell survival rate of the TCEP-SSAD group using specific Example 1 at different concentrations and time periods was shown. The TCEP-SSAD group at a concentration of 0.1 mg / mL showed a stronger tendency to promote the proliferation of BMSCs (bone marrow mesenchymal stem cells). Furthermore, comparing the three hydrolysis systems, the VC-SSAD group at a concentration of 0.2 mg / mL exhibited the strongest proliferation trend.

[0179] The cytotoxicity of three hydrolysis systems on BMSCs was determined using the CCK-8 assay. BMSCs were seeded in 96-well plates and cultured overnight. After treatment with each of the three hydrolysis systems for 24 hours, CCK-8 reagent was added, and absorbance at 450 nm was measured. Cell viability was calculated, and 24-hour cytotoxicity of BMSCs was evaluated to assess the cell biocompatibility of each system. Figure 45 This study compares cell viability at different time points in three hydrolysis systems at a concentration of 0.2 mg / mL; the results show that the CA-SSAD and VC-SSAD groups had the highest survival rates after 3 days. Figure 46 The image shown is a fluorescence micrograph of cell viability and mortality staining using the Calcein-AM / PI double staining method. Figure 46The first column shows the Calcein-AM channel (green fluorescence). All samples exhibited dense, uniform green fluorescence, indicating a high number and viability of cells in the control group, with a very high proportion of live cells. The second column shows the PI (propidium iodide) staining channel (red fluorescence). The red signal was minimal, with almost no visible red fluorescent dots, indicating a very low number of dead cells in the control group. The third column is a combined image of the first two columns, clearly showing the distribution and proportion of live (green) and dead (red) cells; almost all are green, with only occasional scattered red dots. In summary, these results indicate that compared to the Control group, the VC-SSAD, TCEP-SSAD, and CA-SSAD groups all exhibited good cell compatibility.

[0180] 2. Cell scratch test To investigate the migration ability of different hydrolysis systems, cell scratch experiments were performed on dentin slides treated with the Control group and the three hydrolysis systems, following the same procedure as in Specific Example 1. Figure 47 As shown, the results indicate that the VC-SSAD group, TCEP-SSAD group, and CA-SSAD group all promoted BMSC migration. The CA-SSAD group showed a stronger trend in promoting BMSC migration.

[0181] 3. Promotes BMSCs mineralization: Three identical dentin slides were treated with three different hydrolysis systems (all at a concentration of 0.2 mg / mL). The treated slides were then placed in an osteogenic induction solution (an osteogenic induction solution based on complete culture medium supplemented with dexamethasone, sodium β-glycerophosphate, and ascorbic acid) for induction treatment. After 10 days of induction, the slides were removed and stained. The overall staining of the well plates was observed visually (orange-red precipitate indicated a positive result), and the morphology and distribution of calcium nodules were recorded under an inverted microscope at low (4×) and high (10×) magnifications. Complete culture medium was used as a negative control group, and the osteogenic induction solution as a control group. The results of Alizarin Red S (ARS) staining are shown below. Figure 48 As shown. Among them. Figure 48The first row, from left to right, shows panoramic images of the well plates containing complete culture medium, osteogenic induction solution, VC-SSAD group in osteogenic induction solution, TCEP-SSAD group in osteogenic induction solution, and CA-SSAD group in osteogenic induction solution. The second row, from left to right, shows low-power microscopic images of the complete culture medium, osteogenic induction solution, VC-SSAD group in osteogenic induction solution, TCEP-SSAD group in osteogenic induction solution, and CA-SSAD group in osteogenic induction solution. The third row, from left to right, shows high-power microscopic images of the complete culture medium, osteogenic induction solution, VC-SSAD group in osteogenic induction solution, TCEP-SSAD group in osteogenic induction solution, and CA-SSAD group in osteogenic induction solution.

[0182] Alizarin Red S staining results showed that no obvious orange-red calcium nodules formed in the complete culture medium group; a large number of densely distributed orange-red mineralized nodules were visible in the osteogenic induction solution group, and the nodule morphology became clearly visible with increasing magnification. The well plates in the VC-SSAD, TCEP-SSAD, and CA-SSAD groups showed a distinct orange-red color overall, with almost all areas covered by mineralized nodules. The osteogenic induction solution successfully initiated the osteogenic differentiation program of BMSCs, and the extracellular matrix secreted by the cells underwent extensive mineralization, forming dense, fused calcium nodules. With increasing magnification, some areas without orange-red calcium nodules were visible in the VC-SSAD and TCEP-SSAD groups, while the CA-SSAD group showed almost no areas without orange-red calcium nodules. These results indicate that CA-SSAD, TCEP-SSAD, and VC-SSAD can all promote BMSC mineralization, with CA-SSAD showing the best effect.

[0183] Figure 30 The bar chart shows the semi-quantitative results of Alizarin Red S staining (CPC elution). The results indicate that after elution with cetylpyridinium chloride (CPC), the absorbance was measured at 562 nm. The results showed that the OD value of the complete culture medium group was 0.1128, which is relatively low, suggesting no significant spontaneous mineralization in the cells, and can be used as a baseline for subsequent osteogenic induction and intervention groups. The OD value of the osteogenic induction solution group was 0.6532, while the OD values ​​of the VC-SSAD group (1.022) and CA-SSAD group (1.0792) were significantly higher than those of the negative control group (complete culture medium group) and the control experimental group (osteogenic induction solution group). PThe value <0.05 indicates that both CA-SSAD and VC-SSAD can effectively promote BMSCs mineralization and significantly enhance osteogenic differentiation capacity, with CA-SSAD showing the best effect.

[0184] 4. Promotes the mineralization of PDLSCs: Three identical dentin slides were treated with three different hydrolysis systems (all at a concentration of 0.2 mg / mL). The treated slides were then placed in an osteogenic induction solution (an osteogenic induction solution based on complete culture medium supplemented with dexamethasone, sodium β-glycerophosphate, and ascorbic acid) for induction treatment. After 10 days of induction, the slides were removed and stained. The overall staining of the well plates was observed visually (orange-red precipitate indicated a positive result), and the morphology and distribution of calcium nodules were recorded under an inverted microscope at low (4×) and high (10×) magnifications. Complete culture medium was used as a negative control group, and the osteogenic induction solution as a control group. The results of Alizarin Red S (ARS) staining are shown below. Figure 31 As shown, where Figure 31 The first row, from left to right, shows panoramic images of the well plates containing complete culture medium, osteogenic induction solution, VC-SSAD group in osteogenic induction solution, TCEP-SSAD group in osteogenic induction solution, and CA-SSAD group in osteogenic induction solution. The second row, from left to right, shows low-power microscopic images of the complete culture medium, osteogenic induction solution, VC-SSAD group in osteogenic induction solution, TCEP-SSAD group in osteogenic induction solution, and CA-SSAD group in osteogenic induction solution. The third row, from left to right, shows high-power microscopic images of the complete culture medium, osteogenic induction solution, VC-SSAD group in osteogenic induction solution, TCEP-SSAD group in osteogenic induction solution, and CA-SSAD group in osteogenic induction solution.

[0185] Alizarin Red S staining results showed that, compared with the complete culture medium group and the osteogenic induction solution group, the well plates in the VC-SSAD group, TCEP-SSAD group, and CA-SSAD group were generally bright orange-red, with almost all areas covered by mineralized nodules. The osteogenic induction solution successfully initiated the osteogenic differentiation program of PDLSCs, and the extracellular matrix secreted by the cells underwent extensive mineralization, forming dense, fused calcium nodules. With increasing magnification, some areas without orange-red calcium nodules were visible in the VC-SSAD group and TCEP-SSAD group, while the CA-SSAD group had almost no areas without orange-red calcium nodules. The results indicate that CA-SSAD, TCEP-SSAD, and VC-SSAD can all promote the mineralization of PDLSCs, with CA-SSAD showing the best effect.

[0186] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0187] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. Use of a hydrolysate of an Andrias japonicus mucilage extract in the preparation of an agent for preventing dentin hypersensitivity, characterized in that, This dentin hypersensitivity agent comprises a hydrolysate that retains the biological activity of the giant salamander mucus extract. The hydrolysate is a product obtained by reducing the giant salamander mucus extract with an acidic solution at a pH of 1-5. The giant salamander mucus extract is a freeze-dried powder obtained by freeze-drying collected giant salamander skin mucus at -80℃ for 72 hours; the particle size of the giant salamander mucus extract is 1~50μm. The acidic solution includes an aqueous solution of citric acid and / or an aqueous solution of vitamin C and / or a solution of tris(2-carboxyethyl)phosphine hydrochloride; The preparation method of the hydrolysate includes the following specific steps: S1: Use the first solvent to prepare an acidic solution with a pH value of 1~5 as a reducing agent; S2: Add the freeze-dried powder of giant salamander mucus extract and acidic solution to the second solvent, mix well to obtain the first mixture, then add alkaline solution dropwise to the first mixture to adjust the pH value of the first mixture to 5.5~7.5, and let the first mixture stand at 0~8℃ for 12~100h to obtain the second mixture; S3: Centrifuge the second mixture and collect the supernatant, which is the hydrolysate; thus, the pH value of the anti-dentin hypersensitivity agent is 5.5~7.

5.

2. Use according to claim 1, characterized in that, The hydrolysate contains five active proteins: MHC class IA α chain, homeobox protein Hox-D13, axonoderm dynamin aberrant heavy chain 3, ATP synthase protein, and cytochrome b.

3. Use according to claim 2, characterized in that, In steps S1 and S2, the first solvent and the second solvent are both one of ultrapure water, double-distilled water, deionized water, water for injection, and pure water.

4. Use according to claim 2, characterized in that, The acidic solution is an aqueous solution of citric acid with a concentration of 0.2~0.3 mol / L and a pH value of 2~3; the mass ratio of the giant salamander mucus extract to the acidic solution is (1~3):

1.

5. Use according to claim 2, characterized in that, The dentin hypersensitivity agent further includes excipients; the excipients include gel excipients, thickening / thixotropic agents and medical matrix excipients, and the gel excipients include one or more of sodium carboxymethyl cellulose (CMC-Na), hydroxyethyl cellulose (HEC), hydroxypropyl methyl cellulose (HPMC), carbomer, xanthan gum, and sodium alginate. The thickening / thixotropic agent includes one or more of magnesium aluminum silicate, bentonite, polyethylene glycol (PEG), and propylene glycol / glycerin; The medical matrix excipients include one or more of polyvinyl carboxylate, polyvinyl alcohol (PVA), and chitosan.

6. The application according to claim 4, characterized in that, When the acidic solution is a citric acid aqueous solution with a concentration of 0.2~0.3mol / L and a pH value of 2~3, the ratio of the freeze-dried powder of the giant salamander mucus extract to the second solvent in step S2 is 1g:10mL.

7. The application according to claim 6, characterized in that, Both the first solvent and the second solvent are double-distilled water.

8. The application according to claim 7, characterized in that, In step S2, the mass ratio of the freeze-dried powder of the giant salamander mucus extract to the citric acid is 2:

1.

9. The application according to claim 7, characterized in that, The alkaline solution in step S2 is sodium hydroxide or potassium hydroxide, and the pH value of the first mixture is adjusted to 6.

10. The application according to claim 7, characterized in that, In step S3, centrifuge at 1000~8000 rpm for 10 minutes.

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