Use of acidic hydrolysate of andrias davidianus mucus extract in preparation of root surface treatment agent

Abstract

This invention relates to the field of biomaterials, specifically to the application of an acidic hydrolysate of a giant salamander mucus extract in the preparation of a root surface treatment agent. The root surface treatment agent comprises a product obtained by reducing lyophilized powder of salamander skin secretions with an acidic solution at a concentration of 0.2-0.3 mol / L and a pH of 1-5, wherein the mass ratio of the lyophilized salamander skin secretions to the acidic solution is (1-3):1; the resulting root surface treatment agent, after adjustment with an alkaline solution, has a pH of 6-6.5. This root surface treatment agent demonstrates significant potential in promoting natural tooth replantation and restoration, with effects including promoting early load stabilization through natural wet bonding, assisting in the construction of a regenerative microenvironment through active proteins, and promoting adhesion between periodontal ligament stem cells and the matrix by enhancing adhesion signal expression.

Description

Technical Field

[0001] This invention relates to the field of biomaterials, specifically to the application of an acidic hydrolysis product of giant salamander mucus extract in the preparation of root surface treatment agents. Background Technology

[0002] Millions of people worldwide lose their teeth each year for various reasons; it is estimated that approximately [number missing] people lose their teeth annually. 55 million adult teeth are lost for various reasons. Tooth loss is the most difficult and controversial trauma to treat in permanent teeth, accounting for 0.5% to 16% of all traumas. Natural tooth replantation is a surgical procedure that re-implants teeth that cannot be saved due to various reasons into their sockets after external treatment. It is the last resort for preserving natural teeth. This technique can preserve natural teeth lost due to trauma or used for intention tooth replantation, ectopic tooth replantation, etc. The prognosis of replanted teeth is affected by many factors, including the treatment of the lost tooth, the time of loss, and the replantation technique. Under the combined effect of these factors, the prognosis of replanted teeth can generally be divided into five types: direct loss, periodontal ligament healing, surface resorption, inflammatory resorption, and replacement resorption. Among these, periodontal ligament healing is the most ideal healing method, and its success rate varies greatly in different literature reports (5.3%-23%).

[0003] However, existing methods for replanting natural teeth also present certain challenges. For example, after dental trauma, especially in severe cases like tooth avulsion, even with replantation, it's difficult to form a complete and healthy periodontal ligament as in a normal state. During replantation, the inability to seek timely treatment often leads to necrosis of the periodontal ligament on the root surface, significantly reducing the success rate. The high incidence of root / bone resorption further increases the uncertainty after replantation, potentially leading to tooth loss. Although natural tooth replantation remains the primary clinical treatment for completely avulsed teeth, its success rate is limited by factors affecting healing and insufficient periodontal tissue regeneration.

[0004] The uncertainty surrounding the prognosis of tooth replantation significantly limits its widespread application in clinical practice. Since the introduction of tooth replantation technology, how to promote the most ideal healing mode of natural teeth after replantation, namely periodontal ligament healing, has always been a thorny problem that plagues clinical work and urgently needs to be solved. Among the methods to improve periodontal physiological healing, root surface treatment is the most feasible option for dentists and is also the most researched direction.

[0005] Currently, the clinical approach to treating gingival recession involves cleaning the surface of the receding tooth root and treating it with acidic or antibiotic medications to facilitate further reattachment of the gingiva. A common method is to prepare a citric acid-based periodontal root surface treatment agent using a 30%–60% citric acid aqueous solution, buffer salts, and preservatives. This type of root surface treatment agent is highly acidic, with a pH of approximately 1.0–1.8. Therefore, due to its excessive acidity, it is highly irritating and corrosive, particularly damaging to the gingiva, mucosa, and alveolar bone. Furthermore, the strong acid lacks calcium chelation selectivity; large-area, prolonged application can lead to excessive demineralization of cementum and dentin, resulting in a weakened root surface and exacerbated dentin sensitivity over time. This hinders periodontal ligament cell attachment and cementum regeneration, weakening the foundation for repair. In addition, prolonged application can inhibit wound cell proliferation and delay healing.

[0006] Another option is enamel matrix derivatives (EMD) – trade name: Emdogain®. These proteins are extracted from pig tooth germs and are currently the only FDA-approved periodontal regeneration product. Clinically, they are used in root surface treatment for reimplantation, GTR (Gardening Transplantation), and other periodontal and implant surgeries. However, their extraction process is complex, requiring the addition of propylene glycol alginate (PGA) to improve viscosity and solubility; and they are expensive (approximately 8000 RMB per mL). Given that high-end root surface treatment materials and core technologies are currently largely monopolized by foreign companies, limiting industrial development, there is an urgent need to develop root surface treatment materials with independent intellectual property rights in my country.

[0007] The skin secretion of *Andrias davidianus* (SSAD) possesses strong wet bonding and regeneration-promoting effects, making it highly suitable for reimplanted teeth after extraction, where there is significant bleeding in the alveolar socket, making drying difficult and periodontal ligament and even root / bone tissue damage hindering regeneration. 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 active ingredients, 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.

[0008] In summary, there is an urgent need to develop an acidic hydrolysis product of giant salamander mucus extract for use in the preparation of root surface treatment agents, in order to overcome the bottlenecks of existing technologies. Summary of the Invention

[0009] The purpose of this invention is to provide an application of an acidic hydrolysate of the giant salamander mucus extract in the preparation of a root surface treatment agent. This root surface treatment agent has multiple effects and shows significant potential in promoting the replantation and restoration of natural teeth. Its functions include promoting early load stabilization through natural wet bonding, assisting in the construction of a regenerative microenvironment through active proteins, and promoting the adhesion of periodontal ligament stem cells to the matrix by increasing the expression of adhesion signals.

[0010] To address the aforementioned technical problems, this invention provides an application of an acidic hydrolysis product of giant salamander mucus extract in the preparation of a root surface treatment agent.

[0011] Preferably, the root treatment agent comprises a product obtained by reducing lyophilized salamander skin secretions with an acidic solution, wherein the concentration of the acidic solution is 0.2~0.3 mol / L, the pH value is 1~5, and the mass ratio of the lyophilized salamander skin secretions to the acidic substance in the acidic solution is (1~3):1; the root treatment agent obtained after adjustment with an alkaline solution has a pH value of 5.5~6.5.

[0012] Preferably, the pH value of the root surface treatment agent obtained after adjustment with an alkaline solution is 6.4.

[0013] Using the above technical solution, the salamander skin secretions were reduced using a strongly acidic solution, and then the pH was adjusted to a weakly acidic level to obtain an acidic hydrolysate, which serves as the root surface treatment agent. This root surface treatment agent fully retains the regenerative efficacy, antibacterial, antioxidant, and anti-inflammatory effects of the salamander skin secretions. Furthermore, the weakly acidic pH is gentle, does not corrode the gums, subcutaneous connective tissue, or exposed bone surface, and does not alter the root surface's pH balance, providing a suitable microenvironment for cell adhesion and colonization. It truly achieves the goals of cleaning contaminants without harming the plant's own tissues, protecting regenerated proteins, and being low-irritant and highly safe. Firstly, it solves the problem that traditionally extracted salamander skin secretions are solid and inconvenient for use in root surface treatment agents; secondly, it addresses the issue of strong acidic solutions being highly irritating and corrosive, causing significant damage to the plant's own tissues when used directly as root surface treatment agents.

[0014] Preferably, the acidic solution comprises an aqueous solution of citric acid and / or an aqueous solution of vitamin C and / or a solution of tris(2-carboxyethyl)phosphonic acid hydrochloride; the product comprises five active proteins, namely: the α chain of MHC class IA molecules, the homeobox protein Hox-D13, the axonoderm dynamin abnormal heavy chain 3, ATP synthase protein, and cytochrome b.

[0015] Preferably, the acidic solution is an aqueous solution of citric acid with a pH of 2 to 3.

[0016] Preferably, the root surface treatment agent further includes excipients, which include gel excipients, thickening / thixotropic agents, and medical matrix excipients. 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 polycarboxylate, polyvinyl alcohol (PVA), and chitosan.

[0017] Excipients are mainly divided into three groups, among which the most mainstream gel excipients (suitable for oral mucosa / dental surfaces) 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 coat 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" effect. 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 formulations containing fluorine, potassium nitrate, or strontium. Sodium alginate forms a mild film and was commonly used in early root surface treatments.

[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 / root treatments, 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, the preparation method of the root surface treatment 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 salamander skin secretions and acidic solution to the second solvent, mix well to obtain the first mixture, 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 root treatment 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.

[0023] Using the above technical solution, this invention hydrolyzes traditional SSAD powder using an acidic solution (such as citric acid). Specifically, the product obtained by reducing salamander skin secretions with citric acid aqueous solution is then adjusted to a weakly acidic pH using an alkaline solution. After static dissolution and centrifugation, an acidic hydrolysate, namely a liquid SSAD-CA solution, is obtained, thus producing a liquid SSAD-CA root surface treatment agent. This preparation method is simple, has a large yield, 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 biological activity of the salamander skin mucus protein, avoiding protein denaturation or inactivation caused by strong acid or strong alkali treatment. Rich in three carboxyl groups (-COOH), it can promote the formation of a large number of H bonds with aqueous solutions and proteins through deprotonation and protonation, thus promoting protein stability and solubility.

[0024] Preferably, in step S3, an excipient is added to the collected supernatant and mixed evenly to obtain a root surface treatment agent containing the excipient.

[0025] Preferably, step S2 further includes adding an alkaline solution to the first mixture to adjust the pH value of the first mixture to 5.5~6.5.

[0026] 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 0.2~0.3mol / L and a pH of 2~3, the ratio of the lyophilized salamander skin secretion powder to the second solvent in step S2 is 1g:10mL; the particle size of the salamander skin secretion is 1~50μm.

[0027] Preferably, the alkaline solution in step S2 is sodium hydroxide or potassium hydroxide, and the pH value of the first mixture is adjusted to 6.4.

[0028] Preferably, in step S3, the centrifugation is performed at a speed of 1000~8000 rpm for 10 minutes.

[0029] Preferably, in step S3, the centrifugation is performed at 4000 rpm for 10 minutes.

[0030] Beneficial technical effects: (1) This invention applies the product obtained by reducing the skin secretions of giant salamanders with acidic solution using traditional SSAD powder to prepare a liquid root surface treatment agent. This preparation method is simple, has a large yield, and its good biocompatibility can reduce the need for dialysis and other processes to remove toxic solubilizing agents. The mild acidic environment helps to maintain the natural structure and biological activity of the giant salamander skin mucus protein, avoiding protein denaturation or inactivation caused by strong acid or strong alkali treatment. It is rich in three carboxyl groups (-COOH), which can promote the formation of a large number of H bonds with aqueous solution and protein through deprotonation and protonation, thus promoting protein stability and solubility.

[0031] (2) When the acidic solution is an aqueous solution of citric acid, the obtained SSAD-CA root surface treatment agent has multiple effects, surpassing traditional single-effect root surface treatment agents. Experimental verification shows that SSAD-CA root surface treatment agent can not only promote regeneration, but also has better antibacterial, antioxidant and anti-inflammatory effects; SSAD-CA root surface treatment agent can be used as a natural adhesive, with strong wet adhesion in a moist oral environment, which can help reimplanted teeth resist early dynamic loads; SSAD-CA root surface treatment agent has certain osteogenic and anti-bone resorption effects, which can reduce the risk of root / bone resorption. Attached Figure Description

[0032] 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.

[0033] Figure 1 This is a flowchart illustrating the preparation process of the root surface treatment agent of the present invention. Figure 2 A photograph of the root surface treatment agent prepared according to the present invention; Figure 3A This is a particle size distribution diagram of the SSAD-CA root treatment agent of the present invention. Figure 3B This is a particle size distribution diagram of SSAD-S according to the present invention; Figure 3C This is a particle size distribution diagram of SSAD-P of the present invention; Figure 4 Potential diagrams of the SSAD-CA root surface treatment agent prepared in this invention and the control groups SSAD-S and SSAD-P; Figure 5A The infrared spectrum of the SSAD-CA root treatment agent of the present invention is shown below. Figure 5B The infrared spectrum of the root treatment agent SSAD-S of the present invention; Figure 5C The infrared spectrum of the root treatment agent SSAD-P of the present invention; Figure 6A The SSAD-CA root treatment agent, SSAD-S, and SSAD-P of this invention are used at a depth of 2600 cm. -1 Up to 3600cm -1 Raman spectra; Figure 6B The SSAD-CA root treatment agent, SSAD-S, and SSAD-P of this invention are used at a depth of 900 cm. -1 Up to 1700cm -1 Raman spectra; Figure 6C The SSAD-CA root treatment agent, SSAD-S, and SSAD-P of this invention are used at a depth of 200cm. -1 Up to 2200cm -1 Raman spectra; Figure 7A The protein concentration detection results of the SSAD-CA root surface treatment agent of the present invention; Figure 7B The mass spectrometry results are those of the SSAD-CA root surface treatment agent of the present invention. Figure 8 Peptide coverage analysis was performed on the SSAD-CA root surface treatment agent, SSAD-S, SSAD-P and the proteins 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 9The viscosity test results are for the SSAD-CA root treatment agent, SSAD-S, and SSAD-P of this invention. Figure 10A The results show the BTS clearance rates for the SSAD-CA group, EMD group, and Control group of this invention. Figure 10B The results show the DPPH clearance rates for the SSAD-CA group, EMD group, and Control group of this invention. Figure 11A These are photographs of the water contact angles of the SSAD-CA group, EMD group, and Control group of the present invention; Figure 11B The statistical results of water contact angles for the SSAD-CA group, EMD group, and Control group of this invention; Figure 12 A flowchart illustrating the delayed replantation model of the beagle dog for this invention; Figure 13A The results of anterior tooth mobility in the SSAD-CA group, EMD group and Control group of this invention; Figure 13B The results of anterior tooth loss rate in the SSAD-CA group, EMD group and Control group of this invention; Figure 14A The results of the frontal and lateral X-rays of the implantation of the anterior teeth of the Beagle after SSAD-CA treatment following avulsion. Figure 14B The X-ray results of the NS, SSAD-CA, EMD and Control groups of this invention; Figure 15A The HE staining results of the SSAD-CA group, EMD group and Control group of this invention; Figure 15B The statistical results of HE staining in the SSAD-CA group, EMD group and Control group of this invention; Figure 16 The results show the root resorption rates of the SSAD-CA group, EMD group, and Control group of this invention. Detailed Implementation

[0034] 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.

[0035] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0036] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0037] 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%.

[0038] 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.

[0039] Definition of the noun: ABTS: [=2,2'-amino-di(2-ethyl-benzothiazoline sulphonic acid-6)ammonium salt] 2,2'-amino-di(3-ethyl-benzothiazoline sulfonic acid-6)ammonium salt; DPPH: diphenyl picryl hydrazinyl radical, 1,1-diphenyl-2-trinitrophenylhydrazine or 1,1-diphenyl-2-picrylhydrazine; LB broth: Lysogeny Broth (often called Luria-Bertani medium); the most commonly used liquid bacterial culture medium in molecular biology and microbiology laboratories, mainly used for rapid amplification of E. coli, plasmid extraction, protein expression, etc. 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.

[0040] Example: The application of the acidic hydrolysate of the giant salamander mucus extract in the preparation of a root treatment agent, wherein the root treatment agent comprises a product obtained by reducing lyophilized powder of salamander skin secretions with an acidic solution. The concentration of the acidic solution is 0.2-0.3 mol / L, the pH value is 1-5, and the mass ratio of the lyophilized salamander skin secretions to the acidic solution is (1-3):1. After adjustment with an alkaline solution, the pH value of the resulting root treatment agent is 5.5-6.5. The acidic solution comprises an aqueous solution of citric acid and / or an aqueous solution of vitamin C and / or a solution of tris(2-carboxyethyl)phosphonic acid hydrochloride. The product comprises five active proteins: MHCIA class α-chain, Hox-D13 homologous box protein, axonoderm dynamin abnormal heavy chain 3, ATP synthase protein, and cytochrome b.

[0041] In some specific embodiments, the acidic solution is an aqueous solution of citric acid with a pH of 2-3. The root treatment agent obtained after adjustment with an alkaline solution has a pH of 6.4.

[0042] In some specific embodiments, the root surface treatment agent further includes excipients, which include gel excipients, thickening / thixotropic agents, and medical matrix excipients. 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 polycarboxylate, polyvinyl alcohol (PVA), and chitosan.

[0043] 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. 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.

[0044] 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.

[0045] In some specific embodiments, the preparation method of the root surface treatment 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 salamander skin secretions and an acidic solution to a second solvent, mix well to obtain a first mixture, and let the first mixture stand at 0~8℃ for 12~100h to obtain a second mixture; step S2 also includes adding an alkaline solution to the first mixture to adjust its pH value to 5.5~6.5; the ratio of the freeze-dried powder of salamander skin secretions to the second solvent in step S2 is 1g:10mL; the particle size of the salamander skin secretions is 1~50μm; S3: Centrifuge the second mixture and collect the supernatant, which is the root treatment 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. Both the first solvent and the second solvent are double-distilled water. In some specific embodiments, in step S3, an excipient is added to the collected supernatant and mixed evenly to obtain a root surface treatment agent containing the excipient.

[0046] In some specific embodiments, when 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 alkaline solution in step S2 is sodium hydroxide or potassium hydroxide, and the pH value of the first mixture is adjusted to 6.4; in step S3, the mixture is centrifuged at 1000~8000 rpm for 10 min.

[0047] In some specific embodiments, step S3 involves centrifugation at 4000 rpm for 10 minutes.

[0048] Specific Example 1: This example provides a method for preparing SSAD-CA root treatment agent. The specific preparation process is as follows: Figure 1 As shown.

[0049] 1) Obtaining the skin mucus of the giant salamander Healthy adult Chinese giant salamanders (50-110cm in length, 3.0±0.5kg in weight) aged 5-7 years were selected according to standard procedures and cleaned. Afterward, their dorsal skin was repeatedly stimulated using gentle mechanical stimulation methods (such as electrical stimulation and skin scraping) to induce mucus secretion. No harm was caused to the animals throughout the process. The secreted white mucus was collected in 50mL centrifuge tubes and stored. 20℃ refrigerator; The healthy adult Chinese giant salamanders selected were all from the China Giant Salamander Breeding Base in Chongqing, China, and were all artificially bred individuals. All experiments were strictly carried out in accordance with the "Wildlife Protection Law of the People's Republic of China", and no Chinese giant salamanders were euthanized 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.

[0050] 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 the ground product was then sieved through a 300-mesh sieve to obtain 300-mesh SSAD powder. This powder was then properly sealed and stored in a sealed container. It was placed in a refrigerator at 20°C for use in subsequent research.

[0051] 3) Preparation of SSAD-CA root treatment agent: To prepare a citric acid (CA) solution, 1.25g CA is required for 25mL of solution. Add 2500mg SSAD powder and 1.25g CA to 25mL of DD water, and adjust the pH to 6.4 by adding NaOH dropwise. Mix thoroughly and let stand at 4℃ for 48 hours. Centrifuge at 4000rpm for 10 minutes, and collect the supernatant. This supernatant is the liquid root treatment agent SSAD-CA. The actual product is shown below. Figure 2 As shown.

[0052] The root surface treatment agent SSAD-CA prepared in Specific Example 1 was characterized by dynamic light scattering (DLS), infrared spectroscopy (IR), and Raman spectroscopy (RS).

[0053] (a) Materials and Instruments The materials used for characterization are shown in Table 1, and the instruments are shown in Table 2.

[0054] Table 1. Materials used in characterization Table 2. Instruments used for characterization. (II) Experimental methods and procedures 2.1 DLS detection of particle potential and particle size: For comparison, three sets of samples were prepared: the first set was the traditional SSAD freeze-dried powder (SSAD-P), the second set was the SSAD water-soluble protein solution (SSAD-S), and the third set was the root surface treatment agent SSAD-CA (SSAD-CA) prepared in Specific Example 1.

[0055] The first group of traditional SSAD freeze-dried powder preparation involved washing healthy male Chinese giant salamanders aged 3 years (3-6 years, body length 65-110 cm, weight 2.8 ± 0.7 kg) with clean water. Under mild mechanical stimulation, mucus secretion from the dorsal skin was induced and collected in clean tubes. After collection, the SSAD was washed with sterile PBS, shaken, and centrifuged. The SSAD solution was freeze-dried for 24 hours, ground into powder, and separated into different particle sizes (e.g., 60, 100, 300 mesh). To obtain relatively uniform lumps, the powder was passed through sieves of appropriate sizes. Powder that did not pass through smaller sieve sizes was desired in this invention. Higher sieve mesh values ​​correspond to smaller sieve aperture sizes, and therefore, smaller particle sizes. The SSAD-derived powder was stored in… 20°C until further use. The skin is an important respiratory organ for the salamander, containing mucous glands and granular glands. The reason for choosing the Chinese giant salamander's mucus skin extract in this invention is that it represents the mucus secreted by amphibians like the salamander when stimulated. Electrical stimulation and skin scraping can promote mucus secretion.

[0056] The second group of SSAD water-soluble protein solutions was prepared by dissolving SSAD lyophilized powder in water, and the preparation method and proportion were the same as those used in the preparation of SSAD-CA in Specific Example 1.

[0057] The first and second groups serve as a control group.

[0058] Instrument Testing: Close the instrument cover, turn on the instrument, and wait for the indicator light to turn red and flash; start the Zetasizer software and wait for the indicator light to turn green; preheat the machine for 30 minutes; inject the prepared equal amounts of sample into the sample cell. Select the SOP mode (this SOP standardizes the entire process of dynamic light scattering (DLS) particle size measurement and electrophoretic light scattering (ELS) zeta potential measurement, applicable to suspension systems such as nanoparticles, colloids, and emulsions, ensuring accurate and traceable data, and complying with ISO 22412:2025 and GB / T 29022-2021 standards); insert the three sets of sample cells to be tested into the instrument, click test, wait for temperature equilibrium before measuring, and save and analyze the results after the test is completed.

[0059] 2.2 FTIR detection of protein structure: Sample preparation: The first group is the traditional SSAD freeze-dried powder (hereinafter referred to as SSAD-P), the second group is the freeze-dried SSAD water-soluble protein solution (hereinafter referred to as SSAD-S), and the third group is the root surface treatment agent SSAD-CA prepared in Specific Example 1, which is freeze-dried (hereinafter referred to as SSAD-CA). Take 10mg of powder from each of the three groups of samples.

[0060] Adjust test parameters: Set the FTIR instrument to transmission recording mode to accurately acquire the spectral information of the target substance. In the data recording stage, set the spectral data to be recorded in absorbance units, serving as the basis for quantitative analysis. For the wavenumber range, set it to 400–4000 cm⁻¹. -1 This 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 effectively improves experimental efficiency while ensuring data accuracy and clearly presenting spectral details. The number of scans was set to 16.

[0061] Collect and analyze spectra: All FTIR spectra were collected at room temperature, i.e., the background was collected first, and then the FTIR spectra of the samples were collected. The spectra were then analyzed using Origin 2010 software.

[0062] 2.3 RS detection of protein chemical bonds: Sample preparation was the same as for FTIR protein structure detection, with 10 mg of powder from each of the three sample groups.

[0063] Experimental Method: The three sets of samples to be tested were placed on a glass slide, and the slide was moved below the test lens for optical focusing. A second laser focusing was then performed to achieve good photon collection. A 633 laser was selected and switched to an appropriate power level for trial testing. Based on the test results, the integration time and grating size were set. The test button was clicked, and the test results were exported and analyzed using Origin 2010.

[0064] 2.4 Protein composition analysis using proteomic proteometry: Sample preparation was the same as for FTIR protein structure detection, with 10 mg of powder from each of the three sample groups.

[0065] Peptide digestion: Add 40 μL of trypsin buffer (where trypsin is EC 3.4.21.4) to the sample and incubate at 37°C for 16-18 h.

[0066] (1) Chromatographic separation: Solution A in the liquid phase 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 (accounting for 84%). A liquid chromatography column with a specification of 0.15mm×150mm was used in this experiment.

[0067] 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 Zorbax 300SB-C18 peptide traps column via an autosampler, and then the samples were separated by liquid chromatography. The liquid chromatography gradient was dynamically adjusted according to the set parameters: from 0 min to 50 min, the proportion of solution B gradually increased from 4% to 50% in a linear gradient. This gradient change was designed to achieve initial 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, from 50 min to 54 min, 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 components and improving the separation effect. Finally, solution B is maintained at 100% concentration for 54 to 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.

[0068] (2) Mass Spectrometry Identification: The enzymatic digestion products were first separated by high-performance liquid chromatography using capillary chromatography, followed by mass spectrometry analysis using a QExactive mass spectrometer. The entire analysis process lasted for 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 were acquired immediately after each full scan (MS2 scan). The full scan can obtain 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.

[0069] (3) Data Analysis: After the mass spectrometry test is completed, the obtained raw files are 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 are finally generated. The relevant parameters and detailed descriptions involved in the database search process are shown in Table 3. In this invention, a Caudata database is 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.

[0070] Table 3. Relevant parameters involved in database lookup. (III) Experimental Results 3.1 Dynamic Light Scattering Analysis To detect structural changes in the SSAD-CA solution, dynamic light scattering (DLS) was used. DLS is a technique for measuring the motion of nanoparticles and macromolecules in liquids. DLS determines particle size by analyzing fluctuations in the intensity of light scattered by particles in a suspension. Combined with electrophoretic light scattering, it can also be used to measure the velocity of particles moving in an electric field, thereby calculating the particle potential (typically the zeta potential). The zeta potential is an important parameter describing the surface charge characteristics of particles, reflecting their stability and interaction capabilities in liquids. Results are as follows... Figures 3A-3C As shown, Figure 3A The particle size of SSAD-CA obtained was 2029±305.50 nm. Figure 3B The particle size of SSAD-S obtained was 46.93±25.86 nm. Figure 3C The particle size of SSAD-P obtained was 22.70 ± 3.28 nm. Compared with the particle size of SSAD-S and SSAD-P, the particle size of SSAD-S increased by tens of times, which indicates that the treatment of SSAD with CA may lead to enhanced interactions between protein molecules.

[0071] At the same time from Figure 4 Analysis of the potential results shows that the Zeta potential of SSAD-P and SSAD-S solutions is below 10 mV, while the Zeta potential of SSAD-CA solution is between 20 and 30 mV. Therefore, SSAD-CA is more stable than SSAD-P and SSAD-S.

[0072] 3.2 FTIR Analysis Results: To analyze the secondary structure changes of the protein in SSAD-CA, Fourier transform infrared spectroscopy was used for detection. The proportions of each structure were calculated by observing the absorption peak positions in the infrared spectra and combining this with the positions of known characteristic absorption peaks of secondary structures after peak fitting. Figure 5A Infrared spectrum of SSAD-CA group, Figure 5B The infrared spectrum of the SSAD-S group. Figure 5C Infrared spectra of the SSAD-P group. (Source: [Insert Source Here]) Figures 5A-5C The results show that in the SSAD-CA group, the proportion of β-sheets and random coils in the protein secondary structure increased. A deeper analysis from the perspective of the relationship between protein structure and function suggests that this structural change likely leads to enhanced intermolecular interactions. The underlying reason is that β-sheets can bring polypeptide chains closer together and form a more ordered arrangement through intermolecular hydrogen bonds, while random coil regions, lacking a fixed secondary structure pattern, possess relatively high flexibility and variability. Both types of structural regions typically play crucial roles in protein aggregation.

[0073] 3.3 RS analysis results: Raman spectroscopy was used to detect changes in the chemical bonds, side chains, and local conformation of the SSAD-CA protein. Results are as follows: Figures 6A-6C As shown, Figure 6A It can be seen from 3400cm -1 The increased peak height is related to the stretching vibrations of NH and OH, indicating an increase in the number of hydrogen bonds in SSAD-CA; Figure 6B It can be seen from 1200-1300cm -1 The increased peak height is related to the stretching vibration of CN and the variable angle vibration of NH, and this change may be related to the increase in β-folding and random curling. Figure 6C It can be seen from this that 1550-1600cm -1 The increase in peak height is related to the C-C and CN stretching vibrations of aromatic amino acid side chains and peptide bonds. This change may be related to the increase in β-sheet and random coil of aromatic amino acids, while the peak height at 1514 cm⁻¹ is related to the increase in β-sheet and random coil of aromatic amino acids. -1 The decrease in peak intensity is related to the NH bending vibration and CN stretching vibration of the amide II band, indicating a change in the protein's amide bond environment, which is associated with the transformation of the α-helix structure into a β-sheet and random coiling. (1000cm) -1 1003cm -1 The increase in peak height is related to the CH bending vibration or C-C stretching vibration of the benzene ring, indicating a change in the environment of aromatic amino acids. (1300cm) -1 and 1420cm -1The increase in peaks is related to an increase in the symmetric vibrations of COO-, which (carboxylate group) is a negatively charged hydrophilic group. When protein molecules have more COO- groups, these negative charges can enhance the interaction between the protein and water molecules, thereby improving the protein's solubility.

[0074] 3.4. Protein spectroscopy analysis results: Protein concentration of SSAD-CA was detected and analyzed by mass spectrometry. The results are as follows: Figure 7B As shown, the concentration of dissolved proteins increased more than twofold in citric acid-treated solutions compared to untreated solutions. SSAD-P detected 57 proteins, SSAD-S detected 45 proteins, and the citric acid-treated SSAD-CA solution detected 49 proteins, representing an increase in detectable proteins compared to the SSAD-S solution directly dissolved in water. Figure 7A 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 8 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 4 shows that the proportion of dissolved regeneration-related protein peptides Fibroblast growth factor receptor 2 (FGFR-2), Thrombospondin-1, and N-Cadherin increased in the SSAD-CA group; the proportion of dissolved antibacterial protein Lysozyme G (EC 3.2.1.17) increased; the proportion of dissolved antioxidant protein Heat shock protein 70 increased; and the proportion of anti-inflammatory protein Interferon-induced GTP-binding protein Mx3 increased.

[0075] Table 4. Coverage of relevant protein peptides (%) Note: Underlined lines indicate that the relevant protein has the highest peptide coverage in SSAD-CA.

[0076] Specific Example 2: Multi-effect Characterization of SSAD-CA Root Treatment Agent Material The SSAD-CA root surface treatment agent prepared in Specific Example 1 was characterized by multiple effects, including rheometer testing of viscosity, free radical scavenging test to test antioxidant properties, SEM imaging of SSAD-CA-treated extracted tooth root sections, water antennal test to test hydrophilicity, and SEM analysis of the effect of SSAD-CA in vitro mineralized dentin sections.

[0077] I. Experimental Materials and Instruments The materials involved in this embodiment are shown in Table 5, and the instruments involved are shown in Table 6.

[0078] Table 5. Materials used in characterization Table 6. Instruments used for characterization II. Experimental Methods and Procedures 2.1 Rheometer testing of viscosity: Sample preparation: The samples are divided into three groups. Prepare equal amounts of SSAD-CA, PBS, and deionized water (blank control group, i.e., Control group) to be tested. There are 3 samples in each group to ensure that the samples are uniform and free of air bubbles.

[0079] Instrument check: Ensure the instrument is in good working order, and check the power supply, cables, and measuring components. Clean the measuring components to ensure there are no residues.

[0080] Installation and Setup: Select the measurement assembly, use the CP25 rotor, and install it onto the instrument. Spread the sample evenly between the measurement assemblies, ensuring there are no air bubbles. Set the temperature to 25°C and wait for it to stabilize.

[0081] Test: Select the test template "Viscosity Curve", set the default parameters, and perform the test.

[0082] 2.2 Free radical scavenging experiment to detect antioxidant activity: ABTS free radical scavenging assay: ABTS was dissolved in phosphate-buffered saline (PBS) at pH 7.4, with a concentration of approximately 7 mM. The ABTS solution was then mixed with 3% hydrogen peroxide (H₂O₂) at a volume ratio of 1:1 to generate ABTS free radicals. Vitamin C was used as a standard solution. The standard solution was reacted with ABTS free radicals, and the absorbance was measured to plot a standard curve. Test sample solutions (SSAD-CA, EMD) were mixed with ABTS free radicals at different concentrations and reacted for 10 min. During the reaction, the antioxidants in the samples reacted with ABTS free radicals, reducing the number of free radicals. The absorbance was measured at 734 nm, and the absorbance value of each sample was recorded. Antioxidant activity was calculated as follows: Free radical scavenging rate (%) = [1 - (Sample A / AABTS control)] × 100, where Sample A is the absorbance of the sample well, and AABTS control is the absorbance of the ABTS control group.

[0083] DPPH free radical scavenging assay: DPPH was dissolved in ethanol to prepare a 0.1 mM working solution. The test samples (SSAD-CA group, EMD group), blank control group (solvent only, no sample or DPPH), and DPPH control group (DPPH solution only, no sample) were prepared in 96-well plates. The mixed samples were placed at room temperature in the dark for 30 min to allow the antioxidants in the samples to react with DPPH free radicals. The absorbance of each well was measured at 517 nm. The free radical scavenging rate was calculated as follows: Free radical scavenging rate (%) = [1 - (A sample / ADPPH control)] × 100, where A sample is the absorbance of the sample well, and ADPPH control is the absorbance of the DPPH control group.

[0084] 2.3 Water antennal probe test for hydrophilicity: Root section preparation: The third molar of a healthy person was obtained clinically and a 1cm×1cm×0.5cm root bone section was prepared along the root.

[0085] Surface treatment and testing: After 3 days of treatment with SSAD-CA (SSAD-CA group), EMD (EMD group, an enamel matrix derivative, commercially available Emdogain® root surface treatment agent), and physiological saline (NS group), the hydrophilicity changes of the root bone surface were detected using a water antennal spectrometer.

[0086] III. Analysis of Experimental Results 3.1 Viscosity test results Experimental results are as follows Figure 9As shown, the viscosity was measured by a rheometer. The results showed that the SSAD-CA group had a viscosity of 12447.33±1342 mPa·s, which was about 36 times higher than the PBS group (348.89±33.90 mPa·s), 49 times higher than DD water (255.58±40.56 mPa·s), and 3-4 times higher than the 3000-4000 mPa·s reported in the literature for commercially available Emdogain®.

[0087] 3.2 Free radical scavenging experiment: Excessive reactive oxygen species (ROS) exist in the periodontal injury environment, leading to cell damage and inflammation, hindering the healing process. Materials with antioxidant properties can effectively neutralize these ROS, reduce oxidative stress, thereby protecting periodontal ligament cells, promoting cell proliferation and migration, and enhancing tissue regeneration. Furthermore, antioxidant materials can improve the local microenvironment, promote angiogenesis, and further support periodontal ligament healing. For example... Figure 10A As shown, the left side is a statistical chart, and the right side is a photograph of sample changes during the clearance process. The clearance rate of BTS in the SSAD-CA group was as high as 76.36%, while the clearance rate in the EMD group was 45% ± 0.04%, and the difference was statistically significant. Figure 10B As shown, the left side is a statistical chart, and the right side is a photograph of the sample changes during the clearance process. The clearance rate of DPPH in the SSAD-CA group was 74.37%±0.04, and the clearance rate in the EMD group was 45.3%±0.03, with statistically significant differences.

[0088] 3.3 Contact angle test results: The water contact angle reflects the surface wettability and hydrophilicity of a material. A water contact angle <90° generally indicates... The surface of the material exhibits good hydrophilicity, which facilitates cell adhesion and growth, thereby promoting periodontal regeneration. Conversely, a water contact angle >90° may indicate a more hydrophobic surface, potentially leading to poor cell adhesion and affecting the restorative outcome. Results are as follows... Figure 11A and Figure 11B As shown, from Figure 11A It can be clearly seen that the water contact angles of the SSAD-CA group and the EMD group are greater than the water contact angle of Ns; from Figure 11B The results showed that the water contact angle of the SSAD-CA group was 70.18±5.73° and that of the EMD group was 79.27±1.16°, both of which increased the water contact angle of the root surface compared to the Ns group (47.44±8.96°). However, the root surface remained hydrophilic (<90°), suggesting that the SSAD-CA obtained after CA treatment may not directly change the properties of the root surface to promote cell adhesion, but rather that its active proteins mainly play a role in promoting cell adhesion, thereby promoting periodontal regeneration.

[0089] Specific Implementation Example 3: Verification of the periodontal regeneration effect of SSAD-CA and its clinical application using a large animal model.

[0090] Beagles are ideal large animal models for reimplantation experiments because their tooth structure and periodontal tissue physiological characteristics are highly similar to those of humans, allowing them to effectively simulate the actual clinical situation of tooth reimplantation. Therefore, this invention uses beagles to construct a simulated clinical reimplantation model, aiming to further verify the effect of SSAD-CA on promoting periodontal regeneration in animals, as studied in previous research. By rigorously simulating clinical procedures, this study provides a more comprehensive evaluation of the role of SSAD-CA in a physiological environment closer to that of humans, offering a more solid and reliable experimental basis for its eventual application in clinical tooth reimplantation treatment.

[0091] I. Experimental Instruments and Materials: The materials used in this embodiment are shown in Table 7, and the experimental instruments used are shown in Table 8.

[0092] Table 7 Materials used Table 8. Instruments Used II. Experimental Methods and Procedures 2.1 Beagle animal experiments: In this invention, a delayed replantation model for beagle dogs was successfully constructed, such as... Figure 12 The experiment was conducted to investigate the effects of tooth replantation. Six 10-month-old beagles (average weight 10.2g) were used in the experiment. All six were obtained from Zhenjiang Wanwei Experimental Animal Breeding Co., Ltd., and were housed in a strictly monitored free-range environment at the Institute of Traditional Chinese Medicine Preparations, Chongqing Academy of Traditional Chinese Medicine, to ensure the accuracy and reliability of the experiment. These beagles had free access to food and water to maintain their health. Prior to the experiment, all animal experimental protocols were approved by the Ethics Committee of the Institute of Traditional Chinese Medicine Preparations, Chongqing Academy of Traditional Chinese Medicine (DSW20232-04) to ensure that the experimental process met ethical standards. During the experiment, we used combined intravenous and inhalation anesthesia, with professional veterinary assistance during the anesthesia and surgical procedure, to ensure that the beagles did not experience pain during the surgery.

[0093] Experimental groups: The experiment was conducted on 12 upper and lower anterior teeth of each dog. After tooth extraction, root canal treatment was performed according to standard clinical procedures, and physiological saline was placed in vitro for 25 minutes. Before implantation, the teeth were treated with SSAD-CA (SSAD-CA group), EMD (EMD group), or physiological saline (NS group) for 5 minutes. The untreated group served as the control group for comparison.

[0094] Tooth extraction: During the surgery, canine anterior teeth are extracted using a canine anterior tooth extraction elevator and stored in normal saline solution.

[0095] Root canal treatment: A hole is made in the anterior tooth requiring treatment using a dental drill to access the pulp chamber. Infected pulp tissue is removed from the pulp chamber. The root canal is cleaned and enlarged using tools such as root canal files to facilitate disinfection and filling. The length of the root canal is determined using a root canal measuring instrument. Gutta-percha points are dipped in i-Root paste, and the root canal is filled with disinfectant. The opening is restored with resin, and the occlusion is appropriately lowered.

[0096] Root surface treatment: Before implantation, the tooth is treated with SSAD-CA, EMD, and normal saline for 5 minutes. After treatment, the alveolar socket is gently rinsed with saline, and then the treated tooth is re-implanted into its original position.

[0097] Fixation: To fix the replanted tooth, we use periodontal fiber bandage to fix the surgical tooth and the two adjacent teeth.

[0098] Sampling: In the fourth week of the experiment, we collected samples from three groups and assessed the rate of tooth loosening and loss in detail during the sampling period.

[0099] 2.2 CBCT and X-ray examination: Extracted teeth were performed at the Department of Radiology, Affiliated Stomatological Hospital of Chongqing Medical University, after following the proper application process. CBCT and X-ray imaging are used to assess alveolar bone and root resorption. Thorough sterilization is performed during imaging to ensure no cross-contamination occurs.

[0100] 2.3. Histological analysis of HE sections: Material collection: After the experiment, the dogs were euthanized by the Institute of Traditional Chinese Medicine Preparations, Chongqing Academy of Traditional Chinese Medicine, according to relevant standard procedures. The upper and lower jawbones with teeth were removed.

[0101] Fixation: The removed tooth and surrounding tissue were fixed in a 10% neutral buffered formaldehyde solution for 24 hours.

[0102] Decalcification: Decalcify in Plank-Rychlo decalcification solution for 6 months, and place on a shaker at room temperature during decalcification.

[0103] Dehydration: The fixed sample was dehydrated sequentially by passing it through 70%, 80%, 90% and 100% ethanol solutions, with each concentration held for 1 hour.

[0104] Transparency: The dehydrated sample is placed in xylene for transparency treatment to remove residual ethanol.

[0105] Embedding: A transparent sample is placed into an embedding mold, and molten paraffin is poured in. After it cools and solidifies, an embedding block is formed.

[0106] Sectioning: Use a microtome to cut the embedded block into 6-micrometer-thick slices. Attach the slices to a glass slide, ensuring they are flat.

[0107] Dewaxing: The sections are placed in xylene for dewaxing, and then rehydrated with a series of ethanol.

[0108] Staining: Hematoxylin was used for nuclear staining, and eosin was used for cytoplasmic staining. Specifically, after passing through distilled water for 5 seconds, hematoxylin was stained for 6 minutes, followed by rinsing with water for 1 minute, then hydrochloric acid alcohol for 1 second, rinsing with running water for 15 minutes, and finally eosin staining for 6 minutes.

[0109] Mounting and observation: Sections were sequentially treated with 70%, 80%, and 90% alcohol for 1 second each, followed by 95% alcohol for 5 minutes, and then 100% alcohol I and 100% alcohol II for 5 minutes each. After dehydration, the sections were cleared by treating with xylene I and xylene II for 5 minutes each. Finally, neutral resin was used for mounting. After the mounting medium dried, the tissue morphology and cell structure were observed and recorded under an optical microscope. ImageJ image analysis software was used to measure the periodontal ligament width and count the root resorption rate (periodontal ligament width ratio = experimental group measured width / average measured width of normal group; root resorption was counted by dividing the complete root cross-section into 12 equal parts as 12 statistical points, and if root resorption was observed at a point, it was counted according to the type).

[0110] III. Experimental Results and Analysis 3.1 Results of tooth mobility and loss rate testing in Beagle dogs Results of front tooth mobility in Beagle dogs Figure 13A As shown, there was no statistically significant difference between the SSAD-CA group and the EMD group, but the SSAD-CA group had a significantly lower droplet rate than the Conrtol group. The droplet rate results are as follows: Figure 13B The results showed that SSAD-CA (8%) was 8% lower than the EMD group (16%) and 25% lower than the Control group (33%). Although there was no statistically significant difference, the SSAD-CA group showed a trend toward reduced shedding rate.

[0111] 3.2 Beagle CBCT (cone-beam computed tomography / oral and maxillofacial cone-beam computed tomography) and X-ray results CBCT and X-ray images showed that all teeth underwent complete root canal filling after surgery. Figure 14A The images shown are the frontal and lateral X-ray results of implantation after SSAD-CA treatment following anterior tooth avulsion in a beagle dog; Figure 14B The X-ray results for the NS group (treated with saline only), SSAD-CA group, EMD group, and Control group (i.e., the blank control group, which received no treatment) are as follows: Figure 14A and Figure 14BAs can be seen, there was no significant resorption of the periapical alveolar bone and the periodontal ligament was intact after treatment in the SSAD-CA group and the EMD group. However, the Conrtol group showed more severe resorption of the periapical alveolar bone and root, and the periodontal ligament space was widened, indicating inflammation.

[0112] 3.3 Histological analysis of sections HE staining revealed that the periodontal ligament morphology formed after SSAD-CA treatment was more complete. Statistical analysis of the periodontal ligament width ratio showed the following results: Figure 15A and Figure 15B The results show that there was no statistically significant difference between the SSAD-CA group (0.63±0.24) and the EMD group (0.66±0.19), but the SSAD-CA group was significantly larger than the Contro group (0.34±0.26).

[0113] Statistical results of tooth root resorption rate are as follows Figure 16 As shown, there was no statistically significant difference in surface absorption rate between the SSAD-CA group (0.14±0.07), the EMD group (0.09±0.04), and the Control group (0.18±0.06). However, the inflammatory absorption rate of the SSAD-CA group (0.03±0.07) was significantly lower than that of the EMD group (0.17±0.08) and the Control group (0.35±0.12). All three groups showed some degree of bone resorption: SSAD-CA group (0.03±0.04), EMD group (0.08±0.11), and Control group (0.06±0.09), but the results were not statistically significant.

[0114] In summary, the SSAD-CA group promotes periodontal regeneration by maintaining the integrity of the periodontal ligament structure and inhibiting inflammatory root resorption. Its efficacy is comparable to, and even surpasses, commercially available EMDs in terms of inflammation control. Therefore, the root surface treatment agent prepared in this invention provides a potential novel bioactive treatment option for clinical tooth replantation treatment.

[0115] 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.

[0116] 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. The application of an acidic hydrolysis product of giant salamander mucus extract in the preparation of a root surface treatment agent.

2. The application according to claim 1, characterized in that, The root treatment agent comprises a product obtained by reducing salamander skin secretions with an acidic solution, wherein the concentration of the acidic solution is 0.2~0.3 mol / L, the pH value is 1~5, and the mass ratio of salamander skin secretions to the acidic solution is (1~3):1; the root treatment agent obtained after adjustment with an alkaline solution has a pH value of 5.5~6.

5.

3. The application according to claim 2, characterized in that, 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)phosphonic acid hydrochloride; the product includes five active proteins, namely: the α chain of MHCIA class molecules, the homeobox protein Hox-D13, the axonoderm dynamin abnormal heavy chain 3, ATP synthase protein, and cytochrome b.

4. The application according to claim 2, characterized in that, The acidic solution is an aqueous solution of citric acid with a pH of 2-3.

5. The application according to claim 2, characterized in that, The root surface treatment agent also includes excipients, which include gel excipients, thickening / thixotropic agents and medical matrix excipients. 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 polycarboxylate, polyvinyl alcohol (PVA), and chitosan.

6. The application according to claim 4, characterized in that, The preparation method of the root surface treatment agent 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 salamander skin secretions and acidic solution to the second solvent, mix well to obtain the first mixture, 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 root treatment 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.

7. The application of the root treatment agent according to claim 6, characterized in that, Step S2 further includes adding an alkaline solution to the first mixture to adjust the pH value of the first mixture to 6-6.

5.

8. The application according to claim 6, characterized in that, 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 0.2~0.3mol / L and a pH of 2~3, the ratio of the freeze-dried salamander skin secretion powder to the second solvent in step S2 is 1g:10mL; the particle size of the salamander skin secretion is 1~50μm.

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.

4.

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

Images