Wnt5b promotes tooth differentiation biological product and its application

Abstract

The application provides a WNT5B-promoting tooth differentiation biological product and an application thereof. Compared with the prior art, the application has the following advantages: the patent integrates human embryonic tooth germ spatial transcriptome sequencing and epithelial cell and interstitial cell extracellular protein group sequencing data, screens signal molecules WNT5B and CTNNB1 secreted in the development process of tooth germ epithelium, and confirms the important role of WNT5B protein in tooth germ development for the first time. Compared with other WNT family members, WNT5B has stronger tooth-forming ability of promoting tooth-derived stem cells; the effective concentration of WNT5B is low, and 10 ng / ml is the optimal concentration of WNT5B for promoting tooth-derived stem cells to form teeth; and the role of WNT5B and CTNNB1 in extracellular secretion of tooth germ epithelial cells and the synergistic effect of WNT5B and CTNNB1 in promoting tooth differentiation of tooth germ mesenchymal cells are further determined, that is, the combined low-concentration use effect of the two is better than the single high-concentration use.

Description

Technical Field

[0001] The present application relates to the technical field of tooth development and tooth regenerative medicine, and in particular to a WNT5B-promoting tooth differentiation biological product and its application. Background Art

[0002] Tooth development is a finely regulated biological process involving complex interactions between epithelial and mesenchymal cells. These interactions depend on a variety of signaling molecules, including growth factors, cytokines, and WNT proteins. The WNT signaling pathway plays a key role in many biological processes, especially in embryonic development, tissue regeneration, and tumorigenesis. During tooth development, WNT signaling molecules are secreted by epithelial cells and affect the proliferation, differentiation, and migration of mesenchymal cells. Although studies have identified the roles of multiple WNT family members in tooth development, the exact role and mechanism of WNT5B protein, as a signaling molecule secreted by epithelial cells, in tooth germ development is still not fully understood.

[0003] Currently, one of the major challenges facing dental regenerative medicine is how to mimic and restore the natural signaling pathways during tooth development. Although some therapeutic approaches have attempted to utilize the WNT signaling pathway to promote tooth regeneration, these approaches generally lack a deep understanding of the role of specific WNT members in tooth development, resulting in limited therapeutic effectiveness.

[0004] In addition, model systems commonly used in tooth development research, such as mouse and pig tooth development samples, although providing valuable information, have certain limitations in analyzing the functions and regulatory mechanisms of specific signaling molecules. In particular, during human tooth development, how signaling molecules secreted by epithelial cells precisely regulate the behavior of mesenchymal cells remains an unresolved issue. In recent years, with the development of proteomics and cell biology technologies, researchers have begun to be able to more comprehensively analyze and identify signals secreted by human tooth germ epithelial cells. However, most of these studies have focused on protein expression patterns and functional predictions, and in-depth research on the regulatory role and application potential of specific signaling molecules such as WNT5B in tooth germ development is still insufficient. Summary of the Invention

[0005] In response to the above-mentioned technical limitations, this application identifies and analyzes the exocrine proteome of human embryonic tooth germ epithelial cells, especially the WNT5B protein, to reveal its potential role in tooth germ development; through in-depth research on the function and regulatory mechanism of WNT5B, this application provides new strategies and methods for dental regenerative medicine, and proposes a WNT5B-promoting tooth differentiation biological product and its application; it overcomes the deficiencies and defects mentioned in the background technology.

[0006] To achieve the above objectives, this application adopts the following technical solutions:

[0007] The invention of the present application is to provide a biological preparation containing WNT5B recombinant protein.

[0008] Optionally, the above-mentioned biological preparation further contains CTNNB1 recombinant protein (Catenin Beta1).

[0009] Optionally, in the above-mentioned biological preparation, the amino acid sequence of the WNT5B recombinant protein is shown in SEQ ID No.1, and the amino acid sequence of the CTNNB1 recombinant protein (Catenin Beta 1) is shown in SEQ ID No.2; the nucleotide sequence encoding the WNT5B recombinant protein is shown in SEQ ID No.3, and the nucleotide sequence encoding the CTNNB1 recombinant protein (Catenin Beta 1) is shown in SEQ ID No.4.

[0010] SEQ ID No. 1:

[0011] MPSLLLLFTAALLSSWAQLLTDANSWWSLALNPVQRPEMFIIGAQPVCSQLPGLSPGQRKLCQLYQEHMAYIGEGAKTGIKECQHQFRQRRWNCSTADNASVFGRVMQIGSRETAFTHAVSAAGVVNAISRACREGELSTCSRTARPKDLPRDWLWGGCGDNVEYGYRFAKEFVDARE REKNFAKGSEEQGRVLMNLQNNEAGRRAVYKMADVACKCHGVSGSCSLKTCWLQLAEFRKVGDRLKEKYDSAAAMRVTRKGRLELVNSRFTQPTPEDLVYVDPSPDYCLRNESTGSLGTQGRLCNKTSEGMDGCELMCCGRGYNQFKSVQVERCHCKFHWCCFVRCKKCTEIVDQYICK;

[0012] SEQ ID No. 2:

[0013] MATQADLMELDMAMEPDRKAAVSHWQQQSYLDSGIHSGATTTAPSLSGKGNPEEEDVDTSQVLYEWEQGFSQSFTQEQVADIDGQYAMTRAQRVRAAMFPETLDEGMQIPSTQFDAAHPTNVQRLAEPSQMLKHAVVNLINYQDDAELATRAIPELTKLLNDEDQVVVNKAAVMVHQLSKKEASRHAIMRSPQMVSAIVRTMQNTNDVETARCTAGTLHNLSHHREGLLAIFKSGGIPALVKMLGSPVDSVLFYAITTLHNLLLHQEGAKMAVRLAGGLQKMVALLNKTNVKFLAITTDCLQILAYGNQESKLIILASGGPQALVNIMRTYTYEKLLWTTSRVLKVLSVCSSNKPAIVEAGGMQALGLHLTDPSQRLVQNCLWTLRNLSDAATKQEGMEGLLGTLVQLLGSDDINVVTCAAGILSNLTCNNYKNKMMVCQVGGIEALVRTVLRAGDREDITEPAICALRHLTSRHQEAEMAQNAVRLHYGLPVVVKLLHPPSHWPLIKATVGLIRNLALCPANHAPLREQGAIPRLVQLLVRAHQDTQRRTSMGGTQQQFVEGVRMEEIVEGCTGALHILARDVHNRIVIRGLNTIPLFVQLLYSPIENIQRVAAGVLCELAQDKEAAEAIEAEGATAPLTELLHSRNEGVATYAAAVLFRMSEDKPQDYKKRLSVELTSSLFRTEPMAWNETADLGLDIGAQGEPLGYRQDDPSYRSFHSGGYGQDALGMDPMMEHEMGGHHPGADYPVDGLPDLGHAQDLMDGLPPGDSNQLAWFDTDL;

[0014] SEQ ID No.3:

[0015] atgccgagcctgctgctgctgtttaccgcggcgctgctgagcagctgggcgcagctgctgaccgatgcgaacagctggtggagcctggcgctgaacccggtgcagcgcccggaaatgtttattattggcgcgcagccggtgtgcagccagctgccgggcctgagcccgggccagcgcaaactgtgccagctgtatcaggaacatatggcgtatattggcgaaggcgcgaaaaccggcattaaagaatgccagcatcagtttcgccagcgccgctggaactgcagcaccgcggataacgcgagcgtgtttggccgcgtgatgcagattggcagccgcgaaaccgcgtttacccatgcggtgagcgcggcgggcgtggtgaacgcgattagccgcgcgtgccgcgaaggcgaactgagcacctgcggctgcagccgcaccgcgcgcccgaaagatctgccgcgcgattggctgtggggcggctgcggcgataacgtggaatatggctatcgctttgcgaaagaatttgtggatgcgcgcgaacgcgaaaaaaactttgcgaaaggcagcgaagaacagggccgcgtgctgatgaacctgcagaacaacgaagcgggccgccgcgcggtgtataaaatggcggatgtggcgtgcaaatgccatggcgtgagcggcagctgcagcctgaaaacctgctggctgcagctggcggaatttcgcaaagtgggcgatcgcctgaaagaaaaatatgatagcgcggcggcgatgcgcgtgacccgcaaaggccgcctggaactggtgaacagccgctttacccagccgaccccggaagatctggtgtatgtggatccgagcccggattattgcctgcgcaacgaaagcaccggcagcctgggcacccagggccgcctgtgcaacaaaaccagcgaaggcatggatggctgcgaactgatgtgctgcggccgcggctataaccagtttaaaagcgtgcaggtggaacgctgccattgcaaatttcattggtgctgctttgtgcgctgcaaaaaatgcaccgaaattgtggatcagtatatttgcaaa;

[0016] SEQ ID No.4:

[0017]

[0018] Optionally, in the above biological preparation, the concentration of the WNT5B recombinant protein is 10 ng / ml-100 ng / ml; the concentration of the CTNNB1 recombinant protein is 10 ng / ml-100 ng / ml.

[0019] The preferred concentration is 10 ng / ml.

[0020] The second invention of this application is to provide a method for preparing the above-mentioned biological preparation, comprising the following steps:

[0021] S1. Dissolving PLGA:

[0022] Weigh 250 mg of PLGA (poly(lactic-co-glycolic acid)) and dissolve it in 1 ml of dichloromethane (DCM) to obtain a PLGA solution;

[0023] S2. Protein solution preparation:

[0024] Weigh 10 μg of WNT5B and CTNNB1 recombinant proteins (Catenin Beta 1) and dissolve them in 1 ml of appropriate buffer (PBS or deionized water) to obtain protein solution;

[0025] S3. Mixing protein with PLGA solution:

[0026] Add the protein solution to the PLGA solution and stir evenly to ensure that the protein and PLGA are fully mixed;

[0027] S4. Emulsification process:

[0028] The mixed solution was added to 5 ml of deionized water containing 1% polyvinyl alcohol (PVA) solution and ultrasonicated (20 kHz, 30 s) to form an emulsion;

[0029] If necessary, the emulsifier concentration can be increased to improve the emulsification effect;

[0030] S5. Solvent removal:

[0031] The emulsion was placed in a rotary evaporator (Rotavapor) to remove dichloromethane (DCM) until solid particles were obtained;

[0032] S6. Particle washing:

[0033] The nanoparticles were repeatedly washed with deionized water to remove unencapsulated proteins and emulsifiers to obtain pure nanoparticles;

[0034] S7. Drying treatment:

[0035] The nanoparticles were freeze-dried (lyophilized) for long-term storage and stability evaluation.

[0036] Optional surface modification (to improve biocompatibility and targeting):

[0037] 1) PEGylation: To improve the biocompatibility of nanoparticles, the particle surface can be modified with polyethylene glycol (PEG); PEGylation helps slow the recognition of particles by the immune system and prolong their half-life in vivo;

[0038] 2) Targeted modification: Targeting molecules (such as antibodies, peptides, or small molecule ligands) can be attached to the surface of nanoparticles by covalent binding or non-covalent adsorption to enhance their targeted delivery to specific cells or tissues.

[0039] Characterization and analysis of nanoparticles:

[0040] 1) Particle size and particle size distribution: Dynamic light scattering (DLS) was used to measure the particle size and distribution of nanoparticles.

[0041] 2) Surface charge: Zeta potential analysis was used to determine the surface charge of the nanoparticles and evaluate their stability and dispersibility;

[0042] 3) Drug loading and encapsulation efficiency: Quantitatively determine the drug loading and encapsulation efficiency of WNT5B or CTNNB1 in the nanoparticles by BCA protein quantification or ELISA.

[0043] 4) Protein release rate: Use dialysis bag or membrane filtration method to test the protein release curve to evaluate the sustained release effect.

[0044] Sustained release properties:

[0045] 1) Release rate: By adjusting the molecular weight of PLGA, the emulsification process, and the concentration of the emulsifier, the degradation rate of the nanoparticles can be controlled, thereby achieving different protein release rates. Generally, PLGA has a long degradation cycle and can provide sustained release of WNT5B and CTNNB1 for several days to weeks.

[0046] 2) Protein stability: Through this preparation method, nanoparticles can protect WNT5B and CTNNB1 from environmental influences and improve the stability and activity of the proteins.

[0047] The third invention point of the present application is to provide the use of the above-mentioned biological preparation in the preparation of products that promote tooth differentiation.

[0048] Optionally, in the above application, the prepared product promoting tooth differentiation is selected from one or more of products promoting tooth germ mesenchymal cell odontogenesis, products promoting dental pulp stem cell odontogenesis, products promoting dental pulp regeneration and repair, products promoting periodontal tissue repair, products promoting tooth germ tissue regeneration and products promoting dentin regeneration and repair.

[0049] Specifically, products that promote tooth differentiation may include the following categories:

[0050] 1. Dental pulp regeneration and restoration products:

[0051] 1) Dental pulp regeneration products: Stem cell or growth factor-based therapies used to treat pulp necrosis, pulpitis, and other conditions. These products can repair damaged pulp tissue by promoting the proliferation and differentiation of dental pulp stem cells or other types of stem cells.

[0052] 2) Pulp tissue engineering products: combining biomaterials and growth factors to construct functional pulp substitutes to repair pulp tissue loss or damage;

[0053] 2. Periodontal tissue repair products:

[0054] 1) Periodontal ligament repair products: Help repair periodontal damage, such as periodontitis and periodontal pockets, by stimulating the proliferation and differentiation of periodontal ligament cells;

[0055] 2) Periodontal regenerative materials: Regenerative materials that can promote the regeneration of periodontal tissue, alveolar bone, and gums, and improve periodontal health;

[0056] 3. Multifunctional recycled products:

[0057] 1) Multipotent stem cell products: By combining different types of stem cells (such as dental pulp stem cells and tooth germ mesenchymal stem cells), they can simultaneously promote the repair of multiple aspects of the dental pulp, dentin, and periodontal tissues;

[0058] 2) Self-healing multifunctional products: Combining bioactive molecules (such as growth factors, cytokines, antimicrobial peptides, etc.) with materials science to develop multifunctional products with self-healing capabilities.

[0059] The fourth inventive point of the present application is to provide the use of the above-mentioned biological preparation in the preparation of a tooth-promoting differentiation medium, wherein the tooth-promoting differentiation medium is selected from one or more of a tooth-promoting differentiation medium for culturing tooth germ mesenchymal cells, a tooth-promoting differentiation medium for culturing dental pulp stem cells, a tooth-promoting differentiation medium for tooth germ tissue regeneration, and a tooth-promoting differentiation medium for dentin regeneration and repair.

[0060] The fifth invention point of the present application is to provide a method for promoting odontogenic differentiation in vitro, wherein cells with odontogenic potential are placed in the above-mentioned culture medium containing differentiation promoters for culture, and the differentiation promoters are one or more of WNT5B and CTNNB1.

[0061] Optionally, in the above-mentioned method for promoting odontogenic differentiation in vitro, the cells with odontogenic potential are selected from one or more of tooth germ mesenchymal cells and dental pulp stem cells.

[0062] Compared with the prior art, this application has the following advantages:

[0063] This patent discovered for the first time the important role of WNT5B protein in tooth germ development, providing a new strategy for dental regenerative medicine; compared with other WNT family members (WNT5A), WNT5B has a stronger ability to promote odontogenesis of dental stem cells; WNT5B has a low effective concentration, and 10ng / ml is the optimal concentration of WNT5B to promote odontogenesis of dental stem cells; it also further studied the role of WNT5B and CTNNB1 in the exocrine secretion of tooth germ epithelial cells and their synergistic effect in promoting the odontogenesis of tooth germ mesenchymal cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Sequencing results of exocrine secretory proteins from human tooth germ epithelial cells; experimental flow chart; comparison of up- and down-regulation of secretory proteins between dental epithelium (DE group) and dental mesenchyme (DM group); volcano plot showing the differential expression of secretory proteins between the dental epithelium (DE) and dental mesenchyme (DM) groups; heat map showing the expression levels of key secretory proteins in the dental epithelium (DE) and dental mesenchyme (DM) groups; biological pathways enriched for secretory proteins significantly up-regulated in the dental epithelium (DE); biological pathways enriched for secretory proteins significantly up-regulated in the dental mesenchyme (DM).

[0065] Figure 2 Figure 4 shows the spatial transcriptome results of candidate WNT secreted proteins.

[0066] Figure 3 To investigate the effect of WNT5B knockout in human tooth germ epithelial cells on the odontogenic ability of human tooth germ mesenchymal stem cells.

[0067] Figure 4 To investigate the effect of WNT5B and CTNNB1 overexpression on the odontogenic ability of human tooth germ mesenchymal stem cells.

[0068] Figure 5 To screen the effect of the optimal concentration of WNT5B on the odontogenic ability of human tooth germ mesenchymal stem cells and the effect of WNT5B and CTNNB1 at a concentration of 100 ng / ml on the expression of odontogenic-related genes DMP-1 and DSPP.

[0069] Figure 6 To verify the mineralization ability of WNT5B regulating human tooth germ mesenchymal stem cells by alkaline phosphatase staining.

[0070] Figure 7 To verify that WNT5B regulates the mineralization ability of human tooth germ mesenchymal stem cells through Alizarin red staining and its semi-quantitative experiments.

[0071] Figure 8 The odontogenesis-related genes DMP-1, DSPP and ALP were evaluated for their expression and mineralization effects, and it was determined that the odontogenesis ability of the low-concentration combined use of WNT5B and CTNNB1 was significantly higher than that of the high-concentration use alone. DETAILED DESCRIPTION

[0072] To make the purpose, technical solutions and advantages of this application more clear, this application is further described in detail below. However, it should be understood that the description herein is only used to explain this application and is not intended to limit the scope of this application.

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are intended solely for the purpose of describing specific embodiments and are not intended to limit this application. The reagents and instruments used herein are commercially available, and the characterization methods involved can be found in the relevant descriptions in the prior art and will not be further elaborated herein.

[0074] An overview of the experimental methods used in this study:

[0075] 1. Sample collection and processing:

[0076] This study used human embryonic tooth germ samples. The sample source and processing methods were based on the study (Chen et al., 2024, The temporal protein signature analyses of developing human deciduous molartooth germ - Chen-2024-PROTEOMICS - Wiley Online Library). Briefly, human embryonic tooth germ samples were collected at different developmental stages (from the cap stage to the early bell stage) and appropriately processed and preserved.

[0077] 2. Spatial transcriptome sequencing:

[0078] Human embryonic tooth germ samples were analyzed using spatial transcriptome sequencing. The specific methods were based on the study by Chen et al., 2024. Sequencing was performed using the 10× Genomics Visium spatial gene expression platform, and data were analyzed using the R software package Seurat.

[0079] 3. Exocrine proteome sequencing:

[0080] Culture supernatants from tooth germ epithelial cells and mesenchymal cells were collected and subjected to exocrine proteome sequencing. The specific methods were based on the study of (Chen et al., 2024). Protein identification and quantification were performed using liquid chromatography-mass spectrometry (LC-MS / MS).

[0081] 4. Data integration and screening:

[0082] Spatial transcriptome and exocrine proteome data were integrated and analyzed to identify the most secreted signaling molecules during tooth germ epithelial development. Data integration and statistical analysis were performed using the R software package, and GO enrichment analysis was performed to identify key signaling molecules.

[0083] 5. Cell culture and differentiation induction:

[0084] Human tooth germ mesenchymal cells were isolated and cultured according to the method of (Kilic Bektas et al., 2022, Self-Assembled Hydrogel Microparticle-Based Tooth-Germ Organoids). Cells were treated with different concentrations of WNT5B and CTNNB1, alone or in combination, to observe their effects on odontogenic differentiation.

[0085] 6. Detection of odontogenic differentiation markers:

[0086] Real-time quantitative PCR (qPCR) was further used to detect the expression of odontogenic differentiation markers, including DSPP, DMP1, and ALP. Alkaline phosphatase (ALP) activity detection and Alizarin red staining were also performed to evaluate the mineralization capacity of the cells.

[0087] 7. Statistical analysis:

[0088] All experiments were repeated at least three times. Data are presented as mean ± SD. Statistical analysis was performed using SPSS software, using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test. P < 0.05 was considered statistically significant.

[0089] In order to further understand the present application, the present application is further described in detail below in conjunction with the best embodiment.

[0090] Example 1

[0091] This application screened for the first time the extracellular secretory protein WNT5B of human embryonic tooth germ epithelial cells, and on this basis confirmed the role of epithelial secretion of WNT5B in the development of tooth germ, especially the method and application of it as a factor promoting odontogenic differentiation of dental stem cells.

[0092] The specific technical solutions are:

[0093] In a first aspect, the present application provides a biological preparation containing WNT5B recombinant protein.

[0094] The method for screening the human tooth germ epithelial cell exocytotic protein WNT5B comprises the following steps:

[0095] a. Isolate epithelial cells from human embryonic tooth germs and culture them in vitro;

[0096] b. Collect proteins secreted by epithelial cells and analyze their protein composition using proteomics technology;

[0097] c. Using bioinformatics methods, the collected proteins were quantitatively analyzed to screen out the WNT5B protein with the highest expression level.

[0098] Based on the same or highly similar screening process, the present application also provides a biological preparation containing CTNNB1 recombinant protein (CateninBeta 1), as well as the combined use of the two, that is, a biological preparation containing both WNT5B and CTNNB1 recombinant proteins.

[0099] Secondly, based on the above technology, the application of WNT5B protein to promote odontogenic differentiation of tooth germ mesenchymal cells is proposed. The specific method of use includes the following steps:

[0100] a. adding purified WNT5B protein or its active fragment to the dental germ mesenchymal cell culture medium;

[0101] b. Adjust the concentration of WNT5B protein to determine the optimal conditions for promoting odontogenic differentiation;

[0102] c. Evaluate the effect of WNT5B protein on the odontogenic differentiation of tooth germ mesenchymal cells using cell biological methods, such as alkaline phosphatase staining, Alizarin red staining, and real-time quantitative PCR.

[0103] Similarly, based on similar usage methods, the present application also provides the use of CTNNB1 protein (Catenin Beta 1) to promote the odontogenic differentiation of tooth germ mesenchymal cells, as well as the combined use of the two, that is, the use of both WNT5B and CTNNB1 proteins to promote the odontogenic differentiation of tooth germ mesenchymal cells.

[0104] In a third aspect, the present application further provides a sustained-release preparation of WNT5B protein, which can be used for continuous signal supply during tooth tissue regeneration. The use process includes the following steps:

[0105] a. Select a slow-release gel biodegradable material as a carrier;

[0106] b. Mixing the WNT5B protein with a carrier material to form a sustained-release preparation.

[0107] The carrier can specifically adopt the following preparations:

[0108] 1. Polylactic acid-polyglycolic acid copolymer (PLGA):

[0109] PLGA is a biodegradable and biocompatible copolymer composed of lactic acid (LA) and glycolic acid (GA) monomers; it has good controllable degradability, and its degradation rate and drug release rate can be regulated by adjusting the ratio of lactic acid to glycolic acid.

[0110] PLGA features:

[0111] 1) Adjustability: By changing the polymerization ratio (ratio of lactic acid to glycolic acid), the degradation time and drug release rate can be adjusted;

[0112] 2) Degradation products are non-toxic: The degradation products of PLGA in the body are lactic acid and glycolic acid, which are normal metabolic products in the body and therefore will not cause harmful accumulation;

[0113] 3) High drug loading: Suitable for loading a variety of drugs, including proteins, antibiotics, and gene therapy vectors;

[0114] 4) Application: PLGA can be used to prepare sustained-release drug gels, particularly for the long-term release of bioactive proteins (such as WNT5B and CTNNB1). Its degradation time can be adjusted as needed to ensure sustained drug release in vivo, and it has good clinical application prospects.

[0115] Preparation process of PLGA as sustained-release gel carrier:

[0116] Step 1: Dissolve PLGA in an appropriate organic solvent (such as dichloromethane) to form a homogeneous solution;

[0117] Step 2: Add WNT5B and CTNNB1 recombinant proteins to the PLGA solution and mix well;

[0118] Step 3: Add stabilizers and cross-linking agents (such as PEG, gelatin, etc.) to further enhance the gelling properties;

[0119] Step 4: preparing the mixed solution into a gel by solvent evaporation method or oil-in-water emulsion method;

[0120] Step 5: The gel is freeze-dried to form a controlled-release drug-sustaining gel.

[0121] 2. Sodium alginate

[0122] Sodium alginate is a natural polysaccharide usually extracted from seaweed. It is biodegradable and has good biocompatibility and is widely used in drug delivery systems.

[0123] Characteristics of sodium alginate:

[0124] 1) High biocompatibility: Sodium alginate is a natural material that not only has good biocompatibility but also has no obvious immunogenic reaction;

[0125] 2) Adjustability: The network structure of the gel can be controlled by adjusting the type and concentration of the cross-linking agent (e.g., calcium chloride), thereby adjusting the drug release rate;

[0126] 3) Injectability: Sodium alginate gel can be formed into a form suitable for injection and has good degradability in the body.

[0127] 4) Application: Sodium alginate is commonly used as a carrier material for the delivery of proteins, cells, and genes. It can form a sustained-release gel through ionic crosslinking and solvent evaporation methods and is suitable for sustained-release carriers of proteins such as WNT5B and CTNNB1.

[0128] Preparation process of sodium alginate as sustained-release gel carrier:

[0129] Step 1: Dissolve sodium alginate in distilled water to form an aqueous solution of a certain concentration;

[0130] Step 2: Dissolve WNT5B and CTNNB1 proteins in sodium alginate solution and mix evenly;

[0131] Step 3: Slowly add calcium chloride solution to the mixture to crosslink the sodium alginate and form a gel;

[0132] Step 4: Control the degradation rate of the gel and the drug release rate by adjusting the cross-linking time and cross-linker concentration;

[0133] Step 5: After the gel is prepared, the final product can be obtained by freeze drying or injection molding.

[0134] 3. Gelatin

[0135] Gelatin is a natural protein material derived from animal collagen; it has good biocompatibility and biodegradability and is often used to prepare drug sustained-release carriers.

[0136] Gelatin features:

[0137] 1) Biodegradability: Gelatin will be hydrolyzed and degraded in the body, is non-toxic and easily excreted;

[0138] 2) Adjustability: The solubility and gel strength of gelatin can be adjusted by changing the pH value or adding cross-linking agents (such as glutaraldehyde);

[0139] 3) Gelation properties: Gelatin easily forms hydrogels under temperature changes and is suitable for use as a sustained-release carrier;

[0140] 4) Application: Suitable for sustained-release preparations that require biodegradation, especially suitable as a carrier for protein drugs.

[0141] Preparation process of gelatin as sustained-release gel carrier:

[0142] Step 1: Dissolve gelatin in distilled water at an appropriate temperature (e.g., 50-60°C) to prepare a solution.

[0143] Step 2: Add WNT5B and CTNNB1 proteins to the gelatin solution and mix well;

[0144] Step 3: Add a cross-linking agent (such as glutaraldehyde) to strengthen the gel structure of gelatin and adjust the hardness and degradability of the gel;

[0145] Step 4: Cool the solution to room temperature to form a gel;

[0146] Step 5: The gel is freeze-dried to finally obtain a preparation with a sustained-release effect.

[0147] 4. Polyvinyl alcohol (PVA):

[0148] Polyvinyl alcohol (PVA) is a water-soluble synthetic polymer with good gel-forming properties and biocompatibility. PVA can be combined with other biodegradable materials to serve as a carrier for sustained-release formulations.

[0149] Polyvinyl alcohol (PVA) features:

[0150] 1) Water solubility: PVA dissolves in water to form a transparent solution and can be easily prepared into a hydrogel;

[0151] 2) Good biocompatibility and degradability: PVA generally has good biocompatibility and can be gradually degraded in the body;

[0152] 3) Adjustable mechanical strength and release rate: The structure of the gel and the drug release rate can be changed by adjusting the concentration of PVA and the type of cross-linker;

[0153] 4) Application: PVA is commonly used to prepare oral sustained-release preparations, injectable sustained-release preparations and local delivery systems.

[0154] Preparation process of polyvinyl alcohol (PVA) as sustained-release gel carrier:

[0155] Step 1: Dissolve PVA in distilled water and heat to high temperature to dissolve it completely;

[0156] Step 2: Add WNT5B and CTNNB1 proteins to the PVA solution and mix evenly;

[0157] Step 3: Cross-linking is performed by adding a cross-linking agent (such as glutaraldehyde, calcium chloride, etc.) to convert the PVA solution into a gel;

[0158] Step 4: Soak the gel in a cooling solution to complete the cross-linking and drug release process;

[0159] Step 5: After the gel is formed, it is freeze-dried or spray-dried.

[0160] 5. Chitosan

[0161] Chitosan is a natural polysaccharide extracted from the exoskeletons of crustaceans (such as shrimp and crab) with good biodegradability, low toxicity and biocompatibility.

[0162] Chitosan features:

[0163] 1) Biodegradable: Chitosan will gradually degrade in the body through the action of enzymes;

[0164] 2) Antimicrobial properties: Chitosan has natural antimicrobial properties, making it suitable for topical delivery;

[0165] 3) Strong gel-forming ability: Chitosan can react with cross-linking agents (such as ferric chloride) under acidic conditions to form gel;

[0166] 4) Application: Chitosan is often used for the sustained-release delivery of proteins and gene drugs, and is particularly suitable for local delivery and degradable sustained-release carriers.

[0167] Preparation process of chitosan as sustained-release gel carrier:

[0168] Step 1: dissolving chitosan in an appropriate amount of acidic solution (such as acetic acid solution) to form a solution;

[0169] Step 2: Add WNT5B and CTNNB1 proteins to the solution and mix well;

[0170] Step 3: Forming a controlled-release chitosan gel by adding ferric chloride or other cross-linking agents;

[0171] Step 4: Freeze-dry or freeze-form the gel to form a stable sustained-release preparation.

[0172] Based on a similar usage process, the present application also provides a sustained-release preparation of CTNNB1 protein for continuous signal supply during tooth tissue regeneration, as well as the combined use of the two, namely, a sustained-release preparation of "WNT5B protein + CTNNB1 protein" for continuous signal supply during tooth tissue regeneration.

[0173] In a fourth aspect, the present application can provide a dentin formation promoter, which is mainly composed of WNT5B protein or its derivatives and CTNNB1 protein, and is used to promote the natural formation and repair of dentin.

[0174] In a fifth aspect, the present application provides a tooth germ development research tool, including recombinant proteins, antibodies or small molecule compounds for simulating the functions of WNT5B / CTNNB1 proteins, for screening signal pathways or drugs that may affect tooth germ development.

[0175] In the above-mentioned various preparations, the concentration of WNT5B recombinant protein is 10 ng / ml-100 ng / ml; the concentration of CTNNB1 recombinant protein is 10 ng / ml-100 ng / ml; and the preferred concentration is 10 ng / ml.

[0176] Likewise, a tooth differentiation promoting culture medium containing WNT5B protein and / or CTNNB1 protein also falls within the protection of the present application.

[0177] The odontogenic differentiation medium is selected from one or more of an odontogenic differentiation medium for culturing tooth germ mesenchymal cells, an odontogenic differentiation medium for culturing dental pulp stem cells, an odontogenic differentiation medium for tooth germ tissue regeneration, and an odontogenic differentiation medium for dentin regeneration and repair.

[0178] The method for using the culture medium is as follows: cells with odontogenic potential are placed in a culture medium containing WNT5B protein and / or CTNNB1 protein for culture, and the cells with odontogenic potential are tooth germ mesenchymal cells and / or dental pulp stem cells.

[0179] Example 2

[0180] 1. Screening of WNT5B, an exocrine protein secreted from human tooth germ epithelial cells:

[0181] (1) Sample acquisition and protein extraction:

[0182] Human fetuses that were surgically and medically terminated at approximately 11-12 weeks of gestation were obtained from the Beijing Obstetrics and Gynecology Hospital affiliated to Capital Medical University. This study used three fetuses, whose mandibular tooth germs were meticulously dissected, and dental epithelial tissue and dental mesenchyme were obtained for in vitro culture. The Ethics Committee of Capital Medical University approved the use of human fetuses for this study. Participants provided written informed consent, agreeing that their embryos, which had been terminated by medical means, would be used for scientific research. 300 μL of 8 M urea was added to the exocytted proteins of epithelial cells, and protease inhibitors were added at 10% of the lysate. The cells were centrifuged at 14,100 × g for 20 minutes, and the supernatant was collected. The protein concentration was determined by the Bradford method, and the rest was frozen at -80°C.

[0183] (2) Protein digestion and desalting:

[0184] 100 μg of protein was extracted from each sample and then reduced. 200 mM dithiothreitol (DTT) solution was added and incubated at 37°C for 1 hour. The sample was diluted 4-fold by adding 25 mM ammonium bicarbonate (ABC) buffer. Trypsin was then added (trypsin:protein = 1:50) and incubated at 37°C overnight. The next day, 50 μL of 0.1% FA was added to terminate the digestion. 100 μL of 100% ACN was washed with the C18 column and centrifuged at 1200 rpm for 3 minutes. The column was washed once with 100 μL of 0.1% FA and centrifuged at 1200 rpm for 3 minutes. The EP tube was replaced, the sample was added, and the column was centrifuged at 1200 rpm for 3 minutes. The column was washed twice with 100 μL of 0.1% FA and centrifuged at 1200 rpm for 3 minutes. The column was washed once with 100 μL of pH 10 water. The EP tube was replaced and eluted with 70% ACN. The eluates from each sample were combined and lyophilized. Store at -80°C until loading.

[0185] (3) LC-MS / MS analysis:

[0186] Tryptic peptides were analyzed by nanoflow LC-MS / MS using a quadrupole-Orbitrap mass spectrometer (Q Exactive HF-X, Thermo Fisher Scientific, Bremen, Germany) coupled to an EASY nLC 1200 ultrahigh pressure system (Thermo Fisher Scientific) using a nanoelectrospray ionization source. 500 ng of peptide was loaded onto a 25 cm column (150 μm inner diameter, packed with ReproSil-Pur C18-AQ 1.9-μm silica beads; Beijing Qinglian Biotechnology Co., Ltd., Beijing, China). Peptides were separated using a gradient of 8% to 12% B over 5 minutes, 12% to 30% B over 33 minutes, and 40% B over 7 minutes, followed by a 15-minute wash at 95% B at 600 nl / min. Solvent A consisted of 0.1% formic acid in water and solvent B consisted of 80% ACN and 0.1% formic acid in water. The total run time was 60 minutes. The column temperature was maintained at 60°C using an in-house developed oven. Briefly, the mass spectrometer was operated in "top-40" data-dependent mode, with MS spectra collected on an Orbitrap mass analyzer (120,000 resolution, 350–1500 m / z range) with an automatic gain control (AGC) target of 3E6 and a maximum ion injection time of 80 milliseconds. The most intense ion from the full scan was isolated with an isolation width of 1.6 m / z. Following high-energy collisional dissociation (HCD) with a normalized collision energy (NCE) of 27, MS / MS spectra were collected on an Orbitrap (15,000 resolution), with an AGC target of 5E4 and a maximum ion injection time of 45 milliseconds. Dynamic precursor exclusion was enabled for 16 seconds.

[0187] (4) Identification of secreted proteins and DEP functional analysis:

[0188] All RAW files were analyzed using the Proteome Discoverer suite (version 2.4, Thermo Fisher Scientific). MS2 spectra were searched in the uniprot Homo sapiens SP proteome database (20,407 target sequences downloaded on 2023-03-07). The Sequest HT search engine was used with the following parameters: complete trypsin specificity, a maximum of two missed cleavages, a minimum peptide length of 6, fixed aminomethylation of cysteine ​​residues (+57.02146 Da), variable modification by oxidation of methionine residues (+15.99492 Da), a precursor mass tolerance of 15 ppm, and a fragment mass tolerance of 0.02 Da (for MS2 spectra collected in the Orbitrap). Peptide spectrum matches and peptides were filtered using Percolator to a false discovery rate (FDR) of less than 1%. After spectral assignment, peptides were assembled into proteins and further filtered based on the combined probability of their constituent peptides, with a final FDR of 1%. By default, the best matching protein or "master protein" is the protein with the highest number of unique peptides and the lowest percentage of peptide coverage (i.e., the longest protein). Only unique and most concise peptides are considered for quantification.

[0189] Gene Ontology (GO) and InterPro (IPR) analyses were performed using the interproscan-5 program against the nonredundant protein database. The COG (Clusters of Orthologous Groups) and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases were used to analyze protein families and pathways. Enrichment pipelines were used for GO and KEGG enrichment analyses, respectively.

[0190] A total of 952 differentially expressed proteins were identified between dental epithelium and dental mesenchymal tissues. Among the secreted proteins upregulated in the dental epithelium, WNT5b was the most significantly differentially expressed. To validate these results, WNT5B recombinant protein was overexpressed and knocked out in dental epithelial cells to investigate the effect of WNT5b secretion on the odontogenic capacity of dental mesenchymal stem cells. This suggests that WNT5B is crucial for dentinogenesis.

[0191] 1) Spatial transcriptome analysis of human tooth germs:

[0192] Cryosections were cut into 10 μm thick sections and mounted on GEX arrays. Sections were placed on a Thermocycler Adapter with the active surface facing up, incubated at 37°C for 1 minute, fixed with methanol at -20°C for 30 minutes, and then stained with hematoxylin and eosin (H&E) (Eosin, Dako). Brightfield images were captured on a Leica DMI8 whole-slide scanner at 10× resolution.

[0193] Visium Spatial Gene Expression slides and kits (10x Genomics, PN-1000184) were used for processing. 70 μl of permease was added and incubated at 37°C for 36 minutes. Each well was washed with 100 μl of SSC and 75 μl of reverse transcription master mix was added for cDNA synthesis.

[0194] After first-strand synthesis is complete, remove the RT Master Mix from the wells. Add 75 μl of 0.08 M KOH and incubate at room temperature for 5 minutes. Then remove the KOH from the wells and wash with 100 μl of EB buffer. Add 75 μl of Second Strand Mix to each well for second-strand synthesis. cDNA amplification was performed on a S1000™ Touch Thermal Cycler (Bio-Rad).

[0195] The Visum spatial library was constructed using the Visum spatial library construction kit (10× Genomics, PN-1000184). The library was sequenced using an Illumina Novaseq6000 sequencer with a sequencing depth of at least 100,000 reads per spot using a paired-end 150 bp (PE150) read strategy (performed by Beijing Bio-Biotech Co., Ltd.).

[0196] 2) Isolation of human tooth germ mesenchymal cells:

[0197] Human dental germ mesenchymal cells were isolated and cultured to the third generation, and 5×10 5 The cells were seeded / well in a 12-well plate and divided into odontogenesis induction group, WNT5A group, and WNT5B group.

[0198] The culture medium for each group is as follows:

[0199] Odontogenesis induction group: odontogenesis induction differentiation medium;

[0200] WNT5A group: odontogenic differentiation medium + WNT5A recombinant protein (two concentrations of 10 ng / ml and 100 ng / ml);

[0201] Wnt5B group: odontogenic differentiation medium + WNT5B recombinant protein (two concentrations of 10 ng / ml and 100 ng / ml);

[0202] 3) In vitro odontogenesis differentiation:

[0203] Odontogenic differentiation medium: basal medium supplemented with 10 mmol / L sodium β-glycerophosphate, 10 nmol / L dexamethasone, and 50 mg / L vitamin C. RNA was extracted and stained with alkaline phosphatase and Alizarin red on days 7 and 14 of culture.

[0204] 4) Alkaline phosphatase staining:

[0205] a. Remove the culture medium and wash twice with PBS;

[0206] b. Fix with paraformaldehyde, room temperature, 0.5 h;

[0207] c. Wash twice with double distilled water;

[0208] d. Prepare staining solution: 1ml Sodium Nitrite Solution and 1ml FRV-Alkaline

[0209] Mix the solution gently and wait for 2 minutes. Add it to 4-5 ml of deionized water and finally add 1 ml of Naphthol AS-BI Alkaline Solution and mix thoroughly.

[0210] e. Add 1 ml of staining solution to each well of a 12-well plate and stain at room temperature for 15 minutes. Observe the staining with the naked eye.

[0211] f. Wash 5 times with double distilled water, pipetting gently;

[0212] g. Observe and collect images under the microscope.

[0213] 5) Alizarin red staining:

[0214] a. Remove the culture medium and wash twice with PBS;

[0215] b. Fix with paraformaldehyde, 4°C, 30 min;

[0216] c. Wash twice with double distilled water;

[0217] d. Stain with 40 mM Alizarin Red solution (pH 4.2) at room temperature for 1-10 minutes and observe the staining with the naked eye;

[0218] e. Wash 5 times with double distilled water, gently pipetting;

[0219] f. Observe and collect images under the microscope;

[0220] 6) Realtime qPCR primer sequences:

[0221] a) GAPDH:

[0222] Forward:5′-GCACCGTCAAGGCTGAGAAC-3′(SEQ ID No.5);

[0223] Reverse:5′-ATGGTGGTGAAGACGCCAGT-3′(SEQ ID No.6);

[0224] b) ALP:

[0225] Forward:5′-AACATCAGGGACATTGACGTG-3′(SEQ ID No.7);

[0226] Reverse:5′-GTATCTCGGTTTGAAGCTCTTCC-3′(SEQ ID No.8);

[0227] c) DSPP:

[0228] Forward:5′-GGGATGTTGGCGATGCA-3′(SEQ ID No.9);

[0229] Reverse:5′-CCAGCTACTTGAGGTCCATCTTC-3′(SEQ ID No.10);

[0230] d) BSP:

[0231] Forward:5'-TGAATACGAGGGGGAGTACG-3'(SEQ ID No.11);

[0232] Reverse:5′-TAGCCATCGTAGCCTTGTCC-3′(SEQ ID No.12);

[0233] e)DMP-1:

[0234] Forward:5′-GCAGAGTGATGACCCAGAG-3′(SEQ ID No.13);

[0235] Reverse:5′-GCTCGCTTCTGTCATCTTCC-3′(SEQ ID No.14).

[0236] 7)实验步骤:

[0237] A、RNA Protein Systems:

[0238] a. Preparation of DEPC water: Add 600 μl of DEPC to every 600 ml of water to make DEPC water, shake overnight, and sterilize at high temperature and high pressure for 30 minutes the next day;

[0239] b. Disposable pipette tips, centrifuge tubes and other plastic products, soaked in 0.1% DEPC aqueous solution, autoclaved the next day, and then oven-dried;

[0240] c. Bake the glassware and mortar at 180°C for 4 hours;

[0241] d. Solution preparation: Prepare with 0.1% DEPC-treated water, incubate at 37°C for at least 12 hours, and then sterilize by autoclaving.

[0242] B. RNA extraction:

[0243] a. Sample treatment: Cells were lysed with 1 ml of Trizol lysis buffer;

[0244] b. Mix the homogenate sample vigorously by shaking, and then place it on ice for 10 minutes to completely separate the nucleic acid-protein complex;

[0245] c. Centrifuge at 12,000 g at 4°C for 5 minutes, remove the supernatant, and transfer it to a new RNase-free centrifuge tube;

[0246] d. Add 0.2 ml of chloroform per 1 ml of sample, cap the tube, shake vigorously for at least 15 seconds, and place on ice for 3 minutes;

[0247] e. Centrifuge at 12,000 g for 15 minutes at 4°C and transfer the top layer to a fresh tube.

[0248] f. Slowly add an equal volume of isopropanol, invert to mix, and incubate on ice for 10 minutes to precipitate the RNA;

[0249] Centrifuge at 12000 g for 10 minutes at 4°C and discard the supernatant.

[0250] h. Add 1 ml of 75% ethanol, mix by inversion to wash the precipitate, centrifuge at 7500 g for 5 minutes at 4°C, and discard the waste liquid;

[0251] i. Open the lid and air dry for 5-10 minutes. Dissolve the precipitate in 20 μl of ultrapure water and incubate at 56°C for 10 minutes to aid solubilization.

[0252] j. Detect RNA OD value:

[0253] Use Gen5 software to measure RNA concentration and OD260 / 280 ratio on a microplate reader. Normal values ​​are usually between 1.6 and 2.0.

[0254] C. Reverse transcription PCR:

[0255] 1) Before use, mix each solution, centrifuge; calculate the amount of RNA, 5 μg divided by the concentration of RNA to calculate the required μl number;

[0256] 2) Prepare the template RNA / primer mixture in the Microtube tube, take the template RNA according to the calculated μl number, add 1 μl of random primer or Oligo-(dT), and 1 μl of dNTP (mix) each; the total amount is 5 μl;

[0257] 3) After 70°C incubation for 5 minutes, quickly cool on ice for more than 5 minutes, centrifuge for 10 seconds in a microcentrifuge, and store on ice;

[0258] 4) Prepare the reverse transcription reaction mixture, 15 μl system, prepare on ice. Add 4 μl of 5x PCR reaction buffer, 3 μl of MgCl2, 1 μl of PCR Nucleotide Mix (final concentration of 0.5 mM each dNTP), 0.5 μl of RNAse Inhibit to each sample, and the rest is made up with double distilled water, centrifuge for 10 seconds, and mix well;

[0259] 5) Annealing: 25°C, 5 minutes;

[0260] 6) Extension: 42°C, 1 hour;

[0261] 7) Inactivate the reverse transcriptase, incubate at 70°C for 15 minutes, and cool at 4°C.

[0262] D, Detect the OD value of cDNA:

[0263] Turn on the enzyme marker, measure the DNA concentration and OD260 / 280 ratio using Gen5 software, which is usually between 1.6-2.0.

[0264] E, Real-time PCR:

[0265] 1) Configure the Real-time PCR reaction system components as shown in Table 1.

[0266] Table 1

[0267]

[0268]

[0269] 2) The Real-time PCR reaction system is shown in Table 2.

[0270] Table 2

[0271]

[0272] F, Calculation formula of quantitative PCR:

[0273] The first step is to calculate ΔCt = gene Ct value - internal reference GAPDH Ct value;

[0274] Step 2: Calculate ΔΔCt = ΔCt of treatment - ΔCt of control;

[0275] The third step is to calculate the negative △△Ct power of 2;

[0276] The final result of the control is 1. If the other treatments are greater than 1, it means that the gene expression is upregulated; if it is less than 1, it means that the gene expression is downregulated.

[0277] 8) Experimental results:

[0278] Quantitative proteomic analysis of the exocrine proteins of human tooth germ epithelium and human tooth germ mesenchyme revealed 698 differentially expressed proteins that were significantly upregulated in the exocrine proteins of human tooth epithelium. GO analysis enrichment showed that they were mainly enriched in the WNT signaling pathway.

[0279] Figure 1 It showed that WNT5B and CTNNB1 were significantly upregulated in epithelial secretory proteins, which were closely related to the WNT signaling pathway and cell morphogenesis. There was no significant difference between WNT5A and WNT7A. GO enrichment analysis showed that the upregulated genes in epithelial exocrine proteins were mostly enriched in the WNT signaling pathway, indicating that the WNT signaling pathway plays an important role in the development of tooth germ epithelium.

[0280] Figure 1 The flowchart shows the experimental process. Secretory proteins were first collected from dental epithelium (DE group) and dental mesenchyme (DM group) samples, followed by trypsin digestion and analysis of the secretome using liquid chromatography-tandem mass spectrometry (LC-MS / MS). This flowchart illustrates the complete process from sample collection to secretory protein analysis, aiming to investigate differences in secretory protein composition between dental epithelium and dental mesenchyme.

[0281] Figure 1 Shown is a comparison of up- and down-regulated secretory proteins between dental epithelium (DE group) and dental mesenchyme (DM group). Green bars represent 698 secretory proteins upregulated in the DE group, while pink bars represent 254 secretory proteins downregulated in the DM group. This figure demonstrates significant differences in the number of secretory proteins between the DE and DM groups.

[0282] Figure 1A volcano plot shows the differential expression of secretory proteins between the dental epithelium (DE) and dental mesenchyme (DM) groups. The abscissa represents the log2 fold change (FC) of secretory protein expression, and the ordinate represents the negative logarithm of the p-value (-log10). Green and pink dots indicate proteins significantly upregulated in the DE and DM groups, respectively. Proteins such as WNT5B, CTNNB1, WNT7A, and TGF-β1 are significantly upregulated in the DE group, indicating that expression of these secretory proteins is significantly higher in the DE than in the DM.

[0283] Figure 1 A heat map displays the expression levels of key secretory proteins in the dental epithelium (DE) and dental mesenchyme (DM). The color gradient indicates high and low expression (red indicates high expression, blue indicates low expression). Important proteins, including WNT5B, CTNNB1, and WNT7A, are shown. This map clearly demonstrates the differential expression patterns of these key proteins in the dental epithelium and DM.

[0284] Figure 1 The biological pathways enriched for secretory proteins significantly upregulated in the dental epithelium (DE) are shown. The upregulated secretory proteins were primarily enriched in vesicle-mediated transport, cell morphogenesis, signal transduction, and the WNT signaling pathway. This suggests that secretory proteins in the dental epithelium may play an important role in regulating cell morphology and signal transduction.

[0285] Figure 1 The biological pathways enriched for secretory proteins significantly upregulated in the dental mesenchyme (DM) group were revealed. These proteins were primarily enriched in collagen fibril organization, extracellular matrix organization, the TGF-β receptor signaling pathway, and the BMP signaling pathway. This suggests that the dental mesenchyme group significantly enhanced the expression of secretory proteins involved in regulating the extracellular matrix and cell differentiation.

[0286] pass Figure 1 The results showed that there were differences in secretory proteins and related biological functions between the dental epithelium and dental mesenchyme groups, which may be closely related to tooth development and tissue-specific functions.

[0287] Figure 2 Spatial transcriptome data from the study demonstrated that WNT5B, CTNNB1, WNT5A, and WNT7A are expressed in human tooth germ epithelial tissue. The results showed that WNT5B and CTNNB1 were significantly expressed in the epithelial region, indicating that they play an important role in local cell differentiation and development. WNT5A and WNT7A were expressed at low levels but with local specificity, indicating that the WNT signaling pathway has spatial specificity in tissue development.

[0288] Subsequently, human dental epithelial cells with WNT5B knockout and human dental mesenchymal cells were co-cultured in vitro. Figure 3 As shown, knockout of WNT5B in human tooth germ epithelial cells was found to affect its regulation of odontogenesis in human tooth germ mesenchymal stem cells. ELISA was used to detect the expression of WNT5B in the supernatant and the expression changes of odontogenesis-related genes (DSPP, DMP1, and ALP) in tooth germ mesenchymal cells. The results showed that knockout of epithelial WNT5B significantly reduced the expression levels of DSPP, DMP1, and ALP in tooth germ mesenchymal cells, indicating that WNT5B plays an important role in promoting the odontogenesis of human tooth germ mesenchymal stem cells.

[0289] Human tooth germ mesenchymal cells were isolated and cultured, and odontogenic differentiation was induced. Different concentrations of WNT5A and WNT5B recombinant proteins were added, and RT-qPCR detection was performed. It was found that WNT5B had a stronger ability to promote odontogenic differentiation of human tooth germ mesenchymal cells than WNT5A. The results are as follows Figure 4 As shown in the results, both WNT5B and CTNNB1 promote odontogenesis in human tooth germ mesenchymal stem cells, that is, both WNT5B and CTNNB1 proteins significantly promote the expression of odontogenesis-related genes (DSPP, DMP1, ALP, BSP) in tooth germ mesenchymal cells, indicating that these two signaling pathways play a role in promoting the odontogenic differentiation of tooth germ mesenchymal stem cells.

[0290] Further combined with alkaline phosphatase staining, the results are as follows Figure 6 As shown in the results, it was found that WNT5B has a stronger ability to promote the mineralization of human tooth germ mesenchymal cells than WNT5A. At the same time, when WNT5B and CTNNB1 are used together at low concentrations, the expression and mineralization effects of odontogenic genes (DMP-1, DSPP, ALP) are significantly better than when used alone at high concentrations, indicating that they have a synergistic effect in promoting odontogenic differentiation at low doses.

[0291] Subsequently, the concentrations of WNT5B recombinant protein and CTNNB1 recombinant protein were screened, and the results were as follows: Figure 5As shown, PCR testing revealed that 10 ng / ml of WNT5B significantly promoted the expression of odontogenic genes in human tooth germ mesenchymal cells, and both WNT5B and CTNNB1 exhibited a stronger effect in promoting odontogenic differentiation than WNT5A. Comparison of the effects of WNT5B and CTNNB1 at a concentration of 100 ng / ml on the expression of odontogenic-related genes DMP-1 and DSPP revealed that both WNT5B and CTNNB1 significantly upregulated the expression levels of these two genes, with their effects significantly superior to those of the positive control WNT5A, indicating that WNT5B and CTNNB1 play an important role in promoting odontogenic differentiation of tooth germ cells.

[0292] Further alkaline phosphatase and alizarin red staining revealed that 10 ng / ml was the optimal concentration for WNT5B to promote the mineralization of human tooth germ mesenchymal cells (dark gray represents mineralization, the darker the color, the stronger the mineralization ability) ( Figure 6 、 Figure 8 ).

[0293] Potential mechanism of synergistic effect between low concentrations of WNT5B and CTNNB1:

[0294] 1.1 Signaling pathway interactions:

[0295] 1.1.1 Cross-regulation of non-canonical and canonical WNT pathways:

[0296] Low concentrations of WNT5B may indirectly influence canonical WNT pathway activity by activating downstream non-canonical WNT pathway molecules such as RhoA and JNK. For example, JNK activation may enhance CTNNB1 transcriptional activity by phosphorylating it. This cross-regulation may be more pronounced at low concentrations, thereby enabling synergistic effects between WNT5B and CTNNB1.

[0297] 1.1.2 Regulation of CTNNB1 stability and activity:

[0298] WNT5B may enhance the output of the canonical WNT signaling pathway by affecting the stability or activity of CTNNB1. Low concentrations of WNT5B may increase the intracellular concentration and activity of CTNNB1 by inhibiting its degradation complex or promoting its nuclear translocation. This regulatory mechanism may be more sensitive at low concentrations, thereby achieving a synergistic effect.

[0299] 1.2 Co-regulation of downstream gene expression:

[0300] Low concentrations of WNT5B and CTNNB1 may co-regulate the expression of key genes that play an important role in tooth development and odontoblast differentiation. For example, genes such as SOX2 and AXIN2 have been identified as important target genes of the WNT signaling pathway. Low concentrations of WNT5B may enhance the transcriptional activity of CTNNB1 or recruit other transcriptional cofactors, enabling the effective activation of these target genes even at low levels of stimulation, thereby producing a synergistic effect.

[0301] 1.3 Coordinated regulation of cell behavior:

[0302] During tooth development, cell polarity, migration, and differentiation require precise regulation. Low concentrations of WNT5B may provide a suitable microenvironment for the role of CTNNB1 in cell proliferation and differentiation by regulating cytoskeletal remodeling and polarity establishment. For example, WNT5B may promote the spatial arrangement and orientation of odontoblasts, while CTNNB1 activates related genes, further promoting tooth development.

[0303] 1.4 Regulating cell fate determination:

[0304] During early tooth development, low concentrations of WNT5B may assist CTNNB1 in regulating stem cell fate by influencing their polarity and migration. WNT5B may regulate the initial differentiation state of cells through the non-canonical WNT pathway, while CTNNB1 further guides these cells toward odontoblast differentiation by activating specific gene expression. This synergistic effect may ensure that cells can effectively complete the differentiation process at the appropriate time and under the appropriate circumstances.

[0305] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements or improvements made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. Use of a biological agent in preparing a product that promotes tooth differentiation, characterized in that: The biological preparation contains WNT5B recombinant protein and CTNNB1 recombinant protein; the amino acid sequence of the WNT5B recombinant protein is shown in sequence SEQ ID No. 1, and the amino acid sequence of the CTNNB1 recombinant protein is shown in sequence SEQ ID No. 2; the product promoting tooth differentiation is a product that promotes the odontogenic differentiation of tooth germ mesenchymal cells.

2. The use according to claim 1, characterized in that The nucleotide sequence encoding the WNT5B recombinant protein is shown in SEQ ID No. 3, and the nucleotide sequence encoding the CTNNB1 recombinant protein is shown in SEQ ID No.

4.

3. The use according to claim 1 or 2, characterized in that The concentration of the WNT5B recombinant protein is 10 ng / mL-100 ng / mL; the concentration of the CTNNB1 recombinant protein is 10 ng / mL-100 ng / mL.

Images