Use of chuanqiong saponin B in preparation of a preparation for remodeling the root development microenvironment and promoting apexification

Abstract

The application discloses application of Cyaonoside B in preparation of a preparation for remodeling a tooth root development microenvironment and promoting apexification. The application has the following remarkable advantages and beneficial effects: the application first applies Cyaonoside B to apexification, and molecular dynamics simulation proves that Cyaonoside B is stably combined with a c-Kit receptor, and the mechanism is clear. In-vitro CCK-8 and RTCA experiments show that Cyaonoside B is in a concentration-dependent manner to promote proliferation of a tooth germ epithelial cell in a concentration range of 0-50 muM, and the activity is the strongest at 50 muM and without cytotoxicity. In-vivo mouse apex models show that four weeks after injection of 50 muM Cyaonoside B, microCT and tissue staining prove that the calcified substance of an apical foramen hole is significantly increased, a calcification barrier is complete, and physiological closure is achieved. Compared with traditional drugs, the application directly mediates a c-Kit path to actively regulate an apical foramen microenvironment, and provides a new precise apexification strategy.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to the application of Cyaonoside B in the preparation of formulations that reshape the root development microenvironment / promote apexification. Background Technology

[0002] Young permanent teeth, before their roots are fully developed, often suffer from pulp necrosis or periapical inflammation due to caries, trauma, or developmental malformations, leading to arrested root development. Normally, the apical foramen closes naturally after root development is complete, but in young permanent teeth, the root apex often appears funnel-shaped, parallel, or converging, and the apical foramen has not yet formed a natural barrier. Once the pulp dies, root development ceases, making long-term preservation of the affected tooth extremely difficult.

[0003] Apexification is a clinical treatment technique used for young permanent teeth with incompletely formed roots that have pulp necrosis or periapical periodontitis. It involves controlling infection through medication and surgery and inducing the deposition of hard tissue at the root apex, promoting continued root development. The core of this technique lies in using medication to induce the formation of hard tissue in the apical pulp and / or periapical tissue, thereby narrowing or closing the apical foramen. Currently, commonly used apexification drugs include calcium hydroxide preparations and Vitapex paste. However, these traditional drugs have several drawbacks: calcium hydroxide has poor stability, is easily absorbed, and requires a long treatment period; while Vitapex paste possesses both antibacterial activity and good operability, its mechanism of apexification is not fully understood, and its ability to promote the formation of hard tissue at the root apex is limited. Therefore, developing a novel drug that can efficiently and stably induce the formation of a calcified barrier at the root apex is of significant clinical importance.

[0004] c-Kit (also known as KIT / CD117) is a receptor tyrosine kinase that, upon binding to its ligand stem cell factor (SCF), promotes cell proliferation, differentiation, and progenitor cell recruitment. Studies have shown that PDGFRβ... + / c-kit + Dental pulp cells possess the potential of dental pulp stem cells, enabling them to form dentin-like structures both in vitro and in vivo. However, there are currently no reports of applying the regulation of the c-Kit signaling pathway to apexification.

[0005] Cyaonoside B is a saponin compound that can be isolated from monotypic bacteria. To date, there are no reports on the use of cyaonoside B for root apexification. Summary of the Invention

[0006] To address the aforementioned technical limitations, this application proposes the application of Cyaonoside B in the preparation of formulations that reshape the root development microenvironment / promote apexification; this overcomes the deficiencies and defects mentioned in the background art.

[0007] To achieve the above objectives, this application adopts the following technical solution: The invention of this application is to provide the use of Cyaonoside B in the preparation of formulations that reshape the root development microenvironment / promote apexification.

[0008] Optionally, in the above-described application, Cyaonoside B promotes the formation of the calcification barrier in the root apex region and the closure of the apical foramen by mediating the c-Kit signaling pathway in the root development microenvironment.

[0009] Optionally, in the above application, the preparation is an injection solution used to inject into the apical foramen region after root canal treatment.

[0010] Optionally, in the above-described application, the concentration of Cyaonoside B in the formulation is 5-300 μM, preferably 25-200 μM, and more preferably 50 μM.

[0011] The second inventive point of this application is to provide a pharmaceutical composition for root tip induction shaping, the pharmaceutical composition comprising an effective amount of Cyaonoside B as an active ingredient, and a pharmaceutically acceptable carrier.

[0012] Optionally, the above-described pharmaceutical composition is an injectable formulation for local injection into the apical foramen region.

[0013] Optionally, in the above-mentioned pharmaceutical composition, the effective concentration of Cyaonoside B is 5-300 μM, preferably 25-200 μM, and more preferably 50 μM.

[0014] The third inventive point of this application is to provide a cell model for screening root tip induction agents. The model uses primary dental germ epithelial cells, targets the c-Kit signaling pathway, uses cell proliferation activity as an indicator, and evaluates the activity of the test drug in promoting the remodeling of the root tip development microenvironment mediated by the c-Kit pathway through CCK-8 or real-time cell analysis (RTCA) methods.

[0015] The fourth inventive point of this application is to provide a method for apical induction shaping, the method comprising injecting an effective amount of Cyaonoside B into the apical foramen region of an individual during tooth development to mediate the c-Kit signaling pathway, remodel the root development microenvironment, thereby promoting the deposition of calcified tissue in the apical region and the physiological closure of the apical foramen.

[0016] Optionally, in the above method, the injection volume of Cyaonoside B is 1-5 μL, the injection concentration is 5-300 μM, preferably 25-200 μM, and more preferably 50 μM.

[0017] Compared with the prior art, the present invention has the following significant advantages and beneficial effects: First, this invention is the first to apply Cyaonoside B to the field of root tip induction shaping. There are no prior art reports on the use of Cyaonoside B to promote the formation of the root tip calcification barrier or the closure of the apical foramen. Through systematic molecular dynamics simulations, in vitro cell experiments, and in vivo animal experiments, this invention reveals for the first time the excellent effects of Cyaonoside B in root tip induction shaping.

[0018] Second, the mechanism of action of this invention is clearly defined. Molecular dynamics simulations confirm that Cyaonoside B can form a stable complex conformation with the c-Kit receptor protein, with a binding free energy (dG) of -125.376 kJ / mol, indicating a highly thermodynamically favorable binding. Van der Waals interactions (-181.062 kJ / mol) and total molecular mechanical interactions (-173.743 kJ / mol) are the core factors driving this binding. This clearly defined mechanism provides a solid theoretical foundation for the rational design and clinical application of drugs.

[0019] Third, the pharmaceutical composition of the present invention exhibits excellent in vitro proliferative activity. CCK-8 cell proliferation and cytotoxicity experiments, as well as real-time cell analysis (RTCA), revealed that within the concentration range of 0-50 μM, Cyaonoside B's proliferative effect on primary tooth germ epithelial cells was concentration-dependent, with the fastest cell proliferation rate observed at a concentration of 50 μM, and no significant cytotoxicity. This indicates that Cyaonoside B can effectively activate the c-Kit signaling pathway within a safe range, promoting cell proliferation in the root development microenvironment.

[0020] Fourth, the pharmaceutical composition of this invention exhibits a significant apexification effect in vivo. In a mouse model of apexification of young permanent teeth, after injection of 50 μM Cyaonoside B through the apical foramen and continuous observation for 4 weeks, microCT images showed a significant increase in calcification at the apical foramen in the drug intervention group, with the apical foramen essentially closed. HE and Masson staining further confirmed the formation of abundant calcification at the apical foramen in the drug-induced group, with an intact calcification barrier. In contrast, the size of the apical foramen in the control group did not show a significant change compared to healthy teeth. This result indicates that Cyaonoside B can effectively induce the formation of a calcification barrier in the apical region in vivo, achieving physiological closure of the apical foramen.

[0021] Fifth, this invention provides a novel strategy for apexification. Traditional apexification drugs (such as calcium hydroxide and Vitapex) mainly promote hard tissue formation indirectly by creating an alkaline environment and having antibacterial effects, which is a relatively passive mechanism of action. In contrast, this invention directly mediates the c-Kit signaling pathway through Cyaonoside B, actively reshaping the root development microenvironment and precisely regulating the proliferation and differentiation of cells in the apical region at the molecular level, thus achieving active intervention in apexification. Attached Figure Description

[0022] Figure 1 The electrostatic potential surface map of the c-Kit–Cyaonoside B complex shows that Cyaonoside B is located near the recessed pocket on the surface of the c-Kit protein, embedded in a mixed charge microenvironment composed of neutral, negative, and locally positive charges.

[0023] Figure 2 The two-dimensional Gibbs free energy landscape (PC1–PC2) of the c-Kit–Cyaonoside B complex shows that the low-energy region of the system is mainly concentrated in a narrow channel, and the dark blue area represents the most stable dominant conformation cluster.

[0024] Figure 3 The graph shows the change in the number of hydrogen bonds in the c-Kit–Cyaonoside B complex over time, indicating that the number of protein-ligand hydrogen bonds in the complex remained between 2 and 8 during the simulation.

[0025] Figure 4 The energy decomposition diagram of the c-Kit–Cyaonoside B complex MM / PBSA shows the contribution of each energy term to the total binding free energy.

[0026] Figure 5 The graph shows the change in radius of gyration (Rg) of the c-Kit–Cyaonoside B complex over time, indicating that the complex gradually shrinks and tends to stabilize during the simulation.

[0027] Figure 6 The comparison graph of the RMSD of the c-Kit–Cyaonoside B complex and the free c-Kit main chain shows that the two curves are very close in height and have basically the same trend.

[0028] Figure 7 The RMSF plot of the c-Kit–Cyaonoside B complex shows the amplitude distribution of each residue.

[0029] Figure 8 The graph shows the change in SASA of the c-Kit–Cyaonoside B complex over time, indicating that the solvent-accessible surface area gradually decreases and tends to stabilize during the simulation.

[0030] Figure 9 The three-dimensional free energy landscape (Rg–RMSD) of the c-Kit–Cyaonoside B complex shows the presence of a very prominent deep energy valley in the system.

[0031] Figure 10A This study presents the effects of Cyaonoside B on mouse tooth germ epithelial cells (DEs), comparing the cell viability of primary mouse tooth germ epithelial cells at various concentration gradients as shown in the cell proliferation activity concentration gradient experiment.

[0032] Figure 10B To demonstrate the effect of Cyaonoside B on mouse tooth germ epithelial cells (DEs), the experimental results were verified by observing changes in the expression levels of several key proteins in the cells after the addition of 50 µM Cyaonoside B, which showed that Cyaonoside B upregulates the PI3K pathway through the c-Kit receptor.

[0033] Figure 10C To demonstrate the effect of Cyaonoside B on mouse tooth germ epithelial cells (DEs), the changes in the expression levels of two other key proteins, Ki67 and Sox2, after the addition of 50 µM Cyaonoside B were used to verify the experimental results, which showed that Cyaonoside B upregulates the PI3K pathway through the c-Kit receptor.

[0034] Figure 11 The results of periapical shaping in young permanent teeth of mice after intervention with Cyaonoside B were observed. Figure 11 A shows the staining results of the young maxillary first permanent molar of a mouse 21 days after birth; Figure 11 B shows the staining results of the maxillary first permanent molar of a mouse 49 days after birth; Figure 11 C represents the experimental design for in vivo root apexification in mice; Figure 11 D shows a comparison of microCT images of mice 49 days after birth; Figure 11 E- Figure 11 G represents the HE and Masson staining results for the healthy group, control group, and drug-treated group, respectively. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0037] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0038] Example 1 Molecular dynamics simulations of Cyaonoside B and c-Kit receptor proteins 1. Materials and Methods: c-Kit (KIT / CD117) was selected as the receptor protein, and Cyaonoside B was chosen as the small molecule ligand. Cyaonoside B was purchased from TargetMol (TargetMol Chemicals Inc.), catalog number TN6776. First, the c-Kit protein structure was pretreated by removing water of crystallization and irrelevant heteroatoms, adding hydrogen atoms, and checking the integrity of the receptor structure. Simultaneously, the three-dimensional structure of Cyaonoside B was obtained and its conformation optimized. Then, AutoDock Vina was used to perform molecular docking between c-Kit and Cyaonoside B. Docking cassette parameters were set according to the protein's active pocket or potential binding region, resulting in multiple candidate binding conformations. The optimal complex was selected based on binding energy scores and conformational rationality as the initial structure for subsequent molecular dynamics simulations.

[0039] The optimal c-Kit–Cyaonoside B complex was imported into the GROMACS platform for 100 ns molecular dynamics simulations. The protein fraction was described using the CHARMM36 force field, while the topological parameters and force field files for the small molecule ligands were generated using Sobtop to ensure parameter consistency. A solvation system was then constructed, and an appropriate amount of counterion was added to maintain electroneutrality. Before the formal simulation, energy minimization, NVT equilibration, and NPT equilibration were performed sequentially, followed by 100 ns production-phase kinetic simulations.

[0040] After simulation, a systematic analysis of the trajectory was performed: gmx rms was used to calculate the main chain RMSD of the complex to evaluate overall conformational stability; gmx rmsf was used to assess the flexibility changes of each residue; gmx hbond was used to count the number and dynamic changes of protein-ligand hydrogen bonds; gmx sasa was used to analyze changes in solvent accessible surface area (SASA); and gmx gyrate was used to calculate the radius of gyration (Rg) to characterize the overall compactness of the complex. Furthermore, gmx covar and gmx anaeig were used to perform principal component analysis on the trajectory, and gmx sham was used to construct a Gibbs free energy landscape to reveal the main conformational clusters and their energy distribution characteristics. Simultaneously, APBS was used to calculate the surface electrostatic potential of the complex, and PyMOL / APBS Tools was used to visualize the electrostatic potential map to analyze the charge distribution characteristics around the binding pocket. Finally, gmx_MMPBSA was used to calculate the MM / PBSA binding free energy of the protein-ligand complex to further evaluate the interaction strength and binding stability between Cyaonoside B and c-Kit from an energy perspective.

[0041] 2 Results (1) Analysis of electrostatic potential surface diagram: From the surface electrostatic potential diagram ( Figure 1 As can be seen from the image, the c-Kit protein surface exhibits both distinct positive potential regions (blue) and negative potential regions (red), indicating strong charge heterogeneity at its binding interface. Cyaonoside B is located near a recessed pocket on the protein surface, embedded in a microenvironment composed of neutral, negative, and locally positive charges. This "mixed charge pocket" typically facilitates multimodal recognition of ligands via hydrogen bonding, electrostatic attraction, and van der Waals interactions. The ligand-containing region is not a completely flat surface exposed to the solvent, but rather situated in a relatively confined, groove-like environment, indicating good embedding characteristics at this binding site, which is beneficial for improving the spatial matching and complex stability after ligand binding. Overall, these results support the ability of Cyaonoside B to find a suitable electrically complementary region on the c-Kit surface and form a relatively stable binding conformation.

[0042] (2) Analysis of Gibbs free energy landscape diagrams (PC1–PC2): Two-dimensional Gibbs free energy landscape diagram ( Figure 2 The results show that the low-energy region of the system is mainly concentrated in a narrow channel extending from high PC2 / low PC1 to low PC2 / high PC1, with the dark blue area representing the most stable dominant conformation cluster. This result indicates that during the 100 ns simulation, the c-Kit–Cyaonoside B complex does not fluctuate randomly, but rather undergoes certain conformational adjustments before gradually converging into a finite low-energy conformation space. Although a few light-colored secondary basins are still visible in the figure, their small area and scattered distribution suggest that the system may briefly visit several transition states or local metastable states, but ultimately the main low-energy basin still dominates. Overall, this free energy landscape indicates that the complex has a clearly defined dominant stable conformation, and the system's evolution direction is clear, demonstrating that ligand binding can guide the protein into a relatively stable energy state.

[0043] (3) Analysis of the change in the number of hydrogen bonds: The change in the number of hydrogen bonds over time is shown ( Figure 3 The complex initially exhibited a low number of hydrogen bonds during the initial simulation, which then rapidly increased and remained between 2 and 5 for most of the time, reaching 6 to 8 in some localized periods. Although the number of hydrogen bonds fluctuated somewhat, it did not drop to zero for extended periods, indicating that a continuous polar interaction network was maintained between the ligand and protein. Such fluctuations are normal in molecular dynamics simulations, reflecting a dynamic equilibrium of continuous hydrogen bond breaking and rebuilding, rather than instability. Overall, Cyaonoside B and c-Kit maintained relatively stable hydrogen bond connections throughout the 100 ns process, providing crucial support for the long-term binding stability of the complex.

[0044] (4) Analysis of the MM / PBSA energy decomposition diagram: The results of MM / PBSA indicate that ( Figure 4The overall binding free energy (dG) of the complex is -125.376 kJ / mol, a significantly negative value, indicating that the binding of Cyaonoside B to c-Kit is thermodynamically favorable. Among the individual energies, van der Waals interactions (VDW, -181.062 kJ / mol) and total molecular mechanical interactions (MM, -173.743 kJ / mol) are the most significant favorable contributions, suggesting that hydrophobic matching and close contact are the core factors driving ligand binding. The solvent nonpolar term (SA, -18.540 kJ / mol) and coulombic interactions (COU, -8.128 kJ / mol) also provide positive support. In contrast, the polar solvation energy (PB, 50.839 kJ / mol) is positive, which has a certain offsetting effect on binding, but is insufficient to reverse the overall negative free energy trend. In summary, these results indicate that the binding of Cyaonoside B to c-Kit has a strong energy advantage, mainly dependent on nonpolar interactions and spatial adaptation.

[0045] (5) Analysis of the radius of gyration (Rg) diagram: The Rg curve shows ( Figure 5 The system's radius of gyration was close to 4.9 nm at the start of the simulation, then rapidly decreased to approximately 4.2–4.4 nm within the first 10–20 ns, indicating significant conformational contraction and rearrangement of the complex in the early stages. Although there were some fluctuations in the middle, the overall range was limited, especially stabilizing at approximately 4.05–4.15 nm after 70 ns, indicating that the system gradually entered a more compact and stable conformational state in the later stages. The overall decreasing trend of Rg usually indicates a more compact packing and a more convergent conformation within the protein-ligand complex, consistent with the characteristic of structural stability after binding. Therefore, this result supports the view that Cyaonoside B binding helps maintain the good overall compactness of the c-Kit system.

[0046] (6) RMSD plot analysis: RMSD results show ( Figure 6Both the free c-Kit and the c-Kit–Cyaonoside B complex showed a rapid increase in backbone shift during the initial simulation phase, followed by a relatively stable plateau, indicating that the system successfully adapted from its initial conformation to the solution environment. The RMSD of the complex group was slightly higher than that of the free protein group, but the two curves remained highly similar and trended in the same direction, without significant separation or drastic drift, suggesting that ligand binding did not disrupt the overall stability of the protein backbone. After 60 ns, the RMSD entered a new, higher plateau, suggesting a possible large-scale but still controllable conformational rearrangement, which subsequently remained within a relatively stable range without any instability or divergence. Overall, these results indicate that c-Kit underwent some structural adaptation after Cyaonoside B binding, but the complex maintained good overall stability.

[0047] (7) RMSF chart analysis: RMSF results show that ( Figure 7 The fluctuations of most residues in c-Kit were concentrated at low to moderate levels, indicating that the overall protein structure was relatively stable during the simulation. Higher fluctuations mainly occurred in the N-terminal region, some intermediate long loop regions, and the terminal region. These locations typically correspond to flexible loops, linker regions, or solvent-exposed fragments, which are naturally more mobile regions in the protein molecule. In contrast, the fluctuations of the vast majority of main residues were controlled within a reasonable range, indicating that the core domain did not experience widespread instability due to ligand binding. This result suggests that the effect of Cyaonoside B on c-Kit is more inclined towards local flexible regulation rather than overall skeletal disruption; this "local adaptation, overall stability" characteristic is consistent with stable binding models.

[0048] (8) SASA chart analysis: SASA curve shows ( Figure 8 The system initially had a wavelength of approximately 670 nm in the early stages of the simulation. 2 Rapidly dropped to approximately 530 nm 2 It then continued to decrease slowly, and eventually stabilized at approximately 495–510 nm. 2 The SASA fluctuated within a certain range. This trend indicates that the complex underwent a significant reduction in solvent-exposed area in the early stages, suggesting that the interaction between the protein and ligand caused some surface areas to be re-embedded or masked, thus forming a more compact complex. The smaller changes in SASA in the later stages indicate that the system had reached a good solvent environment adaptation state and did not experience repeated large-scale expansion. Combined with the Rg results, the continuous decrease in SASA further proves that the complex gradually shrank and tended to stabilize during the simulation, suggesting that the binding of Cyaonoside B helps maintain a more compact structural conformation of c-Kit.

[0049] (9) Analysis of the three-dimensional free energy landscape map (Rg–RMSD): 3D free energy landscape diagram ( Figure 9 Using Rg and RMSD as reaction coordinates, the system exhibits a prominent deep energy valley. The lowest energy region is concentrated within a narrow area of ​​lower RMSD and more stable Rg, while the remaining large area is located at higher energy plateaus. This characteristic indicates that although multiple conformations of the c-Kit–Cyaonoside B complex can be sampled during simulation, a few low-energy stable states are truly dominant, and the main stable basin is deep, reflecting good conformational convergence and thermodynamic stability. Conformations near the deep energy valley can generally be considered the most likely dominant conformations of the system, suggesting that the complex has a clear stable binding mode during dynamic processes, rather than being dispersed among multiple unstable conformations. Overall, this result further strengthens the aforementioned conclusion that Cyaonoside B and c-Kit form a relatively stable and sustainably maintained binding state.

[0050] Example 2 The effects of compound Cyaonoside B on mouse dental epithelial cells (DEs) and its potential molecular mechanism: First, the effects of cyaonoside B on cell growth and how it affects the signaling pathways of growth factors (SCFs) were investigated. Figure 10A As shown.

[0051] Experimental procedure: Researchers isolated primary dental epithelial cells from E15.5 mouse tooth germs, treated them with different concentrations of Cyaonoside B, and observed cell proliferation.

[0052] Figure 10A The cell proliferation curve shown in the lower left corner is a dose-response curve, with the horizontal axis representing the concentration of cyanoside B and the vertical axis representing the relative cell proliferation rate. The results show that cell proliferation initially increases and then decreases with increasing drug concentration. The cell proliferation rate reaches its peak (highest point) at a concentration of 50 µM, indicating that the drug has the strongest promoting effect on cell proliferation at this concentration. Above this concentration (e.g., 200 µM, 500 µM), cell viability decreases, showing toxicity or inhibitory effects at high concentrations.

[0053] Figure 10A The study also revealed the molecular mechanism, demonstrating the mechanism of action of Cyanoside B. Cyanoside B acts on the c-Kit receptor on the cell surface, thereby activating the intracellular PI3K pathway and leading to a series of gene alterations. Statistical results of cell viability testing are as follows: Figure 10AAs shown on the right, there were control groups, groups treated with SCF (stem cell factor, c-Kit ligand, 50 ng / mL) alone, and groups treated with different concentrations of Cyanoside B. The statistical results show that Cyanoside B significantly promoted cell viability in the range of 1-50 µM, with 50 µM showing the best effect. At the same time, it also had a high concentration inhibitory effect, that is, when the concentration reached 200 µM and 500 µM, cell viability decreased significantly, even lower than that of the control group.

[0054] Figure 10B Immunofluorescence staining experiments were used to observe the changes in the expression levels of several key proteins in cells after the addition of 50 µM Cyanoside B. It was determined that Cyanoside B can specifically regulate the expression profile of different proteins in dental epithelial cells, promote the expression of certain proteins (such as Creb312), and inhibit the expression of other proteins (such as Gldc).

[0055] Figure 10C The study also showed changes in the expression levels of two other key proteins. Ki67 is a classic marker of cell proliferation, and its increased expression confirmed the improvement in cell proliferation activity. Sox2, as a key stem cell marker, is highly correlated with cell stemness and pluripotency. Its increased expression level indicates that Cyanoside B also maintains or enhances the stemness characteristics of cells.

[0056] Example 3 Application of Cyaonoside B in apexification of young permanent teeth in mice: 1. Materials and Methods: The first permanent maxillary molars of mice aged 21 days (P21) were selected, at which time a large amount of epithelial root sheath remained at the apical foramen. An in vivo periapical induction and shaping experiment was conducted in mice: the pulp chamber of the P21 mice was opened at the midline of the molar, and the vital pulp was removed using an insulin injection. The control group received an intracanal injection of 2 μL PBS, while the experimental group received an intracanal injection of 2 μL Cyaonoside B (50 μM). Observations were conducted for 4 weeks. MicroCT scans were performed on P49 mice, and histological observation was performed using HE and Masson staining.

[0057] 2. Results: like Figure 11 The microCT images shown indicate that the apical foramen size in the control group was significantly larger than that in the drug intervention group. In the drug intervention group, there was an increase in calcification at the apical foramen, and the apical foramen was essentially closed.

[0058] Further observation using HE and Masson staining revealed that the apical foramen of normal healthy teeth was open (the epithelial root sheath of the apical foramen of the maxillary first permanent molar of P49 mice was significantly reduced); the control group had sporadic calcifications in the apical foramen, but the size of the apical foramen did not change significantly compared with healthy teeth; the drug-induced group had a large amount of calcifications in the apical foramen, the apical foramen was basically closed, and the calcification barrier was intact.

[0059] The above results indicate that Cyaonoside B can effectively induce the formation of a calcified barrier in the root apex region in vivo, achieving physiological closure of the apical foramen and demonstrating a good root apex-inducing and shaping effect.

[0060] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Application of Cyaonoside B in the preparation of formulations that promote root tip induction.

2. The application according to claim 1, characterized in that, Cyaonoside B promotes the formation of the calcification barrier in the root apex and the closure of the apical foramen by mediating the c-Kit signaling pathway in the root development microenvironment.

3. The application according to claim 1 or 2, characterized in that, The preparation is an injection solution used to inject into the apical foramen area after root canal treatment.

4. The application according to claim 3, characterized in that, The concentration of Cyaonoside B in the formulation is 5-300 μM.

5. A cell model for screening root tip induction agents, characterized in that, The model uses primary dental germ epithelial cells, targets the c-Kit signaling pathway, and uses cell proliferation activity as an indicator. The activity of the test drug in promoting the remodeling of the root tip development microenvironment mediated by the c-Kit pathway is evaluated by CCK-8 or real-time cell analysis (RTCA) method.

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