Use of apelin-13 as a differentiation promoter for dental pulp stem cells

Abstract

The application relates to application of Apelin-13 as a pulp stem cell differentiation promoter. The application research proves that Apelin-13 has an important role in differentiation of pulp stem cells into odontoblasts and mineralization, and application of gelatin sponge containing Apelin-13 in a pulp capping operation can improve the quality and quantity of reparative dentin formation, and the safety and effectiveness of reparative dentin formation, and will have a wide clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of dental pulp repair materials technology, specifically relating to the application of Apelin-13 as a dental pulp stem cell differentiation promoter, including a pharmaceutical composition of Apelin-13, a biological pulp capping agent, and a dental pulp repair method using the biological pulp capping agent. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Dental caries is a common and prevalent oral disease, and achieving true biological pulp regeneration is the ultimate goal pursued in the field of pulp injury repair research. Currently, pulp capping agents are commonly used in clinical practice to promote the formation of reparative dentin, protect the pulp, and promote pulp regeneration. However, their effectiveness is affected by problems such as easy pulp calcification and poor biological activity.

[0004] Apelin-13 is a short peptide containing 13 amino acids with strong biological activity and is one of the common apelin subtypes in human blood. Apelin-13 is expressed in peripheral tissues and the central nervous system, exhibiting different biological functions and effects. Existing studies have shown that apelin-13 participates in pathophysiological processes of the cardiovascular, respiratory, nervous, digestive, and endocrine systems, and plays a role in regulating cell proliferation, autophagy, oxidative stress, and apoptosis. Therefore, research on apelin-13 will also promote disease prevention and treatment.

[0005] Pulp capping agents are commonly used and important materials in the field of dental endodontics. They are used in the treatment of deep caries to promote the formation of reparative dentin and protect the dental pulp. Currently, calcium hydroxide pulp capping agents are commonly used clinically to maintain a local alkaline environment, promote the differentiation of odontoblast-like cells and the formation of reparative dentin, thereby protecting the dental pulp. However, they have drawbacks such as poor bioactivity and a tendency to cause pulp cavity calcification, which affects their effectiveness. Summary of the Invention

[0006] This invention provides the application of Apelin-13 as a differentiation promoter for dental pulp stem cells (hDPSCs). Studies have confirmed that the addition of Apelin-13 to in vitro culture of dental pulp stem cells promotes odontogenic differentiation, playing a crucial positive regulatory role in promoting dentin formation and pulp regeneration. Specifically, this invention demonstrates that Apelin-13 is upregulated in human odontoblasts and promotes odontoblast differentiation. These results indicate that Apelin-13 has broad application prospects in the repair of dental pulp injuries and is expected to be used in pulp repair surgery to improve clinical efficacy.

[0007] Based on the above research findings, the present invention specifically provides the following technical solution:

[0008] In a first aspect, the present invention provides the application of Apelin-13 as a differentiation promoter of dental pulp stem cells.

[0009] It should be noted that the "application as a dental pulp stem cell differentiation promoter" in the first aspect mentioned above mainly refers to the application of Apelin-13 as an active ingredient to enhance the differentiation of dental pulp stem cells. All applications involving the above mechanism are within the scope of protection of the first aspect of this invention, and the specific application methods include at least the following three:

[0010] (1) Apelin-13 was applied as an active ingredient to individuals with dental pulp stem cell differentiation or repair needs;

[0011] (2) Used in the preparation of drugs, medical devices or dressings related to pulp restoration;

[0012] (3) Apply model drugs for preparing dental pulp stem cell differentiation models.

[0013] In the application of aspect (1) above, the dosage of Apelin-13 as the active ingredient should be a therapeutically effective dose. The clinician should be able to precisely adjust the dosage according to the purpose of use. The specific dosage can be confirmed using conventional methods in the art. The administration method includes oral, injection, patch, or other surgical methods. In a specific embodiment, the application of aspect (1) is to use Apelin-13 in pulp repair surgery or deep caries treatment.

[0014] In the application of aspect (2) above, one example of the pulp repair-related dressing is a gelatin sponge patch, in which a gelatin sponge is used as a carrier to carry an effective dose of Apelin-13 and applied to the affected area.

[0015] In this invention, Apelin-13 was added to the differentiation-induced culture of dental pulp stem cells. It was found that Apelin-13 can promote the expression of mineralization-related factors OCN and Runx2. Based on the above research conclusions, in the above (3) applications, Apelin-13 can also be used as a model agent for the differentiation-induced model of dental pulp stem cells, to obtain a dental pulp stem cell model with a high degree of differentiation or to obtain a model with high expression of mineralization factors such as OCN and Runx2.

[0016] In a second aspect, the present invention provides a pharmaceutical composition wherein the Apelin-13 is an active ingredient.

[0017] Preferably, the pharmaceutical composition described in the second aspect further includes other active ingredients, including but not limited to anti-inflammatory, antibacterial, antiviral, or pulp repair active ingredients.

[0018] Preferably, the pharmaceutical composition further includes pharmaceutically necessary excipients; further, the pharmaceutical composition is one of an oral preparation, a patch, or an injection.

[0019] In a third aspect, the present invention provides a biological operculum occlusion agent comprising an effective dose of Apelin-13 or the pharmaceutical composition described in the second aspect.

[0020] Preferably, the biological medullary capping agent is a medical patch, specifically an example of a gelatin sponge loaded with Apelin-13; the preparation method of the gelatin sponge loaded with Apelin-13 is as follows: a sterile collagen sponge is soaked in a solution of Apelin-13 until the collagen sponge is saturated.

[0021] Furthermore, in the solution of Apelin-13, the concentration of Apelin-13 is 8–12 μM, and more specifically, 10 or 11 μM.

[0022] In a fourth aspect, the present invention provides a method for pulp repair, the method comprising using the biological pulp capping agent described in the third aspect applied to the pulp tissue to be repaired.

[0023] Preferably, the pulp repair surgery is a deep caries treatment surgery, and the specific repair method is as follows: the decayed part of the tooth is decomposed and the cavity is prepared, the biological pulp capping agent described in the third aspect is applied to the pulp wall or pulp hole of the cavity, and then it is filled.

[0024] The beneficial effects of one or more of the above technical solutions are:

[0025] 1. Apelin-13 participates in the physiological processes of various diseases in the human body, exhibiting different activities at different sites. Existing research indicates that Apelin-13 promotes the proliferation of bone marrow mesenchymal stem cells. However, this invention, when applied to the culture of dental pulp stem cells, found that Apelin-13 does not promote the proliferation of dental pulp stem cells, but rather promotes their differentiation in a concentration-dependent manner. The above research results of this invention help to further clarify the physiological activity of Apelin-13 and provide a new approach for its clinical application.

[0026] 2. One technical solution of the present invention provides the application of Apelin-13 as a differentiation promoter of dental pulp stem cells. As a substance produced by the body itself, Apelin-13, as an active ingredient, is used in dental pulp repair-related drugs, medical devices, and surgeries. It has good biocompatibility and can effectively overcome the defects of calcium hydroxide pulp capping agents currently used in clinical practice, such as poor bioactivity and easy calcification of the pulp cavity. Attached Figure Description

[0027] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0028] Figure 1 The results of the investigation on the effect of Apelin-13 on hDPSC proliferation described in Example 1;

[0029] Figure 2 The expression of Apelin-13 in hDPSCs induced by mineralization in Example 1;

[0030] Figure 3 The results of adding Apelin-13 to the hDPSCs that were induced to differentiate in Example 1 and culturing them for 6 hours;

[0031] in, Figure 3 A represents the expression results of Runx2 in culture groups with different concentrations of Apelin-13;

[0032] Figure 3 B represents the expression results of OCN in culture groups with different concentrations of Apelin-13;

[0033] Figure 4 The expression results of OCN in hDPSCs induced to differentiate as described in Example 1 after culturing with Apelin-13 for 12 h;

[0034] Figure 5 This is a schematic diagram of the indirect medullary cap repair described in Example 2;

[0035] Figure 6This is a schematic diagram of the direct medullary capping repair described in Example 2;

[0036] The above Figure 5 and Figure 6 In the diagram, 1 represents the filling material, and 2 represents the pulp capping agent. Detailed Implementation

[0037] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, 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 invention pertains.

[0038] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0039] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments; the Apelin-13 used in the following embodiments was purchased from MedChemExpress (Monmouth Junction, NJ, USA), and the Apelin-13 solution was prepared as follows: 1 mg of Apelin-13 powder was dissolved in 0.6007 ml of sterile water, filtered to obtain a 1 mM stock solution, which was stored in a -80°C refrigerator and diluted to the appropriate concentration before use.

[0040] Example 1

[0041] 1. Investigation on the effect of Apelin-13 on hDPSC proliferation

[0042] To investigate the effect of Apelin-13 on the activity of dental pulp stem cells, this example uses a CCK8 assay, and the results are as follows: Figure 1 As shown in the figure, hDPSCs were cultured with a series of concentrations of Apelin-13 in this embodiment. After 24 hours of culture, it can be seen that different concentrations of Apelin-13 did not produce a significant difference in the number of hDPSCs, which indicates that Apelin-13 did not promote the proliferation of dental pulp stem cells.

[0043] 2. Correlation between Apelin-13 and hDPSCs mineralization

[0044] To investigate the application of Apelin-13 in the mineralization process of hDPSCs, this embodiment cultured and identified dental pulp stem cells. Real-time PCR was used to detect the endogenous expression of Apelin-13 during the differentiation of dental pulp stem cells into odontoblasts and the mineralization process. Results were obtained after 7 and 14 days of differentiation induction culture. Figure 2 As shown, from Figure 2 As can be seen, Apelin-13 expression is significantly upregulated during the differentiation and mineralization of dental pulp stem cells, and the expression level of Apelin-13 is also high during the peak of cell differentiation.

[0045] 3. The differentiating effect of Apelin-13 on hDPSCs

[0046] To investigate the effect of Apelin-13 on hDPSC differentiation, this study used a series of concentrations of Apelin-13 to differentiation-induced dental pulp stem cells, and examined the expression level of Runx2 in the stem cells at 6 h and 12 h of culture. Figure 3 , Figure 4 Real-time PCR results showed that after 6 hours of stimulation with apelin, it significantly promoted the expression of dentin formation-related proteins Runx2 and OCN at the mRNA level. Figure 3 ).

[0047] Example 2

[0048] In this embodiment, a pulp capping agent is provided for use in pulp restoration surgery. The pulp capping agent is a collagen sponge loaded with Apelin-13 protein, and its preparation method is as follows: 0.5 mm... 3 Sterile collagen sponges of various sizes were used as carriers and immersed in 5 μL of Apelin-13 protein solution. When the collagen sponges were saturated, they were removed and ready for use.

[0049] The application of the pulp capping agent is as follows: During the treatment of deep caries, the cavity is first prepared by removing decay. The prepared pulp capping agent is then applied to the pulp wall or perforation of the carious cavity, followed by permanent filling or temporary sealing with filling material. The above application includes both direct and indirect pulp capping methods. Indirect pulp capping is described in the attached... Figure 5 As shown in the attached diagram, direct medullary capping... Figure 6 As shown.

[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of Apelin-13 short peptide in the preparation of dental pulp stem cell differentiation promoter.

2. The application of Apelin-13 short peptide in the preparation of a biological pulp capping agent for promoting the differentiation of dental pulp stem cells, characterized in that: The thallus agent contains an effective dose of the short peptide Apelin-13.

3. The use of the Apelin-13 short peptide as described in claim 2 in the preparation of a biological pulp capping agent for promoting the differentiation of dental pulp stem cells, characterized in that, The biological medullary capping agent is a medical patch, which is a collagen sponge loaded with Apelin-13 short peptides. The preparation method of the collagen sponge loaded with Apelin-13 short peptides is as follows: a sterile collagen sponge is soaked in a solution of Apelin-13 short peptides until the collagen sponge is saturated.

Images