Extracellular matrix and its preparation method and application

Abstract

The present invention discloses an extracellular matrix, its preparation method, and application. The extracellular matrix is ​​prepared by adjusting dental stem cell culture conditions and time, followed by decellularization. This invention provides a microenvironment more suitable for the survival of dental stem cells, improves the timeliness of endogenous dental stem cells' odontoblastic and angiogenic differentiation, and offers an effective strategy for full dental pulp tissue regeneration and repair.

Description

Technical Field

[0001] The present invention belongs to the technical field of extracellular matrix preparation, and in particular relates to an extracellular matrix and a preparation method and application thereof. Background Art

[0002] The dental pulp is the only vascularized connective tissue in the tooth structure and is also a highly innervated tissue. The traditional treatment for pulp diseases is root canal therapy, which is widely used clinically. However, because it removes active pulp tissue and has no nerve activity, it is prone to postoperative complications and increases the risk of tooth loss. The dental pulp tissue has its own unique physiological structure. It is located in a hard space surrounded by dentin. The main blood vessels and lymphatic outflow of the dental pulp pass through the root apex. The apical opening is the main route for the exchange of nutrients and waste for the tooth. Even if there is a lateral root canal opening close to the apical foramen, it is much smaller than the apical foramen. This structure makes the injured dental pulp's potential for self-recovery very limited, making it increasingly difficult to form a new and complete vascular network within the complex and changing root canal system.

[0003] Tissue engineering scaffolds are bioactive matrices that support cell adhesion, regulate their proliferation and differentiation, and promote tissue formation. The ideal scaffold for regenerative endodontic treatment should closely resemble the extracellular matrix of the pulp-dentin complex in terms of biological composition and mechanical properties. It should also provide a three-dimensional microenvironment that allows dental stem cells, such as human dental pulp mesenchymal stem cells (hDPSCs), to bind, migrate, proliferate, and differentiate into odontogenic, angiogenic, and neurogenic lineages in three dimensions, as well as promote dentinogenesis, angiogenesis, and neurogenesis. Previous studies have developed scaffold materials such as simple gel microspheres, platelet lysate, or simvastatin-functionalized gel microspheres for regenerative endodontic treatment. However, these simple or functionally modified gel microspheres have limited functionality, promoting only angiogenesis or dentinogenesis, making it difficult to regenerate the complex dental pulp tissue.

[0004] Therefore, there is still a need for a scaffold material that can promote the regeneration of dental pulp tissue with complex components. Summary of the Invention

[0005] To address at least some of the technical issues in the prior art, the present invention utilizes dental stem cells cultured in complete medium, subjected to multi-stage drug-induced culture, and decellularized to produce an extracellular matrix, which can then be used to prepare dental pulp regeneration products. Specifically, the present invention encompasses the following.

[0006] In a first aspect of the present invention, a method for preparing an extracellular matrix is ​​provided, comprising the steps of culturing dental stem cells with complete medium, multi-stage drug-induced culture, and decellularization, wherein:

[0007] The multi-stage drug addition induction culture comprises:

[0008] The first stage induction culture includes the step of culturing in a serum-free medium containing added drugs;

[0009] a second-stage induction culture, which comprises the step of continuing the culture in a medium containing a drug-added fetal bovine serum-based medium at a first concentration; and

[0010] The third stage of induction culture comprises the step of continuing culture in a medium based on fetal bovine serum at a second concentration, wherein the second concentration is higher than the first concentration.

[0011] In certain embodiments, according to the preparation method of the present invention, the complete culture medium comprises 5-15% fetal bovine serum, 0.1-5% glutamine, 0.1-5% penicillin-streptomycin, 0.1-5% ascorbic acid and MEM Alpha basal medium.

[0012] In certain embodiments, according to the preparation method of the present invention, the culture medium used for the first stage induction culture is DMEM basal culture medium supplemented with 5-15 ng / ml platelet-derived factor, 20-30 ng / ml insulin-like growth factor, 25-35 ng / ml bone morphogenetic protein 2 and 0.1-5 ng / ml bone morphogenetic protein 4.

[0013] In certain embodiments, according to the preparation method of the present invention, the culture medium used for the second stage induction culture is DMEM basal culture medium supplemented with 1-5% fetal bovine serum, 5-15 ng / ml platelet-derived factor, 20-30 ng / ml insulin-like growth factor, 25-35 ng / ml bone morphogenetic protein 2 and 0.1-5 ng / ml bone morphogenetic protein 4.

[0014] In certain embodiments, according to the preparation method of the present invention, the culture medium used for the third stage induction culture is a DMEM basal culture medium supplemented with fetal bovine serum at a concentration greater than 5%.

[0015] In certain embodiments, according to the preparation method of the present invention, decellularization is performed using a decellularization reagent comprising the following components:

[0016] Reagent I: deionized water, ammonia, Triton 100, and phenylmethylsulfonyl fluoride;

[0017] Reagent II: anhydrous magnesium chloride solution, nuclease, and phenylmethylsulfonyl fluoride.

[0018] The second aspect of the present invention provides an extracellular matrix, which is prepared by the preparation method described in the first aspect.

[0019] The third aspect of the present invention provides use of an extracellular matrix in preparing a dental pulp regeneration product, wherein the extracellular matrix is ​​the extracellular matrix described in the present invention.

[0020] In certain embodiments, according to the use of the present invention, the dental pulp regeneration product is a dental pulp regeneration scaffold.

[0021] A fourth aspect of the present invention provides a method for regulating dental stem cells in vitro, wherein the regulation comprises upregulating the expression of at least one factor among COL1A1, DMP1, DSPP, VEGF, and PECAM in dental stem cells, the method comprising:

[0022] Dental stem cells are cultured in complete medium, cultured in multiple stages with drug addition, and then decellularized in three steps, wherein:

[0023] The multi-stage drug addition induction culture comprises:

[0024] The first stage induction culture includes the step of culturing in a serum-free medium containing added drugs;

[0025] a second-stage induction culture, which comprises the step of continuing the culture in a medium containing a drug-added fetal bovine serum-based medium at a first concentration; and

[0026] The third stage of induction culture comprises the step of continuing culture in a medium based on fetal bovine serum at a second concentration, wherein the second concentration is higher than the first concentration.

[0027] The present invention provides a microenvironment that is more suitable for the survival of dental stem cells, better improves the timeliness of odontoblastic differentiation and angiogenic differentiation of endogenous dental stem cells, and provides an effective strategy for the regeneration of full dental pulp tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The morphology and number of dental pulp mesenchymal stem cells 24 hours after plating are shown, where a is an image at 4X the initial cell density, and b is an image at 10X the initial cell density.

[0029] Figure 2 The morphology and number of dental pulp mesenchymal stem cells in the control group and the experimental group are shown, where a is a 4X image of the control group and b is a 4X image of the experimental group.

[0030] Figure 3 The morphology and number of dental pulp mesenchymal stem cells in the control group and the experimental group are shown, where a is a 10X image of the control group and b is a 10X image of the experimental group.

[0031] Figure 4The expression of related markers of dental pulp mesenchymal stem cells detected by RT-PCR in different drug components is shown.

[0032] Figure 5 The expression of related proteins of dental pulp mesenchymal stem cells in different drug components was detected by protein immunoblotting.

[0033] Figure 6 Shown is a proteomic cluster analysis of the extracellular matrix extracted from dental pulp mesenchymal stem cells.

[0034] Figure 7 Volcano plot of differential proteins between the extracellular matrix extracted from dental pulp mesenchymal stem cells cultured in the control group and the experimental group.

[0035] Figure 8 HE staining images of tissues after applying the scaffold material prepared from the extracellular matrix of Example 1 to the dental pulp defect model. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0039] Preparation method

[0040] One aspect of the present invention provides a method for preparing an extracellular matrix, wherein the extracellular matrix is ​​obtained from dental stem cells through a complete medium culture, multi-stage drug-induced culture, and decellularization process. The extracellular matrix prepared by the above-described process has the natural design structure and multiple biochemical components of the original organ, and has a high degree of similarity to normal tissues and organs.

[0041] In the present invention, dental stem cells include at least one of dental pulp stem cells (dental pulp mesenchymal stem cells), periodontal ligament stem cells, deciduous tooth exfoliated dental pulp stem cells, and gingival stem cells. In a preferred embodiment, the dental stem cells are dental pulp mesenchymal stem cells.

[0042] In the present invention, the complete culture medium comprises 5-15% (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15%) fetal bovine serum, 0.1-5% (e.g., 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5%) glutamine, 0.1-5% (e.g., 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5%) penicillin-streptomycin, 0.1-5% (e.g., 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5%) ascorbic acid, and MEM Alpha basal medium. In a preferred embodiment, the complete culture medium comprises 10% fetal bovine serum, 1% glutamine, 1% penicillin-streptomycin, 1% ascorbic acid, and MEM Alpha basal medium.

[0043] In the present invention, MEM Alpha basic medium can be purchased from commercial products (e.g., MEM Alpha basic (1X)) or prepared according to a known formula. In a preferred embodiment, the MEM Alpha basic (1X) contains amino acids (including but not limited to 1-10 mM L-glutamine, 0.1-5 mM L-arginine hydrochloride, etc.), vitamins (including but not limited to 0.1-1 mM ascorbic acid, 0-0.1 mM vitamin B12, 0.01-0.2 mM i-inositol, etc.), inorganic salts (including but not limited to 80-150mM sodium chloride, 10-50mM sodium bicarbonate, 0.5-5mM calcium chloride, 0.1-5mM magnesium sulfate, 1-10mM potassium chloride, 0.1-5mM sodium dihydrogen phosphate), ribonucleosides (including but not limited to 0.01-2mM adenine nucleoside, 0.01-2mM cytosine nucleoside, etc.), deoxyribonucleosides (including but not limited to 0.01-2mM 2'-deoxyadenosine nucleoside, etc.), 1-10mM glucose, 5-20mM lipoic acid, 0.1-5mM sodium pyruvate and 0.01-2mM phenol red.

[0044] In a preferred embodiment, the multi-stage drug-added induction culture includes a three-stage induction culture, which includes: a first-stage induction culture, which includes the step of culturing in a serum-free medium containing a drug for 1-5 days (e.g., 1, 2, 3, 4, 5 days); a second-stage induction culture, which includes the step of continuing to culture in a medium based on a first concentration (low concentration) of fetal bovine serum containing a drug for 1-5 days (e.g., 1, 2, 3, 4, 5 days); a third-stage induction culture, which includes the step of continuing to culture in a medium based on a second concentration (high concentration) of fetal bovine serum that does not contain the drug-added component of the present invention for 1-5 days (e.g., 1, 2, 3, 4, 5 days).

[0045] In the present invention, the drug components added for multi-stage drug addition induction culture include or consist of the following components: platelet-derived factor, insulin-like growth factor, bone morphogenetic protein 2 and bone morphogenetic protein 4.

[0046] In the present invention, the culture medium used for the first stage induction culture is supplemented with 5-15 ng / ml, preferably 6-14 ng / ml, and preferably 7-13 ng / ml, such as 7, 8, 9, 10, 11, 12, 13 ng / ml of platelet-derived factor; 20-30 ng / ml, preferably 21-29 ng / ml, and preferably 22-28 ng / ml, such as 22, 23, 24, 25, 26, 27, 28 ng / ml of insulin-like growth factor; 25 -35ng / ml, preferably 26-34ng / ml, also preferably 27-33ng / ml, for example 27, 28, 29, 30, 31, 32, 33ng / ml of bone morphogenetic protein 2; and 0.1-5ng / ml, preferably 0.5-4.5ng / ml, also preferably 1.0-4.0ng / ml, for example 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0ng / ml of DMEM basal culture medium of bone morphogenetic protein 4.

[0047] In the present invention, the DMEM basal medium can be purchased from commercial products (e.g., DMEM basic (1X)) or prepared according to a known formula. In a preferred embodiment, the DMEM basic (1X) contains amino acids (including but not limited to 2-6 mM L-glutamine, 0.5-1 mM L-lysine hydrochloride), vitamins (including but not limited to 0.01-0.2 mM inositol, 0.01-0.2 mM nicotinamide, etc.), inorganic salts (including but not limited to 80-150 mM sodium chloride, 20-60 mM sodium bicarbonate, 0.5-5 mM calcium chloride), 10-40 mM glucose, 0.1-5 mM sodium pyruvate, and 0.01-0.5 mM phenol red.

[0048] In the present invention, the medium used for the second stage induction culture is the medium used for the first stage drug-added induction culture supplemented with low-concentration fetal bovine serum, wherein the low-concentration fetal bovine serum is fetal bovine serum at a concentration of 1-5% (e.g., 1%, 2%, 3%, 4%, 5%).

[0049] In the present invention, the culture medium used for the third stage induction culture is DMEM basic (1X) without the drug-adding component of the present invention but supplemented with high-concentration fetal bovine serum. The high-concentration fetal bovine serum has a concentration greater than 5%, preferably 6-15%, and more preferably 8-15% (e.g., 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%) of fetal bovine serum.

[0050] In the present invention, a decellularization reagent is used for decellularization treatment, and the decellularization reagent includes the following components: Reagent I: deionized water, ammonia water, Triton 100 and phenylmethylsulfonyl fluoride (PMSF); Reagent II: anhydrous magnesium chloride solution, nuclease and PMSF.

[0051] In a preferred embodiment, reagent I comprises 30-70 ml, preferably 35-65 ml, further preferably 40-60 ml, more preferably 45-55 ml, for example 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 ml of deionized water; 50-100 μl, preferably 55-95 μl, further preferably 60-90 μl, further preferably 65-85 μl, more preferably Select 70-80 μl, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 μl of ammonia water; 400-600 μl, preferably 420-580 μl, also preferably 440-560 μl, more preferably 450-550 μl, such as 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550 μl Triton100; and 400-600 μl, preferably 420-580 μl, also preferably 440-560 μl, more preferably 450-550 μl, for example 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550 μl PMSF (0.1-2 μM, preferably 1-2 μM, for example 1 μM).

[0052] In a preferred embodiment, reagent II comprises 1-10 ml, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ml of anhydrous magnesium chloride solution (1-5 mM, preferably 1-3 mM, for example 2 mM); 0.1-5 μl, preferably 0.2-4.5 μl, also preferably 0.3-4.0 μl, further preferably 0.4-3.5 μl, more preferably 0.5-3.0 μl, for example 0.5, 0.6, 0.8, 10 ml of anhydrous magnesium chloride solution (1-5 mM, preferably 1-3 mM, for example 2 mM); 0.1-5 μl, preferably 0.2-4.5 μl, further preferably 0.3-4.0 μl, further preferably 0.4-3.5 μl, more preferably 0.5-3.0 μl, for example 0.5, 0.6, 0.8, 10 ml of anhydrous magnesium chloride solution (1-5 mM, preferably 1-3 mM, for example 2 mM); 7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 2.7, 3.0 μl nuclease; and 30-70 ml, preferably 35-65 ml, also preferably 40-60 ml, more preferably 45-55 ml, for example 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 ml PMSF (0.1-2 μM, preferably 1-2 μM, for example 1 μM).

[0053] use

[0054] One aspect of the present invention provides the use of an extracellular matrix in the preparation of a dental pulp regeneration product, wherein the dental pulp regeneration product includes a scaffold, such as, but not limited to, a tissue engineering scaffold material for dental pulp regeneration. When used as a scaffold material, the extracellular matrix can be combined with an optional carrier or excipient to form a composite material, which can be selected by those skilled in the art based on their needs and is not particularly limited.

[0055] Example 1

[0056] The method for preparing the dental pulp mesenchymal stem cell extracellular matrix is ​​shown below.

[0057] 1. Culture of Dental Pulp Mesenchymal Stem Cells

[0058] Dental pulp mesenchymal stem cells (derived from an oral stem cell bank) were cultured in complete culture medium (10% FBS, 1% glutamine, 1% double-antibody, 1% ascorbic acid, and MEM Alpha basic (1X) supplement) until the cell confluence reached 80%-90%, and then drug induction was added for further culture.

[0059] 2. Drug-induced culture

[0060] The drug-induced culture was conducted in three stages: the first stage was culturing in serum-free DMEM basic (1X) containing the drug for 3 days to observe cell morphology and number and obtain cells induced in the early stages; the second stage was culturing in DMEM basic (1X) containing the drug and 2% fetal bovine serum for 3 days to maintain induction while accelerating cell growth; the third stage was culturing in 10% fetal bovine serum without the drug-induced agent for another 3 days to prepare a larger amount of extracellular matrix for subsequent experiments. In the above method, the drug components added to DMEM basic (1X) included: 10 ng / ml platelet-derived growth factor (PDGF-bb); 25 ng / ml insulin-like growth factor (IGF-1); 30 ng / ml bone morphogenetic protein 2 (BMP-2); and 3 ng / ml bone morphogenetic protein 4 (BMP-4).

[0061] 3. Decellularization

[0062] After the three-stage induction, a decellularization reagent is added to the cells to obtain the extracellular matrix.

[0063] Decellularization steps: remove old culture medium; wash twice with 10ml PBS and aspirate clean; add prepared reagent 1 and time for 20 minutes; aspirate reagent 1 after the time is up; repeat the above steps twice; turn on the constant temperature shaker and adjust the temperature to 37℃ when starting to wash for the third time; observe under a microscope and take pictures after the third wash; wash twice with 10ml PBS and aspirate clean; add prepared reagent 2; put in a constant temperature shaker at 37℃ and time for 30 minutes; after the time is up, remove the reagent in the culture dish; wash three times with 10ml PBS and aspirate clean; add 3ml PBS to each dish and scrape off the ECM with a cell scraper; use a pipette to aspirate the PBS containing ECM into a 2ml EP tube; centrifuge in a high-speed centrifuge for 20 minutes; pour out the supernatant after centrifugation and collect the ECM.

[0064] Decellularization reagent composition: Reagent 1: 50 ml deionized water + 77 μl ammonia water + 500 μl Triton100 + 500 μl PMSF (1 μM); Reagent 2: 5 ml anhydrous magnesium chloride solution (2 mM) + 1 μl Benzonase nuclease + 50 μl PMSF (1 μM).

[0065] 4. Experimental Results

[0066] This example studies the morphology and quantity of dental pulp mesenchymal stem cells cultured in conventional complete medium and drug-added complete medium, wherein: Figure 1 This is an image of dental pulp mesenchymal stem cells under a microscope. Figure 2 and Figure 3In the experimental group, a large number of cells aggregated at the marked position, indicating that the dental pulp mesenchymal stem cells cultured in the experimental group (complete medium with drug) had a better growth effect than those in the control group (conventional complete medium).

[0067] The expression of six genes, COL1A1, DMP1, DSPP, FN1, VEGF, and PECAM, which showed significant differential expression during pulp regeneration, was analyzed after culturing in different medium with added drug components for 6 days. Figure 4 As shown in the figure, the expression levels of COL1A1, DMP1, DSPP, VEGF, and PECAM were the highest in the selected medication component PIB2B4 (in the figure, P represents the addition of platelet-derived factor PDGF-bb; I represents the addition of insulin-like growth factor IGF-1; B2 represents the addition of bone morphogenetic protein 2 (BMP-2); B4 represents the addition of bone morphogenetic protein 4 (BMP-4), and different letter combinations represent different medication regimens). The expression level of FN1 ranked second in the selected regimen and was also at a high level. Among them, COL1A1 and VEGF were consistent with the proteomic results.

[0068] The organic components of dental pulp are rich in type I collagen and fibrin, corresponding to which COL1A1 and FN1 are highly expressed; DMP1 and DSPP are markers related to the differentiation of dental pulp mesenchymal stem cells into odontoblasts; whether blood vessels regenerate plays a vital role in the process of dental pulp regeneration, corresponding to which VEGF and PECAM markers are highly expressed.

[0069] The extracellular matrix of dental pulp mesenchymal stem cells cultured in conventional complete medium and in drug-added medium was extracted for quantitative proteomic analysis ( Figure 5 ). In addition, a cluster analysis diagram was drawn, and the distances between multiple samples were calculated through protein expression data to form a distance matrix. The two classes with the closest distance were merged into a new class, and the distances between the new class and the current classes were calculated. The new class was merged and calculated again until there was only one class. The expression of the selected differential proteins was used to calculate the direct correlation between samples. Samples of the same class can be in the same cluster in the cluster tree because the data attributes are close. Proteins with similar expression values ​​will also be in the same cluster, which may represent similar biological functions. In the cluster diagram, the horizontal axis represents the sample names between groups, and the vertical axis represents the differential genes. In the diagram, red represents high expression values ​​of differential genes in the grouped samples, and blue represents low expression values ​​of differential proteins in the grouped samples ( Figure 6 ). Combined Figure 7 The differential protein volcano plot data showed that the ECM2 protein in the drug-treated cell group was significantly upregulated, indicating that the drug-treated group could produce more extracellular matrix.

[0070] The volcano plot shows the difference between the extracellular matrix extracted from dental pulp mesenchymal stem cells cultured in regular complete medium and drug-supplemented complete medium. Figure 7 There were 1066 differentially expressed proteins, of which 882 were upregulated and 184 were downregulated. In the volcano plot, the vertical axis displays the -log10 (p-value) value, and the horizontal axis displays the log2 (Fold change) value. The screening group was screened for a Fold change ratio ≥2 or ≤0.5 (i.e., 1 / 2) and a p-value <0.05. Upregulated proteins meeting these criteria were indicated by red dots, downregulated proteins by blue dots, and all other proteins by gray dots. The results for VEGFC and COL1A1 were consistent with those for PT-PCR, and the ECM2 protein was consistent with the aggregation observed in drug-treated cultured cells under a microscope.

[0071] Example 2

[0072] The following shows a scaffold material prepared based on the extracellular matrix prepared in Example 1 and the application of the scaffold material in dental pulp regeneration.

[0073] The extracellular matrix obtained by decellularization was freeze-dried in a freeze dryer for 24 hours to obtain extracellular matrix (ECM) powder. An ECM solution and a photocurable hydrogel solution were prepared using a suitable aqueous solvent (such as distilled water or a buffer with an appropriate pH value). The two were then combined through a photocuring reaction to form an ECM-functionalized hydrogel. The functionalized hydrogel was applied to the constructed in situ pulp defect model of the maxillary first molars of SD rats, and HE staining was performed 30 days after the addition of the ECM hydrogel. The results are shown in Figure 2. Figure 8 As shown, loose connective tissue composed of fibroblast-like cells and blood vessels can be seen, and the black arrows indicate small blood vessels. The results indicate that the scaffold material can be effectively used for dental pulp regeneration.

[0074] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may be modified or some of the technical features thereof may be replaced with equivalents. Such modifications or replacements do not deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for preparing an extracellular matrix, characterized in that: The method comprises three steps: culturing dental stem cells in complete medium, multi-stage drug-added induction culture, and decellularization, wherein: The complete culture medium contains 5-15% fetal bovine serum, 0.1-5% glutamine, 0.1-5% penicillin-streptomycin, 0.1-5% ascorbic acid and MEM Alpha basal medium; The multi-stage drug addition induction culture comprises: The first stage of induction culture comprises the step of culturing in a serum-free medium containing added drugs, wherein the medium used for the first stage of induction culture is a DMEM basal medium supplemented with 5-15 ng / ml platelet-derived factor PDGF-bb, 20-30 ng / ml insulin-like growth factor IGF-1, 25-35 ng / ml bone morphogenetic protein 2 (BMP-2), and 0.1-5 ng / ml bone morphogenetic protein 4 (BMP-4); The second stage induction culture comprises the step of continuing to culture in a medium containing a first concentration of fetal bovine serum with added drugs, wherein the medium used for the second stage induction culture is a DMEM basal medium supplemented with 1-5% fetal bovine serum, 5-15 ng / ml platelet-derived factor PDGF-bb, 20-30 ng / ml insulin-like growth factor IGF-1, 25-35 ng / ml bone morphogenetic protein 2 (BMP-2), and 0.1-5 ng / ml bone morphogenetic protein 4 (BMP-4); and The third stage induction culture includes the step of continuing culture in a culture medium based on a second concentration of fetal bovine serum, wherein the second concentration is higher than the first concentration, and the culture medium used for the third stage induction culture is a DMEM basal culture medium supplemented with a concentration of fetal bovine serum greater than 5%.

2. The preparation method according to claim 1, characterized in that Decellularization was performed using a decellularization reagent comprising the following components: Reagent I: deionized water, ammonia, Triton 100, and phenylmethylsulfonyl fluoride; Reagent II: anhydrous magnesium chloride solution, nuclease, and phenylmethylsulfonyl fluoride.

3. An extracellular matrix, characterized in that It is prepared by the preparation method according to claim 1 or 2.

4. Use of extracellular matrix in preparing dental pulp regeneration products, characterized in that: The extracellular matrix is ​​the extracellular matrix according to claim 3 or is prepared by the preparation method according to claim 1 or 2.

5. The use according to claim 4, characterized in that The dental pulp regeneration product is a dental pulp regeneration scaffold.

6. A method for regulating dental stem cells in vitro, characterized in that: The regulation includes upregulating the expression of at least one factor among COL1A1, DMP1, DSPP, VEGF, and PECAM in dental stem cells, and the method includes: Dental stem cells are cultured in complete medium, cultured in multiple stages with drug addition, and then decellularized in three steps, wherein: The complete culture medium contains 5-15% fetal bovine serum, 0.1-5% glutamine, 0.1-5% penicillin-streptomycin, 0.1-5% ascorbic acid and MEM Alpha basal medium; The multi-stage drug addition induction culture comprises: The first stage of induction culture comprises the step of culturing in a serum-free medium containing added drugs, wherein the medium used for the first stage of induction culture is a DMEM basal medium supplemented with 5-15 ng / ml platelet-derived factor PDGF-bb, 20-30 ng / ml insulin-like growth factor IGF-1, 25-35 ng / ml bone morphogenetic protein 2 (BMP-2), and 0.1-5 ng / ml bone morphogenetic protein 4 (BMP-4); The second stage induction culture comprises the step of continuing to culture in a medium containing a first concentration of fetal bovine serum with added drugs, wherein the medium used for the second stage induction culture is a DMEM basal medium supplemented with 1-5% fetal bovine serum, 5-15 ng / ml platelet-derived factor PDGF-bb, 20-30 ng / ml insulin-like growth factor IGF-1, 25-35 ng / ml bone morphogenetic protein 2 (BMP-2), and 0.1-5 ng / ml bone morphogenetic protein 4 (BMP-4); and The third stage induction culture includes the step of continuing culture in a culture medium based on a second concentration of fetal bovine serum, wherein the second concentration is higher than the first concentration, and the culture medium used for the third stage induction culture is a DMEM basal culture medium supplemented with a concentration of fetal bovine serum greater than 5%.

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