Laminin modified collagen hydrogel material as well as preparation method and application thereof

Abstract

The invention relates to a hydrogel material containing laminin, the hydrogel material comprises a hydrogel body and an effective amount of laminin subunit arranged in the hydrogel body, and the laminin subunit is cross-linked with the hydrogel body. The laminin subunit can be added into the hydrogel, so that the polarity of the dental papilla cell of the miniature pig and the differentiation level of the dentin-forming cell are remarkably improved, the differentiation of the dentin-forming cell is directionally regulated and controlled, the differentiation level of the dentin-forming cell is effectively improved, the operation is simple, and the effect is remarkable. In addition, laminin subunits are added into the collagen-based hydrogel, so that the rigidity of the collagen hydrogel is improved, and the arrangement and mechanical strength of collagen fibers in the hydrogel are improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular, relates to a laminin-modified collagen hydrogel material, a preparation method and an application thereof. Background Art

[0002] Hydrogels are widely used as scaffold materials in tissue engineering because of their soft, biocompatible polymer network system. Currently, hydrogels used for dentin regeneration include collagen-based hydrogels, hyaluronic acid-based hydrogels, gelatin methacryloyl hydrogels, and dentin matrix hydrogels. Compared with other hydrogels, collagen-based hydrogels have good biocompatibility and biodegradability, more controllable mechanical strength, good thermal stability, and have advantages in simulating the natural extracellular matrix environment.

[0003] However, there are still some problems with collagen-based hydrogels in the field of dentin regeneration: 1. Control of mechanical strength: Hydrogels need sufficient mechanical strength to withstand the pressure in the body. The main method for regulating collagen-based hydrogels is to increase the concentration of collagen. This approach increases the consumption of collagen and the improvement in mechanical strength is limited by the initial concentration of collagen; 2. Control of cell behavior: Hydrogels need to be able to accurately control the behavior of stem cells, including their proliferation, differentiation direction, and mineralization process, in order to promote the effective regeneration of dentin. At present, it is not clear whether the stem cell differentiation induced by collagen-based hydrogels is odontoblasts or osteoblasts, which makes the structure and strength of the regenerated hard tissue still have a gap with natural dentin.

[0004] Therefore, how to improve the properties of collagen matrix hydrogels to increase mechanical strength and effectively control the behavior of stem cells so that the structure and strength of the regenerated hard tissue are closer to natural dentin is a technical problem that urgently needs to be solved in this field. Summary of the invention

[0005] In view of the problems existing in the above-mentioned prior art, the present invention provides a hydrogel material containing laminin, wherein the hydrogel material comprises a hydrogel body and an effective amount of laminin subunits disposed in the hydrogel body, wherein the laminin subunits are cross-linked with the hydrogel body.

[0006] Laminin (LN) is a non-collagen sugar that constitutes the intercellular matrix. It is mainly synthesized by endothelial cells and fat-storing cells in the liver. Together with collagen, it constitutes a component of the basement membrane. Its biological function is to act as a medium for cell adhesion to the matrix, and it binds to a variety of basement membrane components to regulate cell growth and differentiation. Laminin is mainly present in the clear layer of the basement membrane, close to the surface of the cell base. Laminin is a heterotrimeric molecule composed of three subunit chains: α chain, β chain, and γ chain. There are five known α chains: α1 to α5, three β chains: β1 to β3, and three γ chains: γ1 to γ3.

[0007] Hydrogel is a type of extremely hydrophilic three-dimensional network structure gel. It swells rapidly in water and can hold a large volume of water without dissolving in this swollen state. Due to the presence of a cross-linked network, hydrogel can swell and hold a large amount of water. The amount of water absorbed is closely related to the degree of cross-linking. The higher the degree of cross-linking, the lower the water absorption. The water content in the hydrogel can be as low as a few percent or as high as 99%. The aggregated state of the gel is neither completely solid nor completely liquid.

[0008] As certain embodiments of the present application, the hydrogel body is selected from at least one of collagen-based hydrogel, hyaluronic acid-based hydrogel, gelatin methacryloyl hydrogel, and dentin matrix hydrogel.

[0009] As certain embodiments of the present application, the hydrogel body is a collagen-based hydrogel.

[0010] As certain embodiments of the present application, the collagen in the collagen-based hydrogel is selected from one or more of type I collagen, type II collagen, type III collagen, type V collagen, and type XI collagen.

[0011] Collagen is a biopolymer and the main component of animal connective tissue. There are many types of collagen, and the common types are type I, type II, type III, type V and type XI. Collagen has good biocompatibility, biodegradability and biological activity. Collagen is a family of proteins. According to their distribution and functional characteristics in the body, collagen can be divided into interstitial collagen, basement membrane collagen and pericellular collagen. Interstitial collagen molecules account for the vast majority of collagen in the whole body, including type I, II and III collagen molecules. Type I collagen is mainly distributed in tissues such as skin and tendons, and type II collagen is produced by chondrocytes; basement membrane collagen usually refers to type IV collagen, which is mainly distributed in the basement membrane; pericellular collagen usually refers to type V collagen, which exists in large quantities in connective tissue.

[0012] As certain embodiments of the present application, the collagen in the collagen-based hydrogel is selected from one or more of type I collagen, type II collagen, and type III collagen.

[0013] As certain embodiments of the present application, the collagen in the collagen-based hydrogel is type I collagen.

[0014] As certain embodiments of the present application, the collagen concentration is 2-5 mg / ml.

[0015] The collagen concentration can be selected from 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5 mg / ml.

[0016] As certain embodiments of the present application, the collagen concentration is 3-4 mg / ml.

[0017] As certain embodiments of the present application, the collagen concentration is 3 mg / ml.

[0018] As certain embodiments of the present application, the laminin subunit is selected from one or more of laminin α1, α2, α3, α4, α5, β1, β2, β3, γ1, γ2, and γ3 subunits. As certain embodiments of the present application, the laminin subunit is laminin β3.

[0019] As certain embodiments of the present application, the concentration of the laminin subunit is ≥5 ng / ml.

[0020] As certain embodiments of the present application, the concentration of the laminin subunit is 5-500 ng / ml.

[0021] 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 ng / ml.

[0022] As certain embodiments of the present application, the concentration of the laminin subunit is 50-500 ng / ml.

[0023] As certain embodiments of the present application, the concentration of the laminin subunit is 50 ng / ml.

[0024] The present application also provides a method for preparing the hydrogel material containing laminin, which comprises the following steps: (1) adding collagen to a solvent, cross-linking the collagen molecules to obtain a collagen cross-linked product; (2) adding laminin subunits to the collagen cross-linked product in step (1) to obtain a hydrogel material containing laminin.

[0025] As certain embodiments of the present application, in step (1), the concentration of collagen is 2-5 mg / ml, and in step (2), the final concentration of the laminin subunit is adjusted to be ≥5 ng / ml.

[0026] As certain embodiments of the present application, in step (1), the pH is 7.0-7.5.

[0027] In step (1), the pH value can be selected from 7.0, 7.1, 7.2, 7.3, 7.4, and 7.5.

[0028] As certain embodiments of the present application, the solvent in step (1) is water or PBS.

[0029] As certain embodiments of the present application, the collagen concentration is 3 mg / ml.

[0030] As certain embodiments of the present application, the final concentration of the laminin subunit is 5-500 ng / ml.

[0031] As certain embodiments of the present application, the final concentration of the laminin subunit is 50 ng / ml.

[0032] The present application also provides a drug, which comprises the hydrogel material containing laminin, and the final concentration of the laminin subunit is 50-500 ng / ml.

[0033] The present application also provides the use of the drug in the preparation of a commercial drug, which has at least any one of the following functions: a. promoting dentin regeneration; b. improving the differentiation level of odontoblasts; c. improving the polarity of dental papilla cells; d. improving the activity of tooth germ cells; and e. increasing the expression of molecules related to odontoblast differentiation.

[0034] As certain embodiments of the present application, the dental papilla cell polarity includes planar cell polarity and apical-basal cell polarity.

[0035] As certain embodiments of the present application, the odontoblast differentiation-related molecules include tooth germ cell polarity molecule RHOA and odontoblast differentiation marker molecules DMP1 and DSPP.

[0036] The present application also provides a hydrogel product, which comprises the hydrogel containing laminin, wherein the hydrogel is a collagen-based hydrogel, and the laminin content is 5-500 ng / ml.

[0037] The present application also provides applications of the hydrogel product, which applications include at least any one of the following: a. used to improve the arrangement and mechanical strength of collagen fibers in the hydrogel; b. used to reduce the pore size distribution differences in the collagen hydrogel; c. used to reduce the heterogeneity of collagen fiber distribution; and d. used to increase fiber bonding strength.

[0038] The present application also provides the use of the hydrogel material containing laminin in the preparation of products, wherein the products include one or more of drugs and hydrogel products, and the products have any of the following effects: a. promoting dentin regeneration; b. improving the differentiation level of odontoblasts; c. improving the polarity of dental papilla cells; d. increasing the expression of molecules related to odontoblast differentiation: including the expression of tooth germ cell polarity molecule RHOA, odontoblast differentiation marker molecule DMP1, and DSPP; e. improving the arrangement and mechanical strength of collagen fibers in the hydrogel; f. reducing the difference in pore size distribution of collagen hydrogel; g. reducing the heterogeneity of collagen fiber distribution; and i. increasing fiber bonding strength.

[0039] As described above, the laminin-modified collagen hydrogel of the present application and its preparation method and application have the following beneficial effects:

[0040] The present application can improve the stiffness of collagen hydrogel and the arrangement and mechanical strength of collagen fibers in the hydrogel by adding only a small amount of one component, namely, laminin subunit recombinant protein.

[0041] The present application found that the polarity of miniature pig dental papilla cells and the differentiation level of odontoblasts can be significantly improved by adding laminin subunits to the hydrogel, which is simple to operate and has significant effects.

[0042] The present application found that by adding laminin subunits to the hydrogel, the differentiation of odontoblasts can be regulated in a direction, thereby effectively improving the differentiation level of odontoblasts. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It was shown that the addition of 5-500ng / ml LAMB3 can significantly increase the stiffness of collagen hydrogels;

[0044] Figure 2 The effect of adding 5-500ng / ml LAMB3 collagen hydrogel on the microstate of collagen hydrogel is shown;

[0045] Figure 3 The cell morphology changes of miniature pig dental papilla cells after being plated on the hydrogel surface are shown;

[0046] Figure 4 The expression of cell polarity and odontoblast differentiation-related molecules in miniature pig dental papilla cells cultured on the above hydrogel surface for 3 days was analyzed using RT-qPCR;

[0047] Figure 5 The figure shows sections of miniature pig bell-shaped early deciduous molar tooth germs cultured in collagen hydrogels containing laminin. After culture in collagen hydrogels, staining shows the effects of laminin subunits on cell activity, cell polarity, and odontoblast differentiation. DETAILED DESCRIPTION

[0048] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] [Experimental Materials]

[0050] Collagen I (Yisheng Company, product number 40125ES50)

[0051] Recombinant human LAMB3 protein (AntibodySystem, catalog number YHG64104, size 67.24 kDa, protein length Thr587-Lys1172)

[0052] Phalloidin (Proteintech, Catalog No. PF00003)

[0053] Hematoxylin and eosin: Hematoxylin staining solution (Harris) (Zhuhai Beso Biotechnology Co., Ltd., BA4097), eosin staining solution (Solarbio, G1100)

[0054] EDU (Biyuntian Company, BeyoClick TM EdU-488 Cell Proliferation Detection Kit, Catalog No. C0071S)

[0055] Immunofluorescence staining:

[0056] RHOA antibody (Proteintech, 66733-1-lg)

[0057] DMP1 antibody (Affinity Biosciences, DF8825)

[0058] DSPP antibody, (abcam, ab216892)

[0059] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0060] Example 1 Preparation of collagen hydrogel containing laminin

[0061] The preparation method of the collagen hydrogel containing laminin in the present application is as follows:

[0062] 1. Prepare the gel in a sterile tube on ice and add sterile 10×PBS (final volume / 10) ml first;

[0063] 2. Calculate the required volume of collagen I (ml) = (final volume × 3) / sales protein concentration;

[0064] 3. Add (volume of collagen to be added × 0.023) ml of sterile ice-cold 1 M NaOH to 10× PBS solution;

[0065] 4. Add the following volume of sterile ice-cold distilled water to the solution in the previous step and mix well. The pH of the gel is 7.0-7.5:

[0066] Volume of distilled water added = V(final) - V(collagen) - V(10×PBS) - V(1M NaOH)

[0067] 5. Add the volume of collagen I calculated in step 2 to the mixed solution in the previous step and mix well on ice;

[0068] 6. Add recombinant human LAMB3 protein on ice and mix well to make the final concentration of LAMB3 50 ng / ml;

[0069] 7. Before use, add the above solution to the culture device and let it gel at 37°C for 30 minutes.

[0070] The amount of collagen used in step 2 of the above preparation method can achieve the present invention as long as it can form a gel. In this application, the final concentration of collagen is selected to be 3-4 mg / ml.

[0071] Example 2 Testing of Mechanical Properties of Collagen Hydrogels Containing Laminin

[0072] 2.1 According to the preparation method of the collagen hydrogel containing laminin in Example 1, collagen hydrogels containing different concentrations of laminin β3 subunit were prepared, and the concentrations of laminin β3 subunit were 5, 50, and 500 ng / ml respectively. The mechanical strength of the collagen hydrogel containing different concentrations of laminin β3 subunit was detected by the following detection method: gel was prepared in 35 mm culture medium, and Young's modulus was measured under atomic force microscope on the day after gelation.

[0073] Test results see Figure 1 , it can be seen that the added laminin subunits can increase the mechanical strength of collagen hydrogel within a certain concentration range, to be precise, within the range of 5 to 500 ng / ml.

[0074] 2.2 The collagen in the collagen hydrogels with different concentrations of laminin subunits was stained. The results of collagen fiber staining showed that the addition of 5-500ng / ml LAMB3 changed the arrangement of collagen fibers ( Figure 2 A), the addition of 5-500ng / ml LAMB3 reduced the pore size distribution difference of collagen hydrogel, especially 50ng / ml LAMB3 significantly reduced the pore size distribution difference of collagen hydrogel, and the collagen fiber distribution heterogeneity was smaller and the fiber binding strength was higher (see Figure 2 B).

[0075] Example 3 Effect of collagen hydrogel containing laminin on the polarity of dental papilla cells

[0076] Miniature pig dental papilla cells were plated on the hydrogel surface and stained with phalloidin for the cytoskeletal protein F-actin to show changes in cell morphology. Figure 3 A is a schematic diagram of plating miniature pig dental papilla cells on the hydrogel surface. Figure 3 The cell morphological changes in B showed that the cell polarity increased with the increase of collagen concentration, and the addition of 5-500ng / ml LAMB3 could achieve the improvement of cell polarity.

[0077] ImageJ was used to measure the cytoskeleton for quantitative analysis of planar cell polarity (i.e., the aspect ratio of the cytoskeleton) and apical-basal cell polarity (i.e., the ratio of the length of the nucleus to the top and bottom of the cell on the long axis). It was found that the addition of 5-500 ng / ml of laminin subunits could simultaneously increase both planar cell polarity and apical-basal cell polarity (see Table 1, Table 2, respectively). Figure 3 C and 3D), when the concentration of laminin subunits is selected to be 50-500 ng / ml, planar cell polarity and apical-basal cell polarity can be significantly improved.

[0078] Compared with the blank control ( Figure 3 As for the gel 3 mg / ml group in B to D, the experimental group of the present application only added laminin subunits (content ≥ 5 ng / ml LAMB3) to achieve the improvement of cell polarity.

[0079] Example 4 Effects of laminin-containing collagen hydrogel on cell polarity and odontoblast differentiation at the molecular level

[0080] 4.1 Miniature pig dental papilla cells cultured on collagen hydrogel surfaces containing different concentrations of laminin subunits for 3 days were collected and the expression of molecules related to cell polarity and odontoblast differentiation was analyzed by RT-qPCR. It was found that a certain concentration of laminin subunits could increase the expression of RHOA, DMP1, and DSPP mRNA. 50 ng / ml was the lowest effective concentration of LAMB3 to significantly increase the expression of RHOA, DMP1, and DSPP mRNA (see Figure 4 ). At the same time, it can be seen that compared with the blank control ( Figure 4 As for the gel 3mg / ml group in A to C, the experimental group of the present application only added laminin subunits (content ≥50ng / mlLAMB3) to increase the expression of RHOA, DMP1, and DSPP mRNA.

[0081] 4.2 The bell-shaped early deciduous molar tooth germ of miniature pigs was sliced ​​and cultured in collagen hydrogel. The gel covered half the height of the tooth germ. See the schematic diagram. Figure 5 A. Staining of the tooth germ sections of bell-shaped early deciduous molars of miniature pigs. HE showed that a layer of cells with higher polarity appeared in the dental papilla after culture in collagen hydrogel supplemented with 50ng / ml LAMB3. EDU staining showed that the tooth germs cultured in collagen hydrogel supplemented with 50ng / ml LAMB3 had good cell activity. Immunofluorescence staining showed that the expression of polarity molecule RHOA and odontoblast differentiation marker molecules DMP1 and DSPP in tooth germ cells cultured in collagen hydrogel supplemented with 50ng / ml LAMB3 was significantly increased (see Figure 5 B). At the same time, it can be seen that compared with the blank control ( Figure 5 As for the gel 3mg / ml group in B), the experimental group of the present application only added laminin subunits (content ≥50ng / mlLAMB3), which can increase the expression of RHOA, DMP1, and DSPP mRNA and improve the cell activity of tooth germ.

[0082] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A hydrogel material containing laminin, characterized in that: The hydrogel material comprises a hydrogel body and an effective amount of laminin subunits arranged in the hydrogel body, wherein the laminin subunits are cross-linked with the hydrogel body.

2. The laminin-containing hydrogel material according to claim 1, characterized in that: The hydrogel body is selected from at least one of collagen-based hydrogel, hyaluronic acid-based hydrogel, gelatin methacryloyl hydrogel, and dentin matrix hydrogel; Preferably, the hydrogel body is a collagen-based hydrogel.

3. The laminin-containing hydrogel material according to claim 2, characterized in that: The collagen in the collagen-based hydrogel is selected from one or more of type I collagen, type II collagen, type III collagen, type V collagen, and type XI collagen; Preferably, the collagen is selected from one or more of type I collagen, type II collagen, and type III collagen; Preferably, the collagen is type I collagen; Preferably, the collagen concentration is 2-5 mg / ml; Preferably, the collagen concentration is 3-4 mg / ml; Preferably, the collagen concentration is 3 mg / ml.

4. The laminin-containing hydrogel material according to claim 1, characterized in that: The laminin subunit is selected from one or more of laminin α1, α2, α3, α4, α5, β1, β2, β3, γ1, γ2, and γ3 subunits; Preferably, the laminin subunit is laminin β3 subunit.

5. The laminin-containing hydrogel material according to claim 1, characterized in that: The concentration of the laminin subunit is ≥5 ng / ml; Preferably, the concentration of the laminin subunit is 5-500 ng / ml; Preferably, the concentration of the laminin subunit is 50-500 ng / ml; Preferably, the concentration of laminin is 50 ng / ml.

6. A method for preparing the laminin-containing hydrogel material according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: (1) adding collagen to a solvent to cross-link the collagen molecules to obtain a collagen cross-linked product; (2) adding laminin subunits to the collagen cross-linked product of step (1) to obtain a laminin-containing hydrogel material.

7. The method according to claim 6, characterized in that In step (1), the concentration of collagen is 2-5 mg / ml, and in step (2), the final concentration of laminin subunit is adjusted to ≥ 5 ng / ml; Alternatively, in step (1), the pH is 7.0-7.5; Alternatively, in step (1), the solvent is water or PBS solution; Preferably, the collagen concentration is 3 mg / ml; Preferably, the final concentration of the laminin subunit is 5-500 ng / ml.

8. A hydrogel product, characterized in that: The hydrogel product comprises the laminin-containing hydrogel material according to any one of claims 1 to 5, wherein the hydrogel is a collagen-based hydrogel, and the laminin content is 5-500 ng / ml.

9. A drug, characterized in that The drug comprises the laminin-containing hydrogel material according to any one of claims 1 to 5, and the final concentration of the laminin subunit is 50-500 ng / ml.

10. Use of the laminin-containing hydrogel material according to any one of claims 1 to 5 in preparing products, characterized in that: The product comprises one or more of a drug and a hydrogel product, and the product has any of the following effects: a. Can promote dentin regeneration; b. Improve the differentiation level of odontoblasts; c. Improve the polarity of dental papilla cells; d. Improve the activity of tooth germ cells; e. Increase the expression of odontoblast differentiation-related molecules: including the tooth germ cell polarity molecule RHOA, odontoblast differentiation marker molecules DMP1 and DSPP; f. Improve the arrangement and mechanical strength of collagen fibers in the hydrogel; g. Reduced the pore size distribution difference of collagen hydrogel; h. Reduce the heterogeneity of collagen fiber distribution; And i. Increase fiber bonding strength.

Images