Biological glue with layer-by-layer progressive function as well as preparation method and application of biological glue

Abstract

The invention discloses a preparation method of biological glue with a layer-by-layer progressive function, which comprises the following steps: 1) uniformly mixing a silk fibroin solution and tadpole-shaped silk fibroin particles, adding a tannic acid solution, and mixing to form a viscous gel material; and 2) adding a cartilage matrix material / bone repair material into the viscous gel material prepared in the step 1), and uniformly mixing to obtain the biological glue. According to the invention, a reactant formed by the silk fibroin and the tannic acid has strong adhesion in an articular cavity wetting state, and rapid adhesion is realized; meanwhile, a viscous gel material formed by the silk fibroin, the tannic acid and the tadpole-shaped silk fibroin particles provides a three-dimensional environment for cell proliferation, so that the functions of adhesion, migration, differentiation, proliferation and the like of related cells are collected favorably, and the extrinsic repair effect of cartilage is realized; the biological glue is mainly used in the field of osteochondral injury repair, and the function of repairing osteochondral layer by layer is realized by adopting a layer-by-layer injection and rapid adhesion technology.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical material preparation, and in particular relates to a biological glue with a layer-by-layer progressive function and a preparation method and application thereof. Background Art

[0002] Cartilage is a tissue without blood vessels, nerves and lymph glands, mainly composed of sparse chondrocytes embedded in a dense extracellular matrix. This special tissue structure makes the cartilage's self-repair ability extremely low. If it is not treated properly, it may cause serious functional disorders. Clinically, cartilage damage caused by various reasons is very common.

[0003] At present, the commonly used methods for articular cartilage repair in clinical practice mainly include microfracture, autologous cartilage transplantation and allogeneic cartilage transplantation. Although these methods reduce the pain caused by cartilage damage to patients to a certain extent and improve some functions of cartilage, they have disadvantages such as lack of donors, immune rejection of implanted tissues by the body, and disease transmission caused by transplantation, which are difficult to meet clinical needs.

[0004] Among the matrices formed by many materials, injectable hydrogels, because their structural morphology is closer to natural cartilage matrix, can better provide a microenvironment suitable for the repair and regeneration of damaged cartilage tissue. At the same time, they have injectable properties such as accurate filling of cartilage damage sites of any shape and depth, tight adhesion to surrounding tissues, and minimally invasive injection, making them one of the most ideal cartilage repair materials at present.

[0005] However, existing injectable gels have low mechanical strength and are fragile; they have poor adhesion in a wet state and cannot integrate well with damaged tissues; their degradation rate does not match the rate of new tissue formation, and they are easily metabolized and eliminated, and cannot provide cells with a long-term three-dimensional support environment. Summary of the invention

[0006] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, the main purpose of the present invention is to provide a biological glue with a layer-by-layer progressive function and a preparation method and application thereof.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] A method for preparing a biological glue with a layer-by-layer progressive function comprises the following steps:

[0009] 1) After the silk fibroin solution and the tadpole-shaped silk fibroin particles are evenly mixed, tannic acid solution is added and mixed to form a viscous gel material;

[0010] 2) Adding cartilage matrix material / bone repair material to the viscous gel material prepared in step 1), and mixing evenly to obtain biological glue.

[0011] In some specific embodiments, the tadpole-shaped silk fibroin particles are prepared by the following preparation method, which specifically comprises the following steps:

[0012] 1) uniformly mixing the silk fibroin solution and the polyurethane resin solution to form a silk fibroin pre-solution;

[0013] 2) Using the silk fibroin pre-solution as the dispersed phase, a mixed solution droplet containing silk fibroin is prepared by microfluidic extrusion and then self-assembled at room temperature for 24-48 hours to form tadpole-shaped silk fibroin particles.

[0014] Furthermore, the microfluidic extrusion method is specifically as follows: after extrusion using a microinjection pump, entering a coagulation bath of a polyethylene glycol aqueous solution, collecting silk fibroin mixed solution droplets, and performing self-assembly at room temperature.

[0015] Furthermore, the mass concentrations of the silk fibroin solution and the polyurethane resin solution are both 4-8%, and the mass ratio of the silk fibroin solution to the polyurethane resin solution is 1:0.1-10.

[0016] Furthermore, the diameter of the extrusion needle of the microinjection pump is 50 μm-300 μm.

[0017] In some specific embodiments, the mass ratio of the silk fibroin solution to the tadpole-shaped silk fibroin particles is 4-8:10-50, and the mass concentration of the tannic acid in the mixed system is 0.15-0.45 g / ml.

[0018] In certain specific embodiments, the cartilage matrix material includes a mixture of one or more of hyaluronic acid and chondroitin sulfate; and the bone repair material is a composite material of silk fibroin and hydroxyapatite.

[0019] Furthermore, the mass ratio of the cartilage matrix material to the viscous gel material is 2:5, and the mass ratio of the bone repair material to the viscous gel material is 1:3.

[0020] The present invention also aims to provide a biological glue with a layer-by-layer progressive function.

[0021] The purpose of the present invention is to provide an application of a biological glue with a layer-by-layer progressive function in the field of osteochondral damage repair, specifically:

[0022] During the injection process, the amount of each component was evaluated according to the defect volume, and the biological glue containing hydroxyapatite was injected first, followed by the biological glue containing chondroitin sulfate and hyaluronic acid.

[0023] During the injection process, a three-way tube mode is used, and the silk fibroin mixed solution and the tannic acid solution are respectively loaded with syringes. The mixture can be used after it becomes gel for 2-10 seconds.

[0024] Compared with the prior art, the present invention has at least the following advantages:

[0025] 1) The biological glue provided by the present invention is prepared by uniformly mixing a silk fibroin solution and tadpole-shaped silk fibroin particles, and then adding a tannic acid solution to the mixture to form a viscous gel material; the reactant formed by the silk fibroin and tannic acid can have strong adhesion in a moist state of the joint cavity, thereby achieving rapid adhesion; at the same time, the viscous gel material formed by the silk fibroin, tannic acid and tadpole-shaped silk fibroin particles provides a three-dimensional environment for cell proliferation, helps to recruit the adhesion, migration, differentiation and proliferation of related cells, and achieves the external accumulation repair effect of cartilage; further, the tadpole-shaped silk fibroin particles in the gel scaffold have a function similar to that of cartilage lacunas, which not only provides a microenvironment for chondrocytes, but also maintains their differentiation function, thereby achieving the internal accumulation repair function of cartilage, and progressively achieving cell proliferation and growth and secretion of cartilage extracellular matrix layer by layer, thereby promoting the formation of extracellular matrix; the part containing hydroxyapatite can promote bone formation, thereby achieving simultaneous repair of bone phase and cartilage phase, and improving interface integration ability.

[0026] 2) The biological glue in this application is mainly used in the field of osteochondral injury repair. Specifically, during the injection process, the amount of each component is first evaluated according to the defect volume, and the biological glue containing hydroxyapatite is injected first, and then the biological glue containing chondroitin sulfate and hyaluronic acid is injected; by adopting the technology of layer-by-layer injection and rapid adhesion, the function of repairing osteochondral layer by layer is achieved; BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the specific embodiments of the present invention, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below.

[0028] Figure 1 This is a microscopic morphology of the tadpole-shaped silk fibroin particles in Example 2 of the present invention;

[0029] Figure 2 A diagram showing the live and dead status of cells after co-culture of the bio-glue and cells in Example 2 of the present invention and a diagram showing the distribution of cells inside the gel after co-culture;

[0030] Figure 3 This is a cytotoxicity test diagram of the biological glue in Example 2 of the present invention;

[0031] Figure 4 This is a graph showing the adhesion test of the gel for promoting the repair of the cartilage layer and the subchondral bone layer in Example 2 of the present invention. DETAILED DESCRIPTION

[0032] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. The following embodiments are only illustrative and not restrictive, and the protection scope of the present invention cannot be limited thereto.

[0033] When a certain amount, concentration or other value or parameter is expressed in the form of a range, a preferred range, or a preferred upper and lower numerical limit, it should be understood that it is equivalent to specifically revealing any range by combining any pair of upper range limits or preferred numerical values ​​with any lower range limit or preferred numerical value, regardless of whether the range is specifically disclosed. Unless otherwise indicated, the numerical range values ​​listed herein include the endpoints of the range, and all integers and fractions within the range.

[0034] Unless otherwise indicated, all percentages, parts, ratios, etc. herein are by weight.

[0035] The materials, methods, and examples herein are illustrative and are not to be construed as limiting unless specifically stated.

[0036] In the following examples, whole silkworm cocoons without silkworm pupae were purchased from Guangxi Sericulture Research Institute, and silk fibroin solution was prepared from silkworm cocoons by the following method: silkworm cocoons were degummed in 0.5% sodium bicarbonate alkaline solution to obtain silk fibroin fibers, the silk fibroin fibers were dissolved in 9.3 M lithium bromide solution, and dialyzed for 3 days to obtain the silk fibroin solution.

[0037] The analytical grade sodium bicarbonate and analytical grade lithium bromide used in the experiment were both from Macklin's reagent, and the water used in the experiment was ultrapure water; the polyurethane resin was purchased from Guangzhou Guanzhi Co., Ltd., model C303.

[0038] The test methods used in the following embodiments include:

[0039] By testing the main properties of each test sample respectively, the biocompatibility, adhesion, etc. of the sample can be reflected; the main properties tested in this application include cell live and dead, cytoskeleton, biological glue peeling experiment, etc.

[0040] 1) Mechanical properties test;

[0041] Carry out mechanical testing according to the tensile method specified in GB / T1040.1-2018;

[0042] 2) Cytotoxicity test

[0043] The samples were tested for cytotoxicity using a microplate reader according to the CCK8 method;

[0044] 3) Tests of cell live / dead and tadpole-shaped particles

[0045] Use confocal microscopy equipment to observe the live and dead cells and tadpole-shaped particles of the samples according to methods such as cell live and dead staining;

[0046] Example 1

[0047] The method for preparing the biological glue with a layer-by-layer progressive function provided in this embodiment comprises the following steps:

[0048] 1) Preparation of tadpole-shaped silk particles

[0049] 11) uniformly mixing a silk fibroin solution (6 wt %) and a polyurethane resin solution (6 wt %) in a mass ratio of 1:1 to form a silk fibroin pre-solution;

[0050] 12) The silk fibroin pre-solution was used as the dispersed phase, and after being extruded by a microinjection pump (the diameter of the extrusion needle of the microinjection pump was 50 μm), it entered a coagulation bath of a polyethylene glycol aqueous solution, and the silk fibroin mixed solution droplets were collected and self-assembled at room temperature for 24 hours to form tadpole-shaped silk fibroin particles;

[0051] 2) After the silk fibroin solution and the tadpole-shaped silk fibroin particles are evenly mixed in a mass ratio of 4:10, tannic acid (the mass concentration of tannic acid in the mixed system is 0.2 g / ml) is added and mixed to form a viscous gel material;

[0052] 3) Adding cartilage matrix material / bone repair material to the viscous gel material prepared in step 2), and mixing evenly to obtain biological glue.

[0053] In step 3), a cartilage matrix material is added to the viscous gel material (wherein the cartilage matrix material is hyaluronic acid and chondroitin sulfate in a mass ratio of 2:1, and the mass ratio of the cartilage matrix material to the viscous gel material is 2:5), and the mixture is evenly mixed to obtain a bioglue containing the cartilage matrix material;

[0054] In step 3), a bone repair material (hydroxyapatite) is added to the viscous gel material, and the mass ratio of hydroxyapatite to the viscous gel material is 1:3. After mixing evenly, a biological glue containing the bone repair material is obtained.

[0055] Example 2

[0056] The method for preparing the biological glue with a layer-by-layer progressive function provided in this embodiment comprises the following steps:

[0057] 1) Preparation of tadpole-shaped silk particles

[0058] 11) uniformly mixing a silk fibroin solution (4 wt %) and a polyurethane resin solution (4 wt %) in a mass ratio of 1:4 to form a silk fibroin pre-solution;

[0059] 12) The silk fibroin pre-solution was used as the dispersed phase, extruded by a microinjection pump (the diameter of the extrusion needle of the microinjection pump was 100 μm), and then entered into a coagulation bath of polyethylene glycol aqueous solution, and the silk fibroin mixed solution droplets were collected and self-assembled at room temperature for 36 hours to form tadpole-shaped silk fibroin particles.

[0060] 2) After the silk fibroin solution and the tadpole-shaped silk fibroin particles are uniformly mixed in a mass ratio of 6:30, tannic acid (the mass concentration of tannic acid in the mixed system is 0.3 g / ml) is added and mixed to form a viscous gel material;

[0061] 3) Adding cartilage matrix material / bone repair material to the viscous gel material prepared in step 2), and mixing evenly to obtain biological glue.

[0062] wherein in step 3), a cartilage matrix material (wherein the cartilage matrix material is hyaluronic acid, and the mass ratio of the cartilage matrix material to the viscous gel material is 2:5) is added to the viscous gel material, and the mixture is evenly mixed to obtain a bioglue containing the cartilage matrix material;

[0063] In step 3), a bone repair material (hydroxyapatite) is added to the viscous gel material, and the mass ratio of hydroxyapatite to the viscous gel material is 1:3. After mixing evenly, a biological glue containing the bone repair material is obtained.

[0064] Example 3

[0065] The method for preparing the biological glue with a layer-by-layer progressive function provided in this embodiment comprises the following steps:

[0066] 1) Preparation of tadpole-shaped silk particles

[0067] 11) uniformly mixing a silk fibroin solution (8 wt %) and a polyurethane resin solution (8 wt %) in a mass ratio of 1:8 to form a silk fibroin pre-solution;

[0068] 12) The silk fibroin pre-solution was used as the dispersed phase and extruded by a microinjection pump (the diameter of the extrusion needle of the microinjection pump was 150 μm), and then entered into a coagulation bath of polyethylene glycol aqueous solution, and the silk fibroin mixed solution droplets were collected and self-assembled at room temperature for 36 hours to form tadpole-shaped silk fibroin particles.

[0069] 2) After the silk fibroin solution and the tadpole-shaped silk fibroin particles with a mass ratio of 8:45 are evenly mixed, tannic acid (the mass concentration of tannic acid in the mixed system is 0.25 g / ml) is added and mixed to form a viscous gel material;

[0070] 3) Adding cartilage matrix material / bone repair material to the viscous gel material prepared in step 2), and mixing evenly to obtain biological glue.

[0071] In step 3), a cartilage matrix material is added to the viscous gel material (wherein the cartilage matrix material is hyaluronic acid and chondroitin sulfate in a mass ratio of 2:1, and the mass ratio of the cartilage matrix material to the viscous gel material is 2:5), and the mixture is evenly mixed to obtain a bioglue containing the cartilage matrix material;

[0072] In step 3), a bone repair material (hydroxyapatite) is added to the viscous gel material, and the mass ratio of hydroxyapatite to the viscous gel material is 1:3. After mixing evenly, a biological glue containing the bone repair material is obtained.

[0073] Performance Testing:

[0074] This application takes Example 2 as an example to test the performance of the prepared biological glue containing bone repair material, specifically:

[0075] 1) Tests of cell live / dead and tadpole-shaped particles

[0076] This test example uses a confocal microscope to observe the live and dead cells and tadpole-shaped particles of the sample according to methods such as cell live and dead staining;

[0077] The results are as follows Figure 1 and Figure 2 As shown in the figure, it can be seen that the biological glue in the present application has obvious tadpole-shaped particle formation, and at the same time shows good cell activity during cell culture and does not inhibit cell proliferation, indicating that the material has good cell compatibility.

[0078] 2) Cytotoxicity test

[0079] This test case uses an ELISA instrument to test the cytotoxicity of the sample according to the CCK8 method;

[0080] The results are as follows Figure 3 As shown in the figure, it can be seen that compared with the control group, the number of cells in the biological glue in the present application did not decrease significantly, and the cell proliferation was not inhibited. After 72 hours of culture, the number of cells doubled, indicating that the material had no obvious cytotoxicity in the short term.

[0081] 3) Mechanical properties test

[0082] This test case conducts mechanical testing according to the tensile method specified in GB / T1040.1-2018;

[0083] The results are as follows Figure 4As shown in the figure, it can be seen that the biological glue in the present application exhibits viscoelastic properties after being mechanically stretched, and continues to undergo plastic deformation after exceeding the elastic range, showing good elongation, and has high adhesion in a small deformation range. The plastic deformation range is close to 15mm, which meets the range of motion in the joint cavity and is conducive to further defect repair.

[0084] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. A method for preparing a biological glue with a layer-by-layer progressive function, characterized in that: The steps include: 1) After the silk fibroin solution and the tadpole-shaped silk fibroin particles are evenly mixed, tannic acid solution is added and mixed to form a viscous gel material; 2) Adding cartilage matrix material / bone repair material to the viscous gel material prepared in step 1), and mixing evenly to obtain biological glue.

2. The method for preparing the biological glue with a layer-by-layer progressive function according to claim 1, characterized in that: The tadpole-shaped silk fibroin particles are prepared by the following preparation method, which specifically comprises the following steps: 1) uniformly mixing the silk fibroin solution and the polyurethane resin solution to form a silk fibroin pre-solution; 2) Using the silk fibroin pre-solution as the dispersed phase, a mixed solution droplet containing silk fibroin is prepared by microfluidic extrusion and then self-assembled at room temperature for 24-48 hours to form tadpole-shaped silk fibroin particles.

3. The method for preparing the biological glue with a layer-by-layer progressive function according to claim 2, characterized in that: The microfluidic extrusion method is specifically as follows: after extrusion by a microinjection pump, the mixture enters a coagulation bath of a polyethylene glycol aqueous solution, collects droplets of a silk fibroin mixed solution, and self-assembles at room temperature.

4. The method for preparing the biological glue with a layer-by-layer progressive function according to claim 2, characterized in that: The mass concentrations of the silk fibroin solution and the polyurethane resin solution are both 4-8%, and the mass ratio of the silk fibroin solution to the polyurethane resin solution is 1:0.1-10.

5. The method for preparing the biological glue with a layer-by-layer progressive function according to claim 2, characterized in that: The diameter of the extrusion needle of the microinjection pump is 50 μm-300 μm.

6. The method for preparing the biological glue with a layer-by-layer progressive function according to claim 4, characterized in that: The mass ratio of the silk fibroin solution to the tadpole-shaped silk fibroin particles is 4-8:10-50, and the mass concentration of the tannic acid in the mixed system is 0.15-0.45 g / ml.

7. The method for preparing the biological glue with a layer-by-layer progressive function according to claim 6, characterized in that: The cartilage matrix material includes one or more of hyaluronic acid and chondroitin sulfate; the bone repair material is a composite material of silk fibroin and hydroxyapatite.

8. A biological glue with a layer-by-layer progressive function prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the biological glue with layer-by-layer progressive function according to claim 8 in the field of osteochondral damage repair.

10. A use according to claim 9, characterized in that The steps include: During the injection process, the amount of each component was evaluated according to the defect volume, and the biological glue containing hydroxyapatite was injected first, followed by the biological glue containing chondroitin sulfate and hyaluronic acid.

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