阅读说明：本技术 一种可快速固化的双网络水凝胶及其制备方法与应用 (Rapidly-curable double-network hydrogel and preparation method and application thereof ) 是由 裴仁军 张雅洁 于 2020-05-22 设计创作，主要内容包括：本发明公开了一种可快速固化的双网络水凝胶及其制备方法与应用。所述制备方法包括：将苯并噻唑修饰到四臂聚乙二醇上,获得苯并噻唑修饰的四臂聚乙二醇；将半胱氨酸修饰到四臂聚乙二醇上,获得半胱氨酸修饰的四臂聚乙二醇；以及,使包含丝素蛋白、苯并噻唑修饰的四臂聚乙二醇和半胱氨酸修饰的四臂聚乙二醇的均匀混合体系进行生物正交反应,形成第一重水凝胶网络,之后通过诱导丝素蛋白形成β折叠获得可快速固化的双网络水凝胶。本发明制备的双网络水凝胶固化时间短、内部孔隙分布均匀、机械性能显著增强、生物相容性好,能够为干细胞的存活和增殖提供良好的三维支撑生存环境,可广泛应用于细胞培养或组织工程等领域。(The invention discloses a rapidly-curable double-network hydrogel and a preparation method and application thereof. The preparation method comprises the following steps: modifying benzothiazole onto four-arm polyethylene glycol to obtain benzothiazole modified four-arm polyethylene glycol; modifying cysteine to the four-arm polyethylene glycol to obtain cysteine-modified four-arm polyethylene glycol; and carrying out bio-orthogonal reaction on a uniform mixing system containing silk fibroin, benzothiazole modified four-arm polyethylene glycol and cysteine modified four-arm polyethylene glycol to form a first heavy hydrogel network, and then inducing the silk fibroin to form beta folding to obtain the rapidly solidified double-network hydrogel. The double-network hydrogel prepared by the invention has the advantages of short curing time, uniform internal pore distribution, remarkably enhanced mechanical property and good biocompatibility, can provide a good three-dimensional supporting living environment for survival and proliferation of stem cells, and can be widely applied to the fields of cell culture or tissue engineering and the like.)

1. A method for preparing a rapidly curable double-network hydrogel, which is characterized by comprising the following steps:

(1) providing silk fibroin;

(2) modifying benzothiazole onto four-arm polyethylene glycol to obtain benzothiazole modified four-arm polyethylene glycol;

(3) modifying cysteine to the four-arm polyethylene glycol to obtain cysteine-modified four-arm polyethylene glycol;

(4) performing bio-orthogonal reaction on a uniform mixing system containing silk fibroin, benzothiazole modified four-arm polyethylene glycol and cysteine modified four-arm polyethylene glycol to form a first heavy hydrogel network, and then inducing the silk fibroin to form beta folding to obtain the rapidly solidified double-network hydrogel.

2. The method according to claim 1, wherein the step (1) specifically comprises: and (2) reacting the first mixed reaction system containing the natural silk fibroin and the neutral salt solution at 50-60 ℃ for 4-6 h, and then carrying out post-treatment to obtain the pure water-soluble silk fibroin.

3. The method of claim 2, wherein step (1) further comprises: and after the reaction is finished, dialyzing the obtained reaction mixture for 1-3 days, wherein the adopted dialysis bag has the molecular weight cutoff of 7-14 KDa, and then freeze-drying to obtain pure silk fibroin.

4. The preparation method according to claim 2, wherein the step (1) specifically comprises: degumming natural silkworm cocoons to obtain the natural silk fibroin; and/or the salt contained in the neutral salt solution comprises any one or the combination of more than two of magnesium nitrate, calcium chloride and lithium bromide; and/or the concentration of the neutral salt solution is 9-10 mol/L; and/or the mass-volume ratio of the natural silk fibroin to the neutral salt solution is 1-3: 10 w/v%.

5. The method according to claim 1, wherein the step (2) specifically comprises: and uniformly mixing the benzothiazole and the four-arm polyethylene glycol, and reacting for 10-20 h at 15-30 ℃ to obtain the benzothiazole modified four-arm polyethylene glycol.

6. The preparation method according to claim 1 or 5, wherein the structural formula of the benzothiazole-modified four-arm polyethylene glycol is shown as formula (1):

wherein the value of n is 50-130.

7. The method according to claim 5, wherein the step (2) specifically comprises: enabling a second mixed reaction system containing the four-arm polyethylene glycol, a condensing agent and a first solvent to react for 10-30 min at 0-8 ℃, then adding benzothiazole, uniformly mixing to form a third mixed reaction system, and then enabling the third mixed reaction system to react for 10-20 h at 15-30 ℃ to obtain the benzothiazole modified four-arm polyethylene glycol.

8. The method of claim 7, wherein: the molar ratio of the benzothiazole to the four-arm polyethylene glycol is 1-3: 1; and/or, the step (2) further comprises the following steps: and after the reaction of the third mixed reaction system is finished, adding the obtained reaction mixture into a poor solvent, collecting and purifying the precipitate, and then freeze-drying to obtain the benzothiazole modified four-arm polyethylene glycol.

9. The method according to claim 1, wherein the step (3) specifically comprises: and (2) reacting a fourth mixed reaction system containing a second solvent, the amino-sulfydryl-protected cysteine and a condensing agent at 0-8 ℃ for 10-30 min, adding four-arm polyethylene glycol, uniformly mixing to form a fifth mixed reaction system, and reacting the fifth mixed reaction system at 15-30 ℃ for 10-20 h to obtain the amino-sulfydryl-protected cysteine-modified four-arm polyethylene glycol.

10. The method of claim 9, wherein: the molar ratio of the four-arm polyethylene glycol to the cysteine is 1: 1-3; and/or, the step (3) further comprises: and after the reaction of the fifth mixed reaction system is finished, adding the obtained reaction mixture into a poor solvent, collecting and purifying the precipitate, and then freeze-drying to obtain the amino-sulfydryl-protected cysteine-modified four-arm polyethylene glycol.

11. The production method according to claim 7 or 9, characterized in that: the molar ratio of the condensing agent to the four-arm polyethylene glycol is 1-3: 1; and/or the molar ratio of the condensing agent to the amino sulfydryl protected cysteine is 1-3: 1; and/or the condensing agent comprises any one or the combination of more than two of dicyclohexylcarbodiimide, benzotriazole-1-oxytris (dimethylamino) phosphonium hexafluorophosphate, N-methylmorpholine, 1-hydroxybenzotriazole, isobutyl chloroformate, 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; and/or the first solvent or the second solvent comprises any one or the combination of more than two of N, N-dimethylformamide, dichloromethane and tetrahydrofuran.

12. The production method according to claim 8 or 10, characterized in that: the poor solvent comprises n-hexane and/or diethyl ether; and/or the volume ratio of the poor solvent to the third mixed reaction system or the fifth mixed reaction system is 10-20: 1.

13. the method of claim 9, wherein step (3) further comprises: uniformly mixing amino-sulfydryl-protected cysteine-modified four-arm polyethylene glycol, trifluoroacetic acid and a third solvent to form a sixth mixed reaction system, reacting at 10-30 ℃ for 1-4 h, dialyzing the obtained reaction mixture for 1-3 days, and then freeze-drying to obtain sulfydryl-protected cysteine-modified four-arm polyethylene glycol; preferably, the third solvent includes any one or a combination of two or more of N, N-dimethylformamide, dichloromethane, and tetrahydrofuran.

14. The method of manufacturing according to claim 13, wherein: the mass ratio of the trifluoroacetic acid to the amino sulfydryl protected cysteine modified four-arm polyethylene glycol is 30-100: 1; and/or the intercepted molecular weight of a dialysis bag adopted by dialysis is 3500-14000 Da.

15. The method of claim 13, further comprising: and uniformly mixing the sulfhydryl protected cysteine modified four-arm polyethylene glycol and dithiothreitol in a phosphate buffer solution to form a seventh mixed reaction system, and reacting for 5-20 min at 10-30 ℃ to obtain the cysteine modified four-arm polyethylene glycol.

16. The method of claim 15, wherein: the molar ratio of dithiothreitol to sulfhydryl protected cysteine modified four-arm polyethylene glycol is 3-5: 1.

17. the method of claim 1 or 15, wherein the cysteine-modified four-arm polyethylene glycol has a structural formula shown in formula (2):

wherein the value of m is 50-130.

18. The method according to claim 1, wherein the step (4) specifically comprises:

mixing silk fibroin with phosphate buffer salt solution to form silk fibroin solution;

mixing the silk fibroin solution with benzothiazole modified four-arm polyethylene glycol and cysteine modified four-arm polyethylene glycol respectively to form benzothiazole modified four-arm polyethylene glycol precursor solution and cysteine modified four-arm polyethylene glycol precursor solution; and the number of the first and second groups,

and mixing the benzothiazole modified four-arm polyethylene glycol precursor solution and the cysteine modified four-arm polyethylene glycol precursor solution for bio-orthogonal reaction to form a first heavy hydrogel network, and then forming beta folding by inducing silk fibroin to obtain the rapidly solidified double-network hydrogel.

19. The method of claim 18, wherein: the concentration of silk fibroin in the silk fibroin solution is 5-10 w/v%; and/or, the preparation method comprises the following steps: ultrasonically dispersing benzothiazole modified four-arm polyethylene glycol and cysteine modified four-arm polyethylene glycol in the silk fibroin solution respectively; preferably, the conditions of the ultrasound are: the ultrasonic power is 180-240W, the ultrasonic time is 2-5 s, the ultrasonic is suspended for 2-5 s, and the ultrasonic is circulated for 6-10 times; and/or the molar ratio of the benzothiazole modified four-arm polyethylene glycol to the cysteine modified four-arm polyethylene glycol is 1: 1 to 3.

20. A rapidly curable double-network hydrogel prepared by the method of any one of claims 1 to 19, which has a storage modulus of 2 to 20KPa, a maximum tensile strength of 0.01 to 0.1MPa, a compressive strength of 0.05 to 0.5MPa at a compressive strain of 65%, and a porous structure having pores with a diameter of 100 to 200 μm.

21. Use of the rapidly curable double-network hydrogel according to claim 20 in the field of cell culture or tissue engineering.

22. A three-dimensional cell culture support comprising the rapidly curable double-network hydrogel according to claim 20.

23. A cell culture method, comprising:

culturing stem cells by using the rapidly solidified double-network hydrogel as a three-dimensional cell culture carrier, and promoting the stem cells to proliferate and differentiate; preferably, the load capacity of the stem cells on the double-network hydrogel is 100-1000 ten thousand/mL.

Technical Field

The invention relates to a double-network hydrogel, in particular to a rapidly solidified double-network hydrogel which is used for culturing three-dimensional stem cells and promoting the stem cells to proliferate and differentiate, and a preparation method and application thereof, belonging to the technical field of tissue engineering material preparation.

Background

Tissue, organ defects and dysfunction due to disease, genetics, aging, etc. are one of the major risks facing human health, and are the leading causes of human disease and death. In order to solve the problems of tissue, organ defect and dysfunction, the concept of tissue engineering is proposed, which means to research the relationship between tissue structure and function under normal and pathological conditions, develop biological substitutes, repair, maintain and improve tissue function by applying the principles and methods of engineering and life science. With the development of recent decades, the tissue engineering technology surpasses the traditional 'east wall removal and west wall supplement' therapy, so that the tissue injury repair step into a new era of 'reconstruction, regeneration and replacement' of tissue and organs, and becomes a third effective treatment way after drug treatment and surgical treatment.

The main method of tissue engineering is to inoculate the living cells related to functions on the extracellular matrix substitute, the substitute can provide a space structure for the cells, the cells can grow on the substitute, the compound of the cells and the substitute is formed after a certain period of in vitro culture, and then the obtained compound is transplanted to the damaged tissue in vivo to repair the damaged tissue. In recent years, the research of tissue engineering has mainly focused on the development and research of biomaterials, growth factors, seed cell culture, and compounding and shaping of cells and scaffold materials.

Currently, the common methods for cell inoculation in tissue engineering include: cells are inoculated on the scaffold material and the cells and the material are blended to form the hydrogel scaffold, wherein the blending of the cells and the material can better control the distribution of the cells and has a plurality of advantages in the aspects of cell adhesion, proliferation, migration and three-dimensional structure; in addition, the precision and accuracy of tissue repair can be improved by controlling the shape of the blended hydrogel scaffold. But to ensure the viability of the cells it is often necessary to find materials with a higher biocompatibility.

The stem cell has the characteristics of high proliferation rate, multi-differentiation potential, low immunogenicity and the like, and is the most ideal seed cell for tissue engineering. The hydrogel materials for embedding cells commonly used in tissue engineering include gelatin, collagen, hyaluronic acid, chitosan, alginate, polylactic acid, polyethylene glycol, polycaprolactone, and the like. The high molecular compound has more active functional groups, can be chemically modified to form hydrogel by different methods, and in addition, by adjusting the properties of the hydrogel scaffold, for example, doping extracellular matrix in the hydrogel scaffold, the adhesion of cells and the migration of chemotactic host cells can be increased, and the differentiation capacity of seed cells can also be increased. However, most artificially synthesized polymer materials have low biocompatibility and incomplete degradation, while natural polymer materials have high degradation rate and poor mechanical properties; therefore, it is important to find a material with good biocompatibility and degradability as a scaffold for three-dimensional cell culture.

Disclosure of Invention

The invention mainly aims to provide a double-network hydrogel capable of being rapidly cured and a preparation method thereof, so as to overcome the defects in the prior art.

It is another object of the present invention to provide the use of the rapidly curable double-network hydrogel.

In order to achieve the purpose, the invention adopts the following technical scheme:

the embodiment of the invention provides a preparation method of a rapidly-curable double-network hydrogel, which comprises the following steps:

(1) providing silk fibroin;

(2) modifying benzothiazole onto four-arm polyethylene glycol to obtain benzothiazole modified four-arm polyethylene glycol;

(3) modifying cysteine to the four-arm polyethylene glycol to obtain cysteine-modified four-arm polyethylene glycol;

(4) performing bio-orthogonal reaction on a uniform mixing system containing silk fibroin, benzothiazole modified four-arm polyethylene glycol and cysteine modified four-arm polyethylene glycol to form a first heavy hydrogel network, and then inducing the silk fibroin to form beta folding to obtain the rapidly solidified double-network hydrogel.

The embodiment of the invention also provides the rapidly-curable double-network hydrogel prepared by the method, the double-network hydrogel has the storage modulus of 2-20 KPa, the maximum tensile strength of 0.01-0.1 MPa, the compressive strength of 0.05-0.5 MPa when the compressive strain is 65%, and a porous structure, wherein the aperture of holes contained in the double-network hydrogel is 100-200 mu m.

The embodiment of the invention also provides application of the rapidly-solidified double-network hydrogel in the field of cell culture or tissue engineering.

The embodiment of the invention also provides a three-dimensional cell culture carrier, which comprises the rapidly solidified double-network hydrogel.

The embodiment of the invention also provides a cell culture method, which comprises the following steps:

the rapidly solidified double-network hydrogel is used as a three-dimensional cell culture carrier to culture stem cells, and the stem cells are promoted to proliferate and differentiate.

Compared with the prior art, the invention has the beneficial effects that:

1) the method for constructing the rapidly solidified double-network hydrogel based on the silk fibroin and the functionalized polyethylene glycol system provided by the invention is applied to the research on the proliferation and chondrogenic differentiation of cells, and realizes the blending gelation with the cells. Firstly, respectively modifying benzothiazole and cysteine which are biological orthogonal reaction functional groups onto four-arm polyethylene glycol to obtain benzothiazole modified four-arm polyethylene glycol and cysteine modified four-arm polyethylene glycol, and constructing a first heavy fast crosslinking network; meanwhile, extracting silk fibroin from the silkworm cocoon, and forming a second slow cross-linked network by utilizing ultrasonic-induced beta folding; mixing the two, and preparing the silk fibroin/polyethylene glycol double-network hydrogel under the bioorthogonal reaction and ultrasonic induction;

2) the rapidly-curable double-network hydrogel provided by the invention combines two action modes of physical ultrasound and bio-orthogonal click chemical reaction, and has the advantages that the hydrogel system is rapidly cured due to the rapid performance characteristic of the bio-orthogonal reaction; then, as time goes on, beta sheet is gradually formed to improve the mechanical property of the gel, and meanwhile, the preparation method is simple and can be used for mass preparation;

3) the invention relates to a fast-curing double-network hydrogel, which is prepared by functionally modifying common artificially-synthesized polyethylene glycol to obtain benzothiazole-modified four-arm polyethylene glycol and cysteine-modified four-arm polyethylene glycol, performing ultrasonic treatment on silk fibroin from natural silkworm cocoons, then dissolving the benzothiazole-modified four-arm polyethylene glycol and the cysteine-modified four-arm polyethylene glycol as solvents respectively to obtain two precursor solutions, blending the precursor solutions with cells, and combining the silk fibroin, the benzothiazole-modified four-arm polyethylene glycol and the cysteine-modified four-arm polyethylene glycol, so that the curing speed of the hydrogel system is obviously improved, the mechanical property of the hydrogel system is enhanced, the curing time of the obtained double-crosslinked hydrogel is short, the internal pores are uniformly distributed, the biocompatibility is good, the toxicity is low, and the internal pore diameter is 100-200 mu m, the prepared double-network hydrogel is formed by blending stem cells, and chondrogenic differentiation growth factors (TGF-beta 1/bFGF) are added to effectively promote differentiation of the stem cells into chondrocytes.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a schematic diagram showing the mechanism of preparation of the double-network hydrogel obtained in an exemplary embodiment of the present invention.

FIG. 2 is a microscopic structure view of the double-network hydrogel obtained in an exemplary embodiment of the present invention.

FIGS. 3a to 3c are graphs showing the mechanical properties of the double-network hydrogel obtained in an exemplary embodiment of the present invention, respectively.

FIGS. 4 a-4 c are a confocal, three-dimensional and proliferation map of stem cells in a double-network hydrogel obtained according to an exemplary embodiment of the present invention.

FIGS. 5a to 5c are graphs showing mRNA expression levels of stem cells differentiated into chondrocytes under in vitro culture conditions in a double-network hydrogel obtained in an exemplary embodiment of the present invention.

Detailed Description

As described above, in view of the defects of the prior art, the inventors of the present invention have made extensive studies and extensive practices to propose a technical solution of the present invention. The present invention will be more fully understood from the following detailed description, which should be read in conjunction with the accompanying drawings. Detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which can be embodied in various forms. Therefore, specific functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriately detailed embodiment.

As one aspect of the technical solution of the present invention, there is provided a method for preparing a rapidly curable double-network hydrogel, comprising:

(1) providing silk fibroin;

(2) modifying benzothiazole onto four-arm polyethylene glycol to obtain benzothiazole modified four-arm polyethylene glycol;

(3) modifying cysteine to the four-arm polyethylene glycol to obtain cysteine-modified four-arm polyethylene glycol;

(4) performing bio-orthogonal reaction on a uniform mixing system containing silk fibroin, benzothiazole modified four-arm polyethylene glycol and cysteine modified four-arm polyethylene glycol to form a first heavy hydrogel network, and then inducing the silk fibroin to form beta folding to obtain the rapidly solidified double-network hydrogel.

In some embodiments, step (1) specifically comprises: the mass-to-volume ratio of the components is 1-3: 10, reacting the natural silk fibroin with a first mixed reaction system of neutral salt solution at 50-60 ℃ for 4-6 h, and performing post-treatment to obtain pure water-soluble silk fibroin.

Further, the step (1) further comprises: and after the reaction is finished, dialyzing the obtained reaction mixture for 1-3 days, wherein the adopted dialysis bag has the molecular weight cutoff of 7-14 KDa, and then freeze-drying to obtain pure silk fibroin.

Further, the step (1) specifically comprises: degumming natural silkworm cocoon to obtain the natural silk fibroin.

In some preferred embodiments, step (1) specifically comprises: adding 0.02mol/L Na into selected clean silkworm cocoon₂CO₃Boiling the solution in a water bath kettle at 100 ℃ for 2 times, wherein each time lasts for at least 40min, washing the solution for multiple times by using deionized water, wringing the solution to remove sericin, drying the solution in an oven at 60 ℃ overnight, putting the dried fibroin protein into a neutral salt solution to form a first mixed reaction system for the reaction, and maintaining the temperature of the reaction system at 50-60 ℃.

Further, the salt contained in the neutral salt solution includes any one or a combination of two or more of magnesium nitrate, calcium chloride, lithium bromide, and the like, but is not limited thereto.

Further, the concentration of the neutral salt solution is 9-10 mol/L.

Further, the mass-volume ratio of the natural silk fibroin to the neutral salt solution is 1-3: 10 w/v%.

In some embodiments, step (2) specifically comprises: and uniformly mixing the benzothiazole and the four-arm polyethylene glycol, and reacting for 10-20 h at 15-30 ℃ to obtain the benzothiazole modified four-arm polyethylene glycol.

Further, the structural formula of the benzothiazole modified four-arm polyethylene glycol is shown as the formula (1):

wherein the value of n is 50-130.

In some preferred embodiments, step (2) specifically comprises:

enabling a second mixed reaction system containing the four-arm polyethylene glycol, a condensing agent and a first solvent to react at 0-8 ℃ for 10-30 min to activate carboxyl, then adding benzothiazole, uniformly mixing to form a third mixed reaction system, and then enabling the third mixed reaction system to react at 15-30 ℃ for 10-20 h to obtain the benzothiazole modified four-arm polyethylene glycol.

In a preferred embodiment, the molar ratio of the condensing agent to the four-arm polyethylene glycol is 1 to 3: 1.

further, the condensing agent includes any one or a combination of two or more of dicyclohexylcarbodiimide, benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate, N-methylmorpholine, 1-hydroxybenzotriazole, isobutyl chloroformate, 2- (7-oxybenzotriazole) -N, N' -tetramethylurea hexafluorophosphate, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the like, but is not limited thereto.

Further, the molar ratio of the benzothiazole to the four-arm polyethylene glycol is 1-3: 1.

further, the first solvent includes any one or a combination of two or more of N, N-dimethylformamide, dichloromethane, tetrahydrofuran, and the like, but is not limited thereto.

Further, the step (2) further comprises: after the reaction of the third mixed reaction system is finished, adding the obtained reaction mixture into a large amount of poor solvent, and collecting the precipitate; purifying by a sephadex column G-15, and freeze-drying to obtain the benzothiazole modified four-arm polyethylene glycol.

Further, the poor solvent includes n-hexane, diethyl ether, etc., but is not limited thereto. And the volume ratio of the poor solvent to the third mixed reaction system is 10-20: 1.

in some embodiments, step (3) specifically comprises: and (2) reacting a fourth mixed reaction system containing a second solvent, the amino-sulfydryl-protected cysteine and a condensing agent at 0-8 ℃ for 10-30 min to activate carboxyl, then adding four-arm polyethylene glycol, uniformly mixing to form a fifth mixed reaction system, and then reacting the fifth mixed reaction system at 15-30 ℃ for 10-20 h to obtain the amino-sulfydryl-protected cysteine-modified four-arm polyethylene glycol.

Further, the molar ratio of the amino mercapto protected cysteine to the four-arm polyethylene glycol is 1-3: 1.

further, the molar ratio of the condensing agent to the amino mercapto group-protected cysteine is 1-3: 1.

further, the condensing agent includes any one or a combination of two or more of dicyclohexylcarbodiimide, benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate, N-methylmorpholine, 1-hydroxybenzotriazole, isobutyl chloroformate, 2- (7-oxybenzotriazole) -N, N' -tetramethylurea hexafluorophosphate, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the like, but is not limited thereto.

Further, the second solvent includes any one or a combination of two or more of N, N-dimethylformamide, dichloromethane, tetrahydrofuran, and the like, but is not limited thereto.

Further, the step (3) further comprises: and after the reaction of the fifth mixed reaction system is finished, adding the obtained reaction mixture into a poor solvent, collecting and purifying the precipitate, and then freeze-drying to obtain the amino-sulfydryl-protected cysteine-modified four-arm polyethylene glycol.

Further, the poor solvent includes n-hexane, diethyl ether and the like, but is not limited thereto, and the volume ratio of the poor solvent to the fifth mixed reaction system is 10-20: 1.

in some preferred embodiments, step (3) further comprises: uniformly mixing amino-sulfydryl-protected cysteine-modified four-arm polyethylene glycol, trifluoroacetic acid and a third solvent to form a sixth mixed reaction system, reacting at 10-30 ℃ for 1-4 h, dialyzing the obtained reaction mixture for 1-3 days, and then freeze-drying to obtain sulfydryl-protected cysteine-modified four-arm polyethylene glycol.

Further, the mass ratio of the trifluoroacetic acid to the amino sulfydryl protected cysteine modified four-arm polyethylene glycol is 30-100: 1.

further, the third solvent includes any one or a combination of two or more of N, N-dimethylformamide, dichloromethane, tetrahydrofuran, and the like, but is not limited thereto.

As one of preferable embodiments, the preparation method further comprises: and after the reaction of the sixth mixed reaction system is finished, dialyzing the obtained reaction mixture for 1-3 days by using a dialysis bag with the molecular weight cutoff of 3500-14000 Da, purifying by using a sephadex column G-15, and freeze-drying to obtain the sulfydryl-protected cysteine-modified four-arm polyethylene glycol.

In some preferred embodiments, step (3) further comprises: and uniformly mixing the sulfhydryl protected cysteine modified four-arm polyethylene glycol and dithiothreitol in a phosphate buffer solution to form a seventh mixed reaction system, and reacting for 5-20 min at 10-30 ℃ to obtain the cysteine modified four-arm polyethylene glycol.

Further, the molar ratio of dithiothreitol to thiol-protected cysteine-modified four-arm polyethylene glycol is 3-5: 1.

further, the structural formula of the cysteine-modified four-arm polyethylene glycol is shown as the formula (2):

wherein the value of m is 50-130.

In some embodiments, step (4) specifically comprises:

mixing silk fibroin with phosphate buffer salt solution to form silk fibroin solution;

mixing the silk fibroin solution with benzothiazole modified four-arm polyethylene glycol and cysteine modified four-arm polyethylene glycol respectively to form benzothiazole modified four-arm polyethylene glycol precursor solution and cysteine modified four-arm polyethylene glycol precursor solution; and the number of the first and second groups,

and mixing the benzothiazole modified four-arm polyethylene glycol precursor solution and the cysteine modified four-arm polyethylene glycol precursor solution for bio-orthogonal reaction to form a first heavy hydrogel network, and then forming beta folding by inducing silk fibroin to obtain the rapidly solidified double-network hydrogel.

In some preferred embodiments, step (4) specifically includes:

dissolving silk fibroin in Phosphate Buffered Saline (PBS) to form a solution with the concentration of 5-10 w/v%, and then performing ultrasonic treatment on the silk fibroin solution to serve as a solvent to dissolve a four-arm polyethylene glycol-oxazine polymer and a four-arm polyethylene glycol-cyclooctene polymer respectively to obtain two precursor solutions;

and uniformly mixing the precursor solution in the same volume, transferring the precursor solution into a mould to form a primary cured and formed hydrogel, placing the hydrogel in an incubator for 30min, and slowly forming beta folding by inducing silk fibroin to obtain a secondary cured double-network hydrogel.

Further, the concentration of the silk fibroin in the silk fibroin solution is 5-10 w/v%.

Further, the ultrasonic conditions are: the ultrasonic power is 180-240W, the ultrasonic time is 2-5 s, the ultrasonic is suspended for 2-5 s, and the ultrasonic is circulated for 6-10 times.

Further, the molar ratio of the benzothiazole modified four-arm polyethylene glycol to the cysteine modified four-arm polyethylene glycol is 1: 1 to 3.

The invention adopts bioorthogonal reaction rapid solidification molding, and further adopts physical ultrasound to induce silk fibroin to form beta folding.

In some more specific embodiments, the method for preparing the rapidly curable double-network hydrogel comprises the following steps:

(1) degumming natural silkworm cocoon, and mixing the degummed silkworm cocoon with lithium bromide to react to obtain silk fibroin;

(2) at least modifying benzothiazole onto four-arm polyethylene glycol to obtain benzothiazole modified four-arm polyethylene glycol;

(3) modifying cysteine to the four-arm polyethylene glycol to obtain cysteine-modified four-arm polyethylene glycol;

(4) at least dissolving benzothiazole modified four-arm polyethylene glycol and cysteine modified four-arm polyethylene glycol in silk fibroin after ultrasonic treatment to obtain two precursor solutions;

(5) at least mixing the two precursor solutions uniformly, forming a first heavy hydrogel network through a bioorthogonal reaction, then placing the first heavy hydrogel network in an incubator for 30min, and obtaining the double-network hydrogel by inducing silk fibroin to form beta folding.

In another aspect of the embodiment of the present invention, there is provided a rapidly curable double-network hydrogel prepared by the method, wherein the double-network hydrogel has a storage modulus of 2 to 20KPa, a maximum tensile strength of 0.01 to 0.1MPa, a compressive strength of 0.05 to 0.5MPa when a compressive strain is 65%, and has a porous structure, and pores contained in the double-network hydrogel have a pore size of 100 to 200 μm.

In another aspect of the embodiments of the present invention, there is also provided a use of the rapidly curable double-network hydrogel in the field of cell culture or tissue engineering.

In another aspect of the embodiments of the present invention, there is also provided a three-dimensional cell culture carrier comprising the rapidly curable double-network hydrogel.

In another aspect of the embodiments of the present invention, there is provided a cell culture method including:

the rapidly solidified double-network hydrogel is used as a three-dimensional cell culture carrier to culture stem cells, and the stem cells are promoted to proliferate and differentiate.

In some embodiments, the cell is a rat bone marrow mesenchymal stem cell.

Furthermore, the load capacity of the stem cells on the double-network hydrogel is 100-1000 ten thousand/mL.

By the technical scheme, the double-network hydrogel of the invention carries out functional modification on common synthetic material polyethylene glycol to obtain benzothiazole modified four-arm polyethylene glycol and cysteine modified four-arm polyethylene glycol, carries out ultrasonic treatment on silk fibroin from natural silkworm cocoons, then is used as a solvent to respectively dissolve the benzothiazole modified four-arm polyethylene glycol and the cysteine modified four-arm polyethylene glycol to obtain two precursor solutions, then is mixed with cells, combines the silk fibroin with the benzothiazole modified four-arm polyethylene glycol and the cysteine modified four-arm polyethylene glycol, obviously improves the curing speed of the hydrogel system on the one hand, enhances the mechanical property of the hydrogel system on the other hand, has short curing time, uniform distribution of internal pores, good biocompatibility and low toxicity, and the internal pore diameter is 100-200 mu m, the prepared double-network hydrogel is formed by blending with stem cells by adopting a method of forming, and effectively promotes the stem cells to be differentiated into the chondrocytes by adding the angiogenesis promoting growth factor (TGF-beta 1/bFGF). Meanwhile, the preparation method is simple and can be used for mass preparation.

The technical solutions of the present invention will be described in further detail below with reference to several preferred embodiments and accompanying drawings, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The conditions used in the following examples may be further adjusted as necessary, and the conditions used in the conventional experiments are not generally indicated.

Example 1

The method comprises the following steps: adding 0.02mol/L Na into selected clean silkworm cocoon₂CO₃Boiling the solution in a water bath kettle at 100 ℃ for 2 times, wherein each time lasts for at least 40min, washing the solution for multiple times by using deionized water, wringing the solution to remove sericin, drying the solution in an oven at 60 ℃ overnight, putting dried silk fibroin into a LiBr solution to form a first mixed reaction system for reaction, maintaining the temperature of the reaction system at 60 ℃, and reacting for 4h, wherein the concentration of the LiBr solution is 9.3mol/L, and the mass ratio of the LiBr to the silk fibroin is 10: 1.

and step one, after the reaction is finished, dialyzing for 3 days by using 7KDa cut-off amount to remove impurities, freezing at the temperature of 80 ℃ overnight, and freeze-drying at the temperature of 50 ℃ for 3 days to obtain pure silk fibroin.

Step two: dissolving four-arm polyethylene glycol in 1% anhydrous N, N-dimethylformamide solution, adding N-methylmorpholine and isobutyl chloroformate, and reacting at 4 deg.C for 20 min. Wherein the molar ratio of the N-methylmorpholine to the four-arm polyethylene glycol is 1: 1, the molar ratio of isobutyl chloroformate to four-arm polyethylene glycol is 1: 1.

step three: and (3) after the reaction in the first step is finished, adding benzothiazole, and reacting for 20 hours at 15 ℃. Wherein the molar ratio of the benzothiazole to the four-arm polyethylene glycol is 1.5: 1.

and after the reaction in the third step is finished, precipitating in diethyl ether, wherein the volume ratio of the diethyl ether to the mixed system is 10: 1, obtaining white flocculent precipitate, centrifuging at 12000rpm for 5min, collecting the precipitate, dissolving in water, purifying by a sephadex column G-15, and freeze-drying to obtain benzothiazole modified four-arm polyethylene glycol, wherein the structural formula of the four-arm polyethylene glycol is shown as a formula (1):

wherein the value of n is 113.

The benzothiazole-modified four-arm polyethylene glycol is mainly characterized in that benzothiazole molecules are modified on the four-arm polyethylene glycol, so that the water solubility of the four-arm polyethylene glycol is improved, and the benzothiazole molecules which are only insoluble in water are dissolved in a water phase.

Step four: dissolving aminothiol protected cysteine in an anhydrous N, N-dimethylformamide solution, adding 1-hydroxybenzotriazole and 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate, mixing, reacting at 4 ℃ for 15min, adding four-arm polyethylene glycol, and reacting at 15 ℃ for 20 h. Wherein the mol ratio of the 1-hydroxybenzotriazole to the amino mercapto group protected cysteine is 1.5: the molar ratio of 1, 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate to amino mercapto protected cysteine is 1.5: 1, the molar ratio of the four-arm polyethylene glycol to the amino mercapto protected cysteine is 0.667: 1.

and after the reaction in the fourth step is finished, precipitating in diethyl ether, wherein the volume ratio of the diethyl ether to the mixed system is 10: 1, obtaining white flocculent precipitate, centrifuging at 12000rpm for 5min, collecting the precipitate, dissolving in water, purifying by a sephadex column G-15, and freeze-drying to obtain the amino-sulfhydryl-protected cysteine-modified four-arm polyethylene glycol.

Step five: the four-arm polyethylene glycol modified by the amino sulfydryl protected cysteine, trifluoroacetic acid and dichloromethane are uniformly mixed and reacted for 2h at the temperature of 20 ℃. Wherein the mass ratio of trifluoroacetic acid to amino-sulfydryl-protected cysteine-modified four-arm polyethylene glycol is 100: 1.

and after the fifth reaction step is finished, dialyzing the obtained reaction mixture for 3 days by using a dialysis bag with the molecular weight cutoff of 3500Da, purifying by using a sephadex column G-15, and freeze-drying to obtain the sulfhydryl protected cysteine modified four-arm polyethylene glycol.

Step six: and (3) uniformly mixing the sulfhydryl protected cysteine modified four-arm polyethylene glycol and dithiothreitol in a phosphate buffer solution, and reacting for 15min at 15 ℃. Wherein, the molar ratio of dithiothreitol to sulfhydryl protected cysteine modified four-arm polyethylene glycol is 4: 1.

after the sixth step is finished, freeze-drying to obtain cysteine-modified four-arm polyethylene glycol, wherein the structural formula of the cysteine-modified four-arm polyethylene glycol is shown as a formula (2):

wherein the value of m is 113.

The cysteine modified four-arm polyethylene glycol is mainly characterized in that a bio-orthogonal reaction functional group cysteine is modified on a synthetic polyethylene glycol macromolecule and is used for quick crosslinking and gelling.

Step seven: dissolving silk fibroin in PBS to form a solution with the concentration of 5 w/v%, then performing ultrasonic treatment on the silk fibroin solution (with the power of 240w, ultrasonic treatment for 5s, pausing for 5s, and circulating for 6 times) to serve as a solvent to respectively dissolve benzothiazole-modified four-arm polyethylene glycol and cysteine-modified four-arm polyethylene glycol to obtain two precursor solutions (5% SF/2.5% PEG-CBT solution and 5% SF/2.5% PEG-Cys), then uniformly mixing the precursor solutions in an equal volume, transferring the mixture into a mold, performing primary curing molding, placing the mold in an incubator for 30min, and inducing the silk fibroin to slowly form beta-sheet to obtain the secondary-cured double-network hydrogel.

Example 2

The method comprises the following steps: adding 0.02mol/L Na into selected clean silkworm cocoon₂CO₃Boiling the solution in a water bath kettle at 100 ℃ for 2 times, wherein each time lasts for at least 40min, washing the solution for multiple times by using deionized water, wringing the solution to remove sericin, drying the solution in an oven at 60 ℃ overnight, putting the dried silk fibroin into a LiBr solution to form a first mixed reaction system for carrying out the reaction, and keeping the temperature of the reaction system at 60 ℃ for 5 h; wherein the concentration of the LiBr solution is 9.3mol/L, and the mass ratio of the LiBr to the silk fibroin is 10: 1.

and step one, after the reaction is finished, dialyzing for 2 days by using 7KDa cut-off amount to remove impurities, freezing at the temperature of 80 ℃ overnight, and freeze-drying at the temperature of 50 ℃ for 3 days to obtain pure silk fibroin.

Step two: dissolving four-arm polyethylene glycol in 1% anhydrous N, N-dimethylformamide solution, adding N-methylmorpholine and isobutyl chloroformate, and reacting at 4 deg.C for 20 min. Wherein the molar ratio of the N-methylmorpholine to the four-arm polyethylene glycol is 1: 1, the molar ratio of isobutyl chloroformate to four-arm polyethylene glycol is 1: 1.

step three: and after the reaction in the second step is finished, adding benzothiazole, and reacting for 20 hours at 15 ℃. Wherein the molar ratio of the benzothiazole to the four-arm polyethylene glycol is 1: 1.

and after the reaction in the third step is finished, precipitating in diethyl ether, wherein the volume ratio of the diethyl ether to the mixed system is 15: 1, obtaining white flocculent precipitate, centrifuging at 12000rpm for 5min, collecting the precipitate, dissolving in water, purifying by a sephadex column G-15, and freeze-drying to obtain the benzothiazole modified four-arm polyethylene glycol, wherein the structural formula is shown as a formula (1). Wherein n is 50.

Step four: dissolving aminothiol protected cysteine in anhydrous N, N-dimethylformamide solution, adding 1-hydroxybenzotriazole and benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate, mixing, reacting at 6 deg.C for 20min, adding four-arm polyethylene glycol, and reacting at 15 deg.C for 20 h. Wherein the mol ratio of the 1-hydroxybenzotriazole to the amino mercapto group protected cysteine is 1.5: 1, the molar ratio of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate to amino mercapto protected cysteine is 1.5: 1, the molar ratio of the four-arm polyethylene glycol to the amino mercapto protected cysteine is 0.667: 1.

and after the reaction in the fourth step is finished, precipitating in diethyl ether, wherein the volume ratio of the diethyl ether to the mixed system is 15: 1, obtaining white flocculent precipitate, centrifuging at 12000rpm for 5min, collecting the precipitate, dissolving in water, purifying by a sephadex column G-15, and freeze-drying to obtain the amino-sulfhydryl-protected cysteine-modified four-arm polyethylene glycol.

Step five: the four-arm polyethylene glycol modified by the amino sulfydryl protected cysteine, trifluoroacetic acid and dichloromethane are uniformly mixed and reacted for 4 hours at 10 ℃. Wherein the mass ratio of trifluoroacetic acid to amino-sulfydryl-protected cysteine-modified four-arm polyethylene glycol is 100: 1.

and after the fifth reaction step is finished, dialyzing the obtained reaction mixture for 3 days by using a dialysis bag with the molecular weight cutoff of 3500Da, purifying by using a sephadex column G-15, and freeze-drying to obtain the sulfhydryl protected cysteine modified four-arm polyethylene glycol.

Step six: and (3) uniformly mixing the sulfhydryl protected cysteine modified four-arm polyethylene glycol and dithiothreitol in a phosphate buffer solution, and reacting for 20min at 10 ℃. Wherein, the molar ratio of dithiothreitol to sulfhydryl protected cysteine modified four-arm polyethylene glycol is 4: 1.

and sixthly, after the step six is finished, freeze-drying to obtain the cysteine-modified four-arm polyethylene glycol, wherein the structural formula of the cysteine-modified four-arm polyethylene glycol is shown as the formula (2). Wherein m is 50.

Step seven: dissolving silk fibroin in PBS to form a solution with the concentration of 5 w/v%, then performing ultrasonic treatment on the silk fibroin solution (with the power of 180w, performing ultrasonic treatment for 5s, pausing for 5s, and circulating for 6 times) to serve as a solvent to respectively dissolve benzothiazole-modified four-arm polyethylene glycol and cysteine-modified four-arm polyethylene glycol to obtain two precursor solutions (3% SF/3.5% PEG-CBT solution and 3% SF/3.5% PEG-Cys), then uniformly mixing the precursor solutions in an equal volume, transferring the mixture into a mold, performing primary curing molding, placing the mold in an incubator for 30min, and inducing the silk fibroin to slowly form beta-sheet to obtain the secondary-cured double-network hydrogel.

Example 3

The method comprises the following steps: adding 0.02mol/L Na into selected clean silkworm cocoon₂CO₃Boiling the solution in a water bath kettle at 100 ℃ for 2 times, wherein each time lasts for at least 40min, washing the solution for multiple times by using deionized water, wringing the solution to remove sericin, drying the solution in an oven at 60 ℃ overnight, putting the dried silk fibroin into a LiBr solution to form a first mixed system for the reaction, and reacting for 4h while maintaining the temperature of the reaction system at 60 ℃; wherein the concentration of the LiBr solution is 9.3mol/L, and the mass ratio of the LiBr to the silk fibroin is 10: 1.

and step one, after the reaction is finished, dialyzing for 1 day by using 7KDa cut-off quantity to remove impurities, freezing at the temperature of 80 ℃ overnight, and freeze-drying at the temperature of 50 ℃ for 3 days to obtain pure silk fibroin.

Step two: dissolving four-arm polyethylene glycol in 1% anhydrous N, N-dimethylformamide solution, adding N-methylmorpholine and isobutyl chloroformate, and reacting at 4 deg.C for 20 min. Wherein the molar ratio of the N-methylmorpholine to the four-arm polyethylene glycol is 1.5: 1, the molar ratio of isobutyl chloroformate to four-arm polyethylene glycol is 1.5: 1.

step three: and after the reaction in the second step is finished, adding benzothiazole, and reacting for 20 hours at 15 ℃. Wherein the molar ratio of the benzothiazole to the four-arm polyethylene glycol is 1.5: 1.

and after the reaction in the third step is finished, precipitating in diethyl ether, wherein the volume ratio of the diethyl ether to the mixed system is 18: 1, obtaining white flocculent precipitate, centrifuging at 12000rpm for 5min, collecting the precipitate, dissolving in water, purifying by a sephadex column G-15, and freeze-drying to obtain the benzothiazole modified four-arm polyethylene glycol, wherein the structural formula is shown as a formula (1). Wherein n takes the value of 130.

Step four: dissolving aminothiol protected cysteine in anhydrous N, N-dimethylformamide solution, adding 1-hydroxybenzotriazole and benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate, mixing, reacting at 5 deg.C for 18min, adding four-arm polyethylene glycol, and reacting at 15 deg.C for 20 h. Wherein the mol ratio of the 1-hydroxybenzotriazole to the amino mercapto group protected cysteine is 1.5: 1, the molar ratio of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate to amino mercapto protected cysteine is 1: 1, the molar ratio of the four-arm polyethylene glycol to the amino mercapto protected cysteine is 0.667: 1.

and after the reaction in the fourth step is finished, precipitating in diethyl ether, wherein the volume ratio of the diethyl ether to the mixed system is 15: 1, obtaining white flocculent precipitate, centrifuging at 12000rpm for 5min, collecting the precipitate, dissolving in water, purifying by a sephadex column G-15, and freeze-drying to obtain the amino-sulfhydryl-protected cysteine-modified four-arm polyethylene glycol.

Step five: the four-arm polyethylene glycol modified by the amino sulfydryl protected cysteine, trifluoroacetic acid and dichloromethane are uniformly mixed and reacted for 1h at the temperature of 30 ℃. Wherein the mass ratio of trifluoroacetic acid to amino-sulfydryl-protected cysteine-modified four-arm polyethylene glycol is 100: 1.

and after the fifth reaction step is finished, dialyzing the obtained reaction mixture for 3 days by using a dialysis bag with the molecular weight cutoff of 3500Da, purifying by using a sephadex column G-15, and freeze-drying to obtain the sulfhydryl protected cysteine modified four-arm polyethylene glycol.

Step six: and (3) uniformly mixing the sulfhydryl protected cysteine modified four-arm polyethylene glycol and dithiothreitol in a phosphate buffer solution, and reacting for 5min at 15 ℃. Wherein, the molar ratio of dithiothreitol to sulfhydryl protected cysteine modified four-arm polyethylene glycol is 4: 1.

and sixthly, after the step six is finished, freeze-drying to obtain the cysteine-modified four-arm polyethylene glycol, wherein the structural formula of the cysteine-modified four-arm polyethylene glycol is shown as the formula (2). Wherein m is 130.

Step seven: dissolving silk fibroin in PBS to form a solution with the concentration of 10 w/v%, then performing ultrasonic treatment on the silk fibroin solution (power is 240w, ultrasonic treatment is 2s, pause is 2s, and circulation is performed for 8 times) to serve as a solvent to respectively dissolve benzothiazole modified four-arm polyethylene glycol and cysteine modified four-arm polyethylene glycol to obtain two precursor solutions (5% SF/2.5% PEG-CBT solution and 5% SF/2.5% PEG-Cys), then uniformly mixing the precursor solutions in equal volumes, and transferring the precursor solutions into a mold, wherein the molar ratio of the tetrabenzothiazole modified four-arm polyethylene glycol to the cysteine modified four-arm polyethylene glycol is 1: and 1, primarily curing and forming, placing in an incubator for 30min, and inducing silk fibroin to slowly form beta folding to obtain secondary cured double-network hydrogel.

Example 4

The method comprises the following steps: adding 0.02mol/L Na into selected clean silkworm cocoon₂CO₃Boiling the solution in a water bath at 100 deg.C for 2 times, each time for at least 40min, washing with deionized water for multiple times, wringing to remove sericin, oven drying at 60 deg.C overnight, placing dried silk fibroin into LiBr solution to form a first mixed reaction system for the reaction, and maintaining the reactionThe temperature of the reaction system is 55 ℃, and the reaction is carried out for 5 hours; wherein the concentration of the LiBr solution is 9mol/L, and the mass volume ratio of the silk fibroin to the lithium bromide is 2:10(w/v,%).

And step one, after the reaction is finished, dialyzing for 1 day by using a 14KDa cut-off amount to remove impurities, freezing at the temperature of 80 ℃ overnight, and freeze-drying at the temperature of 50 ℃ for 3 days to obtain pure water-soluble silk fibroin.

Step two: dissolving the four-arm polyethylene glycol in a 1% anhydrous N, N-dimethylformamide solution, adding N-methylmorpholine and isobutyl chloroformate, and reacting on ice at 8 ℃ for 30 min. Wherein the molar ratio of the N-methylmorpholine to the four-arm polyethylene glycol is 1: 1, the molar ratio of isobutyl chloroformate to four-arm polyethylene glycol is 2: 1.

step three: and after the reaction in the second step is finished, adding benzothiazole, and reacting for 10 hours at 25 ℃. Wherein the molar ratio of the four-arm polyethylene glycol to the benzothiazole is 1: 3.

and after the reaction in the third step is finished, precipitating in diethyl ether, wherein the volume ratio of the diethyl ether to the mixed system is 20: 1, obtaining white flocculent precipitate, centrifuging at 12000rpm for 5min to collect the precipitate, dissolving the precipitate with water, and purifying through a sephadex column G-15 to obtain a benzothiazole modified four-arm polyethylene glycol solution, wherein the structural formula of the benzothiazole modified four-arm polyethylene glycol is shown as the formula (1). Wherein n has a value of 60.

Step four: dissolving aminothiol protected cysteine in N, N-dimethylformamide solution, adding 1-hydroxybenzotriazole and benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate, mixing, reacting at 8 deg.C for 10min, adding four-arm polyethylene glycol, and reacting at 25 deg.C for 10 h. Wherein the molar ratio of the 1-hydroxybenzotriazole to the amino mercapto-protected cysteine is 21: 1, the molar ratio of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate to amino mercapto protected cysteine is 2:1, the molar ratio of the four-arm polyethylene glycol to the amino-sulfydryl-protected cysteine is 1: 1.

and after the reaction in the fourth step is finished, precipitating in diethyl ether, wherein the volume ratio of the diethyl ether to the mixed system is 18: 1, obtaining white flocculent precipitate, centrifuging at 12000rpm for 5min, collecting the precipitate, dissolving in water, purifying by a sephadex column G-15, and freeze-drying to obtain the amino-sulfhydryl-protected cysteine-modified four-arm polyethylene glycol.

Step five: the four-arm polyethylene glycol modified by the amino sulfydryl protected cysteine, trifluoroacetic acid and dichloromethane are uniformly mixed and reacted for 1h at the temperature of 18 ℃. Wherein the mass ratio of trifluoroacetic acid to dichloromethane to amino sulfydryl protected cysteine modified four-arm polyethylene glycol is 50: 1.

and after the fifth reaction step is finished, dialyzing the obtained reaction mixture for 1 day by using a dialysis bag with the molecular weight cutoff of 14000Da, purifying by using a sephadex column G-15, and freeze-drying to obtain the sulfhydryl protected cysteine modified four-arm polyethylene glycol.

Step six: and (3) uniformly mixing the sulfhydryl protected cysteine modified four-arm polyethylene glycol and dithiothreitol in a phosphate buffer solution, and reacting for 10min at 20 ℃. Wherein, the molar ratio of dithiothreitol to sulfhydryl protected cysteine modified four-arm polyethylene glycol is 3: 1.

and sixthly, after the step six is finished, freeze-drying to obtain the cysteine-modified four-arm polyethylene glycol, wherein the structural formula of the cysteine-modified four-arm polyethylene glycol is shown as the formula (2). Wherein the value of m is 115.

Step seven: dissolving silk fibroin in PBS to form a solution with the concentration of 10 w/v%, then performing ultrasonic treatment on the silk fibroin solution (with the power of 200w, ultrasonic treatment for 3s, pause for 3s, and cycle for 6 times) to respectively dissolve benzothiazole-modified four-arm polyethylene glycol and cysteine-modified four-arm polyethylene glycol as solvents to obtain two precursor solutions (7% SF/1.5% PEG-CBT solution and 7% SF/1.5% PEG-Cys), then uniformly mixing the precursor solutions, and transferring the precursor solutions into a mold, wherein the molar ratio of the tetrabenzothiazole-modified four-arm polyethylene glycol to the cysteine-modified four-arm polyethylene glycol is 1: and 2, primarily curing and forming, placing in an incubator for 30min, and inducing the silk fibroin to slowly form beta folding to obtain the secondary cured double-network hydrogel.

Example 5

The method comprises the following steps: adding selected clean silkworm cocoon0.02mol/L Na₂CO₃Boiling the solution in a water bath kettle at 100 ℃ for 2 times, wherein each time lasts for at least 40min, washing the solution for multiple times by using deionized water, wringing the solution to remove sericin, drying the solution in an oven at 60 ℃ overnight, putting the dried silk fibroin into a LiBr solution to form a first mixed reaction system for reaction, and reacting for 6h while maintaining the temperature of the reaction system at 50 ℃; wherein the concentration of the LiBr solution is 10mol/L, and the mass volume ratio of the silk fibroin to the lithium bromide is 3:10(w/v,%).

After the reaction of the first step, dialyzing with 10KDa cut-off amount for 2 days to remove impurities, freezing at-80 ℃ overnight, and freeze-drying at-50 ℃ for 1 day to obtain pure silk fibroin.

Step two: dissolving four-arm polyethylene glycol in 1% anhydrous N, N-dimethylformamide solution, adding N-methylmorpholine and isobutyl chloroformate, and reacting on ice at 0 deg.C for 10 min. Wherein the molar ratio of the N-methylmorpholine to the four-arm polyethylene glycol is 2:1, the molar ratio of isobutyl chloroformate to four-arm polyethylene glycol is 1: 1.

step three: and after the reaction in the second step is finished, adding benzothiazole, and reacting for 15 hours at 30 ℃. Wherein the molar ratio of the four-arm polyethylene glycol to the benzothiazole is 1: 2.

after the reaction in the third step is finished, precipitating in normal hexane, wherein the volume ratio of the normal hexane to the mixed system is 20: 1, obtaining white flocculent precipitate, centrifuging at 12000rpm for 5min to collect the precipitate, dissolving the precipitate with water, and purifying through a sephadex column G-15 to obtain a benzothiazole modified four-arm polyethylene glycol solution, wherein the structural formula of the benzothiazole modified four-arm polyethylene glycol is shown as the formula (1). Wherein the value of n is 115.

Step four: dissolving aminothiol protected cysteine in N, N-dimethylformamide solution, adding 1-hydroxybenzotriazole and benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate, mixing, reacting at 0 deg.C for 30min, adding four-arm polyethylene glycol, and reacting at 30 deg.C for 15 h. Wherein the mol ratio of the 1-hydroxybenzotriazole to the amino mercapto group protected cysteine is 1: 1, the molar ratio of benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate to amino mercapto protected cysteine is 1: 1, the molar ratio of the four-arm polyethylene glycol to the amino-sulfydryl-protected cysteine is 1: 3.

after the reaction in the fourth step is finished, precipitating in normal hexane, wherein the volume ratio of the normal hexane to the mixed system is 20: 1, obtaining white flocculent precipitate, centrifuging at 12000rpm for 5min, collecting the precipitate, dissolving in water, purifying by a sephadex column G-15, and freeze-drying to obtain the amino-sulfhydryl-protected cysteine-modified four-arm polyethylene glycol.

Step five: the four-arm polyethylene glycol modified by the amino sulfydryl protected cysteine, trifluoroacetic acid and dichloromethane are uniformly mixed and reacted for 3h at 25 ℃. Wherein the mass ratio of trifluoroacetic acid to dichloromethane to amino sulfydryl protected cysteine modified four-arm polyethylene glycol is 30: 1.

and after the fifth reaction step is finished, dialyzing the obtained reaction mixture for 2 days by using a dialysis bag with the molecular weight cutoff of 10000Da, purifying by using a sephadex column G-15, and freeze-drying to obtain the sulfhydryl protected cysteine modified four-arm polyethylene glycol.

Step six: and (3) uniformly mixing the sulfhydryl protected cysteine modified four-arm polyethylene glycol and dithiothreitol in a phosphate buffer solution, and reacting for 5min at 30 ℃. Wherein, the molar ratio of dithiothreitol to sulfhydryl protected cysteine modified four-arm polyethylene glycol is 5: 1.

and sixthly, after the step six is finished, freeze-drying to obtain the cysteine-modified four-arm polyethylene glycol, wherein the structural formula of the cysteine-modified four-arm polyethylene glycol is shown as the formula (2). Wherein the value of m is 115.

Step seven: dissolving silk fibroin in PBS to form a solution with the concentration of 8 w/v%, then performing ultrasonic treatment on the silk fibroin solution (power is 220w, ultrasonic treatment is 5s, pause is 5s, and circulation is performed for 10 times), dissolving benzothiazole modified four-arm polyethylene glycol and cysteine modified four-arm polyethylene glycol respectively as solvents to obtain two precursor solutions (5% SF/2.5% PEG-CBT solution and 5% SF/2.5% PEG-Cys), then uniformly mixing the precursor solutions, and transferring the mixture into a mold, wherein the molar ratio of the tetrabenzothiazole modified four-arm polyethylene glycol to the cysteine modified four-arm polyethylene glycol is 1: and 3, primarily curing and forming, placing in an incubator for 30min, and inducing the silk fibroin to slowly form beta folding to obtain the secondary cured double-network hydrogel.

The steps one to seven described above can be represented by fig. 1.

Performance test one

The internal structure and the pore size of the double-network hydrogel obtained in the embodiment are tested on a field ring scanning electron microscope tester, and the operation method comprises the following steps:

freezing the double-network hydrogel with liquid nitrogen, freeze-drying at-50 deg.C for 24 hr, spraying gold at 0.2mA for 3min, and observing the microstructure of the hydrogel by scanning electron microscope (as shown in FIG. 2). As can be seen by a scanning electron microscope, the microstructure of the double-network hydrogel is porous, and the aperture is about 100-200 microns.

Performance test 2

Dissolving the silk fibroin into PBS to form a solution with the concentration of 5 w/v%, then carrying out ultrasonic treatment on the silk fibroin solution (power is 240w, ultrasonic treatment is 5s, pause is 5s, and circulation is carried out for 6 times), using the solution as a solvent to respectively dissolve benzothiazole modified four-arm polyethylene glycol and cysteine modified four-arm polyethylene glycol to obtain two precursor solutions (5% SF/2.5% PEG-CBT solution and 5% SF/2.5% PEG-Cys), then uniformly mixing the precursor solutions in equal volume, transferring the mixture into a mold, carrying out primary curing molding, placing the mold in an incubator for 30min, and inducing the silk fibroin to slowly form beta-folding to obtain the secondary cured double-network hydrogel. The storage modulus of the double-network hydrogel obtained in the embodiment is tested on a rheological tester, and as can be seen from a rheological result shown in fig. 3a, G '> G ″ is in a linear relationship, which indicates that the hydrogel is in a gel state, and G' is about 7.5 KPa; the tensile property of the double-network hydrogel obtained in this example was measured by the double-network hydrogel material tester, and as can be seen from the tensile property results in fig. 3b, the maximum tensile stress of the hydrogel was about 90kPa, and the compressive property of the double-network hydrogel obtained in this example was measured by the double-network hydrogel material tester, and as can be seen from the compressive property results in fig. 3c, the compressive stress of the hydrogel was about 0.37MPa when the hydrogel was compressed to 65%.

Performance test three

The conformational structure of the double-network hydrogel obtained in this example was tested by an X-ray diffractometer and a fourier infrared spectrometer, and the procedure included:

and (3) freezing the double-network hydrogel by using liquid nitrogen, carrying out freeze drying at-50 ℃ for 24h, and analyzing the crystal structure by using an X-ray powder diffractometer. Monochromatic CuKa rays are used as a target, the current is 30mA, the voltage is 40kV, the scanning speed is 0.5 DEG/min, the diffraction angle range is 10-50 DEG, and absorption peaks near 2 theta (21 DEG and 24 DEG) can be seen through figures 4 a-4 c, and represent beta-sheet structures.

Performance test four

Cell proliferation assay Using the Dual-network hydrogel obtained in this example

The survival and proliferation of rat bone marrow mesenchymal stem cells (BMSCs) embedded in the double-network hydrogel of this example were determined by calcein staining and tetrazolium salt colorimetry (WST method) by the following procedures:

dissolving silk fibroin in PBS to form a solution with the concentration of 5 w/v%, then performing ultrasonic treatment on the silk fibroin solution (power is 240w, ultrasonic treatment is 5s, pause is 5s, and circulation is performed for 6 times) to serve as a solvent to respectively dissolve benzothiazole modified four-arm polyethylene glycol and cysteine modified four-arm polyethylene glycol to obtain two precursor solutions (5% SF/2.5% PEG-CBT solution and 5% SF/2.5% PEG-Cys), digesting, counting and centrifuging BMSC cells cultured in a basic culture medium at the 3 rd generation to the 6 th generation for 3min at 1000 rpm; mixing with the above two precursor solutions to ensure cell concentration of 2 × 10⁶Per mL; taking 50 mu L of each blending liquid, uniformly mixing the blending liquid in a mould to form hydrogel formed by primary curing, placing the hydrogel in an incubator for 30min, inducing silk fibroin to slowly form beta folding to obtain double-network hydrogel loaded with BMSC (BMSC), blending cells and the double-network hydrogel, transferring the hydrogel into a 24-hole plate, adding a basal medium, and adding 5% CO₂And cultured in an incubator at 37 ℃.

After culturing for 1 day and 7 days, taking out the culture medium, washing with PBS for 3 times, measuring by using a Live/dead kit, and observing the cell activity under the excitation of laser confocal 488/561 nm; viable cells stained with calcein fluoresce green, dead cells stained red.

As shown in FIGS. 5a and 5b, BMSCs survived well in the photo-cured hydrogels obtained in this example and showed three-dimensional structures and significant proliferation, indicating that the present invention had no effect on cell proliferation and provided a three-dimensional growth environment for cells.

Culturing for 1 day, 3 days and 7 days, taking out the culture medium, adding 450 μ L fresh culture medium into each well, adding 50 μ L WST-1, mixing, adding 5% CO₂And incubating for 4h in an incubator at 37 ℃, and taking 100 mu L to test the OD value in a 96-well plate at 450nm of an enzyme-labeling instrument.

As shown in fig. 5c, after the BMSC and the double-network hydrogel obtained in this example are blended, the cells survived better after 1 day of culture, and the cells proliferated obviously after 7 days of culture, which indicates that the double-network hydrogel obtained in this example has low toxicity and good biocompatibility.

Performance test five

Rat bone marrow mesenchymal Stem cells differentiation assay into chondrocytes in the Dual-network hydrogel obtained in this example

RT-PCR is used to detect the expression level of mRNA of the cartilage-specific gene to judge whether stem cells are differentiated.

The 3-6 generation BMSC cells were divided into two groups:

in the first group, BMSC cells of 3 rd to 6 th generations and the double-network hydrogel obtained in the embodiment are mixed and then cultured in a chondrogenic differentiation medium to be used as a control group;

the second group is to culture the 3 rd-6 th generation BMSC cells in culture bottles by using complete culture medium as a control group;

two groups of cell-loaded hydrogels were placed in 5% CO₂Culturing in an incubator at 37 ℃, changing fresh culture medium every other day, culturing for 28 days, removing the culture medium, washing with PBS for 3 times, and extracting total cellular RNA from the BMSC-respectively-loaded double-network hydrogel through a TRIzol Plus RNA purification kit at each time point. RNA purity was assessed using A260/280 nm. Thereafter, 500ng of RNA was reverse transcribed into cDNA using PrimeScriptTM RT kit. RT-PCR detection was performed using SYBR Green I PCR kit. Culture medium without vascular differentiation in culture dishBMSC cells from 3-6 passages in culture were used as calibrator controls and the target gene expression was normalized by non-regulated reference gene expression (Gapdh).

As shown in fig. 5a to 5c, the expression of stem cells in the double-network hydrogel blended with the cells was significantly increased under the chondrogenic differentiation culture conditions compared to the stem cells of the control group in chondrogenic differentiation specific gene type II collagen (Col II), proteoglycan (AGG) and sex determining region Y-box protein 9(Sox 9), which suggests that BMSCs can normally differentiate on the three-dimensional structure of the double-network hydrogel obtained in this example, indicating that the double-network hydrogel obtained in this example has very good biocompatibility and safety.

Comparative example 1

Generally, pure silk fibroin is used for forming high-strength hydrogel by means of organic chemical reagents, but the organic reagents have cytotoxicity and are not beneficial to cell embedding, so that the application of the organic reagents in biomedicine is limited.

Compared with the comparative example 1, the hydrogel obtained in the examples 1 to 5 of the present invention adopts simple physical ultrasound to induce the silk fibroin to form beta-sheet and further solidify to form high strength hydrogel, compared with the above method, the hydrogel formed by physical ultrasound crosslinking has wider biological application, for example, the present invention realizes blending gelation with cells, and is easier for cell embedding compared with the above three-dimensional scaffold material.

Comparative example 2

Generally, the speed of inducing the silk fibroin to form beta folding and further to solidify to form the high-strength hydrogel by utilizing pure silk fibroin through simple physical ultrasound is slow, the gelling time of the silk fibroin is different from dozens of minutes to hours or even days according to the intensity of ultrasound, the rapid formation of the scaffold and the blending of load cells are not facilitated, and the inner pore diameter of the hydrogel formed by the pure silk fibroin is small, so that the exchange of nutrient substances and metabolic wastes is not facilitated.

Compared with the comparative example 2, the double-network hydrogel obtained in the examples 1 to 5 of the present invention obtains a first heavy hydrogel network through bioorthogonal fast crosslinking, and further constructs a second heavy hydrogel network by inducing slow formation of beta-sheet of silk fibroin, so as to enhance the performance of the hydrogel to meet the requirement of cartilage tissue engineering, and the formed double-network hydrogel has stronger mechanical properties, more suitable pore size, three-dimensional microenvironment and biological application.

Comparative example 3

Generally, the hydrogel can be formed by fast crosslinking through a simple bioorthogonal reaction, but the formed hydrogel has low mechanical properties, so that the application of the hydrogel in biomedicine is limited.

Compared with the comparative example 3, the double-network hydrogel obtained in the examples 1 to 5 of the present invention obtains a first heavy hydrogel network through bioorthogonal fast crosslinking, and further constructs a second heavy hydrogel network by inducing slow formation of beta-sheet of silk fibroin, so as to enhance the mechanical properties of the hydrogel to meet the requirements of cartilage tissue engineering.

Comparative example 4

This comparative example lacks steps four through six, as compared to example 1.

In general, physical ultrasound is adopted to induce silk fibroin to form beta folding to be compounded with pure benzothiazole modified four-arm polyethylene glycol, so that bio-orthogonal reaction cannot occur, and double-network hydrogel cannot be formed.

Compared with the comparative example 4, the double-network hydrogel obtained in the example 1 of the invention obtains a first heavy hydrogel network which is rapidly cured by utilizing bioorthogonal rapid crosslinking, and further constructs a second heavy hydrogel network by inducing slow formation of beta-sheet of silk fibroin, so as to improve the mechanical properties of the hydrogel to meet the requirements of cartilage tissue engineering.

Comparative example 5

In this comparative example, step two to step three were omitted compared with example 1.

In general, physical ultrasound is adopted to induce silk fibroin to form beta folding to be compounded with pure cysteine modified four-arm polyethylene glycol, so that bio-orthogonal reaction cannot occur, and double-network hydrogel cannot be formed.

Compared with the comparative example 5, the double-network hydrogel obtained in the example 1 of the invention obtains a first heavy hydrogel network through bioorthogonal rapid crosslinking, and further constructs a second heavy hydrogel network by inducing slow beta-sheet formation of silk fibroin, so as to improve the mechanical properties of the hydrogel to meet the requirements of cartilage tissue engineering.

In conclusion, by the technical scheme, the double-network hydrogel disclosed by the invention is short in curing time, uniform in pore distribution inside the hydrogel, good in biocompatibility and low in toxicity, can provide a three-dimensional living environment for cells, improves the adhesion and proliferation of stem cells on a three-dimensional scaffold, and realizes differentiation to chondrocytes; meanwhile, the preparation method is simple and can be used for mass preparation.

The aspects, embodiments, features and examples of the present invention should be considered as illustrative in all respects and not intended to be limiting of the invention, the scope of which is defined only by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.

The use of headings and chapters in this disclosure is not meant to limit the disclosure; each section may apply to any aspect, embodiment, or feature of the disclosure.

Throughout this specification, where a composition is described as having, containing, or comprising specific components or where a process is described as having, containing, or comprising specific process steps, it is contemplated that the composition of the present teachings also consist essentially of, or consist of, the recited components, and the process of the present teachings also consist essentially of, or consist of, the recited process steps.

Unless specifically stated otherwise, use of the terms "comprising", "including", "having" or "having" is generally to be understood as open-ended and not limiting.

The use of the singular herein includes the plural (and vice versa) unless specifically stated otherwise. Furthermore, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. In addition, where the term "about" is used before a quantity, the present teachings also include the particular quantity itself unless specifically stated otherwise.

It should be understood that the order of steps or the order in which particular actions are performed is not critical, so long as the teachings of the invention remain operable. Further, two or more steps or actions may be performed simultaneously.

In addition, the inventors of the present invention have also made experiments with other materials, process operations, and process conditions described in the present specification with reference to the above examples, and have obtained preferable results.

While the invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Moreover, unless specifically stated any use of the terms first, second, etc. do not denote any order or importance, but rather the terms first, second, etc. are used to distinguish one element from another.