CN113956506A

CN113956506A - Double-network hydrogel and preparation method and application thereof

Info

Publication number: CN113956506A
Application number: CN202010629830.6A
Authority: CN
Inventors: 裴仁军; 倪添雨
Original assignee: Suzhou Institute of Nano Tech and Nano Bionics of CAS
Current assignee: Suzhou Institute of Nano Tech and Nano Bionics of CAS
Priority date: 2020-07-03
Filing date: 2020-07-03
Publication date: 2022-01-21
Anticipated expiration: 2040-07-03
Also published as: CN113956506B

Abstract

The invention discloses a double-network hydrogel and a preparation method and application thereof. The preparation method comprises the following steps: reacting hydroxypropyl methylcellulose with methacrylic anhydride to obtain a hydroxypropyl methylcellulose-methacrylate polymer; inducing the silk fibroin to form beta-folding by adopting ultrasonic treatment, and then mixing the beta-folding with the hydroxypropyl methyl cellulose-methacrylate polymer to perform photopolymerization reaction to obtain the double-network hydrogel. The 3D printing biological ink of the double-network hydrogel prepared by the invention has good printability, can be printed into various shapes together with stem cells, has short curing time, uniform internal pore distribution, remarkably enhanced mechanical property and good biocompatibility, can provide good three-dimensional supporting living environment for survival and proliferation of the stem cells, and can be widely applied to the fields of cell culture or tissue engineering and the like.

Description

Double-network hydrogel and preparation method and application thereof

Technical Field

The invention belongs to the technical field of tissue engineering material preparation, relates to a double-network hydrogel and a preparation method and application thereof, and particularly relates to a double-network hydrogel for culturing three-dimensional stem cells and promoting the stem cells to proliferate and differentiate, and a preparation method and application thereof.

Background

Tissue, organ defects and dysfunction caused by diseases, genetics, aging and the like are one of the major risks faced by 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 and a preparation method and application thereof, so as to overcome the defects of the prior art.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:

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

(1) providing silk fibroin;

(2) reacting hydroxypropyl methylcellulose with methacrylic anhydride to obtain a hydroxypropyl methylcellulose-methacrylate polymer;

(3) inducing the silk fibroin to form beta-folding by adopting ultrasonic treatment, and then mixing the beta-folding with the hydroxypropyl methyl cellulose-methacrylate polymer to perform photopolymerization reaction to obtain the double-network hydrogel.

The embodiment of the invention also provides the double-network hydrogel prepared by the method, the compression modulus of the double-network hydrogel is 20-80 KPa, the double-network hydrogel has a porous structure, and the aperture of holes contained in the double-network hydrogel is 100-300 mu m.

The embodiment of the invention also provides a preparation method of the double-network hydrogel 3D printing biological ink, which comprises the following steps:

providing the silk fibroin and hydroxypropyl methylcellulose-methacrylate polymer described previously;

and inducing the silk fibroin to form beta-folding by adopting ultrasonic treatment, and then mixing the beta-folding with the hydroxypropyl methyl cellulose-methacrylate polymer to obtain the 3D printing biological ink of the double-network hydrogel.

The embodiment of the invention also provides the 3D printing biological ink of the double-network hydrogel prepared by the method.

The embodiment of the invention also provides a 3D printing preparation method of the double-network hydrogel, which comprises the following steps:

providing the 3D printing biological ink of the double-network hydrogel;

and 3D printing is carried out by adopting a nozzle extrusion 3D printing method under illumination to obtain the double-network hydrogel.

The embodiment of the invention also provides the double-network hydrogel prepared by the 3D printing method, the compression modulus of the double-network hydrogel is 20-80 KPa, the double-network hydrogel has a porous structure, and the aperture of holes contained in the double-network hydrogel is 100-300 mu m.

The embodiment of the invention also provides application of the pre-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 double-network hydrogel.

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

and (3) culturing cells by using the double-network hydrogel as a three-dimensional cell culture carrier, and promoting the cells to proliferate and differentiate.

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

(1) the double-network hydrogel constructed based on silk fibroin and a functional cellulose system provided by the invention is simultaneously applied to the research of cell proliferation and cartilage differentiation, and realizes blending printing and gelation with cells. Firstly, modifying methacrylate groups on hydroxypropyl methyl cellulose to obtain hydroxypropyl methyl cellulose-methacrylate polymer, then mixing silk fibroin and the hydroxypropyl methyl cellulose-methacrylate polymer, and preparing silk fibroin/cellulose double-network hydrogel under light initiation and ultrasonic induction;

(2) the double-network hydrogel provided by the invention is a hydrogel, combines two action modes of physical ultrasound and photochemical reaction, and has the advantages that the hydrogel system is rapidly cured due to the characteristic of rapid photopolymerization; then, as the time is prolonged, beta-folding of the silk fibroin 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) according to the double-network hydrogel provided by the invention, the commonly used natural material cellulose is functionalized and modified to obtain a hydroxypropyl methyl cellulose-methacrylate polymer, the silk fibroin from natural silkworm cocoons is subjected to ultrasonic treatment, then the functionalized cellulose solution and the silk fibroin solution after ultrasonic induction are mixed to form 3D printing bio-ink, the stem cells are encapsulated, and 3D printing is carried out, so that the stem cell blending hydrogel with a three-dimensional shape can be manufactured, and the silk fibroin and the functionalized cellulose are combined, so that the curing speed of the hydrogel system is obviously improved on one hand, and the mechanical property of the hydrogel system is enhanced on the other hand; the double-network hydrogel obtained through the ultrasonic-optical curing or 3D printing has the advantages of short curing time, uniform internal pore distribution, good biocompatibility and low toxicity, the internal pore diameter of the double-network hydrogel is 100-300 mu m, and the double-network hydrogel is suitable for circulation of nutrient substances and cell metabolic wastes and provides a good three-dimensional supporting living environment for survival and proliferation of stem cells; meanwhile, the prepared method for co-culturing the double-network hydrogel and the stem cells can effectively promote the differentiation of the stem cells into the chondrocytes to construct the cartilage tissue repair substance by adding the cartilage differentiation promoting growth factors.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be 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 application, and other drawings can be obtained by those skilled in the art without creative efforts.

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

FIG. 2 is an SEM scan of a dual-network hydrogel obtained in an exemplary embodiment of the present invention;

FIGS. 3a-3c are graphs of the mechanical properties of the resulting double-network hydrogels according to an exemplary embodiment of the present invention;

FIG. 4 is a Raman spectrum of a printable dual network hydrogel obtained in an exemplary embodiment of the present invention;

FIGS. 5a-5D are graphs showing the 3D printing effect of the double-network hydrogel obtained in an exemplary embodiment of the present invention;

FIGS. 6a-6b are a proliferation map and a growth confocal map of bone marrow mesenchymal stem cells in a double-network hydrogel obtained in an exemplary embodiment of the present invention, respectively.

FIGS. 7a to 7c are graphs showing mRNA expression levels of bone marrow mesenchymal stem cells differentiated into chondrocytes under in vitro culture conditions in a double-network hydrogel obtained in an exemplary embodiment of the present invention;

FIG. 8 is an SEM scan of differentiation of bone marrow mesenchymal stem cells into chondrocytes under 2-week in vitro culture in a double-network hydrogel obtained in an exemplary embodiment of the present invention.

Detailed Description

In view of the defects of the prior art, the inventor of the present invention has long studied and largely practiced to propose the technical solution of the present invention, which will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

One aspect of the embodiments of the present invention provides a method for preparing a double-network hydrogel, including:

(1) providing silk fibroin;

In some more specific embodiments, step (1) specifically comprises: and (2) reacting the first mixed reaction system containing the natural silk fibroin and the neutral salt solution at 40-50 ℃ for 1-2 h, and then carrying out post-treatment to obtain the pure water-soluble silk fibroin.

Further, the step (1) further comprises: and after the reaction is finished, dialyzing the obtained reaction mixture for 3-4 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 system for the reaction, and maintaining the temperature of the reaction system at 40-50 ℃.

Further, the salt contained in the neutral salt solution includes any one or a combination of two or more of magnesium nitrate, calcium chloride, and lithium bromide, 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 more specific embodiments, step (2) specifically includes: and (2) reacting a second mixed reaction system containing hydroxypropyl methyl cellulose, methacrylic anhydride and 4-dimethylaminopyridine at the temperature of 10-50 ℃ for 12-30 h to obtain the hydroxypropyl methyl cellulose-methacrylate polymer.

Further, the step (2) further comprises: after the reaction is finished, the obtained reaction mixture is subjected to precipitation, purification, centrifugation, dialysis and freeze drying treatment to obtain the hydroxypropyl methyl cellulose-methacrylate polymer.

Further, the precipitating agent used in the precipitation treatment includes diethyl ether or acetone, and is not limited thereto.

Furthermore, the mass ratio of the hydroxypropyl methyl cellulose, the methacrylic anhydride and the 4-dimethylaminopyridine is 1 (0.06-0.2) to (0.06-0.2).

In some preferred embodiments, step (2) further comprises: after the reaction is finished, mixing the obtained reaction mixture with anhydrous ether according to the volume ratio of 1: 10-20, centrifuging at 8000rpm for 5min, collecting precipitate, and mixing the precipitate according to the mass ratio of: dissolving the precipitate in deionized water according to a volume ratio of 1: 50-100, filling the deionized water into a dialysis bag with the volume ratio of 7-14 kDa, changing water every 2-4 hours, dialyzing for 3 days, freezing the obtained solution at-80 ℃ overnight, and vacuum-drying at-40-60 ℃ for 3 days to obtain the pure hydroxypropyl methyl cellulose-methacrylate polymer.

In some more specific embodiments, the hydroxypropyl methylcellulose-methacrylate polymer has the structural formula shown in formula (I):

wherein R is selected from H, CH₃、

Any one of n is 80 to 100, m is 1 to up to3。

In some more specific embodiments, step (3) specifically includes:

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

carrying out ultrasonic treatment on the silk fibroin solution to induce the silk fibroin to form beta-folding;

and mixing the silk fibroin solution subjected to ultrasonic treatment with hydroxypropyl methyl cellulose-methacrylate polymer and a photoinitiator for carrying out photopolymerization reaction to obtain the double-network hydrogel.

In some preferred embodiments, step (3) specifically includes: dissolving silk fibroin in the silk fibroin solution in PBS to form a solution with the concentration of 5-15 w/v%, and then carrying out ultrasonic induction to form a beta-folded structure.

Further, the step (3) further comprises: and rapidly and uniformly mixing the silk fibroin solution and the hydroxypropyl methyl cellulose-methacrylate polymer solution according to the volume ratio of 1: 0.1-10.

Further, the concentration of silk fibroin in the silk fibroin solution is 5-15 w/v%.

Further, the ultrasonic treatment conditions are as follows: 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; the concentration of the hydroxypropyl methyl cellulose-methacrylate polymer used for mixing is 1-5 w/v%; the concentration of the added photoinitiator is 0.15-1 w/v%

Further, the photoinitiator includes Lithium Aryl Phosphinate (LAP) or I2959, preferably lithium aryl phosphinate, and is not limited thereto.

Furthermore, the wavelength used in the photopolymerization reaction is 350-410 nm, and the illumination time is 5-10s, preferably 5-8 s.

In some more specific embodiments, the method for preparing the 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 regenerated silk fibroin;

(2) mixing and reacting at least hydroxypropyl methyl cellulose and methacrylic anhydride to obtain hydroxypropyl methyl cellulose-methacrylate polymer;

(3) at least ultrasonically inducing a silk fibroin solution to form beta-folded powder, mixing the beta-folded powder with a hydroxypropyl methyl cellulose-methacrylate polymer solution, and adding a photoinitiator lithium aryl phosphinate;

(4) irradiating the solution at least under 365nm light, and standing at 37 ℃ to obtain the double-network hydrogel.

The embodiment of the invention also provides the double-network hydrogel prepared by the method, the compression modulus of the double-network hydrogel is 20-80 KPa, the double-network hydrogel has a porous structure, and the aperture of holes contained in the double-network hydrogel is 100-300 mu m, preferably 150-250 mu m.

Another aspect of the embodiments of the present invention also provides a method for preparing a bio-ink for 3D printing of a double-network hydrogel, including:

In some more specific embodiments, the preparation method comprises:

and mixing the silk fibroin solution subjected to ultrasonic treatment with hydroxypropyl methyl cellulose-methacrylate polymer and a photoinitiator to obtain the 3D printing biological ink of the double-network hydrogel.

Further, the ultrasonic treatment conditions are as follows: 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; the concentration of the hydroxypropyl methyl cellulose-methacrylate polymer used for mixing is 1-5 w/v%; the concentration of the added photoinitiator is 0.15-1 w/v%.

In some specific embodiments, the 3D printing bio-ink obtained after the ultrasonic treatment is subjected to 3D printing within 20-60 min, preferably 30-40 min.

In some more specific embodiments, the preparation method further comprises: and mixing the silk fibroin solution subjected to ultrasonic treatment with hydroxypropyl methyl cellulose-methacrylate polymer, a photoinitiator, a growth factor and cells to obtain the 3D printing biological ink of the double-network hydrogel.

Further, the cell includes a stem cell, preferably a bone marrow mesenchymal stem cell, and is not limited thereto.

Further, the growth factor includes a chondroprogressive differentiation growth factor, preferably transforming growth factor-beta 1 (TGF-beta 1) transforming growth factor-beta (TGF-beta 3), and is not limited thereto.

Another aspect of the embodiments of the present invention also provides a 3D printing bio-ink of the double-network hydrogel prepared by the foregoing method.

Another aspect of the embodiments of the present invention also provides a method for preparing a double-network hydrogel through 3D printing, including:

providing the 3D printing biological ink of the double-network hydrogel;

Further, the process conditions adopted by the nozzle extrusion 3D printing method include: the inner diameter of the printing nozzle is 150-250 mu m, the pneumatic pressure is 50-80 kPa, the printing speed is 15-25 mm/s, and the distance between printing lines is 0.5-1.5 mm.

Further, the wavelength of light used for the illumination treatment is 365-410 nm, and the illumination intensity is 1.5-2.5 mW/cm²The irradiation time of each layer of lines for 3D printing is 5-10s, preferably 5-8 s.

In some more specific embodiments, the method for preparing the double-network hydrogel by 3D printing comprises the following steps:

(3) at least ultrasonically inducing a silk fibroin solution to form beta-folded powder, mixing the beta-folded powder with a hydroxypropyl methyl cellulose-methacrylate polymer solution, and adding a photoinitiator lithium aryl phosphinate to form the 3D printing biological ink of the double-network hydrogel;

(4) and 3D biological printing is carried out on the 3D printing biological ink of the double-network hydrogel to obtain the double-network hydrogel, namely the cell scaffold.

The other aspect of the embodiment of the invention also provides the double-network hydrogel prepared by the 3D printing method, wherein the compression modulus of the double-network hydrogel is 20-80 KPa, the double-network hydrogel has a porous structure, and the pore diameter of pores contained in the double-network hydrogel is 100-300 μm, preferably 150-250 μm.

In another aspect of the embodiments of the present invention, there is also provided a use of the above-mentioned 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 double-network hydrogel described above.

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

Further, the cell is a mesenchymal stem cell, and is not limited thereto.

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

Further, the cell culture method comprises: and culturing the cells by using the double-network hydrogel as a three-dimensional cell culture carrier, and culturing for 7-28 days in a cartilage induced differentiation culture medium.

According to the technical scheme, the commonly used hydroxypropyl methyl cellulose is functionalized to obtain hydroxypropyl methyl cellulose-methacrylate polymer, silk fibroin from natural silkworm cocoons is subjected to ultrasonic treatment, then is mixed with the hydroxypropyl methyl cellulose-methacrylate polymer, is added with photoinitiator lithium aryl phosphinate, and then is mixed with cells, so that the silk fibroin can be printed into various shapes in a 3D mode. The natural silk fibroin and the hydroxypropyl methyl cellulose-methacrylate are mixed, on one hand, a double hydrogel network is provided, the mechanical property of the hydrogel can be obviously improved, on the other hand, the printing performance of the hydroxypropyl methyl cellulose is better, the mixture has good printability, the hydrogel obtained by the method is short in curing time, uniform in internal pore distribution, good in biocompatibility and low in toxicity, the internal pore diameter of the hydrogel is 100-300 mu m, the hydrogel is suitable for circulation of nutrient substances and cell metabolic waste, and a good three-dimensional supporting living environment is provided for survival and proliferation of stem cells. Meanwhile, the biological ink and the stem cells can be printed into various shapes together, and meanwhile, the growth factors are added, so that the stem cells can be regulated and controlled to be differentiated towards the cartilage direction, cartilage repair is effectively promoted, and meanwhile, the preparation method is simple and can be used for large-scale preparation.

The technical solutions of the present invention are further described in detail below with reference to several preferred embodiments and the accompanying drawings, which are implemented on the premise of the technical solutions of the present invention, and a detailed implementation manner and a specific operation process are provided, but the scope of the present invention is not limited to the following embodiments.

The experimental materials used in the examples used below were all available from conventional biochemical reagents companies, unless otherwise specified.

Example 1

The method comprises the following steps: shredding selected clean silkworm cocoon, adding 0.02mol/L Na₂CO₃In solution in 9Boiling for 2 times in a water bath at 5 ℃ for at least 30min each time, washing for multiple times by using deionized water, wringing to remove sericin, drying overnight in a 60 ℃ oven, then putting the dried silk fibroin into a LiBr solution to form a first mixed reaction system for reaction, maintaining the temperature of the reaction system at 40 ℃ and reacting for 1h, wherein the concentration of the LiBr solution is 9.3mol/L, and the mass of the silk fibroin and the LiBr solution is as follows: the volume ratio is 1: 10; after the reaction is finished, dialyzing with 7-14 kDa cut-off quantity to remove impurities, dialyzing for 3 days, freezing at minus 80 ℃ overnight, and freeze-drying at minus 50 ℃ for 3 days to obtain pure Silk Fibroin (SF);

step two: dissolving 1g hydroxypropyl methylcellulose (HPMC, 400 mPas) in 100mL anhydrous N, N-Dimethylformamide (DMF), stirring, adding 60 μ L (corresponding to 0.375mmol) of Methacrylic Anhydride (MA) and 0.2g of 4-Dimethylaminopyridine (DMAP) into the solution after the HPMC is dissolved, and reacting at room temperature for 16 h;

after the reaction in the second step is finished, the mixture is dripped into excessive ether for precipitation, the volume ratio of the mixture to the ether is 1:10, the mixture is centrifuged for 5min at 8000rpm, and the generated precipitate is collected. The collected precipitate was dissolved in DMF and precipitated twice more in excess ether. And dissolving the precipitate in deionized water, dialyzing with a cut-off of 7-14 kDa to remove impurities, and dialyzing for 3 days. Finally, the product is frozen at minus 80 ℃ overnight and then is frozen and dried at minus 50 ℃ for 3 days to obtain pure hydroxypropyl methyl cellulose-methacrylate polymer which is marked as HPMC-MA;

step three: dissolving the prepared SF in PBS to prepare 8 wt% solution for later use; HPMC-MA was dissolved in PBS to make a 5 wt% solution for further use. Treating the SF solution with an ultrasonic generator in ice bath (the diameter of an ultrasonic probe is 3mm, the power is 240W, the ultrasonic time is 5s, the suspension is 5s, the circulation is carried out for 8 times), mixing the SF solution with the HPMC-MA solution according to the volume ratio of 15:8, and simultaneously adding a photoinitiator lithium arylphosphinate (LAP, the final concentration is 0.15 wt%);

and after the third step, transferring the solution into a mold within 20min, carrying out 365nm photocuring for 8s for forming, and standing at 37 ℃ for 30min to obtain the secondary-cured double-network hydrogel.

Example 2

The method comprises the following steps: shredding selected clean silkworm cocoon, adding 0.02mol/L Na₂CO₃Boiling the solution in a water bath at 95 ℃ for 2 times, each time for at least 30min, 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 calcium nitrate solution to form a first mixed reaction system for reaction, maintaining the temperature of the reaction system at 40 ℃, and reacting for 2h, wherein the concentration of the calcium nitrate solution is 10mol/L, and the mass of the silk fibroin and LiBr solution is as follows: the volume ratio is 1: 10; after the reaction is finished, dialyzing with a 7-14 kDa cut-off amount to remove impurities, dialyzing for 3 days, freezing at minus 80 ℃ overnight, and freeze-drying at minus 50 ℃ for 3 days to obtain pure SF;

step two: dissolving 1g HPMC (400 mPas) in 100mL anhydrous N, N-Dimethylformamide (DMF), stirring, adding 120 μ L (corresponding to 0.75mmol) of Methacrylic Anhydride (MA) and 0.2g of 4-Dimethylaminopyridine (DMAP) into the solution after the HPMC is dissolved, and reacting at 50 ℃ for 12 h;

step three: dissolving the prepared SF in PBS to prepare 8 wt% solution for later use; HPMC-MA was dissolved in PBS to make a 5 wt% solution for further use. The SF solution was treated with an ultrasonic generator in an ice bath (ultrasonic probe diameter 3mm, power 180W, ultrasonic time 5s, pause 5s, cycle 6 times), mixed with HPMC-MA solution in a volume ratio of 15:8, and added with photoinitiator lithium arylphosphinate (LAP, final concentration 0.15 wt%).

And after the third step, transferring the solution into a mold within 20min, carrying out 365nm photocuring for 5s for forming, and standing at 37 ℃ for 30min to obtain the secondary-cured double-network hydrogel.

Example 3

The method comprises the following steps: shredding selected clean silkworm cocoon, adding 0.02mol/L Na₂CO₃Boiling the solution in a water bath at 95 ℃ for 2 times, each time for at least 30min, 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 calcium chloride solution to form a first mixed reaction system for reaction, maintaining the temperature of the reaction system at 50 ℃, and reacting for 1h, wherein the concentration of the calcium chloride solution is 10mol/L, and the mass of the silk fibroin and LiBr solution is as follows: the volume ratio is 1: 10;

after the reaction is finished, dialyzing with a 7-14 kDa cut-off amount to remove impurities, dialyzing for 4 days, freezing at minus 80 ℃ overnight, and freeze-drying at minus 50 ℃ for 3 days to obtain pure SF;

step two: 1g HPMC (400 mPas) was dissolved in 100mL anhydrous N, N-Dimethylformamide (DMF) with constant stirring. After dissolving HPMC, 180. mu.L (corresponding to 1.125mmol) of Methacrylic Anhydride (MA) and 0.2g of 4-Dimethylaminopyridine (DMAP) were added to the solution and reacted at 10 ℃ for 30 hours;

step three: dissolving the prepared SF in PBS to prepare 8 wt% solution for later use; dissolving HPMC-MA in PBS to prepare a 5 wt% solution for later use, treating the SF solution with an ultrasonic generator in ice bath, mixing the solution with the HPMC-MA solution according to the volume ratio of 15:8, and simultaneously adding a photoinitiator lithium aryl phosphinate (LAP with the final concentration of 0.15 wt%) with an ultrasonic probe with the power of 240W and the ultrasonic time of 2s, pausing for 2s and circulating for 10 times;

and after the third step, transferring the solution into a mold within 20min, carrying out 365nm photocuring for 10s for forming, and standing at 37 ℃ for 30min to obtain the secondary cured double-network hydrogel.

Example 4

The method comprises the following steps: shredding selected clean silkworm cocoon, adding 0.02mol/L Na₂CO₃Boiling the solution in a water bath at 95 ℃ for 2 times, each time for at least 30min, 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, maintaining the temperature of the reaction system at 45 ℃, and reacting for 1.5h, wherein the concentration of the LiBr solution is 9.3mol/L, and the mass of the silk fibroin and the LiBr solution is as follows: the volume ratio is 1: 10;

after the reaction is finished, dialyzing with a 7-14 kDa cut-off amount to remove impurities, dialyzing for 3.5 days, freezing at-80 ℃ overnight, and freeze-drying at-50 ℃ for 3 days to obtain pure SF;

step two: 1g HPMC (400 mPas) was dissolved in 100mL anhydrous N, N-Dimethylformamide (DMF) with constant stirring. After dissolving HPMC, 60. mu.L (corresponding to 0.375mmol) of Methacrylic Anhydride (MA) and 0.2g of 4-Dimethylaminopyridine (DMAP) were added to the solution and reacted for 16h at room temperature;

after the reaction in the second step is finished, the mixture is dripped into excessive ether for precipitation, the volume ratio of the mixture to the ether is 1:10, the mixture is centrifuged for 5min at 8000rpm, and the generated precipitate is collected. The collected precipitate was dissolved in DMF and precipitated twice in excess diethyl ether; dissolving the precipitate in deionized water, dialyzing with a cut-off of 7-14 kDa to remove impurities, dialyzing for 3 days, and finally freezing the product at-80 ℃ overnight and freeze-drying at-50 ℃ for 3 days to obtain a pure hydroxypropyl methyl cellulose-methacrylate polymer, which is recorded as HPMC-MA;

step three: dissolving the prepared SF in PBS to prepare 8 wt% solution for later use; dissolving HPMC-MA in PBS to obtain 5 wt% solution, treating SF solution with ultrasonic generator in ice bath (ultrasonic probe diameter is 3mm, power is 200W, ultrasonic time is 4s, pause is 4s, circulation is 8 times), mixing with HPMC-MA solution at volume ratio of 5:8, and adding photoinitiator lithium arylphosphinate (LAP, final concentration is 0.15 wt%);

Example 5

The method comprises the following steps: shredding selected clean silkworm cocoon, adding 0.02mol/L Na₂CO₃Boiling the solution in a water bath at 95 ℃ for 2 times, each time for at least 30min, 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, maintaining the temperature of the reaction system at 40 ℃, and reacting for 1h, wherein the concentration of the LiBr solution is 9.3mol/L, and the mass of the silk fibroin and the LiBr solution is as follows: the volume ratio of the components is 1:10,

after the reaction is finished, dialyzing with a 7-14 kDa cut-off amount to remove impurities, dialyzing for 3 days, freezing at minus 80 ℃ overnight, and freeze-drying at minus 50 ℃ for 3 days to obtain pure SF;

step two: 1g HPMC (400 mPas) was dissolved in 100mL anhydrous N, N-Dimethylformamide (DMF) with constant stirring. After dissolution of HPMC, 60. mu.L (corresponding to 0.375mmol) of Methacrylic Anhydride (MA) and 0.2g of 4-Dimethylaminopyridine (DMAP) were added to the solution and reacted at room temperature for 16 hours,

step three: dissolving the prepared SF in PBS to prepare 8 wt% solution for later use; HPMC-MA was dissolved in PBS to make a 5 wt% solution for further use. Treating SF solution with ultrasonic generator (ultrasonic probe diameter of 3mm, power of 240W, ultrasonic time of 5s, pause for 5s, circulation for 10 times) in ice bath, mixing with HPMC-MA solution at a volume ratio of 24:5, adding photoinitiator lithium arylphosphinate (LAP, final concentration of 0.15 wt%),

The above steps one to three 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 150-250 microns.

Performance test 2

Dissolving the SF into PBS to form a solution with the concentration of 8 wt%, then carrying out ultrasonic treatment on the SF solution in an ice bath (the diameter of an ultrasonic probe is 3mm, the power is 240w, the ultrasonic time is 5s, pausing is 5s, circulating is carried out for 6 times), adding HPMC-MA solution and photoinitiator LAP which are prepared in advance after the treatment is finished, wherein the final solution contains 3 wt% SF, 1 wt% HPMC-MA and 0.15 wt% LAP, uniformly mixing the solutions, transferring the mixed solution to a mold, and using the strength of 2.5mW/cm²Irradiating the hydrogel for 8s by using light with the wavelength of 365nm, primarily curing and forming, and then placing the hydrogel at 37 ℃ for 30min to obtain the double-network hydrogel.

The hydrogel was then subjected to rheological testing. The hydrogel was formed into a cylinder having a diameter of 8mm and a height of 4mm using a mold, and tested by performing a frequency sweep pattern (0.1-10 Hz, 1% strain) of a parallel plate having a diameter of 8 mm. From the rheological results, FIG. 3a shows that G ' > G ' is in a linear relationship, indicating that the gel state has been achieved, and that G ' is around 20 kPa.

The double-network hydrogel material testing machine is used for testing the compression performance of the double-network hydrogel obtained in the embodiment, and a uniaxial compression testing method is adopted. The hydrogel was formed into a cylindrical shape with a diameter of 8mm and a height of 4mm by a mold. Each hydrogel was compressed to 50% of its original height at a rate of 1mm/min, and subjected to 3 compression cycles. Compressive properties results as shown in figure 3b, it can be seen that the compressive modulus of the hydrogel was about 250kPa when the hydrogel was cycled to 50% compression.

The tensile properties of the double-network hydrogel obtained in this example were measured using the above double-network hydrogel material testing machine. The hydrogel is made into cuboid strips with the length of 15mm, the thickness of 2mm and the width of 5mm by using a mould. The distance between the clamps was measured to be 10mm and the stretching rate was fixed to be 10mm/min, and as a result, as shown in FIG. 3c, it can be seen that the breaking strength in the stretching was about 22kPa and the tensile modulus was about 60 kPa.

Performance test three

Dissolving the SF into PBS to form a solution with the concentration of 8 wt%, then carrying out ultrasonic treatment on the SF solution in an ice bath (the diameter of an ultrasonic probe is 3mm, the power is 240w, the ultrasonic time is 5s, pausing is 5s, and circulating is carried out for 6 times), adding HPMC-MA solution prepared in advance and photoinitiator LAP after the treatment is finished, and finally, the solution contains 3 wt% of SF, 1 wt% of HPMC-MA and 0.15 wt% of LAP. The solution is mixed evenly and transferred to a mould with the intensity of 2.5mW/cm²Irradiating the hydrogel for 8s by using light with the wavelength of 365nm, primarily curing and forming, and then placing the hydrogel at 37 ℃ for 30min to obtain the double-network hydrogel. The hydrogel was frozen in liquid nitrogen and lyophilized for 2 days. The lyophilized product was formed into a thin film, focused using a RENISHAW inVia raman microscope, excited with a helium neon laser (785nm, 10mW) and raman spectral data recorded. As a result, as shown in FIG. 4, it was observed that the hydrogel had a characteristic peak of 1665cm in the amide I region^-1Amide III region with characteristic peak 1230cm^-1And 1275cm^-1And the three peaks all belong to characteristic peaks of beta-sheet, which shows that the main conformation of SF in the double-network hydrogel is a beta-sheet structure.

Performance test four

Dissolving the SF into PBS to form a solution with the concentration of 8 wt%, then carrying out ultrasonic treatment on the SF solution in an ice bath (the diameter of an ultrasonic probe is 3mm, the power is 240w, the ultrasonic time is 5s, pausing is 5s, and circulating is carried out for 6 times), adding HPMC-MA solution and photoinitiator LAP which are prepared in advance after the treatment is finished, and finally obtaining the biological ink containing 3 wt% SF, 1 wt% HPMC-MA and 0.15 wt% LAP;

the modeling was performed using CAD software and the printing experiments were performed on bio-ink using a 3D bio-printer BioScaffolder 3.2 from GESIM, germany. A4-channel LED driver from THORLABS, USA, was equipped to select a 365nm light source with 0.5mW output (100 mA selected for output current and 5V rated for output voltage) and 8cm spacing between the light source and the pre-polymerization solution. The actual light source has a focal diameter of about 0.5cm and the power of the light source for curing the stent is about 2.5mW/cm assuming no loss of output light²The light irradiation time of each point in each layer of the printing is set to be less than 8s so as to prevent the cell from being excessively damaged by the UV light when the cell is mixed in later period. A nozzle having an inner diameter of 160 μm was provided, and the length of the nozzle was fixed to 13 mm. After the parameters are determined, the extrusion flow of the bio-ink is controlled by adjusting the pneumatic pressure, and the pneumatic pressure is selected to be 30-80 kPa in consideration of the viscosity of the bio-ink and the possibility of cell damage in the extrusion process. The width of the printed lines is controlled by the printing speed in addition to the extrusion flow control, the whole size of the model is controlled by selecting 15mm/s and 0.5mm for the line spacing, and the 3D printing effect graph is shown in figures 5 a-5D. It can be observed that the error of the whole size is not more than 1mm, the error rate is not more than 10%, and the printing property is better.

Performance test five

Dissolving the SF into PBS to form a solution with the concentration of 8 wt%, then carrying out ultrasonic treatment on the SF solution in an ice bath (the diameter of an ultrasonic probe is 3mm, the power is 240w, the ultrasonic time is 5s, pausing is 5s, and circulating is carried out for 6 times), adding HPMC-MA solution and photoinitiator LAP which are prepared in advance after the treatment is finished, and finally obtaining the bio-ink containing 3 wt% SF, 1 wt% HPMC-MA and 0.15 wt% LAP. 200 ten thousand mesenchymal stem cells per ml of bio-ink was added, and printing was performed using the bio-ink containing stem cells under the above conditions. The printed cell scaffolds were cultured in complete medium for 10 days, with medium changes every two days. The proliferation of cells in the scaffolds was assessed by the WST-1 method: the printed scaffolds were crushed, incubated for 3 hours with WST-1 reagent at a concentration of 10% v/v, and the absorbance at 450nm was read using a microplate reader for comparison of cell viability. Cytotoxicity was assessed by live/dead staining and samples were imaged and observed using a laser confocal microscope. As shown in fig. 6a and 6b, it can be observed that the mesenchymal stem cells proliferate slowly in the early stage and rapidly accelerate after 7 days, and there is an inevitable early cell death phenomenon due to the irradiation with 365nm light, but the dead cells decrease significantly with the increase of the number of culture days, the proportion of dead cells is less than 15% in 7 days and less than 3% in 10 days. At the same time, the number of viable cells proliferated, especially on day 10. The high proliferation rate of the cells is promoted by the good biocompatibility of the biological ink, and the good cell microenvironment for the bone marrow mesenchymal stem cells is further proved.

Performance test six

Dissolving the SF into PBS to form a solution with the concentration of 8 wt%, then carrying out ultrasonic treatment on the SF solution in an ice bath (the diameter of an ultrasonic probe is 3mm, the power is 240w, the ultrasonic time is 5s, pausing is 5s, and circulating is carried out for 6 times), adding HPMC-MA solution and photoinitiator LAP which are prepared in advance after the treatment is finished, and finally obtaining the bio-ink containing 3 wt% SF, 1 wt% HPMC-MA and 0.15 wt% LAP. 1000 ten thousand mesenchymal stem cells per ml of bio-ink was added, and printing was performed using the bio-ink containing stem cells under the above conditions.

After printing was completed, the hydrogel containing stem cells was cultured in complete medium (containing 10 v/v% medium fetal bovine serum, 1 v/v% cyan/streptomycin, and DMEM/F12 medium) and induction medium (containing 10 v/v% fetal bovine serum, 1 v/v% cyan/streptomycin, 10ng/mL transforming growth factor-beta 1, insulin-transferrin-selenium supplement, 1mM sodium pyruvate, 100nM dexamethasone sodium phosphate, 50. mu.g/mL ascorbic acid, 2mM L-glutamic acid, 40. mu.g/mL hydroxyproline, and DMEM/F12 medium), respectively. RT-PCR is used for detecting the expression level of mRNA of the cartilage generation related gene to judge whether stem cells are differentiated: placing the cell-loaded hydrogel in 5% CO₂Culturing in 37 deg.C incubator, changing fresh culture medium every other day, culturing for 14 days, discarding culture medium, washing with PBS 3 times, and culturing at each time pointTotal cellular RNA was extracted from the double-network hydrogel by TRIzol Plus RNA purification kit. RNA purity was assessed using A260/280 nm. Thereafter, 500ng of RNA was reverse transcribed into cDNA using PrimeScriptTM RT kit. Using SYBR Green I PCR kit RT-PCR detection, selecting three markers: aggrecan (Agg), type II collagen (Col II), high mobility grouping gene 9(SOX 9). Cells cultured in medium that did not undergo induced differentiation in the culture dish were set as calibrator controls and target gene expression was normalized by non-regulated reference gene expression (β -actin). As a result, as shown in FIGS. 7a to 7c, the expression levels of all the markers showed a gradual increase. The expression of each marker gene can be effectively promoted when the double-network hydrogel obtained in the embodiment is co-cultured, namely, the biocompatibility and the capability of promoting cartilage repair of the bio-ink are very excellent.

Performance test seven

The SF solution was dissolved in PBS to form a solution with a concentration of 8 wt%, and then the SF solution was sonicated in an ice bath (sonication probe diameter 3mm, power 240w, sonication time 5s, pause 5s, cycle 6 times). After the treatment, the pre-prepared HPMC-MA solution and photoinitiator LAP are added, and the final solution contains 3 wt% SF, 1 wt% HPMC-MA and 0.15 wt% LAP of biological ink. 1000 ten thousand mesenchymal stem cells per ml of bio-ink was added, and printing was performed using the bio-ink containing stem cells under the above conditions. After printing, the hydrogel containing the stem cells is placed in an induction medium for culturing for 14 days, dehydrated by 10%, 30%, 50%, 70%, 90% and 100% ethanol in sequence, then freeze-dried, and observed for cell morphology by a scanning electron microscope in a field emission environment. As shown in fig. 8, after 2 weeks of hydrogel induced differentiation culture, the mesenchymal stem cells were rounded, showing good state and differentiation tendency of the stem cells, fully demonstrating the good biocompatibility of the bio-ink.

Comparative example 1

In general, pure SF is used to form high strength hydrogels by means of organic chemical reagents, but the organic reagents are cytotoxic and not conducive to cell encapsulation, thereby limiting their application in biomedicine.

Compared with the comparative example 1, the biological ink obtained in the embodiment of the invention adopts simple physical ultrasound to induce SF to form beta-sheet and further solidify to form high-strength hydrogel, compared with the method, the hydrogel formed by physical ultrasound crosslinking has wider biological application, for example, the biological ink can be mixed with stem cells for printing, and the biological ink is easier for stem cell loading compared with other three-dimensional scaffold materials.

Comparative example 2

Generally, the speed of inducing SF to form beta-sheet by pure SF and further solidifying to form high-strength hydrogel is low, the gelling time is 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 internal pore diameter of the hydrogel formed by the pure SF 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 embodiment of the invention slowly forms a beta-folded structure, namely a layer of network, by inducing SF, forms another layer of network by utilizing the broad-light crosslinking reaction of HPMC-MA, the performance of the hydrogel is improved to meet the requirement of cartilage, the formed double-network hydrogel has stronger mechanical property, more suitable aperture and three-dimensional microenvironment and biological application, for example, the invention combines the advantages of single-network hydrogel to construct the double-network hydrogel which can be rapidly cured and has stronger mechanical property, and realizes blending printing with stem cells, and compared with other three-dimensional scaffold materials, the crosslinking speed is higher, and the performance is better.

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.

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.

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.

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.

Claims

1. A preparation method of a double-network hydrogel is characterized by comprising the following steps:

(1) providing silk fibroin;

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 40-50 ℃ for 1-2 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 3-4 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 (2) reacting a second mixed reaction system containing hydroxypropyl methyl cellulose, methacrylic anhydride and 4-dimethylaminopyridine at the temperature of 10-50 ℃ for 12-30 h to obtain the hydroxypropyl methyl cellulose-methacrylate polymer.

6. The method of claim 5, wherein step (2) further comprises: after the reaction is finished, carrying out precipitation, purification, centrifugation, dialysis and freeze drying treatment on the obtained reaction mixture to obtain the hydroxypropyl methyl cellulose-methacrylate polymer; preferably, the precipitating agent adopted in the precipitation treatment comprises diethyl ether and/or acetone;

and or the mass ratio of the hydroxypropyl methyl cellulose, the methacrylic anhydride and the 4-dimethylaminopyridine is 1 (0.06-0.2) to (0.06-0.2).

7. The method according to claim 1 or 5, wherein the hydroxypropyl methylcellulose-methacrylate polymer has a structural formula represented by formula (I):

wherein R is selected from H, CH₃、

N is 80 to 100, and m is 1 to 3.

8. The method according to claim 1, wherein the step (3) specifically comprises:

9. The method of claim 8, wherein: the concentration of silk fibroin in the silk fibroin solution is 5-15 w/v%; preferably, the ultrasonic treatment conditions are as follows: 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; the concentration of the hydroxypropyl methyl cellulose-methacrylate polymer used for mixing is 1-5 w/v%; the concentration of the added photoinitiator is 0.15-1 w/v%; preferably, the photoinitiator comprises lithium aryl phosphinate; preferably, the wavelength used in the photopolymerization reaction is 350-410 nm, and the illumination time is 5-10s, and particularly preferably 5-8 s.

10. The double-network hydrogel prepared by the method of any one of claims 1 to 9, which has a compressive modulus of 20 to 80KPa and a porous structure, wherein pores contained therein have a pore diameter of 100 to 300 μm, preferably 150 to 250 μm.

11. A preparation method of 3D printing biological ink of double-network hydrogel is characterized by comprising the following steps:

providing silk fibroin and a hydroxypropyl methylcellulose-methacrylate polymer of claim 1;

12. The method according to claim 11, characterized by comprising:

13. The method of manufacturing according to claim 12, wherein: the concentration of silk fibroin in the silk fibroin solution is 5-15 w/v%; preferably, the ultrasonic treatment conditions are as follows: 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; the concentration of the hydroxypropyl methyl cellulose-methacrylate polymer used for mixing is 1-5 w/v%; the concentration of the added photoinitiator is 0.15-1 w/v%; preferably, the photoinitiator comprises lithium arylphosphinate.

14. The production method according to claim 11 or 12, characterized in that: 3D printing is carried out on the 3D printing biological ink obtained after ultrasonic treatment within 20-60 min; preferably 30-40 min.

15. The method of claim 11, further comprising: mixing the silk fibroin solution subjected to ultrasonic treatment with hydroxypropyl methyl cellulose-methacrylate polymer, a photoinitiator, growth factors and cells to obtain the 3D printing biological ink of the double-network hydrogel; preferably, the cells comprise stem cells, particularly preferably bone marrow mesenchymal stem cells; preferably, the growth factor comprises a chondroprotective growth factor.

16. 3D printing bio-ink of a double network hydrogel prepared by the method of any one of claims 11-15.

17. A3D printing preparation method of double-network hydrogel is characterized by comprising the following steps:

providing a 3D printed bio-ink of the double-network hydrogel of claim 16;

18. The 3D printing preparation method according to claim 17, wherein the process conditions adopted by the nozzle extrusion 3D printing method include: the inner diameter of the printing nozzle is 150-250 mu m, the pneumatic pressure is 50-80 kPa, the printing speed is 15-25 mm/s, and the distance between printing lines is 0.5-1.5 mm.

19. The 3D printing preparation method according to claim 17, wherein the printing is performed by a printerThe wavelength of light for illumination treatment is 350-410 nm, and the illumination intensity is 1.5-2.5 mW/cm²The irradiation time of each layer of lines for 3D printing is 5-10s, preferably 5-8 s.

20. The double-network hydrogel prepared by the 3D printing method according to any one of claims 17 to 19, which has a compressive modulus of 20 to 80KPa and a porous structure, wherein pores contained in the double-network hydrogel have a pore diameter of 100 to 300 μm, preferably 150 to 250 μm.

21. Use of the double-network hydrogel of claim 10 or 20 in the field of cell culture or tissue engineering.

22. A three-dimensional cell culture support comprising the double-network hydrogel of claim 10 or 20.

23. A cell culture method, comprising:

culturing cells by using the double-network hydrogel of claim 10 or 20 as a three-dimensional cell culture carrier, and promoting the cells to proliferate and differentiate; preferably, the cell is a mesenchymal stem cell; preferably, the load capacity of the cells on the double-network hydrogel is 100-1000 ten thousand/mL; preferably, the cell culture method comprises: culturing cells by using the double-network hydrogel of claim 20 as a three-dimensional cell culture carrier, and culturing for 7-28 days in a cartilage induced differentiation medium.