WO2025015691A1 - Silk fibroin-based ink and use thereof in preparation of tissue engineering scaffold by 3d printing - Google Patents

Abstract

The present invention relates to the technical field of three-dimensional (3D) printing materials, and relates to silk fibroin-based ink and a use thereof in preparation of a tissue engineering scaffold by 3D printing. A small-molecule plasticizer and a mechanical property regulator are added into a silk fibroin aqueous solution; and the concentration of the silk fibroin aqueous solution is adjusted to obtain silk fibroin-based ink. The silk fibroin-based ink is used as a raw material, and an extrusion 3D printer is used to perform 3D printing on a freezing platform to obtain a 3D scaffold; and low-temperature freezing is performed on the obtained 3D scaffold, so that silk fibroin is self-assembled to obtain a tissue engineering scaffold. Compared with the prior art, the method for preparing a tissue engineering scaffold of the present invention uses a mode in which the silk fibroin is self-assembled to generate a β-sheet, so that the tissue engineering scaffold is solidified and qualitative, and a chemical cross-linking agent or other thickening agents do not need to be added, thereby ensuring the biocompatibility of the scaffold. By regulating the content of the plasticizer and the content of the mechanical property regulator in the ink, the mechanical properties of the silk fibroin 3D scaffold can be accurately regulated, meeting the mechanical requirements of various tissue engineering scaffolds.

Description

Silk protein-based ink and its application in 3D printing of tissue engineering scaffolds Technical Field

The present invention belongs to the technical field of 3D printing materials, and in particular relates to silk protein-based ink and its application in 3D printing to prepare tissue engineering scaffolds.

Background Art

In recent years, three-dimensional additive manufacturing (also known as 3D printing) technology has developed rapidly and has become one of the important means of manufacturing materials with complex three-dimensional structures. 3D printing technology can be used to construct biomimetic biomaterials with complex structures for application in the biomedical field, such as artificial blood vessels, stents, organ chips, etc. In addition, by regulating the printing ink and printing parameters, precise control of the material structure and performance can be achieved. In the field of tissue engineering scaffolds, 3D printing can be used to prepare tissue engineering scaffolds for different clinical applications. Currently, commonly used 3D printing materials include gelatin, alginate, hyaluronic acid, collagen, polylactic acid, etc. However, these materials have certain defects, such as poor bioactivity and easy degradation of gelatin, harsh printing conditions and low mechanical strength of collagen, and acidic degradation of polylactic acid. Ideal 3D printing materials must not only meet the requirements of biocompatibility, bioactivity, and mechanical strength matching the target tissue required by tissue engineering scaffolds, but also have excellent printability.

Silk protein is a structural protein extracted from natural silk. It has excellent biocompatibility and biodegradability. Its degradation products are harmless to the human body and can be easily metabolized. In addition, silk protein has strong processability and can be prepared into films, foams, gels and other forms. Therefore, it has been widely used in the biomedical field. The aqueous regenerated silk protein solution has the property of shear thinning, that is, its viscosity decreases with the increase of shear stress, which can reduce the driving force required for printing, thereby improving the printing accuracy. At the same time, post-processing of the scaffold obtained by 3D printing can regulate the secondary structure of silk protein, thereby accurately regulating the mechanical properties and degradation time of the scaffold so that it can match the time and space requirements of different tissue repair processes.

Existing silk protein-based inks usually need to be mixed with other thickeners to improve their printability, or the silk protein molecular chains need to be modified and formed through chemical cross-linking.

For example, Chinese patent CN116118177A discloses a 3D printed hydrogel scaffold based on high molecular weight regenerated silk protein and its preparation method. A thixotropic hydrogel is obtained by mixing and heating a high molecular weight regenerated silk protein (HMWRSF) solution, a hydroxypropyl methylcellulose (HPMC) solution and urea (Urea), and the thixotropic hydrogel can be directly used for 3D printing; the 3D printed HMWRSF/HPMC/Urea hydrogel is ripened with ethanol and the solvent is replaced with deionized water to obtain a 3D printed HMWRSF/HPMC hydrogel scaffold. This patent is a HMWRSF/HPMC blending system.

Chinese patent CN109666302A discloses a method for preparing 3D printed silk protein hydrogel, comprising the following steps: preparing pre-crosslinked silk protein hydrogel and 3D printed silk protein hydrogel, adding double-bond modified cyclodextrin and photoinitiator to the silk protein solution, stirring to obtain single network crosslinked silk protein hydrogel, irradiating with ultraviolet light to form pre-crosslinked silk protein hydrogel, and then crosslinking the cyclodextrin in the fiber and the tyramine root on the molecular chain of the silk protein again by 3D printing, stacking layer by layer to form 3D printed silk protein hydrogel, and also providing 3D printed silk protein hydrogel. The patent uses the host-guest interaction between cyclodextrin and the tyramine root on the silk protein molecular chain to construct a three-dimensional network structure of the hydrogel.

However, these methods will reduce the mechanical strength of silk protein on the one hand, and chemical cross-linking is not conducive to the encapsulation of bioactive substances or cells on the other hand. Therefore, it is urgent to develop a new type of silk protein-based ink that does not require the addition of thickeners and is non-chemically cross-linked for the preparation of tissue engineering scaffolds.

Summary of the invention

Based on the current situation that the prior art lacks silk protein-based 3D printing ink that does not require the addition of a thickener and is non-chemically cross-linked, the present invention provides a silk protein-based ink and its application in 3D printing to prepare tissue engineering scaffolds.

The present invention first provides a novel silk protein-based ink, wherein a small molecule plasticizer and a mechanical property regulator are added to give the silk protein-based ink excellent printability and excellent mechanical properties of a 3D printed tissue engineering scaffold.

The freezing platform can make the silk protein-based ink undergo sol-gel transformation to maintain its morphology, and the small molecule plasticizer can interact with the hydroxyl groups in the silk protein molecular chain through hydrogen bonds, thereby weakening the interaction force between the silk protein molecular chains, thereby improving the mobility of the silk protein molecular chains, and enabling the silk protein-based scaffold to undergo conformational transformation at low temperatures to generate β-folds to stabilize the scaffold morphology. Since the mechanical properties and biodegradability of the scaffold are related to the β-fold content and crystallinity of the silk protein, generally the more β-folds, the higher the crystallinity, the better the mechanical properties of the scaffold, and the longer its degradation time, the present invention can regulate the mechanical properties and biodegradability of the silk protein-based scaffold by adding a mechanical property regulator, thereby expanding its application in the field of tissue engineering.

Based on the silk protein-based ink provided by the present invention, a silk protein-based tissue engineering scaffold can be prepared by 3D printing. The preparation of the silk protein-based tissue engineering scaffold does not require the addition of a thickener (generally referring to a substance that increases the viscosity of the printing ink, such as hydroxypropyl cellulose in Chinese patent CN116118177A or cyclodextrin in Chinese patent CN109666302A, which is a polymer with a large molecular weight) or chemical cross-linking, and can maintain the excellent mechanical properties and biocompatibility of silk protein, while providing the possibility for the loading of bioactive substances, drugs and cells.

One of the objects of the present invention is to provide a silk protein-based ink for 3D printing.

Another object of the present invention is to provide a tissue engineering scaffold based on silk protein-based ink 3D printing.

Another object of the present invention is to provide an application of a tissue engineering scaffold based on silk protein-based ink 3D printing.

The purpose of the present invention can be achieved by the following technical solutions:

The present invention first provides a method for preparing a silk protein-based ink, the method comprising the following steps:

(1) adding a small molecule plasticizer and a mechanical property regulator to the silk protein aqueous solution; and

(2) adjusting the concentration of the silk protein solution to obtain a silk protein-based ink;

Wherein, the small molecule plasticizer is a polyol, and the mechanical property regulator is selected from inorganic compounds of metal ions.

In one embodiment of the present invention, the small molecule plasticizer is selected from one or a combination of ethylene glycol, propylene glycol, glycerol, sorbitol, erythritol, sorbitol, and erythritol.

In one embodiment of the present invention, the mechanical property regulator is selected from one or a combination of calcium chloride, hydroxyapatite, lithium chloride or lithium bromide.

The small molecule plasticizers selected in the present invention are all polyols, and the hydroxyl groups in their structures can interact with the hydroxyl groups on the silk protein molecular chain through hydrogen bonds, thereby weakening the intermolecular interaction force of the silk protein molecular chain, thereby improving the mobility of the silk protein molecular chain and playing a plasticizing role. The small molecule plasticizers in the present invention are different from the thickeners used in the prior art to increase the viscosity of the viscosity printing ink.

The mechanical property regulators selected in the present invention contain metal ions such as calcium ions and lithium ions, which can chelate with the silk protein molecular chains to destroy the hydrogen bond network between the silk protein molecules and thus regulate the mechanical properties of the silk protein scaffold.

In some embodiments of the present invention, the silk protein in step (1) is prepared by the following steps:

(a): adding silkworm cocoons to an aqueous solution of sodium carbonate, heating and boiling for 30-120 minutes, removing sericin, rinsing with water for multiple times to remove sodium carbonate, and then drying at room temperature to obtain degummed silk;

(b): Add the degummed silk into an aqueous solution of lithium bromide, heat it, and after the silk is dissolved, dialyze the solution to obtain silk protein.

In some embodiments of the present invention, in step (1), the mass ratio of the silk protein in the silk protein aqueous solution to the small molecule plasticizer is 100:0.1 to 100:50, preferably 100:1 to 100:30.

In some embodiments of the present invention, in step (1), the mass ratio of the silk protein in the silk protein aqueous solution to the mechanical property modifier is 100:1 to 100:10, preferably 100:2 to 100:5.

In some embodiments of the present invention, in step (2), bioactive substances, drugs or cells may be added to the silk protein solution.

In some embodiments of the present invention, in step (2), bioactive substances such as HRP, BSA, BMP-2, bFGF, decellularized matrix, etc. are added to the silk protein solution.

In some embodiments of the present invention, in step (2), before adjusting the concentration of the silk protein solution, suspended cells are added to the silk protein solution.

In some embodiments of the present invention, the suspension cells are selected from stem cells, fibroblasts, osteoblasts, and the like.

In some embodiments of the present invention, in step (2), the concentration of the silk protein solution is adjusted by adding water to obtain a diluent.

In some embodiments of the present invention, in step (2), the method for adjusting the concentration of the silk protein solution is: obtaining a concentrated solution by natural air drying.

In some embodiments of the present invention, in step (2), the method for adjusting the concentration of the silk protein solution is: obtaining a concentrated solution by reverse dialysis.

In some embodiments of the present invention, in step (2), the method for adjusting the concentration of the silk protein solution is: obtaining a concentrated solution by vacuum centrifugation.

The present invention further provides silk protein-based ink obtained based on the above preparation method.

The present invention further provides an application of the silk protein-based ink, wherein the silk protein-based ink is used for 3D printing to obtain a tissue engineering scaffold.

The present invention further provides a method for preparing a tissue engineering scaffold, using the silk protein-based ink as a raw material and obtaining a tissue engineering scaffold by a freezing 3D printing method, the method comprising the following steps:

(S1) using the silk protein-based ink as a raw material, and performing 3D printing on a freezing platform using an extrusion 3D printer to obtain a three-dimensional scaffold;

(S2) cryogenically freezing or vacuum freeze-drying the three-dimensional scaffold obtained in step (S1) to allow the silk protein to self-assemble to obtain a tissue engineering scaffold.

In some embodiments of the present invention, in step (S1), the concentration of silk protein in the silk protein-based ink is 1 wt% to 40 wt%.

In some embodiments of the present invention, in step (S1), the temperature of the freezing platform used is 0°C to -25°C.

In some embodiments of the present invention, in step (S1), the printing needle used in the extrusion 3D printer is a dispensing needle of 18G to 34G.

In some embodiments of the present invention, in step (S2), the conditions for low-temperature freezing are: low-temperature freezing is performed at a freezing temperature of 0°C to -25°C for at least 6 hours.

In some embodiments of the present invention, in step (S2), the conditions for vacuum freeze drying are: transferring the obtained three-dimensional scaffold to liquid nitrogen, freezing for one hour, and then transferring to a vacuum freeze dryer for freeze drying.

The present invention further provides a tissue engineering scaffold prepared based on the above method.

The present invention further provides the application of the tissue engineering scaffold prepared based on the above method, wherein the tissue engineering scaffold is used for cell culture and tissue regeneration.

Compared with the prior art, the beneficial effects of the present invention are embodied in the following aspects:

(1) The silk protein required by the scheme of the present invention is directly extracted from silkworm cocoons, which has a wide source, low price, and high economic benefits; the added plasticizer and mechanical property regulator are common chemical raw materials, which are cheap and non-biotoxic, and do not affect the biocompatibility of silk protein, and meet the basic requirements of tissue engineering scaffolds.

(2) The silk protein-based ink of the present invention has a simple preparation method, good biocompatibility, low viscosity, good fluidity, and can be applied to different 3D printing methods, such as extrusion or inkjet. It has strong printability and fast curing, and can be used to prepare tissue engineering scaffolds with complex structures.

(3) The present invention uses the silk protein-based ink as a raw material, uses an extrusion-type 3D printer to perform 3D printing on a freezing platform to obtain a three-dimensional scaffold; and then freezes the obtained three-dimensional scaffold at low temperature to allow the silk protein to self-assemble to obtain a tissue engineering scaffold. Low temperature enables the ink to undergo a sol-gel transition (refer to FIG. 2) to generate a three-dimensional scaffold. However, if the silk protein-based ink does not contain other additives, the generated three-dimensional scaffold will melt once it is out of the freezing environment. To solve this technical problem, the present invention adds a small molecule plasticizer to the silk protein-based ink, so that the silk protein can self-assemble to generate β-folds in a freezing environment, so that the scaffold does not dissolve even if it is out of the freezing environment, and the solidification of the tissue engineering scaffold is maintained.

(4) The present invention does not need to add chemical crosslinking agents or other thickeners (thickeners refer to substances that increase the viscosity of printing inks, such as hydroxypropyl cellulose in Chinese patent CN116118177A or cyclodextrin in Chinese patent CN109666302A, which are polymers with relatively large molecular weights), while the small molecule plasticizer in the present invention is a small molecule compound whose purpose is to promote the movement of silk protein molecular chains rather than to increase viscosity, thereby ensuring the biocompatibility of the scaffold. By regulating the content of plasticizers and mechanical property regulators in the ink, the mechanical properties of the silk protein three-dimensional scaffold can be precisely regulated to meet the mechanical requirements of different tissue engineering scaffolds.

(5) The scheme of the present invention can add bioactive substances to the silk protein solution, and the low-temperature 3D printing environment and frozen storage can effectively maintain the biological activity of these bioactive substances.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of the silk protein-based ink prepared in Example 1 of the present invention.

FIG. 2 is a photograph of a tissue engineering scaffold obtained by freeze-printing silk protein-based ink in Example 1 of the present invention.

FIG. 3 is a graph showing the rheological properties of the silk protein-based ink prepared in an embodiment of the present invention at different temperatures.

FIG. 4 is a photograph of the silk protein-based tissue engineering scaffolds prepared in Example 1 of the present invention and the comparative example and a photograph of the two scaffolds immersed in water for 24 hours.

FIG5 is a graph showing the mechanical properties of the silk protein-based tissue engineering scaffolds prepared in Example 1 and Example 5 of the present invention, wherein A represents the tensile strength and elongation at break, and B represents the Young's modulus (dry state).

FIG6 is a characterization of the cell compatibility of the silk protein-based tissue engineering scaffolds prepared in Example 1 and Example 5 of the present invention.

FIG. 7 is a fluorescence image of cytoskeleton staining of cells adherent to the surface of the silk protein-based tissue engineering scaffold prepared in Example 5 of the present invention.

FIG. 8 shows the reaction process between the silk protein-based tissue engineering scaffold prepared in Example 8 of the present invention and TMP.

FIG. 9 is a photograph of the silk protein-based tissue engineering scaffolds prepared in Example 12 and Example 13 of the present invention.

DETAILED DESCRIPTION

The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

Example 1

This embodiment provides a method for preparing a silk protein-based tissue engineering scaffold, comprising the following steps:

(1) Prepare a sodium carbonate aqueous solution with a concentration of 0.02 mol/L, heat it to boiling, add the chopped cocoons into the boiling sodium carbonate aqueous solution and continue boiling for 30 minutes to remove the sericin. Rinse the degummed silk in clean water to remove the sodium carbonate, and air-dry it at room temperature to obtain degummed silk. Prepare a lithium bromide solution with a concentration of 9.3 mol/L, add the degummed silk, keep it at 60°C for 4 hours to fully dissolve the silk, and then dialyze to obtain a silk protein aqueous solution.

(2) Glycerol was added to the silk protein aqueous solution to obtain a solution with a silk protein:glycerol mass ratio of 100:20, and the solution was concentrated to a silk protein mass fraction of 30 wt % by a vacuum concentrator to obtain a silk protein-based ink (silk protein 3D printing ink).

(3) The silk protein 3D printing ink was loaded into the barrel of the 3D printer, and a mesh scaffold program was written in the printing device. A 27G TT dispensing head, an air pressure of 0.25 MPa, a printing rate of 10 mm/s, a layer height of 0.15 mm, a filling spacing of 0.5 mm, and a freezing platform temperature of -18°C were used for 3D printing to prepare a mesh scaffold. The 3D printed scaffold was frozen and stored at -18°C for 24 hours, and the scaffold was solidified and characterized by the self-assembly of silk protein to generate β-folds.

A photograph of the silk protein-based ink prepared in Example 1 is shown in FIG1 , which shows that the silk protein-based ink prepared in the present invention is clear and transparent and has good uniformity.

A photograph of the tissue engineering scaffold obtained by freeze-printing the silk protein-based ink in Example 1 is shown in FIG2 , indicating that the silk protein-based tissue engineering scaffold obtained by the present invention can maintain a complete morphology without post-treatment.

Example 2

The difference from Example 1 is that:

In step (2), the solution was concentrated to 35 wt % by a vacuum concentrator.

The rest is consistent with Example 1.

Example 3

The difference from Example 1 is that:

In step (2), glycerol is added to the silk protein aqueous solution to obtain a solution with a silk protein:glycerol mass ratio of 100:10.

The rest is consistent with Example 1.

Example 4

The difference from Example 1 is that:

In step (2), glycerol is added to the silk protein aqueous solution to obtain a solution with a silk protein:glycerol mass ratio of 100:30.

The rest is consistent with Example 1.

Example 5

The difference from Example 1 is that:

In step (2), in addition to glycerol, calcium chloride is added to the silk protein aqueous solution to obtain a solution with a mass ratio of silk protein: glycerol: calcium chloride of 100:20:2.

The rest is consistent with Example 1.

Example 6

The difference from Example 1 is that:

In step (2), in addition to glycerol, calcium chloride is added to the silk protein aqueous solution to obtain a solution with a mass ratio of silk protein: glycerol: calcium chloride of 100:20:5.

The rest is consistent with Example 1.

Example 7

The difference from Example 1 is that:

In step (2), in addition to glycerol, calcium chloride is added to the silk protein aqueous solution to obtain a solution with a mass ratio of silk protein: glycerol: calcium chloride of 100:20:10.

The rest is consistent with Example 1.

Example 8

The difference from Example 1 is that:

In step (2), in addition to glycerol, horseradish peroxidase (HRP) is added to the silk protein aqueous solution to obtain a solution with an HRP concentration of 10 u/mL.

The rest is consistent with Example 1.

Example 9

The difference from Example 1 is that:

In step (3), use a 25G TT dispensing tip.

The rest is consistent with Example 1.

Example 10

The difference from Example 1 is that:

In step (3), use a 30G TT dispensing tip.

The rest is consistent with Example 1

Embodiment 11

The difference from Example 1 is that:

In step (3), a cylindrical support program is written in the printing device.

The rest is consistent with Example 1, and a cylindrical bracket is obtained.

Example 12

The difference from Example 1 is that:

In step (3), a nose shape support program is written in the printing device.

The rest is consistent with Example 1, and a nose-shaped stent is obtained.

Embodiment 13

The difference from Example 1 is that:

In step (3), an ear morphology support program is written in a printing device.

The rest is consistent with Example 1, and an ear-shaped bracket is obtained.

Comparative Example 1

In order to contrast with the silk protein-based bio-ink prepared by the present invention, a silk protein scaffold without adding a small molecule plasticizer and a mechanical property regulator was prepared according to the following method:

(1) Prepare a sodium carbonate aqueous solution with a concentration of 0.02 mol/L, heat it to boiling, add the chopped cocoons into the boiling sodium carbonate aqueous solution and continue boiling for 30 minutes to remove the sericin. Rinse the degummed silk in clean water to remove the sodium carbonate, and air-dry it at room temperature to obtain degummed silk. Prepare a lithium bromide solution with a concentration of 9.3 mol/L, add the degummed silk, keep it at 60°C for 4 hours to fully dissolve the silk, and then dialyze to obtain a silk protein aqueous solution.

(2) A certain amount of silk protein aqueous solution is taken and the solution is concentrated by a vacuum concentrator to a solution having a silk protein mass fraction of 30 wt %.

(3) The solution in step (2) was loaded into the barrel of a 3D printer, and a mesh-shaped scaffold program was written in the printing device. A 27G TT dispensing head, an air pressure of 0.25 MPa, a printing rate of 10 mm/s, a layer height of 0.15 mm, a filling spacing of 0.5 mm, and a freezing platform temperature of -18°C were used for 3D printing to prepare a mesh-shaped scaffold. The 3D printed scaffold was frozen at -18°C for 24 hours, and no small molecule plasticizer and mechanical property regulator were added to the silk protein scaffold.

Test Example 1

The silk protein ink prepared in Example 1 was characterized for its rheological properties using a rotational rheometer. The silk protein ink was placed between the fixtures of the rotational rheometer to test the changes in the storage modulus and loss modulus of the silk protein ink with temperature. The rheological properties graph is shown in FIG3 .

Test Example 2

The water stability of the silk protein scaffolds prepared in Example 1 and Comparative Example 1 was tested by immersing in water. The silk protein scaffolds prepared in Example 1 and Comparative Example 1 were immersed in 5 mL of deionized water for 24 hours and then photographed and observed. The photographs are shown in FIG4 .

Test Example 3

The dry mechanical properties of the silk protein scaffolds prepared in Example 1 and Example 5 were tested by a mechanical testing machine equipped with a 50N load cell. The samples were cut into dumbbell-shaped specimens according to the ASTM standard, and then the samples were loaded onto the furniture of the machine. For each test, the stretching rate of all samples was 100% strain/min, and the stretching was stopped until the sample broke, and at least 5 replicates were tested for each group of samples. The cross-sectional area of each sample was calculated by multiplying the thickness by the gauge width. Stress and strain were calculated based on the original cross-sectional area and length, respectively. Young's modulus, elongation at break, and breaking strength were determined by the stress-strain curve. The mechanical property characterization results are shown in Figure 5.

Test Example 4

The cell compatibility of the silk protein scaffolds prepared in Example 1 and Example 5 was characterized by co-culturing with L929 cells and testing the cell activity at specific time points. 2×10 ⁴ cell/well L292 cells were inoculated in a 24-well plate. After the cells adhered to the wall, the samples were added. Each group was equipped with at least 6 duplicate wells. The wells without samples were used as blank controls. They were incubated at 37°C and 5% CO ₂ concentration. The liquid was changed every 2 days. Before all samples were added to the well plate, they were sterilized at high temperature using a high-pressure steam sterilizer. After incubation for 3 and 5 days, the CCK8 kit was used to detect cell viability. The results of cell compatibility are shown in Figure 6.

Test Example 5

The cell adhesion performance of the silk protein scaffold prepared in Example 5 was observed by co-culturing with L929 cells and observing the cell adhesion by fluorescence staining at a specific time point. Samples were added to a 24-well plate, with at least 6 replicates per group, and then 2×10 ⁴ cell/well of L292 cells were inoculated and incubated at 37°C in an atmosphere of 5% CO2 concentration, with the solution changed every 2 days. Before all samples were added to the well plate, they were sterilized at high temperature using a high-pressure steam sterilizer. On the 7th day of incubation, the culture medium was discarded, and a 4% paraformaldehyde solution was added for fixation. After fixation for 2 hours, the fixative was discarded, 0.1% Triton X-100 was added for permeabilization, and the permeabilization solution was discarded after 15 minutes of permeabilization. The phalloidin working solution was added and incubated for two hours to stain the cytoskeleton. The cells adhering to the surface of the scaffold were observed using a laser confocal microscope. The fluorescent staining image of the cytoskeleton is shown in Figure 7.

Test Example 6

The functionality of the silk protein scaffold prepared in Example 8 was tested by reacting with 3,3'5,5'-tetramethylbenzidine (TMB). The silk protein scaffold prepared in Example 8 was added to 1 mL of TMB solution, and a photo was taken to record the color change of the solution. The color change diagram of the solution is shown in FIG8 .

Result analysis:

It can be seen from Figure 3 that in the temperature range of 25°C to -10°C, the loss modulus of the silk protein-based ink is higher than its storage modulus, indicating that the silk protein ink exhibits liquid fluidity at this time. When the temperature reaches -10°C, the storage modulus and loss modulus rise suddenly, and the storage modulus is higher than the loss modulus, indicating that the silk protein ink undergoes a sol-gel transition and exhibits solid properties. This indicates that the silk protein-based ink can solidify rapidly when in contact with the freezing platform, thereby maintaining its shape.

As can be seen from Figure 4, the addition of glycerol makes the structure of the silk protein scaffold more stable and can maintain its shape in water. However, the pure silk scaffold without the addition of plasticizer melts immediately after being frozen and thawed, dissolves in large quantities in water, and cannot maintain its complete shape.

As can be seen from Figure 5, the silk protein scaffold prepared in Example 1 has good mechanical properties, and the addition of calcium chloride significantly improves the tensile strength, elongation at break and Young's modulus of the scaffold, indicating that calcium chloride has the function of regulating the mechanical properties of the silk protein scaffold.

As can be seen from FIG6 , the cell viability of the silk protein scaffolds prepared in Example 1 and Example 5 on the third and fifth days was not significantly different from that of the blank control group, indicating that the silk protein scaffolds have excellent biocompatibility.

As can be seen from FIG. 7 , the silk protein scaffold prepared in Example 5 can support the adhesion and growth of cells.

As can be seen from FIG. 8 , the silk protein scaffold prepared in Example 8 has the ability to catalyze TMB, indicating that the silk protein-based ink and printing method developed in the present invention will not affect the function of the active substance.

The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims (19)

A method for preparing a silk protein-based ink, characterized in that the method comprises the following steps: (1) adding a small molecule plasticizer and a mechanical property regulator to the silk protein aqueous solution; and (2) adjusting the concentration of the silk protein solution to obtain a silk protein-based ink; Wherein, the small molecule plasticizer is a polyol, and the mechanical property regulator is an inorganic compound containing metal ions. The method for preparing a silk protein-based ink according to claim 1, characterized in that the small molecule plasticizer is selected from one or a combination of ethylene glycol, propylene glycol, glycerol, sorbitol, erythritol, sorbitol, and erythritol. The method for preparing a silk protein-based ink according to claim 1, characterized in that the mechanical property regulator is selected from one or a combination of calcium chloride, hydroxyapatite, lithium chloride or lithium bromide. The method for preparing a silk protein-based ink according to claim 1, characterized in that the silk protein in step (1) is prepared by the following steps: (a): adding silkworm cocoons to an aqueous solution of sodium carbonate, heating and boiling for 30-120 minutes, removing sericin, rinsing with water for multiple times to remove sodium carbonate, and then drying at room temperature to obtain degummed silk; (b): Add the degummed silk into an aqueous solution of lithium bromide, heat it, and after the silk is dissolved, dialyze the solution to obtain silk protein. The method for preparing a silk protein-based ink according to claim 1 is characterized in that, in step (1), the mass ratio of the silk protein in the silk protein aqueous solution to the small molecule plasticizer is 100:0.1 to 100:50, preferably 100:1 to 100:30. The method for preparing a silk protein-based ink according to claim 1, characterized in that, in step (1), the mass ratio of the silk protein in the silk protein aqueous solution to the mechanical property regulating agent is 100:1 to 100:10, preferably 100:2 to 100:5. The method for preparing a silk protein-based ink according to claim 1 is characterized in that, in step (2), before adjusting the concentration of the silk protein solution, bioactive substances, drugs or cells are added to the silk protein solution. The method for preparing a silk protein-based ink according to claim 7 is characterized in that the bioactive substance is selected from one or more of HRP, BSA, BMP-2, bFGF or acellular matrix; and the cells are selected from stem cells, fibroblasts or osteoblasts. The method for preparing a silk protein-based ink according to claim 1 is characterized in that the method for adjusting the concentration of the silk protein solution is: obtaining a diluent by adding water, or obtaining a concentrated solution by natural air drying, or obtaining a concentrated solution by reverse dialysis, or obtaining a concentrated solution by vacuum centrifugation. A silk protein-based ink obtained by the preparation method according to any one of claims 1 to 9. The use of the silk protein-based ink according to claim 10 is characterized in that the silk protein-based ink is used for 3D printing to obtain a tissue engineering scaffold. A method for preparing a tissue engineering scaffold, characterized in that the silk protein-based ink according to claim 10 is used as a raw material to obtain a tissue engineering scaffold by a freezing 3D printing method, and the method comprises the following steps: (S1) using the silk protein-based ink as a raw material, and performing 3D printing on a freezing platform using an extrusion 3D printer to obtain a three-dimensional scaffold; (S2) cryogenically freezing or vacuum freeze-drying the three-dimensional scaffold obtained in step (S1) to allow the silk protein to self-assemble to obtain a tissue engineering scaffold. The method for preparing a tissue engineering scaffold according to claim 12, characterized in that, in step (S1), the concentration of silk protein in the silk protein-based ink is 1wt% to 40wt%. The method for preparing a tissue engineering scaffold according to claim 12, characterized in that in step (S1), the temperature of the freezing platform used is 0°C to -25°C. The method for preparing a tissue engineering scaffold according to claim 12, characterized in that in step (S1), the printing needle used in the extrusion 3D printer is a dispensing needle of 18G to 34G. The method for preparing a tissue engineering scaffold according to claim 12 is characterized in that, in step (S2), the conditions for low-temperature freezing are: low-temperature freezing is performed at a freezing temperature of 0°C to -25°C for at least 6 hours. The method for preparing a tissue engineering scaffold according to claim 12 is characterized in that, in step (S2), the conditions for vacuum freeze drying are: transferring the obtained three-dimensional scaffold to liquid nitrogen, freezing for one hour, and then transferring it to a vacuum freeze dryer for freeze drying. A tissue engineering scaffold prepared by the method according to any one of claims 12 to 17. The use of the tissue engineering scaffold according to claim 18 is characterized in that the tissue engineering scaffold is used for cell culture and tissue regeneration.