Silk fibroin-based ink and use thereof in preparation of tissue engineering scaffold by 3D printing

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

[0001] 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

[0002] In recent years, three-dimensional additive manufacturing (also known as 3D printing) technology has rapidly developed and has become a key method for fabricating materials with complex three-dimensional structures. 3D printing technology can be used to construct biomimetic biomaterials with complex structures for biomedical applications, such as artificial blood vessels, stents, and organ-on-chips. Furthermore, by manipulating the printing ink and printing parameters, precise control of the material structure and properties can be achieved. In the field of tissue engineering scaffolds, 3D printing can be used to prepare tissue engineering scaffolds for various clinical applications. Currently, commonly used 3D printing materials include gelatin, alginate, hyaluronic acid, collagen, and polylactic acid. However, these materials all have certain drawbacks. For example, gelatin has poor bioactivity and is easily degraded; collagen requires harsh printing conditions and has low mechanical strength; and polylactic acid degrades into an acidic state. Ideal 3D printing materials must meet the requirements for tissue engineering scaffolds, including biocompatibility, bioactivity, and mechanical strength that matches the target tissue, while also exhibiting excellent printability.

[0003] 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 is highly processable and can be prepared into films, foams, gels, and other forms. Therefore, it has been widely used in the biomedical field. Aqueous regenerated silk protein solutions have the property of shear thinning, that is, their viscosity decreases with increasing shear stress. This can reduce the driving force required for printing, thereby improving printing accuracy. At the same time, post-processing of the 3D-printed scaffolds can regulate the secondary structure of silk protein, thereby precisely controlling the mechanical properties and degradation time of the scaffold to match the time and space requirements of different tissue repair processes.

[0004] Existing silk protein-based inks usually need to be blended with other thickeners to improve their printability, or the silk protein molecular chains need to be modified and shaped through chemical cross-linking.

[0005] For example, Chinese patent CN116118177A discloses a 3D-printed hydrogel scaffold based on high molecular weight regenerated silk protein and its preparation method. A high molecular weight regenerated silk protein (HMWRSF) solution, hydroxypropyl methylcellulose (HPMC) solution, and urea (Urea) are blended and heated to produce a thixotropic hydrogel that can be directly used for 3D printing. The 3D-printed HMWRSF / HPMC / Urea hydrogel is then cured in ethanol and the solvent is replaced with deionized water to produce a 3D-printed HMWRSF / HPMC hydrogel scaffold. This patent describes an HMWRSF / HPMC blend system.

[0006] Chinese patent CN109666302A discloses a method for preparing a 3D-printed silk protein hydrogel, comprising the following steps: preparing a pre-crosslinked silk protein hydrogel and a 3D-printed silk protein hydrogel; adding a double-bond-modified cyclodextrin and a photoinitiator to a silk protein solution; stirring to obtain a single-network crosslinked silk protein hydrogel; irradiating the solution with ultraviolet light to form a pre-crosslinked silk protein hydrogel; and then 3D printing to further crosslink the cyclodextrin in the fiber with the tyramine groups on the silk protein molecular chains, resulting in layer-by-layer stacking to form a 3D-printed silk protein hydrogel. The patent also provides a 3D-printed silk protein hydrogel. This patent utilizes the host-guest interaction between the cyclodextrin and the tyramine groups on the silk protein molecular chains to construct a three-dimensional network structure for the hydrogel.

[0007] However, these methods reduce the mechanical strength of silk fibroin, while chemical crosslinking hinders the encapsulation of bioactive substances or cells. Therefore, there is an urgent need to develop a new silk fibroin-based ink that does not require the addition of thickeners and is non-chemically crosslinked for the preparation of tissue engineering scaffolds.

[0008] Summary of the Invention

[0009] Based on the current situation that the prior art lacks silk protein-based 3D printing ink that does not require the addition of thickeners 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.

[0010] The present invention first provides a novel silk protein-based ink, to which a small molecule plasticizer and a mechanical property regulator are added to give the silk protein-based ink excellent printability and excellent mechanical properties for 3D printed tissue engineering scaffolds.

[0011] The freezing platform allows the silk protein-based ink to undergo a sol-gel transition, thereby maintaining its morphology. The small molecule plasticizer can interact with the hydroxyl groups in the silk protein molecular chains through hydrogen bonds, thereby weakening the interaction between the silk protein molecular chains and improving the mobility of the silk protein molecular chains. This allows the silk protein-based scaffold to undergo a conformational transition at low temperatures to form β-sheets, stabilizing the scaffold's morphology. Since the mechanical properties and biodegradability of the scaffold are related to the β-sheet content and crystallinity of the silk protein, generally, the more β-sheets, the higher the crystallinity, the better the scaffold's mechanical properties, and also its longer degradation time. Therefore, by adding a mechanical property modifier, the present invention can modulate the mechanical properties and biodegradability of the silk protein-based scaffold, thereby expanding its application in the field of tissue engineering.

[0012] 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 refers 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 of loading bioactive substances, drugs and cells.

[0013] One of the purposes of the present invention is to provide a silk protein-based ink for 3D printing.

[0014] Another object of the present invention is to provide a tissue engineering scaffold based on silk protein-based ink 3D printing.

[0015] Another object of the present invention is to provide an application of a tissue engineering scaffold based on 3D printing of silk protein-based ink.

[0016] The purpose of the present invention can be achieved by the following technical solutions:

[0017] The present invention first provides a method for preparing a silk protein-based ink, the method comprising the following steps:

[0018] (1) adding a small molecule plasticizer and a mechanical property regulator to the silk protein aqueous solution; and

[0019] (2) adjusting the concentration of the silk protein solution to obtain a silk protein-based ink;

[0020] Wherein, the small molecule plasticizer is a polyol, and the mechanical property regulator is selected from inorganic compounds of metal ions.

[0021] 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.

[0022] In one embodiment of the present invention, the mechanical property modifier is selected from one or a combination of calcium chloride, hydroxyapatite, lithium chloride or lithium bromide.

[0023] The small molecule plasticizers used in the present invention are all polyols. The hydroxyl groups in their structures can interact with the hydroxyl groups on the silk protein chains through hydrogen bonds, thereby weakening the intermolecular interactions of the silk protein chains, improving the mobility of the silk protein chains 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 printing inks.

[0024] The mechanical property modifiers selected in the present invention contain metal ions such as calcium ions and lithium ions. These ions can chelate with the silk protein molecular chains to destroy the hydrogen bond network between silk protein molecules, thereby regulating the mechanical properties of the silk protein scaffold.

[0025] In some embodiments of the present invention, the silk protein in step (1) is prepared by the following steps:

[0026] (a) Silkworm cocoons are added to an aqueous solution of sodium carbonate and heated to boil for 30-120 minutes to remove sericin. The silkworm cocoons are then rinsed with water multiple times to remove the sodium carbonate and then dried at room temperature to obtain degummed silk.

[0027] (b) The degummed silk is added to an aqueous solution of lithium bromide, heated, and after the silk is dissolved, the solution is dialyzed to obtain silk protein.

[0028] 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.

[0029] 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.

[0030] In some embodiments of the present invention, in step (2), bioactive substances, drugs or cells may be added to the silk protein solution.

[0031] In some embodiments of the present invention, in step (2), bioactive substances such as HRP, BSA, BMP-2, bFGF, acellular matrix, etc. are added to the silk protein solution.

[0032] 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.

[0033] In some embodiments of the present invention, the suspension cells are selected from stem cells, fibroblasts, osteoblasts, and the like.

[0034] 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 dilution solution.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] The present invention further provides silk protein-based ink obtained based on the above preparation method.

[0039] 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.

[0040] 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 the tissue engineering scaffold by a cryo-3D printing method, the method comprising the following steps:

[0041] (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;

[0042] (S2) The three-dimensional scaffold obtained in step (S1) is subjected to low-temperature freezing or vacuum freeze-drying to allow the silk protein to self-assemble to obtain a tissue engineering scaffold.

[0043] 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%.

[0044] In some embodiments of the present invention, in step (S1), the temperature of the freezing platform used is 0°C to -25°C.

[0045] 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.

[0046] 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.

[0047] In some embodiments of the present invention, in step (S2), the vacuum freeze-drying is performed under the following conditions: the obtained three-dimensional scaffold is transferred to liquid nitrogen, and after freezing for one hour, is transferred to a vacuum freeze dryer for freeze-drying.

[0048] The present invention further provides a tissue engineering scaffold prepared based on the above method.

[0049] The present invention further provides the use of a tissue engineering scaffold prepared based on the above method, wherein the tissue engineering scaffold is used for cell culture and tissue regeneration.

[0050] Compared with the prior art, the beneficial effects of the present invention are embodied in the following aspects:

[0051] (1) The silk protein required by the present invention is directly extracted from silk cocoons, which is widely available, cheap, and has 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, thus meeting the basic requirements of tissue engineering scaffolds.

[0052] (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.

[0053] (3) The present invention uses the silk protein-based ink as a raw material and uses an extrusion-type 3D printer to perform 3D printing on a freezing platform to obtain a three-dimensional scaffold; the obtained three-dimensional scaffold is then cryogenically frozen to allow the silk protein to self-assemble, thereby obtaining a tissue engineering scaffold. The low temperature enables the ink to undergo a sol-gel transition (see Figure 2), thereby generating 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 removed from 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 into β-folds in a freezing environment, so that the scaffold does not dissolve even after being removed from the freezing environment, thereby maintaining the solidification and qualitative properties of the tissue engineering scaffold.

[0054] (4) The present invention does not require the addition of chemical crosslinkers or other thickeners (thickeners are 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 high molecular weights). In contrast, the present invention uses a small molecule plasticizer, a small molecule compound whose purpose is to promote the movement of silk protein molecular chains rather than increase viscosity, thereby ensuring the biocompatibility of the scaffold. By regulating the content of the plasticizer and mechanical property modifier in the ink, the mechanical properties of the silk protein three-dimensional scaffold can be precisely controlled to meet the mechanical requirements of different tissue engineering scaffolds.

[0055] (5) 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

[0056] FIG1 is a photograph of the silk protein-based ink prepared in Example 1 of the present invention.

[0057] FIG2 is a photograph of a tissue engineering scaffold obtained by freeze-printing silk protein-based ink in Example 1 of the present invention.

[0058] FIG3 is a graph showing the rheological properties of the silk protein-based ink prepared in an embodiment of the present invention at different temperatures.

[0059] FIG4 is a photograph of the silk protein-based tissue engineering scaffolds prepared in Example 1 of the present invention and the comparative example, and photographs of the two scaffolds immersed in water for 24 hours.

[0060] 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).

[0061] 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.

[0062] FIG7 is a fluorescence image of cytoskeleton staining of cells adhered to the surface of the silk protein-based tissue engineering scaffold prepared in Example 5 of the present invention.

[0063] FIG8 shows the reaction process of the silk protein-based tissue engineering scaffold prepared in Example 8 of the present invention and TMP.

[0064] FIG9 is a photograph of the silk protein-based tissue engineering scaffolds prepared in Example 12 and Example 13 of the present invention. DETAILED DESCRIPTION

[0065] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0066] Example 1

[0067] This embodiment provides a method for preparing a silk protein-based tissue engineering scaffold, comprising the following steps:

[0068] (1) Prepare a 0.02 mol / L sodium carbonate aqueous solution, heat it to boiling, add the chopped cocoons to 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 9.3 mol / L lithium bromide solution, 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.

[0069] (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).

[0070] (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. 3D printing was performed using 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 fill spacing of 0.5 mm, and a freezing platform temperature of -18°C to prepare a mesh scaffold. The 3D printed scaffold was frozen at -18°C for 24 hours to allow the silk protein to self-assemble into β-folds to solidify the scaffold.

[0071] 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.

[0072] 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 its intact morphology without post-processing.

[0073] Example 2

[0074] The difference from Example 1 is that:

[0075] In step (2), the solution was concentrated to 35 wt % by a vacuum concentrator.

[0076] Other details are the same as those in Example 1.

[0077] Example 3

[0078] The difference from Example 1 is that:

[0079] 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.

[0080] Other details are the same as those in Example 1.

[0081] Example 4

[0082] The difference from Example 1 is that:

[0083] 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.

[0084] Other details are the same as those in Example 1.

[0085] Example 5

[0086] The difference from Example 1 is that:

[0087] 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.

[0088] Other details are the same as those in Example 1.

[0089] Example 6

[0090] The difference from Example 1 is that:

[0091] 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.

[0092] Other details are the same as those in Example 1.

[0093] Example 7

[0094] The difference from Example 1 is that:

[0095] 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.

[0096] Other details are the same as those in Example 1.

[0097] Example 8

[0098] The difference from Example 1 is that:

[0099] 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.

[0100] Other details are the same as those in Example 1.

[0101] Example 9

[0102] The difference from Example 1 is that:

[0103] In step (3), a 25G TT dispensing tip was used.

[0104] The rest is consistent with Example 1.

[0105] Example 10

[0106] The difference from Example 1 is that:

[0107] In step (3), a 30G TT dispensing tip was used.

[0108] The rest is consistent with Example 1

[0109] Example 11

[0110] The difference from Example 1 is that:

[0111] In step (3), a cylindrical support program is written in the printing device.

[0112] The rest is consistent with Example 1, and a cylindrical stent is obtained.

[0113] Example 12

[0114] The difference from Example 1 is that:

[0115] In step (3), a nose morphology support program is written in the printing device.

[0116] The rest is consistent with Example 1, and a nose-shaped stent is obtained.

[0117] Example 13

[0118] The difference from Example 1 is that:

[0119] In step (3), an ear morphology support program is written in the printing device.

[0120] The rest is consistent with Example 1, and an ear-shaped bracket is obtained.

[0121] Comparative Example 1

[0122] In order to contrast with the silk protein-based bio-ink prepared by the present invention, a silk protein scaffold without adding small molecule plasticizers and mechanical property modifiers was prepared according to the following method:

[0123] (1) Prepare a 0.02 mol / L sodium carbonate aqueous solution, heat it to boiling, add the chopped cocoons to 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 9.3 mol / L lithium bromide solution, 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.

[0124] (2) A certain amount of silk protein aqueous solution was taken and concentrated to a solution with a silk protein mass fraction of 30 wt % using a vacuum concentrator.

[0125] (3) The solution in step (2) was loaded into the barrel of a 3D printer, and a mesh scaffold program was written in the printing device. 3D printing was performed using 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 to prepare a mesh scaffold. The 3D printed scaffold was frozen at -18°C for 24 hours. The silk protein scaffold was not added with small molecule plasticizers and mechanical property modifiers.

[0126] Test Example 1

[0127] The silk fibroin ink prepared in Example 1 was characterized for its rheological properties using a rotational rheometer. The silk fibroin ink was placed between the grips of the rotational rheometer, and the storage modulus and loss modulus of the silk fibroin ink were measured as a function of temperature. The rheological properties are shown in Figure 3.

[0128] Test Example 2

[0129] The water stability of the silk fibroin scaffolds prepared in Example 1 and Comparative Example 1 was tested by immersion in water. The silk fibroin scaffolds prepared in Example 1 and Comparative Example 1 were immersed in 5 mL of deionized water for 24 hours and then photographed. The photographs are shown in Figure 4.

[0130] Test Example 3

[0131] The dry mechanical properties of the silk protein scaffolds prepared in Example 1 and Example 5 were tested using a mechanical testing machine equipped with a 50N load cell. The samples were cut into dumbbell-shaped specimens according to ASTM standards, and then loaded onto the machine's furniture. For each test, the stretching rate of all samples was 100% strain / min 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 from the stress-strain curve. The mechanical property characterization results are shown in Figure 5.

[0132] Test Example 4

[0133] 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 L292 cells were plated in a cell / well format. After the cells adhered, samples were added. Each well was plated with at least six replicates, and wells without sample served as blank controls. The cells were incubated at 37°C in a 5% CO2 atmosphere, with the medium changed every two days. All samples were sterilized in an autoclave before addition. Cell viability was assessed using a CCK8 assay at 3 and 5 days of incubation. The cell compatibility results are shown in Figure 6.

[0134] Test Example 5

[0135] The cell adhesion performance of the silk protein scaffold prepared in Example 5 was investigated by co-culturing with L929 cells and observing cell adhesion by fluorescence staining at specific time points. Samples were added to a 24-well plate, with at least 6 replicates per group, and 2×10 4 L292 cells were cultured in a cell / well format at 37°C in an atmosphere of 5% CO2, with the medium changed every two days. All samples were sterilized in an autoclave before being added to the well plates. On the seventh day of incubation, the culture medium was discarded and fixed with 4% paraformaldehyde solution. After fixation for 2 hours, the fixative was discarded and permeabilized with 0.1% Triton X-100. After permeabilization for 15 minutes, the permeabilization solution was discarded and phalloidin working solution was added. The cells were incubated for two hours to stain the cytoskeleton. Cells adhered to the scaffold surface were observed using a laser confocal microscope. The fluorescent staining image of the cytoskeleton is shown in Figure 7.

[0136] Test Example 6

[0137] The functionality of the silk scaffold prepared in Example 8 was tested by reacting it with 3,3'5,5'-tetramethylbenzidine (TMB). The silk scaffold prepared in Example 8 was added to 1 mL of TMB solution, and the color change of the solution was photographed. The color change diagram of the solution is shown in Figure 8.

[0138] Result analysis:

[0139] It can be seen from Figure 3 that the loss modulus of the silk protein-based ink is higher than its storage modulus in the temperature range of 25°C to -10°C, 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 rapidly solidify when in contact with the freezing platform, thereby maintaining its shape.

[0140] 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 plasticizer melts immediately after being frozen and reheated, dissolves in water in large quantities, and cannot maintain its complete shape.

[0141] 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 effect of regulating the mechanical properties of the silk protein scaffold.

[0142] 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.

[0143] As can be seen from FIG. 7 , the silk protein scaffold prepared in Example 5 can support cell adhesion and growth.

[0144] As can be seen from FIG8 , 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.

[0145] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. 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

1. 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.

2. 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.

3. The method for preparing a silk protein-based ink according to claim 1, characterized in that: The mechanical property regulator is selected from calcium chloride, hydroxyapatite, lithium chloride or lithium bromide, or a combination of the two or more thereof.

4. The method for preparing a silk protein-based ink according to claim 1, characterized in that: The silk protein described 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.

5. 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 small molecule plasticizer is 100:0.1 to 100:50, preferably 100:1 to 100:

30.

6. 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.

7. The method for preparing a silk protein-based ink according to claim 1, 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.

8. The method for preparing a silk protein-based ink according to claim 7, characterized in that: The bioactive substance is selected from one or more of HRP, BSA, BMP-2, bFGF or decellularized matrix; the cells are selected from stem cells, fibroblasts or osteoblasts.

9. The method for preparing a silk protein-based ink according to claim 1, characterized in that: The method for adjusting the concentration of the silk protein solution is: obtaining a diluent by adding water, obtaining a concentrated solution by natural air drying, obtaining a concentrated solution by reverse dialysis, or obtaining a concentrated solution by vacuum centrifugation.

10. A silk protein-based ink obtained by the preparation method according to any one of claims 1 to 9.

11. The use of the silk protein-based ink according to claim 10, characterized in that: The silk protein-based ink is used for 3D printing to obtain tissue engineering scaffolds.

12. A method for preparing a tissue engineering scaffold, characterized in that: Using the silk protein-based ink according to claim 10 as a raw material, a tissue engineering scaffold is obtained 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.

13. 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 1 wt% to 40 wt%.

14. 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.

15. 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.

16. The method for preparing a tissue engineering scaffold according to claim 12, characterized in that: In step (S2), the low-temperature freezing is performed under the conditions of: performing low-temperature freezing at a freezing temperature of 0°C to -25°C for at least 6 hours.

17. The method for preparing a tissue engineering scaffold according to claim 12, characterized in that: In step (S2), the vacuum freeze-drying is performed under the following conditions: the obtained three-dimensional scaffold is transferred to liquid nitrogen, and after freezing for one hour, it is transferred to a vacuum freeze dryer for freeze drying.

18. A tissue engineering scaffold prepared by the method according to any one of claims 12 to 17.

19. The use of the tissue engineering scaffold according to claim 18, characterized in that: The tissue engineering scaffold is used for cell culture and tissue regeneration.

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