WO2020237414A1

WO2020237414A1 - Modified biopolymer and application thereof in 3d printing

Info

Publication number: WO2020237414A1
Application number: PCT/CN2019/088283
Authority: WO
Inventors: 阮长顺; 何慧敏; 林子锋; 吴明明; 李铎
Original assignee: 深圳先进技术研究院
Priority date: 2019-05-24
Filing date: 2019-05-24
Publication date: 2020-12-03

Abstract

A modified biopolymer, comprising a biopolymer backbone, and a ureido pyrimidone group and a tyramine group which are grafted on the backbone. A bio-ink comprises a solvent and the modified biopolymer dissolved in the solvent, and further comprises a biological functional component loaded by the modified biopolymer. The viscosity of the modified biopolymer is adjustable with temperature, and the modified polymer can be directly solidified by itself at room temperature to form a gel having good mechanical properties. The bio-ink has good viscosity and biocompatibility, is applicable to 3D printing, can be used for direct printing and forming of a biological scaffold, and avoids additional addition of components harmful to biological components, such as a cross-linking agent.

Description

Modified biopolymer and its application in 3D printing

Technical field

The invention belongs to the technical field of biological materials, and specifically relates to a modified biological polymer and a preparation method thereof, biological ink and its application.

Background technique

On the basis of 3D printing technology, 3D bioprinting has great development potential in the fields of preparing artificial organs and tissues, realizing personalized treatment and drug screening by loading cells and growth factors. As the most widely used bioprinting method, micro-extrusion printing is one of the most important problems that limit its rapid development and clinical application is the lack of bio-inks with both excellent printing performance and biological activity. Generally, the bio-ink used for 3D bioprinting needs to have the following properties:

a). Biological activity: to ensure that the cells have a high survival rate, good spreading, proliferation and differentiation capabilities;

b). Rapid prototyping capability: Rapid prototyping capability is the basis for ensuring high-precision printing;

c). Controllable degradation performance: to ensure that the bioprinted organoids are well used in the field of tissue engineering.

Collagen is widely present in animal tissues as one of the main components of extracellular matrix. The biopolymer obtained from collagen has a structure similar to that of collagen, which ensures good cell compatibility, and at the same time has the advantages of low cost and easy acquisition. Therefore, collagen is widely used as the basic material of bio-ink, the most representative of which is methacrylic acid-modified biopolymer bio-ink. However, such biopolymer-based inks have very low viscosity and are not suitable for the construction of precise three-dimensional structures, and the subsequent crosslinking requires the addition of toxic initiators and UV radiation that is harmful to cells.

At present, in order to improve the printing performance of biopolymer-based bioprinting inks and the problem of molding under mild conditions, it has been disclosed that researchers add thickeners to biopolymer inks to realize the printability of biopolymer inks. However, the photo-crosslinking system brings uncertain factors to the subsequent function of cells, and the biological activity is still insufficient. In view of the biological activity of the cross-linking method, the researchers achieved good maintenance of cell activity during the cross-linking process by grafting tyramine on the biopolymer and using a gentle enzymatic cross-linking method at room temperature, but only by this The cross-linking method is difficult to achieve the printability of the material.

It can be seen from the above that the prior art bio-ink still causes the following disadvantages:

a). Can not solve the good printability of biopolymer-based bio-ink and the subsequent good biological activity at the same time;

b). Unable to print complex biopolymer biostents;

c). It is impossible to pack different cells for precise printing for further tissue engineering applications.

Therefore, how to develop a bio-ink that can effectively solve these drawbacks is a technical problem that those skilled in the art have been trying to solve.

technical problem

The purpose of the present invention is to overcome the shortcomings of the prior art, and provide a modified biopolymer and a preparation method thereof, so as to solve the problem that the ink viscosity caused by the existing biopolymer as a bio-ink substrate is very low and is not suitable for precise use. The technical problem of reducing biological activity during the construction of three-dimensional structure and cross-linking.

Another object of the present invention is to provide a modified biopolymer bio-ink and its application, in order to overcome the existing biopolymer-based ink can not simultaneously solve the problem of biopolymer-based bio-ink's good precision printing and subsequent good biological activity technical problem.

Technical solutions

In order to achieve the objective of the present invention, one aspect of the present invention provides a modified biopolymer. The modified biopolymer includes a main chain of a biopolymer body, on which ureidopyrimidinone groups and tyramine groups are grafted.

Preferably, the ureidopyrimidinone group is grafted onto the main chain through the ureido group contained in the ureidopyrimidinone group.

Preferably, the tyramine group is grafted onto the main chain by -CO-NH-.

Preferably, the biopolymer body is a biopolymer containing amine groups and/or carboxyl groups.

Further preferably, the ureidopyrimidinone group is grafted onto the main chain by reacting the ureidopyrimidinone containing an isocyanate group with the biopolymer.

Further preferably, the ureidopyrimidinone group is grafted to the main chain by the condensation reaction of the biopolymer grafted with the ureidopyrimidinone group and a compound containing a tyramine group on.

Preferably, the content of the ureidopyrimidinone group in the modified biopolymer is 0.1-0.2 mM g-1.

Preferably, the content of the tyramine group in the modified biopolymer is 0.1-0.5 mM g-1.

Preferably, the biopolymer body includes at least one of gelatin, polyvinyl alcohol, alginic acid, hyaluronic acid, and carboxymethyl chitosan.

In another aspect of the present invention, a method for preparing a modified biopolymer is provided. The preparation method of the modified biopolymer includes the following steps:

Ureapyrimidinone containing isocyanate groups and biopolymer body containing amine groups and/or carboxyl groups are subjected to a ureylation reaction in the first reaction solvent to generate ureidopyrimidinone grafted biopolymers;

The ureidopyrimidinone grafted biopolymer and the tyramine group-containing compound are subjected to a condensation reaction in a second reaction solvent containing a catalyst to obtain a modified ureidopyrimidinone group and a tyramine group Biopolymers.

Preferably, the conditions for the ureidolation reaction of the ureidopyrimidinone with isocyanate groups and the biopolymer in the first reaction solvent meet at least one of the following conditions:

The mass ratio of the ureidopyrimidinone containing isocyanate groups to the biopolymer is 1: (10-30);

The mass ratio of the biopolymer to the first reaction solvent is 1: (15-20);

The first reaction solvent is at least one of dimethyl sulfoxide, dimethyl formamide, carbon tetrachloride, and ether.

Preferably, the conditions for the condensation reaction of the ureidopyrimidinone grafted biopolymer and the compound containing a tyramine group in a second reaction solvent containing a catalyst satisfy at least one of the following conditions:

The mass ratio of the ureidopyrimidinone grafted biopolymer to the tyramine group-containing compound is 1: (0.3-0.6);

The mass ratio of the biopolymer grafted with ureidopyrimidinone to the second reaction solvent is 1: (15-20);

The catalyst is a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, 4-(4,6-dimethoxy) At least one of triazine-2-yl)-4-methylmorpholine hydrochloride;

The compound containing a tyramine group is at least one of tyramine hydrochloride, tyramine, dopamine hydrochloride, and 6-hydroxydopamine hydrochloride.

In yet another aspect of the present invention, a biological ink is provided. The bio-ink includes a solvent and a biopolymer dissolved in the solvent, and the bio-ink also includes a biofunctional component supported by the biopolymer, wherein the biopolymer is a modified biopolymer of the present invention. Things.

Preferably, the biological functional component is a cell, and the content of the cell in the biological ink is 2.1×10 ⁵ cells mL ^-1 .

Preferably, the mass concentration of the modified biopolymer in the bio-ink is 15%-25%.

Preferably, the biological functional component is at least one of cells, growth factors, and drugs.

Preferably, the solvent is at least one of phosphate buffer solution and cell culture medium.

At the same time, the invention also provides an application method of the biological ink of the invention. Specifically, the application of the bio-ink in 3D printing.

In yet another aspect of the present invention, a biological scaffold is provided. The biological scaffold is prepared from the biological ink of the present invention.

At the same time, the present invention also provides a method for preparing the biological scaffold. The preparation method of the biological scaffold includes the following steps:

The 3D printing process is performed with the biological ink of the present invention as a raw material.

Preferably, the conditions of the 3D printing are as follows:

The printing temperature is 10-43℃;

Printing rate is 4-15 mm/s.

Preferably, after the step of 3D printing treatment, it further comprises the step of enzymatic cross-linking reaction treatment of the bio-scaffold formed by printing in a solution containing hydrogen peroxide and horseradish peroxidase.

Beneficial effect

Compared with the prior art, the modified biopolymer of the present invention is modified by using ureidopyrimidinone groups and tyramine groups, so that the modified biopolymer has a good viscosity, and the viscosity is adjustable with temperature. At the same time, the modified biopolymer also has the characteristic of directly coagulating itself at room temperature, and the gel formed by coagulation has good mechanical properties. Therefore, the modified biopolymer is particularly suitable as a bio-ink for 3D printing, which effectively avoids the addition of additional initiators harmful to biologically active ingredients and harmful ultraviolet light irradiation, and effectively improves the modified biopolymerization. The biocompatibility and activity of biologically active ingredients.

The preparation method of the modified biopolymer of the present invention can effectively graft the ureidopyrimidinone group and the tyramine group on the main chain of the biopolymer body, and realize the modification and modification of the biopolymer body, thereby giving the generated The modified biopolymer has good viscosity and self-coagulation characteristics and good mechanical properties of the gel formed by coagulation. Moreover, the method for preparing the modified biopolymer can ensure that the performance of the modified biopolymer is stable, the conditions are easy to control, and the efficiency is high.

Since the bio-ink of the present invention contains the modified bio-polymer of the present invention, the bio-ink has good viscosity, and the viscosity is adjustable with temperature, and it also has self-coagulation characteristics. Therefore, the bio-ink has good viscosity at the same time. It has biocompatibility and is especially suitable for 3D printing. It can be directly printed and molded, avoiding the addition of additional components such as cross-linking agents that are harmful to biological components.

Since the biological scaffold of the present invention is formed by 3D direct printing using the biological ink of the present invention, the biological scaffold has high precision and the loaded bioactive components have high activity. Therefore, the biological scaffold has high biological activity.

Description of the drawings

The present invention will be further described below in conjunction with the accompanying drawings and embodiments. In the accompanying drawings:

Fig. 1 is a schematic diagram of the process flow of a method for preparing a modified biopolymer according to an embodiment of the present invention;

Figure 2 shows the urea pyrimidinone containing isocyanate groups and the biopolymer in the preparation method of the modified biopolymer according to the embodiment of the present invention, the ureidopyrimidinone grafted with the biopolymer and the biopolymer Schematic diagram of the chemical formula of the condensation reaction of compounds with tyramine groups;

3 is a schematic diagram of the structure of a biological scaffold printed with biological ink and a biological scaffold after cross-linking reaction of the biological scaffold according to an embodiment of the present invention; wherein, FIG. 3A is a schematic view of the structure of a biological scaffold printed with biological ink and the biological scaffold according to an embodiment of the present invention Fig. 3B is a schematic diagram showing the cross-linked structure of modified biopolymer molecules in the printed bio-stent shown in Fig. 3A after cross-linking treatment; Fig. 3C shows the printed bio-stent shown in Fig. 3A Schematic diagram of the molecular structure of the modified biopolymer after cross-linking treatment;

Fig. 4 is a graph of the temperature-sensitive response and mechanical properties of the solution of the modified biopolymer provided in Example 11 of the present invention; wherein, Fig. 4A is a graph of the mechanical properties of the gel formed after the solution of the modified biopolymer is solidified. 4B is the temperature sensitive response performance diagram of the modified biopolymer solution;

Figure 5 is a photograph of the bio-ink provided in Examples 21-23 of the present invention and the respective printability conclusion diagram; among them, Figure 5A is a photo of the biological ink provided in Examples 21-23, and Figure 5B is provided in Example 21-23 The printability conclusion map of the bio-ink at the corresponding temperature;

Figure 6 is a schematic diagram of the two-dimensional structure of the biological scaffold printed in Example 31; among them, Figure 6B is a partial enlarged view of a in Figure 6A, Figure 6D is a partial enlarged view of b in Figure 6C, and Figure 6E is a two-dimensional view shown in Figure 6C Structured biological scaffold for active cell staining;

7 is a schematic diagram of the three-dimensional structure of the biological scaffold printed in Example 31; wherein, FIG. 7B is a partial enlarged view of FIG. 7A, and FIG. 7C is a view of active cell staining of the three-dimensional structure of the biological scaffold shown in FIG. 7A;

8 is a schematic diagram of a three-dimensional bionic organ model scaffold structure printed in Example 31;

9 is a diagram showing the survival, spreading, and proliferation of cells in the three-dimensional biological scaffold provided in Example 32 after being stained to death.

The best mode of the invention

In order to make the technical problems, technical solutions, and beneficial effects to be solved by the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, but not to limit the present invention.

In one aspect, embodiments of the present invention provide a modified biopolymer. The molecular structure diagram of the modified biopolymer is shown as compound B in FIG. 2, which includes the main chain 1 of the biopolymer body, and the main chain 1 is grafted with ureidopyrimidinone group 2 and phenol Amine group 3.

In one embodiment, the ureidopyrimidinone group 2 is grafted onto the body of the biopolymer through the ureido group (-NH-CO-NH-) contained in the ureidopyrimidinone group 2. On the main chain 1. In a specific embodiment, the ureidopyrimidinone group 2 is grafted onto the main chain 1 of the biopolymer body by reacting the ureidopyrimidinone containing isocyanate groups with the body of the biopolymer. . By designing the linking group of the ureidopyrimidinone group 2 and the main chain 1 of the biopolymer body, the ureidopyrimidinone group 2 can be stably grafted on the main chain 1 of the biopolymer body, In this way, the modification of biopolymers can be achieved, which can increase the viscosity of the aqueous biopolymer solution, thereby giving the modified biopolymer aqueous solution a relatively high viscosity, increasing the application range of the modified biopolymer, especially Application in 3D precision printing. In an embodiment, the content of the ureidopyrimidinone group 2 in the modified biopolymer is 0.1-0.2 mM g ^-1 , specifically 0.14 mM g ^-1 . By controlling the grafting amount of the ureidopyrimidinone group 2, the viscosity of the aqueous solution of the modified biopolymer is controlled, and its application range is improved, especially its applicability in three-dimensional precision printing.

In one embodiment, the tyramine group 3 is grafted onto the biopolymer backbone 1 through an imide group (-CO-NH-). In a specific embodiment, the ureidopyrimidinone group 3 is grafted to the ureidopyrimidinone group 3 by the condensation reaction of the biopolymer grafted with the ureidopyrimidinone group 2 and the compound containing the tyramine group 3. The main chain 1 of the biopolymer body. By designing the linking group between the tyramine group 3 and the main chain 1 of the biopolymer body, the tyramine group 3 can be stably grafted onto the main chain 1 of the biopolymer body to realize the tyramine group 3 The synergistic modification of the amine group 3 and the ureidopyrimidinone group 2 on the body of the biopolymer, while giving the aqueous solution of the modified biopolymer a relatively high viscosity, the modified biopolymer The viscosity of the aqueous solution has the characteristics of being adjustable with temperature, and has the characteristics of automatic coagulation at room temperature, and the mechanical properties of the gel formed by coagulation have been significantly improved. In this way, the biocompatibility of the modified biopolymer has been significantly improved and enhanced, thereby increasing the application range of the biopolymer, especially the application in three-dimensional precision printing. In an embodiment, the content of the tyramine group 3 in the modified biopolymer may be 0.1-0.5 mM g ^-1 , preferably 0.11 mM g ^-1 . By controlling the grafting amount of the tyramine group 3, the modified biopolymer is endowed with further enzymatic crosslinking characteristics and its ability to carry out enzymatic crosslinking under mild conditions is improved, so that the modified biopolymer contains Further enzymatic cross-linking of the printed scaffold of the material provides the possibility of cross-linking the cell-encased scaffold under mild conditions, thereby improving the stability of the entrapped scaffold, that is, the biological scaffold under physiological conditions.

In an embodiment, the main chain of the biopolymer body contained in the modified biopolymer in each of the foregoing embodiments may be provided by a biopolymer containing an amine group and/or a carboxyl group. As in a specific embodiment, the biopolymer body includes at least one of gelatin, polyvinyl alcohol, alginic acid, hyaluronic acid, and carboxymethyl chitosan. These biopolymers contain abundant amine groups and/or carboxyl groups, so that the ureidopyrimidinones containing isocyanate groups react with the amine groups contained in the biopolymers to form ureidopyrimidinone groups 2 , And grafted onto the main chain 1 of the biopolymer body. The compound containing the tyramine group 3 reacts with the carboxyl group contained in the biopolymer body to form the ureidopyrimidinone group 3, which is grafted onto the main chain 1 of the biopolymer body.

Therefore, the modified biopolymer through the synergistic modification effect of ureidopyrimidinone group 2 and tyramine group 3, its solution has good viscosity and viscosity adjustable with temperature characteristics, and it has room temperature solidification Characteristics, the gel formed by solidification has excellent mechanical properties and excellent biocompatibility, and can carry out a gentle enzymatic cross-linking reaction in the presence of cells to improve the thermal stability of the material under physiological conditions. Therefore, the modified biopolymer is particularly suitable as a bio-ink for 3D (three-dimensional) precision printing.

Correspondingly, the embodiment of the present invention also provides a method for preparing the modified biopolymer described above. The process flow of the preparation method of the modified biopolymer is shown in Figure 1, which includes the following steps:

S01: The ureidopyrimidinone containing isocyanate groups and the biopolymer body containing amine groups and/or carboxyl groups are subjected to a ureylation reaction in the first reaction solvent to generate ureidopyrimidinone grafted biopolymers ；

S02: The ureidopyrimidinone grafted biopolymer and the tyramine group-containing compound are subjected to a condensation reaction in a second reaction solvent containing a catalyst to obtain a ureidopyrimidinone group and a tyramine group modified Modified biopolymers.

Wherein, in the step S01, the ureidopyrimidinone containing the isocyanate group and the biopolymer body are ureidolated in the first reaction solvent as shown in FIG. 2. During the urea reaction between the biopolymer body and the ureidopyrimidinone containing isocyanate groups, the isocyanate groups contained in the ureidopyrimidinone reactant containing isocyanate groups and the The amine group on the main chain of the biopolymer reacts to form a urea group (-NH-CO-NH-), that is, the ureidopyrimidinone group 2 is grafted onto the biopolymer body through the urea group On the main chain 1, the compound A in Figure 2 is shown in detail.

In one embodiment, in the reaction system in step S01, the ureidopyrimidinone containing isocyanate groups and the biopolymer body can be in a mass ratio of 1: (10-30), specifically as It is mixed in the first reaction solvent at a ratio of 1:20. By controlling the reaction concentration ratio of the two, the ureidopyrimidinone containing isocyanate groups can fully react with the body of the biopolymer, specifically, the urea reaction is carried out, so that the body The main chain 1 is grafted with ureidopyrimidinone group 2 to modify the biopolymer. In addition, the concentration of the reactants in the first reaction solvent and the kind of the first reaction solvent can be further controlled to increase the ureidolation reaction rate and increase the yield of the target compound A, such as In an embodiment, the mass ratio of the biopolymer body to the first reaction solvent is 1: (15-20), specifically 1:17; the first reaction solvent can be dimethyl sulfoxide. In addition, based on the ureidolation reaction in step S01, preferably, the ureidolation reaction is performed in a protective atmosphere, such as a nitrogen atmosphere, to ensure the yield of the target product.

The urea reaction in step S01 can be carried out under isothermal conditions at room temperature, and the reaction time should be component, such as 24 hours. After the urea reaction in step S01 is completed, it also includes the step of generating ureidopyrimidinone grafted biopolymer for purification. In a specific embodiment, the biopolymer grafted with ureidopyrimidinone is purified, and the biopolymer grafted with ureidopyrimidinone is precipitated by precipitation separation, and then the precipitate is dried. Obtain pure ureidopyrimidinone grafted biopolymer.

In addition, the biopolymer body containing amine and/or carboxyl groups in step S01 includes at least one of gelatin, polyvinyl alcohol, alginic acid, hyaluronic acid, and carboxymethyl chitosan as described above. . The ureidopyrimidinone containing isocyanate groups can be but not only 2(6-isocyanatohexylaminocarbonylamino)-6-methyl-4[1H]pyridone, as long as it can be biological Compounds in which the amino groups of the polymer backbone react to generate ureidopyrimidinone groups are all within the scope of the present invention.

The ureidopyrimidinone grafted biopolymer in the step S02 is a condensation reaction between the compound A and the compound containing a tyramine group, as shown in FIG. 2. During the condensation reaction between the compound A and the tyramine group-containing compound, the amine group contained in the tyramine group-containing compound reacts with the carboxyl group on the main chain of the biopolymer to form an imide The amine group (-CO-NH-), that is, the tyramine group 3 is grafted onto the main chain 1 of the biopolymer body through the imide group, as shown in compound B in FIG. 2.

In one embodiment, in the reaction system in step S02, the ureidopyrimidinone grafted biopolymer and the tyramine group-containing compound may have a mass ratio of 1:(0.3-0.6), specifically Mix in the second reaction solvent at a ratio of 1:0.43. By controlling the reaction concentration ratio of the two, the compound containing the tyramine group can fully react with the compound A, specifically, the condensation reaction is carried out, so that the main chain 1 of the biopolymer is grafted with tyramine Group 3 realizes the modification of the biopolymer body. In addition, it is possible to further increase the condensation reaction rate and increase the yield of the target compound B by controlling the concentration of the reactants in the second reaction solvent and the type of the second reaction solvent, as in an implementation In an example, the mass ratio of the ureidopyrimidinone grafted biopolymer to the second reaction solvent is 1: (15-20); the second reaction solvent can be water (preferably double distilled water) . In another embodiment, the catalyst is a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS) , At least one of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride. In addition, the added amount of the catalyst can be added according to the conventional amount of the corresponding catalyst according to the type of catalyst. For example, in the specific implementation, the catalyst is 1-(3-dimethylaminopropyl)-3-ethyl carbon. In the case of a mixture of diimine hydrochloride and N-hydroxysuccinimide (EDC/NHS), the mass ratio of the EDC/NHS to the tyramine group-containing compound is (0.35-0.5):1. The compound containing a tyramine group can be, but not only tyramine hydrochloride, as long as it is a compound capable of reacting with the carboxyl group of the main chain of the biopolymer to form an imide group (-CO-NH-). Within the scope of disclosure.

The condensation reaction in the step S02 can be carried out under isothermal conditions at room temperature, and the reaction time should be component, such as 6 hours. After the condensation reaction in step S02 is completed, it also includes the step of generating a modified biopolymer modified with ureidopyrimidinone groups and tyramine groups for purification. In a specific embodiment, the modified biopolymer modified with ureidopyrimidinone groups and tyramine groups can be purified by dialysis, and the retentate collected by the dialysis is freeze-dried to obtain pure urea groups. A modified biopolymer modified with pyrimidinone groups and tyramine groups.

In addition, in addition to the ratio and concentration of the reactants in the above step S01 and step S02, the mass ratio between the reactants or the concentration of the reactants in the solution can also be based on the reaction target to reactant dosage ratio and reaction If the concentration of the substance in the solution is adjusted adaptively, then any adaptive adjustment under the premise of the technical solution disclosed in the present invention is within the scope of the technical enlightenment that the technical solution of the present invention can give, that is, it is all in the original The invention is within the scope of the disclosure.

Therefore, the method for preparing the modified biopolymer can effectively graft the ureidopyrimidinone group 2 and the tyramine group 3 on the main chain 1 of the biopolymer body, thereby realizing the modification of the biopolymer body. , Thereby giving the generated modified biopolymer good viscosity and good mechanical properties of the gel formed by solidification, and further enzymatic cross-linking reaction can be carried out. In addition, the method for preparing the modified biopolymer can ensure that the performance of the modified biopolymer is stable, and the conditions are easy to control and efficient, thereby reducing the production cost of the modified biopolymer.

On the other hand, based on the above-mentioned modified biopolymer and its preparation method, embodiments of the present invention also provide a bio-ink. The bio-ink includes a solvent and a biopolymer dissolved in the solvent, and also includes a biofunctional component carried by the biopolymer.

Wherein, the solvent contained in the bio-ink may be a solvent used for the bio-ink, for example, it may be at least one of a phosphate buffer solution and a cell culture medium. On the one hand, the selected solvent can effectively dissolve the biopolymer to form a uniform and stable dispersion system; on the other hand, it can effectively ensure the activity of the loaded biological functional components.

The biopolymer contained in the bioink is the modified biopolymer described above. The modified biopolymer is used as a matrix component of the bio-ink, giving the bio-ink good viscosity, making the bio-ink good 3D (three-dimensional) precision printing characteristics, and giving the bio-ink solidification characteristics at room temperature And the gel formed by solidification has good mechanical properties. In one embodiment, the mass concentration of the modified biopolymer in the bio-ink is 15%-25%; by controlling the content of the modified biopolymer matrix in the bio-ink, optimization The viscosity of the biological ink improves the 3D (three-dimensional) precision printing characteristics of the biological ink and improves the quality of the 3D (three-dimensional) precision printing device.

The biological function contained in the biological ink imparts corresponding biological activity to the biological ink. The biological functional component can be selected according to needs. As in an embodiment, the biological functional component can include at least one of cells, growth factors, and drugs. In addition, the loading amount of the biological functional component in the biological ink may be an effective dose. The "effective dose" refers to the effective amount of the device formed by the bio-ink printing that can effectively exert the corresponding target biological activity. Those skilled in the art will understand that the "effective dose" should also depend on the type of the biological functional ingredient loaded and the corresponding device type. As in an embodiment, the content of the biological functional component in the biological ink may be but not only 2.1×10 ⁵ cells mL ^-1 .

Therefore, since the bio-ink is based on the above-mentioned modified biopolymer as the matrix, the bio-ink has a good viscosity, the viscosity is adjustable with temperature, and it also has the property of self-coagulation at room temperature.

It is precisely because the bio-ink has good viscosity and biocompatibility at the same time, therefore, the bio-ink can be used in printing, especially in 3D precision printing. In this way, the bio-ink is directly printed and formed, and can be cross-linked and solidified by itself at room temperature such as physiological temperature (about 37°C), thereby effectively avoiding the addition of additional components such as cross-linking agents that are harmful to biological components. The activity of the loaded biological functional components is guaranteed, thereby effectively improving the biocompatibility of the biological ink. At the same time, the cured bio-ink has good mechanical properties, thereby effectively ensuring the stability of the bio-device formed by printing.

In another aspect, based on the characteristics and applications of the bio-ink, an embodiment of the present invention also provides a bio-scaffold. The biological scaffold is prepared from the biological ink described above. Specifically, it is formed by the above-mentioned biological ink through printing such as 3D precision printing. In this way, the biological scaffold has high biological activity, stable structure, and high precision. In a specific embodiment, the biological scaffold may be a bionic organ structure, and can realize the function of a bionic organ structure. In a specific embodiment, the biological scaffold may have a two-dimensional structure as shown in FIG. 5, a three-dimensional porous structure as shown in FIG. 6, or a three-dimensional bionic organ model as shown in FIG.

At the same time, the embodiment of the present invention also provides a method for preparing the above-mentioned biological scaffold. The preparation method of the biological scaffold includes the following steps:

The above-mentioned biological ink is used as a raw material for 3D printing, as shown in A in Figure 3.

Wherein, in one embodiment, the conditions of the 3D printing in the method for preparing the biological scaffold are as follows:

Printing temperature is 10-43℃; and/or printing speed is 4-15 mm/s. The specific printing temperature is 37°C, and the printing speed is 8 mm/s. By controlling the printing temperature and speed, the accuracy, quality and biological activity of the biological scaffold can be effectively improved.

In a further embodiment, after the step of using the above bio-ink as a raw material for 3D printing, it further includes the step of enzymatic cross-linking reaction of the bio-scaffold formed by printing in a solution containing hydrogen peroxide and horseradish peroxidase. . In this way, the biological scaffold is further subjected to enzymatic cross-linking reaction treatment in the horseradish peroxidase solution containing hydrogen peroxide to improve the cross-linking stability of the biological scaffold. Specifically, when the modified biopolymer contained in the bio-scaffold formed by printing is subjected to enzymatic cross-linking reaction in the solution containing hydrogen peroxide and horseradish peroxidase, the modified biopolymer The ureidopyrimidinone group grafted on the main chain (ureidopyrimidinone group 2 as shown in Figure 2) undergoes a crosslinking reaction, and the tyramine group grafted on the main chain of the modified biopolymer (Tyramine group 3 shown in FIG. 2) cross-linking reaction occurs, specifically as shown in B in FIG. 3, thereby forming a cross-linked bioscaffold shown in C in FIG.

The present invention will now be described in further detail with reference to specific examples.

1. Modified biopolymer and its preparation method embodiment

Example 11

This embodiment provides a modified biopolymer and a preparation method thereof. The modified biopolymer includes a gelatin backbone to which ureidopyrimidinone groups and tyramine groups are grafted.

The preparation method of the modified biopolymer includes the following steps:

Step S11: Under the protection of nitrogen, dissolve 6 g of gelatin at 100 mL of dimethyl sulfoxide, and then cooled to room temperature; weigh 0.3g of 2(6-isocyanatohexylaminocarbonylamino)-6-methyl-4[1H]pyridone into the gelatin solution and add React for 24 h at room temperature; the reacted solution is precipitated 3 times with 1 L of ethanol solution, and then dried in vacuum for 24 hours to obtain 5.1 g of light yellow fixed, which is to produce ureidopyrimidinone grafted gelatin, its yield Calculated as 85%;

Step S12: Weigh 1.5 g of the light yellow solid obtained in step S11 and dissolve in 100 mL of deionized water, then gradually add 1.3 g tyramine hydrochloride, 0.45 g 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.7 g N-hydroxysuccinimide (NHS); adjust the pH of the solution to 4.7 and react overnight To obtain modified gelatin modified with ureidopyrimidinone groups and tyramine groups;

Step S13: The solution containing the modified gelatin modified by the ureidopyrimidinone group and the tyramine group is dialyzed for 3 days through a dialysis bag with a molecular weight cutoff of 7000, and the white spongy solid obtained by freeze drying is the ureidopyrimidinone /Tyramine modified gelatin.

Example 12

The preparation method of the modified biopolymer includes the following steps:

Step S11: Under the protection of nitrogen, dissolve 6 g of gelatin at 100 mL dimethyl sulfoxide, and then cooled to room temperature; Weigh 0.15 g of 2(6-isocyanatohexylaminocarbonylamino)-6-methyl-4[1H]pyridone into the gelatin solution and add React for 24 hours at room temperature; the reacted solution is precipitated 3 times with 1 L of ethanol solution, and then dried in vacuum for 24 hours to obtain 5.1 g Light yellow fixed, that is, gelatin grafted with ureidopyrimidinone, the yield is calculated as 85%;

Example 13

The preparation method of the modified biopolymer includes the following steps:

Step S11: Under the protection of nitrogen, dissolve 6 g of gelatin at 100 mL dimethyl sulfoxide, then cool to room temperature; weigh 0.6 g of 2(6-isocyanatohexylaminocarbonylamino)-6-methyl-4[1H]pyridone into the gelatin solution and add it to the React for 24 hours at room temperature; the reacted solution is precipitated 3 times with 1 L of ethanol solution, and then dried in vacuum for 24 hours to obtain 5.1 g Light yellow fixed, that is, gelatin grafted with ureidopyrimidinone, the yield is calculated as 85%;

Step S12: Weigh 1.5 g of the light yellow solid obtained in step S11 and dissolve in 100 mL of deionized water, then gradually add 1.3 g tyramine hydrochloride, 0.45 g 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.7 g N-hydroxysuccinimide (NHS); adjust the pH of the solution to 4.7 and react Overnight, a modified gelatin modified with ureidopyrimidinone groups and tyramine groups is obtained;

Example 14

The preparation method of the modified biopolymer includes the following steps:

Step S11: Under the protection of nitrogen, dissolve 6 g of gelatin at 100 mL of dimethyl sulfoxide, and then cooled to room temperature; weigh 1.2 g of 2(6-isocyanatohexylaminocarbonylamino)-6-methyl-4[1H]pyridone into the gelatin solution and add React for 24 hours at room temperature; the reacted solution is precipitated 3 times with 1 L of ethanol solution, and then dried in vacuum for 24 hours to obtain 5.1 g Light yellow fixed, that is, gelatin grafted with ureidopyrimidinone, the yield is calculated as 85%;

Step S12: Weigh 1.5 g of the light yellow solid obtained in step S11 and dissolve in 100 mL of deionized water, then gradually add 1.3 g tyramine hydrochloride, 0.45 g 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.7 g N-hydroxysuccinimide (NHS); adjust the pH of the solution to 4.7 and react overnight , To obtain modified gelatin modified with ureidopyrimidinone groups and tyramine groups;

Example 15

This embodiment provides a modified biopolymer and a preparation method thereof. The modified biopolymer includes a main chain of hyaluronic acid, on which an ureidopyrimidinone group and a tyramine group are grafted.

The preparation method of the modified biopolymer includes the following steps:

Step S11: Under the protection of nitrogen, 1 g hyaluronic acid was dissolved in 100 In mL DMSO, then cool to room temperature; weigh 0.05 g of 2(6-isocyanatohexylaminocarbonylamino)-6-methyl-4[1H]pyridone into the hyaluronic acid solution and react at room temperature 24 h; the reacted solution was precipitated 3 times with 1 L of ethanol solution, and then dried in vacuum for 24 hours to obtain 0.9 g white solid, that is, hyaluronic acid grafted with ureidopyrimidinone, the yield is calculated to be 88.6%;

Step S12: Weigh 3g of the light yellow solid obtained in step S11 and dissolve in 100 mL of deionized water, then gradually add 1.3 g tyramine hydrochloride, 0.45 g 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.65 N-hydroxysuccinimide (NHS); adjust the pH of the solution to 4.7 and react overnight, Obtain hyaluronic acid modified with ureidopyrimidinone group and tyramine group;

Step S13: The solution containing the hyaluronic acid modified by the ureidopyrimidinone group and the tyramine group is dialyzed for 3 days through a dialysis bag with a molecular weight cut-off of 7000, and the white spongy solid obtained by freeze drying is the ureidopyrimidinone /Tyramine modified hyaluronic acid.

Example 16

This embodiment provides a modified biopolymer and a preparation method thereof. The modified biopolymer includes a carboxymethyl chitosan backbone, on which ureidopyrimidinone groups and tyramine groups are grafted.

The preparation method of the modified biopolymer includes the following steps:

Step S11: Under the protection of nitrogen, dissolve 1 g carboxymethyl chitosan in 100 mL DMSO at 25°C by magnetic stirring, then cool to room temperature; weigh out 0.05 g 2(6-isocyanatohexylaminocarbonyl Amino)-6-methyl-4[1H]pyridone was added to the carboxymethyl chitosan solution and reacted at room temperature for 24 h; the reacted solution was precipitated 3 times with 1 L of ethanol solution, and then vacuum Dry for 24 hours to get 0.9 g Light yellow fixed, that is, carboxymethyl chitosan grafted with ureidopyrimidinone is produced, and its yield is calculated as 89%;

Step S12: Weigh 1.5 g of the light yellow solid obtained in step S11 and dissolve in 100 mL of deionized water, then gradually add 0.65 g tyramine hydrochloride, 0.23 g 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.3 N-hydroxysuccinimide (NHS); adjust the pH of the solution to 4.7 and react overnight , To obtain carboxymethyl chitosan modified with ureidopyrimidinone group and tyramine group;

Step S13: The solution containing the carboxymethyl chitosan modified by the ureidopyrimidinone group and the tyramine group is dialyzed for 3 days through a dialysis bag with a molecular weight cutoff of 7000, and the white spongy solid obtained by freeze drying is urea Pyrimidone/tyramine modified carboxymethyl chitosan.

Related performance tests for the modified biopolymers provided in Example 11 to Example 16:

The modified biopolymers provided in Examples 11 to 16 were tested for solubility, temperature sensitivity and mechanical properties, respectively. Among them, the modified biopolymer obtained in Examples 13 and 14 is not dissolved in an aqueous solution. The temperature sensitive performance and mechanical properties of the modified biopolymer provided in Example 11 are shown in FIG. 4. Among them, the temperature sensitive performance of the solution of the modified biopolymer provided in Example 11 is shown in Figure 4B. It can be seen from Figure 4B that the viscosity of the solution increases significantly with the increase of temperature and has good temperature sensitive performance. . The mechanical properties of the gel formed after the solidification of the modified biopolymer solution provided in Example 11 are shown in FIG. 4A. As can be seen from FIG. 4A, the mechanical properties of the gel have also been significantly improved.

In addition, the temperature-sensitive response performance and mechanical properties of the modified biopolymers provided in other embodiments were tested respectively, and the results were similar to those shown in FIG. 4B and FIG. 4A. Therefore, the modified biopolymers provided in the embodiments of the present invention It has stable and excellent viscosity, and its gel has stable and excellent mechanical properties.

2. Examples of biological ink and its preparation method

Examples 21-23

Embodiments 21-23 respectively provide a biological ink and a preparation method thereof. The biological ink includes a phosphate buffered saline (PBS) solvent and a modified biopolymer dissolved in the PBS solvent and a cell component with biological activity; the mass-volume ratio of the modified biopolymer to the PBS Respectively 2 g: 8 mL (Example 21), 2 g: 10 mL (Example 22), 2 g: 13.3 mL (Example 23); the content of the biologically active cells in the bio-ink is 2.1 ×10 ⁵ cells mL ^-1 . Wherein, the modified biopolymer is the modified biopolymer provided in Example 11. The biological ink provided by the embodiments 21-23 is shown in Figure 5A.

The method for preparing bio-ink includes the following steps:

Weigh 2g of the ureidopyrimidinone/tyramine modified modified biopolymer solid provided in Example 11, dissolve it in 8 mL of PBS at 50°C and cool to 37°C, then add the required cells and mix well to obtain Bio-ink contained in cells.

Example 24

This embodiment provides a biological ink and a preparation method thereof. The bio-ink includes a PBS solvent, a modified biopolymer dissolved in the PBS solvent and a cell component with biological activity; the mass-volume ratio of the modified biopolymer to the PBS is 2g:11.3mL The content of the biologically active cells in the biological ink is 2.1×10 ⁵ cells mL ^-1 . Wherein, the modified biopolymer is the modified biopolymer provided in Example 12.

The method for preparing bio-ink includes the following steps:

Weigh g of the ureidopyrimidinone/tyramine modified modified biopolymer solid provided in Example 12, dissolve it in 11.3 mL of PBS solution at 50°C and cool it down to 37°C, then add the required cells and mix well. Obtain the cell-encapsulated bio-ink.

Examples 25-28

This Example 25-2? A biological ink and a preparation method thereof are respectively provided. The bio-ink includes a PBS solvent, a modified biopolymer dissolved in the PBS solvent, and a cell component with biological activity; the mass-volume ratio of the modified biopolymer to the PBS solvent is 2g:6mL The content of the biologically active cells in the biological ink is 2.1×10 ⁵ cells mL ^-1 . Wherein, the modified biopolymers are the modified biopolymers provided in Examples 13-16, respectively.

The method for preparing bio-ink includes the following steps:

Weigh g of the ureidopyrimidinone/tyramine-modified modified biopolymer solids provided in Examples 13-18 and dissolve them in 6 mL of cell culture solution at 50°C and cool to 37°C, and then add the required The cells are evenly mixed to obtain the bio-ink carried by the cells.

Perform a printing performance test on the bio-ink provided in Example 21 to Example 28:

The biological inks provided in Examples 21 to 28 were tested for printing performance at 34°C, 37°C, 40°C, and 43°C, respectively. Among them, the printability of the biological inks obtained in Examples 21 to 23 with temperature is shown in FIG. 5B. It can be seen from FIG. 5B that the printability of the bio-ink is affected by the content of the modified biopolymer and the printing temperature. In other embodiments, the printability test results of the bio-ink are similar to those shown in Figure 5B.

3. Examples of biological scaffolds and preparation methods thereof

Example 31

This embodiment provides a biological scaffold and a preparation method thereof. The biological scaffold is formed by 3D printing using the modified biopolymer bio-ink provided in Example 21.

The preparation method of the biological scaffold includes the following steps:

S11: Take 10 mL of the modified biopolymer bio-ink provided in Example 21 into the barrel of the 3D printer and fix it on the printing shaft, and control the barrel temperature at 37°C through a constant temperature heater;

S12: The three-dimensional printer prints through the specified model. The diameter of the gun head used for printing is 300 μm, the printing pressure is 60 kPa, and the printing speed is 8 mm/s.

Example 32

This embodiment provides a biological scaffold and a preparation method thereof. The bio-scaffold is formed by using the modified biopolymer bio-ink provided in Example 21 and 3D printing, followed by enzymatic cross-linking reaction treatment with horseradish peroxidase reaction solution.

The preparation method of the biological scaffold includes the following steps:

S11: Take 10 mL of the modified biopolymer bio-ink provided in Example 24 into the barrel of the 3D printer and fix it on the printing shaft, and control the barrel temperature at 37°C through a constant temperature heater;

S12: The three-dimensional printer prints through the specified model, the diameter of the gun head used for printing is 300 μm, the printing pressure is 60 kPa, and the printing speed is 8 mm/s;

S13: Prepare 20 units mL-1 of horseradish peroxidase reaction solution with phosphate buffer; soak the active stent printed in step S12 in 10 mL of enzyme solution and add 6 μL of 30% hydrogen peroxide solution at room temperature React for 10 min; take out the stent after reaction, wash it with phosphate buffer solution, put it into the culture medium, and culture it in the incubator. After long-term cultivation, the pore structure of the three-dimensional scaffold remains intact.

Example 33

The preparation method of the biological scaffold includes the following steps:

S11: Take 10 mL of the modified biopolymer bio-ink provided in Example 25 into the barrel of the 3D printer and fix it on the printing shaft, and control the barrel temperature at 37°C through a constant temperature heater;

Related performance tests of the biological scaffolds provided in Examples 31 to 33:

The two-dimensional structure biological scaffold, the three-dimensional porous structure and the three-dimensional biomimetic organ model were printed into the embodiments 31 to 33 respectively. The two-dimensional structure biological scaffold printed in Example 31 is shown in FIG. 6. Fig. 6B is a partial enlarged view of a in Fig. 6A, and Fig. 6D is a partial enlarged view of b in Fig. 6C. It can be seen from FIGS. 6A to 6D that the two-dimensional bio-scaffold formed by printing in Example 31 has a stable structure. The biological scaffold with the two-dimensional structure shown in Figure 6C is stained with active cells. The micrograph after the staining process is shown in Figure 6E. It can be seen from Figure 6E that the biological scaffold with the two-dimensional structure has high cell activity and good performance. Biocompatibility and biological activity.

The three-dimensional structure biological scaffold printed in Example 31 is shown in FIG. 7. Fig. 7B is a partial enlarged view of Fig. 7A. It can be seen from FIGS. 7A to 7B that the three-dimensional bio-scaffold printed in Example 31 has a stable structure. The biological scaffold with the three-dimensional structure shown in Fig. 7A is subjected to active cell staining. The micrograph after the staining process is shown in Fig. 7C. It can be seen from Fig. 7C that the biological scaffold with a three-dimensional structure has high cell activity and good biological properties. Compatibility and biological activity.

The three-dimensional bionic organ model scaffold printed in Example 31 is shown in FIG. 8. It can be seen from FIG. 8 that the three-dimensional bionic organ model scaffold printed in Example 31 has a stable structure and excellent mechanical properties.

In addition, the performances of the two-dimensional structured biological scaffolds, three-dimensional porous structures, and three-dimensional bionic organ models formed by printing in other embodiments are similar to those of the biological scaffolds in embodiment 31. Therefore, the biological scaffold provided by the embodiments of the present invention has a stable structure, excellent mechanical properties, and excellent biocompatibility.

Furthermore, the three-dimensional biological scaffold provided in Example 32 was stained with living dead cells to observe the survival rate of the cells after printing, and the scaffold cultured in the incubator was changed to medium every two days, and the cells inside the scaffold were observed Survival and spreading and proliferation. The results of the cell experiment are shown in Figure 9. Figure 9 shows that the printed three-dimensional biological scaffold has a high survival rate of cells, and the cells show good spreading and proliferation in the later culture process.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement and improvement made within the spirit and principle of the present invention shall be included in the protection of the present invention. Within range.

Claims

A modified biopolymer includes a main chain of the main body of the biopolymer, and is characterized in that an ureidopyrimidinone group and a tyramine group are grafted on the main chain.
The modified biopolymer according to claim 1, wherein the ureidopyrimidinone group is grafted onto the main chain through the ureido group contained in the ureidopyrimidinone group; and /or

The tyramine group is grafted onto the main chain by -CO-NH-; and/or

The biopolymer body is a biopolymer containing amine groups and/or carboxyl groups.
The modified biopolymer according to claim 2, wherein the ureidopyrimidinone group is grafted onto the biopolymer by reacting the ureidopyrimidinone containing isocyanate groups with the biopolymer The main chain.
The modified biopolymer according to claim 2, wherein the ureidopyrimidinone group is obtained by combining the biopolymer grafted with the ureidopyrimidinone group with a tyramine group. The compound of the group undergoes a condensation reaction and is grafted onto the main chain.
The modified biopolymer according to any one of claims 1 to 4, wherein the content of the ureidopyrimidinone group in the modified biopolymer is 0.1-0.2 mM g -1 ;

The content of the tyramine group in the modified biopolymer is 0.1-0.5 mM g -1 ;

The biopolymer body includes at least one of gelatin, polyvinyl alcohol, alginic acid, hyaluronic acid, and carboxymethyl chitosan.
A method for preparing modified biopolymers includes the following steps:

Ureapyrimidinone containing isocyanate groups and the biopolymer body containing amine groups and/or carboxyl groups are subjected to a ureylation reaction in the first reaction solvent to generate ureidopyrimidinone grafted biopolymers;

The ureidopyrimidinone grafted biopolymer and the tyramine group-containing compound are subjected to a condensation reaction in a second reaction solvent containing a catalyst to obtain a modified ureidopyrimidinone group and a tyramine group Biopolymers.
The preparation method according to claim 6, wherein the conditions for the ureidolation reaction of the ureidopyrimidinone with isocyanate groups and the biopolymer in the first reaction solvent at least satisfy the following At least one condition:

The mass ratio of the ureidopyrimidinone containing isocyanate groups to the biopolymer is 1: (10-30);

The mass ratio of the biopolymer to the first reaction solvent is 1: (15-20);

The first reaction solvent is at least one of dimethyl sulfoxide, dimethyl formamide, carbon tetrachloride, and ether.
The preparation method according to claim 6, characterized in that the conditions for the condensation reaction of the ureidopyrimidinone grafted biopolymer and the tyramine group-containing compound in a second reaction solvent containing a catalyst are at least Meet at least one of the following conditions:

The mass ratio of the ureidopyrimidinone grafted biopolymer to the tyramine group-containing compound is 1: (0.3-0.6);

The mass ratio of the biopolymer grafted with ureidopyrimidinone to the second reaction solvent is 1: (15-20);

The catalyst is a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, 4-(4,6-dimethoxy) At least one of triazine-2-yl)-4-methylmorpholine hydrochloride;

The compound containing a tyramine group is at least one of tyramine hydrochloride, tyramine, dopamine hydrochloride, and 6-hydroxydopamine hydrochloride.
A bio-ink comprising a solvent and a biopolymer dissolved in the solvent, and is characterized in that it also includes a biofunctional component supported by the biopolymer, wherein the modified biopolymer is according to claim 1. -The modified biopolymer according to any one of 5 or the modified biopolymer prepared by the preparation method according to any one of claims 6-8.
The biological ink according to claim 9, wherein the biological functional component is cells, and the content of the cells in the biological ink is 2.1×10 5 cells mL -1 ; or/and

The mass concentration of the modified biopolymer in the bio-ink is 15%-25%.
The bio-ink according to claim 9 or 10, wherein the biological functional component is at least one of cells, growth factors, and drugs; or/and

The solvent is at least one of phosphate buffer solution and cell culture medium.
The application of the biological ink of any one of claims 9-11 in 3D printing.
A biological scaffold, characterized in that the biological scaffold is prepared from the biological ink according to any one of claims 9-11.
A method for preparing a biological scaffold includes the following steps:

The 3D printing process is performed with the biological ink of any one of claims 9-11 as a raw material.
The preparation method according to claim 14, wherein the conditions of the 3D printing are as follows:

The printing temperature is 10-43℃; and/or

The printing speed is 4-15 mm/s.
The preparation method according to claim 14 or 15, characterized in that: after the step of 3D printing processing, it further comprises enzymatic cross-linking of the bio-scaffold formed by printing in a solution containing hydrogen peroxide and horseradish peroxidase Reaction processing steps.