Modified polymer, preparation method of modified polymer, bio-ink and application of bio-ink

Abstract

The invention discloses a modified polymer, a preparation method of the modified polymer, bio-ink and application of the bio-ink, and relates to the field of 3D printing. The modified polymer mainly comprises a biopolymer, a first group and a second group, the first group and the second group are connected with the biopolymer, the biopolymer is subjected to methacrylic modification, pyrimidone is connected to the biopolymer, and therefore the modified polymer has good printability, and the printing performance is improved. The photocuring property is fast; the mechanical property after curing is relatively good. Meanwhile, as the temperature rises to 37 DEG C, multiple internal hydrogen bonds are opened, a large space is generated, cells can survive better and longer, and the relation of printability, mechanical strength and biological activity is successfully coordinated.

Description

Technical Field

[0001] The present application relates to the field of 3D printing, and in particular to a modified polymer and a preparation method thereof, biological ink and applications thereof. Background Art

[0002] Bio-3D printing is a manufacturing method that uses 3D printing to create personalized biological functional structures based on the biomimetic morphology, biological functions, cell growth microenvironment, etc. of biological materials (hydrogels, etc.) and cells.

[0003] The material used in biological 3D printing is called bio-ink. The important indicators that affect the quality of bio-ink include printability, mechanical properties and bioactivity. Among them, printability mainly depends on a variety of physical and chemical properties, such as good rheological properties; mechanical properties can provide sufficient mechanical support to maintain the original printed structure without fusion and collapse; bioactivity is not only reflected in the retention of the activity of the biological functional components (such as cells, etc.) carried during the printing and cross-linking stages, but also in the survival time of the biological functional components after the scaffold is formed.

[0004] At present, the base materials of bio-ink mainly include alginate, collagen, biopolymer (Gel), etc., but these materials cannot have good printability, mechanical properties and biological activity at the same time, and it is difficult to meet the needs of bio-ink. Summary of the invention

[0005] In view of this, the present application provides a modified polymer and a preparation method thereof, a bio-ink and an application thereof, aiming to provide a bio-ink substrate having excellent printability, mechanical strength and biological activity.

[0006] The embodiment of the present application is implemented as follows:

[0007] In a first aspect, the present application provides a modified polymer, comprising a biopolymer and a first group and a second group connected to the biopolymer, wherein the first group comprises a structure shown in the following structural formula (I), and the second group comprises a structure shown in the following structural formula (II); wherein * represents a connectable site.

[0008]

[0009] Optionally, in some embodiments of the present application, the biopolymer has a main chain and a connectable group connected to the main chain, and the connectable group includes at least one of a carboxyl group, a hydroxyl group, and an amino group;

[0010] The second group further includes -NH-CO-, and the second group is connected to the amino group through the -NH-CO-; and / or,

[0011] The first group is connected to the connectable group; and / or,

[0012] The biopolymer includes any one of gelatin, alginate, hyaluronic acid, chitosan, silk fibroin and polylysine.

[0013] Optionally, in some embodiments of the present application, the structural formula of the second group is as shown in formula (III).

[0014]

[0015] Wherein, R is a C1-C8 alkylene group.

[0016] Optionally, in some embodiments of the present application, in the modified polymer, the grafting rate of the first group is 5.0% to 95.0%.

[0017] Optionally, in some embodiments of the present application, in the modified polymer, the grafting rate of the second group is 5.0% to 95.0%.

[0018] In a second aspect, the present application also proposes a method for preparing a modified polymer, comprising the following steps:

[0019] providing a methacrylated biopolymer, a pyrimidinone derivative, and a first solvent;

[0020] mixing the methacrylated biopolymer, the pyrimidone derivative and the first solvent to react to obtain a modified polymer;

[0021] The pyrimidone derivatives include those having the structure shown in the following structural formula (II).

[0022]

[0023] Optionally, in some embodiments of the present application, the first solvent includes one or more of dimethyl sulfoxide, N,N-dimethylformamide, and tetrahydrofuran; and / or,

[0024] The mass ratio of the methacrylated biopolymer to the pyrimidone derivative is 1:0.008-0.6; and / or,

[0025] The reaction temperature is 10 to 60° C.; and / or,

[0026] The reaction time is 8 to 48 hours; and / or,

[0027] The reaction is carried out in an environment where the water content by mass is less than 0.1%; and / or,

[0028] The reaction is carried out in an oxygen-free environment; and / or,

[0029] The methacrylated biopolymer comprises methacrylated gelatin; and / or,

[0030] The pyrimidone derivatives include isocyanate-terminated 2-urea-4[H]-pyrimidone.

[0031] In a third aspect, the present application also proposes a bio-ink comprising a modified polymer, a photoinitiator, a biofunctional component and a solvent, wherein the modified polymer comprises the modified polymer described above, or is prepared by the preparation method described above.

[0032] Optionally, in some embodiments of the present application, in every 100 ml of the bio-ink, the mass of the modified polymer is 3 to 90 g; and / or,

[0033] In every 100 ml of the bio-ink, the mass of the photoinitiator is 0.0005 to 0.01 g; and / or,

[0034] The biological functional component includes cells, and the content of the cells in the biological ink is less than or equal to 1×10 8 cells / ml; and / or,

[0035] The solvent includes at least one of a buffer solution with a pH of 7 to 8 and a cell culture medium.

[0036] In a fourth aspect, the present application also proposes an application of the above-mentioned bio-ink in 3D printing.

[0037] The technical solution provided by the present application, by modifying the biopolymer with methacrylic acid, enables the modified polymer to be cross-linked and cured under photocatalysis, and the cross-linking method is mild, which helps to maintain the activity of the biological functional components in the cross-linking stage, and also enables the biological ink to adapt to most 3D printing processes, expanding its application range; at the same time, pyrimidone is connected to the biopolymer, and the grafted product has a ureido pyrimidone structure, so that a multiple hydrogen bond system can be formed between the modified polymer molecules, and a reversible hydrogen bond network that can be broken and reorganized is constructed, so that the modified polymer has good viscosity and good mechanical properties after curing. At the same time, the hydrogen bonds in the multiple hydrogen bond system remain connected at room temperature, and after being placed in a temperature environment suitable for the cultivation of biological functional components, the temperature rises, resulting in increased thermal motion, and the hydrogen bonds are destroyed, resulting in a larger space inside, which is convenient for the biological functional components to survive better and longer, effectively improving the biological activity of the biological functional components in the scaffold, and well coordinating the relationship between printability, mechanical strength and biological activity, solving the problem that the printability-mechanical strength and biological activity-mechanical strength of traditional biological ink materials are incompatible. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0039] Figure 1 is a comparison of H NMR spectra of GelMA and GelMA-UPy in Experimental Example 1;

[0040] Figure 2 is a comparison of infrared spectra of Gel, GelMA, UPy-NCO and GelMA-UPy in Experimental Example 1;

[0041] Figure 3 This is a schematic diagram of the synthesis route of GelMA proposed in one embodiment of the present application;

[0042] Figure 4 Schematic diagram of the synthesis route of GelMA-UPy proposed in one embodiment of the present application;

[0043] Figure 5 is a schematic diagram of the formation of a hydrogen bond network of GelMA-UPy in one embodiment of the present application;

[0044] Figure 6 is the viscosity-shear rate diagram of GelMA and GelMA-UPy in Experimental Example 2;

[0045] Figure 7 is the compressive stress-strain diagram of GelMA and GelMA-UPy hydrogels in Experimental Example 2;

[0046] Figure 8 is the compression modulus graph of GelMA and GelMA-UPy in Experimental Example 2;

[0047] Fig. 9 is the compressive strength diagram of GelMA and GelMA-UPy in Experimental Example 2;

[0048] Fig.10 is the FDA staining image of hydrogel-encapsulated BMSCs in Experimental Example 3;

[0049] Fig.11 This is a comparison chart of the number of viable cells during FDA staining of hydrogel-encapsulated BMSCs in Experimental Example 3;

[0050] Fig.12 This is the FDA / PI staining picture of the BMSCs-loaded tissue engineering scaffold in Experimental Example 3;

[0051] Fig.13The stent prepared in Example 2 of Example 1;

[0052] Fig.14 A stent prepared in Example 3 of Example 1;

[0053] Fig.15 This is another bracket prepared in Example 3 of Example 1. DETAILED DESCRIPTION

[0054] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. In addition, it should be understood that the specific implementation methods described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0055] In this application, unless otherwise specified, the directional words used, such as "upper" and "lower", refer specifically to the directions of the drawings in the accompanying drawings. In addition, in the description of the specification of this application, the term "including" means "including but not limited to".

[0056] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0057] In this application, "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0058] In the present application, "at least one" means one or more, and "plurality" means two or more. "At least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0059] Explanation of terms

[0060] In the present application, "gelatin" is a macromolecular hydrophilic colloid, which is a product of partial hydrolysis of collagen. The type (source) of gelatin is not particularly limited, and can be derived from connective tissues such as bones, cartilages and skins of animals such as pigs, fish, and cattle, as well as tanning industry waste, which is not limited in the present application. The molecular weight of the gelatin can be 40,000 to 100,000, 40,000 to 80,000, or 60,000 to 80,000. Figure 3 This is a schematic diagram that can reflect the structural characteristics of gelatin to a certain extent. As shown in the figure, Gel has a main chain, and the main chain is connected to multiple amino groups, multiple hydroxyl groups, multiple carboxyl groups, etc.

[0061] In the present application, "alkylene" refers to a group formed by the loss of two hydrogen atoms from an alkane, and may be a straight chain alkylene or a branched chain alkylene. C1-C8 alkylene refers to an alkylene with 1 to 8 carbon atoms, such as C1 alkylene, C2 alkylene, C3 alkylene, C4 alkylene, C5 alkylene, C6 alkylene, C7 alkylene or C8 alkylene, and may include, but are not limited to, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH(CH3)CH2-, -CH2(CH2)2CH2-, -CH2(CH2)3CH2-, -CH2(CH2)4CH2-, -CH2(CH2)5CH2-, -CH2(CH2)6CH2-, and the like.

[0062] Not only can a mild printing and cross-linking method be used to maintain cell activity, but also the long-term growth of cells inside the scaffold can be maintained. Existing bio-ink substrates, such as Gel, have insufficient performance and are difficult to meet the needs of bio-ink. At present, the method of adding other ingredients is often used to improve certain performances, but this method has the following disadvantages: on the one hand, it will increase the complexity of the ink system, and on the other hand, it cannot simultaneously meet the coordination of bio-ink with excellent printability, mechanical properties and bioactivity. However, studies have found that the scaffolds printed with bio-inks with high mechanical properties can provide sufficient mechanical support and have good printability, but at the same time, such scaffolds often limit the growth of cells inside them, which is not conducive to the long-term activity of cells, and the bioactivity of cells in the scaffolds. In other words, the printability-mechanical strength and bioactivity-mechanical strength of bio-inks are often contradictory. Therefore, the development of a bio-ink with excellent printability, mechanical strength and bioactivity is of great significance in the field of biological 3D printing.

[0063] In view of this, in a first aspect, the present application provides a modified polymer, which includes a biopolymer and a first group and a second group connected to the biopolymer, wherein the first group includes a structure shown in the following structural formula (I), and the second group includes a structure shown in the following structural formula (II); wherein * represents a connectable site.

[0064]

[0065] It can be understood that the structure represented by formula (I) and the structure represented by formula (II) can be directly connected to the biopolymer or connected through other connecting groups.

[0066] In some embodiments, the first group is connected to the connectable group. In actual preparation, methacrylic anhydride reacts with the biopolymer, and the first group is grafted onto the biopolymer to obtain a methacrylated biopolymer.

[0067] The biopolymer includes any one of polysaccharides, proteins and polypeptides such as gelatin, alginate, hyaluronic acid, chitosan, silk fibroin and polylysine; in comparison, gelatin has better biocompatibility. When the biopolymer is gelatin, the methacrylated biopolymer includes methacrylated gelatin.

[0068] In some embodiments, in the modified polymer, the grafting rate of the first group is 5.0% to 95.0%, for example, it can be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 90%, 99.9%. The grafting rate refers to the percentage of the content of the amidated connectable groups in the biopolymer to the total amount of connectable groups in the biopolymer. In some embodiments, the biopolymer is gelatin, and the first group is controlled to be grafted on the amino group of the gelatin by controlling the reaction conditions, such as the reaction temperature, so that the grafting rate is obtained by detecting the amino content and then calculating according to the following formula:

[0069]

[0070] In some embodiments, the second group further comprises -NH-CO-, and the second group is connected to the amino group through the -NH-CO-. Specifically, in some embodiments, the structural formula of the second group is as shown in formula (III).

[0071]

[0072] Wherein, R is a C1-C8 alkylene group.

[0073] Furthermore, in some embodiments, in the modified polymer, the grafting rate of the second group is 5.0% to 95.0%, for example, it can be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 90%, 99.9%. By controlling the grafting rate of the second group, the proportion of the hydrogen bond network can be controlled, thereby effectively regulating the viscosity, mechanical properties and biological activity of the biological ink.

[0074] Furthermore, in some embodiments, the biopolymer is gelatin, and in the modified polymer, the grafting rate of the first group is 60% to 65%, and the grafting rate of the second group is 20% to 34.9%. By controlling the ratio of the first group to the second group, it is helpful to coordinate the relationship between printability, mechanical strength and biological activity, and achieve a better balance.

[0075] By grafting the first group and the second group onto the biopolymer, a modified polymer can be prepared. In some embodiments, the modified polymer has a structure shown in the following structural formula (IV).

[0076]

[0077] In the present application, the biopolymer is modified by methacrylation so that the modified polymer can be cross-linked and cured under photocatalysis, so that the bio-ink can adapt to most 3D printing processes and expand its application range; at the same time, pyrimidone is connected to the biopolymer, and the grafted product has a ureido pyrimidone structure, such as Figure 5 As shown, multiple hydrogen bond systems can be formed between the modified polymer molecules to construct a reversible hydrogen bond network that can be broken and recombined, so that the modified polymer has good viscosity and good mechanical properties after curing; at the same time, the hydrogen bonds in the multiple hydrogen bond system remain connected at room temperature, and after being placed in a temperature environment suitable for the cultivation of biological functional components, the temperature rises, resulting in increased thermal motion, and the hydrogen bonds are destroyed, resulting in a larger space inside, which is convenient for the biological functional components to survive better and longer. In this application, not only are the two groups easy to graft onto biopolymers, the synthesis path is simple, and the modified polymer obtained is safe and non-toxic; the bio-ink prepared based on the modified polymer can achieve complex structure construction and long-term internal cell growth through biological 3D printing, and well coordinates the relationship between printability, mechanical strength and bioactivity, solving the problem that the printability-mechanical strength and bioactivity-mechanical strength of traditional bio-ink materials are incompatible.

[0078] In a second aspect, the present application also proposes a method for preparing a modified polymer, see Figure 4 , the preparation method comprises the following steps:

[0079] S10, providing a methacrylated biopolymer, a pyrimidone derivative, and a first solvent;

[0080] S20, mixing the methacrylated biopolymer, the pyrimidone derivative and the first solvent to react to obtain a modified polymer;

[0081] The pyrimidone derivatives include those having the structure shown in the following structural formula (II).

[0082]

[0083] In S10:

[0084] The methacrylated biopolymer can be prepared by the following steps: reacting a biopolymer solution with methacrylic anhydride to obtain a reaction solution; precipitating the reaction solution with an alcohol solvent, washing, and purifying the reaction solution to obtain a methacrylated biopolymer; wherein the biopolymer solution includes a biopolymer and a second solvent.

[0085] In some embodiments, the second solvent includes one or more of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), and tetrahydrofuran.

[0086] In some embodiments, the methacrylated biopolymer includes methacrylated gelatin. Methacrylated gelatin (GelMA) refers to gelatin modified with methacrylate. In the present application, in methacrylated gelatin, the degree of substitution of the first group can be 5.0% to 95.0% (i.e., 5.0% to 95.0% of the connectable groups on the gelatin molecule are amidated). The GelMA can be purchased directly on the market and has a wide range of sources. Using GelMA as a raw material is conducive to the industrialization of modified polymer production and facilitates the promotion and application of materials. GelMA can also be prepared by itself. When preparing the GelMA, the biopolymer is gelatin; and the reaction temperature is 30 to 60°C, and the reaction time is 1 to 6 hours.

[0087] In some embodiments, the reaction solution is precipitated with an alcohol solvent, and then washed and purified to obtain methacrylated gelatin, the steps comprising: mixing the reaction solution with an alcohol solvent to obtain a precipitate; mixing the precipitate with deionized water, heating at 30 to 60° C. to dissolve it, then dialyzing it with a dialysis bag with a molecular weight cutoff of 8000D to 14000D, and then centrifuging and drying to obtain methacrylated gelatin.

[0088] In some embodiments, the pyrimidone derivative has the following structural formula.

[0089]

[0090] Further, in a specific embodiment, the pyrimidone derivative includes isocyanate-terminated 2-urea-4[H]-pyrimidone (UPy-NCO), and UPy-NCO has the following structural formula.

[0091]

[0092] The pyrimidone derivative can be purchased on the market or prepared by itself. In some embodiments, the preparation method of the UPy-NCO comprises: mixing AMH (2-amino-4-hydroxy-6-methylpyrimidine, CAS: 3977-29-5) and HDI (1,6-hexamethylene diisocyanate, CAS: 822-06-0), and performing a reflux reaction under an inert gas environment to obtain UPy-NCO. The molar ratio of HDI to AMH is 1 to 20:1.

[0093] In some embodiments, the first solvent includes one or more of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), and tetrahydrofuran.

[0094] In step S20:

[0095] In some embodiments, the mass ratio of the methacrylated biopolymer to the pyrimidone derivative is 1:0.008-0.6; for example, it can be 1:0.008, 1:0.01, 1:0.015, 1:0.02, 1:0.024, 1:0.025, 1:0.026, 1:0.028, 1:0.03, 1:0.035, 1:0.04, 1:0.05, 1:0.055, 1:0.06, 1:0.07, 1:0.08, 1:0.09, 1:0.1, 1:0.15, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6 and values ​​between any two of the above values, so that the grafting rate of the second group can be effectively regulated. Furthermore, the mass ratio of the methacrylated biopolymer to the pyrimidone derivative is preferably 1:0.025 to 1:0.1.

[0096] In some embodiments, the reaction temperature is 10-60°C; for example, it can be 10°C, 15°C, 20°C, 23°C, 25°C, 27°C, 30°C, 32°C, 35°C, 40°C, 50°C, 60°C and values ​​between any two of the above values.

[0097] In some embodiments, the reaction time is 8 to 48 hours; for example, it can be 8 h, 10 h, 15 h, 18 h, 20 h, 22 h, 24 h, 25 h, 27 h, 30 h, 32 h, 36 h, 40 h, 48 h, and values ​​between any two of the above values.

[0098] In some embodiments, the reaction is carried out in an environment with a water content of less than 0.1% by mass; in other embodiments, the reaction is carried out in an oxygen-free environment. Controlling the water and oxygen content in the reaction environment can prevent hydrolysis or oxidation and promote the effective synthesis of the modified polymer. In specific implementation, a non-aqueous solvent can be used to provide a liquid environment for the reaction, and at the same time, an inert gas is introduced into the reaction system to place it in an anhydrous and oxygen environment; the inert gas includes but is not limited to nitrogen, helium, argon, etc.

[0099] In some embodiments, the step S20 specifically comprises: mixing the methacrylated biopolymer, the pyrimidone derivative and the first solvent, reacting to obtain a reaction liquid; mixing the reaction liquid and the third solvent to obtain a solid, washing the solid with anhydrous ethanol, and drying to obtain a modified polymer. The modified polymer can be stored away from light for later use. The third solvent includes but is not limited to one or two of acetone, pyridine, and dioxane.

[0100] In a third aspect, the present application proposes a bio-ink, which can be used for bio-printing, such as for making tissue regeneration scaffolds, biomimetic tissues, or biomimetic devices. Specifically, the bio-ink includes a modified polymer, a photoinitiator, a biofunctional component, and a solvent, and the modified polymer includes the modified polymer described above, or is prepared by the preparation method described above.

[0101] In some embodiments, the mass of the modified polymer in each 100 ml of the bio-ink is 3 to 90 g; for example, it can be 3 to 10 g, 8 to 15 g, 14 to 20 g, 18 to 30 g, 25 to 40 g, 35 to 50 g, 45 to 60 g, 55 to 70 g, 65 to 80 g, 78 to 90 g, etc. The mass of the photoinitiator in each 100 ml of the bio-ink is 0.0005 to 0.01 g; for example, it can be 0.0005 to 0.001 g, 0.0008 to 0.005 g, 0.003 to 0.01 g, etc.

[0102] In some embodiments, the photoinitiator can accelerate the cross-linking and curing of the bio-ink under light irradiation, including but not limited to one or more of phenyl (2,4,6-trimethylformyl) phosphate lithium salt (LAP), IC-2959 and VA086. The solvent includes at least one of a buffer solution of pH 7 to 8 and a cell culture medium; the buffer solution of pH 7 to 8 can be a PBS buffer; and the cell culture medium can be a serum-free α-MEM culture medium.

[0103] In some embodiments, the biofunctional component may include at least one of cells, growth factors, and drugs. In a specific embodiment, the biofunctional component includes cells, and the content of the cells in the bio-ink is 0×10 5 ~1×10 8 cells / ml; for example, 0.005×10 5 ~1×10 5 cells / ml、0.08×10 5 ~10×10 5 cells / ml、8×10 5 ~15×10 5 cells / ml、14×10 5 ~20×10 5 cells / ml、15×10 5 ~30×10 5 cells / ml、16×10 5 ~50×10 5 cells / ml、45×10 5~100×10 5 cells / ml、80×10 5 ~150×10 5 cells / ml、130×10 5 ~180×10 5 cells / ml、170×10 5 ~500×10 5 cells / ml、500×10 5 ~1×10 8 cells / ml, etc.

[0104] In a fourth aspect, the present application also proposes an application of the bio-ink as described above in 3D printing.

[0105] In some embodiments, 3D printing using the above-mentioned bio-ink can be achieved according to the following steps:

[0106] The bio-ink is loaded into the silo of the 3D printer, and the ink is printed to form a scaffold according to the pre-set modeling path, and then the scaffold is irradiated with light to be photo-crosslinked and cured. The light can be the light emitted by a blue-violet flashlight (wavelength of 365-405nm), and the irradiation time can be 20s-200s.

[0107] The technical scheme and technical effects of the present application are described in detail below through specific embodiments and comparative examples. The following embodiments are only some embodiments of the present application and do not specifically limit the present application.

[0108] Experimental Materials

[0109] (1) Porcine gelatin Type A and methacrylic anhydride (MA) were purchased from Sigma-Aldrich.

[0110] (2) Hexyl diisocyanate (HDI), 2-amino-4-hydroxy-6-methylpyrimidine (AHM), and sodium hydroxide (NaOH) were purchased from Aladdin Company;

[0111] (3) Dimethylformamide (DMF), n-pentane, and pyridine (anhydrous) were purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd., and phosphate buffer solution (PBS) was purchased from Hyclone Company, USA;

[0112] (4) Human BMSCs were purchased from Saiye Company;

[0113] (5) α-MEM medium was purchased from Hyclone Company;

[0114] (6) Fetal bovine serum (FBS) was purchased from Gibco.

[0115] Example 1

[0116] (1) Synthesis of GelMA: Weigh 20 g of Gel into a three-necked flask, add 400 mL of PBS, and stir at 50 °C for 1 h until the Gel is completely dissolved; add 160 mL of DMF to the flask; then add 30 mL of DMF and 1.6 mL of MA was added to the separatory funnel and gradually and slowly added to the three-necked flask, mixed, protected from light, and reacted at 40°C for 2h to obtain a mixture; the mixture was precipitated with 3L of anhydrous ethanol, and then the collected precipitate was squeezed dry, placed in a clean and dry beaker, deionized water was added, heated at 50°C and stirred continuously to completely dissolve it; a dialysis bag MD77 and a large beaker were prepared, the above solution was added to the dialysis bag, the dialysis bag was placed in a large beaker filled with deionized water, and dialyzed in a water bath at 50°C for 1 day and 40°C for 3 days; a 50mL centrifuge tube was prepared, the solution in the dialysis bag was poured into the centrifuge tube, centrifuged at 4000rpm / min for 10min, the supernatant was poured into a cylindrical drying dish with a diameter of 15cm; frozen at -20°C for 2 days; vacuum freeze-dried at -80°C for 3 days, sealed and stored in a drying oven for later use. After testing, the grafting rate of MA in the prepared GelMA was 62.50%±1.92%.

[0117] (2) Synthesis of UPy-NCO: Take a total of 100 g (593.5 mmol, 12.4 eq) of HDI into a flask and heat the oil bath to 100°C; introduce nitrogen into the reaction flask to replace the air therein, then add 6 g (48 mmol, 1 eq) of AHM into the reaction flask, turn on the mechanical stirrer for stirring, condense and reflux; heat at 100°C for 16 h with continuous mechanical stirring; then remove the oil bath, continue to pass nitrogen, and cool to room temperature; the resulting reaction solution is washed with n-pentane 3 times, 500 mL each time, and ultrasonically treated for 5 min; and washed with acetone 3 times, 500 mL each time; finally, the obtained white powder is filtered under reduced pressure and dried at 50°C for 2 days to obtain a white powder, which is stored in a vacuum drying oven for later use.

[0118] (3) Synthesis of GelMA-UPy: Add appropriate amount of molecular sieves to DMSO to remove water for at least 2 days; dissolve GelMA (6 g) in anhydrous DMSO (100 mL) at 55°C under nitrogen atmosphere by magnetic stirring, and cool after complete dissolution; after cooling the solution to room temperature, add 0.45 g of UPy-NCO and stir at room temperature for 24 h; after the reaction is completed, take a clean beaker, pour 500 mL of acetone into it, slowly pour the above reaction solution into the beaker, filter, add 500 mL of acetone to wash, ultrasonicate for 5 min, and repeat the operation three times; after the last acetone wash, dry it slightly, add 500 mL of anhydrous ethanol, filter, and repeat three times; put the obtained solid into a drying dish, vacuum dry it at 40°C for 24 h, and store it in a drying oven away from light for later use.

[0119] (4) Preparation of bio-ink: Weigh LAP, dissolve LAP in a mixture of PBS and serum-free α-MEM medium = 2:1 (v / v) on a clean bench, filter and sterilize to obtain LAP solution. Weigh GelMA-UPy material, irradiate it in a UV sterilizer for 90 min; then dissolve GelMA-UPy material in LAP solution on a clean bench, in a 65°C water bath for 30 min; adjust the pH of the solution to about 7.2 with prepared 1M NaOH to obtain a mixed solution.

[0120] Perform cell digestion and cell counting; take a certain amount of human BMSCs cells and add them to a new centrifuge tube, digest them at 1000rpm / min for 5min, and remove the supernatant; add LAP solution to resuspend the cells, calculate the volume of cells and residual liquid in advance, ensure the content of each component in the final biological ink, and prepare a cell suspension; take the mixed solution and keep it warm in a 37°C water bath, take the above cell suspension and mix it with the material solution to obtain a biological ink encapsulating cells, wherein the number of cells is 1.5 million / mL, the mass volume fraction of GelMA-UPy material is 15% (w / v, g / ml), and the mass volume fraction of LAP is 0.2% (w / v, g / ml).

[0121] (5) 3D printing:

[0122] Example 1: The bio-ink prepared above was placed into a cylindrical (inner diameter 5 mm, length 5 mm) transparent plastic mold, and a blue-violet flashlight (wavelength 365-405 nm) was used to irradiate the mold from opposite sides for 60 seconds to obtain a cylinder.

[0123] Example 2: Extrusion-type biological 3D printing: biological ink or cell-loaded biological ink is loaded into the barrel; a printing needle (diameter 350 μm) is installed to avoid light, and then loaded into the light-curing nozzle, and a scaffold is constructed by an extrusion-type biological 3D printer, and then light-cured to form, such as Fig.13 As shown, a model with clear lines and fine structure can be constructed.

[0124] Example 3: DLP 3D printing scaffold: Re-prepare the bio-ink, containing 0.25% (w / v) LAP and 0.025% (w / v) tartrazine, mix thoroughly, and protect from light. Add to the DLP printer platform, set the printing parameters, and perform light-curing printing. The result is as follows: Fig.14 and Fig.15 As shown, a model with clear lines and fine structure can be constructed.

[0125] Example 2

[0126] This example is basically the same as Example 1, except that in step (3) of this example: the amount of UPy-NCO added is changed from 0.45 g to 0.05 g, and other parameters and steps remain unchanged.

[0127] Example 3

[0128] This example is basically the same as Example 1, except that in step (3) of this example: the amount of UPy-NCO added is changed from 0.45 g to 0.15 g, and other parameters and steps remain unchanged.

[0129] Example 4

[0130] This example is basically the same as Example 1, except that in step (3) of this example: the amount of UPy-NCO added is changed from 0.45 g to 0.3 g, and other parameters and steps remain unchanged.

[0131] Example 5

[0132] This example is basically the same as Example 1, except that in step (3) of this example: the amount of UPy-NCO added is changed from 0.45 g to 0.6 g, and other parameters and steps remain unchanged.

[0133] Example 6

[0134] This example is basically the same as Example 1, except that in step (3) of this example: the amount of UPy-NCO added is changed from 0.45 g to 1.2 g, and other parameters and steps remain unchanged.

[0135] Comparative Example 1

[0136] This comparative example is basically the same as Example 1, except that in this comparative example, in step (3), GelMA is replaced by Gel. Accordingly, step (1) is omitted.

[0137] Results: In step (5), after being irradiated with light having a wavelength of 365 to 405 nm, no solidification to form a cylinder was possible.

[0138] Comparative Example 2

[0139] This comparative example is basically the same as Example 1, except that the product of this comparative example is GelMA. Accordingly, steps (2) and (3) are omitted.

[0140] Experimental Example 1

[0141] (I) GelMA and GelMA-UPy prepared in Example 1 were subjected to nuclear magnetic resonance testing. 1 HNMR spectrum see Figure 1 .

[0142] Figure 1In the figure, GelMA-UPy has a characteristic peak at 2.55ppm and 5.76ppm, which correspond to the d-methyl hydrogen on UPy-NCO (2.55ppm) and the e-hydrogen on UPy-NCO (5.76ppm), respectively, while GelMA has no characteristic peaks at these two locations, indicating that GelMA-UPy was successfully prepared.

[0143] (ii) Gel, GelMA, UPy-NCO and GelMA-UPy in Example 1 were subjected to infrared spectroscopy detection respectively. The results are as follows Figure 2 shown.

[0144] from Figure 2 It can be seen that in the spectrum of UPy-NCO, the wave number is 2275cm -1 A strong and sharp absorption peak appears at 3300 cm, which is caused by the stretching vibration absorption of the isocyanate group. In the spectrum of GelMA-UPy monomer, there is no absorption peak at this wave number, indicating that the isocyanate group has been completely reacted to generate the corresponding carbamate. -1 At the -NH characteristic peak, the absorbance of GelMA-UPy is smaller than that of GelMA, indicating that the remaining amino groups on GelMA are replaced by UPy-NCO.

[0145] (III) The GelMA-UPy obtained in step (3) of Examples 1 to 6 was taken and its UPy-NCO grafting rate was detected. The results are recorded in Table 1.

[0146] The detection method of grafting rate is: determination by ninhydrin colorimetry. Determination is carried out according to the Regen amino acid detection kit. Ninhydrin colorimetry is a method commonly used to determine the content of amino acids. This article uses the Regen amino acid detection kit for detection. Its detection principle is: under weak acid conditions, amino acids (Amino acid, AA) and ninhydrin can generate diketoindeneamine after co-heat reaction. The product diketoindeneamine is blue-purple with an absorption peak at a wavelength of 570nm, so the content of free amino groups can be calculated based on the absorbance value of the solution.

[0147] Table 1

[0148] Grafting rate Example 1 32.31％±0.94％ Example 2 20.19％±0.24％ Example 3 23.43％±0.62％ Example 4 29.17％±1.55％ Example 5 31.79％±3.23％ Example 6 31.3％±0.63％

[0149] Experimental Example 2

[0150] Rheological test: The GelMA-UPy bio-ink prepared in Example 1 and the GelMA bio-ink prepared in Comparative Example 2 were respectively subjected to rheological tests using the "viscosity-shear rate" test mode. The parameters were set as follows: temperature was 25°C; test gap was 0.2 mm; a PP25 plate was used for rotation test, and the shear rate was from 0.1 to 100 s-1 The test was performed according to logarithmic changes, and at least 3 replicate samples were set for each group. The prepared GelMA and GelMA-UPy-0.45 bio-inks were tested using an oscillating temperature scan. The temperature scan range was 15-40°C, 40 points were taken for testing, and at least 3 replicate samples were set for each group. The results are shown in Figure 2. Figure 6 shown.

[0151] Mechanical test: Soak the sample in PBS and perform compression test on the hydrogel at a compression speed of 0.1 mm / s until the gel block breaks. Each group of samples has at least 3 parallel samples. The curve is drawn using OriginPro software, and the slope is calculated based on the first 10% of the compressed sample to obtain the compression modulus of the sample, and the data is analyzed and summarized. The results are shown in the figure below. Figure 7-9 shown.

[0152] Figure 6 It can be seen that the maximum viscosity of GelMA and GelMA-UPy bio-inks is significantly different. The maximum viscosity of GelMA is 602Pa·s, while the maximum viscosity of GelMA-UPy can reach 2890Pa·s. This shows that compared with GelMA, the introduction of UPy can significantly improve the viscosity and shear thinning properties.

[0153] Figure 7 It can be seen that compared with GelMA, GelMA-UPy has higher compressive strength and toughness. GelMA breaks when compressed to 60%, while GelMA-UPy breaks when compressed to 70%. Figure 8 It can be seen that the compression modulus of GelMA-UPy (83.48±11.93 kPa) is significantly higher than that of GelMA (48.98±8.94 kPa). Fig. 9 As shown in Figure 2, the compressive strength of GelMA-UPy (398.00±123.32 kPa) is three times that of GelMA (129.42±30.88 kPa). The results show that the mechanical properties of GelMA-UPy are significantly improved.

[0154] Experimental Example 3

[0155] (1) Preparation of hydrogel-encapsulated cell scaffolds: 50 μL of cell-laden bio-ink was squeezed into a sterile dish and irradiated with a blue-violet flashlight (wavelength 365-405 nm) for 35 seconds to obtain a gel. The gel was transferred to a 24-well plate with a stainless steel spoon and cultured in the well plate containing α-MEM complete medium. FDA staining was performed after 14 days of culture. The results are as follows: Fig.10 , 11As shown in Table 2, the horizontal mark in the figure is the depth value of the gel from the surface to the inside, for example, 40-80 μm refers to the depth of 40-80 μm from the gel surface. The bio-ink is the bio-ink prepared in Example 1 and the bio-ink prepared in Comparative Example 2.

[0156] from Fig.10 and 11 It can be seen that after 14 days of culture, the number of cells inside GelMA gradually decreased from the outside to the inside, basically dropping to 25 / mm 2 The deep layer of GelMA-UPy still maintained a high number of cells, with the average number of cells per unit area basically maintained at 50 / mm 2 The above results indicate that compared with GelMA, the interior of GelMA-UPy is more suitable for long-term cell growth and survival.

[0157] (2) Referring to the method of experiment (1), the cell survival of the bio-inks of Examples 2 to 6 was investigated. The results are recorded in Table 2.

[0158] It can be seen from the table that in the scaffolds made of the bio-ink of each embodiment, after 14 days of culture, the deep layer of the gel still maintains a high number of cells, indicating that the interior of the GelMA-UPy proposed in the present application is more suitable for long-term cell growth and survival.

[0159] Table 2

[0160]

[0161]

[0162] The modified polymers and their preparation methods, bio-inks and their applications provided in the embodiments of the present application are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the methods and core ideas of the present application. At the same time, for technicians in this field, according to the ideas of the present application, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. A modified polymer, characterized in that It comprises a biopolymer and a first group and a second group connected to the biopolymer, wherein the first group comprises a structure shown in the following structural formula (I), and the second group comprises a structure shown in the following structural formula (II); wherein * represents a connectable site; 2. The modified polymer according to claim 1, characterized in that The biopolymer has a main chain and a connectable group connected to the main chain, wherein the connectable group includes at least one of a carboxyl group, a hydroxyl group and an amino group; The second group further includes -NH-CO-, and the second group is connected to the amino group through the -NH-CO-; and / or, The first group is connected to the connectable group; and / or, The biopolymer includes any one of gelatin, alginate, hyaluronic acid, chitosan, silk fibroin and polylysine.

3. The modified polymer according to claim 2, characterized in that The structural formula of the second group is shown in formula (III): Wherein, R is a C1-C8 alkylene group.

4. The modified polymer according to claim 1, characterized in that In the modified polymer, the grafting rate of the first group is 5.0% to 95.0%.

5. The modified polymer according to claim 1, characterized in that In the modified polymer, the grafting rate of the second group is 5% to 95%.

6. A method for preparing a modified polymer, characterized in that: The following steps are involved: providing a methacrylated biopolymer, a pyrimidinone derivative, and a first solvent; mixing the methacrylated biopolymer, the pyrimidone derivative and the first solvent to react to obtain a modified polymer; Wherein, the pyrimidone derivative includes a group structure shown in the following structural formula (II):

7. The preparation method according to claim 6, characterized in that: The first solvent includes one or more of dimethyl sulfoxide, N,N-dimethylformamide, and tetrahydrofuran; and / or, The mass ratio of the methacrylated biopolymer to the pyrimidone derivative is 1:0.008-0.6; and / or, The reaction temperature is 10 to 60° C.; and / or, The reaction time is 8 to 48 hours; and / or, The reaction is carried out in an environment where the water content by mass is less than 0.1%; and / or, The reaction is carried out in an oxygen-free environment; and / or, The methacrylated biopolymer comprises methacrylated gelatin; and / or, The pyrimidone derivatives include isocyanate-terminated 2-urea-4[H]-pyrimidone.

8. A biological ink, characterized in that: The invention comprises a modified polymer, a photoinitiator, a biological functional component and a solvent, wherein the modified polymer comprises the modified polymer according to any one of claims 1 to 5, or is prepared by the preparation method according to claim 6 or 7.

9. The biological ink according to claim 8, characterized in that: In every 100 ml of the bio-ink, the mass of the modified polymer is 3 to 90 g; and / or, In every 100 ml of the bio-ink, the mass of the photoinitiator is 0.0005 to 0.01 g; and / or, The biological functional component includes cells, and the content of the cells in the biological ink is less than or equal to 1×10 8 cells / ml; and / or, The solvent includes at least one of a buffer solution with a pH of 7 to 8 and a cell culture medium.

10. Use of the bio-ink according to claim 8 or 9 in 3D printing.

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