Conductive bio-ink with controllable cell distribution function and preparation method of conductive bio-ink

Abstract

The invention relates to bio-ink, microspheres for bio-ink, and preparation methods of the bio-ink and the microspheres. The bio-ink comprises: a microsphere comprising a methacrylated gelatin and a first conductive polymer, the microsphere optionally comprising a first cell; a hydrogel comprising a methacrylated gelatin and a second conductive polymer, the hydrogel optionally comprising second cells, the microspheres having pores. The bio-ink provided by the invention has good biocompatibility, conductivity, rheological property and mechanical property, and can promote interaction among biological materials.

Description

Technical Field

[0001] The present application relates to the field of biomaterials, in particular to bio-ink, microspheres for bio-ink, and methods for preparing the same. Background Art

[0002] 3D bioprinting technology enables precise and controlled printing of tissue structures. By creating 3D cell-laden structures in vitro, it offers a novel approach for regenerative medicine and tissue engineering. In recent years, it has also seen a variety of applications in organ reconstruction and tissue regeneration. There are two main approaches to 3D bioprinting technology. The first strategy, 3D printing, involves printing materials such as plastics, metals, composites, and polymers. Engineered scaffolds are typically developed by seeding cells onto porous, acellular 3D scaffolds after printing. The second strategy, 3D bioprinting, involves mixing cells with biomaterials to co-print tissue structure scaffolds. During the bioprinting process, the bioink must ensure that the biomaterials contained within it maintain biological viability or proliferation capacity. Ideally, the bioink should possess good rheological and biological properties, as well as mechanical properties. The bioink should be cross-linked or stabilized during or immediately after bioprinting to achieve the final shape and structure of the designed structure.

[0003] There are many types of bio-inks currently used in 3D bioprinting, with different functions, but they generally lack the following two functions:

[0004] First, after printing, it can provide cells with a stable three-dimensional spatial structure, creating an extracellular microenvironment to achieve orderly cell arrangement and provide conditions for multiple cell interactions. Regarding the first requirement, most current studies simply mix a single cell type into the bio-ink. During the printing process, three-dimensional scaffolds are printed layer by layer using bio-inks loaded with different cells, achieving a layered distribution of cells at different printing levels. However, this method has certain limitations. The cells are distributed between different layers at long distances, and only a small number of cells at the contact surface can interact. The majority of cells in the middle of the layer do not interact, which fails to truly simulate the physiological distribution of cells in the body. Another method is to mix two types of cells into a bio-ink simultaneously for printing to achieve a co-distribution of the two types of cells. However, this method often results in a disordered arrangement of cells, unable to form a specific spatial distribution, and weak cell interaction. Therefore, there is a lack of an ideal bio-ink with a unique internal spatial structure that can achieve the orderly arrangement and distribution of one or more cells, enhance the interaction between different cells, and thus construct a more biomimetic macro-micro dual spatial structure 3D printed scaffold.

[0005] Second, they must possess specialized properties to provide the necessary physical and biochemical signals. Regarding the second requirement, tissue regeneration requires a variety of physiological signals, including biochemical, mechanical, and electrical signals. Tissue damage often alters local physiological electrical signals, thereby interfering with intercellular electrical signal transmission. By introducing exogenous electrical signals, altered intercellular electrical signaling can be compensated for, promoting stem cell adhesion, migration, proliferation, and differentiation, thereby promoting tissue regeneration. However, currently available hydrogel matrices are generally non-conductive or have very low conductivity, which hinders intercellular electrical signal communication. This has prompted researchers to focus on conductive bioinks. Conductive hydrogels are prepared by incorporating conductive polymers, conductive nanoparticles, or other conductive elements into hydrogel systems through various strategies to create three-dimensional conductive hydrogels with electrical conductivity and high water content. However, these composite conductive hydrogels suffer from drawbacks such as uncertain polymerization residues, high biotoxicity, and non-biodegradability, limiting their biomedical applications. Therefore, there is a need to identify suitable conductive hydrogels for the preparation of conductive bioinks that can facilitate intercellular electrical signal communication, mimic the physiological microenvironment of electroactive tissues, and ultimately effectively promote cell activity and tissue regeneration.

[0006] Taking the above factors into consideration, the present invention proposes to synthesize a hydrogel with conductive functionality by polymerizing methacrylated gelatin (GelMA), which has good biocompatibility, with conductive polymer monomers. The hydrogel is prepared into microspheres using microfluidics technology, and then the microspheres and hydrogel are uniformly mixed to form a conductive bio-ink with a microsphere-gel composite spatial structure. The microspheres and hydrogel can be combined with various cells, tissues, and other biomaterials. This unique spatial structure can enhance the interaction between biomaterials, thereby achieving in vitro reconstruction of biomaterials, and therefore has great potential in tissue engineering repair and reconstruction. Summary of the Invention

[0007] In a first aspect, the present application provides a bio-ink, which may comprise:

[0008] microspheres comprising methacrylated gelatin and a first conductive polymer, the microspheres optionally comprising first cells;

[0009] a hydrogel comprising methacrylated gelatin and a second conductive polymer, the hydrogel optionally comprising a second cell, and

[0010] The microspheres have pores.

[0011] In a second aspect, the present application provides a method for preparing microspheres, which may include:

[0012] adding a first initiator to a solution comprising methacrylated gelatin and monomers of a conductive polymer to form a conductive prepolymer-grafted methacrylated gelatin;

[0013] drying the conductive prepolymer-grafted methacryloyl gelatin to form a porous conductive prepolymer-grafted methacryloyl gelatin,

[0014] adding the porous conductive prepolymer-grafted methacrylated gelatin to a solvent and then adding a second initiator to form a first hydrogel comprising the second initiator, the first hydrogel optionally comprising cells;

[0015] The first hydrogel and an oil phase solution containing mineral oil and a surfactant are mixed and formed into a microsphere-type gel,

[0016] irradiating the microsphere-type gel with ultraviolet light to form a microsphere precursor;

[0017] further polymerizing the conductive prepolymer in the microsphere precursor by a third initiator to form microspheres,

[0018] The microspheres have pores.

[0019] In a third aspect, the present application provides a method for preparing a bio-ink, which may include:

[0020] adding a first initiator to a solution comprising methacrylated gelatin and monomers of a conductive polymer to form methacrylated gelatin comprising a conductive prepolymer graft;

[0021] drying the conductive prepolymer-grafted methacryloyl gelatin to form a porous conductive prepolymer-grafted methacryloyl gelatin,

[0022] adding the porous conductive prepolymer-grafted methacrylated gelatin in a solvent and then adding a second initiator to form a first hydrogel comprising the second initiator, the first hydrogel optionally comprising cells;

[0023] Mixing the microspheres according to the present application with the first hydrogel, and then irradiating with ultraviolet light to form a second hydrogel;

[0024] A third initiator is added to the second hydrogel to further polymerize the conductive prepolymer in the second hydrogel, thereby obtaining the bio-ink.

[0025] In a fifth aspect, the present application provides a method for preparing biomaterials for tissue repair, cell culture or organ reconstruction, which includes 3D printing the bio-ink according to the present application.

[0026] In a sixth aspect, the present application provides the use of the bio-ink according to the present application in tissue repair, cell culture, and organ reconstruction. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The embodiments illustrated herein will be further described below with reference to the accompanying drawings. However, the accompanying drawings are only for allowing those skilled in the art to better understand the concept of the present invention and are not intended to limit the scope of the present invention.

[0028] Figure 1 A photograph of dry, pure conductive polymer-grafted methacrylated gelatin is shown ( Figure 1 A) and its Fourier transform infrared spectrum ( Figure 1 B).

[0029] Figure 2 The results show that the 5% methacrylated gelatin-polypyrrole (GelMA-PPy) hydrogel ( Figure 2 B) and 5% GelMA hydrogel ( Figure 2 A) When connected to LED light; the resistivity of 5% GelMA-PPy and 5% GelMA hydrogel ( Figure 2 C); and the conductivity of 5% GelMA-PPy and 5% GelMA hydrogels ( Figure 2 D).

[0030] Figure 3 The microscopic images of GelMA-PPy microspheres formed at different flow rates (ie, different volume ratios of oil phase solution to water phase solution) are shown. Figure 3 Scale bar in = 200 μm.

[0031] Figure 4 The particle size of GelMA-PPy microspheres in the oil phase is shown ( Figure 4 A and B); Particle size of GelMA-PPy microspheres in aqueous phase ( Figure 4 C and D); and the particle size comparison of GelMA-PPy microspheres in oil phase solution and aqueous phase solution ( Figure 4 E), where Figure 4 Scale bar in = 200 μm.

[0032] Figure 5 The effect of different concentrations of FeCl3 on biocompatibility is shown, where Figure 5 Scale bar in = 200 μm.

[0033] Figure 6 The rheological test results of the bio-ink according to the present application are shown ( Figure 6 of A); and the effect of shear rate on viscosity ( Figure 6 B).

[0034] Figure 7 A: pure GelMA-PPy microspheres; B: cell-loaded GelMA-PPy microspheres, where Figure 7 Scale bar in = 100 μm.

[0035] Figure 8 The picture of live / dead staining of cell-loaded GelMA-PPy microspheres is shown. Figure 8 Scale bar in = 200 μm.

[0036] Figure 9 A: GelMA-PPy microgel bio-ink extruded in a 3D printer ( Figure 9 A); and structures of different shapes formed by 3D printing ( Figure 9 B, C and D), where Figure 9 The ruler in the figure = 1 cm. DETAILED DESCRIPTION

[0037] Hereinafter, the concept of the present invention will be further elaborated according to specific embodiments. However, the specific embodiments listed are only for illustrative purposes and are not intended to limit the scope of the present invention. Those skilled in the art will recognize that the specific features in any of the following embodiments can be used for any other embodiment, as long as it does not deviate from the gist of the present invention.

[0038] The following description is provided to better define this application and to guide those skilled in the art in practicing this application. Unless otherwise specified, terms are to be understood according to conventional usage by those skilled in the relevant art. All patent documents, academic papers, and other publications cited herein are incorporated herein by reference in their entirety.

[0039] definition

[0040] Where a numerical range is given herein, the range includes its endpoints, and all individual integers and fractions within the range, and also includes each narrower range formed by all possible combinations of those endpoints and internal integers and fractions to form subgroups of the larger numerical group within the range to the same extent, just as each of those narrower ranges is explicitly given. For example, the ultraviolet light irradiation time of 10 min to 15 min means that the ultraviolet light irradiation time can be 10 min, 11 min, 12 min, 13 min, 14 min, or 15 min, etc., and ranges formed therefrom, etc.

[0041] As used herein, the terms “about,” “approximately,” or “approximately” when modifying a numerical value refer to values ​​within a range of ±5% of the modified numerical value.

[0042] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance can, but need not, occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0043] In this application, for the sake of simplicity, an ink is always defined as a bioink, even if it does not contain biological material.

[0044] Bioink

[0045] The present application provides a bio-ink, which may include:

[0046] microspheres comprising methacrylated gelatin and a first conductive polymer;

[0047] a hydrogel comprising methacrylated gelatin and a second conductive polymer, and

[0048] The microspheres have pores.

[0049] In embodiments, the microspheres may further comprise first cells. In embodiments, the hydrogel may further comprise second cells.

[0050] In an embodiment, the first conductive polymer and the methacrylated gelatin in the microspheres may be connected to each other via a chemical bond. In an embodiment, the second conductive polymer and the methacrylated gelatin in the hydrogel may be connected to each other via a chemical bond.

[0051] In embodiments, at least a portion of the pores of the microspheres may be filled with hydrogel. In some embodiments, all of the pores of the microspheres may be filled with hydrogel.

[0052] In an embodiment, the monomer of the first conductive polymer and the monomer of the second conductive polymer can be each independently selected from pyrrole, aniline, thiophene, fluorene, carbazole, and derivatives thereof. In an embodiment, the monomer of the first conductive polymer and the monomer of the second conductive polymer can be the same as or different from each other. For example, the monomer of the first conductive polymer can be pyrrole and the monomer of the second conductive polymer can be aniline, or the monomer of the first conductive polymer and the monomer of the second conductive polymer can both be pyrrole.

[0053] In embodiments, the first cell and the second cell may be the same or different from each other. In embodiments, the first cell and the second cell may be each independently selected from a neuronal cell, a vascular endothelial cell, a stem cell, a fibroblast, or any combination thereof. For example, the first cell may be a vascular endothelial cell and the second cell may be a neuronal cell, or the first cell and the second cell may both be neuronal cells, or the first cell may be a neuronal cell and the second cell may be a vascular endothelial cell, or the first cell and the second cell may both be vascular endothelial cells.

[0054] In an embodiment, the content of the methacryloylated gelatin and the second conductive polymer in the hydrogel can be 5% w / v to 7.5% w / v, 5.5% w / v to 7.0% w / v, or 6.0% w / v to 6.5% w / v, relative to the volume of the hydrogel, for example, 5% w / v, 5.2% w / v, 5.4% w / v, 5.5% w / v, 5.6% w / v, 5.8% w / v, 6.0% w / v, 6.2% w / v, 6.4% w / v, 6.5% w / v, 6.6% w / v, 6.8% w / v, 7.0% w / v, 7.2% w / v, 7.4% w / v, or 7.5% w / v.

[0055] In an embodiment, the particle size of the microspheres can be 150 μm to 500 μm, 160 μm to 480 μm, 180 μm to 450 μm, 200 μm to 400 μm, 250 μm to 350 μm, or 280 μm to 320 μm, for example, 150 μm, 155 μm, 160 μm, 165 μm, 170 μm, 175 μm, 180 μm, 185 μm, 190 μm. , 195μm, 200μm, 210μm, 215μm, 220μm, 225μm, 230μm, 235μm, 240μm, 245μm, 250μm, 260μm, 280μm, 300μm, 320μm, 340μm, 360μm, 380μm, 400μm, 420μm, 440μm, 460μm, 480μm, or 500μm.

[0056] In an embodiment, the content of microspheres can be 80% to 90%, or 83% to 88%, for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% relative to the total weight of the bio-ink.

[0057] In an embodiment, the hydrogel content can be 10% to 20%, or 13% to 18%, for example 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, relative to the total weight of the bio-ink.

[0058] In embodiments, the microspheres and the hydrogel may each independently have an electrical conductivity of 1.2 to 1.8 S / m, or 1.4 to 1.6 S / m, such as 1.2 S / m, 1.3 S / m, 1.4 S / m, 1.5 S / m, 1.6 S / m, 1.7 S / m, or 1.8 S / m. In embodiments, the size of the pores in the microspheres may be 5 to 20 μm, or 10 to 15 μm, such as 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, or 20 μm.

[0059] In an embodiment, the first conductive polymer may be present in an amount of 20% to 35% relative to the total weight of the microspheres. In an embodiment, the first conductive polymer may be present in an amount of 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% relative to the total weight of the microspheres.

[0060] In an embodiment, the second conductive polymer may be present in an amount of 20% to 35% relative to the total weight of the hydrogel. In an embodiment, the second conductive polymer may be present in an amount of 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% relative to the total weight of the microspheres.

[0061] In some embodiments, the conductivity of the bio-ink can be 1.2 to 1.8 S / m, or 1.4 to 1.6 S / m, for example, 1.2 S / m, 1.3 S / m, 1.4 S / m, 1.5 S / m, 1.6 S / m, 1.7 S / m, or 1.8 S / m.

[0062] Preparation of microspheres

[0063] The present application provides a method for preparing microspheres for bio-ink, which may include:

[0064] adding a first initiator to a solution comprising methacrylated gelatin and monomers of a conductive polymer to form a conductive prepolymer-grafted methacrylated gelatin;

[0065] The conductive prepolymer-grafted methacryloyl gelatin is dried to obtain a porous conductive prepolymer-grafted methacryloyl gelatin.

[0066] adding a porous conductive prepolymer-grafted methacrylated gelatin to a solvent, and then adding a second initiator to form a first hydrogel containing the second initiator;

[0067] The first hydrogel and an oil phase solution containing mineral oil and a surfactant are mixed and formed into a microsphere-type gel,

[0068] irradiating the microsphere-type gel with ultraviolet light to form a microsphere precursor;

[0069] The conductive prepolymer in the microsphere precursor is further polymerized by a third initiator to form microspheres,

[0070] The microspheres have pores.

[0071] In an embodiment, the second initiator is a photoinitiator. In an embodiment, the second initiator is selected from lithium phenyl-2,4,6-trimethylbenzoyl phosphite (LAP), dibenzoyl ketone, benzil, α,α'-ethoxyacetophenone, benzophenone, 4-methylbenzophenone, phenyl bis(2,4,6-trimethylbenzoyl)phosphine oxide, 2,4,6-trimethylbenzoyl-diphenylphosphine oxide, 1-hydroxy-cyclohexyl-phenyl ketone, or any combination thereof.

[0072] In an embodiment, the first initiator and the third initiator may be the same as or different from each other. In an embodiment, the first initiator and the third initiator are each independently selected from: a persulfate selected from ammonium persulfate, sodium persulfate and potassium persulfate; an organic peroxide initiator selected from benzoyl peroxide, lauroyl peroxide, cumene hydroperoxide, tert-butyl hydroperoxide, di-tert-butyl peroxide, diisopropyl peroxide, tert-butyl perbenzoate, methyl ethyl ketone peroxide, cyclohexanone peroxide, diisopropyl peroxydicarbonate and dicyclohexyl peroxydicarbonate; an azo initiator selected from azobisisobutyronitrile, azobisisovaleronitrile and azobisisoheptonitrile; ferric chloride; or any combination thereof.

[0073] In an embodiment, the monomers of the conductive polymer are selected from pyrrole, aniline, thiophene, styrene, fluorene, carbazole, and derivatives thereof.

[0074] In an embodiment, the method may further comprise adding cells to the first hydrogel, such that the first hydrogel comprises both the second initiator and the cells.

[0075] In some embodiments, the cells can be selected from neuronal cells, vascular endothelial cells, stem cells, fibroblasts, or any combination thereof.

[0076] In an embodiment, the particle size of the microspheres can be 150 μm to 500 μm, 160 μm to 480 μm, 180 μm to 450 μm, 200 μm to 400 μm, 250 μm to 350 μm, or 280 μm to 320 μm, for example, 150 μm, 155 μm, 160 μm, 165 μm, 170 μm, 175 μm, 180 μm, 185 μm, 190 μm, 195μm, 200μm, 210μm, 215μm, 220μm, 225μm, 230μm, 235μm, 240μm, 245μm, 250μm, 260μm, 280μm, 300μm, 320μm, 340μm, 360μm, 380μm, 400μm, 420μm, 440μm, 460μm, 480μm, or 500μm. In some embodiments, the size of the pores in the microspheres can be 5 to 20 μm, or 10 to 15 μm, for example 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, or 20 μm.

[0077] In an embodiment, the solvent may be selected from PBS solution and DMEM culture medium.

[0078] In embodiments, the volume ratio of solvent to second initiator can be 8:1 to 10:1, or 8.5:1 to 9.5:1, e.g., 8:1, 8.2:1, 8.5:1, 8.8:1, 9.0:1, 9.2:1, 9.5:1, 9.8:1, or 10:1.

[0079] In an embodiment, the volume ratio of mineral oil to surfactant in the oil phase solution may be 96:4 to 90:10.

[0080] In an embodiment, the volume ratio of the second hydrogel to the oil phase solution in the microsphere-type gel can be 1:2 to 1:10, 1:3 to 1:8, or 1:4 to 1:6, for example, 1:2.0, 1:2.5, 1:3.0, 1:3.5, 1:4.0, 1:4.5, 1:4.8, 1:5.0, 1:5.2, 1:5.4, 1:5.5, 1:5.8, 1:6.0, 1:6.5, 1:7.0, 1:7.5, 1:8.0, 1:8.5, 1:9.0, 1:9.5, or 1:10.

[0081] In an embodiment, the first initiator may be present in an amount of 15% to 30%, or 18% to 20%, for example 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, relative to the weight of the monomers of the conductive polymer.

[0082] In an embodiment, the content of the second initiator is 8% to 12.5%, or 9% to 11%, relative to the weight of the methacrylated gelatin, for example 8.2%, 8.5%, 8.8%, 9.0%, 9.2%, 9.5%, 9.8%, 10.0%, 10.0%,.

[0083] In an embodiment, the third initiator is present in an amount of 0.05M to 0.2M, or 0.10M to 0.15M, such as 0.05M, 0.08M, 0.10M, 0.12M, 0.15M, 0.18M, ​​or 0.2M.

[0084] In an embodiment, the microsphere-shaped gel can be formed by microfluidics technology. In an embodiment, the microsphere-shaped gel is formed by a microfluidic chip.

[0085] Preparation of bio-ink

[0086] The present application provides a method for preparing a bio-ink, which may include:

[0087] adding a first initiator to a solution comprising methacrylated gelatin and monomers of a conductive polymer to form methacrylated gelatin comprising a conductive prepolymer graft;

[0088] drying the conductive prepolymer-grafted methacryloyl gelatin to obtain porous conductive prepolymer-grafted methacryloyl gelatin;

[0089] adding a porous conductive prepolymer-grafted methacrylated gelatin to a solvent, and then adding a second initiator to form a first hydrogel containing the second initiator;

[0090] The microspheres according to the present application are mixed with a first hydrogel, and then irradiated with ultraviolet light to form a second hydrogel;

[0091] A third initiator is added to the second hydrogel to further polymerize the conductive prepolymer in the second hydrogel, thereby obtaining a bio-ink.

[0092] In an embodiment, the bio-ink may comprise cells. In an embodiment, the microspheres may further comprise first cells. In an embodiment, the first hydrogel may comprise second cells. In an embodiment, the first cells and the second cells may be the same or different from each other. In an embodiment, the first cells and the second cells may each be independently selected from neurons, vascular endothelial cells, stem cells, fibroblasts, or any combination thereof. For example, the first cell may be an endothelial cell and the second cell may be a neuronal cell, or the first cell and the second cell may both be neuronal cells, or the first cell may be a neuronal cell and the second cell may be a vascular endothelial cell, or the first cell and the second cell may both be vascular endothelial cells.

[0093] In an embodiment, the first initiator and the third initiator may be the same or different from each other. In some embodiments, the first initiator and the third initiator may be independently selected from: a persulfate selected from ammonium persulfate, sodium persulfate, and potassium persulfate; an organic peroxide initiator selected from benzoyl peroxide, lauroyl peroxide, cumene hydroperoxide, tert-butyl hydroperoxide, di-tert-butyl peroxide, diisopropyl peroxide, tert-butyl perbenzoate, methyl ethyl ketone peroxide, cyclohexanone peroxide, diisopropyl peroxydicarbonate, and dicyclohexyl peroxydicarbonate; an azo initiator selected from azobisisobutyronitrile, azobisisovaleronitrile, and azobisisoheptonitrile; FeCl3; or any combination thereof. In some embodiments, the second initiator is a photoinitiator. In an embodiment, the second initiator can be selected from lithium phenyl-2,4,6-trimethylbenzoyl phosphite, dibenzoyl ketone, benzil, α,α'-ethoxyacetophenone, benzophenone, 4-methylbenzophenone, phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide, 2.4,6-trimethylbenzoyl-diphenylphosphine oxide, 1-hydroxy-cyclohexyl-phenyl ketone, or any combination thereof.

[0094] In an embodiment, the first conductive polymer and the methacrylated gelatin in the microspheres may be connected to each other via a chemical bond. In an embodiment, the second conductive polymer and the methacrylated gelatin in the hydrogel may be connected to each other via a chemical bond.

[0095] In embodiments, at least a portion of the pores of the microspheres may be filled with hydrogel. In some embodiments, all of the pores of the microspheres may be filled with hydrogel.

[0096] In an embodiment, the formation of the methacryloylated gelatin containing the conductive prepolymer grafts may be carried out at a temperature in the range of 35° C. to 42° C., e.g., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., or 42° C. In an embodiment, the formation of the methacryloylated gelatin containing the conductive prepolymer grafts may be carried out for 2 to 4 hours, e.g., 2 hours, 2.5 hours, 3 hours, 3.5 hours, or 4 hours.

[0097] In an embodiment, the UV light exposure time can be 25 to 40 seconds, or 30 to 35 seconds, for example 25 seconds, 28 seconds, 30 seconds, 32 seconds, 34 seconds, 35 seconds, 36 seconds, 38 seconds or 40 seconds.

[0098] In embodiments, polymerization of the conductive prepolymer may be carried out at a temperature of 20 to 37°C, or 25 to 30°C, for example 20°C, 22°C, 25°C, 28°C, 30°C, 32°C, 35°C, or 37°C.

[0099] In an embodiment, the polymerization time of the conductive prepolymer may be 2 to 10 minutes, for example 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes.

[0100] In an embodiment, the monomer of the conductive polymer can be selected from pyrrole, aniline, thiophene, styrene, fluorene, carbazole, and derivatives thereof. For example, the monomer of the conductive polymer can be pyrrole. For example, the monomer of the conductive polymer can be aniline. For example, the monomer of the conductive polymer can be thiophene.

[0101] In an embodiment, the particle size of the microspheres is 150 μm to 500 μm, 160 μm to 480 μm, 180 μm to 450 μm, 200 μm to 400 μm, 250 μm to 350 μm, or 280 μm to 320 μm, for example, 150 μm, 155 μm, 160 μm, 165 μm, 170 μm, 175 μm, 180 μm, 185 μm, 190 μm, 195μm, 200μm, 210μm, 215μm, 220μm, 225μm, 230μm, 235μm, 240μm, 245μm, 250μm, 260μm, 280μm, 300μm, 320μm, 340μm, 360μm, 380μm, 400μm, 420μm, 440μm, 460μm, 480μm, or 500μm.

[0102] In embodiments, the size of the pores in the microspheres can be 5 to 20 μm, or 10 to 15 μm, for example 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, or 20 μm.

[0103] In an embodiment, the content of microspheres relative to the total weight of the bio-ink is 80% to 90%, for example 80%, 81%, 82%, 83%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%.

[0104] In an embodiment, the first hydrogel comprises 10% to 20% by weight relative to the total weight of the bio-ink, for example, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.

[0105] In an embodiment, the solvent may be selected from PBS solution and DMEM culture medium.

[0106] In embodiments, the volume ratio of solvent to second initiator can be 8:1 to 10:1, or 8.5:1 to 9.5:1, e.g., 8:1, 8.2:1, 8.5:1, 8.8:1, 9.0:1, 9.2:1, 9.5:1, 9.8:1, or 10:1.

[0107] In embodiments, the conductivity of the first hydrogel may be 1.2 to 1.8 S / m, or 1.4 to 1.6 S / m, e.g., 1.2 S / m, 1.3 S / m, 1.4 S / m, 1.5 S / m, 1.6 S / m, 1.7 S / m, or 1.8 S / m.

[0108] In an embodiment, the present application provides microspheres for bio-ink, which are formed by the method for preparing microspheres for bio-ink according to an embodiment of the present application.

[0109] In an embodiment, the present application provides uses of the bio-ink according to the present application in tissue repair, cell culture, and organ reconstruction.

[0110] In an embodiment, the bio-ink according to the present application has good biocompatibility. In an embodiment, the bio-ink according to the present application has good electrical conductivity, which can help accelerate the bioelectrical conduction of neuronal cells and stimulate cell proliferation. In an embodiment, the bio-ink according to the present application has good rheological properties. In an embodiment, the bio-ink according to the present application has good mechanical properties. In an embodiment, the bio-ink according to the present application has a complex spatial structure, which can achieve biomimetic distribution of carried neural and vascular cells, strengthen the interaction between the two cells, and achieve in vitro reconstruction of the neuro-vascular network.

[0111] Example

[0112] To better illustrate the objectives, technical solutions, and advantages of the present invention, the present invention will be further described below with reference to specific examples and comparative examples. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified. The following examples are for illustrative purposes only and are not intended to limit the scope of this application.

[0113] Example 1. Preparation of bio-ink

[0114] Preparation of Porous Conductive Prepolymer Grafted Methacrylated Gelatin

[0115] Dissolve 1g of GelMA in 50ml of PBS and incubate at 60°C for 2 hours to allow for complete dissolution. Then, add 400μl of pyrrole (Py) and stir magnetically at 40°C for 2 hours. Then, slowly add 0.1g of ammonium persulfate to the reaction mixture and stir overnight at 40°C to obtain methacryloyl-grafted gelatin (GelMA-PrePy) with pyrrole prepolymer.

[0116] Then, GelMA-PrePy was placed in a dialysis bag (MWCO: 8000-14000) and dialyzed in deionized water for 3 days. Finally, the dialyzed solution was frozen at -80°C overnight and then placed in a freeze dryer and freeze-dried for 2 days to obtain pure GelMA-PrePy foam material. Figure 1 Shown are photos of the gray-black porous pure GelMA-PPy obtained after freeze-drying, and infrared spectra of GelMA-PPy and GelMA.

[0117] Preparation of GelMA-PPy microspheres

[0118] The above-mentioned pure GelMA-PrePy foam material was dissolved in PBS solution, and the photoinitiator LAP (phenyl-2.4.6-trimethylbenzoyl lithium phosphite) was added and allowed to stand at 60°C for 2 hours to fully dissolve it, wherein the volume ratio of PBS solution to photoinitiator LAP was 9:1 to obtain a 5% (w / v) GelMA-PrePy aqueous solution. Neuronal cells (an immortalized rat pheochromocytoma cell) were then added to the GelMA-PrePy aqueous solution to obtain a GelMA-PrePy aqueous solution containing neuronal cells. The GelMA-PrePy aqueous solution containing neuronal cells was injected into a 10ml syringe and used as an aqueous phase channel. Mineral oil and surfactant SP80 were blended in a volume ratio of 96:4 and injected into a 50ml syringe and used as an oil phase channel.

[0119] The aqueous and oil channel syringes were separately mounted on micropumps. The syringe outlets were connected to the aqueous and oil channel inlets of a microfluidic chip via transparent hoses. The aqueous channel pump rate was 1 ml / h, and the oil channel pump rate was 5 ml / h. After the aqueous and oil phases merged, they were sheared into microsphere-shaped gels. The microsphere-shaped gels were then irradiated with a 405 nm UV lamp at the outlet of the microfluidic chip for 35 seconds for crosslinking, yielding crosslinked microsphere precursors.

[0120] The microsphere precursors were allowed to settle, the top oil layer removed, and 4°C PBS added and repeatedly pipetted through the microsphere precursors to wash the mineral oil and surfactant from the microsphere precursor surface. The tubes were then centrifuged at 1200 rpm for 5 minutes. After centrifugation, the tubes separated into three layers from top to bottom: mineral oil, PBS, and microspheres. The top layer of mineral oil and PBS was removed, and PBS was added again to wash the mixture, followed by centrifugation. This process was repeated three times to obtain pure microsphere precursors.

[0121] Add 5 ml of 0.05 M FeCl3 to the pure microsphere precursor for chemical cross-linking polymerization for 2 minutes. Then, centrifuge at 800 rpm for 3 minutes to remove the upper layer of FeCl3, and then add PBS to wash and remove unreacted Fe. 3+ , and washed three times to obtain methacrylated gelatin-polypyrrole (GelMA-PPy) microspheres with a particle size of 180-200 μm.

[0122] Preparation of Methacrylated Gelatin-Polypyrrole Hydrogel

[0123] The pure GelMA-PrePy foam material was added to a PBS solution, and the photoinitiator LAP was added. The mixture was then allowed to stand at 60°C for 2 hours. The volume ratio of the PBS solution to the photoinitiator LAP was 9:1, resulting in a 5% (w / v) GelMA-PrePy hydrogel. Vascular endothelial cells (self-cultured vascular endothelial cells) were then added to the GelMA-PrePy hydrogel to obtain a GelMA-PrePy hydrogel containing vascular endothelial cells.

[0124] Preparation of bio-ink

[0125] The cell-containing GelMA-PrePy hydrogel and GelMA-PPy microspheres were thoroughly mixed, then centrifuged at 1200 rpm for 5 minutes to remove excess liquid from the upper layer. This process was repeated at least three times until the interstices between the microspheres were largely, or even completely, filled with GelMA-PrePy hydrogel. Finally, the mixture was irradiated with a 405 nm UV lamp for 35 seconds, followed by the addition of 5 ml of 0.05 M FeCl₃ for chemical cross-linking polymerization for 2 minutes, resulting in a conductive microgel bioink with a stable, complex spatial structure.

[0126] Example 2 to Example 13

[0127] In substantially the same manner as in Example 1, by adjusting the flow rates of the oil phase and the aqueous phase and the concentration of FeCl3 in the microfluidic process, the bio-inks of Examples 2 to 13 were prepared. The specific conditions of Examples 1 to 13 are summarized in Table 1 below.

[0128] Table 1

[0129]

[0130]

[0131] 2. Performance Evaluation of Bio-ink

[0132] 2.1 Effects of water phase flow rate and oil phase flow rate on microsphere morphology

[0133] The morphology of the microspheres obtained in Examples 1 to 10 was observed by electron microscopy at different water phase flow rates and oil phase flow rates. Figure 3 As shown, Figure 3 (a), (b), (c), (d), (e), (f), (g), (h), (i) and (j) correspond to Examples 2 to 5, Example 1 and Examples 6 to 10, respectively. It can be seen that the faster the oil phase flow rate and the slower the water phase flow rate, the smaller the microsphere particle size. Figure 4A shows the microspheres of Example 1 in mineral oil observed by electron microscopy. Figure 4 B shows the particle size distribution of the microspheres of Example 1 in mineral oil. Figure 4 C shows the microspheres of Example 1 in PBS observed by electron microscopy. Figure 4 D shows the particle size distribution of the microspheres of Example 1 in PBS. Figure 4 Figure E shows that the microspheres of Example 1 swelled after being transferred from mineral oil to PBS, and the particle size increased.

[0134] 2.2 Conductivity

[0135] The conductivity of Example 1 and unmodified GelMA was tested, as shown in Figure 2 As shown, it shows the conductivity of 5% (w / v) GelMA-Ppy hydrogel and 5% GelMA hydrogel, where Figure 2 Figure A shows that the LED light connected to 5% (w / v) GelMA-PPy hydrogel is significantly brighter than the LED light connected to 5% (w / v) GelMA hydrogel. Figure 2 Figure B shows that the 5% GelMA-PPy hydrogel has a lower resistivity and Figure 2 C in Figure 3 shows that the 5% GelMA-PPy hydrogel has a higher conductivity.

[0136] 2.3 Biocompatibility

[0137] The effects of bio-inks obtained with different initiator concentrations on cell viability were tested by cell live-dead staining experiments. Figure 5 As shown, it shows the blank control group (no bio-ink added) ( Figure 5 A) and Example 11 ( Figure 5 B), Example 1 ( Figure 5 C), Example 12 ( Figure 5 D) and Example 13 ( Figure 5 E) Effect of the bio-ink on the activity of rat pheochromocytoma cells. The experimental results show that the bio-ink prepared with 0.025M and 0.05M FeCl3 concentrations has better cell compatibility.

[0138] 2.4 Rheological properties

[0139] The temperature-shear modulus and shear rate-viscosity properties of the GelMA-Ppy bio-ink and pure GelMA hydrogel in Example 1 were tested by rheometer.

[0140] refer to Figure 6For A, it can be seen that the elastic modulus of the bioink according to the present application is always greater than the loss modulus (G’>G”) within the range of 4-37°C, indicating that the ink always remains in the gel state, proving that its shear modulus is basically independent of temperature and has temperature independence, and can be printed under various temperature conditions. While for the pure GelMA hydrogel, the elastic modulus is less than the loss modulus (G’<G”) after the temperature is greater than 15°C, indicating that the pure GelMA hydrogel undergoes a gel-sol transition at 15-16°C and becomes liquid, and cannot be printed at this temperature. Reference Figure 6 For B, as the shear rate continuously increases, the shear viscosity of the GelMA-Ppy bioink shows a significant downward trend. Compared with the pure GelMA hydrogel, the GelMA-PPy microgel bioink has a higher viscosity and is more suitable for extrusion-based bioprinting.

[0141] 2.5 Observation of microspheres loaded with cells

[0142] The microspheres without loaded cells and the microspheres loaded with cells were observed through an ordinary microscope. Figure 7 Figure A shows the microspheres without loaded cells. Figure 7 Figure B shows the microspheres loaded with cells. It can be seen that the microspheres according to the present application still maintain a complete microsphere morphology after loading cells.

[0143] 2.8 Biocompatibility

[0144] Figure 8 The biocompatibility of the GelMA-PPy bioink loaded with cells is shown. The GelMA-PPy bioink loaded with cells was cultured in vitro for 10 days, and then cell viability and dead staining were performed. It can be seen that a large number of cells survived, indicating that the bioink is suitable for cell growth and proliferation. At the same time, the volume of the microspheres loaded with cells in the bioink also increased significantly, indicating that the bioink is beneficial for the outward growth of cells after a large number of proliferations.

[0145] 2.9 Printability

[0146] The printability of the bioink of Example 1 was tested by a 3D bioprinter. The temperature of the printing syringe was set at 15°C, the printing speed was 2 mm / s, the extrusion speed was 0.4 mm 3 / s, and the platform temperature was 4°C. Reference Figure 9 It can be seen that the GelMA-PPy microgel bioink is extruded in a filamentous form in the 3D printer, and the diameter is close to the diameter of the needle. In the three-dimensional grid, the junction of two perpendicular filaments shows good interconnectivity between adjacent layers, and there are obvious microspheres and the surrounding hydrogel morphology, proving that the bioink has a composite spatial structure.

Claims

1. Bio-ink, including: microspheres comprising methacrylated gelatin and a first conductive polymer, the microspheres optionally comprising first cells; a hydrogel comprising methacrylated gelatin and a second conductive polymer, the hydrogel optionally comprising a second cell, The microspheres have pores.

2. The bio-ink according to claim 1, wherein the monomers of the first conductive polymer and the monomers of the second conductive polymer are each independently selected from pyrrole, aniline, thiophene, fluorene, carbazole, and derivatives thereof, Optionally, the first conductive polymer and the methacrylated gelatin in the microspheres are connected to each other via chemical bonds, Optionally, the second conductive polymer and the methacrylated gelatin in the hydrogel are connected to each other via a chemical bond, Optionally, at least a portion of the pores of the microspheres are filled with hydrogel.

3. The bio-ink of claim 1, wherein the first cell and the second cell are independently selected from neurons, vascular endothelial cells, stem cells, fibroblasts, or any combination thereof, Optionally, the first cell and the second cell are identical or different from each other, Optionally, the content of the methacrylated gelatin and the second conductive polymer in the hydrogel is 5% w / v to 7.5% w / v relative to the volume of the hydrogel. Optionally, the particle size of the microspheres is 150 μm to 500 μm. Optionally, the content of the microspheres is 80% to 90% relative to the total weight of the bio-ink, Optionally, the content of the hydrogel is 10% to 20% relative to the total weight of the bio-ink.

4. A method for preparing microspheres, comprising: adding a first initiator to a solution comprising methacrylated gelatin and monomers of a conductive polymer to form a conductive prepolymer-grafted methacrylated gelatin; drying the conductive prepolymer-grafted methacryloyl gelatin to obtain porous conductive prepolymer-grafted methacryloyl gelatin; adding the porous conductive prepolymer-grafted methacrylated gelatin to a solvent and then adding a second initiator to form a first hydrogel comprising the second initiator, the first hydrogel optionally comprising cells; Mixing the first hydrogel and an oil phase solution containing mineral oil and a surfactant to form a microsphere-type gel; irradiating the microsphere-type gel with ultraviolet light to form a microsphere precursor; further polymerizing the conductive prepolymer in the microsphere precursor by a third initiator to form microspheres; The microspheres have pores.

5. The method of claim 4, wherein the second initiator is a photoinitiator, Optionally, the monomer of the conductive polymer is selected from pyrrole, aniline, thiophene, styrene, fluorene, carbazole, and derivatives thereof, Optionally, the first initiator and the third initiator are the same as or different from each other, Optionally, the first initiator and the third initiator are independently selected from: persulfates selected from ammonium persulfate, sodium persulfate and potassium persulfate; organic peroxide initiators selected from benzoyl peroxide, lauroyl peroxide, cumene hydroperoxide, tert-butyl hydroperoxide, di-tert-butyl peroxide, diisopropyl peroxide, tert-butyl perbenzoate, methyl ethyl ketone peroxide, cyclohexanone peroxide, diisopropyl peroxydicarbonate and dicyclohexyl peroxydicarbonate; azo initiators selected from azobisisobutyronitrile, azobisisovaleronitrile and azobisisoheptonitrile; FeCl3; or any combination thereof, Optionally, the second initiator is selected from phenyl-2,4,6-trimethylbenzoyl lithium phosphite, benzoyl ketone, benzil, α,α'-ethoxyacetophenone, benzophenone, 4-methylbenzophenone, phenyl bis (2,4,6-trimethylbenzoyl) phosphine oxide, 2,4,6-trimethylbenzoyl-diphenylphosphine oxide, 1-hydroxy-cyclohexyl-phenyl ketone or any combination thereof, Optionally, the particle size of the microspheres is 150 μm to 500 μm, Optionally, the solvent is selected from PBS solution and DMEM culture medium, Optionally, the volume ratio of the solvent to the second initiator is 8:1 to 10:1, Optionally, the volume ratio of the mineral oil to the surfactant in the oil phase solution is 96:4 to 90:10, Optionally, the volume ratio of the first hydrogel to the oil phase solution in the microsphere gel is 1:2 to 1:10, Optionally, the content of the first initiator is 15% to 30% relative to the weight of the monomer of the conductive polymer, Optionally, the content of the second initiator is 8% to 12.5% ​​relative to the weight of the methacrylated gelatin, Optionally, the concentration of the third initiator is 0.05M to 0.2M.

6. The method of claim 5, wherein the method further comprises adding cells to the first hydrogel so that the first hydrogel simultaneously contains the second initiator and the cells, optionally, the cells are selected from neuronal cells, vascular endothelial cells, stem cells, fibroblasts or any combination thereof.

7. A method for preparing a bio-ink, comprising: adding a first initiator to a solution comprising methacrylated gelatin and monomers of a conductive polymer to form methacrylated gelatin comprising a conductive prepolymer graft; drying the conductive prepolymer-grafted methacryloyl gelatin to form a porous conductive prepolymer-grafted methacryloyl gelatin; adding the porous conductive prepolymer-grafted methacrylated gelatin in a solvent and then adding a second initiator to form a first hydrogel comprising the second initiator, the first hydrogel optionally comprising cells; mixing the microspheres prepared by the method of any one of claims 4 to 6 with the first hydrogel, and then irradiating with ultraviolet light to form a second hydrogel; A third initiator is added to the second hydrogel to further polymerize the conductive prepolymer in the second hydrogel, thereby obtaining the bio-ink.

8. The method of claim 7, wherein the second initiator is a photoinitiator, Optionally, the monomer of the conductive polymer is selected from pyrrole, aniline, thiophene, styrene, fluorene, carbazole, and derivatives thereof, Optionally, the first initiator and the third initiator are the same as or different from each other, Optionally, the first initiator and the third initiator are independently selected from: persulfates selected from ammonium persulfate, sodium persulfate and potassium persulfate; organic peroxide initiators selected from benzoyl peroxide, lauroyl peroxide, cumene hydroperoxide, tert-butyl hydroperoxide, di-tert-butyl peroxide, diisopropyl peroxide, tert-butyl perbenzoate, methyl ethyl ketone peroxide, cyclohexanone peroxide, diisopropyl peroxydicarbonate and dicyclohexyl peroxydicarbonate; azo initiators selected from azobisisobutyronitrile, azobisisovaleronitrile and azobisisoheptonitrile; FeCl3: or any combination thereof, Optionally, the second initiator is selected from phenyl-2,4,6-trimethylbenzoyl lithium phosphite, benzoyl ketone, benzil, α,α'-ethoxyacetophenone, benzophenone, 4-methylbenzophenone, phenyl bis (2,4,6-trimethylbenzoyl) phosphine oxide, 2,4,6-trimethylbenzoyl-diphenylphosphine oxide, 1-hydroxy-cyclohexyl-phenyl ketone or any combination thereof, Optionally, the particle size of the microspheres is 150 μm to 500 μm, Optionally, the solvent is selected from PBS solution and DMEM culture medium, Optionally, the volume ratio of the solvent to the second initiator is 8:1 to 10:1, Optionally, the content of the first initiator is 15% to 30% relative to the weight of the monomer of the conductive polymer, Optionally, the content of the second initiator is 8% to 12.5% ​​relative to the weight of the methacrylated gelatin, Optionally, the concentration of the third initiator is 0.05M to 0.2M, Optionally, the cells are selected from neuronal cells, vascular endothelial cells, stem cells or any combination thereof, Optionally, the content of the microspheres is 80% to 90% relative to the total weight of the bio-ink, Optionally, the content of the hydrogel is 10% to 20% relative to the total weight of the bio-ink, Optionally, at least a portion of the pores of the microspheres are filled with hydrogel.

9. A method for preparing a biomaterial for tissue repair, cell culture or organ reconstruction, comprising 3D printing the bio-ink according to any one of claims 1 to 3.

10. Use of the bio-ink according to any one of claims 1 to 3 in preparing biomaterials for tissue repair, cell culture, and organ reconstruction.

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