Method for constructing anisotropic artificial tissue based on direct writing of cell-loaded micro-nano fibers

Abstract

The invention relates to a method for constructing an anisotropic artificial tissue based on direct writing of cell-loaded micro-nano fibers, and belongs to the technical field of tissue engineering. According to the method, living cells are directly loaded into various types of bioactive materials through a near-field direct writing technology, and micro-nano biological fibers with specific structures and functions are formed by utilizing a visible light crosslinking technology. The fibers can simulate a network structure of a natural extracellular matrix, provide an excellent growth environment for cells and realize artificial tissue construction, so that repair and regeneration of tissues are promoted.

Description

Technical Field

[0001] The invention relates to a method for constructing anisotropic artificial tissue based on direct writing of cell-carrying micro-nano fibers, and belongs to the technical field of tissue engineering. Background Art

[0002] With the rapid development of biomedical engineering, tissue engineering has attracted extensive attention as an important means of repairing and regenerating damaged tissues. In this field, constructing artificial tissues with excellent biological activity has become one of the key technologies. Anisotropy is a common property of many tissues in the human body, such as skeletal muscle, connective tissue, cornea, skin, blood vessels, nerves and cartilage. The fibers of skeletal muscle are arranged along the length of the muscle, providing a specific contractile force; the arrangement of collagen fibers in connective tissues such as skin and ligaments gives them direction-dependent mechanical behavior; the collagen fiber layer structure of the cornea gives it different optical and mechanical properties in different directions; the mechanical properties of the skin will change with the change of the test direction, affecting the selection and healing of surgical incisions; in the multi-layer structure of the vascular wall, the specific arrangement of endothelial cells and smooth muscle cells is crucial to the function of blood vessels; the axons of peripheral nerves are arranged along specific directions to support long-distance transmission of signals; the nerve fiber bundles of central nervous systems such as the spinal cord and brain also show obvious anisotropy. These anisotropic properties are not only crucial to the normal physiological function of tissues, but also provide challenges for tissue engineering to simulate the structure and function of natural tissues, promoting the development of scaffold material design and manufacturing technology to better promote tissue repair and regeneration.

[0003] The anisotropic properties of human tissues provide important guidance for the application of micro-nano biofibers in tissue engineering. Micro-nano biofibers can be used to construct artificial tissues with structures and functions similar to natural tissues due to their unique size effect, high specific surface area and good biocompatibility. Among the preparation technologies of micro-nano biofibers, electrospinning technology, microfluidics technology and three-dimensional bioprinting technology are the three main methods. Electrospinning can produce fibers with diameters ranging from nanometers to micrometers, with good controllability and scalability. However, electrospinning technology causes disordered fiber arrangement due to the jet whipping effect, and manufacturing conditions such as high temperature, strong electric field, and toxic solvents are more harmful to cells. The mechanism of microfiber electrospinning suitable for living cells has not been fully elucidated, and how to ensure the high survival rate of living cells is a major challenge. Three-dimensional bioprinting technology provides more precise control of fiber structure and can construct complex three-dimensional tissue structures on demand, but printing speed and cell survival rate are still challenges. Biological 3D printing technology has been able to use living cells, biomolecular materials and growth factors to construct functional tissues and organs. The main biological 3D printing technologies include inkjet bioprinting, microextrusion bioprinting and laser-assisted bioprinting. These technologies achieve the construction of bionic tissues and organs by layer-by-layer deposition, and can print out tissues with simple structures and single functions, such as bones, skin, heart valves, etc. However, these technologies mainly operate on a macroscopic scale, making it difficult to achieve the orderly arrangement of cells and the construction of microscopic topological structures.

[0004] In traditional tissue engineering, scaffold planting is usually adopted, that is, cells are inoculated on a pre-prepared scaffold to form tissues through self-assembly and proliferation of cells. However, this method has some limitations, such as uneven distribution of cells on the scaffold, difficulty in nutrient delivery and metabolic waste removal, and the influence of scaffold material selection and degradation rate on cell behavior. In addition, the removal process of the scaffold may cause damage to the new tissue, affecting the integrity and function of the tissue. In addition, in tissue engineering, it is an important challenge to ensure that the constructed artificial tissue has good structural stability while maintaining cell activity and promoting cell growth. Traditional cross-linking methods, such as the use of high temperature, strong acid or strong alkali, ultraviolet light and other conditions, may cause damage to cells. Among them, high temperature affects the structural function and enzyme activity of intracellular proteins. Acidic or alkaline environments can destroy the pH balance inside and outside the cell, affecting the normal progress of various biochemical reactions in the cell. The photochemical reaction of ultraviolet light initiators produces highly reactive free radicals, which react with macromolecules such as lipids, proteins and nucleic acids in the cell, resulting in lipid peroxidation of the cell membrane, cross-linking or breakage of proteins, and damage to DNA. These adverse effects limit their application in encapsulation of living cells. In order to ensure the structural stability of these micro-nano bio-fibers and maintain the biological activity of encapsulated cells, a mild cross-linking method is needed to fix the fiber structure. Visible light initiators generate free radicals under light of a specific wavelength, which can react with the polymer chains in the fiber to form stable chemical bonds. This process occurs under relatively mild conditions, avoiding thermal or chemical damage to the cells.

[0005] Cell electrospinning is an advanced micro-nanoscale fiber manufacturing technology. It forms fibers by stretching cell-containing bio-inks using electric field forces, and achieves the direct embedding of living cells into micron or nanoscale fiber structures. This method can produce fibers with diameters ranging from nanometers to micrometers, and optimizes the electric field effect to improve cell survival. Cell electrospinning technology has a wide range of application potential in tissue engineering. Geun Hyung Kim used cell electrospinning technology to directly embed cells into micro / nano fibers to manufacture cell-loaded fiber structures, achieving uniform cell distribution and high cell survival, providing new ideas for subsequent tissue regeneration and functional recovery. He Jiankang used alginate as the main biomaterial to explore its application in microscale electrohydrodynamic bioprinting and its potential in tissue engineering. However, this material selection limits the diversity of materials, especially in providing biocompatibility and bioactivity. Jos Maldà facilitated the rapid curing of two new photoresponsive hydrogel bio-inks based on gelatin and silk fibroin through a light-mediated cross-linking mechanism, achieving micron-scale resolution of cell-loaded fiber structures, although the tissue specificity and bioactivity of the materials need to be improved. These studies collectively point to the core elements of cell spinning technology: (1) achieving high-resolution and precisely controlled three-dimensional structure fabrication; (2) developing bioactive materials with broad applicability and tissue specificity, ensuring high cell survival and biocompatibility; and (3) optimizing long-term stability and degradation control, and further verifying its potential for tissue formation. Solving these problems will promote the application of cell spinning technology in tissue engineering and regenerative medicine, and provide new possibilities for constructing artificial tissues that more accurately mimic natural tissues. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a method for constructing anisotropic artificial tissue based on direct writing of cell-laden micro-nano fibers. This method directly loads living cells into various types of bioactive materials through near-field direct writing technology, and uses visible light cross-linking technology to form micro-nano bio-fibers with specific structures and functions. These fibers can simulate the network structure of natural extracellular matrix, provide cells with a superior growth environment, realize artificial tissue construction, and thus promote tissue repair and regeneration. The technical solution of the present invention has broad application prospects in organ chips, personalized organ printing, transplantable tissue engineering construction, and the manufacture of high-sensitivity detection biosensors, and is expected to significantly promote the development of the field of biomedical engineering.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows.

[0008] A method for constructing anisotropic artificial tissue based on direct writing of cell-carrying micro-nano fibers, the method steps comprising:

[0009] (1) Preparation of bio-ink: mixing a bioactive material with a conductivity of less than 2 mS / cm, polyethylene oxide (PEO), glycerol, a visible light initiator, and water with a purity higher than deionized water to obtain a bio-ink;

[0010] (2) Preparation of cell-loaded micro-nanofibers: After mixing bioactive cells with bio-ink, near-field direct writing method is used to prepare cell-loaded micro-nanofibers with cells evenly distributed inside the fibers;

[0011] (3) Visible light crosslinking: Under visible light irradiation, the cell-laden micro-nano fibers are crosslinked to obtain micro-nano bio-fibers;

[0012] (4) Construction of anisotropic artificial tissue: Micro-nano biofibers are cultured in vitro, and cells proliferate, migrate, and differentiate to construct artificial tissue.

[0013] Preferably, in step (1), in the bio-ink, the mass fraction of the bioactive material is 2% to 6%, the mass fraction of PEO is 1% to 3%, and the mass fraction of glycerol is 5% to 25%.

[0014] Preferably, in step (1), the bioactive material includes acellular matrix with a conductivity of <2 mS / cm, collagen, fibrinogen, and gelatin.

[0015] Preferably, in step (1), the visible light initiator is tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate / sodium persulfate (Ru / SPS) or lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). More preferably, the concentration of Ru / SPS in the bio-ink is 0.25 / 2.5-1.0 / 10.0 mM; the mass fraction of LAP in the bio-ink is 0.05% to 1.5%.

[0016] Preferably, in step (2), when the bioactive cells and the bio-ink are mixed, the density of the bioactive cells is 1×10 6 ~1×10 7 More preferably, the bioactive cells include skeletal muscle cells, vascular smooth muscle cells, and neural stem cells.

[0017] Preferably, in step (2), during near-field direct writing, the ambient temperature is 25-37°C, the ambient humidity is 50%-70%, the applied voltage is 1.5-3.0 kV, the collection distance is 3-6 mm, the nozzle moving speed is 20-60 mm / s, and the extrusion speed is set to 0.01-0.03 mm. 3 / s, and the current generated during near-field direct writing is <0.1mA.

[0018] Preferably, in step (2), the diameter of the cell-laden micro-nanofiber is 5 to 60 μm.

[0019] Preferably, in step (2), the arrangement of the cell-laden micro-nano fibers includes parallel arrangement, perpendicular intersection arrangement, axial arrangement and circumferential arrangement.

[0020] Preferably, in step (3), the wavelength of visible light is 405 to 450 nm, and the illumination time is 10 to 90 s.

[0021] An anisotropic artificial tissue is constructed by the above method.

[0022] Beneficial Effects

[0023] The present invention improves the spinning performance and moisture retention of bio-ink by preparing it, obtains micro-nano bio-fibers by using near-field direct writing technology, realizes the stability of the structure and the active encapsulation of cells by gentle visible light cross-linking of cells, simulates the natural extracellular matrix, and promotes cell attachment and growth. Cultivation is carried out in a controlled in vitro culture environment to promote cell proliferation, migration and differentiation, and form anisotropic artificial tissues with specific functions. This method not only improves the cell survival rate, but also constructs a highly bionic tissue structure by precisely controlling the deposition and arrangement of micro-nano bio-fibers. These artificial tissues are similar to the natural extracellular matrix in size, composition, structure and material properties, thereby providing a more suitable growth microenvironment for cells. Specifically:

[0024] (1) High cell survival rate and biocompatibility: The near-field direct writing technology combined with the mild visible light cross-linking method effectively avoids the damage to cells caused by strong electric fields, high temperatures and toxic solvents. This gentle direct writing method ensures the high survival rate of cells during the preparation of micro-nano biofibers, while maintaining the biocompatibility of the bio-ink, providing a safe and effective method for the direct encapsulation of living cells.

[0025] (2) Construction of microscopic topological structure: The fibers obtained by near-field direct writing after mixing cells and bio-ink simulate the structure of natural tissues. This not only improves the orientation of cell attachment and growth, but also provides cells with a more bionic microenvironment, thereby promoting the expression of specific cell functions and the formation of tissues.

[0026] (3) Ability to simulate the heterogeneous structure of natural tissue cells: The cells are evenly distributed and their activity is maintained in the micro-nano bio-fibers. This uniform cell distribution and activity maintenance makes the constructed artificial tissue closer to natural tissue in structure and function, providing a new technical solution for tissue engineering.

[0027] (4) Chemical composition regulation and rheological property optimization: The chemical components are carefully selected to carefully regulate the rheological properties of the bio-ink, such as the spinning window and shear thinning behavior, to ensure the stability and printability of the micro-nano bio-fiber construction. This optimization of chemical components enables the bio-ink to adapt to different printing requirements and conditions, improving the controllability of the printing process and the accuracy of the printed structure.

[0028] (5) Wide range of material applicability: Applicable to a variety of bioactive materials, including natural and synthetic polymers, which provides a wide range of material options for tissue engineering. This wide range of material applicability enables the present invention to select the most suitable biomaterials for different tissue and organ needs to achieve specific biological functions and mechanical properties.

[0029] (6) Improving the efficiency and quality of tissue construction: The present invention not only improves the uniform distribution and activity of cells in the three-dimensional structure, but also avoids the limitations of traditional scaffold implantation and realizes the integrated manufacturing of cells and materials. This integrated manufacturing method is expected to improve the efficiency and quality of tissue construction and bring breakthrough progress to the field of regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a staining diagram of the acellular matrix tissue of the porcine tibialis anterior muscle according to an example of the present invention.

[0031] Figure 2 Graph showing pH and conductivity of an example of the present invention.

[0032] Figure 3 Schematic diagram of the cross-linking of the bio-ink of the present invention and the effect of Ru / SPS on the conductivity of the bio-ink.

[0033] Figure 4 The conductivity of the bio-ink with different cell densities embedded in the examples of the present invention.

[0034] Figure 5 This is a graph showing the relationship between fiber diameter and applied voltage in an example of the present invention.

[0035] Figure 6 This is a graph showing the relationship between fiber diameter and collection distance in an example of the present invention.

[0036] Figure 7 Schematic diagram of near-field direct writing and a single-layer near-field direct writing fiber according to an example of the present invention.

[0037] Figure 8 These are near-field direct writing fibers with different topological structures according to the examples of the present invention.

[0038] Fig. 9 This is a picture of a single-layer micro-nano bio-fiber encapsulated with living cells as an example of the present invention.

[0039] Fig.10 This is a muscle fiber bundle structure formed by stacking micro-nano biological fibers encapsulating living cells in an example of the present invention. DETAILED DESCRIPTION

[0040] The present invention will be further described in detail below in conjunction with specific embodiments.

[0041] (1) Selection and performance optimization of bioactive materials: The present invention is applicable to a variety of bioactive materials, including but not limited to natural polymers such as decellularized matrix, collagen, alginate, gelatin, etc. These materials have good biocompatibility. Further requirement that the conductivity of the bioactive material is less than 2mS / cm can ensure that the selected bioactive material can adapt to the electrospinning process, thereby preparing high-quality micro-nano fibers and constructing ideal anisotropic artificial tissues.

[0042] (2) Composition of bio-ink: Bio-ink is composed of bioactive materials, polyethylene oxide (PEO), glycerol, visible light initiator and water. The selection of bioactive materials is to provide the necessary bioactivity to support cell attachment, proliferation and differentiation; PEO is added to regulate the rheological properties of bio-ink to ensure its printability during near-field direct writing; glycerol is added to improve the moisture retention of bio-ink and further maintain the living environment of cells; and the use of visible light initiator provides a mild cross-linking curing method, which can achieve rapid curing of bio-ink without damaging living cells, thereby improving the survival rate of cells during printing. By optimizing the chemical composition, the flow and deformation characteristics of bio-ink when subjected to external forces, as well as the response characteristics of bio-ink in electric fields, are further adjusted, so that the formation of fibers is more uniform and controllable, ensuring its stability and printability during near-field direct writing.

[0043] (3) Cell encapsulation and formation of micro-nano biofibers: In the process of constructing anisotropic artificial tissues with micro-nano fibers, cell encapsulation and the formation of micro-nano biofibers is a key step. The process involves mixing living cells with bioactive materials and directly writing them into micro-nano biofibers using near-field direct writing technology. In order to ensure the molding stability and structural continuity of the fibers, and to avoid negative effects such as electroporation, electrolytic damage, and thermal damage to cells under high voltage and high current, the threshold range of key process parameters must be further precisely controlled. These parameters include but are not limited to voltage, collection distance, extrusion speed, and fiber arrangement and trajectory control. By precisely controlling these parameters, the natural tissue structure can be simulated, and cell growth and differentiation along a specific direction can be promoted, thereby achieving uniform distribution and functionalization of the cell-fiber composite structure.

[0044] (4) Visible light crosslinking: The present invention is applicable to a variety of visible light initiators, including but not limited to tris(2,2′-bipyridyl)dichloro-ruthenium(II)hexahydrate / sodium persulfate Ru / SPS), phenyl-2,4,6-trimethylbenzoyldi-Phenylphosphinate (2,4,6-Trimethylbenzoyldi-Phenylphosphinate, LAP), etc. The formed micro-nano biofibers are crosslinked using visible light initiators to enhance the structural stability of the fibers. The crosslinking process is carried out under visible light irradiation, avoiding the conditions of high temperature, strong acid, strong alkali or ultraviolet light used in traditional methods, thereby reducing potential damage to cells.

[0045] (5) Anisotropic artificial tissue construction: The micro-nano biofiber structure is placed in a controlled culture environment that simulates the in vivo environment to promote cell proliferation, migration and differentiation. After an appropriate culture time, cell activity and functional performance are evaluated using methods such as live / dead cell staining, CCK-8 proliferation assay, and immunofluorescence staining. During the culture process, cells proliferate, migrate and differentiate inside the fiber, eventually forming tissues with specific functions.

[0046] Example 1

[0047] (1) The preparation process of the bioactive material is as follows (taking gelatinized porcine tibialis anterior muscle acellular matrix as an example):

[0048] Step 1: Tissue pretreatment. Cut fresh pig tibialis anterior muscle into slices, remove fascia and fat tissue. Add slices of tissue to deionized water, and use magnetic stirring to remove dust, blood, and residual fascia and fat cells.

[0049] Step 2: SDS cleaning. Sodium dodecyl sulfate (SDS) is a powerful surfactant that can destroy cell membranes and dissolve proteins, removing cellular components from tissues. Ethylenediaminetetraacetic acid (EDTA) can chelate metal ions, helping SDS penetrate tissues more effectively. Add SDS and EDTA to deionized water to prepare a PBS solution of SDS and EDTA, and wash the tissue treated in step 1 with magnetic stirring.

[0050] Step 3: Triton X-100 treatment. Triton X-100 is a non-ionic surfactant that helps to more effectively remove cell components, especially the dissolution of nucleic acids. Add Triton X-100 and EDTA to the PBS solution to prepare the Triton X-100 decellularization working solution, and wash the tissue treated in step 2 with magnetic stirring. Change the Triton X-100 solution regularly.

[0051] Step 4: Nuclease (DNase) treatment. Nucleic acid molecules, especially DNA, can activate pattern recognition receptors in the immune system and trigger inflammatory responses. Nucleases can specifically degrade nucleic acids in tissues, including DNA and RNA, effectively removing cellular components while helping to reduce inflammatory responses caused by later materials. Add DNase to the PBS solution to prepare a DNase decellularization working solution, and wash the tissue treated in step 3 with magnetic stirring.

[0052] Step 5: Isopropanol washing. Isopropanol can be used as a cleaning agent to help remove residual SDS, TritonX-100 and other surfactants in the tissue, thereby reducing the toxicity of the final material. The tissue treated in step 4 is placed in isopropanol for magnetic stirring and washing.

[0053] Step 6: Washing with deionized water. The tissue treated in step 5 is placed in deionized water and washed multiple times with magnetic stirring to remove isopropanol and reduce the toxicity of the final obtained material.

[0054] Step 7: Sterilization and disinfection: The tissue treated in step 6 is placed in a disinfection solution containing peracetic acid and anhydrous ethanol for washing to remove residual bacteria, viruses and other microorganisms.

[0055] Step 8: Freeze drying. The tissue obtained in step 7 is frozen and then freeze dried in a freeze dryer to obtain a decellularized matrix. The decellularized matrix tissue of the pig tibialis anterior muscle is stained as shown in FIG. Figure 1 As shown, the cell nucleus is removed, and proteins, polysaccharides, and collagen fibers are retained.

[0056] Step 9: Grinding. The tissue obtained in step 8 is ground with the aid of liquid nitrogen and filtered through a mesh to obtain acellular matrix powder.

[0057] Step 10: Dissolving: The decellularized matrix powder obtained in step 9 is added to acetic acid and pepsin for digestion to obtain a decellularized matrix gel.

[0058] Step 11: Hydrolysis: The decellularized matrix gel obtained in step 10 is subjected to a hydrolysis reaction, and the collagen is fully hydrolyzed into gelatin while retaining other effective components of the decellularized matrix, so as to achieve the purpose of making the material water-soluble and reduce the conductivity of the material.

[0059] Step 12 Dialysis. Put the gelatinized acellular matrix obtained in Step 11 into a dialysis bag and dialyze it with deionized water, changing the deionized water regularly to remove the ions introduced in Step 10 until the pH value and conductivity reach the standards (6.5 < pH < 7.5, σ < 2 mS / cm), as Figure 2 shown.

[0060] Step 13 Freeze-drying. After freezing the material obtained in Step 12, perform freeze-drying in a freeze dryer to obtain a water-soluble low-conductivity gelatinized acellular matrix solid.

[0061] (2) The preparation process of the cell-laden bioink is as follows:

[0062] Step 1 Bioink preparation: Dissolve the low-conductivity gelatinized acellular matrix of porcine tibialis anterior muscle in deionized water to form a 2 wt% solution, add 2 wt% PEO to increase the feasibility of near-field direct writing, add 10 wt% glycerol to increase the moisture retention, and add 0.25 / 2.5 mM Ru / SPS visible light photoinitiator to ensure the visible light crosslinking of the directly written fibers to obtain the bioink. The crosslinking schematic diagram of the bioink and the effect of Ru / SPS on the conductivity of the bioink are as Figure 3 shown.

[0063] Step 2 Characterization of bioink properties: Rheological properties affect the printing performance and fiber morphology of the ink, and electrical properties are related to the efficiency of electrospinning and the uniformity of the fibers. Characterize the rheological and electrical properties of the prepared bioink. Through characterization and optimization, ensure the stability of the bioink during the printing process and the quality of the fibers, and optimize the spinning window.

[0064] Step 3 Preparation of cell-laden bioink: Prepare a bioink containing living cells for subsequent tissue engineering and bioprinting applications to promote cell growth and tissue regeneration. Embed C2C12 cells with a density of 5×10 6 cells / mL into the above bioink to obtain the cell-laden bioink. The phospholipid bilayer inside the cell membrane is filled with electrolytes, which will affect the conductivity of the bioink. The conductivity of the bioink embedded with different cell densities is as Figure 4 shown. The C2C12 living cells are evenly dispersed in the bioink, providing nutrition and protection for the cells. The cell viability and CCK-8 proliferation experiments show that the bioink has good biocompatibility and can effectively support the growth and proliferation of C2C12 cells.

[0065] (3) The process of near-field direct writing of micro-nano biofibers is as follows:

[0066] Step 1: Regulate key process parameters: Build a multi-module biological 3D printing platform including a micron-level high-precision motion platform and near-field direct writing. Regulate the key process parameters of near-field direct writing. The relationship between fiber diameter and applied voltage is shown in Figure 5, and the relationship between fiber diameter and collection distance is shown in Figure 6. Figure 6 The final process parameter range is as follows: the cell-carrying bio-ink is loaded into the barrel and a voltage of 1.5 kV is applied for near-field direct writing. The collection distance is set to 5 mm, the nozzle movement speed is set to 25 mm / s, and the extrusion speed is set to 0.01 mm. 3 / s, the obtained fiber diameter is 37.75μm, and the schematic diagram of near-field direct writing is similar to that of single-layer near-field direct writing fiber. Figure 7 shown.

[0067] Step 2 Conductive glass receiving: Conductive glass, as a carrier, can evenly distribute the charge, which helps to control the deposition and curing process of the fiber. Conductive glass receives near-field direct writing fibers of different topological structures, such as Figure 8 shown.

[0068] Step 3: Visible light irradiation curing: Through light curing, the fiber structure is quickly fixed to maintain its shape and strength, while ensuring the activity and function of the cells. Use 450nm visible light irradiation for 30s to obtain stable near-field direct writing micro-nano bio-fibers, such as Fig. 9 shown.

[0069] (4) In vitro cultivation process of skeletal muscle fibers:

[0070] Step 1: Cultivation in growth medium: stacking micro-nano bio-fibers encapsulating living cells to form a muscle fiber bundle structure, such as Fig.10 As shown, cells were cultured in a growth medium at 37°C in a humidified environment with 5% CO2, and the cells gradually adapted to the microfiber structure, proliferated and migrated therein, laying the foundation for subsequent differentiation.

[0071] Step 2: Cell activity and proliferation assessment: The results of live / dead cell staining and CCK-8 proliferation assay showed that the cells grew and proliferated well.

[0072] Step 3: Inducing myotube formation: After 5 days, the culture medium was replaced with differentiation medium (high glucose DMEM supplemented with 2% horse serum and 1% P / S) to induce myotube formation. The cells began to express specific markers, showing a tendency to differentiate into mature tissues.

[0073] Step 4: Evaluation of cell differentiation and myotube formation: Cell RNA was extracted on day 14, and cDNA was prepared using a cDNA synthesis kit. The expression levels of relative myogenic differentiation genes (Myf5, MyoG, MyoD, and MHC) were analyzed using SYBR-Green using the Real-Time PCR system. Immunofluorescence staining was performed to evaluate the formation of myotubes in the muscle structure. The results showed that living cells will form an orderly arrangement on the microfibers and eventually mature into tissues with specific functions. At the end of the cultivation period, the constructed micro-nano biofiber tissues showed high cell activity and good biological functions.

[0074] Example 2

[0075] Step 1: Preparation of bio-ink: dissolve gelatin and PEO powder in water, stir magnetically at 37°C and 500 rpm for 24 h to obtain a 2% gelatin-1.5% PEO aqueous solution, add 15% glycerol to increase moisture retention, add 0.5 / 5.0 mM Ru / SPS visible light photoinitiator to ensure visible light crosslinking of the direct-writing fiber, and obtain bio-ink.

[0076] Step 2 Preparation of cell-loaded bio-ink: Prepare bio-ink containing living cells for subsequent tissue engineering and bio-printing applications to promote cell growth and tissue regeneration. 7 HFF cells / mL were embedded in the above bio-ink to obtain cell-laden bio-ink. HFF living cells were evenly dispersed in the bio-ink, providing nutrition and protection for the cells. Cell live-death and CCK-8 proliferation experiments showed that the bio-ink had good biocompatibility and could effectively support the growth and proliferation of HFF cells.

[0077] (3) The process of near-field direct writing of micro-nano biofibers is as follows:

[0078] Step 1: Regulate key process parameters: Build a multi-module biological 3D printing platform including a micron-level high-precision motion platform and near-field direct writing. Regulate the key process parameters of near-field direct writing, and finally determine the process parameter range as follows: load the above-mentioned cell-carrying bio-ink into the barrel and apply a voltage of 1.8kV for near-field direct writing, the barrel heating temperature is 37℃, the collection distance is set to 7mm, the nozzle movement speed is set to 40mm / s, and the extrusion speed is set to 0.02mm 3 / s, and the obtained fiber diameter is 24.61μm.

[0079] Step 2 Conductive glass receiving: Conductive glass acts as a carrier that evenly distributes the charge and helps control the fiber deposition and curing process.

[0080] Step 3: Visible light curing: Through light curing, the fiber structure is quickly fixed to maintain its shape and strength while ensuring the activity and function of the cells. Use 450nm visible light for 40s to obtain stable near-field direct writing micro-nano bio-fibers.

[0081] (4) In vitro cultivation process of cell-laden micro-nano fibers:

[0082] Step 1: Cultivate in growth medium: Place the muscle fiber bundle structure formed by stacking micro-nano bio-fibers encapsulating living cells in a humid environment at 37° C. and a 5% CO 2 atmosphere, and culture in growth medium. The cells gradually adapt to the micro-fiber structure, and proliferate and migrate therein.

[0083] Step 2: Cell activity and proliferation assessment: The results of live / dead cell staining and CCK-8 proliferation assay showed that the cells grew and proliferated well.

[0084] In summary, the invention includes but is not limited to the above embodiments. Any equivalent substitution or partial improvement made under the spirit and principle of the invention shall be deemed to be within the protection scope of the invention.

Claims

1. A method for constructing anisotropic artificial tissue based on direct writing of cell-laden micro-nano fibers, characterized in that: The method steps include: (1) Preparation of bio-ink: mixing a bioactive material with a conductivity of less than 2 mS / cm, polyethylene oxide (PEO), glycerol, a visible light initiator, and water with a purity higher than deionized water to obtain a bio-ink; (2) Preparation of cell-loaded micro-nanofibers: After mixing bioactive cells with bio-ink, near-field direct writing method is used to prepare cell-loaded micro-nanofibers with cells evenly distributed inside the fibers; (3) Visible light crosslinking: Under visible light irradiation, the cell-laden micro-nano fibers are crosslinked to obtain micro-nano bio-fibers; (4) Construction of anisotropic artificial tissue: Micro-nano biofibers are cultured in vitro, and cells proliferate, migrate, and differentiate to construct artificial tissue.

2. A method for constructing anisotropic artificial tissue based on direct writing of cell-laden micro-nano fibers as claimed in claim 1, characterized in that: In step (1), in the biological ink, the mass fraction of the bioactive material is 2% to 6%, the mass fraction of PEO is 1% to 3%, and the mass fraction of glycerol is 5% to 25%.

3. A method for constructing anisotropic artificial tissue based on direct writing of cell-laden micro-nano fibers as claimed in claim 1 or 2, characterized in that: In step (1), the bioactive material includes acellular matrix with a conductivity of <2 mS / cm, collagen, fibrinogen, and gelatin.

4. A method for constructing anisotropic artificial tissue based on direct writing of cell-laden micro-nano fibers as claimed in claim 1 or 2, characterized in that: In step (1), the visible light initiator is tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate / sodium persulfate (Ru / SPS) or lithium phenyl-2,4,6-trimethylbenzoylphosphite (LAP); preferably, the concentration of Ru / SPS in the bio-ink is 0.25 / 2.5-1.0 / 10.0 mM; the mass fraction of LAP in the bio-ink is 0.05% to 1.5%.

5. The method for constructing anisotropic artificial tissue based on direct writing of cell-laden micro-nano fibers according to claim 1, characterized in that: In step (2), when the bioactive cells and the bio-ink are mixed, the density of the bioactive cells is 1×10 6 ~1×10 7 More preferably, the bioactive cells include skeletal muscle cells, vascular smooth muscle cells, and neural stem cells.

6. A method for constructing anisotropic artificial tissue based on direct writing of cell-laden micro-nano fibers as claimed in claim 1 or 5, characterized in that: In step (2), during near-field direct writing, the ambient temperature is 25-37°C, the ambient humidity is 50%-70%, the applied voltage is 1.5-3.0 kV, the collection distance is 3-6 mm, the nozzle moving speed is 20-60 mm / s, and the extrusion speed is set to 0.01-0.03 mm. 3 / s, and the current generated during near-field direct writing is <0.1mA.

7. A method for constructing anisotropic artificial tissue based on direct writing of cell-laden micro-nano fibers as claimed in claim 1 or 5, characterized in that: In step (2), the diameter of the cell-laden micro-nanofiber is 5 to 60 μm.

8. A method for constructing anisotropic artificial tissue based on direct writing of cell-laden micro-nano fibers as claimed in claim 1 or 5, characterized in that: In step (2), the arrangement of the cell-laden micro-nano fibers includes parallel arrangement, vertical intersection arrangement, and axial arrangement.

9. The method for constructing anisotropic artificial tissue based on direct writing of cell-laden micro-nano fibers according to claim 1, characterized in that: In step (3), the wavelength of visible light is 405 to 450 nm, and the illumination time is 10 to 90 s.

10. An anisotropic artificial tissue, characterized in that: It is obtained by the method according to any one of claims 1 to 9.

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