A multi-jet 3D bioprinted scaffold for a biomimetic valve and uses thereof

Abstract

This invention belongs to the technical field of 3D printing of biological tissues and organs, and discloses a biomimetic valve multi-nozzle 3D bioprinted scaffold and its application. The scaffold, from bottom to top, comprises a firmly connected elastin layer, a polysaccharide layer, and a collagen fiber layer. Each layer is constructed by alternating 3D printing of at least two materials; one of these materials is a photocurable hydrogel material providing mechanical support, and the other is a photocurable hydrogel material mixed with bioactive substances. Furthermore, the bioactive substances used in the elastin layer, polysaccharide layer, and collagen fiber layer are different from each other. This invention improves the structure and composition of the 3D bioprinted scaffold, creating a biomimetic three-layer structure and composition for a valve. The scaffold, designed with a three-layer structure of elastin, polysaccharide, and collagen fiber layers, provides cells with a specific sensory microenvironment, stimulating them to secrete corresponding extracellular matrix and completing extracellular matrix remodeling.

Description

Technical Field

[0001] This invention belongs to the technical field of 3D printing of biological tissues and organs, and more specifically, relates to a multi-nozzle 3D bioprinting scaffold for a biomimetic valve and its application. Background Technology

[0002] Heart valve disease has always been a major threat to human health. The main causes of heart valve disease include congenital defects, rheumatic fever, infective endocarditis, and valvular calcification. Among these, valvular stenosis and / or regurgitation caused by valvular calcification are the most common valvular lesions. Currently, the mechanism of valvular calcification is not fully understood and requires further investigation. There is also no effective way to prevent valvular calcification, and treatment mainly relies on valve replacement surgery. Existing valve replacements are mostly mechanical valves and bioprosthetic valves. Both types of valves have their drawbacks: patients who undergo heart valve replacement are more prone to thrombosis, thus requiring lifelong anticoagulation, which severely impacts their quality of life. Furthermore, for some infants with congenital valvular defects, mechanical valves may be unusable due to factors such as being too large or not growing with the child. Bioprosthetic valves, on the other hand, degenerate over time and are prone to valvular calcification, requiring replacement approximately every 10-15 years, increasing patient suffering. When studying the calcification mechanism of bioprosthetic valves, using living bioprosthetic valves would be costly for researchers, as there could be significant differences between living valves, and the procedure would be very complicated. Furthermore, there is an urgent need for tissue-engineered valves that are readily available in large quantities, exhibit minimal variability, are low-cost, and easy to operate when screening for methods or drugs to prevent or treat valve calcification. In addition, tissue-engineered valves are currently considered by researchers to be one of the most promising valve replacements.

[0003] Tissue-engineered valves, similar to natural tissues, are created by implanting seed cells into bioprinted scaffold materials. Through the proliferation, migration, degradation, and remodeling of extracellular matrix components by these seed cells, valve tissues with high durability, no need for anticoagulation, resistance to calcification, low immunogenicity, and strong regenerative capacity are constructed. Therefore, 3D bioprinted scaffolds need to possess good biocompatibility, biodegradability, and the ability to provide suitable mechanical support. Multi-arm polyethylene glycol exhibits colorless, odorless, low-toxicity, low immunogenicity, and good water solubility, making it a widely used, highly biocompatible, multifunctional, and biodegradable polymer. Its adjustable molecular weight, ease of modification, and ability to be combined with other materials to adjust stiffness and swelling properties endow the scaffold with excellent and controllable mechanical properties.

[0004] 3D bioprinting technology allows for customized printing schemes to meet diverse needs, enabling individualized designs. This facilitates spatial control of complex tissues to reproduce their heterogeneity and allows for strict structural control, ensuring the manipulability of material mechanical and degradation properties. Extrusion 3D printing, a key branch of 3D bioprinting, has become one of the most commonly used printing technologies in tissue engineering research due to its simple process and high cell loading capacity. Bioprinting inks, containing biocompatible components and possessing excellent shear-thinning properties, play a crucial role in 3D bioprinting. Different compositions of bioprinting inks are suitable for various tissue engineering applications, and living cells can be encapsulated within them. This allows for better and more uniform dispersion of cells within the hydrogel formed by the ink.

[0005] Currently, researchers using bioprinting inks to print human tissues or organs mainly focus on skin, blood vessels, and bones, with very few researching the printing and fabrication of biomimetic artificial valves. Furthermore, in the printing and fabrication of skin, blood vessels, and bones, single-nozzle printing is often used, which can only print a single material to build a scaffold, making it difficult to combine multiple materials in local structures. Therefore, it is difficult to construct complex tissues and organs. Summary of the Invention

[0006] To address the aforementioned deficiencies or improvement needs of existing technologies, the present invention aims to provide a biomimetic valve multi-nozzle 3D bioprinted scaffold and its application. This scaffold is modified in its structure and composition, incorporating a biomimetic valve with a three-layer structure: an elastin layer, a polysaccharide layer, and a collagen fiber layer (the three layers are firmly connected). Each layer includes at least two photocurable hydrogel materials, one providing mechanical support and the other mixed with bioactive substances (which may also contain cells). The resulting cross-linked bioactive hydrogel provides a suitable living environment for valvular interstitial cells and other cells. Furthermore, different bioactive substances are distributed across different layers during printing (i.e., different bioactive substances can be used to construct the elastin, polysaccharide, and collagen fiber layers), providing cells with a specific sensory microenvironment, stimulating them to secrete corresponding extracellular matrix, and completing extracellular matrix remodeling.

[0007] To achieve the above objectives, according to one aspect of the present invention, a multi-nozzle 3D bioprinted scaffold for biomimetic valves is provided, characterized in that it comprises, from bottom to top, an elastin layer, a polysaccharide layer, and a collagen fiber layer that are firmly connected to each other.

[0008] For any one of the elastin layer, polysaccharide layer and collagen fiber layer, they are all 3D printed by alternating at least two materials; for any one of the at least two materials used, one of these materials is a photocurable hydrogel material used to provide mechanical support, and the other is a photocurable hydrogel material mixed with bioactive substances;

[0009] Furthermore, the bioactive substances used in the elastin layer, polysaccharide layer, and collagen fiber layer are different from each other.

[0010] As a further preferred embodiment of the present invention, the bioactive substance used in the elastin layer is at least one of chondroitin sulfate (ChS) and fibronectin (FN);

[0011] The corresponding bioactive substance used in the polysaccharide layer is polyaspartic acid (PASP);

[0012] The bioactive substances used in the collagen fiber layer are at least one of fibroblast growth factor bFGF, recombinant human type III collagen (rhCol III), and ascorbic acid (AA);

[0013] Preferably, the photocurable hydrogel material used to provide mechanical support is specifically a mixture of multi-arm polyethylene glycol diacrylate (PEGDA) and methacrylamide gelatin (GelMA); the photocurable hydrogel material mixed with bioactive substances is specifically GelMA mixed with bioactive substances.

[0014] More preferably, the photocurable hydrogel material containing bioactive substances also contains cells.

[0015] According to another aspect of the present invention, the present invention provides a method for preparing the above-mentioned biomimetic valve multi-nozzle 3D bioprinted scaffold, characterized by comprising the following steps:

[0016] S1. Mix multi-arm PEGDA with GelMA in phosphate buffered solution (PBS) to obtain printing ink one; mix GelMA with ChS in PBS to obtain printing ink two; mix GelMA with PASP in PBS to obtain printing ink three; mix GelMA with bFGF in PBS to obtain printing ink four.

[0017] S2. Load printing ink into the channels of each nozzle of the 3D bioprinter, wherein the first nozzle cartridge is loaded with printing ink one, the second nozzle cartridge is loaded with printing ink two, the third nozzle cartridge is loaded with printing ink three, and the fourth nozzle cartridge is loaded with printing ink four.

[0018] S3. Using 3D printing, firstly, an elastin layer is prepared by alternating printing with the first and second nozzles; then, a polysaccharide layer is prepared by alternating printing with the first and third nozzles; next, a collagen fiber layer is prepared by alternating printing with the first and fourth nozzles; cross-linking and curing are performed using ultraviolet light to obtain a biomimetic valve multi-nozzle 3D bioprinted scaffold; or:

[0019] Using 3D printing, a collagen fiber layer is first prepared by alternating printing with the first and fourth nozzles; then a polysaccharide layer is prepared by alternating printing with the first and third nozzles; next, an elastin layer is prepared by alternating printing with the first and second nozzles; and finally, cross-linking and curing are performed with ultraviolet light to obtain a biomimetic valve multi-nozzle 3D bioprinted scaffold.

[0020] As a further preferred embodiment of the present invention, step S1 specifically includes the following sub-steps:

[0021] Preparation of Printing Ink 1: Multi-arm PEG is dissolved in dichloromethane (DCM) or toluene, nitrogen or argon is introduced, and the mixture is stirred at a temperature of 0-30°C. Triethylamine and allyl organic compounds are added to react. After the reaction is complete, the reaction solution is precipitated with an organic solvent, filtered and dried to obtain multi-arm PEGDA. Then, multi-arm PEGDA and GelMA are dissolved in PBS, a photoinitiator solution is added, the mixture is mixed evenly, and air bubbles are removed to obtain Printing Ink 1.

[0022] Preparation of Printing Ink II: Gelatin was placed in deionized water or PBS and stirred at 40℃~70℃ until completely dissolved. Then, methacrylic anhydride or glycidyl methacrylate was added, and the reaction was continued at 40℃~70℃. The product was dialyzed through a dialysis bag for several days and then freeze-dried to obtain GelMA. Then, GelMA and ChS were mixed in PBS, a photoinitiator solution was added, and the mixture was stirred evenly and the air bubbles were removed to obtain Printing Ink II.

[0023] Preparation of Printing Ink III: Gelatin was placed in deionized water or PBS and stirred at 40℃~70℃ until completely dissolved. Then, methacrylic anhydride or glycidyl methacrylate was added, and the reaction was continued at 40℃~70℃. The product was dialyzed through a dialysis bag for several days and then freeze-dried to obtain GelMA. Then, GelMA and PASP were mixed in PBS buffer, a photoinitiator solution was added, and the mixture was stirred evenly to remove air bubbles, thus obtaining Printing Ink III.

[0024] Preparation of Printing Ink IV: Gelatin was placed in deionized water or PBS and stirred at 40℃~70℃ until completely dissolved. Then, methacrylic anhydride or glycidyl methacrylate was added, and the reaction was continued at 40℃~70℃. The product was dialyzed through a dialysis bag for several days and then freeze-dried to obtain GelMA. Then, GelMA was dissolved in PBS, and fibroblast growth factor bFGF and photoinitiator solution were added. The mixture was stirred evenly, and air bubbles were removed to obtain Printing Ink IV.

[0025] As a further preferred embodiment of the present invention, in step S3, the number of printing layers of the elastin layer, the polysaccharide layer, and the collagen fiber layer is 1 to 6, and each printing layer is printed alternately by corresponding dual printheads;

[0026] The printing parameters used in the 3D printing are as follows:

[0027] The first nozzle uses a 23-27G diameter needle, with a nozzle temperature of 18℃-25℃, a moving speed of 3-8mm / s, and an extrusion speed of 0.3-0.6mm. 3 / s, the nozzle's no-load backflow is 0.2-0.5mm, and the lift is 1-2mm;

[0028] The second, third, and fourth nozzles use 23–27G diameter needles, with a nozzle temperature of 16℃–24℃, a moving speed of 3–7 mm / s, and an extrusion speed of 0.25–0.7 mm / s. 3 / s, the nozzle's no-load backflow is 0.2-0.6mm, and the lift is 1-2mm;

[0029] UV irradiation is performed after each alternating layer is printed, i.e., a pre-crosslinking process is carried out once; the UV light intensity used for UV irradiation is 5–15 mW / cm². 2 Each UV irradiation lasts for 3 to 15 seconds; and after the last printed layer is pre-crosslinked by UV irradiation, it is irradiated again to ensure complete crosslinking; more preferably, the UV wavelength used for UV irradiation is 405 nm.

[0030] As a further preferred embodiment of the present invention, for the preparation of printing ink one, the multi-arm PEGDA is one or more of 2arm-PEGDA, 3arm-PEGDA, 4arm-PEGDA, 6arm-PEGDA and 8arm-PEGDA with an average molecular weight of 10,000 to 20,000.

[0031] Accordingly, the multi-arm PEG component in the multi-arm PEGDA corresponds to one or more combinations of two-arm polyethylene glycol (2arm-PEG), three-arm polyethylene glycol (3arm-PEG), four-arm polyethylene glycol (4arm-PEG), six-arm polyethylene glycol (6arm-PEG), and eight-arm polyethylene glycol (8arm-PEG).

[0032] As a further preferred embodiment of the present invention, for the preparation of printing ink one, the molar ratio of the multi-arm PEG to triethylamine and allyl organic compound is satisfied as 1:(2-8):(2-8), and the reaction time is 8-24h; the organic solvent used for precipitation is one of diethyl ether, methyl tert-butyl ether and isopropyl ether.

[0033] In the obtained printing ink, the concentration of multi-arm PEGDA was 0.02–0.1 g / mL, and the concentration of GelMA was 0.05–0.15 g / mL;

[0034] Preferably, the allyl organic compound is one of acryloyl chloride and methacryloyl chloride;

[0035] For the preparation of printing ink II, the stirring speed is 400-800 rpm; the amount of methacrylic anhydride or glycidyl methacrylate used is 0.4-1.2 mL per gram of gelatin; the reaction time is 1-24 h; the molecular cutoff of the dialysis bag is 8000-14000 Da; and the dialysis time is 2-14 days.

[0036] In the second printing ink obtained, the concentration of GelMA was 0.05–0.15 g / mL, and the concentration of ChS was 0.005–0.02 g / mL;

[0037] For the preparation of printing ink three, the concentration of GelMA in the obtained printing ink three is 0.05-0.15 g / mL, and the concentration of PASP is 0.005-0.02 g / mL;

[0038] For the preparation of printing ink four, the concentration of GelMA in the obtained printing ink four is 0.05-0.15 g / mL, and the concentration of bFGF is 1-100 ng / mL;

[0039] In the preparation of printing ink one, printing ink two, printing ink three and printing ink four, the air bubbles are removed specifically by centrifugation.

[0040] The photoinitiator in printing ink 1, printing ink 2, printing ink 3 and printing ink 4 is one or more of α-ketoglutarate (α-KGA), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (I2959) and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP); preferably lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP).

[0041] As a further preferred embodiment of the present invention, in step S1, valve interstitial cells are additionally added to printing inks two, three, and four, with the corresponding concentration of valve interstitial cells in these printing inks being 1–5 × 10⁻⁶. 6 per ml.

[0042] According to another aspect of the present invention, the present invention provides the application of the above-described biomimetic valve multi-nozzle 3D bioprinting scaffold in the preparation of valve tissue and / or valve organoids.

[0043] Compared with existing technologies, this invention utilizes a biomimetic three-layer valve structure and composition, and obtains an artificial valve through 3D printing and cultivation. To address the challenges of balancing mechanical properties and cell culture in existing scaffolds, and the difficulty in achieving integrated molding of the three-layer valve structure, this invention designs and selects at least two photocurable hydrogel materials to print each 3D printed layer. One material provides mechanical support (e.g., a hydrogel obtained by mixing and curing multi-arm PEGDA and GelMA can be used to provide mechanical support for the 3D bioprinted scaffold), while the other material contains bioactive substances and cells (the cells are optional; they can be added to the printing ink or not, but rather injected into the printed scaffold using a "dispensing" method for cultivation; for example, GelMA can be used as a photocurable bioprinting ink to encapsulate bioactive substances and valvular interstitial cells during printing). The initial morphology of the valve tissue is obtained through alternating multi-nozzle printing, layer-by-layer printing, and photocuring, followed by culture in an incubator. This invention particularly utilizes a mixture of PEGDA and GelMA as a photocurable hydrogel material to provide mechanical support, and GelMA as a photocurable hydrogel material for mixing bioactive substances. The multi-armed PEGDA, depending on its molecular weight and number of arms, can be inter-doped to possess various properties such as swelling capacity, curing time, and adjustable mechanical properties. The combination of multi-armed PEGDA and GelMA provides good mechanical properties for the scaffold while satisfying printability. The bioactive hydrogel can provide a suitable living environment for cells such as valvular interstitial cells, and different bioactive substances are distributed in different layers during printing, providing cells with a specific sensory microenvironment, stimulating them to secrete corresponding extracellular matrix, and completing extracellular matrix remodeling. This invention combines the advantages of multiple materials, possesses high designability, and provides a new approach for the production and preparation of tissue-engineered valves, which is of great significance in 3D printing research of biological tissues and organs.

[0044] The 3D bioprinting scaffold of this invention can be printed alternately. For example, it can be printed alternately using multiple nozzles according to a preset pattern using existing 3D bioprinting processes. Each layer of the scaffold includes at least two materials, referred to as the first material and the second material. The first material can support bioactive substances, active factors, or biocompatible hydrogels containing cells; the second material is a hydrogel providing mechanical support. The first and second materials are printed alternately to construct an integrated 3D bioprinting scaffold. This invention can particularly design four types of printing inks, each containing at least one photocurable hydrogel material, prepared by modifying a polymer with an olefinic carbon group; for example, printing ink one with multi-arm PEGDA and GelMA as the main functional components; printing ink two with GelMA and ChS as the main functional components; printing ink three with GelMA and PASP as the main functional components; and printing ink four with GelMA and bFGF as the main functional components. This invention allows for flexible selection of the printing ink providing mechanical support hydrogels and bioactive substances according to actual needs. Based on this invention, fibroblast growth factor bFGF, PASP, ChS, etc. can be preferred as bioactive substances for 3D bioprinting scaffolds, and GelMA can be selected as a bioactive substrate to encapsulate valve interstitial cells and different bioactive substances respectively. Bioactive hydrogels are obtained by ultraviolet light crosslinking, thereby creating a more efficient biomimetic three-layer valve structure.

[0045] Specifically, the present invention can achieve the following beneficial effects:

[0046] (1) This invention utilizes a biomimetic valve structure and composition, selecting a photocurable hydrogel that provides mechanical support and a photocurable hydrogel mixed with bioactive substances and cells. A three-layer structure (corresponding to the elastin layer, polysaccharide layer, and collagen fiber layer of the valve) is designed using a bio-3D printer. The initial morphology of the valve tissue is obtained through alternating multi-nozzle printing, layer-by-layer printing, and photocuring. Culture medium is then added and cultured in an incubator. This invention uses alternating multi-nozzle printing to obtain the scaffold, allowing the mechanical properties of the mechanical support hydrogel and the cell culture performance of the bioactive hydrogel to be adjusted separately, avoiding the need to balance mechanical and cell culture performance with a single printing ink. In particular, this invention can use a mixture of PEGDA and GelMA as the photocurable hydrogel material to provide mechanical support. The swelling properties, curing time, and mechanical properties of the scaffold can be optimized by adjusting the molecular weight and number of arms of PEGDA.

[0047] (2) The biomimetic valve multi-nozzle 3D bioprinting scaffold material prepared by the present invention has good mechanical properties and biodegradability, and promotes the proliferation, migration and extracellular matrix secretion of valve interstitial cells.

[0048] (3) This invention uses multi-nozzle collaborative printing with different printing inks to biomimetically prepare a three-layer valve structure, and uses bioactive substances to induce valve interstitial cells to secrete corresponding extracellular matrix in different structural layers, thereby achieving layered or integrated printing of the valve.

[0049] (4) Furthermore, in the 3D printing process, the present invention can, in particular, perform pre-crosslinking with ultraviolet light after each alternating layer is printed. Unlike complete crosslinking after each alternating layer is printed, the present invention enables the already printed m-th layer to bond more tightly with the newly printed m+1-th layer through pre-crosslinking, thereby obtaining a scaffold with a tightly bonded three-layer structure of a biomimetic valve.

[0050] (5) The present invention prepares valve bioprinted scaffolds by a bio-3D printer. The entire preparation process is short, simple, integrates multiple advantages, and is easy to mass-produce. Moreover, the size, geometry, and spatial heterogeneous structure of the scaffold can be designed according to requirements, such as to meet the different shapes of mitral and tricuspid valves.

[0051] In summary, the 3D bioprinted scaffold of this invention has multiple functions such as good mechanical strength, high bioactivity and biodegradability. It can promote cell proliferation, migration and extracellular matrix remodeling, and has significant application value in valve repair, research on the pathogenesis of valve calcification and screening of drugs for the treatment of valve calcification. Attached Figure Description

[0052] Figure 1 The image shows the hydrogen nuclear magnetic resonance (NMR) analysis results of the 8arm-PEGDA preparation product in the printing ink obtained in Example 1.

[0053] Figure 2 The image shows the hydrogen nuclear magnetic resonance (NMR) analysis results of the product GelMA prepared in Example 4.

[0054] Figure 3 The images show the physical specimen of the 3D-printed elastin layer scaffold obtained in Example 10 and the staining image of the cell culture substrate secretion. Figure 3 (a) in the image corresponds to the actual object (the scale in the image represents 2mm). Figure 3 (b) in the image corresponds to the cell culture medium secretion staining pattern (the scale bar in the image represents 250 μm).

[0055] Figure 4 The images shown are of the 3D-printed polysaccharide layer obtained in Example 10 and the cell culture substrate secretion staining image; wherein, Figure 4 (a) in the image corresponds to the actual object (the scale in the image represents 2mm). Figure 4 (b) in the image corresponds to the cell culture medium secretion staining pattern (the scale bar in the image represents 250 μm).

[0056] Figure 5 The images shown are of the 3D-printed collagen fiber layer obtained in Example 10 and the cell culture substrate secretion staining image; wherein, Figure 5 (a) in the image corresponds to the actual object (the scale in the image represents 2mm). Figure 5 (b) in the image corresponds to the cell culture medium secretion staining pattern (the scale bar in the image represents 250 μm).

[0057] Figure 6 This is a physical image of the 3D bioprinter used in an embodiment of the present invention.

[0058] Figure 7 This is a multi-nozzle 3D bioprinted scaffold for a biomimetic valve obtained in Example 12 (the scale bar in the figure indicates 2mm).

[0059] Figure 8 The image shows the cell viability and death staining after three-dimensional culture of cells encapsulated in GelMA hydrogel obtained in Example 13; wherein, Figure 8 (a) corresponds to 0.05 g / mL GelMA. Figure 8 (b) in the text corresponds to 0.1 g / mL GelMA.

[0060] Figure 9 This is a staining image of the cells after three-dimensional culture encapsulated in ElaMA hydrogel, obtained in Comparative Example 1.

[0061] Figure 10 This is a staining image of the live and dead cells after three-dimensional culture of cells encapsulated in ChSMA hydrogel, obtained in Comparative Example 2.

[0062] Figure 11 This is a staining image of the cells after three-dimensional culture using HAMA hydrogel encapsulated in Comparative Example 3. Detailed Implementation

[0063] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0064] This invention relates to four types of printing inks, which can be synthesized, for example, by following these steps:

[0065] (1) Printing Ink 1: Dissolve multi-arm PEG in dichloromethane (DCM), introduce nitrogen gas, stir at room temperature, and slowly add triethylamine and allyl organic compound in a certain molar ratio to react. After the reaction is completed, precipitate the reaction solution with an organic solvent, filter and dry to obtain multi-arm PEGDA (i.e., polyethylene glycol diacrylate); dissolve multi-arm PEGDA and GelMA in PBS in a certain proportion, add photoinitiator solution, mix evenly, remove air bubbles, and obtain printing ink 1.

[0066] (2) Printing ink II: Place gelatin in deionized water and stir at a constant temperature to dissolve it completely. Then slowly add methacrylic anhydride or glycidyl methacrylate and continue the reaction for a period of time. Dialyze the product with a dialysis bag for a few days and freeze-dry it to obtain GelMA. Mix GelMA and ChS in PBS in a certain proportion, add photoinitiator solution, mix evenly, remove air bubbles, and obtain printing ink II.

[0067] (3) Printing ink three: GelMA and PASP obtained in step (2) are mixed in PBS in proportion, photoinitiator solution is added, mixed evenly, and air bubbles are removed to obtain printing ink three.

[0068] (4) Printing Ink Four: Dissolve the GelMA obtained in step (2) in PBS, add a small amount of fibroblast growth factor bFGF and photoinitiator solution, mix well, remove air bubbles, and obtain printing ink four.

[0069] Specifically, in printing ink one, the molar ratio of multi-arm PEG to triethylamine and allyl organic compound can be 1:(2-8):(2-8), the reaction time is 8-24h, and the organic solvent used for precipitation can be one of diethyl ether, methyl tert-butyl ether, and isopropyl ether; the concentration of multi-arm PEGDA in printing ink one can be 0.02-0.1g / mL, the concentration of GelMA can be 0.05-0.15g / mL, and the photoinitiator can be one of α-ketoglutaric acid (α-KGA), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (I2959), and lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (LAP).

[0070] Allyl organic compounds can be, for example, one of acryloyl chloride and methacryloyl chloride.

[0071] For printing ink II, the constant temperature for gelatin can be 40℃~70℃, the rotation speed can be 400~800rpm, the amount of methacrylic anhydride or glycidyl methacrylate can be 0.4~1.2mL / g gelatin, the reaction time can be 1~24h, the molecular cutoff of the dialysis bag can be 8000~14000Da, and the dialysis time can be 2~14 days; the concentration of GelMA in printing ink II can be 0.05~0.15g / mL, and the concentration of ChS can be 0.005~0.02g / mL.

[0072] In the third type of printing ink, the concentration of GelMA can be 0.05–0.15 g / mL, and the concentration of PASP can be 0.005–0.02 g / mL.

[0073] In the fourth type of printing ink, the concentration of GelMA can be 0.05–0.15 g / mL, and the concentration of bFGF can be 1–100 ng / mL.

[0074] Then, the printing ink prepared in steps (1)-(4) can be loaded into the corresponding printing nozzle barrels, the printing pattern and printing parameters can be set, cross printing can be performed, and UV light can be used for curing to obtain a biomimetic valve multi-nozzle 3D printed bioprinting scaffold.

[0075] For example, the printed pattern can be a series of layers in the shape of a cuboid or a triangular prism. Taking the cuboid shape as an example, the pattern design and printing parameters can be as follows:

[0076] On one hand, the pattern design can be generated by a bio-3D printer. For example, the length and width of the cuboid can be 10-30mm. The first and second nozzles can alternately print 1-6 layers to obtain an elastin layer (each layer is printed alternately, that is, the first nozzle completes the printing, and then the second nozzle completes the printing within the same layer; taking the ink lines printed by different nozzles in an alternating arrangement as an example, specifically, the first nozzle prints m ink lines parallel to the X direction, and the second nozzle then continues to print n ink lines parallel to the X direction between any two adjacent ink lines, finally obtaining m+n ink lines, the length of any ink line is the length of the cuboid, and the total span of m+n ink lines is the width of the cuboid). The first and third nozzles can alternately print 1-6 layers to obtain a polysaccharide layer, and the first and fourth nozzles can alternately print 1-6 layers to obtain a collagen fiber layer. By stacking the entire layer, the target scaffold can be obtained.

[0077] On the other hand, the printing parameters for a bio-3D printer can be set as follows: the first nozzle uses a 23-27G diameter needle (corresponding to a 3D printing layer height of 0.1-0.35mm), the nozzle temperature is 18℃-25℃, the moving speed is 3-8mm / s, and the extrusion speed is 0.3-0.6mm. 3 The nozzle's empty run retraction is 0.2–0.5 mm / s, and the lift is 1–2 mm. The second, third, and fourth nozzles use 23–27G needles, with a nozzle temperature of 16℃–24℃, a moving speed of 3–7 mm / s, and an extrusion speed of 0.25–0.7 mm / s. 3 / s, the printhead retraction during idle travel is 0.2–0.6 mm, and the lift-off is 1–2 mm. The UV lamp used for UV treatment in the following embodiments is integrated into the printhead, with a wavelength of 405 nm and a light intensity of 5–15 mW / cm². 2 (e.g., 5mW / cm) 2 10mW / cm 2 15mW / cm 2 Each time an alternating layer is printed, it is pre-crosslinked by UV light irradiation once, with each UV treatment lasting 3 to 15 seconds. After the last printed layer is pre-crosslinked by UV light irradiation, it is irradiated by UV light again to ensure complete crosslinking.

[0078] The following are specific examples:

[0079] Example 1: A method for preparing printing ink for a multi-nozzle 3D bioprinting scaffold with a biomimetic valve, comprising the following steps:

[0080] (1) Dissolve 20,000 molecular weight 8arm-PEG in anhydrous DCM to obtain a solution with a concentration of 0.4 g / mL.

[0081] (2) Introduce nitrogen gas and perform magnetic stirring at 500 rpm at room temperature.

[0082] (3) Triethylamine and acryloyl chloride are added sequentially and slowly in a ratio of 1:8:8, and the reaction is carried out for 12 hours.

[0083] (4) After the reaction is complete, the product is precipitated with diethyl ether, and then re-dissolved with DCM and precipitated with diethyl ether three times. The precipitate is dried under vacuum to obtain 8arm-PEGDA.

[0084] (5) Dissolve 8arm-PEGDA and GelMA in PBS at concentrations of 0.02 g / mL and 0.05 g / mL, respectively. Add photoinitiator LAP at a concentration of 0.0003 g / mL, mix well, and remove air bubbles to obtain printing ink one. Figure 1The figure shows the proton NMR results of the prepared product 8arm-PEGDA. It can be seen from the figure that 8arm-PEGDA has three more peaks than 8arm-PEG in the range of 6 to 6.5 ppm, indicating that acryloyl chloride has been successfully modified onto 8arm-PEG.

[0085] Example 2: A method for preparing printing ink for a multi-nozzle 3D bioprinting scaffold with a biomimetic valve, comprising the following steps:

[0086] (1) Dissolve 4arm-PEG with a molecular weight of 15,000 in anhydrous DCM to obtain a solution with a concentration of 0.3 g / mL.

[0087] (2) Introduce nitrogen gas and perform magnetic stirring at 800 rpm in an ice-water bath.

[0088] (3) Triethylamine and methacryloyl chloride are added sequentially and slowly in a ratio of 1:5:5, based on molar amounts, and the reaction is carried out for 24 hours.

[0089] (4) After the reaction is complete, the product is precipitated with methyl tert-butyl ether, and then redissolved with DCM and precipitated with methyl tert-butyl ether 4 times. The precipitate is dried under vacuum to obtain 4arm-PEGDA.

[0090] (5) Dissolve 4arm-PEGDA and GelMA in PBS at concentrations of 0.05 g / mL and 0.10 g / mL, respectively. Add photoinitiator LAP at a concentration of 0.0004 g / mL, mix well, and remove air bubbles under vacuum to obtain printing ink.

[0091] Example 3: A method for preparing printing ink for a multi-nozzle 3D bioprinting scaffold with a biomimetic valve, comprising the following steps:

[0092] (1) Dissolve 10,000 molecular weight 2arm-PEG in anhydrous DCM to obtain a solution with a concentration of 0.5 g / mL.

[0093] (2) Introduce nitrogen gas and perform magnetic stirring at 700 rpm at 30°C.

[0094] (3) Triethylamine and acryloyl chloride are added sequentially and slowly in a ratio of 1:3:3, based on molar amounts, and the reaction is carried out for 8 hours.

[0095] (4) After the reaction is complete, the product is precipitated with diethyl ether, and then redissolved with DCM and precipitated with diethyl ether again. After repeating this process twice, the precipitate is dried under vacuum to obtain 2arm-PEGDA.

[0096] (5) Dissolve 2arm-PEGDA and GelMA in PBS at concentrations of 0.10 g / mL and 0.15 g / mL, respectively. Add photoinitiator LAP at a concentration of 0.0005 g / mL, mix well, and remove air bubbles under vacuum to obtain printing ink one.

[0097] Examples 1-3 described above were carried out at room temperature. In addition to room temperature, they can also be carried out at other temperatures ranging from 0 to 30°C.

[0098] Example 4: A method for preparing GelMA from printing inks two, three, and four of a multi-nozzle 3D bioprinting scaffold for a biomimetic valve, comprising the following steps:

[0099] (1) Place 10g of Gelatin in 100mL of deionized water and stir at 450rpm at 60℃ to dissolve it, thus obtaining a homogeneous solution.

[0100] (2) While stirring continuously, slowly add 4 mL of methacrylic anhydride.

[0101] (3) After reacting for 10 hours, add deionized water to dilute.

[0102] (4) The product was dialyzed for 7 days using a dialysis bag with a molecular cutoff of 8000 Da.

[0103] (5) The dialysis solution was freeze-dried to obtain GelMA.

[0104] Figure 2 The figure shows the proton NMR analysis results of the prepared product GelMA. It can be seen from the figure that GelMA has two more peaks than Gelatin at around 5.5 ppm. This represents two different types of hydrogen on both sides of the double bond of methacrylic anhydride, proving that the olefin group was successfully modified onto Gelatin.

[0105] In addition to the preparation method of Example 4, GelMA can also be prepared using other known methods in the prior art, or commercially available products can be used.

[0106] Example 5: A method for preparing printing ink two, printing ink three, and printing ink four for a multi-nozzle 3D bioprinting scaffold for a biomimetic valve, comprising the following steps:

[0107] (1) Printing ink 2: Dissolve GelMA and ChS in PBS at concentrations of 0.1 g / mL and 0.01 g / mL, respectively. After pasteurization, add sterilized photoinitiator LAP to a final concentration of 0.0003 g / mL. After mixing the solution evenly, remove air bubbles under vacuum.

[0108] (2) Printing ink 3: Dissolve GelMA and PASP in PBS at concentrations of 0.1 g / mL and 0.01 g / mL, respectively. After pasteurization, add sterilized photoinitiator LAP to a final concentration of 0.0003 g / mL. After mixing the solution evenly, remove air bubbles under vacuum.

[0109] (3) Printing ink 4: Dissolve GelMA and bFGF in PBS at concentrations of 0.1 g / mL and 10 ng / mL, respectively. After pasteurization, add the sterilized photoinitiator LAP to a final concentration of 0.0003 g / mL. After the solution is mixed evenly, remove the air bubbles under vacuum.

[0110] Example 6: A method for preparing printing ink two, printing ink three, and printing ink four for a multi-nozzle 3D bioprinting scaffold for a biomimetic valve, comprising the following steps:

[0111] (1) Printing ink 2: Dissolve GelMA and ChS in PBS at concentrations of 0.05 g / mL and 0.02 g / mL, respectively. After pasteurization, add sterilized photoinitiator LAP to a final concentration of 0.0002 g / mL. After mixing the solution evenly, remove air bubbles under vacuum.

[0112] (2) Printing ink 3: Dissolve GelMA and PASP in PBS at concentrations of 0.05 g / mL and 0.02 g / mL, respectively. After pasteurization, add sterilized photoinitiator LAP to a final concentration of 0.0002 g / mL. After the solution is mixed evenly, remove air bubbles under vacuum.

[0113] (3) Printing ink 4: Dissolve GelMA and bFGF in PBS at concentrations of 0.05 g / mL and 2 ng / mL, respectively. After pasteurization, add the sterilized photoinitiator LAP to a final concentration of 0.0002 g / mL. After the solution is mixed evenly, remove the air bubbles under vacuum.

[0114] Example 7: A method for preparing printing ink two, printing ink three, and printing ink four for a multi-nozzle 3D bioprinting scaffold for a biomimetic valve, comprising the following steps:

[0115] (1) Printing ink 2: Dissolve GelMA and ChS in PBS at concentrations of 0.05 g / mL and 0.005 g / mL, respectively. After pasteurization, add sterilized photoinitiator LAP to a final concentration of 0.0004 g / mL. After mixing the solution evenly, remove air bubbles under vacuum.

[0116] (2) Printing ink 3: Dissolve GelMA and PASP in PBS at concentrations of 0.15 g / mL and 0.02 g / mL, respectively. After pasteurization, add sterilized photoinitiator LAP to a final concentration of 0.0004 g / mL. After mixing the solution evenly, remove air bubbles under vacuum.

[0117] (3) Printing ink 4: Dissolve GelMA and bFGF in PBS at concentrations of 0.15 g / mL and 100 ng / mL, respectively. After pasteurization, add sterilized photoinitiator LAP to a final concentration of 0.0004 g / mL. After the solution is mixed evenly, remove air bubbles under vacuum.

[0118] Example 8: A printing scheme for a multi-nozzle 3D bioprinting scaffold for a biomimetic valve, using a SUNP BIOMAKER4 3D bioprinter (actual object image shown). Figure 6 As shown), it includes the following aspects:

[0119] (1) Printing pattern: The bottom surface of the cuboid is 14mm long and 14mm wide, the printing layer height is 0.20mm, the extrusion needle diameter is 25G, the printing nozzle one and the printing nozzle two alternately print 1 and 2 layers; the printing nozzle one and the printing nozzle three alternately print 3 and 4 layers; the printing nozzle one and the printing nozzle four alternately print 5 and 6 layers.

[0120] (2) Printing parameters: Print head temperature set to 25℃, moving speed 5.5mm / s, extrusion speed 0.5mm. 3 The nozzle retraction and lift during idle travel are 0.5mm and 1mm respectively; the temperature of printhead 2 is set to 24℃, the temperature of printhead 4 is set to 24℃, the moving speed is 7mm / s, and the extrusion speed is 0.7mm / s. 3 / s, the nozzle's no-load retraction and lift are 0.35mm and 1mm respectively, and the UV lamps attached to the nozzles are selected with the following parameters: wavelength 405nm, light intensity 15mW / cm². 2 The illumination time is 3 seconds.

[0121] (3) Printing of a three-layer structure and cross-linking with ultraviolet light in cell culture well plates.

[0122] Example 9: A printing scheme for a multi-nozzle 3D bioprinting scaffold for a biomimetic valve, using a SUNP BIOMAKER4 3D bioprinter, includes the following aspects:

[0123] (1) Printing pattern: The bottom surface of the cuboid is 20mm long and 20mm wide, the printing layer height is 0.25mm, the extrusion needle diameter is 23G, the printing nozzle one and the printing nozzle two alternately print 1 and 2 layers; the printing nozzle one and the printing nozzle three alternately print 3 and 4 layers; the printing nozzle one and the printing nozzle four alternately print 5 and 6 layers.

[0124] (2) Printing parameters: Print head temperature set to 20℃, moving speed 3mm / s, extrusion speed 0.3mm. 3 The nozzle retraction and lift during idle travel are 0.5mm and 1mm respectively; the temperature of printhead 2 is set to 16℃, the temperature of printhead 4 is set to 16℃, the moving speed is 3mm / s, and the extrusion speed is 0.25mm / s. 3 / s, the nozzle's no-load retraction and lift are 0.2mm and 2mm respectively, and the UV lamps attached to the nozzles are selected with the following parameters: wavelength 405nm, light intensity 5mW / cm². 2 The illumination time is 12 seconds.

[0125] (3) Printing of a three-layer structure and cross-linking with ultraviolet light in cell culture well plates.

[0126] Example 10: Layered printing of a multi-nozzle 3D bioprinting scaffold for a biomimetic valve, comprising the following steps:

[0127] (1) Elastin layer: The printing ink I obtained in Example 1 was placed in the printing nozzle barrel; the printing ink II obtained in Example 5 was mixed with valve interstitial cells (i.e., third-generation porcine valve interstitial cells, VIC; the same below), wherein the concentration of valve interstitial cells was 1×10 5 The ink was placed in the cartridge of print head two according to the corresponding printing parameters in Example 8. Print head one and print head two were used to print alternately. The resulting elastin scaffold was immersed in the culture medium and cultured in a cell culture incubator. Figure 3 The image shows a 3D-printed elastin layer scaffold and fluorescent staining of elastin secreted by valvular cells after 14 days of culture. The image shows that the scaffold is intact and that the addition of ChS can promote the expression of elastin matrix by valvular cells.

[0128] (2) Polysaccharide layer: The printing ink obtained in Example 1 is placed in the printing nozzle cartridge; the printing ink obtained in Example 5 is mixed with valvular interstitial cells, wherein the concentration of valvular interstitial cells is 1×10⁻⁶. 5 The ink was placed in the printing nozzle three cartridge at a density of 1 / mL. Printing was performed alternately by printing nozzle one and printing nozzle three according to the corresponding printing parameters in Example 8. The resulting polysaccharide scaffold was immersed in the culture medium and cultured in a cell culture incubator. Figure 4The image shows a 3D-printed polysaccharide layer scaffold and a fluorescent staining image of polysaccharide secreted by valvular cells after 14 days of culture. The image shows that the scaffold is intact and that the addition of PASP can promote the expression of polysaccharide matrix by valvular cells.

[0129] (3) Collagen fiber layer: The printing ink obtained in Example 1 is placed in the printing nozzle cartridge; the printing ink obtained in Example 5 is mixed with valve interstitial cells, wherein the concentration of valve interstitial cells is 1×10⁻⁶. 5 The ink was placed in the printing nozzle four cartridge at a density of 1 / mL. Printing was performed alternately by printing nozzle one and printing nozzle four according to the corresponding printing parameters in Example 8. The resulting collagen fiber layer scaffold was immersed in the culture medium and cultured in a cell culture incubator. Figure 5 The image shows a 3D-printed collagen scaffold and a fluorescence staining image of collagen secreted by valvular cells after 14 days of culture. The image shows that the scaffold is intact and the addition of bFGF can promote the expression of collagen matrix by valvular cells.

[0130] Example 11: Layered printing of a multi-nozzle 3D bioprinting scaffold for a biomimetic valve, comprising the following steps:

[0131] (1) Elastin layer: The printing ink I obtained in Example 2 was placed in the printing nozzle barrel; the printing ink II obtained in Example 6 was mixed with valve interstitial cells, wherein the concentration of valve interstitial cells was 2×10 5 The ink was placed in the cartridge of print head two at a density of 1 / mL. Print head one and print head two were used to print alternately according to the corresponding printing parameters in Example 9. The resulting elastin scaffold was immersed in the culture medium and cultured in a cell culture incubator.

[0132] (2) Polysaccharide layer: The printing ink obtained in Example 2 was placed in the printing nozzle cartridge; the printing ink obtained in Example 6 was mixed with interstitial valve cells, wherein the concentration of interstitial valve cells was 2 × 10⁻⁶. 5 The ink was placed in the printing nozzle three cartridge at a density of 1 / mL. Printing was performed alternately by printing nozzle one and printing nozzle three according to the corresponding printing parameters in Example 9. The resulting polysaccharide scaffold was immersed in the culture medium and cultured in a cell culture incubator.

[0133] (3) Collagen fiber layer: The printing ink obtained in Example 2 is placed in the printing nozzle cartridge; the printing ink obtained in Example 6 is mixed with valve interstitial cells, wherein the concentration of valve interstitial cells is 2×10⁻⁶. 5 The ink was placed in the printing nozzle four cartridge at a density of 1 / mL. Printing was performed alternately by printing nozzle one and printing nozzle four according to the corresponding printing parameters in Example 9. The resulting collagen fiber layer scaffold was immersed in the culture medium and cultured in a cell culture incubator.

[0134] Example 12: A one-time molding printing of a biomimetic valve multi-nozzle 3D bioprinting scaffold, comprising the following steps:

[0135] (1) Place the printing ink obtained in Example 1 into the printing nozzle cartridge.

[0136] (2) Printing inks II, III, and IV obtained in Example 5 were respectively mixed with valve interstitial cells, wherein the concentration of valve interstitial cells was 1×10⁻⁶. 5 The mixture is prepared by mixing each sample at a rate of 1 / mL, and then placing the mixture into the cartridges of printheads 2, 3, and 4, respectively.

[0137] (3) Printing nozzles 1 and 2 alternately print 1 and 2 layers as elastin layers according to the corresponding printing parameters in Example 8; then printing nozzles 1 and 3 alternately print 3 and 4 layers as polysaccharide layers; then printing nozzles 1 and 4 alternately print 5 and 6 layers as collagen fiber layers; the resulting three-layer structure scaffold is immersed in the culture medium. Figure 7 This is a physical image of a multi-nozzle 3D printed bio-printed scaffold for a biomimetic valve. As can be seen from the image, the scaffold has a clear morphology and a complete structure.

[0138] Example 13

[0139] VIC cells were encapsulated in GelMA solutions at concentrations of 0.05 g / mL and 0.1 g / mL for three-dimensional culture, followed by viability and death staining. The results are as follows: Figure 8 As shown, the GelMA solution exhibits excellent three-dimensional cell culture performance, producing a large number of viable cells that can spread out. Furthermore, due to the shear-thinning properties of the GelMA material itself, the printing effect is excellent with high fidelity.

[0140] Comparative Example 1

[0141] ElaMA was prepared in this comparative example: 2g of elastin was dissolved in 40mL of deionized water and stirred at 50℃ until dissolved. 10mL of methacrylic anhydride was slowly added, and the reaction was continued for 24h with stirring. Then, 40mL of deionized water was added to terminate the reaction. After dialysis and lyophilization, ElaMA was obtained. A 0.2g / mL ElaMA solution was then prepared to encapsulate VIC cells for three-dimensional culture. Figure 9 The live / dead staining patterns obtained at a relatively good concentration for cell culture are still far inferior to those obtained at other cell cultures. Figure 8 The number and spreading state of live cells in the solution. Furthermore, the ElaMA solution is relatively dilute and lacks shear-thinning properties, making it difficult to perform extrusion printing.

[0142] Comparative Example 2

[0143] 2 g of ChS was dissolved in 100 mL of deionized water and stirred at room temperature until dissolved. 10 mL of methacrylic anhydride was slowly added, while the pH of the solution was adjusted to 8 using sodium hydroxide. The reaction was stirred for 24 h. The product was precipitated and washed with ethanol, reconstituted, dialyzed, and lyophilized to obtain ChSMA. A ChSMA solution with a concentration of 0.02 g / mL was then prepared to encapsulate VIC cells for three-dimensional culture. Figure 10 The live / dead staining patterns obtained at optimal cell culture concentrations are far inferior to those obtained at other cell cultures. Figure 8 The number and spreading state of live cells in the medium. Furthermore, the ChSMA solution is relatively dilute and lacks shear-thinning properties, making it difficult to perform extrusion printing.

[0144] Comparative Example 3

[0145] 2g of hyaluronic acid was dissolved in 100mL of deionized water and stirred in an ice-water bath until dissolved. 8mL of methacrylic anhydride was slowly added, while simultaneously adjusting the pH of the solution to 8.5 with sodium hydroxide. The reaction was stirred for 24 hours. The product was then dialyzed and lyophilized to obtain HAMA. A 0.02g / mL HAMA solution was then prepared to encapsulate VIC cells for three-dimensional culture. Figure 11 The live / dead staining patterns obtained at optimal cell culture concentrations are far inferior to those obtained at other cell cultures. Figure 8 The number and spreading state of live cells. Although the HAMA solution concentration can be viscous, providing some single-head printing capability, its lack of shear-thinning properties results in very poor performance when printing with multiple nozzles.

[0146] By comparing Example 13 with Comparative Examples 1, 2, and 3, it is evident that, compared to methacrylamide elastin (ElaMA), chondroitin sulfate (ChSMA), and hyaluronic acid (HAMA), the present invention preferably uses GelMA mixed with bioactive substances and cells. Due to its temperature-sensitive and shear-thinning properties, GelMA plays a crucial role in extrusion 3D printing. When ElaMA or ChSMA is dissolved in PBS, the solution is similar to water with very low viscosity, making extrusion printing difficult. Although HAMA can be formulated into a higher viscosity solution, its extrusion printing performance is still inferior to GelMA; in most cases, HAMA is difficult to use in multi-nozzle 3D extrusion printing. Furthermore, although some researchers have used ElaMA, ChSMA, and HAMA to encapsulate cells for culture, the cell culture results are far inferior to those of GelMA. This is because GelMA contains RGD sequences, which greatly promote cell adhesion, cell proliferation, and migration.

[0147] The above embodiments are merely examples. For instance, in addition to using third-generation porcine valvular interstitial cells (VIC), commercially available heart valve cells from other animal sources can also be used. In the alternating printing of the above embodiments, the ink lines are parallel to the X-direction (and parallel to the Y-direction for the next layer). Besides this latitude and longitude arrangement, other alternating methods can be used (e.g., parallel to the X-axis at a certain angle, such as 30°, 45°, 60°, etc., so that the two printing layers form an "X" intersection), as long as each 3D printing layer is printed by two nozzles.

[0148] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A multi-nozzle 3D bioprinted scaffold for a biomimetic valve, characterized in that, From bottom to top, it consists of a layer of elastin, a layer of polysaccharides, and a layer of collagen fibers that are firmly connected to each other; For any one of the elastin layer, polysaccharide layer and collagen fiber layer, they are all 3D printed by alternating at least two materials; for any one of the at least two materials used, one of these materials is a photocurable hydrogel material used to provide mechanical support, and the other is a photocurable hydrogel material mixed with bioactive substances; The bioactive substance used in the elastin layer is at least one of chondroitin sulfate (ChS) and fibronectin (FN). The corresponding bioactive substance used in the polysaccharide layer is polyaspartic acid (PASP). The bioactive substances used in the collagen fiber layer are at least one of fibroblast growth factor bFGF, recombinant human type III collagen rhCol III, and ascorbic acid AA. The photocurable hydrogel material used to provide mechanical support is specifically a mixture of multi-arm polyethylene glycol diacrylate (PEGDA) and methacrylamide gelatin (GelMA); the photocurable hydrogel material mixed with bioactive substances is specifically GelMA mixed with bioactive substances.

2. The multi-nozzle 3D bioprinted scaffold for a biomimetic valve as described in claim 1, characterized in that, Photocurable hydrogel materials containing bioactive substances also contain cells.

3. The method for fabricating a multi-nozzle 3D bioprinted scaffold for a biomimetic valve as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Mix multi-arm PEGDA with GelMA in phosphate-buffered saline (PBS) to obtain printing ink one; mix GelMA with ChS in PBS to obtain printing ink two; mix GelMA with PASP in PBS to obtain printing ink three; mix GelMA with bFGF in PBS to obtain printing ink four. S2. Load printing ink into the channels of each nozzle of the 3D bioprinter, wherein the first nozzle cartridge is loaded with printing ink one, the second nozzle cartridge is loaded with printing ink two, the third nozzle cartridge is loaded with printing ink three, and the fourth nozzle cartridge is loaded with printing ink four. S3. Using 3D printing, firstly, an elastin layer is prepared by alternating printing with the first and second nozzles; then, a polysaccharide layer is prepared by alternating printing with the first and third nozzles; next, a collagen fiber layer is prepared by alternating printing with the first and fourth nozzles; and then, cross-linking and curing are performed with ultraviolet light to obtain a biomimetic valve multi-nozzle 3D bioprinted scaffold. or: Using 3D printing, a collagen fiber layer is first prepared by alternating printing with the first and fourth nozzles; then a polysaccharide layer is prepared by alternating printing with the first and third nozzles; next, an elastin layer is prepared by alternating printing with the first and second nozzles; and finally, cross-linking and curing are performed with ultraviolet light to obtain a biomimetic valve multi-nozzle 3D bioprinted scaffold.

4. The method for fabricating the multi-nozzle 3D bioprinted scaffold of the biomimetic valve as described in claim 3, characterized in that, Step S1 specifically includes the following sub-steps: Preparation of Printing Ink 1: Multi-arm PEG is dissolved in dichloromethane (DCM) or toluene, nitrogen or argon is introduced, and the mixture is stirred at a temperature of 0-30°C. Triethylamine and allyl organic compounds are added to react. After the reaction is complete, the reaction solution is precipitated with an organic solvent, filtered and dried to obtain multi-arm PEGDA. Then, multi-arm PEGDA and GelMA are dissolved in PBS, a photoinitiator solution is added, the mixture is stirred evenly, and air bubbles are removed to obtain Printing Ink 1. Preparation of Printing Ink II: Gelatin was placed in deionized water or PBS and stirred at 40℃~70℃ until completely dissolved. Then, methacrylic anhydride or glycidyl methacrylate was added, and the reaction was continued at 40℃~70℃. The product was dialyzed through a dialysis bag for several days and then freeze-dried to obtain GelMA. Then, GelMA and ChS were mixed in PBS, a photoinitiator solution was added, and the mixture was stirred evenly to remove air bubbles, thus obtaining Printing Ink II. Preparation of Printing Ink III: Gelatin was placed in deionized water or PBS and stirred at 40℃~70℃ until completely dissolved. Then, methacrylic anhydride or glycidyl methacrylate was added, and the reaction was continued at 40℃~70℃. The product was dialyzed through a dialysis bag for several days and then freeze-dried to obtain GelMA. Then, GelMA and PASP were mixed in PBS buffer, a photoinitiator solution was added, and the mixture was stirred evenly to remove air bubbles, thus obtaining Printing Ink III. Preparation of Printing Ink IV: Gelatin was placed in deionized water or PBS and stirred at 40℃~70℃ until completely dissolved. Then, methacrylic anhydride or glycidyl methacrylate was added, and the reaction was continued at 40℃~70℃. The product was dialyzed through a dialysis bag for several days and then freeze-dried to obtain GelMA. Then, GelMA was dissolved in PBS, and fibroblast growth factor bFGF and photoinitiator solution were added. The mixture was stirred evenly, and air bubbles were removed to obtain Printing Ink IV.

5. The preparation method according to claim 3, characterized in that, In step S3, the number of printing layers for the elastin layer, the polysaccharide layer, and the collagen fiber layer is 1 to 6, and each printing layer is printed alternately by corresponding dual printheads; The printing parameters used in the 3D printing are as follows: The first nozzle uses a 23~27G diameter needle, with a nozzle temperature of 18℃~25℃, a moving speed of 3~8mm / s, and an extrusion speed of 0.3~0.6mm. 3 / s, the nozzle's no-load backflow is 0.2~0.5mm, and the lift is 1~2mm; The second, third, and fourth nozzles use 23-27G diameter needles, with a nozzle temperature of 16℃-24℃, a moving speed of 3-7mm / s, and an extrusion speed of 0.25-0.7mm. 3 / s, the nozzle's no-load backflow is 0.2~0.6mm, and the lift is 1~2mm; UV irradiation is performed after each alternating layer is printed, i.e., a pre-crosslinking process is carried out once; the UV light intensity used for UV irradiation is 5~15mW / cm². 2 Each UV irradiation lasted 3-15 seconds; and after the last printed layer was pre-crosslinked by UV irradiation, it was irradiated again to ensure complete crosslinking.

6. The preparation method according to claim 5, characterized in that, The ultraviolet light used in the ultraviolet irradiation is 405nm wavelength.

7. The preparation method according to claim 4, characterized in that, For the preparation of printing ink one, the multi-arm PEGDA is one or more combinations of 2-arm-PEGDA, 3-arm-PEGDA, 4-arm-PEGDA, 6-arm-PEGDA and 8-arm-PEGDA with an average molecular weight of 10,000 to 20,000; Accordingly, the multi-arm PEG component in the multi-arm PEGDA corresponds to one or more combinations of two-arm polyethylene glycol 2 arm-PEG, three-arm polyethylene glycol 3 arm-PEG, four-arm polyethylene glycol 4 arm-PEG, six-arm polyethylene glycol 6 arm-PEG, and eight-arm polyethylene glycol 8 arm-PEG.

8. The preparation method according to claim 4, characterized in that, For the preparation of printing ink one, the molar ratio of the multi-arm PEG to triethylamine and allyl organic compound is 1:(2~8):(2~8), and the reaction time is 8~24h; the organic solvent used for precipitation is one of diethyl ether, methyl tert-butyl ether and isopropyl ether; In the obtained printing ink, the concentration of multi-arm PEGDA is 0.02~0.1g / mL, and the concentration of GelMA is 0.05~0.15g / mL; For the preparation of printing ink II, the stirring speed is 400~800 rpm; the amount of methacrylic anhydride or glycidyl methacrylate used is 0.4~1.2 mL per gram of gelatin; the reaction time is 1~24 h; the molecular cutoff of the dialysis bag is 8000~14000 Da, and the dialysis time is 2~14 days; In the second printing ink obtained, the concentration of GelMA was 0.05~0.15 g / mL, and the concentration of ChS was 0.005~0.02 g / mL; For the preparation of printing ink three, the concentration of GelMA in the obtained printing ink three is 0.05~0.15g / mL, and the concentration of PASP is 0.005~0.02g / mL; For the preparation of printing ink four, the concentration of GelMA in the obtained printing ink four is 0.05~0.15g / mL, and the concentration of bFGF is 1~100ng / mL; In the preparation of printing ink one, printing ink two, printing ink three and printing ink four, the air bubbles are removed specifically by centrifugation. The photoinitiators in Printing Ink 1, Printing Ink 2, Printing Ink 3 and Printing Ink 4 are one or more of α-ketoglutaric acid, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone and phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

9. The preparation method according to claim 8, characterized in that, The allyl organic compound is one of acryloyl chloride and methacryloyl chloride; The photoinitiator in Printing Ink 1, Printing Ink 2, Printing Ink 3 and Printing Ink 4 is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

10. The preparation method according to claim 3, characterized in that, In step S1, valve interstitial cells are additionally added to printing inks two, three, and four, with the concentration of valve interstitial cells in each ink being 1~5×10⁻⁶. 6 per ml.

11. The application of the multi-nozzle 3D bioprinted scaffold of the biomimetic valve as described in claim 1 or 2 in the preparation of valve tissue and / or valve organoids.

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