Gelatin composite hydrogel ink for 3D coaxial printing as well as preparation method and application of gelatin composite hydrogel ink

Abstract

The invention provides gelatin composite hydrogel ink for 3D coaxial printing and a preparation method and application thereof.The gelatin composite hydrogel ink for 3D coaxial printing comprises PGA bio-ink and a CaCl2 aqueous solution, the PGA bio-ink comprises methylacryloylated gelatin, sodium alginate and polyoxyethylene, and through combination of sodium alginate and Ca < 2 + > and photo-crosslinking gelation, the gelatin composite hydrogel ink for 3D coaxial printing is formed. The gelatin composite hydrogel ink for 3D coaxial printing forms a hollow tubular unit, and is prepared by the following steps: (S10) preparing methacrylated gelatin; (S20) preparing the PGA bio-ink; and (S30) preparation of a CaCl2 aqueous solution, printing is accurate, tensile yield is easy, and the prepared biological scaffold has good biocompatibility.

Description

Technical Field

[0001] The present invention relates to, in particular to, a 3D coaxially printed gelatin composite hydrogel ink and a preparation method and application thereof. Background Art

[0002] Tissue engineering is the development of tissue and organ substitutes to repair, improve and reconstruct injured tissues and organs, with the ultimate goal of replacing organ transplantation. In recent years, bioprinting technology has become a hot research direction in tissue engineering. It can effectively encapsulate cells in bio-inks, effectively solving the problems of uneven cell distribution and cell loss in the past printing process. Among them, extrusion bioprinting is a relatively simple technology. It is expected to print functional three-dimensional biological structures and tissue models through precise 3D control of various components (such as cells, support media, etc.). In order to produce anisotropic structures that simulate the biological functions of anisotropic tissues, the methods for manufacturing tissue engineering scaffolds include: inducing the orientation of polymer chains or hydrogel chains using electric / magnetic fields, ion flow direction and diffusion, stretching, biological templates, and drying under restricted conditions. These resulting scaffolds have oriented patterns that enable them to mimic the mechanical properties of related tissues. However, these methods have some disadvantages, such as: (1) the biological properties of these processes and scaffolds are poor; (2) the cell seeding efficiency is low, and it is difficult to prepare anisotropic tissues in vitro.

[0003] Tubular tissue structures are very common in the human body, such as blood vessels, ureters, and urethra. Their diameters vary from large blood vessels of several millimeters to capillaries of several microns, with different diameters and different tissue structures. Therefore, it is very necessary to construct a method that can print hollow tubes of different diameters. Most of the traditional tubular structure printing technologies are based on sacrificial material methods, that is, after printing the complete structure, the sacrificial material is removed by adjusting the temperature, pH, etc. to obtain a hollow vascular structure to support the fusion of multicellular spheres or cylinders into vascular structures. Due to the complexity of the sacrificial bioprinting process, the size, morphology, and function of the obtained vascular-like structure may be limited. Although the recent development of multilayer coaxial extrusion systems has made technological progress, making it possible to directly manufacture hollow hydrogel tubes, it is still not easy to produce complex 3D cell-filled hollow structures, and it has not been proven. Therefore, one-step printing of hollow tubes with high precision, isotropic specificity, and excellent cell survival environment is urgently needed. Summary of the invention

[0004] One advantage of the present invention is that it provides a 3D coaxially printed gelatin composite hydrogel ink and a preparation method and application thereof, which has good biocompatibility, accurate printing, and easy stretching and yielding.

[0005] Another advantage of the present invention is that it provides a 3D coaxially printed gelatin composite hydrogel ink and its preparation method and application, which has high mechanical tensile properties and a double or multiple cross-linking mechanism, and the printed tubular tissue has good isotropic specificity.

[0006] Another advantage of the present invention is that it provides a 3D coaxially printed gelatin composite hydrogel ink and its preparation method and application, uses natural material gelatin as raw material to construct a double cross-linked network, and uses methacrylic anhydride and natural materials to prepare methacrylic gelatin (GelMA). The use of sodium alginate as biological ink is based on the rapid gelation of sodium alginate and calcium ions to achieve the construction of a hollow structure.

[0007] Another advantage of the present invention is that it provides a 3D coaxially printed gelatin composite hydrogel ink and its preparation method and application. In order to improve the cell adhesion and growth performance of the ink, GelMA is added to the ink to form a physical network cross-linked by calcium ions for extrusion. After extrusion, chemical cross-linked hydrogel is formed by photocrosslinking to further stabilize the structure. In order to achieve a directional fiber structure, polyethylene oxide (PEO), a classic thickener and spinning agent, is introduced. After directional shearing, the hydrogel chains will be distributed along the axial direction.

[0008] Another advantage of the present invention is to provide a 3D coaxially printed gelatin composite hydrogel ink and its preparation method and application. The ink is injectable, can be smoothly extruded, will not clog the needle, and has a shear thinning property, which is crucial for a 3D printing platform used as an ink.

[0009] Another advantage of the present invention is that it provides a 3D coaxially printed gelatin composite hydrogel ink and its preparation method and application, which are non-cytotoxic. The results show that the cells in the large-size vascular stent show a highly active and orderly growth morphology along the circumferential direction of the tube, and are therefore suitable for use as a stent in the human body.

[0010] According to one aspect of the present invention, the present invention provides a method for preparing a 3D coaxially printed gelatin composite hydrogel ink, comprising the following steps: (S10) Preparation of methacryloyl gelatin; (S20) Preparation of PGA bio-ink; and (S30)CaCl 2 Preparation of aqueous solution.

[0011] The step (S20) comprises the following steps: (S201) dissolving methacrylated gelatin monomer in H 2 O; (S202) adding LAP, oscillating to mix, and protecting from light; (S203) then adding sodium alginate and polyethylene oxide, mixing, and centrifuging.

[0012] In the step (S20), the concentration of methacrylated gelatin is 10%, the concentration of sodium alginate is 0.5%-2.0%, the concentration of polyethylene oxide is 0.8%, and the concentration of CaCl 2 The concentration of the solution is 0.8%.

[0013] In the step (S10), gelatin and methacrylic anhydride are reacted in a phosphate buffer solution with a pH of 7.4, dialyzed in a 40° C. water bath, and freeze-dried to obtain methacrylated gelatin.

[0014] 1 g of methacryloyl gelatin monomer was weighed and dissolved in 10 mL of dHO. 2 O, and then fully dissolve the methacryloyl gelatin at 50°C, then add 0.3% (m / w) LAP, shake and mix again, protect from light with tin foil, then add 0.1g sodium alginate and 0.08g polyethylene oxide to the gelatin solution, mix again in a 50°C water bath, centrifuge at 3000 rpm for 3min, remove excess bubbles for subsequent use.

[0015] The preparation of methacryloyl gelatin is as follows: 50 g of gelatin and 3.2 mL of methacrylic anhydride are fully reacted in 500 mL of phosphate buffer (pH = 7.4) at 50°C for 3 hours, the above solution is dialyzed in a 40°C water bath for 5 days, the solution is changed 3 times a day, and then freeze-dried for 48 hours to synthesize methacryloyl gelatin.

[0016] Among them, PGA bio-ink and CaCl 2 The aqueous solutions were placed in different channels. When they were squeezed out, sodium alginate and Ca 2+ They are combined and rapidly cross-linked to form hollow tubular units, which are then printed under a predetermined tensile stress.

[0017] According to another aspect of the present invention, the present invention also provides a 3D coaxially printed gelatin composite hydrogel ink, wherein the 3D coaxially printed gelatin composite hydrogel ink comprises PGA bio-ink and CaCl 2 The aqueous solution, wherein the PGA bio-ink comprises methacrylated gelatin, sodium alginate and polyethylene oxide, is used as a 3D coaxially printed gelatin composite hydrogel ink, and the PGA bio-ink and CaCl 2 The aqueous solution was in situ cross-linked in different channels at a flow rate ratio of 5:4, and the sodium alginate and Ca 2+ The combined, photo-crosslinked and gelled to form hollow tubular units.

[0018] The outer axis is the PGA bio-ink channel during printing, and the inner axis is 0.8% CaCl 2 Solution, when sodium alginate and Ca 2+ Combined and quickly cross-linked, a hollow tubular unit is formed. The pulling stress is changed by changing the spindle speed and the spindle size. The spindle speed is 66 rpm and the spindle diameter is 5 mm. After printing, it is cured under 405 nm ultraviolet light for 30 seconds.

[0019] According to another aspect of the present invention, the present invention also provides an application of 3D coaxial printing gelatin composite hydrogel ink, wherein the composite hydrogel ink comprises PGA bio-ink and CaCl 2 Aqueous solution, suitable for printing biomimetic directional muscular hollow tubular scaffolds.

[0020] The 3D coaxially printed gelatin composite hydrogel ink is used for coaxial printing and combined with stress traction to manufacture a hundred-micron tubular unit. The coaxial printing method comprises the following steps: (A) placing PGA bio-ink in the outer axis channel; (B) placing CaCl 2 The aqueous solution is placed in the inner shaft channel; (C) the tensile stress is adjusted, photo-crosslinked and gelled to form a hollow tubular unit; and (D) after printing is completed, UV light curing is performed.

[0021] The inner shaft size is 22G, the outer shaft size is 17G, the flow rates of the inner and outer channels are set to 0.4mL / min and 0.5mL / min respectively, the shaft speed is 66rpm, the shaft diameter is 5mm, and after printing is completed, it is cured under 405nm ultraviolet light for 30s.

[0022] The preparation method of PGA bio-ink is as follows: dissolve methacrylated gelatin monomer in H 2 O; add LAP, shake and mix, protect from light;) then add sodium alginate and polyethylene oxide, mix and centrifuge.

[0023] The concentration of methacryloyl gelatin is 10%, the concentration of sodium alginate is 0.5%-2.0%, the concentration of polyethylene oxide is 0.8%, and the concentration of CaCl 2 The concentration of the solution is 0.8%.

[0024] According to another aspect of the present invention, a method for preparing a 3D coaxially printed gelatin composite hydrogel ink provided by the present invention comprises the following steps: (S10) preparing methacryloyl gelatin, reacting gelatin with methacrylic anhydride in a phosphate buffer solution, dialyzing in a water bath, and freeze-drying to obtain methacryloyl gelatin; (S20) Preparation of PGA bio-ink, wherein the step (S20) comprises the following steps: (S201) dissolving methacrylated gelatin monomer in H 2O; (S202) adding LAP, oscillating and mixing, and protecting from light; (S203) then adding sodium alginate and polyethylene oxide, mixing, centrifuging, and preparing PGA bio-ink; and (S30)CaCl 2 Preparation of aqueous solution; PGA bio-ink and CaCl 2 The aqueous solutions were placed in different channels. When PGA and CaCl 2 When the aqueous solution is squeezed out at a flow rate ratio of 5:4, the sodium alginate and Ca 2+ Coaxial in-situ cross-linking and light curing form a hollow tubular unit. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the synthesis of methacryloyl gelatin according to the above preferred embodiment of the present invention.

[0026] Figure 2 1 is a hydrogen nuclear magnetic resonance spectrum of methacryloyl gelatin according to the above preferred embodiment of the present invention.

[0027] Figure 3 Schematic diagram of rheological characterization of seaweed yogurt when the concentration is 1% according to the above preferred embodiment of the present invention.

[0028] Figure 4 It is a schematic diagram of coaxial printing and material formula screening according to the above preferred embodiment of the present invention.

[0029] Figure 5 It is a schematic diagram of live-dead staining of smooth muscle cells in tubular units under different components according to the above preferred embodiment of the present invention.

[0030] Figure 6 It is a schematic diagram of electron microscopy, pore size statistics and cell survival rate statistics of hydrogels of different components according to the above preferred embodiment of the present invention.

[0031] Figure 7 It is a detection schematic diagram of manufacturing micron-level directional tubular units by coaxial printing and coordinated stress traction according to the above preferred embodiment of the present invention.

[0032] Figure 8 Schematic diagram of the biocompatibility of the PGA composite hydrogel according to the above preferred embodiment of the present invention.

[0033] Fig. 9 It is a detection schematic diagram of manufacturing micron-level directional tubular units by coaxial printing and coordinated stress traction according to the above preferred embodiment of the present invention.

[0034] Fig.10 It is a schematic diagram of printing a millimeter-level directional tubular stent according to the above preferred embodiment of the present invention.

[0035] Fig.11 It is a schematic diagram of printing and dyeing of a cell-laden PGA directional tubular scaffold according to the above preferred embodiment of the present invention.

[0036] Fig.12 It is a schematic diagram of the 3D printing structure of a multi-layer tubular stent according to the ink in the above preferred embodiment of the present invention. DETAILED DESCRIPTION

[0037] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art can think of other obvious variations. The basic principles of the present invention defined in the following description can be applied to other embodiments, variations, improvements, equivalents, and other technical solutions that do not deviate from the spirit and scope of the present invention.

[0038] The present invention focuses on the key issue of regulating the directional growth of smooth muscle cells. By introducing polyethylene oxide (PEO), based on the phase separation and shear orientation characteristics of the viscous polymer compound mixture, the pre-shearing in-situ coaxial bioprinting of highly oriented porous hydrogel microfibers is designed to print tubular units. Specifically, Figure 4 As shown in (ad), GELMA, sodium alginate, PEO, and cells are uniformly mixed together to form a two-phase viscous bio-ink, which is transported through a silicone tube. After the directional shearing action of the silicone tube, the hydrogel chains will be distributed along the axial direction, and then in situ cross-linked in the coaxial needle through the calcium chloride solution on the inner axis to form a temporarily stable hollow tube, and then further chemically cross-linked through UV light curing to form a stable hollow tubular unit.

[0039] Reference Figure 1 To Attachment Fig.12 As shown, a 3D coaxially printed gelatin composite hydrogel ink according to a preferred embodiment of the present invention is schematically shown, and a method for preparing a 3D coaxially printed gelatin composite hydrogel ink comprises the following steps: (S10) Preparation of methacryloyl gelatin; (S20) Preparation of PGA bio-ink; and (S30)CaCl 2 Preparation of aqueous solution.

[0040] In the step (S10), 50 g of gelatin (GELATIN) and 3.2 mL of methacrylic anhydride (MA) were fully reacted in 500 mL of phosphate buffer (pH = 7.4) at 50°C for 3 hours. The above solution was dialyzed in a 40°C water bath for 5 days, the solution was changed 3 times a day, and then freeze-dried for 48 hours. The chemical reaction equation is as follows Figure 1 shown.

[0041] The synthesized methacryloyl gelatin (GelMA) was detected by hydrogen nuclear magnetic resonance spectroscopy. 1 H NMR (500M) PROTON nuclear magnetic resonance instrument, recording the relationship between GELATIN and GelMA at room temperature. 2 O is the solvent 1 H NMR spectrum, see Appendix Figure 2 shown.

[0042] The step (S20) includes the following steps: (S201) Dissolve the methacrylated gelatin monomer in H 2 O in; (S202) adding LAP, vortexing to mix, and protecting from light; and (S203) Then, sodium alginate and polyethylene oxide are added, mixed, and centrifuged.

[0043] The concentration of methacryloyl gelatin is 10%, the concentration of sodium alginate is 0.5%-2.0%, and the concentration of CaCl 2 The concentration of the solution was 0.8%, the concentration of polyethylene oxide was 0.8%, which was used as the experimental group, and the concentration of polyethylene oxide was 0% as the control group.

[0044] In order to evaluate the influence of methacrylated gelatin (GelMA), sodium alginate (Alg) and polyethylene oxide (PEO) on the performance of ink, this project changed the concentration of Alg and PEO under the condition of GelMA concentration. The concentration of hydrogel ink components is set as shown in Table 1: Table 1. Setting of hydrogel ink component concentrations The present invention prepares a detailed formula of a PGA hydrogel mixed system with different sodium alginate concentrations by ultraviolet crosslinking. When the sodium alginate concentration is 1%, 1.5% and 2%, it is represented by PGA-1, PGA-1.5 and PGA-2, respectively, collectively referred to as PGA hydrogel, wherein G represents GelMA, A represents Alg, and P represents PEO. At this concentration, those without PEO are marked as GA-1, GA-1.5 and GA-2. The following examples are described by taking PGA-1 as an example. Example

[0045] Weigh 1 g of GelMA monomer and dissolve it in 10 mL of dHO. 2O, and then fully dissolve GelMA at 50°C, then add 0.3% (m / w) LAP (Lithium Phenyl (2,4,6-trimethylbenzoyl) phosphinate), shake again to mix, and pay attention to tin foil to protect from light.

[0046] Then, 0.1 g of sodium alginate and 0.08 g of polyethylene oxide were added to the gelatin solution, and the mixture was mixed again in a 50° C. water bath, and centrifuged at 3000 rpm for 3 min to remove excess bubbles to obtain PGA-1 for subsequent use.

[0047] In the step (S30), 0.8% CaCl 2 The aqueous solution was prepared as follows: 0.08 g CaCl 2 The monomer was dissolved in 10 mL dH 2 O, shake thoroughly to mix for subsequent use. Example

[0048] Weigh 1 g of GelMA monomer and dissolve it in 10 mL of dHO. 2 O, and then fully dissolve GelMA at 50°C, followed by the addition of 0.3% (m / w) LAP (Lithium Phenyl (2,4,6-trimethylbenzoyl) phosphinate), shake again to mix, and protect the tin foil from light.

[0049] Then, 0.1 g of sodium alginate was added to the gelatin solution without adding polyethylene oxide, and the mixture was mixed again in a 50° C. water bath, and centrifuged at 3000 rpm for 2.5 min to remove excess bubbles to obtain GA-1 for subsequent use.

[0050] In the step (S30), 0.8% CaCl 2 The aqueous solution was prepared as follows: 0.08 g CaCl 2 The monomer was dissolved in 10 mL dH 2 O, shake thoroughly to mix for subsequent use. Example

[0051] Weigh 1 g of GelMA monomer and dissolve it in 10 mL of dHO. 2 O, and then fully dissolve GelMA at 50°C, followed by the addition of 0.3% (m / w) LAP (Lithium Phenyl (2,4,6-trimethylbenzoyl) phosphinate), shake again to mix, and protect the tin foil from light.

[0052] Then, 0.15 g of sodium alginate and 0.08 g of polyethylene oxide were added to the gelatin solution, and the mixture was mixed again in a 50° C. water bath, and centrifuged at 3000 rpm for 3.5 min to remove excess bubbles to obtain PGA-1.5 for subsequent use.

[0053] In the step (S30), 0.8% CaCl 2 The aqueous solution was prepared as follows: 0.08 g CaCl 2 The monomer was dissolved in 10 mL dH 2 O, shake thoroughly to mix for subsequent use. Example

[0054] Weigh 1 g of GelMA monomer and dissolve it in 10 mL of dHO. 2 O, and then fully dissolve GelMA at 50°C, followed by the addition of 0.3% (m / w) LAP (Lithium Phenyl (2,4,6-trimethylbenzoyl) phosphinate), shake again to mix, and protect the tin foil from light.

[0055] Then, 0.15 g of sodium alginate was added to the gelatin solution without adding polyethylene oxide, and the mixture was mixed again in a 50° C. water bath, and centrifuged at 3000 rpm for 3.5 min to remove excess bubbles to obtain GA-1.5 for subsequent use.

[0056] In the step (S30), 0.8% CaCl 2 The aqueous solution was prepared as follows: 0.08 g CaCl 2 The monomer was dissolved in 10 mL dH 2 O, shake thoroughly to mix for subsequent use. Example

[0057] Weigh 1g of GelMA monomer and dissolve it in 10mL of dHO. 2 O, and then fully dissolve GelMA at 50°C, followed by the addition of 0.3% (m / w) LAP (Lithium Phenyl (2,4,6-trimethylbenzoyl) phosphinate), shake again to mix, and protect the tin foil from light.

[0058] Then, 0.2 g of sodium alginate and 0.08 g of polyethylene oxide were added to the gelatin solution, and the mixture was mixed again in a 50° C. water bath, and centrifuged at 3000 rpm for 4 min to remove excess bubbles to obtain PGA-2 for subsequent use.

[0059] In the step (S30), 0.8% CaCl 2 The aqueous solution was prepared as follows: 0.16 g CaCl 2 The monomer was dissolved in 20 mL dH 2 O, shake thoroughly to mix for subsequent use. Example

[0060] Weigh 1g of GelMA monomer and dissolve it in 10mL of dHO. 2 O, and then fully dissolve GelMA at 50°C, followed by the addition of 0.3% (m / w) LAP (Lithium Phenyl (2,4,6-trimethylbenzoyl) phosphinate), shake again to mix, and protect the tin foil from light.

[0061] Then 0.2 g of sodium alginate was added to the gelatin solution without adding polyethylene oxide, and the mixture was mixed again in a 50°C water bath, centrifuged at 3000 rpm for 3 min, and excess bubbles were removed to obtain GA-2 for subsequent use. The polyethylene oxide was 0%, which was used as the control group.

[0062] In the step (S30), 0.8% CaCl 2 The aqueous solution was prepared as follows: 0.08 g CaCl 2 The monomer was dissolved in 10 mL dH 2 O, shake thoroughly to mix for subsequent use.

[0063] The bio-ink manufactured by the above embodiment can be applied to coaxial printing combined with stress traction to manufacture tubular units of the order of 100 micrometers. The coaxial printing method of the application process includes the following steps: Place PGA bio-ink in the outer axis channel; Place CaCl2 aqueous solution in the inner shaft channel; Adjusting tensile stress, photo-crosslinking and gelation to form hollow tubular units; and After printing, it is cured with UV light.

[0064] Specifically, the outer axis of the printing device is 17G, the inner axis is 22G, the outer axis and the inner axis are coaxial needles, the outer axis is the bio-ink channel, and the inner axis is 0.8% CaCl 2 Solution, when sodium alginate and Ca 2+When combined, they can be quickly cross-linked to form a hollow tubular unit. The flow rates of the inner and outer channels are set to 0.4 mL / min and 0.5 mL / min respectively. The traction device is a self-designed and assembled three-dimensional tubular bracket 3D printing device. The coaxial printing synergistic stress traction device is as follows Fig.10 As shown in (a) in the figure. The tubular bracket printer has both horizontal linear movement and rotation axis. By changing the rotation axis speed and the rotation axis size, the pulling stress can be easily changed. The rotation axis speed selected in this study is 66 rpm and the rotation axis diameter is 5 mm. After printing, it is cured under 405nm ultraviolet light for 30s.

[0065] In the present invention, the 3D coaxially printed gelatin composite hydrogel ink comprises PGA bio-ink and CaCl 2 The aqueous solution, wherein the PGA bio-ink comprises methacrylated gelatin, sodium alginate and polyethylene oxide, and the sodium alginate and Ca 2+ Combined with photo-cross-linking gelation, it is used as 3D coaxially printed gelatin composite hydrogel ink to form hollow tubular units, which are suitable for printing bionic directional muscular tubular scaffolds.

[0066] The hydrogel ink components of the above embodiment were tested. Figure 3 As shown, the rheological characterization of sodium alginate at a concentration of 1%: (a) shear rate test; (b) temperature oscillation frequency test; (c) oscillation frequency test when not cross-linked; (d) Ca 2+ Oscillation frequency test after cross-linking; (e) Ca 2+ Photocuring process after cross-linking; (f) Oscillation frequency test after double cross-linking The results are as follows: (1) Shear rate scanning test: The injectability of hydrogel is a very important property, which determines whether it can be extruded smoothly during printing, otherwise it will clog the needle. The present invention uses the change of shear rate to observe the change of hydrogel viscosity, such as Figure 3 As shown in (a), it can be observed that the viscosity of both GA hydrogel and PGA hydrogel decreases with the increase of shear rate, which shows that it has the property of shear thinning, which is crucial for 3D printing platform used as ink. At the same time, the viscosity of PGA hydrogel is always greater than that of GA hydrogel, which is consistent with the phenomenon observed in the experiment.

[0067] (2) Temperature oscillation frequency test: Gelatin methacrylate (GelMA) is a temperature-sensitive hydrogel. The appropriate temperature is extremely critical for printing. If the temperature is too low, the composite hydrogel will be in a gel state and will not be easily squeezed out from the coaxial needle, resulting in rough and uneven printed lines. If the temperature is too high, the viscosity of the composite hydrogel will be too low and it will not be able to osmotic pressure with Ca. 2+The cross-linking is timely and sufficient, but it is in the form of droplets and cannot form a line. Figure 3 In (b), it can be found that at 5-27℃, the storage modulus (G') of GA and PGA hydrogels is greater than the loss modulus (G''), which means that at low temperatures, the composite hydrogel is solid and difficult to extrude through the coaxial needle; at 27-40℃, the storage modulus (G') of GA and PGA hydrogels is less than the loss modulus (G''), which means that after the temperature rises, the composite hydrogel changes from solid to liquid, and is suitable for extrusion from the needle. Therefore, we selected 37℃, the same as the human body temperature, as the printing temperature.

[0068] (3) Oscillation frequency test before cross-linking: Oscillation frequency test was performed on GA and PGA hydrogels at 37°C to observe the changes in their moduli. Figure 3 As shown in (c), it is observed that the loss modulus of both is greater than the storage modulus, indicating that at this temperature, both are liquid, and the storage modulus and loss modulus of PGA hydrogel are greater than those of GA hydrogel.

[0069] (4) Ca 2+ Oscillation frequency test after cross-linking: Figure 3 As shown in (d), in Ca 2+ After cross-linking, the storage modulus of GA hydrogel and PGA hydrogel was greater than the loss modulus, indicating that 2+ After cross-linking, the hydrogel has solid properties. At the same time, it can be seen that the modulus of PGA hydrogel is greater than that of GA hydrogel, indicating that the stability of PGA hydrogel blocks is greater than that of GA hydrogel. As the oscillation frequency increases, the moduli of both increase accordingly. The maximum modulus of PGA hydrogel is 2260 Pa, and the maximum modulus of GA hydrogel is 1329.65 Pa, indicating that Ca 2+ The cross-linked hydrogel blocks can temporarily maintain a stable structure.

[0070] (5) Photocuring process: In Ca 2+ After cross-linking, the cross-linked hydrogel was then subjected to in situ photocross-linking, and the modulus recording process was as follows: Figure 3 As shown in (e), the storage modulus of the photocrosslinked PGA hydrogel is 13239.8 Pa, and that of the GA hydrogel is 12496.3 Pa, which is comparable to that of the Ca-only hydrogel. 2+ Compared with the modulus after cross-linking, the modulus is significantly increased, which indicates that the stability of the chemical structure of the hydrogel after photo-cross-linking is significantly improved, which is the basis for us to continue 3D printing.

[0071] (6) Oscillation frequency test after double cross-linking: The oscillation frequency test of the hydrogel block after double cross-linking is performed, such as Figure 3As shown in (f), the storage modulus of PGA hydrogel is slightly larger than that of GA hydrogel, but both are above 10,000 Pa, which indicates that the double cross-linked structure is very stable.

[0072] The tubular units manufactured by the above-mentioned embodiments were subjected to biological tests.

[0073] 1. Cytocompatibility assessment Reference Figure 4 To Attachment Figure 6 Coaxial printing and material formulation screening.

[0074] Figure 4 (a) is a schematic diagram of coaxial extrusion of hydrogel ink; (b) is a real picture of coaxial printing; (c) is the effect of printing temperature on molding; (d) is the injection of red ink into the tubular unit. Figure 5 (a) is the live-dead staining image of smooth muscle cells cultured in tubular sheets under different components on the 10th day; (b) is the cell spreading rate of smooth muscle cells in tubular sheets under different components on the 10th day. Figure 6 (a) is an electron micrograph of the pore sizes of hydrogels with different components; (b) is a statistical graph of the pore sizes of hydrogels with different components.

[0075] Cell live and dead staining: Use the live and dead staining kit according to the manufacturer's instructions. In short, use Calcein-AM and iodide (PI) for staining. Calcein-AM introduces the acetylmethoxymethyl (AM) group on the basis of traditional Calcein, which increases the hydrophobicity and enables it to easily penetrate the membrane of living cells. It can be reduced to Calcein inside the cell, which can make living cells emit green fluorescence; PI can only pass through the disordered area of ​​the cell membrane of dead cells to reach the nucleus, and embed into the DNA double helix of the cell to produce red fluorescence, so PI can make dead cells show red fluorescence. Use confocal laser scanning microscopy (CLSM, STELLARIS 5, Leica, Germany) to observe fluorescence. Use Image J to analyze cell viability and roundness. The formula for calculating cell viability is as follows: Cell Viability (%) =number of green / (number of green + number of red)×100%.

[0076] Cell proliferation assessment: Immediately after printing, take a 5 mm long tube from the coaxial printing and place it in a 96-well plate. Add 100 μL complete medium and 10 μL CCK-8 solution to each well and incubate at 37°C and 5% CO. 2Incubate in an incubator for 2 hours. After the sample is taken out, place the 96-well plate in a microplate reader at 450 nm to measure the absorbance (OD), and record it as the number of cells on Day 0. Continue to culture in the incubator to measure the absorbance on the 1st, 3rd, and 10th days of culture, and calculate cell survival and proliferation (Cellproliferation) by the following formula. The obtained data are statistically analyzed using GraphPad. ODt is the CCK-8 absorbance value at the set sampling time point; ODblank is the CCK-8 absorbance value of the control group; OD0 is the CCK-8 absorbance value on Day 0.

[0077] Cell proliferation (%)=(ODt-ODblank) / (OD0-ODblank)×100% That is, in the present invention, by introducing polyethylene oxide (PEO), according to the phase separation and shear orientation characteristics of the viscous polymer compound mixture, the pre-shearing in situ coaxial bioprinting of highly oriented porous hydrogel microfibers was designed to print tubular units. Specifically, Figure 4 As shown in (ad), GELMA, sodium alginate, PEO, and cells are uniformly mixed to form a two-phase viscous bio-ink, which is transported through a silicone tube. After the directional shearing action of the silicone tube, the hydrogel chains will be distributed along the axial direction, and then in situ cross-linked in the coaxial needle through the calcium chloride solution on the inner axis to form a temporarily stable hollow tube, and then further chemically cross-linked by UV curing to form a stable hollow tubular unit. The connectivity of the tubular unit is proved by injecting red ink into it; Figure 4 Observations under high-power optical microscopes in (e) and (f) show that the structure without PEO has no obvious rules and the hydrogel chains are chaotically distributed, while the fiber filaments distributed along the axial direction can be observed after the introduction of PEO, which provides a structural basis for the directional growth of cells. Fluorescent gelatin was further used to observe the pores and fiber orientation under a confocal fluorescence microscope. The tubular unit without PEO has no directional fibers, but after the introduction of PEO, directional fibers with the same axial direction can be observed, and a larger pore structure can be seen, which provides space for cell proliferation and spreading.

[0078] Reference Figure 6 As shown in the figure, when the GELMA concentration is 10%, the concentration of sodium alginate is changed, the smooth muscle cells and the bio-ink are evenly mixed, the corresponding tubular units are printed by the above printing method, the cells are stained for live and dead, the survival and spreading of the smooth muscle cells are observed, and the roundness is counted. Figure 6It can be clearly observed that regardless of the sodium alginate concentration, the number of surviving cells is high, indicating that the material has good biocompatibility for cells; at the same time, as the sodium alginate concentration decreases, the number of smooth muscle spreads significantly increases on the 10th day, which is in line with theory and logic. When the sodium alginate concentration is high, the hydrogel structure is denser and the space for cell spread is smaller. As the sodium alginate concentration decreases, the hydrogel pores increase and the cells spread accordingly; and under the same sodium alginate concentration, the smooth muscle cells in the bio-ink introduced with PEO spread more than the smooth muscle without PEO, and grow along the axial direction. Therefore, this study will design the control group and experimental group with gelatin as 10%, sodium alginate as 1%, and PEO as variables.

[0079] Figure 6 (ce) shows the live-death staining and proliferation of smooth muscle cells in the tubular units on days 1, 3, and 10. (c) shows the live-death staining of smooth muscle cells on GA / PGA tubular units; (d) shows the proliferation rate of smooth muscle cells; and (e) shows the survival rate of smooth muscle cells.

[0080] On the first day, the survival rate of smooth muscle in the GA tubular unit was only 63.5%, while the survival rate in the PGA tubular unit could reach 85.85%. It has been shown in the previous article that the composite hydrogel has no obvious cytotoxicity to cells. The possible reason for this result is the cell damage caused during the actual printing process. The reason why the survival rate of smooth muscle cells in PGA is higher than that in GA tubular units is that the larger hydrogel pores can provide more sufficient material exchange space. By the 10th day of culture, the survival rate of smooth muscle cells in both tubular units reached more than 90%, which fully demonstrated the high biocompatibility of the tubular units printed by the composite hydrogel for cells. At the same time, we can observe that the expansion rate of smooth muscle cells in PGA tubular units is higher than that in GA tubular units, and in PGA tubular units, smooth muscle cells are expanded along the long longitudinal direction of the tubule.

[0081] Figure 7 Coaxial printing and stress traction are used to manufacture micron-scale oriented tubular units. (a) is a schematic diagram of the coordination of coaxial printing and traction device; (b) is a schematic diagram of molecular chain orientation; (c) is the live-dead staining of smooth muscle cells under different pulling forces on the first day; (d) is the cell survival rate of smooth muscle cells under different pulling forces on the first day; (e) is the structural statistics of tubular units under different extrusion rates (OD: outer wall; ID: inner wall; WT: wall thickness); (f) is the optical microscope and fluorescence microscope imaging of GA tubular units after pulling (i 4 times under optical microscope; ii 20 times under optical microscope; iii structural imaging under fluorescence microscope): (g) is the optical microscope and fluorescence microscope imaging of PGA tubular units after pulling (i 4 times under optical microscope; ii 20 times under optical microscope; iii structural imaging under fluorescence microscope) 2. Skeleton and antibody staining of smooth muscle in GA and PGA composite hydrogel In order to observe the changes in the morphology and function of smooth muscle cells in the printed tubular units over time, the F-actin, α-SMA, DES, OPN and nuclei of smooth muscle cells were fluorescently stained. In short, after fixation with 3mL 4% paraformaldehyde for 30min, 0.3% Triton X-100 3mL was used to permeabilize the cells for 30min. For the skeleton of smooth muscle cells, actin was then incubated in the dark for 60min with Phalloidin-labeled phalloidin; for α-SMA staining, 5% BSA was blocked for 6h, primary antibody was added and incubated overnight at room temperature, and then FITC-labeled fluorescent secondary antibody was added and incubated for 6h; for DES and OPN staining, 5% BSA was blocked for 6h. The primary antibody was prepared by diluting mouse smooth muscle Desmin antibody at 1:400 and rabbit smooth Osteopontin antibody at 1:150 in PBS, and incubated overnight at room temperature. The secondary antibody was incubated for 6 h with green fluorescent Desmin and red fluorescent Osteopontin fluorescent secondary antibodies diluted at 1:500; the nucleus was counterstained with ready-to-use DAPI solution for 30 min. Finally, the cell orientation angle and the length-to-length ratio of the nucleus were observed by fluorescence microscopy and analyzed using Image J.

[0082] Figure 8 . Biocompatibility of PGA composite hydrogel: (a) Live / dead staining of smooth muscle cells on tubular units at corresponding concentrations; (b) Statistical analysis of smooth muscle cell proliferation rate; (c) Statistical analysis of smooth muscle cell survival rate; (d) Skeleton staining of smooth muscle cells on tubular units corresponding to the 10th day; (e) Orientation distribution of smooth muscle cells on tubular units corresponding to the 10th day; (f) Schematic diagram of orientation distribution of smooth muscle cells; (g) Schematic diagram of length-to-length ratio of cell nuclei; (h) Statistical diagram of length-to-length ratio of cell nuclei.

[0083] We further performed skeleton staining on the smooth muscle cells in the tubular unit and observed the cell angle distribution and the nucleus aspect ratio (NAR). Figure 8(d) in the figure shows the fluorescent image of the smooth muscle cell skeleton and cell nucleus after staining on the 10th day. GA is the control group, PGA-unstretched group is a group containing orientation factor PEO but not subjected to small stress traction, and PGA-stretched group is a group subjected to stress traction. It can be seen intuitively that the smooth muscle cells in the group without PEO and the group not subjected to traction have chaotic orientation, while the smooth muscle cells in the group containing PEO and subjected to stress traction have obvious cell orientation characteristics. Image.J was further used to quantify the orientation angles of the smooth muscle cells and the aspect ratios of the cell nuclei in the above three groups. Figure 8 As shown in (d), in the PGA tubular unit containing PEO and subjected to stress traction, the smooth muscle cells feel the effect of the PEO oriented fibers, and the cell orientation is concentrated at 0°. The smooth muscle cells in the other two groups are randomly distributed between -90° and 90°, with no orientation. Figure 8 (f) in the figure is a schematic diagram of cell orientation and the aspect ratio of the nucleus. Further, we counted the aspect ratios of the nucleus of smooth muscle cells in three different groups, such as Figure 8 As shown in (h), the average length-to-width ratio of the smooth muscle cell nuclei in the GA group was 1.480, the length-to-width ratio of the cell nuclei in the PGA group that was not subjected to traction was 1.900, and the length-to-width ratio of the cell nuclei in the PGA group that was subjected to traction was 2.707. After analysis, it was found that the P value between the GA group and the PGA-unstretched group was 0.0352, and the P value between the PGA-unstretched and PGA-stretched groups was <0.0001, which was statistically significant. It was confirmed that in order to induce the oriented growth of smooth muscle cells, it was necessary to combine the designed hydrogel structure with the corresponding process means, and to induce the oriented arrangement of smooth muscle cells on a microscopic scale by constructing a PEO oriented microstructure.

[0084] Fig. 9 .(a) α-SMA antibody staining; (b) double staining fluorescence image; (c) DES / OPN fluorescence intensity statistics (DES refers to Desmin, OPN refers to Osteopontin) 3. Printing of small diameter directional tubular stents Based on the tubular unit, we independently designed and assembled a device that can print small-diameter tubular brackets with rollers that can both roll and translate. Fig.10As shown in (a), the printing device includes a PGA composite hydrogel extrusion control device and a calcium chloride extrusion control device. The printed tubular unit is received by a roller. The roller diameter can be easily changed according to the printing requirements. The motor speed that controls the roller rotation speed can also be changed to obtain tubular units of different thicknesses. The roller motor is fixed to the translation base, and the translation speed and distance are adjusted to obtain tubular brackets of different lengths. The key point of printing the bracket is to match the extrusion rate, roller diameter, roller rotation speed and translation speed.

[0085] Fig.10 . Millimeter-level directional tubular stent printing (a) Actual image of tubular stent printing; (b) Printing speed adjustment; (c) Printing of 3mm and 5mm tubular stents respectively; (d) Fluorescence image of directional tubular stent; (e) Stent elasticity demonstration; (f) Simulated vascular suture of tubular stent; (g) Schematic diagram of bursting pressure test; (h) Initial state of bursting pressure test; (i) Maximum pressure value Morphological characterization of PGA tubular scaffolds: PGA oriented tubular scaffolds with diameters of 3 mm and 5 mm were printed respectively. After curing with 405 nm UV light for 30 s, they were removed from the roller and the printed scaffolds were observed under macroscopic and fluorescence microscopes.

[0086] Burst pressure of PGA directional tubular stent: This study independently designed and assembled a burst pressure test device, such as Fig.10 As shown in (h) in the figure. The right side is a syringe connected to the syringe pump, the middle is an electronic pressure device, the left side is connected to the tubular bracket printed by this research institute, and the middle is connected to the rubber tube through a three-way pipe. The technical key and difficulty of this experiment lies in ensuring the airtightness of all connection points in the experiment, so the experiment was carried out after being tightly sealed with a sealing film.

[0087] like Fig.10 As shown in (a), a large-scale annular structure printing device is constructed based on a tubular unit. The tubular unit is printed out through a coaxial device, and the printed hollow tube is received by a roller. It includes a lateral translation structure and a rotating roller. By changing the diameter of the roller and the speed of the motor, annular structures of different sizes can be obtained simply and efficiently. Fig.10 In (c), we show two kinds of printed images with different diameters (3mm and 5mm). We further use fluorescent gelatin printing and 3D shaping under confocal microscope. The top view shows the annular fibers, which are the structural basis for the annular growth of smooth muscle cells. The printed lumen can still restore its original structure after being squeezed, indicating that our tubular structure has good elasticity. Fig.10In (f), the printed tubular unit was sutured end to end with 9-0 absorbable suture to simulate the vascular suture process in clinical practice. The results showed that the tubular structure was suitable for the suture process and had potential value for clinical application. We conducted a burst pressure test on the side wall pressure that the PGA tubular stent could withstand, as shown in Figure 2. Fig.10 The (gh) in it can withstand a maximum pressure of 32.7kPa, which is approximately equal to a pressure of 240mmHg, which is completely suitable for blood vessel replacement in the human body.

[0088] 4. Printing PGA directional tubular scaffolds with cells Digested smooth muscle cells were plated at 1×10 7 / mL was evenly mixed with sterilized bio-ink, and the ink extrusion speed and calcium chloride extrusion speed were set to 0.5mL / min. A 5mm diameter shaft was used, the shaft speed was 66rpm, the translation distance was set to 20,000 pulses, and the translation rate was set to 130 pulses per second. After printing, 405nm ultraviolet light was used for cross-linking for 30s, and then the scaffold was removed from the mold, rinsed twice with sterile PBS, and 20mL of complete culture medium was added and placed in a cell culture incubator for culture. Live and dead staining was performed on the first day after printing to observe the survival and compatibility of cells in the printed scaffold. On the 5th and 10th days, skeleton staining was performed in the same way as before. The spreading and orientation of smooth muscle cells in the corresponding tubular scaffolds were observed. The results showed that the cells showed a morphology of high activity and orderly growth along the circumference of the tube in large-size vascular scaffolds.

[0089] Fig.11 .(a) GA tubular scaffold; (b) PGA tubular scaffold (live / dead cell staining is shown on day 1, and framework staining is shown on days 5 and 10).

[0090] 5. Construction of complex tubular scaffolds 3D printing of multi-layer tubular scaffolds: Prepare transparent, black, red and blue bio-inks respectively, and print the transparent layer-black layer-red layer-blue layer in sequence.

[0091] 3D printing of multi-branched tubular stents: Print several directional tubular structures of 3 mm and 5 mm respectively. After printing, use the same bio-ink to bond and solidify them to construct a vascular network branch structure.

[0092] Fig.12 . The construction of complex tubular stents (ac) The construction of multi-layer tubular stents for ureters; (de) The construction of multi-branched tubular stents for blood vessels.

[0093] Through the above biological detection, it is found that the present invention uses natural material gelatin as raw material to construct a double cross-linked network, and uses methacrylic anhydride and natural materials to prepare methacrylic gelatin (GelMA). The use of sodium alginate as biological ink is based on the rapid gelation of sodium alginate and calcium ions to achieve the construction of a hollow structure. The biological ink provided by the present invention has good biocompatibility, accurate printing, easy tensile yield, high mechanical tensile properties, and a double or multiple cross-linking mechanism. The printed tubular tissue has good isotropic specificity and no cytotoxicity. The cells in the large-size vascular stent show a highly active and orderly growth morphology along the circumferential direction of the tube, so it is suitable for use as a stent in the human body. In addition, the ink is injectable, can be extruded smoothly, will not clog the needle, and has the property of shear thinning, which is crucial for a 3D printing platform used as ink.

[0094] It should be understood by those skilled in the art that the embodiments of the present invention described above and shown in the accompanying drawings are only examples and do not limit the present invention. The purpose of the present invention has been fully and effectively achieved. The functional and structural principles of the present invention have been demonstrated and explained in the embodiments, and the embodiments of the present invention may be deformed or modified in any way without departing from the principles.

Claims

1. A method for preparing 3D coaxially printed gelatin composite hydrogel ink, characterized in that: The following steps are involved: (S10) preparing methacryloyl gelatin, reacting gelatin with methacrylic anhydride in a phosphate buffer solution, dialyzing in a water bath, and freeze-drying to obtain methacryloyl gelatin; (S20) Preparation of PGA bio-ink, wherein the step (S20) comprises the following steps: (S201) dissolving methacrylated gelatin monomer in H2O; (S202) adding LAP, oscillating and mixing, and protecting from light; (S203) subsequently adding sodium alginate and polyethylene oxide, mixing, and centrifuging to obtain PGA bio-ink; and (S30) Preparation of CaCl2 aqueous solution; wherein the PGA bio-ink and the CaCl2 aqueous solution are placed in different channels, respectively. When the PGA and CaCl2 aqueous solutions are extruded at a flow rate ratio of 5:4, the sodium alginate and CaCl2 aqueous solution are 2+ Coaxial in-situ cross-linking and light curing form a hollow tubular unit.

2. The method for preparing a 3D coaxially printed gelatin composite hydrogel ink according to claim 1, wherein in the step (S20), the concentration of methacrylated gelatin is 10%, the concentration of sodium alginate is 0.5%-2.0%, the concentration of polyethylene oxide is 0.8%, and the concentration of CaCl2 solution is 0.8%.

3. The method for preparing a 3D coaxially printed gelatin composite hydrogel ink according to claim 1 or 2, wherein in the step (S10), gelatin and methacrylic anhydride are reacted in a phosphate buffer solution with a pH of 7.4, dialyzed in a 40°C water bath, and freeze-dried to obtain methacrylated gelatin.

4. The 3D coaxially printed gelatin composite hydrogel ink according to claim 3, wherein in the step (S20), 1 g of methacrylated gelatin monomer is weighed and dissolved in 10 mL of dH2O, and then the methacrylated gelatin is fully dissolved at 50°C, followed by adding 0.3% (m / w) LAP, shaking and mixing, and protecting from light with tin foil, and then adding 0.1 g of sodium alginate and 0.08 g of polyethylene oxide to the gelatin solution, mixing in a 50°C water bath, and centrifuging at 3000 rpm for 3 min to remove excess bubbles for subsequent use.

5. The 3D coaxial printing gelatin composite hydrogel ink according to claim 4, wherein the preparation of methacrylated gelatin is as follows: 50 g of gelatin and 3.2 mL of methacrylic anhydride are fully reacted in 500 mL of phosphate buffer at 50° C. for 3 h, the solution is dialyzed in a 40° C. water bath for 5 days, the solution is changed 3 times a day, and then freeze-dried for 48 h to synthesize methacrylated gelatin.

6. A 3D coaxially printed gelatin composite hydrogel ink, characterized in that: The 3D coaxially printed gelatin composite hydrogel ink comprises PGA bio-ink and CaCl2 aqueous solution, wherein the PGA bio-ink comprises methacrylated gelatin, sodium alginate and polyethylene oxide. As the 3D coaxially printed gelatin composite hydrogel ink, the PGA bio-ink and the CaCl2 aqueous solution are in situ cross-linked in different channels at a flow rate ratio of 5:4, and the sodium alginate and CaCl2 aqueous solution are cross-linked. 2+ The combined, photo-crosslinked and gelled to form hollow tubular units.

7. The 3D coaxially printed gelatin composite hydrogel ink according to claim 6, wherein the 3D coaxially printed gelatin composite hydrogel ink is prepared by the preparation method described in any one of claims 1 to 5.

8. The 3D coaxially printed gelatin composite hydrogel ink according to claim 6, wherein during printing, the outer axis is a PGA bio-ink channel, the inner axis is a 0.8% CaCl2 solution, and when sodium alginate and CaCl2 are mixed, the outer axis is a PGA bio-ink channel, the inner axis is a 0.8% CaCl2 solution, and the inner axis is a 0.8% CaCl2 solution. 2+ When combined, they cross-link quickly to form a hollow tubular unit. The tensile stress can be changed by changing the spindle speed and spindle size. The spindle speed is 66 rpm and the spindle diameter is 5 mm. After printing, it is cured under 405 nm ultraviolet light for 30 seconds.

9. An application of 3D coaxial printing gelatin composite hydrogel ink, characterized in that: The composite hydrogel ink comprises PGA bio-ink and CaCl2 aqueous solution, and is suitable for printing biomimetic directional muscle tubular scaffolds. The coaxial printing method of the 3D coaxially printed gelatin composite hydrogel ink for coaxial printing combined with stress traction to manufacture tubular units of 100 micrometers comprises the following steps: (A) placing PGA bio-ink in an outer axial channel; (B) placing a CaCl2 aqueous solution in an inner axial channel; (C) adjusting the pulling stress, photo-crosslinking and gelling to form a hollow tubular unit; and (D) UV curing after printing is completed.

10. The application of 3D coaxially printed gelatin composite hydrogel ink according to claim 9, wherein the inner shaft size is 22G, the outer shaft size is 17G, the flow rates of the inner and outer channels are set to 0.4mL / min and 0.5mL / min respectively, the shaft speed is 66rpm, the shaft diameter is 5mm, and after printing is completed, it is cured under 405nm ultraviolet light for 30s.

11. The use of the 3D coaxially printed gelatin composite hydrogel ink according to claim 9 or 10, wherein the preparation method of the PGA bio-ink is as follows: dissolving methacrylated gelatin monomer in H2O; adding LAP, oscillating and mixing, and protecting from light;) then adding sodium alginate and polyethylene oxide, mixing, and centrifuging.

12. The use of the 3D coaxially printed gelatin composite hydrogel ink according to claim 11, wherein the concentration of methacrylated gelatin is 10%, the concentration of sodium alginate is 0.5%-2.0%, the concentration of polyethylene oxide is 0.8%, and the concentration of CaCl2 The concentration of the solution is 0.8%.

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