3D coaxial printing gelatin composite hydrogel ink, and preparation method and application thereof

Abstract

The present application provides a kind of 3D coaxial printing gelatin composite hydrogel ink and its preparation method and application, the 3D coaxial printing gelatin composite hydrogel ink includes PGA biological ink and CaCl2 aqueous solution, wherein the PGA biological ink includes methacrylated gelatin, sodium alginate and polyethylene oxide, by the combination of sodium alginate and Ca 2+ Photo-crosslinking gelation, as 3D coaxial printing gelatin composite hydrogel ink, forms hollow tubular unit, and is prepared by the following steps: (S10) preparation of methacrylated gelatin; (S20) preparation of PGA biological ink; and (S30) preparation of CaCl2 aqueous solution, printing precision, easy to stretch yield, the prepared biological scaffold has good biocompatibility.

Description

TECHNICAL FIELD

[0001] The present application relates to, in particular to a 3D coaxial printing gelatin composite hydrogel ink and its preparation method and application. BACKGROUND

[0002] Tissue engineering is a development of tissue and organ substitutes to repair, improve and reconstruct injured tissues and organs, and the ultimate goal is to replace organ transplantation. In recent years, bioprinting technology as a hot research direction of tissue engineering, it can effectively wrap cells in biological ink, effectively solve the problem of uneven distribution of cells and cell loss in the past printing process. Among them, extrusion bioprinting is a relatively simple technology, which can print three-dimensional biological structures and tissue models with functional through precise 3D control of various components (such as cells, support media, etc.). In order to manufacture anisotropic structures that simulate the biological functions of anisotropic tissues, the manufacturing methods of tissue engineering scaffolds are: using electric field / magnetic field, ion flow direction and diffusion, stretching, biological template, drying under limited conditions to induce the oriented arrangement of polymer chains or hydrogel chains. These generated scaffolds have directional patterns, which enable them to mimic the mechanical properties of related tissues. However, these methods have some disadvantages, such as: (1) these processes and scaffolds have poor biological performance; (2) low cell seeding efficiency, 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, urethra, etc. Their diameters vary from a few millimeters of large blood vessels to a few micrometers of capillaries, and the diameters and tissue structures are different. Therefore, it is necessary to construct a method that can print hollow pipes of different diameters. Traditional tubular structure printing technology is mostly based on the method of sacrificing materials, that is, after printing the complete structure, the hollow vascular structure is obtained by removing the sacrificial material through adjusting the temperature, PH, etc. to support the fusion of multicellular spheroids or cylinders into a vascular structure. Due to the complexity of the sacrificial bioprinting process, the size, shape and function of the obtained vascular-like structure may be limited. Although recent developments in multi-layer coaxial extrusion systems have made technical progress, making it possible to directly manufacture hollow hydrogel pipes, it is still not easy to achieve and has not been proven that complex 3D cell-filled hollow structures can be achieved. Therefore, it is urgent to print hollow pipes with high precision, anisotropy and excellent cell survival environment in one step. SUMMARY

[0004] An advantage of the present application is to provide a 3D coaxial printing gelatin composite hydrogel ink and its preparation method and application, which has good biocompatibility, printing precision and easy stretching yield.

[0005] Another advantage of the present application is to provide a 3D coaxial printing gelatin composite hydrogel ink and its preparation method and application, which has high mechanical tensile performance, double or multiple crosslinking mechanism, and good each direction specificity of the printed tubular tissue.

[0006] Another advantage of the present application is to provide a 3D coaxial printing gelatin composite hydrogel ink and its preparation method and application, which uses natural material gelatin as raw material to construct a double crosslinking network, uses selective chemical reaction of methacrylic anhydride and natural material to prepare methacrylated gelatin (GelMA), and uses sodium alginate as a biological ink based on rapid gelation of sodium alginate and calcium ions to realize construction of a hollow structure.

[0007] Another advantage of the present application is to provide a 3D coaxial printing gelatin composite hydrogel ink and its preparation method and application, in order to improve cell adhesion and growth performance of the ink, GelMA is added to the ink, thereby forming a physical network crosslinked by calcium ions to realize extrusion, and after extrusion, a chemical crosslinking hydrogel is formed by light crosslinking to further stabilize the structure; in order to realize a directional fiber structure, polyethylene oxide (PEO) is introduced, which is a classic thickening agent and spinning agent, and after directional shearing, the hydrogel chain will be distributed along the axial direction.

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

[0009] Another advantage of the present application is to provide a 3D coaxial printing gelatin composite hydrogel ink and its preparation method and application, which has no cytotoxicity, and results show that cells in a large-size vascular stent present a high-activity, ordered growth morphology along the circumferential direction of the tube, and thus is suitable for being applied to the human body as a stent.

[0010] According to one aspect of the present application, the present application provides a preparation method of a 3D coaxial printing gelatin composite hydrogel ink, comprising the following steps:

[0011] (S10) Preparation of methacrylated gelatin;

[0012] (S20) Preparation of PGA biological ink; and

[0013] (S30) Preparation of CaCl2 aqueous solution.

[0014] The step (S20) comprises the following steps: (S201) dissolving the methacrylated gelatin monomer in H2O; (S202) adding LAP, oscillating and mixing uniformly, and avoiding light; (S203) then adding sodium alginate and polyethylene oxide, mixing uniformly, and centrifuging.

[0015] In the step (S20), the concentration of the methacrylated gelatin is 10%, the concentration of the sodium alginate is 0.5%-2.0%, the concentration of the polyethylene oxide is 0.8%, and the concentration of the CaCl2 solution is 0.8%.

[0016] In the step (S10), the gelatin is reacted with methacrylic anhydride in a phosphate buffer solution with pH=7.4, and then dialyzed in a water bath at 40°C, and freeze-dried to obtain the methacrylated gelatin.

[0017] In the step (S10), the gelatin is reacted with methacrylic anhydride in a phosphate buffer solution with pH=7.4, and then dialyzed in a water bath at 40°C, and freeze-dried to obtain the methacrylated gelatin.

[0018] In the step (S10), the gelatin is reacted with methacrylic anhydride in a phosphate buffer solution with pH=7.4, and then dialyzed in a water bath at 40°C, and freeze-dried to obtain the methacrylated gelatin.

[0019] In the step (S10), the gelatin is reacted with methacrylic anhydride in a phosphate buffer solution with pH=7.4, and then dialyzed in a water bath at 40°C, and freeze-dried to obtain the methacrylated gelatin. 2+ The PGA bio-ink and the CaCl2 aqueous solution are placed in different channels, and when they are extruded, the sodium alginate and Ca

[0020] According to another aspect of the present application, the present application also provides a 3D coaxial printing gelatin composite hydrogel ink, which comprises a PGA bio-ink and a CaCl2 aqueous solution, wherein the PGA bio-ink comprises methacrylated gelatin, sodium alginate and polyethylene oxide, and as the 3D coaxial printing 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 Ca 2+ The PGA bio-ink and the CaCl2 aqueous solution are placed in different channels, and when they are extruded, the sodium alginate and Ca

[0021] Wherein the outer shaft is the PGA bio-ink channel and the inner shaft is the 0.8% CaCl2 solution when printing, and the sodium alginate and Ca 2 + Combined, cross-linked quickly, and formed hollow tubular units, the pulling stress was changed by changing the rotation speed and the size of the rotating shaft, wherein the rotation speed of the rotating shaft was 66 rpm and the diameter of the rotating shaft was 5 mm, and the printed product was cured under 405 nm ultraviolet light for 30 s.

[0022] According to another aspect of the present application, the present application also provides an application of a 3D coaxial printing gelatin composite hydrogel ink, which comprises a PGA bio-ink and a CaCl2 aqueous solution, and is suitable for printing a biomimetic directional muscular hollow tubular scaffold.

[0023] The 3D coaxial printing gelatin composite hydrogel ink is used in a coaxial printing method for manufacturing a micrometer-level tubular unit under combined stress traction, which comprises the following steps: (A) placing the PGA bio-ink in the outer shaft channel; (B) placing the CaCl2 aqueous solution in the inner shaft channel; (C) adjusting the pulling stress, photo-crosslinking and gelation to form a hollow tubular unit; and (D) curing the printed product under ultraviolet light.

[0024] Wherein the size of the inner shaft is 22G, the size of the outer shaft is 17G, the flow rates of the inner and outer channels are set to be 0.4 mL / min and 0.5 mL / min respectively, the rotation speed of the rotating shaft is 66 rpm, the diameter of the rotating shaft is 5 mm, and the printed product is cured under 405 nm ultraviolet light for 30 s after printing.

[0025] The PGA bio-ink is prepared by the following method: dissolving the methacrylated gelatin monomer in H2O; adding LAP, oscillating and mixing uniformly, and avoiding light; then adding sodium alginate and polyethylene oxide, mixing uniformly, and centrifuging.

[0026] Wherein the concentration of the methacrylated gelatin is 10%, the concentration of the sodium alginate is 0.5%-2.0%, the concentration of the polyethylene oxide is 0.8%, and the concentration of the CaCl2 solution is 0.8%.

[0027] According to another aspect of the present application, the present application provides a preparation method of a 3D coaxial printing gelatin composite hydrogel ink, which comprises the following steps:

[0028] (S10) Preparation of methacrylated gelatin, reacting gelatin and methacrylic anhydride in a phosphate buffer solution, water bath dialysis, and freeze-drying to obtain methacrylated gelatin;

[0029] (S20) Preparation of PGA bio-ink, wherein step (S20) includes the following steps: (S201) dissolving methacrylamide gelatin monomer in H2O; (S202) adding LAP, shaking to mix, and protecting from light; (S203) subsequently adding sodium alginate and polyethylene oxide, mixing, centrifuging, and obtaining PGA bio-ink; and

[0030] (S30) Preparation of CaCl2 aqueous solution; wherein PGA bio-ink and CaCl2 aqueous solution are placed in different channels, and when PGA and CaCl2 aqueous solution are extruded at a flow rate ratio of 5:4, sodium alginate and Ca... 2+ Coaxial in-situ crosslinking, followed by photocuring, forms hollow tubular units. Attached Figure Description

[0031] Figure 1 This is a schematic diagram illustrating the synthesis of methacrylamide gelatin according to the above-described preferred embodiment of the present invention.

[0032] Figure 2 This is the 1H NMR spectrum of methacrylamide gelatin according to the above-described preferred embodiment of the present invention.

[0033] Figure 3 This is a schematic diagram of the rheological characterization of seaweed yogurt at a concentration of 1% according to the above-described preferred embodiment of the present invention.

[0034] Figure 4 This is a schematic diagram of coaxial printing and material formulation screening according to the above-described preferred embodiment of the present invention.

[0035] Figure 5 This is a schematic diagram of live and dead staining of smooth muscle cells in tubular units under different components according to the above-described preferred embodiment of the present invention.

[0036] Figure 6 This is a schematic diagram of electron microscopy, pore size statistics, and cell viability statistics of different components of hydrogel according to the above-described preferred embodiments of the present invention.

[0037] Figure 7 This is a schematic diagram of the detection process for manufacturing micron-sized oriented tubular units using coaxial printing and stress traction according to the above-described preferred embodiment of the present invention.

[0038] Figure 8 This is a schematic diagram illustrating the biocompatibility of the PGA composite hydrogel according to the above-described preferred embodiment of the present invention.

[0039] Figure 9 This is a schematic diagram of the detection process for manufacturing micron-sized oriented tubular units using coaxial printing and stress traction according to the above-described preferred embodiment of the present invention.

[0040] Figure 10is a printing diagram of a millimeter-scale oriented tubular scaffold according to the above preferred embodiment of the present application.

[0041] Figure 11 is a printing and dyeing diagram of a cell-laden PGA oriented tubular scaffold according to the above preferred embodiment of the present application.

[0042] Figure 12 is a 3D printing structure diagram of a multi-layer tubular scaffold according to the above preferred embodiment of the present application. DETAILED DESCRIPTION

[0043] The following description is presented to enable any person skilled in the art to practice the present application as claimed. The preferred embodiments disclosed herein are only examples of the present application and alternative embodiments will be apparent to those skilled in the art upon reading the following description. The essential characteristics of the present application defined in the following description are not to be limited to the specific embodiments disclosed in the following description, but encompass all alternatives, modifications, and equivalents falling within the spirit and scope of the present application.

[0044] The present application is around the key problem of regulating the directional growth of smooth muscle cells, by introducing polyethylene oxide (PEO), according to the phase separation and shear orientation characteristics of the mixture of viscous polymer compounds, a pre-sheared in-situ coaxial bioprinting of highly oriented porous hydrogel microfibers is designed to print tubular units. Specifically, as shown in (a-d) of FIG. 1, GELMA, sodium alginate, PEO, and cells are uniformly mixed to form a two-phase viscous bio-ink, which is delivered through a silica gel tube. After directional shearing through the silica gel tube, the hydrogel chains will be distributed along the axial direction. Then, a temporary stable hollow tubular structure is formed by in-situ cross-linking of calcium chloride solution on the inner shaft in the coaxial needle. Finally, further chemical cross-linking is performed by ultraviolet light curing to form a stable hollow tubular unit. Figure 4

[0045] Referring to FIGS. 1-4, a 3D coaxial printing gel composite hydrogel ink according to the preferred embodiment of the present application is shown, and a preparation method of the 3D coaxial printing gel composite hydrogel ink includes the following steps: Figure 1 Figure 12 (S10) Preparation of methacrylated gelatin;

[0046] (S20) Preparation of PGA bio-ink; and

[0047] (S30) Preparation of CaCl2 aqueous solution.

[0048]

[0049] ​​​Wherein in the step (S10), 50 g of gelatin (GELATIN) is fully reacted with 3.2 mL of methacrylic anhydride (MA) in 500 mL of a phosphate buffer (pH = 7.4) at 50°C for 3 h. The above solution is dialyzed in a 40°C water bath for 5 days, with liquid change 3 times a day, and then freeze-dried for 48 h. The chemical reaction equation is as shown in Figure 1 .

[0050] The synthesized methacrylated gelatin (GelMA) is detected by nuclear magnetic resonance hydrogen spectrum. The nuclear magnetic resonance hydrogen spectrum is recorded at room temperature by using 1 H NMR (500M) PROTON nuclear magnetic resonance instrument, recording GELATIN and GelMA with D2O as the solvent 1 H NMR spectrum, as shown in the accompanying Figure 2 .

[0051] The step (S20) comprises the following steps:

[0052] (S201) dissolving the methacrylated gelatin monomer in H2O;

[0053] (S202) adding LAP, oscillating and mixing uniformly, and avoiding light; and

[0054] (S203) then adding sodium alginate and polyethylene oxide, mixing uniformly, and centrifuging.

[0055] Wherein the concentration of the methacrylated gelatin is 10%, the concentration of the sodium alginate is 0.5%-2.0%, the concentration of the CaCl2 solution is 0.8%, and the concentration of the polyethylene oxide is 0.8%, and the concentration of the polyethylene oxide is 0% as the control group.

[0056] In order to evaluate the influence law of the composition of methacrylated gelatin (GelMA), sodium alginate (Alg) and polyethylene oxide (PEO) on the ink performance, the concentration of Alg and PEO is changed under the condition of the concentration of GelMA in this project, and the concentration setting of the hydrogel ink components is shown in Table 1:

[0057] Table 1. Concentration setting of hydrogel ink components

[0058] The PGA hydrogel mixed system with different concentrations of sodium alginate is prepared by the ultraviolet light crosslinking method, and the PGA hydrogel is represented by PGA-1, PGA-1.5 and PGA-2 when the concentration of sodium alginate is 1%, 1.5% and 2%, respectively, and is collectively referred to as PGA hydrogel, wherein G represents GelMA, A represents Alg, and P represents PEO. Under 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

[0059] Weigh 1g of GelMA monomer and dissolve it in 10mL of dH2O. Then, dissolve GelMA completely at 50℃. Next, add 0.3% (m / w) of LAP (Lithium Phenyl(2,4,6-trimethylbenzoyl) phosphinate), shake and mix again, and protect from light with aluminum foil.

[0060] Add 0.1g sodium alginate and 0.08g polyethylene oxide to the gelatin solution, mix again in a 50°C water bath, centrifuge at 3000rpm for 3min to remove excess air bubbles, and obtain PGA-1 for subsequent use.

[0061] In step (S30), the 0.8% CaCl2 aqueous solution is prepared as follows: Weigh 0.08g of CaCl2 monomer, dissolve it in 10mL of dH2O, shake thoroughly to mix, and prepare for subsequent use. Example

[0062] Weigh 1g of GelMA monomer and dissolve it in 10mL of dH2O. Then, dissolve the GelMA completely at 50℃. Subsequently, add 0.3% (m / w) of LAP (Lithium Phenyl(2,4,6-trimethylbenzoyl)).

[0063] (Phosphine), shake again to mix well, and keep away from light by using aluminum foil.

[0064] Add 0.1g of sodium alginate to the gelatin solution (without adding polyethylene oxide), mix again in a 50°C water bath, centrifuge at 3000rpm for 2.5min to remove excess air bubbles, and obtain GA-1 for subsequent use.

[0065] In step (S30), the 0.8% CaCl2 aqueous solution is prepared as follows: Weigh 0.08g of CaCl2 monomer, dissolve it in 10mL of dH2O, shake thoroughly to mix, and prepare for subsequent use. Example

[0066] Weigh 1g of GelMA monomer and dissolve it in 10mL of dH2O. Then, dissolve the GelMA completely at 50℃. Subsequently, add 0.3% (m / w) of LAP (Lithium Phenyl(2,4,6-trimethylbenzoyl)).

[0067] (Phosphine), shake again to mix well, and keep away from light by using aluminum foil.

[0068] To the gelatin solution, 0.15 g sodium alginate and 0.08 g polyethylene oxide were added again, and mixed again in a 50 °C water bath. Centrifugation was performed at a speed of 3000 rpm for 3.5 min to remove excess air bubbles, and PGA-1.5 was obtained for subsequent use.

[0069] In the step (S30), the 0.8% CaCl2 aqueous solution was prepared as follows: 0.08 g CaCl2 monomer was dissolved in 10 mL dH2O, and mixed well by shaking. The solution was ready for subsequent use. Example

[0070] 1 g of GelMA monomer was weighed into 10 mL dH2O, and then GelMA was fully dissolved at 50 °C. Then 0.3% (m / w) LAP (Lithium Phenyl(2,4,6-trimethylbenzoyl) phosphinate) was added, and mixed again by shaking. Note that tin foil was used to avoid light.

[0071] phosphinate), and mixed again by shaking. Note that tin foil was used to avoid light.

[0072] To the gelatin solution, 0.15 g sodium alginate and 0.08 g polyethylene oxide were added again, and mixed again in a 50 °C water bath. Centrifugation was performed at a speed of 3000 rpm for 3.5 min to remove excess air bubbles, and PGA-1.5 was obtained for subsequent use.

[0073] In the step (S30), the 0.8% CaCl2 aqueous solution was prepared as follows: 0.08 g CaCl2 monomer was dissolved in 10 mL dH2O, and mixed well by shaking. The solution was ready for subsequent use. Example

[0074] 1 g of GelMA monomer was weighed into 10 mL dH2O, and then GelMA was fully dissolved at 50 °C. Then 0.3% (m / w) LAP (Lithium Phenyl(2,4,6-trimethylbenzoyl) phosphinate) was added, and mixed again by shaking. Note that tin foil was used to avoid light.

[0075] phosphinate), and mixed again by shaking. Note that tin foil was used to avoid light.

[0076] To the gelatin solution, 0.15 g sodium alginate and 0.08 g polyethylene oxide were added again, and mixed again in a 50 °C water bath. Centrifugation was performed at a speed of 3000 rpm for 3.5 min to remove excess air bubbles, and PGA-1.5 was obtained for subsequent use.

[0077] In the step (S30), the configuration method of the 0.8% CaCl2 aqueous solution is as follows: 0.16 g of CaCl2 monomer is weighed and dissolved in 20 mL of dH2O, and fully shaken and mixed, for subsequent use. Embodiment

[0078] 1 g of GelMA monomer is weighed and dissolved in 10 mL of dH2O, and then the GelMA is fully dissolved at 50°C, followed by the addition of 0.3% (m / w) of LAP (Lithium Phenyl(2,4,6-trimethylbenzoyl)

[0079] phosphinate), and shaken and mixed again, paying attention to tin paper to avoid light.

[0080] 0.2 g of sodium alginate is further added to the gelatin solution, and polyethylene oxide is not added, and the mixture is mixed again in a 50°C water bath, and centrifuged at a speed of 3000 rpm for 3 min to remove excess bubbles, to obtain GA-2, for subsequent use. The polyethylene oxide is 0%, as a control group.

[0081] In the step (S30), the configuration method of the 0.8% CaCl2 aqueous solution is as follows: 0.16 g of CaCl2 monomer is weighed and dissolved in 20 mL of dH2O, and fully shaken and mixed, for subsequent use.

[0082] The bio-ink manufactured by the above embodiment can be applied to coaxial printing combined with stress traction to manufacture a micrometer-level tubular unit, and the coaxial printing method in the application process comprises the following steps:

[0083] The PGA bio-ink is placed in the outer shaft channel;

[0084] The CaCl2 aqueous solution is placed in the inner shaft channel;

[0085] The traction stress is adjusted, the photo-crosslinking gelation is performed, and a hollow tubular unit is formed; and

[0086] After printing, the ultraviolet light is cured.

[0087] Specifically, the outer shaft size of the printing device is 17G, and the inner shaft size is 22G, and the outer shaft and the inner shaft are coaxial needles, wherein the outer shaft is a bio-ink channel, and the inner shaft is a 0.8% CaCl2 solution, which can be quickly crosslinked to form a hollow tubular unit when the sodium alginate is combined with Ca 2+ 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 three-dimensional tubular stent 3D printing device independently designed and assembled, and the coaxial printing combined with stress traction device is as shown in Figure 10The tubular scaffold printer has linear movement and rotating shaft in horizontal direction, and can conveniently change the pulling stress by changing the rotating speed and rotating shaft size. The rotating speed of the rotating shaft is 66 rpm, and the rotating shaft diameter is 5 mm. After printing, the gel is cured under 405 nm ultraviolet light for 30 s.

[0088] In the present application, the 3D coaxial printing gelatin composite hydrogel ink comprises PGA biological ink and CaCl2 aqueous solution, wherein the PGA biological ink comprises methacrylated gelatin, sodium alginate and polyethylene oxide, and the CaCl2 aqueous solution is used for cross-linking the PGA biological ink through sodium alginate and Ca 2+ In combination, photo-crosslinking gelation, as a 3D coaxial printing gelatin composite hydrogel ink, forms a hollow tubular unit, which is suitable for application in printing of a biomimetic directional muscular tubular scaffold.

[0089] The hydrogel ink components of the above-mentioned embodiments are tested, and the test is as shown in Figure 3 The rheological characterization of the sodium alginate concentration of 1% is as shown in (a) shear rate test; (b) temperature oscillation frequency test; (c) oscillation frequency test before cross-linking; (d) oscillation frequency test after Ca 2+ cross-linking; (e) photo-curing process after Ca 2+ cross-linking; (f) oscillation frequency test after double cross-linking

[0090] The results are as follows:

[0091] (1) Shear rate scanning test: the injectability of the hydrogel is a very important property, which determines whether the hydrogel can be smoothly extruded during printing, otherwise the needle will be blocked. In the present application, the change of the shear rate is used to observe the change of the viscosity of the hydrogel, as shown in (a) of Figure 3 It can be observed that the viscosity of both the GA hydrogel and the PGA hydrogel decreases with the increase of the shear rate, which shows that it has the property of shear thinning, which is crucial for the application of the 3D printing platform. At the same time, the viscosity of the PGA hydrogel is always greater than that of the GA hydrogel, which is consistent with the observed phenomenon in the experiment.

[0092] (2) Temperature oscillation frequency test: methacrylated gelatin (GelMA) is a temperature-sensitive hydrogel, and the appropriate temperature is extremely critical for printing. If the temperature is too low, the composite hydrogel is in a gel state and is not easy to be extruded from the coaxial needle, resulting in rough and uneven lines; if the temperature is too high, the viscosity of the composite hydrogel is too low, and the Ca 2+ cannot be cross-linked in time and sufficiently, and the composite hydrogel is in the form of droplets and cannot form a line. From Figure 3As shown in (b), within the temperature range of 5-27℃, the storage modulus (G') of the GA and PGA hydrogels is greater than the loss modulus (G''). This indicates that at lower temperatures, the composite hydrogel is in a solid state, making it difficult to extrude through a coaxial needle. Within the temperature range of 27-40℃, the storage modulus (G') of the GA and PGA hydrogels is less than the loss modulus (G''). This indicates that as the temperature increases, the composite hydrogel changes from a solid state to a liquid state, making it suitable for extrusion through a needle. Therefore, we selected 37℃, which is the same as human body temperature, as the printing temperature.

[0093] (3) Vibration frequency test before crosslinking: The vibration frequency of GA and PGA hydrogels was tested at 37℃, and the modulus changes of the two were observed. Figure 3 As shown in (c), it was observed that the loss modulus of both was greater than the storage modulus, indicating that both were in a liquid state at this temperature, and that the storage modulus and loss modulus of the PGA hydrogel were greater than those of the GA hydrogel.

[0094] (4) Ca 2+ Post-crosslinking oscillation frequency test: such as Figure 3 As shown in (d) in Ca 2+ After crosslinking, the storage modulus of both GA hydrogel and PGA hydrogel was greater than their loss modulus, indicating that in Ca... 2+ After cross-linking, the hydrogels exhibited solid properties. Furthermore, the modulus of the PGA hydrogel was greater than that of the GA hydrogel, indicating that the stability of the PGA hydrogel block was greater than that of the GA hydrogel. With increasing oscillation frequency, the moduli of both increased. The maximum modulus of the PGA hydrogel was 2260 Pa, while the maximum modulus of the GA hydrogel was 1329.65 Pa, indicating that Ca... 2+ The cross-linked hydrogel block can temporarily maintain a stable structure.

[0095] (5) Photocuring process: In Ca 2+ After crosslinking, the crosslinked hydrogel is then subjected to in-situ photocrosslinking, and the modulus recording process is 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, compared to that of the Ca hydrogel alone. 2+ Compared to the previous method, the modulus of the cross-linked hydrogel increased significantly, indicating that the chemical structure stability of the hydrogel after photocrosslinking was significantly improved, which is the basis for our continued 3D printing.

[0096] (6) Oscillation frequency test after double cross-linking: The oscillation frequency of the double-cross-linked hydrogel block was tested, such as... Figure 3 As shown in (f), the storage modulus of PGA hydrogel is slightly greater than that of GA hydrogel, but both are above 10,000 Pa, which indicates that the structure after double cross-linking is very stable.

[0097] Biological tests were performed on the tubular units manufactured in the above examples.

[0098] 1. Cell compatibility evaluation

[0099] Reference is made to the drawings Figure 4 to the drawings Figure 6 Coaxial printing and material formulation screening.

[0100] Figure 4 (a) is a schematic diagram of hydrogel ink coaxial extrusion; (b) is a coaxial printing physical diagram; (c) is the influence of printing temperature on molding; (d) is the injection of red ink into the tubular unit. Figure 5 (a) is a live and dead staining diagram of smooth muscle cells cultured in the tubular unit for 10 days under different components; (b) is the cell spreading rate of smooth muscle cells in the tubular unit for 10 days under different components. Figure 6 (a) is an electron microscope diagram of the pore size of hydrogels of different components; (b) is a statistical diagram of the pore size of hydrogels of different components.

[0101] Cell live and dead staining: according to the manufacturer's instructions, using a live and dead staining kit, briefly, using Calcein-AM and iodine pyridine (PI) for staining, Calcein-AM is introduced into the traditional Calcein acetylmethoxy methyl (AM) group, which increases the hydrophobicity, so that it can easily penetrate the cell membrane, and it can be reduced to Calcein in the cell, which can make the living cells emit green fluorescence; PI can only penetrate the disordered region of the cell membrane of dead cells to reach the cell nucleus, and embed in the DNA double helix of the cell to produce red fluorescence, so PI can make dead cells show red fluorescence. The fluorescence was observed using a confocal laser scanning microscope (CLSM, STELLARIS 5, Leica, Germany). Image J was used to analyze the cell viability (Cell Viability) and roundness (Roundness). The cell viability calculation formula is as follows:

[0102] Cell Viability (%) =number of green / (number of green + number of red)×100%.

[0103] Cell proliferation assessment: Immediately after printing, take a 5mm length of the coaxially printed tube and place it in a 96-well plate. Add 100μL of complete culture medium and 10μL of CCK-8 solution to each well. Incubate at 37℃ and 5% CO2 for 2 hours. After removing the samples, place the 96-well plate in a microplate reader at 450nm to measure the absorbance (OD). Record the number of cells on Day 0. Continue culturing in the incubator and measure the absorbance on days 1, 3, and 10. Cell proliferation is calculated using the following formula. The 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.

[0104] Cell proliferation (%)=(ODt-ODblank) / (OD0-ODblank)×100%

[0105] In other words, in this invention, by introducing polyethylene oxide (PEO), and based on the phase separation and shear orientation characteristics of viscous polymer mixtures, a pre-shearing in-situ coaxial bioprinting method for highly oriented porous hydrogel microfibers was designed for printing tubular units. Specifically, as... Figure 4 As shown in (ad), GELMA, sodium alginate, PEO, and cells are uniformly mixed to form a biphasic adhesive bio-ink. Delivered through a silicone tube, the hydrogel chains distribute axially after being directionally sheared by the silicone tube. Then, through a calcium chloride solution within the inner axis, they crosslink in situ within a coaxial needle to form a temporarily stable hollow tubular structure. Further chemical crosslinking is achieved through UV curing, forming stable hollow tubular units. The connectivity of these units is demonstrated by injecting red ink into them. Figure 4 Observations under a high-power optical microscope (e) and (f) reveal that the structure without PEO has no obvious regularity, and the hydrogel chains are randomly distributed. In contrast, the structure with PEO shows axially distributed filaments, providing a structural basis for directional cell growth. Further observation of pores and fiber orientation under a confocal fluorescence microscope using fluorescent gelatin shows that the tubular units without PEO have no oriented fibers, while those with PEO can be observed to have axially oriented fibers and larger pore structures, providing space for cell proliferation and spread.

[0106] Reference Appendix Figure 6 As shown, with a GELMA concentration of 10%, the concentration of sodium alginate was varied, and smooth muscle cells were uniformly mixed with bio-ink. Corresponding tubular units were printed using the aforementioned printing method. Cell viability and mortality were stained to observe the survival and spread of smooth muscle cells, and their roundness was statistically analyzed. Figure 6It can be clearly observed that the number of cell survival is high regardless of the concentration of sodium alginate, which indicates that the material is good for the biocompatibility of cells; at the same time, with the decrease of the concentration of sodium alginate, the number of smooth muscle spreading is significantly improved at the 10th day, which is consistent with the theory and logic. When the concentration of sodium alginate is high, the hydrogel structure is relatively dense, and the space for cell spreading is small. With the decrease of the concentration of sodium alginate, the pore of hydrogel increases, and the cell spreads accordingly. In the case of the same concentration of sodium alginate, the smooth muscle cells in the bio-ink with PEO are more spread than those without PEO, and grow along the axial direction. Therefore, the control group and the experimental group will be designed with gelatin as 10%, sodium alginate as 1%, and PEO as a variable.

[0107] Figure 6 (c-e) in the present application are the live and dead staining and proliferation of smooth muscle cells in the tubular unit on the 1st, 3rd, and 10th day. (c) is the live and dead staining of smooth muscle cells on the GA / PGA tubular unit; (d) is the proliferation rate statistics of smooth muscle cells; (e) is the survival rate statistics of smooth muscle cells.

[0108] On the 1st day, the survival rate of smooth muscle in the GA tubular unit is only 63.5%, while the survival rate in the PGA tubular unit can reach 85.85%. It has been proved in the foregoing that the composite hydrogel has no obvious cytotoxicity to cells. Considering the possible reasons for this result is the cell damage caused by the actual printing process, and the reason why the survival rate of smooth muscle cells in PGA is higher than that in GA tubular unit is that the larger hydrogel pore can provide more sufficient material exchange space. On the 10th day of culture, the survival rate of smooth muscle cells in both tubular units reaches more than 90%, which fully illustrates the high biocompatibility of the composite hydrogel printed tubular unit to cells. At the same time, we can observe that the spreading rate of smooth muscle cells in the PGA tubular unit is higher than that in the GA tubular unit, and the smooth muscle cells in the PGA tubular unit spread along the longitudinal direction of the tubular.

[0109] Figure 7 Coaxial printing and stress traction to manufacture micron-scale directional tubular units, (a) is a schematic diagram of coaxial printing and traction device; (b) is a schematic diagram of molecular chain orientation; (c) is the live and dead staining of smooth muscle cells on the 1st day under different traction forces; (d) is the cell survival rate of smooth muscle cells on the 1st day under different traction forces; (e) is a structural statistical diagram of tubular units under different extrusion rates (OD: outer wall; ID: inner wall; WT: wall thickness) (f) is the imaging of GA tubular unit after traction under optical microscope and fluorescence microscope (i 4 times under light microscope; ii 20 times under light microscope; iii structure imaging under fluorescence microscope); (g) is the imaging of PGA tubular unit after traction under optical microscope and fluorescence microscope (i 4 times under light microscope; ii 20 times under light microscope; iii structure imaging under fluorescence microscope).

[0110] 2. Smooth muscle cytoskeleton and antibody staining in GA and PGA composite hydrogels

[0111] To observe the morphology and function of smooth muscle cells in the printed tubular units over time, the smooth muscle cells were fluorescently stained for F-actin, a-SMA, DES, OPN and nuclei. Briefly, after 3 mL of 4% paraformaldehyde fixation for 30 min, the cells were permeabilized with 0.3% Triton X-100 3 mL for 30 min. For cytoskeleton of smooth muscle cells, the actin was then incubated with Phalloidin-labeled phalloidin for 60 min in the dark; for a-SMA staining, 5% BSA was used to block for 6 h, and the primary antibody was added for incubation overnight at room temperature, followed by the addition of FITC-labeled secondary antibody for incubation for 6 h; for DES, OPN staining, 5% BSA was used to block for 6 h. The mouse smooth muscle Desmin antibody was diluted to 1 :400 and the rabbit smooth Osteopontin antibody was diluted to 1 : 150 in PBS to prepare the primary antibody, which was incubated overnight at room temperature, and the secondary antibody was green-fluorescent Desmin and red-fluorescent Osteopontin secondary antibody diluted to 1 : 500 for incubation for 6 h; for nuclei, the ready-to-use DAPI solution was used for restaining for 30 min. Finally, the imaging was observed by fluorescence microscopy and the cell orientation angle and nucleus aspect ratio were analyzed using Image J.

[0112] Figure 8 Biocompatibility of PGA composite hydrogels: (a) Live / dead staining of smooth muscle cells on the corresponding tubular units at the corresponding concentrations; (b) Statistical analysis of the proliferation rate of smooth muscle cells; (c) Statistical analysis of the survival rate of smooth muscle cells; (d) Cytoskeleton staining of smooth muscle cells on the corresponding tubular units on day 10; (e) Orientation distribution map of smooth muscle cells on the corresponding tubular units on day 10; (f) Schematic diagram of the orientation distribution of smooth muscle cells; (g) Schematic diagram of the nucleus aspect ratio; (h) Statistical diagram of the nucleus aspect ratio.

[0113] We further performed cytoskeleton staining of smooth muscle cells in the tubular units to observe the cell angle distribution and nucleus aspect ratio (NAR). In the tubular units with GA, the cells were arranged in a spiral pattern, and the NAR was 1.5. In the tubular units with PGA, the cells were arranged in a spiral pattern, and the NAR was 1.2. Figure 8(d) shows the fluorescent images of the smooth muscle cell skeleton and the nucleus staining at day 10. GA is the control group, PGA-Unstretched group is the group containing the orientation factor PEO but without small stress traction, and PGA-Stretched is the group after stress traction. It can be seen intuitively that the smooth muscle cells in the no PEO group and the unstretched group are disordered, and the smooth muscle cells in the PEO-containing and stress-traction group have obvious cell orientation characteristics. Further, the orientation angle of the smooth muscle cells in the above three groups and the length ratio of the nucleus are quantitatively analyzed using Image.J. As shown in (e) of FIG. 6, the smooth muscle cells in the PEO-containing and stress-traction group are concentrated at 0°, and the smooth muscle cells in the other two groups are randomly distributed at -90°-90°, without orientation. Figure 8 (d) shows that in the PEO-containing and stress-traction PGA tubular unit, the smooth muscle cells are affected by the PEO directional fibers, and the cell orientation is concentrated at 0°, and the smooth muscle cells in the other two groups are randomly distributed at -90°-90°, without orientation. Figure 8 (f) is a diagram of cell orientation and nucleus length ratio. Further, the length ratio of the nucleus of the smooth muscle cells in the three different groups is statistically analyzed, as shown in (g) of FIG. 6. Figure 8 As shown in (h) of FIG. 6, the average length ratio of the nucleus of the smooth muscle cells in the GA group is 1.480, the length ratio of the nucleus of the smooth muscle cells in the unstretched PGA group is 1.900, and the length ratio of the nucleus of the smooth muscle cells in the stretched PGA group is 2.707. It is found by analysis that the P value between the GA group and the PGA-unstretched group is 0.0352, and the P value between the PGA-unstretched group and the PGA-stretched group is less than 0.0001, which has obvious statistical significance. It is confirmed that in order to induce the oriented growth of smooth muscle cells, it is necessary to combine the designed hydrogel structure with the corresponding process means, to induce the directional arrangement of smooth muscle cells on the microscale by constructing the PEO directional microstructure.

[0114] Figure 9 (a) α-SMA antibody staining; (b) double staining fluorescent image; (c) DES / OPN fluorescence intensity statistical diagram (DES refers to Desmin, and OPN refers to Osteopontin)

[0115] 3. Printing of small-diameter directional tubular stents

[0116] On the basis of the tubular unit, a roller printing small-diameter tubular stent device with rolling and translation is independently designed and assembled, as shown in FIG. 7. Figure 10The printing device includes a PGA composite hydrogel extrusion control device and a calcium chloride extrusion control device, receives the printed tubular unit through a roller, the diameter of the roller can be conveniently changed according to the printing requirement, the speed of the motor controlling the rotation of the roller can also be changed to obtain tubular units with different diameters, and the roller motor is fixed on a translation base, and the speed and distance of translation are adjusted to obtain tubular supports with different lengths. The key point of printing the support is to match the extrusion rate, the diameter of the roller, the rotation speed of the roller and the translation speed.

[0117] Figure 10 . Millimeter-scale directional tubular support printing (a) tubular support printing physical map; (b) printing speed debugging; (c) printing tubular supports with diameters of 3 mm and 5 mm respectively; (d) fluorescence map of directional tubular support; (e) elasticity demonstration of the support; (f) simulation of blood vessel suture of the tubular support; (g) schematic diagram of burst pressure test; (h) initial state of burst pressure test; (i) maximum pressure value

[0118] Morphological characterization of PGA tubular support: PGA directional tubular supports with diameters of 3 mm and 5 mm were printed respectively, and after 30 s of ultraviolet light curing at 405 nm, they were taken off the roller and observed under a macroscopic and fluorescence microscope.

[0119] Burst pressure of PGA directional tubular support: In this study, a burst pressure testing device was designed and assembled, as shown in Figure 10 (h). The right side is a syringe connected with a syringe pump, the middle is an electronic pressure device, and the left side is connected with the tubular support printed in this study, and the middle is connected with a rubber tube through a three-way tube. The key points and difficulties of this experiment are to ensure the airtightness of all connection points, so the experiment is carried out after being tightly sealed with sealing film.

[0120] As shown in Figure 10 (a), based on the tubular unit, a large-size ring structure printing device is constructed, which prints the tubular unit through coaxial device, and receives the hollow pipe printed through the roller, including a horizontal moving translation structure and a rotating roller, by changing the diameter of the roller and the rotation speed of the motor, different sizes of ring structure can be simply and efficiently obtained. In Figure 10 (c), we show the printing physical map of two diameters (3 mm and 5 mm), further use fluorescence gel printing and do 3D shaping under confocal, the ring-shaped fiber can be seen in the overhead view, which is the structural basis for the ring-shaped growth of smooth muscle cells. The tubular knot structure has good elasticity, as the lumen obtained by printing can still recover the original structure after extrusion. In Figure 10In (f) of FIG. 1, the printed tubular unit was end-to-end sutured with 9-0 absorbable thread to simulate the process of blood vessel suturing in clinic. The results showed that the tubular structure could be applied to the suturing process and had potential value for clinical application. For the side wall pressure that the PGA tubular scaffold could withstand, we did a burst pressure test, as shown in Figure 10 In (g-h) of FIG. 1, the maximum pressure that could be withstood was 32.7 kPa, which was approximately equal to 240 mmHg of pressure, which was fully applicable to blood vessel replacement in the human body.

[0121] 4. Cell-laden printing of PGA directional tubular scaffolds

[0122] The digested smooth muscle cells were mixed with the sterilized bio-ink at 1 x 10 7 / mL. The extrusion speed of the bio-ink and the calcium chloride was set to 0.5 mL / min, a 5 mm diameter shaft was used, the shaft rotation speed was 66 rpm, the translation distance was set to 20,000 pulses, and the translation rate was set to 130 pulses per second. After printing, the scaffold was crosslinked using 405 nm ultraviolet light for 30 s, and then removed from the mold. After being washed with sterile PBS for 2 times, 20 mL of complete medium was added and placed in a cell culture incubator for culture. On the first day after printing, live / dead staining was performed to observe the survival and compatibility of the cells in the printed scaffold. On the 5th and 10th days, skeleton staining was performed in the same way. The spreading and orientation of the smooth muscle cells in the corresponding tubular scaffold were observed. The results showed that the cells in the large-size blood vessel scaffold presented a high activity and ordered growth along the circumferential direction.

[0123] Figure 11 (a) GA tubular scaffold; (b) PGA tubular scaffold (live / dead cell staining on the 1st day, skeleton staining on the 5th and 10th days).

[0124] 5. Construction of complex tubular scaffolds

[0125] 3D printing of multi-layer tubular scaffolds: transparent, black, red, and blue bio-inks were configured respectively, and transparent layer-black layer-red layer-blue layer were printed in turn.

[0126] 3D printing of multi-branch tubular scaffolds: several directional tubular structures of 3 mm and 5 mm were printed respectively, and then the same bio-ink was used for bonding and solidification after printing to construct a branched structure of the vascular network type.

[0127] Figure 12 Construction of complex tubular scaffolds (a-c) Construction of multi-layer tubular scaffolds for ureters; (d-e) Construction of multi-branch tubular scaffolds for blood vessels.

[0128] Through the above biological detection, it is found that the present application constructs a double cross-linking network by using natural material gelatin as raw material, and uses the selective chemical reaction of methacrylic anhydride and natural material to prepare methacrylated gelatin (GelMA), and uses sodium alginate as a biological ink based on the rapid gelation of sodium alginate and calcium ions to realize the construction of hollow structure. The biological ink provided by the present application has good biocompatibility, accurate printing, easy to stretch yield, high mechanical tensile property, double or multiple cross-linking mechanism, and the printed tubular tissue has good each direction specificity, no cytotoxicity, and cells in the large-size vascular stent present a high-activity, ordered growth morphology along the circumferential direction, so it is suitable for being applied to human body as a stent. Moreover, the ink has injectability, can be smoothly extruded, and will not block the needle, and has shear thinning property, which is crucial for the application of 3D printing platform as ink.

[0129] Those skilled in the art will understand that the embodiments of the application shown in the above description and drawings are only examples and do not limit the application. The purpose of the present application has been fully and effectively achieved. The function and structural principle of the present application has been shown and explained in the embodiments, and the embodiments of the present application can have any modification or modification without departing from the principle.

Claims

1. A method for fabricating a small-diameter tubular scaffold using 3D coaxial printing, characterized in that, Includes the following steps: (S10) Preparation of methacrylamide gelatin: gelatin is reacted with methacrylic anhydride in a phosphate buffer solution, dialyzed in a water bath, and freeze-dried to obtain methacrylamide gelatin. (S20) Preparation of PGA bio-ink, wherein step (S20) includes the following steps: (S201) dissolving methacrylamide gelatin monomer in H2O; (S202) adding LAP, shaking to mix, and protecting from light; (S203) subsequently adding sodium alginate and polyethylene oxide, mixing, centrifuging, and obtaining PGA bio-ink; and (S30) Preparation of CaCl2 aqueous solution; wherein PGA bio-ink and CaCl2 aqueous solution are placed in different channels. PGA bio-ink is first delivered through a silicone tube. After directional shearing by the silicone tube, the hydrogel chains are distributed axially. Then, PGA and CaCl2 aqueous solutions in different channels are extruded at flow rates of 0.5 mL / min and 0.4 mL / min, respectively. PGA bio-ink and CaCl2 aqueous solution are mixed in a common channel before being extruded onto a roller. Sodium alginate and CaCl2 are then mixed. 2+ In situ crosslinking of the rollers was performed to construct PEO-oriented microstructures, which were then printed at 37°C and photocured to form small-diameter tubular scaffolds that promote oriented cell growth.

2. The method for preparing a small-diameter tubular scaffold by 3D coaxial printing according to claim 1, wherein in step (S20), the concentration of methacrylamide 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 small-diameter tubular scaffold according to claim 1 or 2, wherein in step (S10), gelatin and methacrylic anhydride are reacted in a phosphate buffer solution with pH=7.4, dialyzed in a water bath at 40°C, and freeze-dried to obtain methacrylamide gelatin.

4. The method for preparing a small-diameter tubular scaffold for 3D coaxial printing according to claim 3, wherein in step (S20), 1g of methacrylamide gelatin monomer is weighed and dissolved in 10mL of dH2O, and then the methacrylamide gelatin is fully dissolved at 50°C. Subsequently, 0.3% (m / w) of LAP is added, the mixture is shaken and mixed, and then protected from light with aluminum foil. 0.1g of sodium alginate and 0.08g of polyethylene oxide are added to the gelatin solution, and the mixture is mixed in a 50°C water bath. The solution is centrifuged at 3000rpm for 3min to remove excess air bubbles for subsequent use.

5. The method for preparing a small-diameter tubular scaffold for 3D coaxial printing according to claim 4, wherein the preparation of methacrylamide gelatin is as follows: 50g of gelatin and 3.2mL of methacrylic anhydride are reacted in 500mL of phosphate buffer at 50°C for 3h, the above solution is dialyzed in a 40°C water bath for 5 days, with the solution changed 3 times a day, and then freeze-dried for 48h to synthesize methacrylamide gelatin.

6. The small-diameter tubular stent prepared by the preparation method according to any one of claims 1 to 5, characterized in that, The gelatin composite hydrogel ink for preparing the small-diameter tubular scaffold comprises PGA bio-ink and CaCl2 aqueous solution. The PGA bio-ink includes methacrylamide gelatin, sodium alginate, and polyethylene oxide. As a 3D coaxial printing gelatin composite hydrogel ink, the PGA bio-ink and CaCl2 aqueous solution are mixed in different channels at a flow rate ratio of 5:4 and then extruded onto a roller for in-situ crosslinking. This crosslinking is achieved through the interaction of sodium alginate and CaCl2. 2+ Combined, photocrosslinking and gelation occur, forming a hollow, small-diameter tubular scaffold.

7. The small-diameter tubular scaffold according to claim 6, wherein during printing, the outer shaft is a PGA bio-ink channel, the inner shaft is a 0.8% CaCl2 solution, the PGA bio-ink and CaCl2 solution are extruded into a common channel connected to the outer and inner shafts for mixing, and then extruded again, when sodium alginate and CaCl2... 2+ When combined, they crosslink rapidly to form hollow tubular units. The tensile stress is changed by altering the rotation speed and size of the shaft, with the shaft speed being 66 rpm and the shaft diameter being 5 mm. After printing, the units are cured under 405 nm ultraviolet light for 30 seconds.

8. An application of a small-diameter tubular stent, characterized in that, The composite hydrogel ink comprises PGA bio-ink and CaCl2 aqueous solution, suitable for printing biomimetic directional muscular tubular scaffolds. The coaxial printing method using the 3D coaxial printing gelatin composite hydrogel ink for coaxial printing of combined stress traction to manufacture hundreds-micron-scale tubular units includes the following steps: (A) Placing PGA bio-ink in an outer axial channel, the outer axial channel comprising a silicone tube, through which the PGA bio-ink is first transported, and after directional shearing by the silicone tube, the hydrogel chains are distributed axially; (B) Placing CaCl2 aqueous solution in an inner axial channel, wherein the ends of the outer axial channel and the inner axial channel are connected and communicate in a common channel, the PGA bio-ink and CaCl2 aqueous solution are extruded into the common channel for mixing, and then further extruded; (C) Adjusting the tensile stress, photocrosslinking and gelling to form a small-diameter tubular scaffold; and (D) Printing at 37°C, followed by UV curing after printing, wherein the flow rates of the inner and outer channels are set to 0.4 mL / min and 0.5 mL / min, respectively.

9. The application of the small-diameter tubular support according to claim 8, wherein the inner shaft dimension is 22G, the outer shaft dimension is 17G, the shaft rotation speed is 66rpm, the shaft diameter is 5mm, and the printing is cured under 405nm ultraviolet light for 30s after printing.

10. The application of the small-diameter tubular stent according to claim 8 or 9, wherein the PGA bio-ink is prepared as follows: dissolve methacrylamide gelatin monomer in H2O; add LAP, shake to mix, and protect from light; then add sodium alginate and polyethylene oxide, mix, and centrifuge.

11. The application of the small-diameter tubular stent according to claim 10, wherein the concentration of methacrylamide 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%.

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