A mechanically responsive hollow hydrogel scaffold, its preparation method and application in loading cells

Abstract

The application provides a mechanically responsive hollow hydrogel scaffold and a preparation method and application in cell loading thereof. The shell of the hollow hydrogel scaffold is obtained by coaxial printing of a bio-ink for 3D printing, and the core is a hollow structure; wherein the bio-ink for 3D printing comprises methacrylated gelatin, N-acryloylglycineamide and nanoclay; the methacrylated gelatin, N-acryloylglycineamide and nanoclay in the shell of the hollow hydrogel scaffold form physical interpenetration, and the N-acryloylglycineamide forms intermolecular hydrogen bonds, and the methacrylated gelatin and N-acryloylglycineamide form a double crosslinking network. The hydrogel scaffold can respond to mechanical stimulation, quickly and uniformly and accurately load bioactive components (such as cells) by regulating strain and mechanical loading times, and in this process, damage to cells caused by extrusion mechanical shear force can be avoided, so that the loaded cells have high viability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a mechanical response hollow hydrogel scaffold, a preparation method thereof and application thereof in cell loading. BACKGROUND

[0002] Tissue engineering generally contains biological scaffolds, cells and growth factors as main factors, but how to realize effective construction and uniform compounding of these factors is the key challenge limiting the development of tissue engineering. Biological 3D printing takes cells or active biological materials as basic structural units, combines computer precise control and 3D printing technology, and reconstitutes in vitro a tissue / organ with biological function, which provides an effective solution for the function construction of tissue engineering scaffolds and the uniform compounding of cells, and can be applied to the fields of in vivo tissue damage repair, organ transplantation, in vitro pathological, pharmacological and cancer research. Since biological 3D printing was first reported, the field has developed rapidly. In the traditional 3D printing, the cells are difficult to be uniformly and accurately loaded in the internal scaffold by the method of printing the scaffold first and then inoculating the surface, while the biological 3D printing scaffold can realize the uniform and accurate loading of cells.

[0003] At present, extrusion type biological 3D printing is mainly used in biological 3D printing. Extrusion type biological 3D printing can be divided into three processes: 1. biological ink is extruded from a pipe with a small caliber; 2. instantaneous structural stability; 3. long-term structural stability. Therefore, the biological ink needs to have shear thinning, controllable gelation, structural fidelity and high stability to realize smooth connection of the above three processes.

[0004] As the raw material of biological 3D printing, biological ink (with hydrogel material as the main matrix) is the material basis for structure shaping and internal cell growth, but for a long time, the hydrogel materials that can be used for 3D printing shaping are limited, which greatly restricts the development and application of biological 3D printing technology. Secondly, many commonly used hydrogel biological materials have good biological activity, but these materials are often difficult to be directly applied to general biological 3D printing process due to low viscosity and low mechanical strength, and the printing of cell-loaded hydrogel materials also needs to consider the problem of maintaining cell activity, especially for extrusion biological 3D printing, the mechanical shear force generated in the printing process is easy to cause damage to the cells, which increases the difficulty of printing.

[0005] CN112516324A discloses an additive for reducing shear injury of stem cell suspension injection, characterized by being composed of the following raw materials in parts by weight: hyaluronic acid 0.1-5 parts, collagen type II hydrolysate 1-5 parts, cholesterol 0.01-0.1 parts, glutathione 0.1-2 parts and ceramide 0.1-1 part. A stem cell suspension is added with the additive. The invention is to protect cells and reduce the damage of needle shearing force by adding the additive to the stem cell suspension, but this method does not construct a 3D printed tissue engineering scaffold.

[0006] CN113444264A discloses a preparation method and application method of a double-network hydrogel for three-dimensional culture of cells. First, a methyl methacrylated hyaluronic acid conjugate grafted with a short peptide specific to Sortase A enzyme is synthesized. The substrate is cross-linked with a certain concentration of Sortase A enzyme to obtain an injectable hyaluronic acid single-network hydrogel. Then, an enzyme and light double-crosslinked hyaluronic acid-gelatin double-network hydrogel is prepared. The enzyme cross-linked hyaluronic acid hydrogel is used as the first network to form a gel quickly, and the ultraviolet light cross-linked methyl methacrylated gelatin hydrogel is used as the second reinforcing network. The extrusion type biological 3D printing needs to balance the printing property of biological ink and the activity of cells. In order to reduce the damage of shear force of biological ink to cells during the printing process, the mechanical strength of the hydrogel is often low, which greatly restricts the application range of the construct, especially in the stress or load bearing parts.

[0007] Therefore, reducing the damage of mechanical shear force in the printing process to cells and improving the mechanics of biological 3D printed hydrogel scaffold are crucial to the development of biological 3D printing. SUMMARY

[0008] In view of the deficiencies of the prior art, the purpose of the present application is to provide a mechanically responsive hollow hydrogel scaffold and a preparation method and application thereof. The mechanically responsive hollow hydrogel scaffold can respond to mechanical stimulation, quickly and uniformly load cells. The present application provides a biological 3D printing method, which constructs tissues / organs in vitro for tissue damage repair or replacement, etc. The method can avoid the damage of cells during the biological 3D printing process, to some extent, solves the dilemma of extrusion type biological 3D printing limited by biological ink, and improves the mechanics of biological 3D printing construct, and expands its application range.

[0009] To achieve the purpose of the present application, the following technical solutions are adopted:

[0010] In a first aspect, the present application provides a mechanically responsive hollow hydrogel scaffold, wherein the shell of the mechanically responsive hollow hydrogel scaffold is obtained by coaxial printing with biological 3D printing ink, and the core is a hollow structure.

[0011] The ink for bio-inspired 3D printing comprises methacrylated gelatin, N-acryloylglycineamide and nanoclay; the methacrylated gelatin, N-acryloylglycineamide and nanoclay in the shell of the hollow hydrogel scaffold form physical interpenetration, and the N-acryloylglycineamide forms intermolecular hydrogen bonds, and the methacrylated gelatin and N-acryloylglycineamide form a double crosslinking network.

[0012] In the present application, the ink for bio-inspired 3D printing is configured by methacrylated gelatin (GelMA), N-acryloylglycineamide (NAGA) and nanoclay (LNP), which is used as the shell, and the core is a hollow structure, and after coaxial core / shell 3D printing, crosslinking and curing are performed, wherein the physical interpenetration of GelMA and NAGA and LNP, the intermolecular hydrogen bonds formed by NAGA, and the double crosslinking network formed by GelMA and NAGA, endow the mixed ink with excellent printing properties, self-supporting and high strength characteristics, and can maintain the hollow structure without adding a core support.

[0013] In addition, the present application can also provide an effective means for loading and delivering other bioactive ingredients (drugs, active factors, etc.). At the same time, the mechanical response characteristics of the 3D printed hollow hydrogel scaffold can be used to recruit cells in vivo and promote tissue damage repair.

[0014] Preferably, the mass ratio of the methacrylated gelatin, N-acryloylglycineamide and nanoclay is (1-10):(1-10):(1-10), for example, it can be 1:1:1, 1:2:3, 1:4:5, 1:6:6, 1:8:9, 1:8:10, 2:1:1, 2:2:3, 2:4:5, 2:6:6, 2:8:9, 2:8:10, 5:1:1, 5:2:3, 5:4:5, 5:6:6, 5:8:9, 5:8:10, 10:1:1, 10:2:3, 10:4:5, 10:9:9, etc.

[0015] Preferably, the solvent of the ink for bio-inspired 3D printing comprises any one or a combination of at least two of water, phosphate buffer, glucose solution or sodium chloride solution.

[0016] Preferably, the ink for bio-inspired 3D printing further comprises a photoinitiator.

[0017] Preferably, the content of the photoinitiator accounts for 0.1-0.5% of the total mass of the ink for bio-inspired 3D printing, for example, it can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, etc.

[0018] Preferably, the photoinitiator comprises any one or a combination of at least two of phenyl-2,4,6-trimethylbenzoyl phosphinic acid lithium, photoinitiator 2959 or photoinitiator 1173, preferably phenyl-2,4,6-trimethylbenzoyl phosphinic acid lithium (LAP).

[0019] Preferably, the method for preparing the bio-inspired 3D printing ink comprises the following steps:

[0020] (1) mixing nano-clay and part of solvent to obtain solution A; mixing methacrylated gelatin, N-acryloylglycylamide, optional photoinitiator and the rest of solvent to obtain solution B;

[0021] (2) mixing solution A and solution B obtained in step (1) to obtain the bio-inspired 3D printing ink.

[0022] Preferably, in step (1), the volume ratio of the part of solvent and the rest of solvent is (1-5):(1-5), for example, it can be 1:1, 1:2, 1:3, 1:4, 1:5, 5:1, 4:1, 3:1, 2:1, etc.

[0023] Preferably, in step (1), the mixing temperature for obtaining solution A is 20-40℃, for example, it can be 20℃, 25℃, 30℃, 35℃, 40℃, etc., and the mixing time is 10-60 min, for example, it can be 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, etc.

[0024] Preferably, in step (1), the mixing temperature for obtaining solution B is 55-65℃, for example, it can be 55℃, 56℃, 58℃, 60℃, 61℃, 63℃, 65℃, etc., and the mixing time is 1-3 h, for example, it can be 1 h, 1.5 h, 2 h, 2.5 h, 3 h, etc.

[0025] Preferably, in step (2), the mixing temperature of solution A and solution B is 10-40℃, for example, it can be 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc., and the mixing time is 30-120 min, for example, it can be 30 min, 60 min, 80 min, 100 min, 120 min, etc.

[0026] Preferably, the hollow hydrogel scaffold is formed by stacking at least one layer (e.g., 1, 2, 3, 4, 5, 8, 10, 15, 20, 30, 40, 50, 100, 200, etc.) of hydrogel fiber layers, and each hydrogel fiber layer contains at least one basic unit of hollow cylindrical structure (e.g., 1, 2, 3, 4, 5, 6, 8, 10, 15, 20, 25, 30, 50, etc.).

[0027] Preferably, the stacking method of each layer of the hollow hydrogel scaffold includes any one of orthogonal, oblique, or parallel stacking.

[0028] Preferably, the inner diameter of the basic unit of the hollow cylindrical structure is 200-600 μm, for example, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, etc., and the outer diameter is 400-1000 μm, for example, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, etc.

[0029] Preferably, the gap width between each hollow cylindrical fiber basic unit is 200-1000 μm, for example, it can be 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, etc.

[0030] Secondly, the present invention provides a method for preparing the mechanically responsive hollow hydrogel scaffold, the method comprising the following steps:

[0031] The bio-like 3D printing ink is loaded into a printing barrel to serve as the outer shell, while the core is left unfilled. Using coaxial core / shell 3D printing technology, a hollow hydrogel scaffold is printed by one-step extrusion. Finally, it is cured by ultraviolet cross-linking to obtain the mechanically responsive hollow hydrogel scaffold.

[0032] Preferably, the energy for the ultraviolet cross-linking curing is 0.1-8 W / cm². 2 For example, it could be 0.1 W / cm 2 0.5 W / cm 2 1 W / cm 2 2 W / cm 2 3 W / cm 2 4 W / cm 2 5 W / cm 2 6 W / cm 2 7 W / cm2 8 W / cm 2 temperature is 10-40℃, for example, it can be 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc., and the time is 5-120 min, for example, it can be 5 min, 10 min, 20 min, 40 min, 60 min, 80 min, 100 min, 120 min, etc.

[0033] In a third aspect, the present application provides a use of the mechanically responsive hollow hydrogel scaffold in the preparation of a product for loading and / or delivering bioactive components.

[0034] In the present application, the bioactive components include drugs, active factors, cells, etc.

[0035] In a fourth aspect, the present application provides a method for loading cells, which comprises the following steps:

[0036] The mechanically responsive hollow hydrogel scaffold is immersed in a cell suspension, an external force is applied to compress the scaffold to cause a specific deformation, and after the compression force is removed, the scaffold recovers to the original shape while loading the cells.

[0037] Preferably, the loading time of the cells is within 1 min, for example, it can be 1 min, 50 s, 40 s, 30 s, 20 s, 10 s, etc.

[0038] Under the conventional strip, the material is directly immersed in the cell suspension for 1 min under static conditions (basically no cell loading or a small amount of cell loading), while under the dynamic condition of mechanical stimulation, the cells loaded within 1 min can be more than 20 times of the static loading, and here 1 min is the calculated time of 10 compression cycles, and one compression recovery can be completed within 5 s.

[0039] Compared with the prior art, the present application has the following beneficial effects:

[0040] (1) The present application prepares a size-controllable high-strength hollow hydrogel scaffold by a coaxial core / shell 3D printing one-step method, and utilizes the mechanical response characteristics of the hollow hydrogel scaffold to quickly and uniformly and accurately load cells;

[0041] (2) The present application utilizes the mechanical response characteristics of the 3D printed hollow hydrogel scaffold to load drugs, active factors, etc., and utilizes the mechanical response characteristics of the 3D printed hollow hydrogel scaffold to recruit cells in vivo and promote the repair of tissue damage;

[0042] (3) The hollow support in the application has excellent compression strength, and can restore the initial state after compression strain of 80% or more without breaking, while the solid support breaks obviously at strain of about 40%;

[0043] (4) The mechanically responsive loaded cells in the application have high activity, and can proliferate massively after 3 days, which avoids the damage of mechanical shear force in the extrusion printing process to the cells. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 The schematic diagram of the double cross-linked network in the hollow hydrogel support provided by the application is shown.

[0045] Figure 2 The flow chart of printing and loading cells of the hollow hydrogel support provided by the application is shown.

[0046] Figure 3 The hollow structure diagram of different inner and outer diameters in the hydrogel support provided by the application is shown.

[0047] Figure 4 The stress-strain diagram of the hydrogel support provided by the application is shown.

[0048] Figure 5A The hollow hydrogel loaded cell diagram after 1 day without mechanical stimulation is shown.

[0049] Figure 5B The hollow hydrogel loaded cell diagram after 1 day under mechanical stimulation is shown.

[0050] Figure 5C The hollow hydrogel loaded cell diagram after 3 days under mechanical stimulation is shown. DETAILED DESCRIPTION

[0051] The technical solutions of the application will be further described through specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the application, and should not be regarded as specific limitation on the application.

[0052] The sources of raw materials in the following examples and comparative examples are shown as follows:

[0053]

[0054] Example 1

[0055] The embodiment provides a mechanically responsive hollow hydrogel support, which is prepared by the following method:

[0056] S1 1 g of LNP was dissolved in 5 mL of deionized water, stirred at room temperature for 20 min to dissolve completely, to prepare solution A for standby; 1 g of methyl methacrylate gelatin (GelMA), 1 g of N-acryloyl glycineamide (NAGA) and 10 mg of photoinitiator (LAP) were added to 5 mL of deionized water, stirred at 60°C for 2 h to dissolve completely, to prepare solution B; the above solution A and solution B were mixed in equal volume, stirred uniformly, to prepare 10 mL of ink required for printing;

[0057] S2 The above prepared ink was loaded into a printing cartridge as a shell, and no ink was added to the core, and coaxial core / shell 3D printing was adopted, and a hollow structure hydrogel scaffold was printed by one-step extrusion through a coaxial needle of a certain size (specifically shown in Table 1 below, “-” represents that the test is not performed), and the printed scaffold was subjected to ultraviolet crosslinking curing (energy 2 W / cm 2 , temperature 25°C, time 40 min) to obtain hydrogel scaffolds with hollow structures of different sizes;

[0058] Table 1

[0059]

[0060] As shown in Figure 1 , by adjusting the size of the coaxial needle, a size-controllable hollow hydrogel scaffold was obtained. The physical interpenetration of GelMA and NAGA, the intermolecular hydrogen bond formed by NAGA and the double crosslinking network formed by GelMA and NAGA endow the mixed ink with excellent printing properties, self-supporting and high strength characteristics, which can maintain the hollow structure without adding an internal core support.

[0061] The prepared hydrogel was cut into a thin piece of about 1 mm in thickness, immersed in a rhodamine B solution for 5 min, and then washed with deionized water for 3 times, and the hollow structure inside the hydrogel was observed under a fluorescence microscope. As shown in Figure 3 , the hollow structure of different inner and outer diameters inside the hydrogel scaffold can be observed. The hollow hydrogel scaffold is stacked by at least 5 layers of hydrogel fiber layers, and each layer of hydrogel fiber layer contains at least 3 basic units of hollow cylindrical structure, and the stacking mode of each layer of the basic unit of the hollow hydrogel scaffold is orthogonal.

[0062] The prepared hydrogel (serial numbers 4 and 9) was printed into a hollow scaffold of 1 cm × 1 cm × 1 cm, and the mechanical tester was used to test the mechanical properties of the scaffold, as shown in Figure 4 , the hollow scaffold has excellent compressive strength and can still recover to the initial state after a compressive strain of 80%, without breaking, while the solid scaffold breaks obviously at a strain of about 40%.

[0063] Example 2

[0064] The present example provides a mechanically responsive hollow hydrogel scaffold, which is prepared by the following method:

[0065] S1 0.8 g of LNP was dissolved in 4 mL of deionized water, stirred at 30°C for 15 min to dissolve completely, to prepare solution A for standby; 1.2 g of methacrylated gelatin (GelMA), 1.2 g of N-acryloylglycineamide (NAGA) and 12 mg of photoinitiator (LAP) were added to 6 mL of deionized water, stirred at 58°C for 2.5 h to dissolve completely, to prepare solution B; the above solution A and solution B were mixed in equal volume, stirred uniformly, to prepare 10 mL of ink required for printing;

[0066] S2 The above prepared ink was loaded into the printing cartridge as the shell, and the core was not added with ink, and coaxial core / shell 3D printing was adopted, and a hollow structure hydrogel scaffold was printed by one-step extrusion through a coaxial needle of a certain size (specifically shown in Table 2), and the printed scaffold was subjected to ultraviolet crosslinking curing (energy 2W / cm 2 , temperature 25°C, time 30 min), to obtain hydrogel scaffolds with hollow structure of different sizes;

[0067] Table 2

[0068]

[0069] Example 3

[0070] The present example provides a mechanically responsive hollow hydrogel scaffold, which is prepared by the following method:

[0071] S1 1.5 g of LNP was dissolved in 5 mL of deionized water, stirred at 37°C for 30 min to dissolve completely, to prepare solution A for standby; 0.8 g of methacrylated gelatin (GelMA), 0.8 g of N-acryloylglycineamide (NAGA) and 8 mg of photoinitiator (LAP) were added to 5 mL of deionized water, stirred at 65°C for 1.5 h to dissolve completely, to prepare solution B; the above solution A and solution B were mixed in equal volume, stirred uniformly, to prepare 10 mL of ink required for printing;

[0072] S2 The above prepared ink was loaded into the printing cartridge as the shell, and the core was not added with ink, and coaxial core / shell 3D printing was adopted, and a hollow structure hydrogel scaffold was printed by one-step extrusion through a coaxial needle of a certain size (specifically shown in Table 3), and the printed scaffold was subjected to ultraviolet crosslinking curing (energy 1W / cm2 temperature 25℃, time 50 min);

[0073] Table 3

[0074]

[0075] Example 4

[0076] The present example provides a mechanically responsive hollow hydrogel scaffold, which is prepared by the following method:

[0077] S1 0.5 g of LNP was dissolved in 5 mL of deionized water, stirred at 25℃ for 10 min to fully dissolve, to prepare solution A for standby; 1.2 g of methacrylated gelatin (GelMA), 1.3 g of N-acryloylglycineamide (NAGA) and 10 mg of photoinitiator (LAP) were added to 5 mL of deionized water, stirred at 60℃ for 2 h to fully dissolve, to prepare solution B; the above solution A and solution B were mixed in equal volume, stirred uniformly, to prepare 10 mL of ink required for printing;

[0078] S2 The prepared ink was loaded into the printing cartridge as the shell, and the core was not added with ink. Coaxial core / shell 3D printing was adopted, and the hollow structure hydrogel scaffold was printed by one-step extrusion through a coaxial needle of a certain size (specifically shown in Table 4 below), and the printed scaffold was subjected to ultraviolet crosslinking curing (energy 4W / cm 2 temperature 25℃, time 10 min), to obtain hydrogel scaffolds with hollow structure of different sizes;

[0079] Table 4

[0080]

[0081] Example 5

[0082] The present example provides a mechanically responsive hollow hydrogel scaffold, which is prepared by the following method:

[0083] S1 1.5 g of LNP was dissolved in 6 mL of deionized water, stirred at 37℃ for 30 min to fully dissolve, to prepare solution A for standby; 0.5 g of methacrylated gelatin (GelMA), 0.5 g of N-acryloylglycineamide (NAGA) and 10 mg of photoinitiator (LAP) were added to 4 mL of deionized water, stirred at 60℃ for 2 h to fully dissolve, to prepare solution B; the above solution A and solution B were mixed in equal volume, stirred uniformly, to prepare 10 mL of ink required for printing;

[0084] S2 The prepared ink is loaded into a printing cartridge as a shell, and the core is not added with ink. A coaxial core / shell 3D printing is adopted to extrude and print the hydrogel scaffold with a hollow structure through a coaxial needle with a certain size (specifically shown in Table 5 below). The printed scaffold is subjected to ultraviolet cross-linking curing (energy is 2 W / cm 2 , temperature is 25℃, and time is 30 min) to obtain hydrogel scaffolds with a hollow structure of different sizes;

[0085] Table 5

[0086]

[0087] Comparative Example 1

[0088] The present comparative example provides a mechanically responsive hollow hydrogel scaffold, which is prepared by the following method:

[0089] S1 1.5 g of LNP is dissolved in 5 mL of deionized water, and stirred at 25℃ for 30 min until completely dissolved to prepare solution A for standby; 1.5 g of methacrylated gelatin (GelMA) and 10 mg of a photoinitiator (LAP) are added to 5 mL of deionized water, and stirred at 60℃ for 2 h until completely dissolved to prepare solution B; the above-mentioned solution A and solution B are mixed in equal volume, and stirred uniformly to prepare 10 mL of ink required for printing;

[0090] S2 The prepared ink is loaded into a printing cartridge as a shell, and the core is not added with ink. A coaxial core / shell 3D printing is adopted to extrude and print the hydrogel scaffold with a hollow structure through a coaxial needle with a certain size (specifically shown in Table 6 below). The printed scaffold is subjected to ultraviolet cross-linking curing (energy is 0.5 W / cm 2 , temperature is 25℃, and time is 5 min) to obtain hydrogel scaffolds with a hollow structure of different sizes;

[0091] Table 6

[0092]

[0093] Comparative Example 2

[0094] The present comparative example provides a mechanically responsive hollow hydrogel scaffold, which is prepared by the following method:

[0095] S1 1.5 g of LNP was dissolved in 5 mL of deionized water, stirred at 30℃ for 30 min to fully dissolve, to prepare solution A for standby; 1.5 g of N-acryloylglycineamide (NAGA) and 10 mg of a photoinitiator (LAP) were added to 5 mL of deionized water, stirred at 60℃ for 2 h to fully dissolve, to prepare solution B; the above solution A and solution B were mixed in equal volume, stirred uniformly, to prepare 10 mL of ink required for printing;

[0096] S2 The above prepared ink was loaded into a printing cartridge as a shell, and no ink was added to the core, and coaxial core / shell 3D printing was adopted, and a hollow structure hydrogel scaffold was printed by one-step extrusion through a coaxial needle of a certain size (specifically shown in Table 7), and the printed scaffold was subjected to ultraviolet crosslinking curing (energy 5W / cm 2 , temperature 25℃, time 50 min), to obtain hydrogel scaffolds with hollow structures of different sizes;

[0097] Table 7

[0098]

[0099] Application Example 1

[0100] The application example provides a method for loading cells, which comprises the following steps:

[0101] The hollow scaffold (No. 4) provided in Example 1 was immersed in a cell suspension, and the scaffold was compressed to cause a specific deformation amount. After the compression force was removed, the scaffold quickly absorbed the surrounding cells while recovering to the original shape, and this process could be completed within 1 min. By using the mechanical responsiveness of the hollow scaffold, the cells were quickly and uniformly loaded. After the scaffold was continuously cultured at 37℃ for 3 days, the loaded cells proliferated in the scaffold.

[0102] As Figures 5A-5C can be seen, the hollow hydrogel can respond to mechanical stimulation to quickly load a large number of cells, the loaded cells have high activity, and can proliferate in large quantities after 3 days; and the hollow hydrogel without mechanical response only has a small amount of cell loading.

[0103] The applicant declares that the hollow hydrogel scaffold and the preparation method and application thereof of the present application are illustrated by the above examples, but the present application is not limited to the above process steps, that is, it does not mean that the present application must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvement of the present application, equivalent replacement of the materials selected by the present application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. A mechanically responsive hollow hydrogel scaffold, characterized in that, The outer shell of the mechanically responsive hollow hydrogel scaffold is obtained by coaxial printing using bio-like 3D printing ink, and the core is a hollow structure. The hollow hydrogel scaffold is formed by stacking at least one layer of hydrogel fiber, and each layer of hydrogel fiber contains at least one basic unit of hollow fiber structure. The mechanically responsive hollow hydrogel scaffold is prepared by the following method, which includes the following steps: The bio-like 3D printing ink is loaded into a printing barrel as the outer shell, while the core is left unfilled. Using coaxial core / shell 3D printing technology, a hollow hydrogel scaffold is extruded in one step, and finally cured by UV cross-linking to obtain the mechanically responsive hollow hydrogel scaffold. The bio-like 3D printing ink comprises methacrylated gelatin, N-acrylglycolamide, nanoclay, and a photoinitiator. In the outer shell of the hollow hydrogel scaffold, methacrylated gelatin, N-acrylglycolamide, and nanoclay form a physical interpenetration, with N-acrylglycolamide forming intermolecular hydrogen bonds, and methacrylated gelatin and N-acrylglycolamide forming a double cross-linked network. The mass ratio of methacrylated gelatin, N-acrylglycolamide, and nanoclay is (1-10):(1-10):(1-10).

2. The hollow hydrogel scaffold with mechanical response according to claim 1, characterized in that, The solvent for the bio-like 3D printing ink includes any one or a combination of at least two of the following: water, phosphate buffer, glucose solution, or sodium chloride solution.

3. The hollow hydrogel scaffold with mechanical response according to claim 1, characterized in that, The photoinitiator accounts for 0.1-0.5% of the total volume of the bio-like 3D printing ink.

4. The hollow hydrogel scaffold with mechanical response according to claim 1, characterized in that, The photoinitiator includes any one or a combination of at least two of the following: lithium phenyl-2,4,6-trimethylbenzoylphosphonate, photoinitiator 2959, or photoinitiator 1173.

5. The hollow hydrogel scaffold with mechanical response according to claim 4, characterized in that, The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphonate.

6. The hollow hydrogel scaffold with mechanical response according to claim 1, characterized in that, The preparation method of the bio-like 3D printing ink includes the following steps: (1) Mix nano-clay and part of the solvent to obtain solution A; mix methacrylated gelatin, N-acrylglycolamide, optional photoinitiator and the remaining solvent to obtain solution B; (2) Mix solution A and solution B obtained in step (1) to obtain the bio-like 3D printing ink.

7. The hollow hydrogel scaffold with mechanical response according to claim 6, characterized in that, In step (1), the volume ratio of the partial solvent to the remaining solvent is (1-5):(1-5).

8. The hollow hydrogel scaffold with mechanical response according to claim 6, characterized in that, In step (1), the mixing temperature of solution A is 20-40℃ and the mixing time is 10-60 min.

9. The hollow hydrogel scaffold with mechanical response according to claim 6, characterized in that, In step (1), the temperature at which solution B is mixed is 30-65℃ and the mixing time is 0.5-3 h.

10. The hollow hydrogel scaffold with mechanical response according to claim 6, characterized in that, In step (2), the temperature at which solution A and solution B are mixed is 10-40℃, and the mixing time is 30-120 min.

11. The hollow hydrogel scaffold with mechanical response according to claim 1, characterized in that, The hollow hydrogel scaffold can be stacked in any of the following ways: orthogonal, oblique, or parallel stacking.

12. The hollow hydrogel scaffold with mechanical response according to claim 1, characterized in that, The basic unit of the hollow fiber structure has an inner diameter of 200-600 μm and an outer diameter of 400-1000 μm.

13. The hollow hydrogel scaffold with mechanical response according to claim 1, characterized in that, The gap width between the basic units of each hollow fiber structure is 200-1000 μm.

14. A method for preparing a hollow hydrogel scaffold with mechanical response according to any one of claims 1-13, characterized in that, The method for preparing the mechanically responsive hollow hydrogel scaffold includes the following steps: The bio-like 3D printing ink is loaded into the printing barrel to serve as the outer shell, while the core is not filled with ink. Using coaxial core / shell 3D printing technology, a hydrogel scaffold with a hollow structure is printed by one-step extrusion. Finally, it is cured by ultraviolet cross-linking to obtain the mechanically responsive hollow hydrogel scaffold.

15. The method for preparing the mechanically responsive hollow hydrogel scaffold according to claim 14, characterized in that, The energy for the ultraviolet cross-linking curing is 0.1-8 W / cm². 2 The temperature is 10-40℃ and the time is 5-120 min.

16. The use of a hollow hydrogel scaffold with mechanical response according to any one of claims 1-13 in the preparation of products loaded with and / or delivered with bioactive ingredients.

17. A method for loading cells, characterized in that, The method for loading cells includes the following steps: The hollow hydrogel scaffold with mechanical response as described in any one of claims 1-13 is immersed in a cell suspension, and an external force is applied to compress the scaffold to cause a specific deformation. After the compression force is removed, the scaffold restores its initial shape and achieves cell loading.

18. The method for loading cells according to claim 17, characterized in that, The time required to load the cells is within 1 minute.

Images