3D printing hydrogel stent with biological activity as well as preparation method and application of 3D printing hydrogel stent

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a 3D printing hydrogel stent with biological activity and a preparation method and application thereof. The preparation method provided by the invention comprises the following steps: (1) extracting a mesenchymal stem cell exosome; (2) preparing 3D printing ink; (3) designing a 3D printing hydrogel stent model; and (4) 3D printing is conducted. The hydrogel scaffold obtained by the preparation method disclosed by the invention has relatively good mechanical property, moderate swelling property and relatively high biological activity.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a 3D printed hydrogel scaffold with biological activity, a preparation method thereof, and an application thereof. Background Art

[0002] Soft tissue fillers are used in medicine and cosmetology to correct various soft tissue defects or enhance appearance. Soft tissue defects may be caused by various conditions such as soft tissue tumor resection, congenital abnormalities, trauma and aging. Most hydrogels currently used for soft tissue filling on the market are relatively soft, and there are problems such as being unable to be customized according to specific conditions, mechanical properties that are difficult to meet filling requirements, or low biological activity. 3D printing technology provides a solution for shape customization and rapid preparation, and by adjusting the composition of 3D printing ink, the printed product can have certain mechanical properties. Patent No. CN114796604A discloses a 3D printing ink for corneal regeneration. The 3D printing ink uses a certain proportion of PEGDA, GelMA, a photoinitiator and a light blocker to finally prepare a hydrogel with a corneal shape. However, the role of the hydrogel is more of a physical filling or support, and it does not have biological activity itself.

[0003] Exosomes derived from mesenchymal stem cells may contain growth factors, cytokines, DNA, lipids, etc., which can promote tissue regeneration and accelerate the healing process to a certain extent. However, there are some difficulties and challenges in applying exosomes to the field of soft tissue regeneration. For example, as they are taken up and internalized by target cells or captured by macrophages, the concentration of exosomes will drop rapidly, which will limit the timeliness of exosome treatment. When exosomes are loaded into hydrogels, since hydrogels have a sustained release effect, as the hydrogels degrade, the exosomes can continue to act on the filled area for a long time. However, hydrogels used for soft tissue filling often require a certain mechanical strength or support strength, but as the hydrogels degrade, their mechanical strength will also decrease. In addition, the contact between hydrogel-encapsulated exosomes and cells is limited, and it is usually necessary to increase the content of exosomes to improve the therapeutic effect, but the production capacity of exosomes is limited and they cannot be mass-produced at present.

[0004] In summary, there are still some technical barriers in the preparation of bioactive hydrogels for soft tissue filling, so it is necessary to propose new methods and strategies to alleviate or partially alleviate the shortcomings of existing technologies. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a 3D printed hydrogel scaffold with bioactivity and a preparation method thereof, as well as an application in filling soft tissue defects. The specific technical scheme is as follows.

[0006] A method for preparing a 3D printed hydrogel scaffold with biological activity comprises the following steps:

[0007] S01: Extraction of mesenchymal stem cell exosomes;

[0008] S02: preparing a 3D printing ink, wherein the 3D printing ink comprises a hydrogel matrix and the mesenchymal stem cell exosomes; the hydrogel matrix comprises 2%-20% w / v of methacryloyl gelatin, 2%-20% w / v of polyethylene glycol diacrylate and 0.1%-0.5% w / v of a photoinitiator; the methacryloyl gelatin and polyethylene glycol diacrylate are used in a ratio of 1:0.5-1; the 3D printing ink further comprises 0.05-0.3% w / v of the mesenchymal stem cell exosomes;

[0009] S03: Designing a 3D printed hydrogel scaffold model, wherein the hydrogel scaffold model has a uniformly arranged microporous structure; it is understood that the size and dimensions of the hydrogel scaffold model are designed according to the size and shape of the part to be filled;

[0010] S04: Perform 3D printing and perform UV cross-linking on the printed hydrogel scaffold.

[0011] In some preferred embodiments, the size of the hydrogel scaffold model is 20×20×2 cubic millimeters, and the micropore size is about 0.4-0.6 mm. Furthermore, the usage ratio of the methacrylated gelatin and polyethylene glycol diacrylate is 1:1.

[0012] In some preferred embodiments, the hydrogel matrix includes 10% w / v methacrylated gelatin and 10% w / v polyethylene glycol diacrylate.

[0013] Furthermore, the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt.

[0014] Furthermore, the extraction method of the mesenchymal stem cell exosomes includes differential centrifugation; including centrifugation at 300-500g for 5-10 minutes, centrifugation at 1000-5000g for 5-10 minutes, centrifugation at 8000-10000g for 5-10 minutes, and centrifugation at 100000-120000g for 50-70 minutes.

[0015] Furthermore, the extrusion 3D printing technology is used in S03, and the specific operation includes printing at 1.5-3.0 mm / s under 3-4 Bar conditions.

[0016] Furthermore, the 3D printing ink also includes 0.2% w / v of the mesenchymal stem cell exosomes.

[0017] It is understood that the 3D printing ink further comprises 0.05% w / v, 0.1% w / v, 0.15% w / v, 0.25% w / v and / or 0.3% w / v of the mesenchymal stem cell exosomes.

[0018] Furthermore, the methacrylated gelatin is a mixture of methacrylic anhydride and gelatin PBS solution; the volume ratio of the gelatin PBS solution to the methacrylic anhydride is 25:1.

[0019] The 3D printed hydrogel scaffold prepared by the above preparation method.

[0020] Application of the above preparation method in preparing materials for soft tissue defects.

[0021] The application of the above-mentioned 3D printed hydrogel scaffold in the preparation of soft tissue defect materials.

[0022] Beneficial effects of the present invention

[0023] 1. The present invention provides a 3D printed hydrogel scaffold with biological activity and a preparation method thereof. The method of the present invention obtains a 3D printing ink with biological activity by simplifying the types of materials (only 4 materials in total, one of which is a photoinitiator), and then supplemented with a special model structure design (with a uniformly penetrating microporous structure), to obtain a hydrogel scaffold with good mechanical properties, moderate swelling properties and high biological activity, which can be used for filling soft tissue defects. It should be noted that the scaffold designed by the present invention has a uniform porous structure, which can increase the contact amount between cells and exosomes while allowing cells to enter the micropores, so that the biological functions of exosomes can be efficiently utilized. Therefore, the present invention only needs to add a small amount of exosomes to achieve a better repair effect.

[0024] 2. The method of the present invention adjusts the proportion of materials so that the obtained hydrogel scaffold has good mechanical strength and moderate swelling performance. Better mechanical properties can make the material less likely to be damaged when subjected to external forces, and provide effective support for the filled part. Swelling can increase the tightness with the filled cavity and even play a role in compression and hemostasis. However, if the swelling rate is too large, the overall water content of the material will be too high, which will cause the predetermined shape of the material (i.e., the shape of a sheet with a uniform microporous structure) to collapse, thereby causing a decrease in mechanical strength. Therefore, moderate swelling performance is an important consideration.

[0025] 3. The hydrogel scaffold provided by the present invention can not only provide good support and filling for soft tissue defects, but also the microporous structure can provide certain spatial support for cell and soft tissue regeneration, and match the filling and repair of soft tissue. The microporous structure can also allow more cells to enter the hydrogel and contact with exosomes, so that the adhesion and repair effect of exosomes is optimized, and the synergistic effect of exosomes and hydrogel matrix is ​​achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a scanning electron micrograph of a dry GelMA sponge-like solid prepared in one embodiment of the present invention;

[0027] Figure 2 This is a scanning electron micrograph of mesenchymal stem cell exosomes extracted from one of the embodiments of the present invention;

[0028] Figure 3 A physical image of a stent printed using 3D printing inks of different components in one embodiment of the present invention;

[0029] Figure 4 An optical microscope image of a stent printed using a 3D printing ink of a specific composition in one embodiment of the present invention;

[0030] Figure 5 This is a graph showing the mechanical properties and compression modulus results of a stent printed using 3D printing inks of different components in one embodiment of the present invention;

[0031] Figure 6 This is a result diagram of the swelling performance of a stent printed using 3D printing inks of different components in one embodiment of the present invention;

[0032] Figure 7 This is a graph showing the degradation performance of a stent printed using 3D printing inks of different components in one embodiment of the present invention;

[0033] Figure 8 This is a live-dead staining image of mouse fibroblasts (L929) treated with a hydrogel scaffold loaded with and without exosomes in one embodiment of the present invention;

[0034] Fig. 9 This is a graph showing the survival rate of mouse fibroblasts (L929) treated with hydrogel scaffolds loaded with and without exosomes in one of the embodiments of the present invention. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0036] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0037] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0038] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0039] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0040] In this specification, some embodiments may be disclosed in a format of being within a certain range. It should be understood that such description of "being within a certain range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the range The description of should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within this range, for example, 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0041] The following is a detailed description of the technical scheme of a 3D printed exosome-loaded soft tissue filling and repair hydrogel material and a preparation method thereof through specific embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1

[0043] This embodiment provides an example of a 3D printed soft tissue filling and repair hydrogel material loaded with exosomes.

[0044] Step 1: prepare methacryloyl gelatin (GelMA), mix and stir a PBS solution in which a certain amount of gelatin is dissolved and a methacrylic anhydride solution, stir and mix to obtain a methacryloyl gelatin solution, dialyze and freeze-dry the solution to obtain a GelMA sponge-like solid.

[0045] Step 2: Extract mesenchymal stem cell exosomes (EXO) and prepare them by differential centrifugation and ultrafiltration.

[0046] Step 3: Prepare 3D printing ink, including mixing 2%-20% w / v of methacryloyl gelatin (GelMA) sponge solid, 2%-20% w / v of polyethylene glycol diacrylate (PEGDA), 0.1%-0.5% w / v of photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) and 0.05%-3% w / v of mesenchymal stem cell exosomes (EXO).

[0047] Step 4: pre-cool the 3D printing ink at 4°C, then print the formed scaffold in an extrusion 3D printer according to the programmed computer-aided design (CAD) model, and then cross-link and cure the printed scaffold with ultraviolet light. The scaffold has good formability and a uniform microporous structure;

[0048] In this embodiment, the size of the stent model is 20×20×2 cubic millimeters, the whole is in sheet shape, and the micropore size is about 0.6 mm.

[0049] Example 2

[0050] This embodiment provides a specific preparation example of methacryloyl gelatin (GelMA).

[0051] 10 g of gelatin was added to phosphate buffered saline (PBS) at a ratio of 1:10 (w / v, g / mL), and stirred at 60°C for about 30 min until fully dissolved; the temperature was controlled to 50°C, and methacrylic anhydride (4 mL) was added at a ratio of 1:25 (v / v), and stirred for 2 h to obtain a crude product. The crude product was dialyzed in deionized water at 40°C for 3 days to remove excess methacrylic acid (the cutoff of the dialysis bag was 8–14 kDa), and the distilled water was replaced every 12 h. Finally, the dialyzed solution was freeze-dried in a vacuum freeze dryer to obtain a dry GelMA sponge-like solid (such as Figure 1 shown).

[0052] Example 3

[0053] This example provides an example of a method for extracting exosomes from mesenchymal stem cells.

[0054] (1) The culture medium of mesenchymal stem cells was collected and divided into centrifuge tubes, and the tubes were centrifuged three times at 4°C, namely, centrifuged at 300 g for 10 minutes to remove cells in the culture medium, at 2000 g for 10 minutes to remove cell debris in the culture medium, and at 10000 g for 10 minutes to remove large vesicles to obtain a supernatant.

[0055] (2) The obtained supernatant was transferred into an ultracentrifuge tube for crude extraction of exosomes. The centrifugation condition was 110,000 g for 70 minutes. The supernatant of the ultracentrifuge tube was removed and different volumes of pre-cooled saline were added to resuspend the exosomes (to keep the exosome concentration within a certain range; at the same time, to make it easier to collect the exosomes attached to the tube wall). The ultracentrifuge tube wall was gently blown to completely separate the exosomes from the ultracentrifuge tube wall. The suspension was collected in a 50 ml centrifuge tube and the exosomes were filtered using a 0.22 μm filter.

[0056] (3) The filtered exosomes were purified by ultracentrifugation at 110,000 g for 70 minutes. The supernatant of the ultracentrifuge tube was removed and different volumes of pre-cooled saline were added to resuspend the exosomes. The wall of the ultracentrifuge tube was gently blown to completely separate the exosomes from the wall of the ultracentrifuge tube. The suspension was collected and mixed in a centrifuge tube and finally stored at -80°C. The electron microscopy image of the exosomes is shown in Figure 2. Figure 2 shown.

[0057] Example 4

[0058] This embodiment provides examples of preparing different 3D printing inks.

[0059] At room temperature, methacryloyl gelatin (GelMA), polyethylene glycol diacrylate (PEGDA), photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) and mesenchymal stem cell exosomes (EXO) were mixed evenly in different proportions. The experimental group settings are shown in Table 1.

[0060] Table 1 3D printing inks with different components Experimental Grouping GelMA PEGDA EXO LAP A 10％ —— 0.2％ 0.25％ B 10％ 5％ 0.2％ 0.25％ C 10％ 10％ 0.2％ 0.25％ D 10％ 10％ —— 0.25％

[0061] Groups A, B, and C in Table 1 were designed to explore the mechanical properties, swelling properties, and degradation properties of 3D printing inks with different components. Groups C and D were designed to explore the synergistic effects of the hydrogel components and exosomes in 3D printing inks.

[0062] Example 5

[0063] This embodiment provides an example of a method for 3D printing a hydrogel scaffold and performance verification of the printed hydrogel scaffold.

[0064] The bio-ink prepared according to the different proportions in Table 1 of Example 4 was loaded into a 10 mL syringe. The syringe was then placed in a 4°C refrigerator for about 15 minutes to allow the ink to become a gel state. A 24G needle was loaded, the pressure was about 3.5 Bar, the speed was about 1.5 mm / s, the needle and barrel temperature was about 25°C, and printing was performed according to the programmed computer-aided design (CAD) model. After printing, the hydrogel was cross-linked by UV light, the cross-linking time was 3 minutes, and the power of the UV lamp was 25 mW / cm 2 , wavelength is 405nm.

[0065] The hydrogels printed by 3D printing inks formulated with different components are shown in Figure 2. Figure 3 shown.

[0066] from Figure 3 It can be seen that groups A, B, and C can print out sheet-shaped hydrogel scaffolds (hereinafter referred to as scaffolds) as a whole, but careful observation shows that the scaffolds of groups B and C are better than those of group A. Specifically, the hydrogel scaffolds of groups B and C basically maintain the initial printing morphology, and the rectangular shape is relatively regular with clear boundaries, indicating that the addition of PEGDA improves the formability of the bio-ink. In particular, in group C, the hydrogel scaffold not only has a clear outline, but also has a regular arrangement of microporous structures, uniform pores, and micropores that penetrate the hydrogel scaffold; although the arrangement of the microporous structure can also be seen on the hydrogel scaffold of group B, the pores obviously do not penetrate the hydrogel scaffold; this shows that the ratio of GelMA and PEGDA is crucial for the formation of special structures.

[0067] Further, from Figure 4 It can be seen that the internal structure of the hydrogel scaffold printed by group C is arranged in uniform layers, with uniform pores and interconnected pores. This is crucial for cell adhesion, material exchange and metabolism, and is also conducive to the interaction between exosomes loaded in the hydrogel scaffold and cells.

[0068] This example also provides verification of other properties of the hydrogel scaffold.

[0069] Mechanical tests were performed on the hydrogel scaffolds of groups A, B, and C: a universal testing machine equipped with a 500N load cell was used to perform compression performance tests at a compression rate of 1 mm / min. The linear elastic region (3% to 15%) of the compression curve was used to calculate the compression modulus of the hydrogel scaffold. Figure 5It can be seen that the addition of PEGDA can significantly improve the mechanical properties of the hydrogel, and the mechanical properties increase with the increase of the PEGDA content. The compression modulus of group C can reach 1080 kPa, which shows that the hydrogel scaffold can be used for soft tissue filling and can provide higher mechanical support for the soft tissue at the filling site. The specific results are shown in Table 2.

[0070] Table 2 Mechanical properties of 3D printed scaffolds with different components

[0071] The swelling performance of the three groups of hydrogel scaffolds A, B, and C was further tested: the hydrogel scaffolds of different groups were weighed, immersed in deionized water, and then taken out after 4, 8, 12, and 16 hours, respectively. The surface moisture was absorbed with filter paper and the mass was measured. The swelling rate was calculated using the increased mass ratio. The specific results are shown in Table 3 and Figure 6 shown.

[0072] Table 3 Swelling properties of 3D printed scaffolds with different components

[0073] From the data of Group A and Group B, it can be seen that the addition of PEGDA has improved the swelling rate of the hydrogel scaffold to a certain extent. The swelling of the hydrogel scaffold to a certain extent can increase the tightness with the filled cavity, and can even play a role in compression and hemostasis. However, if the swelling rate is too large, the overall water content of the material will be too high, which will cause the predetermined shape of the material (i.e., the shape of a sheet with a uniform microporous structure) to collapse, resulting in the inability of the surrounding tissue to be immersed in the cavity of the scaffold even if it is filled into the cavity, thus affecting the recovery effect. It further shows that the ratio of GelMA and PEGDA is also very important for the performance of special structures.

[0074] The degradation performance of the three groups of hydrogel scaffolds A, B, and C was further tested: the freeze-dried hydrogel scaffolds of different groups were weighed and placed in collagenase solution, and then transferred to a constant temperature air oscillation box (37°C). They were taken out after 7, 14, 21, and 28 days, respectively, rinsed with deionized water, freeze-dried, and weighed to calculate their degradation rates. The results are shown in Figure 7 .

[0075] from Figure 7 It can be seen that the degradation rates of the three groups of hydrogels have increased to varying degrees, indicating that this material can be degraded under the action of enzymes. With the addition of PEGADA, the degradation rate of the hydrogel scaffold gradually decreased; but hydrogels that degrade too quickly cannot provide sufficient support for soft tissue growth. Therefore, adjusting the degradation rate of the hydrogel scaffold by adjusting the ratio of GelMA to PEGDA is of great significance for the effective filling and repair of soft tissue.

[0076] Furthermore, the in vitro cell adhesion performance of mouse fibroblasts (L929) was tested on the hydrogel scaffolds of groups C and D: the hydrogel scaffolds of groups C and D were placed in 24-well plates at 5×10 4 L929 cells were inoculated at a concentration of 10 cells / well and cultured for 3 days before live and dead cell staining. Figure 8 It can be seen that compared with group D, group C has more live cells (green), fewer dead cells (red), and a fuller scaffold structure. Figure 8 The fluorescence shape shows that the cells in group C have a higher degree of spreading, which indicates that the porous structure of the hydrogel increases the number of contacts between EXO and cells, and the addition of EXO is more conducive to cell adhesion and proliferation, playing a synergistic role.

[0077] Furthermore, the cytotoxicity test of mouse fibroblasts (L929) was performed on the hydrogel scaffolds of groups C and D: the hydrogel scaffolds of groups C and D were placed in 96-well plates at 2×10 4 L929 cells were inoculated at a concentration of 10 cells / well. After 1, 3, and 7 days of co-culture, cells without hydrogel scaffolds were used as the control group. The cells were stained with CCK-8 method, and the absorbance at 450 nm was tested with a microplate reader. The cell survival rate was calculated by the absorbance ratio. Fig. 9 As shown, the cell survival rate of the hydrogel scaffold group was above 90% compared with the control group, and the cell survival rate of each group increased significantly with the extension of culture time. This shows that the structure of this hydrogel scaffold promotes cell adhesion, material exchange and metabolism. Compared with group D, the cell survival rate of group C increased significantly, which further shows that the addition of EXO has the effect of cell adhesion and proliferation, making the hydrogel scaffold biologically active.

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that several improvements and modifications may be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention and do not deviate from the scope defined by the claims of the present invention.

[0079] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A method for preparing a 3D printed hydrogel scaffold with biological activity, characterized in that: The following steps are involved: S01: Extraction of mesenchymal stem cell exosomes; S02: preparing a 3D printing ink, wherein the 3D printing ink comprises a hydrogel matrix and the mesenchymal stem cell exosomes; the hydrogel matrix comprises 2%-20% w / v of methacryloyl gelatin, 2%-20% w / v of polyethylene glycol diacrylate and 0.1%-0.5% w / v of a photoinitiator; the methacryloyl gelatin and polyethylene glycol diacrylate are used in a ratio of 1:0.5-1; the 3D printing ink further comprises 0.05-0.3% w / v of the mesenchymal stem cell exosomes; S03: designing a 3D printed hydrogel scaffold model, wherein the hydrogel scaffold model has a uniformly arranged microporous structure; S04: Perform 3D printing and perform UV cross-linking on the printed hydrogel scaffold.

2. The preparation method according to claim 1, characterized in that The usage ratio of the methacrylated gelatin and polyethylene glycol diacrylate is 1:

1.

3. The preparation method according to claim 1, characterized in that: The photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

4. The preparation method according to claim 1, characterized in that: The extraction method of the mesenchymal stem cell exosomes includes differential centrifugation; including centrifugation at 300-500g for 5-10 minutes, centrifugation at 1000-5000g for 5-10 minutes, centrifugation at 8000-10000g for 5-10 minutes, and centrifugation at 100000-120000g for 50-70 minutes.

5. The preparation method according to claim 1, characterized in that: The S03 uses extrusion 3D printing technology, including printing at 1.5-3.0 mm / s under 3-4 Bar conditions.

6. The preparation method according to claim 1, characterized in that: The 3D printing ink also includes 0.2% w / v of the mesenchymal stem cell exosomes.

7. The preparation method according to claim 1, characterized in that: The methacrylated gelatin is a mixture of methacrylic anhydride and gelatin PBS solution; the volume ratio of the gelatin PBS solution to the methacrylic anhydride is 25:

1.

8. A 3D printed hydrogel support prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the preparation method according to any one of claims 1 to 7 in preparing materials for soft tissue defects.

10. Use of the 3D printed hydrogel scaffold according to claim 8 in preparing soft tissue defect materials.

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