Preparation method of collagen scaffold with gradient structure and loaded with bioactive components

Abstract

The invention relates to a preparation method of a collagen scaffold with a gradient structure and loaded with bioactive components, which comprises the following steps: firstly, fully soaking half of a pure collagen scaffold in a sodium alginate solution, then putting half of the pure collagen scaffold in a calcium chloride solution, and carrying out cross-linking reaction until half of the pure collagen scaffold carries calcium alginate; the collagen scaffold with the calcium alginate is obtained; then completely soaking the collagen scaffold with the calcium alginate in a solution containing a bioactive component, and fully soaking under a low temperature condition, so that the bioactive component is loaded on the other half of the pure collagen scaffold to obtain a primary scaffold; finally, the primary scaffold is placed in a citric acid solution and fully soaked to remove calcium alginate, washing and drying are conducted, and the collagen scaffold with the gradient structure and loaded with the bioactive components is obtained.The collagen scaffold prepared through the method is of a gradual integrated gradient structure without obvious layering in the middle, has good mechanical performance and is good in repairing effect.

Description

Technical Field

[0001] The present invention relates to the technical field of collagen scaffolds, and in particular to a method for preparing a collagen scaffold with a gradient structure and loaded with bioactive components. Background Art

[0002] Collagen, as a core component of the extracellular matrix (ECM), is widely used in the preparation of tissue repair scaffolds such as bone, cartilage, and rotator cuff due to its excellent biocompatibility and bioactivity. This repair scaffold can also be called a collagen scaffold.

[0003] In the existing technology, the commonly used traditional collagen scaffolds are mostly homogenized structures, which are difficult to match such physiological gradient requirements (the structure of natural bone cartilage and rotator cuff tissue often has no obvious stratification in cross section and a gradual gradient feature). There are also some layered composite scaffolds. Although these scaffolds try to simulate gradient structures, the interlayer interface bonding is weak, and slippage is likely to occur between layers, resulting in structural instability, stratification, and mechanical mismatch or shedding problems. The overall mechanical imbalance of the scaffold affects the developmental phenotype of cells in the defect, which is not conducive to tissue regeneration and repair. In addition, a single physical structure is difficult to meet the diverse needs of cell differentiation and migration in the microenvironment of different regions of the gradient structure tissue, which seriously limits the repair effect. Summary of the Invention

[0004] The present invention aims to solve the technical problems of mechanical mismatch and poor repair effect in the prior art and provides a method for preparing a collagen scaffold with stable structure, good repair effect and gradient structure and loaded with bioactive components.

[0005] The object of the present invention is achieved by a method for preparing a collagen scaffold with a gradient structure and loaded with bioactive components, comprising the following steps: (1) Preparation of collagen scaffold with calcium alginate: First, half of the pure collagen scaffold is fully immersed in sodium alginate solution, and then half of the pure collagen scaffold is placed in calcium chloride solution for cross-linking reaction. Half of the pure collagen scaffold is coated with calcium alginate, thus obtaining a collagen scaffold with calcium alginate; (2) Preparing a primary scaffold: The collagen scaffold with calcium alginate prepared in step (1) is completely immersed in a solution containing a bioactive component, and is fully immersed under low temperature conditions so that the other half of the pure collagen scaffold is loaded with the bioactive component, that is, a collagen scaffold with half of calcium alginate and the other half loaded with the bioactive component is obtained, that is, a primary scaffold; (3) Preparing a collagen scaffold with a gradient structure and loaded with bioactive components: placing the primary scaffold prepared in step (3) in a citric acid solution, soaking it thoroughly to remove calcium alginate, washing it, and drying it to obtain a collagen scaffold with a gradient structure and loaded with bioactive components.

[0006] Furthermore, step (1) specifically includes the following steps: (1.1) Weigh sodium alginate and mix it with deionized water or ultrapure water to prepare a 1-5% w / v sodium alginate solution. Soak half of the pure collagen scaffold in the sodium alginate solution for 4-10 minutes. (1.2) Soak half of the pure collagen scaffold soaked in sodium alginate in step (1.1) in 80-400 mM calcium chloride solution and allow the cross-linking reaction to proceed for 3-10 minutes to obtain a collagen scaffold with calcium alginate for later use.

[0007] Furthermore, in step (2), the concentration of the bioactive component solution is 0.01 to 0.3 g / ml, and the collagen scaffold with calcium alginate prepared in step (1) is completely immersed in the bioactive component solution, and ultrasonically oscillated at 20 to 25 kHz for 20 to 60 minutes at a low temperature of 0 to 15°C to obtain a primary scaffold; the bioactive component is a drug-like substance for repairing tissue or a biomimetic tissue material-like substance.

[0008] Furthermore, the drug substance for repairing tissue is sodium alendronate or glucosamine, and the bionic tissue material substance is nano-hydroxyapatite.

[0009] Furthermore, in step (2), the concentration of the bioactive component solution is 1 to 50 ug / ml, and the collagen scaffold with calcium alginate prepared in step (1) is completely immersed in the bioactive component solution and immersed in a low temperature environment of 1 to 5°C for 8 to 24 hours to obtain a primary scaffold; the bioactive component is one of an active protein substance, an anti-inflammatory active substance, and an antibacterial active substance.

[0010] Furthermore, the active protein substance is a growth factor, the anti-inflammatory active substance is curcumin, and the antibacterial active substance is penicillin or gentamicin.

[0011] Furthermore, step (3) specifically includes the following steps: (3.1) Soak the half of the primary scaffold with calcium alginate in step (2) in a 20-200 mM citric acid solution at a temperature of 4-25°C for 10-30 min to remove the calcium alginate and obtain a collagen scaffold loaded with bioactive components; (3.2) The bioactive component-loaded collagen scaffold obtained in step (3.1) is washed 1 to 2 times with deionized water or ultrapure water, and freeze-dried to obtain a clean bioactive component-loaded collagen scaffold with a gradient structure.

[0012] Furthermore, in step (1), the pure collagen scaffold can be obtained from collagen-containing tissues in the human body or collagen-containing tissues in animals.

[0013] Furthermore, in step (3.2), the freeze-drying conditions are -54 to -62°C and the air pressure is 0.1 mbar.

[0014] The present invention also provides the use of the collagen scaffold with a gradient structure and loaded with bioactive components in preparing at least one tissue regeneration and repair product among bone repair patches, cartilage repair patches, skin patches, rotator cuff patches, corneal patches, and vascular wall patches.

[0015] The preparation method of the collagen scaffold with a gradient structure and loaded with bioactive components provided by the present invention utilizes the cross-linking principle that sodium alginate and calcium chloride react to form calcium alginate, so that half of the collagen scaffold is loaded with calcium alginate. Then, when the entire collagen scaffold is immersed in a solution containing the bioactive components, the half of the collagen scaffold with calcium alginate is prevented from entering the collagen scaffold due to the barrier effect of calcium alginate, while the half of the collagen scaffold without calcium alginate can be loaded with the bioactive components. Finally, the principle that the binding force of calcium ions of calcium alginate with citric acid is higher than that of alginic acid is utilized to gently remove the calcium alginate without causing protein denaturation in the collagen scaffold, and the removal process is safe and controllable. The collagen scaffold loaded with bioactive components and having a gradient structure prepared by the present invention has good biocompatibility. The bioactive components present a progressive, integrated gradient structure in the collagen scaffold without obvious stratification in the middle. This gradient structure can meet the diverse needs of cell differentiation and migration in the microenvironment of different regions between biological tissues, and improve the tissue repair effect. The present invention combines the excellent biocompatibility, weak antigenicity and biomimetic extracellular matrix structure of collagen with the signal transduction function of specific bioactive molecules, thereby creating a functional regenerative medicine material with good mechanical properties, interlayer interface bonding and repair effect. It can be used to construct tissue scaffold repair materials for different application scenarios and applied in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a flow chart for preparing a collagen scaffold with a gradient structure and loaded with bioactive components according to the present invention.

[0017] Figure 2 These are Alizarin red stained images of the internal cross-sectional slices of the products prepared by the two methods of Comparative Example 1 and Example 1.

[0018] Figure 3 These are appearance pictures of the pure collagen scaffold prepared in comparative example 2 and the collagen scaffold loaded with nano-hydroxyapatite and having a gradient structure prepared in example 1.

[0019] Figure 4 These are stress-strain curves of the pure collagen scaffold prepared in Comparative Example 2 and the nano-hydroxyapatite-loaded collagen scaffold with a gradient structure prepared in Example 1.

[0020] Figure 5 These are cytotoxicity test diagrams of the pure collagen scaffold prepared in Comparative Example 2 and the nano-hydroxyapatite-loaded collagen scaffold with a gradient structure prepared in Example 1.

[0021] Figure 6 These are cell live-death staining images of the pure collagen scaffold prepared in Comparative Example 2 and the nano-hydroxyapatite-loaded collagen scaffold with a gradient structure prepared in Example 1. DETAILED DESCRIPTION

[0022] The present invention is further analyzed, explained and compared through specific examples and comparative examples.

[0023] Example 1 (1) In this embodiment, the bioactive component is selected from biomimetic tissue materials, and the biomimetic tissue material is preferably nanohydroxyapatite. 0.15g of sodium alginate is weighed and dissolved in 10ml of deionized water at 37℃ to prepare a 1.5% w / v sodium alginate solution. Human tissues can be selected from umbilical cord, skin, cartilage, bone, tendon, and nucleus pulposus. In this embodiment, umbilical cord tissue donated by healthy pregnant women is preferred. After removing arteries and veins, cleaning blood and dirt, removing antigens and inactivating viruses, and freeze-drying, the collagen scaffold is obtained. Half of the collagen scaffold is soaked in the sodium alginate solution for 5 minutes; 0.113g of calcium chloride is weighed and dissolved in 10ml of deionized water to prepare a 102mM calcium chloride solution. Immerse half of the collagen scaffold in step (1) in 102 mM calcium chloride solution, ensuring that the part of the collagen scaffold soaked in sodium alginate is just immersed in the calcium chloride solution, and perform ionic crosslinking reaction for 5 minutes. After calcium chloride and sodium alginate are crosslinked, calcium alginate is formed. Half of the collagen scaffold is covered with calcium alginate (which can be regarded as a calcium alginate barrier layer), and a collagen scaffold with calcium alginate is obtained.

[0024] (2) The bioactive component solution is a nanohydroxyapatite solution. 2.5 g of the bioactive component nanohydroxyapatite is weighed and dissolved in 10 ml of deionized water to prepare a 25% w / v nanohydroxyapatite solution. The collagen scaffold with calcium alginate prepared in step (1) is then completely immersed in the nanohydroxyapatite solution. The high-frequency oscillation mode of an ultrasonic cell disruptor is used to oscillate the hydroxyapatite into the other half of the collagen scaffold. The instrument ultrasonic frequency is 20 kHz, the ultrasonic time is 40 min, and the temperature is 15 ° C. Thus, a collagen scaffold with half of the collagen scaffold containing calcium alginate and the other half loaded with bioactive components is obtained, i.e., a primary scaffold.

[0025] (3) Weigh 0.142 g of sodium citrate and dissolve it in 10 ml of deionized water to prepare a 55 mM sodium citrate solution. After the ultrasonic loading of nanohydroxyapatite is completed, soak the primary scaffold prepared in step (2) in a 102 mM citric acid solution at a temperature of 25°C for 20 min. After the calcium alginate in the primary scaffold is removed by the sodium citrate solution, wash it twice with deionized water or ultrapure water, and then place the prepared collagen scaffold in a -80°C refrigerator for half an hour and then place it in a freeze dryer for freeze drying to obtain a collagen scaffold loaded with nanohydroxyapatite with a gradient structure (half of the collagen scaffold is empty and the other half is loaded with nanohydroxyapatite).

[0026] Example 2 The difference between this example and Example 1 is that the concentration of the prepared sodium alginate solution is 2% w / v, half of the collagen scaffold is immersed in the sodium alginate solution for 6 minutes, and a 120 mM calcium chloride solution is prepared to cross-link the 2% w / v sodium alginate to form calcium alginate. The cross-linking time is 5 minutes, resulting in a collagen scaffold with calcium alginate. The collagen scaffold with calcium alginate is then immersed in a 25% w / v nanohydroxyapatite solution and sonicated for 45 minutes to obtain a primary scaffold. Finally, the primary scaffold is immersed in an 80 mM sodium citrate solution for 20 minutes. The various conditions of the other experimental steps remain unchanged, and a collagen scaffold with a gradient structure loaded with nanohydroxyapatite is obtained.

[0027] Example 3 This example differs from Example 1 in that the sodium alginate solution prepared was at a 4% w / v concentration. Half of the collagen scaffold was immersed in the sodium alginate solution for 8 minutes. A 150 mM calcium chloride solution was then prepared to crosslink the 4% w / v sodium alginate to form calcium alginate. The crosslinking time was 6 minutes. This resulted in a collagen scaffold with calcium alginate. The calcium alginate-loaded collagen scaffold was then immersed in a 30% w / v nanohydroxyapatite solution and sonicated at 0°C for 60 minutes. The calcium alginate barrier layer was then immersed in a 100 mM sodium citrate solution for 25 minutes. All other experimental conditions remained unchanged, resulting in a nanohydroxyapatite-loaded collagen scaffold with a gradient structure.

[0028] Example 4 This example differs from Example 1 in that the sodium alginate solution prepared was 5% w / v. Half of the collagen scaffold was immersed in the sodium alginate solution for 10 minutes. A 200 mM calcium chloride solution was then prepared to crosslink the 5% w / v sodium alginate to form calcium alginate. The crosslinking time was 10 minutes. This resulted in a collagen scaffold with calcium alginate. The calcium alginate-loaded collagen scaffold was then immersed in a 10% w / v nanohydroxyapatite solution and sonicated for 30 minutes. The calcium alginate barrier layer was then immersed in a 200 mM sodium citrate solution for 15 minutes. All other experimental conditions remained unchanged, resulting in a nanohydroxyapatite-loaded collagen scaffold with a gradient structure.

[0029] Example 5 The difference between this embodiment and embodiment 1 is that in step (2), the bioactive component is selected from active protein substances, preferably BMP-2 growth factor. 100 μg of BMP-2 growth factor is dissolved in 10 ml of 1% w / v sterilized bovine serum albumin solution, and the collagen scaffold with calcium alginate is immersed in the bovine serum albumin solution. The immersion temperature is 4°C for 12 h, and the conditions of the other experimental steps remain unchanged, thereby obtaining the gradient biomimetic collagen scaffold loaded with growth factors of the present invention.

[0030] Example 6 This example differs from Example 5 in that 200 μg of BMP-2 growth factor was dissolved in 10 ml of a 1% w / v sterilized bovine serum albumin solution. The lower half of the collagen scaffold with a calcium alginate barrier layer was immersed in the bovine serum albumin solution for 24 hours at a temperature of 4°C. All other experimental conditions remained unchanged, resulting in the growth factor-loaded gradient biomimetic collagen scaffold of the present invention.

[0031] Comparative Example 1 This comparative example differs from Example 1 in that step (1) is omitted, and a calcium alginate barrier layer is not prepared on the collagen scaffold. Instead, the collagen scaffold without calcium alginate is directly placed in a 25% w / v nanohydroxyapatite solution. Using the high-frequency oscillation mode of an ultrasonic cell disruptor, the nanohydroxyapatite is oscillated into the collagen scaffold, and the entire collagen scaffold is completely loaded with nanohydroxyapatite, resulting in a collagen scaffold fully loaded with nanohydroxyapatite.

[0032] Comparative Example 2 Umbilical cords of perinatal tissue donated by healthy pregnant women were selected, and after removing arteries and veins, cleaning blood and dirt, removing antigens and inactivating viruses, the collagen scaffold was freeze-dried to obtain a pure collagen scaffold.

[0033] Test Example 1 The main content of this test example is to perform staining and section characterization on the collagen scaffold fully loaded with nanohydroxyapatite (control group), the product of Example 1, and the collagen scaffold loaded with nanohydroxyapatite with a gradient structure (experimental group), the product of Example 1, to observe the internal structures of the two.

[0034] The products of Comparative Example 1 and Example 1 were equilibrated with sucrose in a gradient sucrose solution, embedded in an embedding agent, and cut into 7 μm thick sections using a cryostat. The sample sections were then mounted on glass slides. Alizarin red stain was then added dropwise to the slide where the sample section was located and allowed to stain for 5 minutes. The sections were then thoroughly washed with distilled water to remove any excess color, dehydrated with a gradient of ethanol, transparentized with xylene, and mounted with neutral gum. After mounting, the alizarin red-stained sections were placed under a microscope for observation.

[0035] like Figure 2 The internal cross-sectional slices of the products prepared by the two methods of Comparative Example 1 and Example 1 are stained with Alizarin red (scale bar = 200 μm). Figure 2 As shown, the internal cross-sectional image of the collagen scaffold of the experimental group (the collagen scaffold loaded with nanohydroxyapatite having a gradient structure obtained in Example 1) shows the lower half of the scaffold with a uniform distribution of nanohydroxyapatite gradient. It can be seen that the internal cross-section is progressive, with no obvious stratification in the middle, forming a good gradient structure, while the hydroxyapatite in the collagen scaffold in the control group is randomly distributed inside the scaffold and is obviously stratified.

[0036] Test Example 2 In the static mechanical test, a universal testing machine was used to test the compression resistance of the collagen scaffold. The compression speed was kept constant at 0.2 mm / min, and the stress-strain curves of the two collagen scaffolds were finally obtained.

[0037] Figure 3 The photographs show the appearance of the pure collagen scaffold prepared in Comparative Example 2 and the nano-hydroxyapatite loaded collagen scaffold with a gradient structure prepared in Example 1.

[0038] Figure 4 This is a comparison chart of the mechanical tests of the pure collagen scaffold prepared in comparative example 2 and the collagen scaffold loaded with nano-hydroxyapatite and having a gradient structure prepared in example 1. Compared with the pure collagen scaffold, the collagen scaffold loaded with nano-hydroxyapatite and having a gradient structure has stronger mechanical properties.

[0039] Test Example 3 In order to verify the cell compatibility of the pure collagen scaffold prepared in Comparative Example 2 and the collagen scaffold loaded with nanohydroxyapatite having a gradient structure obtained in Example 1, the extraction method was used for testing. Specific steps: soak the two collagen scaffolds in penicillin-streptomycin (PS) and irradiate under ultraviolet light, and then wash three times with ultrapure water. Subsequently, the two collagen scaffolds were soaked in DMEM culture medium supplemented with 10% fetal bovine serum and 1% PS for 24 hours. The "no scaffold group" group refers to the positive control group of cells cultured in ordinary culture medium that has not been soaked in the scaffold. Rat bone marrow mesenchymal stem cells were cultured at a rate of 2×10 per well. 4 The cells were seeded in a 24-well plate at a density of .3 groups were set up and marked as Group 1, 2 and 3 respectively. Normal culture medium was added to the wells of Group 1 for culture. Culture medium soaked in the pure collagen scaffold prepared in Comparative Example 2 was added to the wells of Group 2, and culture medium soaked in the collagen scaffold loaded with nanohydroxyapatite with a gradient structure prepared in Example 1 was added to the wells of Group 3. After continued culture for a period of time (1, 3, 5 days), the cell proliferation was detected using a cell counting kit (CCK-8). Subsequently, the cells were washed with PBS. The live-dead staining solution (Calcein / PI cell activity detection reagent) was added to the well plates of the above three groups and incubated at 37°C for 30 minutes. Living cells were observed under a fluorescence microscope, and fluorescence showed that the living cells were stained green (corresponding to Figure 6 mid-white highlight).

[0040] like Figure 5 As shown, WST water-soluble tetrazolium salt colorimetric reagent was used to detect cell viability test. From the results, it can be seen that the pure collagen scaffold prepared in Comparative Example 2 and the collagen scaffold loaded with nano-hydroxyapatite with a gradient structure obtained in Example 1 did not leak toxic side effects, had no inhibitory effect on cell growth, and had good biocompatibility.

[0041] Figure 6 The live-death staining images of the pure collagen scaffold prepared in comparative example 2 and the gradient-structured nano-hydroxyapatite-loaded collagen scaffold prepared in example 1 (scale bar = 100 μm) show that the cells in the pure collagen scaffold and the gradient-structured hydroxyapatite-loaded collagen groups are similar to the cells cultured in normal culture medium (without scaffold). The cells grow well and basically show green fluorescence (corresponding to Figure 6 The bright white area in the middle shows that the modification of the scaffold will not affect the normal growth of cells.

Claims

1. A method for preparing a collagen scaffold with a gradient structure and loaded with bioactive components, comprising the following steps: (1) Preparation of collagen scaffold with calcium alginate: First, half of the pure collagen scaffold is fully immersed in sodium alginate solution, and then half of the pure collagen scaffold is placed in calcium chloride solution for cross-linking reaction. Half of the pure collagen scaffold is coated with calcium alginate, thus obtaining a collagen scaffold with calcium alginate; (2) Preparing a primary scaffold: The collagen scaffold with calcium alginate prepared in step (1) is completely immersed in a solution containing a bioactive component, and is fully immersed under low temperature conditions so that the other half of the pure collagen scaffold is loaded with the bioactive component, that is, a collagen scaffold with half of calcium alginate and the other half loaded with the bioactive component is obtained, that is, a primary scaffold; (3) Preparing a collagen scaffold with a gradient structure and loaded with bioactive components: placing the primary scaffold prepared in step (3) in a citric acid solution, soaking it thoroughly to remove calcium alginate, washing it, and drying it to obtain a collagen scaffold with a gradient structure and loaded with bioactive components.

2. The method for preparing a collagen scaffold with a gradient structure and loaded with bioactive components according to claim 1, characterized in that: Step (1) specifically includes the following steps: (1.1) Weigh sodium alginate and mix it with deionized water or ultrapure water to prepare a 1-5% w / v sodium alginate solution. Soak half of the pure collagen scaffold in the sodium alginate solution for 4-10 minutes. (1.2) Soak half of the pure collagen scaffold soaked in sodium alginate in step (1.1) in 80-400 mM calcium chloride solution and allow the cross-linking reaction to proceed for 3-10 minutes to obtain a collagen scaffold with calcium alginate for later use.

3. The method for preparing a collagen scaffold with a gradient structure and loaded with bioactive components according to claim 2, characterized in that: In step (2), the concentration of the bioactive component solution is 0.01 to 0.3 g / ml, and the collagen scaffold with calcium alginate prepared in step (1) is completely immersed in the bioactive component solution, and ultrasonically oscillated at 20 to 25 kHz for 20 to 60 minutes at a low temperature of 0 to 15°C to obtain a primary scaffold; the bioactive component is a drug-like substance for repairing tissue or a biomimetic tissue material-like substance.

4. The method for preparing a collagen scaffold with a gradient structure and loaded with bioactive components according to claim 3, characterized in that: The drug substance for repairing tissue is sodium alendronate or glucosamine, and the bionic tissue material substance is nano-hydroxyapatite.

5. The method for preparing a collagen scaffold with a gradient structure and loaded with bioactive components according to claim 2, characterized in that: In step (2), the concentration of the bioactive component solution is 1 to 50 ug / ml, and the collagen scaffold with calcium alginate prepared in step (1) is completely immersed in the bioactive component solution and immersed in a low temperature environment of 1 to 5°C for 8 to 24 hours to obtain a primary scaffold; the bioactive component is one of an active protein substance, an anti-inflammatory active substance, and an antibacterial active substance.

6. The method for preparing a collagen scaffold with a gradient structure and loaded with bioactive components according to claim 5, characterized in that: The active protein substance is a growth factor, the anti-inflammatory active substance is curcumin, and the antibacterial active substance is penicillin and gentamicin.

7. The method for preparing a collagen scaffold with a gradient structure and loaded with bioactive components according to claim 3 or 5, characterized in that: Step (3) specifically includes the following steps: (3.1) Soak the half of the primary scaffold with calcium alginate in step (2) in a 20-200 mM citric acid solution at a temperature of 4-25°C for 10-30 min to remove the calcium alginate and obtain a collagen scaffold loaded with bioactive components; (3.2) The bioactive component-loaded collagen scaffold obtained in step (3.1) is washed 1 to 2 times with deionized water or ultrapure water, and freeze-dried to obtain a clean bioactive component-loaded collagen scaffold with a gradient structure.

8. The method for preparing a collagen scaffold with a gradient structure and loaded with bioactive components according to claim 3 or 5, characterized in that: In step (1), the pure collagen scaffold can be obtained from collagen-containing tissues in the human body or collagen-containing tissues in animals.

9. The method for preparing a collagen scaffold with a gradient structure and loaded with bioactive components according to claim 7, characterized in that: In step (3.2), the freeze-drying conditions are -54 to -62°C and the air pressure is 0.1 mbar.

10. Use of the bioactive component-loaded collagen scaffold with a gradient structure according to claim 7 in the preparation of at least one tissue regeneration and repair product selected from the group consisting of a bone repair patch, a cartilage repair patch, a skin patch, a rotator cuff patch, a corneal patch, and a vascular wall patch.

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