A ligament regeneration scaffold with layer-by-layer induction performance and its preparation method

Abstract

The present invention discloses a ligament regeneration scaffold with layer-by-layer induction performance and a preparation method thereof. The ligament regeneration scaffold of the present invention has a multi-layer composite structure, which is composed of a gradient-degraded micron fiber reinforcement layer and an oriented nanofiber induction layer overlapped and composited; wherein, the micron fiber reinforcement layer is woven by yarns with different degradation cycles through multi-dimensional and multi-textile molding, and the nanofiber induction layer is prepared by electrospinning of polymer materials and bioactive ingredients. The preparation method of the present invention comprises: preparing a micron fiber reinforcement layer, preparing a nanofiber induction layer, and then compounding layer by layer to obtain a ligament regeneration scaffold with layer-by-layer induction performance. Based on the regulation of the gradient degradation structure, the present invention realizes the gradient degradation of the micron fiber reinforcement layer while ensuring the initial mechanical strength, exposing the nanofiber induction layer layer by layer, so as to accurately regulate the infiltration and remodeling of the internal tissue of the scaffold, and can be used for ligament / tendon regeneration and reconstruction treatment.

Description

Technical Field

[0001] The present invention relates to a ligament regeneration scaffold with layer-by-layer induction performance and a preparation method thereof, belonging to the technical field of medical devices. Background Art

[0002] The anterior cruciate ligament (ACL) is a crucial ligament connecting the femur and tibia in the knee joint. It originates from the back of the femur, runs obliquely within the joint cavity, and inserts at the front of the tibia. The ACL works together with other ligaments to ensure normal movement and maintain knee stability. In recent years, with the increasing number of people participating in sports, the incidence of sports injuries has increased year by year. Anterior cruciate ligament (ACL) injuries are the most common. However, due to the lack of blood vessels and cells, the ACL is difficult to heal on its own after injury, and surgical reconstruction is often performed clinically.

[0003] There are three main methods for anterior cruciate ligament reconstruction: autologous transplantation, allograft transplantation, and artificial ligament. The advantages of autologous transplantation include improved revascularization of the affected area, a lower immune response, and easy integration with surrounding tissue. However, autologous transplantation cannot guarantee complete restoration of pre-injury functional capacity, and various complications and pain may occur at the donor site. Compared to autologous transplantation, allograft transplantation does not pose donor site complications, can shorten surgical time, and has no size restrictions. However, allograft transplantation is subject to immune rejection and the risk of disease transmission. Given the numerous drawbacks of autologous and allograft transplantation, artificial ligaments are increasingly being considered. Artificial ligaments are ligament substitutes made from natural or synthetic polymers through certain molding processes. They can be used to replace or repair damaged ligaments. Compared to autologous and allograft transplantation, artificial ligaments offer advantages such as no donor complications, no immune rejection, and no risk of disease transmission, making them a research hotspot in recent years.

[0004] Most artificial ligaments used clinically are made from non-degradable materials. Although these grafts have sufficient initial tensile strength and may have a good recovery effect in short-term use, they may encounter some problems in long-term use. Tissue has difficulty growing into the interior of the artificial ligament. Even after several months, collagen fibers can only infiltrate the first two layers of the multi-layered graft. Some collagen fibers can penetrate the interior of the artificial ligament, but they cannot form an orderly organization and arrangement, forming disordered scar tissue, which leads to poor development of the new ligament.

[0005] Therefore, in recent years, degradable artificial ligaments have become a research hotspot. For example, the invention patent with authorization number CN104043151B provides a composite artificial ligament and its preparation method. By winding several filament bundles with different degradation properties into wires, then weaving them into a mesh structure, and finally rolling them into an artificial ligament with a rod-like structure, it has good mechanical properties and can promote the attachment of surrounding tissues and the repair and normal growth of ligament tissues. At the same time, some components in the body can be gradually biodegraded without causing a significant decrease in the mechanical properties of the ligament. Although this patent uses gradient degradation space to guide tissue growth, after the tissue grows in, it cannot induce collagen arrangement through contact guidance, and cannot ensure the oriented growth of collagen and tissue after implantation. The invention patent with authorization number CN108653811B provides an artificial ligament and its preparation method. Electrospinning technology is used to spin on the surface of an artificial ligament semi-finished product to form a collagen nanofiber layer to obtain an artificial ligament. The collagen nanofiber layer on the outer surface of this patent can mimic the extracellular matrix and increase the biocompatibility of the artificial ligament. However, it does not form a collagen nanofiber layer inside the ligament scaffold and cannot provide a sustained induction effect after tissue ingrowth. At present, invention patents for artificial ligaments mostly focus on the problem of tissue infiltration into the scaffold, but cannot achieve the orderly arrangement of tissue and collagen after tissue infiltration. Or they focus on the problem of orderly arrangement of tissue and collagen on the surface of the scaffold, but cannot guarantee tissue and collagen infiltration, and even if they can infiltrate, they cannot ensure that they will remain in an orderly arrangement inside the scaffold. Summary of the Invention

[0006] The technical problem to be solved by the present invention is that the existing artificial ligament cannot simultaneously ensure that tissue and collagen infiltrate the interior of the scaffold and maintain orderly arrangement inside the scaffold.

[0007] In order to solve the above technical problems, the present invention provides a ligament regeneration scaffold with layer-by-layer induction performance, wherein the ligament regeneration scaffold has a multi-layer composite structure, which is composed of a micron fiber reinforcement layer whose degradation cycle increases layer by layer from the outside to the inside and an oriented nanofiber induction layer overlapped and composited; wherein, the micron fiber reinforcement layer is woven by yarns with different degradation cycles through multi-dimensional and multi-textile molding, and the nanofiber induction layer is prepared by electrospinning a composite spinning solution with the micron fiber reinforcement layer as the receiving base, and the composite spinning solution contains polymer materials and bioactive components.

[0008] Preferably, the ligament regeneration scaffold has a composite structure of 3 to 30 layers.

[0009] Preferably, the yarns with different degradation cycles are selected from at least two of silk, PGA, PLGA, PPDO, PCL, PLA and P4HB; and the yarns are monofilaments, multifilaments, twisted yarns or braided yarns.

[0010] Preferably, the polymer material is at least one of P4HB, PHBV, PHA, P(LLA-CL), PPDO, PLGA, PGA, PEG, PGCL, PCL and PLA; and the bioactive ingredient is at least one of tilapia collagen, bovine Achilles tendon collagen, porcine collagen, gelatin, silk protein, fibrin, elastin, chitosan, alginate and hyaluronic acid.

[0011] The present invention also provides a method for preparing the above-mentioned ligament regeneration scaffold with layer-by-layer induction performance, comprising the following steps:

[0012] Step 1: selecting yarns with different degradation cycles, determining the scaffold structure, and weaving by machine weaving, knitting, or braiding to prepare multiple groups of micron fiber reinforcement layers, wherein the degradation cycles of the multiple groups of micron fiber reinforcement layers are distributed in a gradient manner;

[0013] Step 2: Cleaning and drying the micron fiber reinforcement layer prepared in step 1;

[0014] Step 3: preparing a composite spinning solution with a polymer material and a bioactive ingredient, and performing electrospinning using the micron fiber reinforcement layer obtained in step 2 as a receiving substrate, preparing a nanofiber induction layer on each group of micron fiber reinforcement layers, and obtaining multiple groups of composite layers consisting of micron fiber reinforcement layers and nanofiber induction layers;

[0015] Step 4: The composite layer obtained in Step 3 is laminated with the microfiber reinforcement layer and the nanofiber induction layer in an overlapping manner, following the degradation cycle of the microfiber reinforcement layer from the outside to the inside, to form a multi-layer composite structure. After cleaning, drying, and sterilization, a ligament regeneration scaffold with layer-by-layer induction performance is obtained. The overlapping and laminating method can be layer by layer.

[0016] Preferably, the cleaning in step 2 is: cleaning with 70-80 wt% ethanol aqueous solution, and the drying temperature is 35-40°C.

[0017] Preferably, the specific preparation method of the composite spinning solution in step 3 is: mixing the polymer material and the bioactive component in a mass ratio of 9:1 to 1:9 and then uniformly dissolving the mixture in a solvent.

[0018] Preferably, the solvent is at least one of hexafluoroisopropanol, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetone, dichloromethane, chloroform, tetrahydrofuran, 1,4-dioxane, methane, trifluoroacetic acid, trifluoroethanol, ultrapure water and aqueous acetic acid solution.

[0019] Preferably, the cleaning method in step 4 is: cleaning with ethanol, and the sterilization method is: sterilization with ethylene oxide.

[0020] Preferably, when weaving is used for weaving in step 1, the micron fiber reinforced layer with the longest degradation cycle is prepared first, and the composite layer composed of a single group of micron fiber reinforced layers and a nanofiber induction layer is prepared in step 3. Step 4 is to weave the next group of micron fiber reinforced layers on the composite layer prepared in step 3, that is, repeat steps 1-3 until the preparation of the ligament regeneration scaffold is completed.

[0021] Among them, the diameter of the yarns with different degradation cycles selected is 0.05-0.5mm; the pore size of the micron fiber reinforcement layer is 10-30μm, and the thickness is 0.1-1.5mm; the pore size of the nanofiber induction layer is 0.5-15μm, and the thickness is 0.005-0.1mm; the length of the prepared ligament regeneration scaffold is 5-70mm, the thickness is 1-10mm, and the width is 1-15mm.

[0022] The present invention's ligament regeneration scaffold with layer-by-layer induction properties utilizes electrospinning technology to create an oriented nanofiber induction layer, inducing the orderly arrangement of cells and collagen. A gradient-degradable micron fiber reinforcement layer is fabricated using multi-dimensional, multi-textile molding and weaving. While maintaining initial mechanical strength, this layer achieves gradient degradation, exposing the nanofiber induction layer layer by layer. The gradient degradation of the micron fiber reinforcement layer allows for the in-growth of tissue and collagen, inducing their infiltration. The oriented arrangement of the nanofiber induction layer provides topographical cues for cell and tissue growth, inducing oriented growth of tissue and collagen within the scaffold. The bioactive components contained in the nanofiber induction layer provide biochemical cues for cell and tissue growth, enhancing their viability and thereby synergistically inducing tissue regeneration.

[0023] The in vitro culture of the ligament regeneration scaffold with layer-by-layer induction performance and the ordinary PET scaffold with fibroblasts showed that more cell proliferation can be observed on the ligament regeneration scaffold with layer-by-layer induction performance, and the cell spreading is good. The cells present an elongated spindle-shaped morphology, the cell viability is increased by 30-70%, and the cell penetration depth inside the scaffold is increased by 20-50%. At the same time, the orderly arrangement of tissues and collagen fibers can be achieved, promoting the remodeling of tissues and collagen fibers.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The ligament regeneration scaffold prepared by the present invention has a layer-by-layer induction performance. The multi-layer structure is composed of a gradient-degradable micron fiber reinforcement layer and an oriented nanofiber induction layer, achieving high initial mechanical strength.

[0026] 2. The present invention prepares a ligament regeneration scaffold with layer-by-layer induction performance. By adjusting the spinning parameters, the nanofibers in the nanofiber induction layer can be oriented, thereby providing contact guidance for cells, promoting cell growth along the fiber orientation direction and the orientation of collagen fibers.

[0027] 3. The ligament regeneration scaffold with layer-by-layer induction performance prepared by the present invention can form a gradient-degradable micron fiber reinforcement layer by adjusting the types and proportions of yarns with different degradation cycles. After implantation, it degrades layer by layer, exposing the surface of the oriented nanofiber induction layer layer by layer, continuously inducing cell growth and its oriented arrangement inside the scaffold. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the structure of the ligament regeneration scaffold with layer-by-layer induction performance of the present invention; I and II respectively represent the oriented nanofiber induction layer and the gradient-degradable microfiber reinforcement layer, wherein 1-a and 1-b represent yarns with different degradation cycles constituting the microfiber reinforcement layer;

[0029] Figure 2 Schematic diagram of the structure of the ligament regeneration scaffold with layer-by-layer induction performance prepared in Example 1 and the micron fiber reinforcement layer prepared by weaving in step 2 of Example 1; 2-a represents a yarn with a long degradation time, and 2-b represents a yarn with a short degradation time;

[0030] Figure 3 Schematic diagram of the structure of the ligament regeneration scaffold with layer-by-layer induction performance prepared in Example 2 and the micron fiber reinforcement layer prepared by knitting in step 2 of Example 2; 3-a represents a yarn with a long degradation time, and 3-b represents a yarn with a short degradation time;

[0031] Figure 4 Schematic diagram of the structure of the ligament regeneration scaffold with layer-by-layer induction performance prepared in Example 3 and the micron fiber reinforcement layer prepared by weaving in step 2 of Example 3; 4-a represents a yarn with a long degradation time, and 4-b represents a yarn with a short degradation time. DETAILED DESCRIPTION

[0032] To make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0033] Example 1

[0034] A method for preparing a ligament regeneration scaffold with layer-by-layer induction performance comprises the following steps:

[0035] Step 1: Silk braided yarn (2-a) and PGA (2-b) monofilament were selected as yarns with different degradation cycles.

[0036] Step 2: Prepare micron fiber reinforcement layer. Determine the scaffold structure and use the weaving method ( Figure 2 ), plain weave was selected, silk was used as warp yarn, silk and PGA were alternately arranged as weft yarn, and two micron fiber reinforced layers (II-1, II-2) with weft yarn ratios of silk:PGA=1:3 and silk:PGA=1:1 were obtained.

[0037] Step 3: Cleaning and Drying: The micron fiber reinforcement layer prepared in step 2 is cleaned with 75% ethanol and then dried at 37°C for later use.

[0038] Step 4: Prepare the nanofiber induction layer (I). Bovine Achilles tendon collagen and PCL were mixed in a mass ratio of 15:85 and uniformly dissolved in hexafluoroisopropanol to obtain a composite spinning solution. Using the two micron fiber reinforcement layers obtained in Step 3 as the receiving substrate, the composite spinning solution was electrospun to produce the nanofiber induction layer. The spinning voltage was 15 kV, the receiving distance was 16 cm, the roller speed was 1600 rpm, the injection rate was 0.8 ml / h, and the spinning time was 1 hour.

[0039] Step 5: The two micron fiber reinforcement layers carrying the nanofiber induction layer are overlapped layer by layer to form a three-layer structure with the upper and lower layers being silk:PGA=1:3 and the middle layer being silk:PGA=1:1. After suturing and reinforcement with silk braided thread, the structure is washed with ethanol and dried, and then sterilized with ethylene oxide to obtain the stent ( Figure 2 ).

[0040] The in vitro culture of the above-prepared ligament regeneration scaffold and ordinary PET scaffold with fibroblasts showed that more cell proliferation could be observed on the ligament regeneration scaffold with layer-by-layer induction performance, and the cells had good spreading properties. The cells showed an elongated spindle-shaped morphology, cell viability increased by 30%, and the cell penetration depth inside the scaffold increased by 40%. At the same time, the orderly arrangement of tissues and collagen fibers could be achieved, promoting the remodeling of tissues and collagen fibers.

[0041] Example 2

[0042] A method for preparing a ligament regeneration scaffold with layer-by-layer induction performance comprises the following steps:

[0043] Step 1: PCL (3-a) monofilament and PLGA (3-b) monofilament were selected as yarns with different degradation cycles.

[0044] Step 2: Prepare micron fiber reinforcement layer. Determine the scaffold structure and use knitting method ( Figure 3), weft plain needle organization, PCL and PLGA interval configuration, weaving on the machine, and obtained three micron fiber reinforced layers (II-1, II-2, II-3) with yarn ratios of PCL:PLGA=1:3, PCL:PLGA=1:2, and PCL:PLGA=1:1 respectively.

[0045] Step 3: Cleaning and Drying: The micron fiber reinforcement layer prepared in step 2 is cleaned with 75% ethanol and then dried at 37°C for later use.

[0046] Step 4: Prepare the nanofiber induction layer (I). Mix gelatin and PGCL in a mass ratio of 30:70 and evenly dissolve in trifluoroethanol to obtain a composite spinning solution. Using the three micron fiber reinforcement layers obtained in Step 3 as a receiving substrate, the composite spinning solution is electrospun to produce the nanofiber induction layer. The spinning voltage is 16 kV, the receiving distance is 14 cm, the roller speed is 1800 rpm, the injection rate is 0.6 ml / h, and the spinning time is 2 hours.

[0047] Step 5: The three micron fiber reinforcements carrying the nanofiber induction layer are overlapped layer by layer to form a five-layer structure with the outermost two layers being PCL:PLGA=1:3, the second outermost two layers being PCL:PLGA=1:2, and the innermost layer being PCL:PLGA=1:1. After being sutured and reinforced with PCL thread, the structure is washed with ethanol and dried, and then sterilized with ethylene oxide to obtain the stent ( Figure 3 ).

[0048] The in vitro culture of the above-prepared ligament regeneration scaffold and ordinary PET scaffold with fibroblasts showed that more cell proliferation could be observed on the ligament regeneration scaffold with layer-by-layer induction performance, and the cells had good spreading properties. The cells showed an elongated spindle-shaped morphology, cell viability was increased by 50%, and the cell penetration depth inside the scaffold was increased by 30%. At the same time, the orderly arrangement of tissues and collagen fibers could be achieved, promoting the remodeling of tissues and collagen fibers.

[0049] Example 3

[0050] A method for preparing a ligament regeneration scaffold with layer-by-layer induction performance comprises the following steps:

[0051] Step 1: PPDO (4-a) monofilament and PGA (4-b) monofilament were selected as yarns with different degradation cycles.

[0052] Step 2: Prepare micron fiber reinforcement layer. Determine the scaffold structure and use weaving method ( Figure 4 ), 12 spindles are woven, PPDO and PGA are arranged at intervals, and the weaving is carried out on the machine to first obtain a micron fiber reinforcement layer (II-3) with PPDO:PGA=1:1.

[0053] Step 3: Cleaning and Drying: The micron fiber reinforcement layer prepared in step 2 is cleaned with 75% ethanol and then dried at 37°C for later use.

[0054] Step 4: Prepare the Nanofiber Inductive Layer (I). Hyaluronic acid and PPDO were mixed in a mass ratio of 20:80 and uniformly dissolved in hexafluoroisopropanol to obtain a composite spinning solution. Using the micronized fiber reinforcement layer obtained in Step 3 as a receiving substrate, the composite spinning solution was electrospun using a solution electrospinning process to produce the nanofiber inductive layer. The spinning voltage was 14 kV, the receiving distance was 16 cm, the roller speed was 1700 rpm, the injection rate was 0.8 ml / h, and the spinning time was 1.5 hours.

[0055] Step 5: The micron fiber reinforced layer with the nanofiber induction layer obtained in step 4 is used as the core yarn and woven with 12 spindles with a spindle ratio of PPDO:PGA=1:2 to obtain a micron fiber reinforced layer (II-2).

[0056] Step 6: Cleaning and Drying: The micron fiber reinforcement layer obtained in step 5 is cleaned with 75% ethanol and then dried at 37°C for later use.

[0057] Step 7: Prepare the nanofiber induction layer (I). Hyaluronic acid and PPDO were mixed in a mass ratio of 20:80 and uniformly dissolved in hexafluoroisopropanol to obtain a composite spinning solution. Using the micronized fiber reinforcement layer obtained in Step 6 as a receiving substrate, the composite spinning solution was electrospun to produce the nanofiber induction layer. The spinning parameters were the same as those described in Step 4.

[0058] Step 8: The micron fiber reinforced layer loaded with the nanofiber induction layer obtained in step 7 is used as the core yarn and woven with 12 spindles with a spindle ratio of PPDO:PGA=1:3 to obtain a micron fiber reinforced layer (II-1).

[0059] Step 9: Cleaning and Drying: The micron fiber reinforcement layer obtained in step 8 is cleaned with 75% ethanol and then dried at 37°C for later use.

[0060] Step 10: Preparation of nanofiber induction layer (I). Hyaluronic acid and PPDO were mixed in a mass ratio of 20:80 and then uniformly dissolved in hexafluoroisopropanol to obtain a composite spinning solution. The micron fiber reinforcement layer obtained in step 9 was used as a receiving substrate. The composite spinning solution was electrospun using a solution electrospinning process to prepare the outermost nanofiber induction layer. The spinning parameters were the same as those described in step 4. After washing with ethanol and drying, the scaffold was sterilized with ethylene oxide to obtain the scaffold ( Figure 4 ).

[0061] The in vitro culture of the above-prepared regenerative ligament scaffold and ordinary PET scaffold with fibroblasts showed that more cell proliferation could be observed on the ligament regeneration scaffold with layer-by-layer induction performance, and the cells had good spreading properties. The cells showed an elongated spindle-shaped morphology, cell viability increased by 40%, and the cell penetration depth inside the scaffold increased by 40%. At the same time, the orderly arrangement of tissues and collagen fibers could be achieved, promoting the remodeling of tissues and collagen fibers.

[0062] The above embodiments are only preferred embodiments of the present invention and are not intended to limit the present invention in any form or substance. It should be noted that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A ligament regeneration scaffold with layer-by-layer induction performance, characterized in that: The ligament regeneration scaffold has a multi-layer composite layer, which is composed of a micron fiber reinforcement layer whose degradation cycle increases layer by layer from the outside to the inside and an oriented nanofiber induction layer overlapped and composited; wherein, the micron fiber reinforcement layer is woven by yarns with different degradation cycles through multi-dimensional and multi-textile molding, and the nanofiber induction layer is prepared by electrostatic spinning of a composite spinning solution with the micron fiber reinforcement layer as a receiving base, and the composite spinning solution contains polymer materials and bioactive components.

2. The ligament regeneration scaffold with layer-by-layer induction performance according to claim 1, characterized in that: The ligament regeneration scaffold has 3 to 30 composite layers.

3. The ligament regeneration scaffold with layer-by-layer induction performance according to claim 1, characterized in that: The yarns with different degradation cycles are selected from at least two of silk, PGA, PLGA, PPDO, PCL, PLA and P4HB; the yarns are monofilaments, multifilaments, twisted yarns or braided yarns.

4. The ligament regeneration scaffold with layer-by-layer induction performance according to claim 1, characterized in that: The polymer material is at least one of P4HB, PHBV, PHA, P(LLA-CL), PPDO, PLGA, PGA, PEG, PGCL, PCL and PLA; the bioactive ingredient is at least one of tilapia collagen, bovine Achilles tendon collagen, porcine collagen, gelatin, silk protein, fibrin, elastin, chitosan, alginate and hyaluronic acid.

5. The method for preparing the ligament regeneration scaffold with layer-by-layer induction performance according to any one of claims 1 to 4, characterized in that: The steps include: Step 1: selecting yarns with different degradation cycles, determining the scaffold structure, and weaving by machine weaving, knitting, or braiding to prepare multiple groups of micron fiber reinforcement layers, wherein the degradation cycles of the multiple groups of micron fiber reinforcement layers are distributed in a gradient manner; Step 2: Cleaning and drying the micron fiber reinforcement layer prepared in step 1; Step 3: preparing a composite spinning solution with a polymer material and a bioactive ingredient, and performing electrospinning using the micron fiber reinforcement layer obtained in step 2 as a receiving substrate, preparing a nanofiber induction layer on each group of micron fiber reinforcement layers, and obtaining multiple groups of composite layers consisting of micron fiber reinforcement layers and nanofiber induction layers; Step 4: The composite layer obtained in step 3 is overlapped and compounded with the micron fiber reinforcement layer and the nanofiber induction layer in the order of increasing degradation cycle from the outside to the inside to form a multi-layer composite layer. After cleaning, drying and sterilization, a ligament regeneration scaffold with layer-by-layer induction performance is obtained.

6. The method for preparing the ligament regeneration scaffold with layer-by-layer induction performance according to claim 5, characterized in that: The cleaning in step 2 is: washing with 70-80 wt % ethanol aqueous solution, and the drying temperature is 35-40° C.

7. The method for preparing the ligament regeneration scaffold with layer-by-layer induction performance according to claim 5, characterized in that: The specific preparation method of the composite spinning solution in step 3 is: mixing the polymer material and the bioactive component in a mass ratio of 9:1 to 1:9 and then uniformly dissolving the mixture in a solvent.

8. The method for preparing the ligament regeneration scaffold with layer-by-layer induction performance according to claim 7, characterized in that: The solvent is at least one of hexafluoroisopropanol, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetone, dichloromethane, chloroform, tetrahydrofuran, 1,4-dioxane, methane, trifluoroacetic acid, trifluoroethanol, ultrapure water and acetic acid aqueous solution.

9. The method for preparing the ligament regeneration scaffold with layer-by-layer induction performance according to claim 5, characterized in that: The cleaning method in step 4 is: cleaning with ethanol, and the sterilization method is: sterilization with ethylene oxide.

10. The method for preparing the ligament regeneration scaffold with layer-by-layer induction performance according to claim 5, characterized in that: When weaving is used for weaving in step 1, the micron fiber reinforced layer with the longest degradation cycle is prepared first. The composite layer composed of a single group of micron fiber reinforced layers and a nanofiber induction layer is prepared in step 3. Step 4 is to weave the next group of micron fiber reinforced layers on the composite layer prepared in step 3, that is, repeat steps 1-3 until the preparation of the ligament regeneration scaffold is completed.

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