Intervertebral disc fibrous ring tissue engineering repair material and application thereof

Abstract

The invention relates to an intervertebral disc fibrous ring tissue engineering repair material and application thereof, and aims to solve the problems of difficult fibrous ring repair, high recurrence rate, complicated operation and the like in the prior art. The repair material provided by the invention has excellent biocompatibility, superelasticity and shape memory effect, and can effectively promote regeneration and repair of the annulus fibrosus, significantly improve the surgical effect and reduce postoperative recurrence. The repairing material comprises one or more compounds of collagen, acellular matrix protein, gelatin polylactic acid and expanded polytetrafluoroethylene. The materials act together to support the growth of a plurality of cells such as chondrocytes, fibroblasts and mesenchymal stem cells, provide extremely high mechanical stability and biological activity, and are suitable for being used in a complex living body environment. Through specific chemical and physical treatment processes, the composite materials are assembled into the intervertebral disc fibrous ring tissue engineering repair material capable of supporting and repairing damaged intervertebral discs. The repair material and method provide a new solution for treatment of intervertebral disc degenerative diseases, and have wide application prospects and clinical values.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedicine, and particularly relates to an intervertebral disc annulus fibrosus tissue engineering repair material and application thereof. BACKGROUND

[0002] Intervertebral disc herniation is one of the more common clinical spinal degenerative diseases, which can occur in each spinal segment of the cervical spine, lumbar spine and thoracic spine. It mainly refers to the different degrees of degeneration of the nucleus pulposus, annulus fibrosus and cartilage plate, especially the nucleus pulposus, and under the action of external force, the annulus fibrosus ruptures and the nucleus pulposus protrudes (or prolapses) into the posterior spinal canal, causing the spinal cord and spinal nerve root to be stimulated or compressed, and causing neck, shoulder, waist and leg pain or corresponding nerve symptoms. If the compression site is in the cervical segment, the main manifestations are neck and shoulder pain, upper limb radiating pain and numbness; if the compression site is in the thoracic segment, the main manifestations are chest and back pain, lower limb sensory disturbance and weakness; if the compression site is in the lumbar segment, the main manifestations are lumbar and leg pain, sciatic nerve (lower limb important nerve) pain and other symptoms.

[0003] The existing treatment methods for intervertebral disc herniation mainly include non-surgical treatment, interventional treatment, nucleus pulposus removal and fusion fixation, but the existing treatment methods have the following shortcomings:

[0004] 1. Non-surgical treatment includes drug treatment, physical therapy and rehabilitation training, etc., which is suitable for patients in early stage or with mild symptoms. However, these methods can only temporarily relieve symptoms and cannot solve the root problem of intervertebral disc herniation, and the symptoms are easy to recur.

[0005] 2. Interventional treatment such as radiofrequency ablation of intervertebral disc and intradiscal injection therapy can relieve pain to a certain extent, but has limited repair effect on the annulus fibrosus.

[0006] 3. The nucleus pulposus removal method includes conventional small incision and transforaminal endoscopic approach, which removes the protruding nucleus pulposus to relieve nerve compression. However, the nucleus pulposus removal method has the following disadvantages: unable to repair the annulus fibrosus rupture, the ruptured part of the annulus fibrosus is difficult to heal after the nucleus pulposus is removed, and is easy to cause recurrence. It is difficult to control the amount of nucleus pulposus removal: too much removal may lead to instability of the intervertebral disc, and too little removal may not be able to fully relieve symptoms. High recurrence rate: the structure of the intervertebral disc is unstable after the operation, and is easy to protrude again.

[0007] 4. Annulus fibrosus suture technique is suitable for more than 90% of patients with simple intervertebral disc herniation, and its operation is simple and has low risk. However, annulus fibrosus suture technique also has certain limitations: slow repair speed: the healing speed of annulus fibrosus is slow, and a long time is needed after operation for complete recovery. Recurrence risk: during the period when the annulus fibrosus is not completely healed, the intervertebral disc structure is still unstable, and the sutured annulus fibrosus has a certain risk of fracture recurrence due to tissue aging.

[0008] In order to solve the above problems and improve the treatment effect of intervertebral disc herniation, a new repair material is needed, which should have the following characteristics:

[0009] 1. Good biocompatibility: compatible with human tissues and does not cause immune rejection.

[0010] 2. Good mechanical properties: can realize tissue filling and annulus fibrosus reinforcement functions at the same time.

[0011] 3. Superelasticity and shape memory effect: can be folded into a small volume during infusion, and restored to the original shape by heating or other methods, filling and stabilizing the intervertebral disc structure.

[0012] 4. Promote cell growth: provide a good cell growth environment to promote tissue regeneration and wound healing.

[0013] 5. Antibacterial property: avoid infection and ensure the smooth progress of the repair process.

[0014] Annulus fibrosus suture technique combined with a new repair material can promote the one-stage closure of damaged annulus fibrosus, accelerate the rapid repair of annulus fibrosus, and reduce the recurrence rate of intervertebral disc herniation. SUMMARY

[0015] The purpose of the present application is to provide a new intervertebral disc annulus fibrosus tissue engineering repair material to solve the problems of difficult annulus fibrosus repair, high recurrence rate and complex operation in the prior art. The repair material has excellent biocompatibility, superelasticity and shape memory effect, can effectively promote the regeneration and repair of annulus fibrosus, significantly improve the operation effect, and reduce postoperative recurrence.

[0016] The present application provides an intervertebral disc annulus fibrosus tissue engineering repair material and its application, which comprises a composite of one or more of collagen, acellular matrix protein, gelatin polylactic acid and expanded polytetrafluoroethylene.

[0017] The present application has biocompatibility, the repair material is highly compatible with human tissues and does not cause immune rejection. The material has excellent elastic properties and can quickly restore shape in the body. The material has shape memory effect and can quickly restore to the original shape by heating to a certain temperature (without stress) under extreme deformation. The repair material can carry human tissue cells, promote cell growth, form tissue connection, realize tissue filling and annulus fibrosus reinforcement at the same time, and achieve the purpose of fixing intervertebral disc.

[0018] The repair material is folded into a spherical or other irregular shape in vitro, the repair material is sterilized by cobalt-60 or electron beam irradiation, mesenchymal stem cells are inoculated on the film at a density of 5x105-2x106 cells per square centimeter, and the cells are allowed to adhere to the film under a microscope for 0.5-6 hours. The repair material is folded and injected into the designated site through the intervertebral mirror, and after 1 minute, the repair material is stretched into an extended state by surrounding body fluid infiltration, and the repair material is fixed to the nucleus pulposus resection site by a fixator, and then the annulus fibrosus is sutured. Autologous cell growth guides tissue regeneration and promotes wound healing, greatly improving the prognosis of patients; the repair material provides a growth environment for human tissue cells, forms tissue connection, and the repair material and tissue are inseparable, achieving the purpose of fixing intervertebral disc.

[0019] The present application also provides a preparation method of an intervertebral disc annulus fibrosus tissue engineering repair material, comprising the following steps:

[0020] Step one: preparation of the expanded polytetrafluoroethylene layer

[0021] 1) Preparation of the expanded polytetrafluoroethylene layer:

[0022] First, prepare the basic expanded polytetrafluoroethylene (ePTFE) layer, which will serve as the basic component of the structural support.

[0023] 2) Layer stacking:

[0024] Stack the prepared two layers of ePTFE according to the fiber direction, and place a layer of polytetrafluoroethylene film between the two layers as an adhesive to enhance the stability of the overall structure.

[0025] 3) Fixing and heat treatment:

[0026] The stacked layers are fixed and compacted after stacking to ensure flatness, wrinkle-free and bubble-free, and are treated by heating and holding to form a durable composite material to provide a base material for subsequent steps.

[0027] Step two: modification of collagen

[0028] Wrap 1-5 nm modified collagen material on the surface of the structural support component.

[0029] 1) Collagen solubilization and modification:

[0030] Type I collagen was solubilized in dimethylformamide and anhydrous triethylamine and acryloyl chloride were added under ice water bath conditions. The mixture was continuously stirred and allowed to react for a long time at room temperature to produce modified collagen product A, which provides improved biocompatibility and mechanical strength;

[0031] 2) Chemical reactions and product preparation:

[0032] Hydroxyethyl isobromobutyrate and anhydrous ε-caprolactone were dissolved in toluene, and stannous octoate was added as a catalyst. The reaction product was precipitated in methanol to obtain product B, which is used to enhance structural stability;

[0033] 3) Polymerization and crosslinking:

[0034] Product A and product B were mixed with cuprous bromide and subjected to freeze-thaw cycle treatment, heated and dialyzed, and decellularized matrix protein was added to obtain modified collagen, which is used to coat structural support materials;

[0035] Step three: gel formation:

[0036] The reinforcement rib was dissolved, the pH was adjusted, and silver nanoparticles were added to make a gel, which was coated with the material prepared in step two to a thickness of 10-20 nm. The gel was solidified by constant temperature incubation to form a repair material.

[0037] 1) Preparation of reinforcement rib gel PLA, PGA, and PLGA fishbone-shaped reinforcement ribs were dissolved, the pH was adjusted, and silver nanoparticles were added to make a gel.

[0038] 2) Repair material formation:

[0039] The reinforcement rib was coated with the material prepared in step two, and the gel was solidified by incubation at a constant temperature of 36-37°C for 2-3 hours to form the final repair material, which has certain mechanical properties and provides additional structural strength to the repair material.

[0040] Step four: integration and application of the final product

[0041] The prepared repair material was cut to the desired size to form the final repair material, which will be directly used for intervertebral disc repair.

[0042] Step five: performance testing:

[0043] The integrated repair material was subjected to a series of biological safety and functional tests, including antibacterial testing, cytotoxicity testing, biofilm growth inhibition testing, and hemolysis testing, to verify its clinical application feasibility and safety.

[0044] The intervertebral disc annulus fibrosus tissue engineering repair material and its preparation method of the present application have the following advantages compared with the prior art

[0045] Advantages:

[0046] 1. Improved intervertebral disc structure and function:

[0047] The repair material can effectively fill the defects of the annulus fibrosus in the intervertebral disc, and through the combined action of physical support and bioactive factors, promote the repair of the intervertebral disc structure and the recovery of the function.

[0048] 2. Excellent biocompatibility:

[0049] The collagen, decellularized matrix protein, gelatin polylactic acid and other biological materials used in the repair material have very high biocompatibility, reducing the immune rejection reaction and inflammatory reaction after operation.

[0050] 3. Promote cell proliferation and migration:

[0051] The bioactive factors in the repair material can stimulate the proliferation and migration of surrounding native cells, accelerate the self-repair process of the damaged intervertebral disc, and help maintain the biomechanical properties of the intervertebral disc.

[0052] 4. Improve the safety and effectiveness of the operation:

[0053] Due to the temperature sensitivity and injectability of the material, the operation is simple and minimally invasive, and the material can quickly gel in the body, reducing the risk of intraoperative and postoperative complications.

[0054] 5. Prolong the duration of intervertebral disc function:

[0055] The design of the structure and composition of the material, such as the use of electrospun fibers and composite polymers, improves the mechanical properties and durability of the material, which helps to maintain the long-term function of the intervertebral disc.

[0056] 6. Reduce the recurrence rate of intervertebral disc degeneration:

[0057] The bioactivity of the material and its good compatibility with the organism can effectively prevent the further development of intervertebral disc degeneration and reduce the possibility of recurrence and reoperation.

[0058] 7. Comprehensive evaluation of safety:

[0059] The comprehensive performance test of the material, including antibacterial test, cytotoxicity test, biofilm inhibition test and hemolysis test, ensures the safety and applicability of the material, and provides sufficient scientific basis for clinical application.

[0060] Through the above beneficial effects, the annulus fibrosus tissue engineering repair material of the application can effectively improve and repair the damaged intervertebral disc due to degenerative diseases, significantly improve the quality of life of patients, and has wide clinical popularization value and application prospect.

[0061] BRIEF DESCRIPTION OF DRAWINGSIn order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, hereinafter, a brief introduction will be given to the drawings needed to be used in the embodiments, and obviously, the drawings described below are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained according to these drawings without creating any creative labor.

[0062] Figure 1 The morphological changes of the repair material before and after infusion;

[0063] Figure 2 The morphological changes of the repair material before and after infusion;

[0064] Figure 3 The cell adhesion diagram of the repair material;

[0065] Figure 4 The preparation process of the repair material.

[0066] In the figure, 1, 2 are marked: (a) is the morphology of the material before infusion, (b) is the extended morphology of the material reaching the designated site, (c) is the adjusted surrounding morphology of the material, and (c) A is the material embedded in the intervertebral disc part. DETAILED DESCRIPTION

[0067] The detailed description of the various exemplary embodiments of the present application should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application. It should be understood that the terms described in the present application are only for the description of the particular embodiments, and are not used to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value within the stated range and any other stated value or intermediate value within the stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art of the present application. Although only preferred methods and materials are described in the present application, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present application. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0068] Many modifications and variations of the present application described in the specific embodiments of the application can be made by those skilled in the art without departing from the spirit or scope of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples of the application are exemplary only. It is intended to include what is within the scope of the present application, including equivalents thereof, along with modifications based on the substitution of elements, combinations with or without other elements, and the like.

[0069] Example One

[0070] Step One

[0071] 1) Preparation of expanded PTFE layer as structural support material;

[0072] 2) Two layers of expanded PTFE from Step 1 are placed in a stack according to the respective fiber orientation, and a PTFE film is placed between the two layers as an adhesive;

[0073] 3) The stacked layers are fixed, ensuring that they are flat, wrinkle-free and bubble-free after placement, and are vertically pressurized along the stacking direction, and are treated by heating and insulation to form a strong and durable composite material, providing a base material for subsequent steps.

[0074] Step Two: Modification of Collagen

[0075] 1) Collagen dissolution and modification:

[0076] Type I collagen is dissolved in dimethylformamide, and anhydrous triethylamine and acryloyl chloride are added under ice water bath conditions. The mixture is continuously stirred and reacted at room temperature for a long time to produce modified collagen product A, which provides improved biocompatibility and mechanical strength;

[0077] 2) Chemical reaction and product preparation:

[0078] Hydroxyethyl isobromobutyrate and anhydrous ε-caprolactone are dissolved in toluene, and stannous octoate is added as a catalyst. The reaction product is precipitated in methanol to obtain product B, which is used to enhance structural stability;

[0079] 3) Polymerization and crosslinking:

[0080] Product A and product B are mixed with cuprous bromide and subjected to freeze-thaw cycle treatment, heated and reacted, and dialyzed, and decellularized matrix protein is added to obtain modified collagen, which is used to coat the structural support material, i.e., wrapping 1-5 nm of modified collagen material on the surface of the structural support assembly;

[0081] Step Three: Gelation molding:

[0082] After the reinforcement is dissolved, pH is adjusted and silver nanoparticles are added to make a gel, which is coated with the material prepared in step two to a thickness of 10-20 nm. The gel is solidified by incubation at a constant temperature to form the repair material.

[0083] 1) Preparation of the reinforcement gel PLA, PGA and PLGA fishbone-shaped reinforcement is prepared, dissolved, pH is adjusted and silver nanoparticles are added to make a gel.

[0084] 2) Formation of the repair material:

[0085] The reinforcement is coated with the material prepared in step two, and the gel is solidified by incubation at a constant temperature of 36-37°C for 2-3 h to form the final repair material, which has certain mechanical properties and provides additional structural strength to the repair material.

[0086] Step four: integration and application of the final product

[0087] The prepared repair material is cut to the required size to form the final repair material, which will be directly used for the repair of intervertebral discs.

[0088] Step five: performance testing:

[0089] The repair material prepared above needs to be tested for antibacterial activity, cytotoxicity, promotion of biofilm growth (crystal violet staining), and hemolysis.

[0090] The specific test methods are as follows:

[0091] 1) Antibacterial test: The antibacterial activity of the repair material against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa was tested. cm 2 of the repair material was taken as the experimental group, and PET film was taken as the control group. OD = 0.1 of the bacterial solution (Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa) was added to the surface of the experimental and control groups, and then incubated for 4 hours before plating. After 24 hours of incubation, the number of colonies was counted.

[0092] 2) Cytotoxicity test: MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyl tetrazolium bromide) method was used to test the toxicity of the repair material to human skin fibroblasts.

[0093] Human skin fibroblasts (1 x 104 CFU / mL) were cultured in 24-well plates with 10% fetal bovine serum medium for 48 h, and cm 2The repair material as a sample and PET film is added to the human skin fibroblast solution at 37℃, and after 72h of culture, 0.1mL of MTT solution (5g / L PBS solution) is added dropwise to the well plate for further culture, and after 4h, the supernatant in the well plate is removed, and 0.75mL of DMSO is added to dissolve the formazan crystals. The OD (490nm) value is tested by an enzyme marker to characterize the release amount of formazan, and the relative growth rate of human skin fibroblasts is tested in parallel for 3 times.

[0094] 3) Test of promoting the growth of biofilm (crystal violet staining):

[0095] The repair material with an area of 1.5×1.5cm 2 is taken as the experimental group, and the PET film is taken as the control group, and OD=0.1 of Pseudomonas aeruginosa (Escherichia coli or Staphylococcus aureus) is added dropwise to the surface of the experimental group and the control group, and cultured in a 37℃ incubator for 24h. The culture medium is sucked off, and washed with PBS for 3 times, and treated with 95% ethanol for 15min, and after air-drying the ethanol, a 0.1% crystal violet solution is added for 15min, and washed with sterile water for 3 times, and finally dissolved with 33% acetic acid and biofilm-acting crystal violet, and the OD (490nm) value is tested by an enzyme marker to characterize the amount of biofilm.

[0096] 4) Hemolysis test:

[0097] Fresh human blood (3mL) is centrifuged at 1500rpm for 15min and washed with PBS until the supernatant is transparent. The red blood cell precipitate is diluted to 2vol% with PBS. The sterilized repair material sample and PET film (area: 1.5×1.5cm 2 ) are immersed in the diluted blood (5mL per tube) solution, and incubated at 37℃ for 3h respectively. After the treated diluted blood sample is centrifuged at 1500rpm for 15min, 100μL of supernatant is transferred to a 96-well plate. The amount of hemoglobin release is evaluated by recording OD576nm with an enzyme marker. Red blood cells in PBS and 2% Triton are used as negative and positive controls respectively.

[0098] Application example:

[0099] The repair material is folded into a spherical shape or other irregular shape in vitro, and the repair material is sterilized by cobalt-60 or electron beam irradiation, and mesenchymal stem cells are inoculated on the repair material at a cell density of 5×105-2×106 per square centimeter, and after 3-7 days of culture, the cells can be observed to adhere to the repair material to form a multi-layered dense cell membrane structure under a microscope, such as Figure 3 .

[0100] The repair material adheres cells and is mechanically folded into a shape that can pass through the intervertebral mirror, and is injected into the designated site through the intervertebral mirror. After 1 min, the repair material reaches a temperature of 36-37℃, the memory effect works, the folded state opens into the extended state of the repair material, and the repair material is fixed to the nucleus pulposus removal site through the fixator, and then the annulus fibrosus is sutured.

[0101] The biomaterial film is filled into the intervertebral annulus fibrosus, and the space left after the nucleus pulposus is removed plays a role in filling and stabilizing the spine. Autologous cell growth guides tissue regeneration and promotes wound healing, greatly improving patient prognosis. The repair material provides a human tissue cell growth environment, forms a tissue connection, and the repair material and the tissue are inseparable, achieving the purpose of fixing the intervertebral disc. Figure 1 、 2 (a) is the shape of the material before infusion, Figure 1 、 2 (b) is the extended shape of the material to the designated site, Figure 1 、 2 (c) is the adjusted surrounding shape of the material, Figure 1 、 2 (c) A is the material embedded in the intervertebral disc portion.

[0102] Preferably, the biomaterial film is mostly filled into the intervertebral annulus fibrosus after the nucleus pulposus is removed, and the biomaterial is partially sutured to the intervertebral annulus fibrosus defect to play a role in fixation and tension reduction.

[0103] The biomaterial gradually fuses and absorbs with the body after surgery. The cells carried by the biomaterial play a role in promoting repair, accelerating the healing of the intervertebral annulus fibrosus, and can effectively reduce the complication of postoperative cracking of the intervertebral annulus fibrosus.

[0104] Although examples of the present application have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these examples without departing from the principles and spirit of the present application, the scope of the present application being defined by the appended claims and their equivalents; the above description is only a specific embodiment of the present application, various examples do not limit the implementation content of the present application, and those of ordinary skill in the art can modify or modify the previously described specific embodiments without departing from the implementation spirit and scope of the application.

Claims

1. An annulus fibrosus tissue engineered repair material for intervertebral discs, characterized by, A composite including one or more of collagen, decellularized matrix protein, gelatin polylactic acid, and expanded polytetrafluoroethylene.

2. The method of claim 1, wherein the method of preparing an annulus fibrosus tissue engineering repair material for an intervertebral disc is characterized by The method comprises the following steps: Step one: Preparation of the expanded polytetrafluoroethylene layer (1) Preparation of the expanded polytetrafluoroethylene layer: First, prepare the basic expanded polytetrafluoroethylene (ePTFE) layer, which will serve as the structural support component; (2) Layer stacking: Stack the two prepared ePTFE layers according to the fiber direction, and place a polytetrafluoroethylene film between them as an adhesive to enhance the stability of the overall structure; (3) Fixing and heat treatment: After stacking, the stack is fixed and compacted to ensure flatness, wrinkle-free, and bubble-free, and is treated by heating and holding to form a durable composite material, providing a basis for subsequent steps; Step two: Modification of collagen (1) Collagen dissolution and modification: Dissolve type I collagen in dimethylformamide, and under ice water bath conditions, add anhydrous triethylamine and acryloyl chloride. After continuous stirring, react at room temperature for a long time to obtain modified collagen product A, which provides improved biocompatibility and mechanical strength; (2) Chemical reaction and product preparation: Dissolve hydroxyethyl isobromoacetate and anhydrous ε-caprolactone in toluene, add stannous octoate as a catalyst, and precipitate the reaction product in methanol to obtain product B, which is used to enhance structural stability; (3) Polymerization and crosslinking: Mix product A and product B with cuprous bromide, then perform freeze-thaw cycle treatment, heat reaction, and dialysis. Add decellularized matrix protein to obtain modified collagen, which is used to coat the structural support material, i.e., wrap 1-5 nm of modified collagen material on the surface of the structural support component; Step three: Gel formation: After the reinforcement rib is dissolved, adjust the pH and add silver nanoparticles to prepare a gel, which is coated with 10-20 nm of the material prepared in step two. The gel is solidified by constant temperature incubation to form a repair material; (1) Preparation and application of reinforcement rib Manufacture fishbone-shaped reinforcement ribs of PLA, PGA, and PLGA. After dissolution, adjust the pH and add silver nanoparticles to prepare a gel; (2) Preparation of collagen film The gel prepared from the reinforcement rib is coated with the material prepared in step two. The gel is solidified by constant temperature incubation to form the final repair material, which is made into a film with certain mechanical properties. These films provide additional structural strength to the repair material; Step four: Integration and application of the final product Cut the prepared repair material into the desired size to form the final repair material, which will be directly used for intervertebral disc repair; Step five: Performance testing: Perform a series of biological safety and functional tests on the integrated repair material, including antibacterial test, cytotoxicity test, biofilm growth inhibition test, and hemolysis test, to verify its clinical application feasibility and safety.

3. The annulus fibrosus tissue engineering repair material according to claim 1 or 2, characterized in that The adhesive layer is a polytetrafluoroethylene film.

4. The method of claim 1, wherein the method is for preparing a tissue engineered annulus fibrosus repair material for intervertebral disc. The mass ratio of type I collagen to anhydrous triethylamine and acryloyl chloride is 10:(0.45-0.48): (0.68-0.70)。 5. The method for preparing an intervertebral disc annulus fibrosus tissue engineering repair material according to claim 1, characterized in that, The mass ratio of the isobromobutyric acid hydroxyethyl ester to the anhydrous epsilon-caprolactone and the stannous octoate is (4.3-4.5):100: (10-11)。 6. The method of claim 1, wherein the method is a method of preparing an annulus fibrosus tissue engineering repair material for an intervertebral disc, characterized in that, The mass ratio of the product A to the product B, the cuprous bromide and the pentamethyl diethylene triamine is 1:2:(0.028-0.03):(0.03-0.04).

7. A method for preparing a tissue-engineered repair material for the annulus fibrosus of an intervertebral disc according to claim 1, characterized in that, The gelation forming stage dissolves the modified collagen in acetic acid solution, prepares a collagen solution, and drops sodium hydroxide solution to adjust pH to 7.

4.

8. A method for preparing a tissue-engineered repair material for the annulus fibrosus of an intervertebral disc according to claim 1, characterized in that, The bio-material film of the reinforcing rib is cut into square films with a length of 5mm-15mm and a width of 3mm-8mm.

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