High-porosity bionic tendon repair patch as well as preparation method and application thereof

Abstract

The invention provides a high-porosity bionic tendon repair patch as well as a preparation method and application thereof. The preparation method comprises the following steps: adding human-derived recombinant collagen into a poly-L-lactic acid solution to prepare an electrospinning solution, carrying out electrostatic spinning, putting into a cross-linking solution, carrying out soaking cross-linking, and finally carrying out freeze forming to obtain the bionic tendon repair patch. A degradable polymer PLLA and a natural high polymer material human-derived recombinant collagen are used for preparing a degradable spinning raw material with high biocompatibility, and the prepared bionic tendon repair patch has good biomechanical properties, large porosity, good biocompatibility, good biocompatibility and good biocompatibility through the cooperation of an electrostatic spinning technology and a controllable freeze-drying process, and can be used for preparing the bionic tendon repair patch for the human-derived recombinant collagen-based bionic tendon repair patch for the human-derived recombinant collagen-based bionic tendon repair patch for the human-derived recombinant collagen-based bionic tendon repair patch for the human-derived recombinant collagen-based bionic tendon repair patch. The bionic tendon transplantation material has excellent water absorption, has a bionic microfiber structure, effectively makes up for the defects of traditional tendon transplantation, and has very wide application prospects.

Description

Technical Field

[0001] The present invention relates to a field of bionic tendon repair with high porosity, and in particular to a preparation method and application thereof. Background Art

[0002] Tendons are primarily composed of parallel bundles of collagen fibers. Their primary function is to connect muscles and bones, transmit muscle contraction force, and maintain body stability and motor function. However, due to various acute and chronic factors, such as sports injuries, inflammation, and aging, tendons may become defective or ruptured, leading to pain, limited motor function, and other problems, significantly impacting quality of life.

[0003] The physiological characteristics of tendons determine that tendon injuries are usually difficult to heal on their own. The main reasons are as follows: First, compared with other soft tissues such as muscles, tendons have a poorer blood supply. When a tendon is injured, due to insufficient blood supply, it is difficult for cells in the injured area to obtain sufficient nutrition and oxygen. At the same time, the metabolic products produced by cell metabolism are difficult to be cleared in time, which makes necrosis and fibrosis prone to occur at the injured tendon site, resulting in difficulties in tendon repair. In addition, the proliferation and regeneration rate of tendon cells is slow, so the regeneration capacity of tendon tissue is relatively poor. In addition, the structure and function of tendons also make it difficult for them to heal on their own: because tendons connect muscles and bones, they are subject to greater stretching and pressure. Under normal circumstances, tendons already bear a large load, so it is even more difficult to bear after injury, which will further aggravate the condition of tendon injury.

[0004] Treatment options for tendon injuries include physical therapy, medication, and surgery. Physical therapy includes ice, heat, ultrasound, electrical stimulation, and other treatments. Medication primarily includes over-the-counter medications such as nonsteroidal anti-inflammatory drugs (NSAIDs) and muscle relaxants. However, physical therapy and medication are generally only suitable for mild to moderate tendon injuries and can only relieve pain and inflammation. These conservative treatments primarily serve as a supplementary aid for the body's repair process.

[0005] Treatment of severe tendon injuries typically requires surgical intervention to restore tendon function. Surgical methods are primarily categorized as direct suturing and tendon grafting. Ideal direct suturing requires six key elements: easy tendon penetration, secure knots, smooth apposition of the stumps, minimal gapping at the repair site, minimal disruption to tendon blood supply, and sufficient strength during healing to withstand the stresses of early postoperative exercise. However, tendon defects larger than 3 cm are often difficult to repair with direct suturing and require tendon grafting.

[0006] Currently, autologous tendon transplantation is a commonly used transplantation method in clinical practice, offering rapid healing, minimal adhesion, and resistance to deformation. However, the availability of autologous tendons is limited, harvesting can compromise the function of the donor site, and the surgery is relatively lengthy. Allograft tendon transplantation has become one of the most widely used tendon transplantation methods in recent years. While allograft tendons share the same biological structure and function as autologous tendons, allografts carry the risks of immune rejection and disease transmission. Xenograft tendon transplantation is less common in clinical practice, primarily due to the increased risk of immune rejection, infection, and disease transmission. Furthermore, the structure of xenograft tendons differs from that of native tendons in the human body, potentially leading to structural instability. This can result in the graft being unable to withstand normal pressure and loads. While xenograft tendon transplantation may be a viable option in certain special circumstances, it is not a routine treatment due to its significant limitations.

[0007] Given the numerous shortcomings and limitations of conventional tendon transplantation, the search for a superior alternative has become a clinically urgent need. In recent years, with the advancement of tissue engineering, tissue-engineered artificial tendons have become increasingly popular in modern medical clinical applications, and related cutting-edge preparation technologies have become a research hotspot that has garnered significant attention.

[0008] Tissue-engineered artificial tendons offer significant advantages, including abundant raw material resources, excellent tissue compatibility, superior biomechanical properties, strong plasticity, and biodegradability—they can fuse with the original tendon tissue, eliminating the need for secondary surgical removal. Therefore, tissue-engineered tendon transplantation can largely address the shortcomings of traditional tendon transplantation and holds a promising future. Summary of the Invention

[0009] In view of this, the purpose of the present invention is to propose a high-porosity bionic tendon repair patch and its preparation method and application, combining nanotechnology with biodegradable polymer materials and natural polymer materials, and through the synergistic effect of electrospinning technology and controllable freeze-drying process, preparing an artificial tendon patch with a natural tendon bionic structure.

[0010] Specifically, the present invention uses electrospinning technology to prepare nano-scale fibers to accurately simulate the microstructure of collagen fiber bundles in natural tendons. Its highly ordered arrangement is highly similar to the parallel fiber bundle structure of natural tendons. However, the fiber materials prepared by traditional electrospinning methods are usually too dense and have low porosity, which is not conducive to the internal migration of cells and the penetration of nutrients, which limits its application in tissue regeneration. To solve this problem, the present invention introduces controllable freeze-drying technology, which is combined with freeze-drying molds for liquid nitrogen freezing. It can effectively prevent the spinning patch from cracking or structural collapse during pre-freezing or liquid nitrogen freeze-drying, and a loose porous structure can be introduced into the fiber network after freeze-drying. The loose porous structure formed by freeze-drying not only retains the microscopic biomimetic characteristics of electrospun fibers, but also significantly improves the porosity and connectivity of the patch, providing an ideal three-dimensional microenvironment for cell adhesion, proliferation and ingrowth.

[0011] The technical solution of the present invention is achieved as follows:

[0012] A method for preparing a high-porosity bionic tendon repair patch comprises the following steps: adding human recombinant collagen to a L-polylactic acid solution to prepare an electrospinning solution, electrospinning, then immersing in a cross-linking solution for cross-linking, and finally freeze-forming to obtain the bionic tendon repair patch;

[0013] The mass ratio of the poly(L-lactic acid) to the human recombinant collagen is 1-10:1-3.

[0014] The present invention uses degradable polymer PLLA and natural polymer material human recombinant collagen as raw materials for bionic tendon repair patches. They have a short degradation cycle, no residue, extremely low immunogenicity, higher safety, are suitable for clinical long-term implantation, and are more in line with the time window for rotator cuff repair.

[0015] A further solution is that the mass concentration of the electrospinning solution is 10% to 20%; and the human recombinant collagen is human recombinant type III collagen.

[0016] A further solution is that when the mass concentration of the electrospinning solution is 12% to 15%, the mass ratio of the poly(L-lactic acid) to the human recombinant type III collagen is 3 to 8:1 to 2;

[0017] When the mass concentration of the electrospinning solution is 15.1% to 19%, the mass ratio of the poly (L-lactic acid) to the human recombinant type III collagen is 1 to 10:1 to 3;

[0018] When the mass concentration of the electrospinning solution is 19.1% to 20%, the mass ratio of the poly(L-lactic acid) to the human recombinant type III collagen is 7 to 9:1 to 3.

[0019] A further solution is to add L-polylactic acid into an organic solvent to prepare the L-polylactic acid solution with a mass concentration of 5% to 15%.

[0020] In a further embodiment, the organic solvent comprises at least one of hexafluoroisopropanol, chloroform, formic acid, tetrafluoroethylene, acetic acid, dichloromethane, ethanol, dimethyl sulfoxide, tetrahydrofuran or dimethylformamide.

[0021] A further solution is that the process parameters of the electrospinning are: voltage 15-30 kV, propulsion rate 5-8 mL / h, and receiving distance 10-20 cm.

[0022] A further solution is that the soaking and cross-linking time is 12 to 24 hours.

[0023] A further solution is that the cross-linking liquid includes a cross-linking agent, an active agent and water in a mass volume ratio of 8-10g:2-3g:150-250mL, the cross-linking agent includes a water-soluble carbodiimide cross-linking agent, and the active agent includes N-hydroxysuccinimide.

[0024] A further solution is that the freezing molding includes pre-freezing or liquid nitrogen freezing, the pre-freezing temperature is -80°C to -70°C, the pre-freezing time is 1 to 2 hours, and the liquid nitrogen freezing time is 1 to 5 minutes.

[0025] The present invention also provides a bionic tendon repair patch prepared by the above preparation method.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention utilizes the degradable polymer PLLA and the natural polymer material human recombinant collagen to prepare a degradable and highly biocompatible spinning raw material. Through the synergy of electrospinning technology and controllable freeze-drying process, the prepared bionic tendon repair patch has good biomechanical properties, large product porosity, excellent water absorption, and a bionic microfiber structure, which effectively makes up for the shortcomings of traditional tendon transplantation and has a very broad application prospect.

[0028] Subcutaneous implantation experiments and animal experiments have verified that the bionic tendon repair patch provided by the present invention is non-toxic to cells and helps cells to grow in, which can promote rapid tendon repair and provide an ideal three-dimensional microenvironment for cell adhesion, proliferation and ingrowth. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a process flow for preparing a high-porosity bionic tendon repair patch of the present invention;

[0030] Figure 2 This is a freeze-drying mold diagram;

[0031] Figure 3 This is a sample photo of the high-porosity bionic tendon repair patch prepared by the present invention;

[0032] Figure 4 This is a graph showing the tensile strength of the high-porosity bionic tendon repair patch of Example 1;

[0033] Figure 5 This is a microstructure diagram of the high-porosity bionic tendon repair patch of Example 1;

[0034] Figure 6 This is a water contact angle diagram of the high-porosity bionic tendon repair patch in Example 1;

[0035] Figure 7 The results of gross observation and pathological HE staining after 12 weeks of subcutaneous implantation in rabbits;

[0036] Figure 8 A: Exposure of the infraspinatus tendon; B: Suture repair control; C: Suture + patch repair results;

[0037] Figure 9 To obtain the material, the general observation result diagram is shown;

[0038] Figure 10 This is the biomechanical failure load result diagram;

[0039] Figure 11 Pathological HE staining and MASSON staining results. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0041] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0042] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0043] The method for testing the performance of the high-porosity bionic tendon repair patch of the present invention is as follows:

[0044] 1. Tensile strength

[0045] Equipment: Universal material testing machine, pneumatic flat fixture

[0046] Material: Rotator cuff patch (20mm×30mm, thickness 2-4mm)

[0047] Experimental steps:

[0048] (1) Equipment calibration

[0049] The load cell was zeroed and the grip spacing was set to 20 mm (gauge length).

[0050] Use standard weights to verify the load accuracy (error ≤ ± 1%).

[0051] (2) Clamping the specimen

[0052] Open the clamp (pressure 0.3-0.5 MPa), clamp the specimen vertically, and ensure that the gauge length is centered. The parameters are set as follows:

[0053] parameter Setting value Stretching speed 10-50mm / min (adjusted according to material toughness) Preload 0.1N

[0054] (3) Test execution

[0055] Start the equipment and apply a preload (0.1 N) for 10 seconds to eliminate initial relaxation. Start stretching and monitor the load-displacement curve in real time until the specimen breaks.

[0056] Record the fracture position (gauge length section / fixture area). If the fracture occurs at the fixture, the data will be invalid.

[0057] (4) Data Collection

[0058] Export raw data: time (s), displacement (mm), load (N).

[0059] Key parameters: peak load (Fmax), fracture displacement (ΔL).

[0060] (5) Data processing

[0061] Calculation of tensile strength: σ = AFmax (MPa), A = width (5 mm) × thickness (mm) Calculation of elongation at break: ε = L0ΔL × 100%, L0 = 20 mm (gauge length).

[0062] 2. Water absorption

[0063] Randomly select 5 samples, record them as m1, immerse them in a beaker containing 20 mL of water for 60 minutes, gently grasp one corner with tweezers and remove them from the water. Hold the tweezers gently on the water surface for 1 minute, then weigh them again, record them as m2, and calculate the water absorption multiple as follows, reporting the average value:

[0064]

[0065] Where:

[0066] A——water absorption multiple of sample;

[0067] m1——mass of the sample before wetting, in grams (g);

[0068] m2——The mass of the sample before soaking, in grams (g).

[0069] 3. Contact angle

[0070] Randomly select samples of appropriate size, place them on the contact angle test platform, use the equipment's automatic titration system to drip pure water, take test photos, and measure the contact angle using the volumetric method / fitting method / angle method.

[0071] 4. Subcutaneous implantation test

[0072] Three New Zealand rabbits that had passed quarantine were selected, with two subcutaneous implantation holes per animal. For subcutaneous implantation, after isoflurane anesthesia, the back was shaved and disinfected. Two incisions were made on either side of the midline of the back. Blunt dissection was used to create two subcutaneous pouches. A 10 mm × 10 mm patch was transferred to the pouch. The skin incisions were sutured with 3-0 suture, and the wounds were disinfected with iodine. The animals were allowed to eat and drink freely after surgery, and the wounds were regularly disinfected with anti-inflammatory drugs. Twelve weeks after implantation, samples were collected for gross observation and photography. Hematoxylin and eosin (HE) staining was performed, and pathological examination was performed to observe cell ingrowth into the patch.

[0073] 5. Animal experiments

[0074] Effectiveness test on rabbit tendon injury: 30 male, 2.5-3.0 kg, common New Zealand rabbits that passed quarantine were randomly divided into two groups of 15 rabbits each. Six rabbits in each group were treated at 6 and 12 weeks after surgery, and the remaining six served as normal controls.

[0075] Model Preparation: After isoflurane anesthesia, New Zealand rabbits were fixed in lateral recumbency on the operating table. The surgical area was routinely disinfected and draped. A 3-cm longitudinal incision was made over the shoulder joint. Blunt dissection was performed to expose the insertion of the infraspinatus muscle on the lesser tuberosity. The infraspinatus tendon was sutured using the Kessler method with non-absorbable 2.0 sutures. The tendon was sharply sectioned, and the cartilage at the tendon insertion footprint was scraped with a blade. Four holes were drilled into the bone bed with 0.8-mm Kirschner wires. The ends of the tendon suture were passed through the bony canal of the humerus, then knotted and securely fixed to the greater tuberosity of the humerus. The muscle and skin were sutured layer by layer, and the wound was disinfected with iodine. Postoperatively, the animals had free access to food and water, and received analgesia and anti-inflammatory medication for 5 consecutive days.

[0076] General observation was performed daily after surgery, and body weight was measured regularly. Gross observation was performed at 6 and 12 weeks after surgery. Half of the tendon-bone tissue was taken for biomechanical testing, and the other half was taken for pathological examination (HE staining and MASSON staining) to comprehensively evaluate the safety and effectiveness of the rotator cuff patch in treating tendon injury in rabbits.

[0077] Example 1 - A high-porosity bionic tendon repair patch

[0078] Biodegradable polymer material: Poly-L-lactic acid (PLLA)

[0079] Natural polymer material: recombinant type III collagen

[0080] The preparation process of the high-porosity bionic tendon repair patch is as follows:

[0081] 1. Solution Preparation

[0082] Electrospinning solution (PLLA: recombinant type III collagen = 2:1, solution concentration 18% w / v).

[0083] The preparation steps of the electrospinning solution are as follows:

[0084] ① PLLA dissolution: 3.6 g PLLA particles were added to 30 mL hexafluoroisopropanol (HFIP) and magnetically stirred at room temperature for 1 h until the solution became colorless and transparent.

[0085] ② Collagen addition: Add 1.8 g of recombinant type III collagen and continue stirring at room temperature for 15 minutes without centrifugation.

[0086] 2. Electrospinning

[0087] The electrospinning solution was prepared into composite fibers by electrospinning to obtain a PLLA / Col tendon repair patch.

[0088] The process parameters of the above electrospinning are as follows:

[0089] Voltage: +27kV (needle) / -7kV (receiver);

[0090] Propulsion rate: 6mL / h (controlled by precision syringe pump);

[0091] Needle diameter: 0.5-0.8mm;

[0092] Receiving distance: 15cm;

[0093] Temperature: 25±1℃;

[0094] Relative humidity: <70%.

[0095] 3. Vacuum drying: vacuum drying at room temperature for 12 hours after electrospinning.

[0096] 4. Cutting: After drying, cut to obtain samples.

[0097] 5. Cross-linking treatment

[0098] The specific steps are as follows:

[0099] (1) Cross-linking solution: 9.2 g water-soluble carbodiimide cross-linker (Error Detection and Correction, EDAC) + 2.3 g N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS) + 200 mL ultrapure water (pH ≈ 7);

[0100] (2) Immersion crosslinking: The above samples were immersed in the crosslinking solution and kept at 25°C in the dark for 24 h.

[0101] 6.Pure water cleaning: Immerse and clean in ultrapure water.

[0102] 7. Freeze forming: After cleaning, the sample is placed in a freeze-drying mold (the freeze-drying mold can be customized according to the sample size) and pre-frozen at -80℃ for 1 hour to form glassy ice crystals.

[0103] 8. Vacuum dry for 24 hours.

[0104] 9. Irradiation sterilization.

[0105] Performance test results

[0106] 1. High mechanical strength of the product

[0107] Electrospinning technology is used to prepare nano-scale fibers to accurately simulate the microstructure of collagen fiber bundles in natural tendons. The polymer components and nanofiber microstructure give the material good mechanical properties. Figure 4 It can be seen that the average tensile strength of the bionic tendon repair patch prepared in Example 1 can reach 80N.

[0108] 2. The product has large porosity and good water absorption

[0109] Depend on Figure 5 As shown in Table 1, the bionic tendon repair patch prepared in Example 1 increases its porosity through controlled freeze-drying without destroying the microfiber structure, and the product has good water absorption.

[0110] Table 1 Water absorption of bionic tendon repair patch

[0111]

[0112] 3. High wettability

[0113] Depend on Figure 6 It can be seen that the pure water contact angle of the bionic tendon repair patch prepared in Example 1 is 50°, the contact angle is small, and the wettability of the product is high.

[0114] 4. Non-cytotoxic and can promote cell growth

[0115] Depend on Figure 7 It can be seen that after 12 weeks of subcutaneous implantation in rabbits, the size of the patch did not change significantly, and cells grew in a large number and in an orderly manner. Judging from the cell morphology, spindle-shaped fibroblasts ( Figure 7 Below, enlarged image on the left) and chondrocytes ( Figure 7 The following, middle and right magnified images) were generated, and no obvious inflammatory reaction was observed around the patch (no inflammatory cells were observed). Example 1 shows that the prepared biomimetic tendon repair patch has good biocompatibility and biological activity that promotes tissue regeneration.

[0116] 5. Good biological activity can promote tendon repair

[0117] Depend on Figures 8-11 It can be seen that through the rabbit tendon injury repair experiment, at 6 and 12 weeks after surgery, gross observation of the samples showed that the tendon tissue at the tendon-bone interface in the patch group was more and thicker. The mechanical results showed that the mechanical strength of the tendon in the patch group was better than that in the suture control group. The results of HE and MASSON staining of the pathological tissue showed that the cells in the tendon-bone fusion site in the patch group were more regularly arranged, there were more collagen fibers, and the interface bonding was stronger. The bionic tendon repair patch prepared in Example 1 has good biological activity and can effectively promote tendon repair.

[0118] Example 2 - A high-porosity bionic tendon repair patch

[0119] The difference between this embodiment and embodiment 1 is that the preparation of the electrospinning solution is different, and the remaining steps are the same, as follows:

[0120] Solution preparation

[0121] Table 2

[0122]

[0123] Note: Tetrafluoroethylene (TEE), dichloromethane (DCM), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), dimethylformamide (N,N-Dimethylformamide,DMF)

[0124] The preparation steps of the electrospinning solution are as follows:

[0125] ① PLLA dissolution: Add PLLA particles to the main solvent according to the ratio in the table above, and stir magnetically at room temperature for 1 hour until the solution becomes colorless and transparent.

[0126] ② Collagen addition: recombinant type III collagen was added and stirred at room temperature for 15 minutes to obtain electrospinning solutions of different concentrations.

[0127] The remaining steps are the same as in Example 1.

[0128] In Example 2, the solvent for preparing the electrospinning solution was adjusted. High-volatility solvents (such as DCM: ethanol = 9:1) can form ultrafine fibers due to rapid phase separation; medium- and low-volatility systems (such as THF: DMF = 7:3) can obtain uniform fibers by delaying solidification, and the mechanical strength is improved; strong polar solvents (such as TFE: acetic acid = 8:2) can optimize the blending compatibility of PLLA and collagen.

[0129] By using different organic solvents to prepare electrospinning solutions, the microfiber structure prepared by electrospinning technology can be adjusted, and the final freeze-dried sample has both bionic microfiber structure and good porosity.

[0130] Example 3

[0131] The difference between this embodiment and embodiment 1 is that the concentration of the spinning solution is different, and the remaining steps are the same, which are as follows:

[0132] Solution preparation

[0133] Table 3

[0134]

[0135] The preparation steps of the electrospinning solution are as follows:

[0136] ① PLLA dissolution: Add PLLA particles into 30 mL HFIP according to the ratio in the table above and stir magnetically at room temperature for 1 h until the solution becomes colorless and transparent.

[0137] ② Collagen addition: recombinant type III collagen was added and stirred at room temperature for 15 minutes to obtain electrospinning solutions of different concentrations.

[0138] The remaining steps are the same as in Example 1.

[0139] Example 3 adjusts the total polymer concentration, wherein the spinning solution with a low concentration (10%-12%) has a low viscosity and is easy to form fine fibers and a high porosity structure, but the fiber continuity is poor and bead defects are easy to occur; the spinning solution with a medium concentration (15%) has an appropriate viscosity, the fibers are uniform and continuous, the porosity is moderate, and the mechanical strength and biological activity are balanced; the spinning solution with a high concentration (20%) has a significantly increased viscosity, the fiber diameter is coarsened, and the porosity is reduced, but the high PLLA ratio (such as 18%) drives the mechanical strength to be significantly improved.

[0140] The difference in ratio at the same total concentration can regulate the performance in a targeted manner. The collagen concentration dominates the biological activity, and the PLLA concentration dominates the mechanical strength. The two need to be balanced through ratio to achieve the target application.

[0141] According to the concentration of the spinning solution provided by the present invention, the ratio of L-polylactic acid and human recombinant type III collagen is adjusted, and the tendon patches prepared have excellent physical and chemical properties and biological properties.

[0142] Example 4 - A high-porosity bionic tendon repair patch

[0143] The difference between this embodiment and embodiment 1 is that the parameters of the electrospinning technology are different, and the remaining steps are the same, which are as follows:

[0144] electrospinning

[0145] Table 4

[0146]

[0147] The electrospinning solution was made into composite fibers through electrospinning with different parameter settings to obtain PLLA / Col tendon repair patches.

[0148] The subsequent steps are the same as in Example 1.

[0149] In Example 4, the parameters of the electrospinning technology were adjusted, and the microstructure of the patch was the same as that in Example 1. The product had good mechanical properties, and the average tensile strength could reach about 80N.

[0150] If the receiving distance of electrospinning is too long or too short, it will affect the fiber diameter of the patch.

[0151] Comparative Example 1

[0152] The difference between this comparative example and Example 1 is that, in step 7, after cleaning, the sample is placed in a freeze-drying mold (the freeze-drying mold can be customized according to the sample size) and pre-frozen at -80°C for 20 minutes. The remaining and subsequent steps are the same as in Example 1.

[0153] The freezing time is too short, the rotator cuff patch sample is not pre-cooled sufficiently, the residual water in the sample is not completely converted into a solid state, and the water cannot completely sublime from a solid state to a gaseous state, which can easily cause the sample to melt and the protein quality to denature.

[0154] In addition, when the freezing time is too long, the rotator cuff patch sample is over-precooled and the sample breaks due to overcooling.

[0155] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for preparing a high-porosity bionic tendon repair patch, characterized in that: The method comprises the following steps: adding human recombinant collagen to a L-lactic acid solution to prepare an electrospinning solution, electrospinning, placing the solution in a cross-linking solution for immersion and cross-linking, and finally freezing and forming the solution to obtain the bionic tendon repair patch; The mass ratio of the poly(L-lactic acid) to the human recombinant collagen is 1-10:1-3.

2. The method for preparing the bionic tendon repair patch according to claim 1, characterized in that: The mass concentration of the electrospinning solution is 10% to 20%; and the human recombinant collagen is human recombinant type III collagen.

3. The method for preparing the bionic tendon repair patch according to claim 2, wherein: When the mass concentration of the electrospinning solution is 12% to 15%, the mass ratio of the poly(L-lactic acid) to the human recombinant type III collagen is 3 to 8:1 to 2; When the mass concentration of the electrospinning solution is 15.1% to 19%, the mass ratio of the poly (L-lactic acid) to the human recombinant type III collagen is 1 to 10:1 to 3; When the mass concentration of the electrospinning solution is 19.1% to 20%, the mass ratio of the poly(L-lactic acid) to the human recombinant type III collagen is 7 to 9:1 to 3.

4. The method for preparing a bionic tendon repair patch according to claim 1, wherein: The poly(L-lactic acid) solution with a mass concentration of 5% to 15% is prepared by adding the poly(L-lactic acid) into an organic solvent.

5. The method for preparing the bionic tendon repair patch according to claim 4, characterized in that: The organic solvent includes at least one of hexafluoroisopropanol, chloroform, formic acid, tetrafluoroethylene, acetic acid, dichloromethane, ethanol, dimethyl sulfoxide, tetrahydrofuran or dimethylformamide.

6. The method for preparing a bionic tendon repair patch according to claim 1, characterized in that: The process parameters of the electrospinning are: voltage 15-30 kV, propulsion rate 5-8 mL / h, and receiving distance 10-20 cm.

7. The method for preparing a bionic tendon repair patch according to claim 1, characterized in that: The soaking and cross-linking time is 12 to 24 hours.

8. The method for preparing a bionic tendon repair patch according to claim 1, characterized in that: The cross-linking liquid comprises a cross-linking agent, an active agent and water in a mass volume ratio of 8-10g:2-3g:150-250mL, the cross-linking agent comprises a water-soluble carbodiimide cross-linking agent, and the active agent comprises N-hydroxysuccinimide.

9. The method for preparing a bionic tendon repair patch according to claim 1, wherein: The freezing molding includes pre-freezing or liquid nitrogen freezing, the pre-freezing temperature is -80°C to -70°C, the pre-freezing time is 1 to 2 hours, and the liquid nitrogen freezing time is 1 to 5 minutes.

10. The bionic tendon repair patch prepared by the method for preparing a bionic tendon repair patch according to any one of claims 1 to 9.

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