Compression-resistant and slow-release type artificial nerve guiding catheter and preparation method thereof

Abstract

The invention belongs to the technical field of medical biological materials capable of being implanted into a human body, and particularly relates to a compression-resistant and slow-release type artificial nerve guiding catheter and a preparation method thereof. The whole artificial nerve guiding catheter provided by the invention is of a sandwich tube wall structure, and compared with a common single-layer structure, the inner layer and the outer layer are made of high polymer materials (such as PBS / PLA films), micropores (with adjustable density) are formed in the inner layer and the outer layer, and nutrient substances and metabolic waste in and out channels are provided for regenerated nerves; and a semi-closed space is provided for the middle-layer structure of the catheter wall, so that the nerve guiding catheter has pressure resistance. The middle layer is made of a gel material (such as a silk fibroin / agarose double-crosslinking gel network), can absorb water to become a compression-resistant core of the nerve guiding catheter, and can serve as a drug-loading slow-release layer to generate antibacterial performance. Furthermore, the mechanical and shape recovery performance of the nerve conduit can be improved by adjusting the ratio of the silk fibroin to the agarose.

Description

Technical Field

[0001] The invention belongs to the technical field of medical biomaterials that can be implanted into the human body, and in particular relates to a pressure-resistant, slow-release artificial nerve guide catheter and a preparation method thereof. Background Art

[0002] Small (<5mm) nerve defects can be regenerated or repaired by end-to-end suture, while large nerve defects must be regenerated with the help of nerve transplantation or artificial nerve catheters. Autologous nerve transplantation is the gold standard for nerve repair, but it has insurmountable disadvantages such as limited donor sources, secondary surgical damage, and donor site complications. Allogeneic nerve transplantation has alleviated the problem of donor sources to a certain extent, but the risk of immune rejection and disease transmission has increased significantly. Nerve guide catheters are expected to fundamentally solve the problem of nerve transplantation. In particular, natural material-based nerve guide catheters are highly favored for their excellent biocompatibility, controllable degradability, wide range of raw material sources, and easy processing. However, the biggest disadvantage of this type of nerve guide catheter is its poor mechanical properties. The regenerated nerves will be squeezed by the surrounding tissues, resulting in failure of nerve repair.

[0003] Research on nerve guide catheters to repair nerve defects has been conducted since the 1980s, and some products have entered clinical practice, such as the one produced by Integra Life Science in the United States. and NeuroWrap TM Collagen nerve guide catheter, NeuroFlex produced by Collagen Matrix, USA, and Collagen nerve guide catheter, produced by AxoGen, USA and Collagen nerve guide catheters, artificial nerve sheaths produced by Beijing Tianxinfu Medical Devices Co., Ltd., and chitosan nerve guide catheters produced by Jiangsu Yitong Biotechnology Co., Ltd., Beijing Huifukang Medical Technology Co., Ltd. and Hayesmore Biotechnology Co., Ltd. With the increase in clinical cases, 0.2%-5% of cases have failed nerve repair due to infection. Summary of the invention

[0004] In order to overcome the defects of the prior art, one of the objects of the present invention is to provide a pressure-resistant, slow-release artificial nerve guide catheter with improved mechanical properties and anti-infection properties.

[0005] A second object of the present invention is to provide a method for preparing a pressure-resistant, slow-release artificial nerve guide catheter.

[0006] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows:

[0007] A pressure-resistant, slow-release artificial nerve guide catheter comprises, from inside to outside, an inner layer of a porous structure polymer material, a gel material layer, and an outer layer of a porous structure polymer material.

[0008] Optionally, the porous structure polymer material is a PBS / PLA composite material. The optional PBS / PLA composite material is prepared by compounding at a mass ratio of PBS:PLA=1:1.

[0009] Optionally, the gel material is a silk fibroin and agar composite material.

[0010] Optionally, the gel material layer is also loaded with antibacterial active ingredients.

[0011] Optionally, the antibacterial active ingredient is ciprofloxacin.

[0012] Optionally, the porous structure polymer material is prepared by a PBS / PLA organic solvent solution containing a porogen;

[0013] Optionally, the PBS / PLA organic solvent solution containing the porogen is a 31.17 wt % to 33.55 wt % PBS / PLA chloroform solution containing 5 to 20 mg / mL of the porogen;

[0014] Optionally, the gel material layer is prepared from a water / glycerol composite solvent solution of silk fibroin, agar, and / or ciprofloxacin.

[0015] Optionally, the mass ratio of silk fibroin to agarose in the water / glycerol composite solvent solution of silk fibroin, agar, and / or ciprofloxacin is 0.75-1.5: the water / glycerol composite solvent is a composite solvent with a volume ratio of water to glycerol of 9:1;

[0016] Optionally, the silk fibroin mass percentage is 4.11wt% to 8.22wt%, the agarose mass percentage is 5.48wt%, and the glycerol mass percentage is 23.05wt%.

[0017] The method for preparing the above-mentioned pressure-resistant and slow-release artificial nerve guide catheter comprises the following steps:

[0018] 1) Prepare a water-soluble mandrel;

[0019] 2) preparing a PBS / PLA organic solvent solution containing a porogen, and preparing an inner PBS / PLA coating on the surface of the mandrel by a leaching-drying cycle treatment method;

[0020] 3) preparing a water / glycerol composite solvent solution of silk fibroin, agar, and / or ciprofloxacin, cooling the solution to a semi-solid state, and further preparing a drug-loaded or non-drug-loaded silk fibroin / agarose coating on the surface of the PBS / PLA coating prepared in step 2) by an extraction-room temperature solidification cycle treatment method;

[0021] 4) preparing a PBS / PLA organic solvent solution containing a porogen, and preparing an outer PBS / PLA coating on the surface of the silk fibroin / agarose coating prepared in step 3) by using an extraction-drying cycle treatment method;

[0022] 5) Soaking the semi-finished product prepared in step 4) in an aqueous solution to remove the maltitol rod and the pore-forming agent, thereby obtaining the pressure-resistant, sustained-release artificial nerve guide catheter.

[0023] Optionally, the specific method for preparing the water-soluble core rod in step 1) is to use 3D printing technology to print out maltitol rods using maltitol as raw material.

[0024] Optionally, the specific method of preparing the PBS / PLA organic solvent solution containing the porogen in step 2) and step 4) is to prepare a chloroform solution of the PBS / PLA mixture and add ammonium bicarbonate as the porogen;

[0025] The specific method for preparing the water / glycerol composite solvent solution of silk fibroin, agar, and / or ciprofloxacin in step 3) is to dissolve silk fibroin, agarose and / or ciprofloxacin in 80°C water / glycerol and stir at constant temperature to fully dissolve.

[0026] Beneficial effects of the present invention:

[0027] 1) The artificial nerve guide catheter provided by the present invention has a sandwich tube wall structure as a whole, which has significant advantages over ordinary single layers (such as collagen nerve guide catheters). On the one hand, the inner and outer layers of the tube wall of the artificial nerve guide catheter of the present invention are composed of polymer materials (such as PBS / PLA films), which have micropores (density adjustable), which will provide channels for the regenerated nerves to enter and exit nutrients and metabolic waste, and provide a semi-enclosed space for the middle layer structure of the tube wall, so that the nerve guide catheter has pressure resistance. On the other hand, the middle layer of the tube wall of the nerve guide catheter is composed of gel material (such as silk fibroin / agarose double cross-linked gel network), which can not only absorb moisture to become the pressure-resistant core of the nerve guide catheter, but also can serve as a storage warehouse for anti-infective drugs, and avoid potential infection risks during clinical implantation by slowly releasing drugs.

[0028] 2) Furthermore, the artificial nerve guide catheter of the present invention can improve the mechanical properties of the nerve guide catheter by adjusting the ratio of silk fibroin and agarose.

[0029] 3) The artificial nerve guide catheter of the present invention has excellent mechanical properties and shape recovery ability, and has excellent antibacterial activity by loading antibacterial drugs through the intermediate gel layer. In the specific embodiment of the present invention, it is verified through experiments that the artificial nerve guide catheter provided by the present invention can guide the repair of broken nerves. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A stereomicroscope photograph of a cross section of the pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter prepared in Example 10 (Figure A), and an infrared spectrum of the inner layer, middle layer, and outer layer structures (Figure B);

[0031] Figure 2 Scanning electron microscope photos of the cross section (A), middle layer (B), inner layer (C) and outer layer (D) of the pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter prepared in Example 8;

[0032] Scanning electron micrographs of the inner layer (E) and outer layer (F) of the pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter prepared in Example 15;

[0033] Figure 3 The mechanical properties test results of the compression-resistant and sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheters prepared in Examples 4 and 10 (A);

[0034] The pressure variation curve of the pressure-resistant and sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter prepared in Example 10 during 15 cycles of continuous compression and relaxation (B);

[0035] Figure 4 The ciprofloxacin release kinetic curves of the pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheters prepared in Examples 4 and 10 in 0.1 U / mL protease XIV phosphate buffer.

[0036] Figure 5 The antibacterial activity of the pressure-resistant, drug-free PBS / PLA-silk fibroin / agarose artificial nerve guide catheter (control sample) prepared in Example 11 and the pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter prepared in Examples 8 and 10 against Escherichia coli (A) and Staphylococcus aureus (B) was tested by the inhibition zone method;

[0037] Figure 6The monitoring results of the effect of using the compression-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter prepared in Example 10 to repair the 10 mm sciatic nerve defect in New Zealand white rabbits are as follows: Figure A is a picture of the nerve guide catheter connected to the broken sciatic nerve through end-to-end anastomosis; Figure B is an electromyogram 16 weeks after the nerve guide catheter is implanted; Figure C is an H&E staining photo of the longitudinal section of the nerve guide catheter and surrounding tissues 12 weeks after the nerve guide catheter is implanted; Figure D is an S100 fluorescent immunohistochemistry photo of the longitudinal section of the nerve guide catheter and surrounding tissues 12 weeks after the nerve guide catheter is implanted. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with specific implementation modes, but the protection scope of the present invention is not limited thereto.

[0039] The PBS / PLA complex used in the following examples is a commercially available product, or a mixture of medical source materials in a mass ratio of PBS:PLA=1:1.

[0040] Example 1

[0041] Maltitol sticks were prepared using 3D printing technology: maltitol was used as the raw material, the melting temperature was set to 150°C, the melting time was 30 minutes, the printing pressure was 100 kPa, and the inner diameter of the print head was 0.4 mm.

[0042] Example 2

[0043] This embodiment provides a pressure-resistant, slow-release artificial nerve guide catheter, and the preparation method thereof is specifically as follows:

[0044] 1) Dissolve PBS / PLA with a weight fraction of 33.55% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on the surface of the maltitol rod, thereby obtaining a maltitol rod-PBS / PLA coating;

[0045] 2) 4.11% silk fibroin by weight, 5.48% agarose by weight and 0.27% ciprofloxacin by weight were dissolved in 80°C water / glycerol (volume ratio = 9:1, glycerol weight fraction 23.05%), stirred at constant temperature to fully dissolve, and then cooled to a semi-solidified state. The above maltitol rod-PBS / PLA coating was subjected to leaching-room temperature curing cycle treatment in the semi-solidified solution for 3 times, and a silk fibroin / agarose coating was prepared on the surface of the PBS / PLA coating, that is, a maltitol rod-PBS / PLA coating-silk fibroin / agarose coating was obtained;

[0046] 4) Dissolve PBS / PLA with a weight fraction of 33.55% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod-PBS / PLA coating-silk fibroin / agarose coating is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on its surface, and obtain an artificial nerve guide catheter with maltitol rod and porogen;

[0047] 5) soaking in an aqueous solution to remove the maltitol rod and the porogen, thereby obtaining a pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter.

[0048] Example 3

[0049] This embodiment provides a pressure-resistant, slow-release artificial nerve guide catheter, and the preparation method thereof is specifically as follows:

[0050] 1) PBS / PLA with a weight fraction of 33.39% was dissolved in a chloroform solution. Ammonium bicarbonate porogen was added and stirred to a concentration of 5 mg / mL. Then, the maltitol rod was subjected to 5 times of leaching-drying cycle in the above chloroform solution, thereby forming a PBS / PLA coating on the surface of the maltitol rod to obtain a maltitol rod-PBS / PLA coating.

[0051] 2) 4.11% silk fibroin by weight, 5.48% agarose by weight and 0.59% ciprofloxacin by weight were dissolved in 80°C water / glycerol (volume ratio = 9:1, glycerol weight fraction 23.05%), stirred at constant temperature to fully dissolve, and then cooled to a semi-solidified state. The above maltitol rod-PBS / PLA coating was subjected to leaching-room temperature curing cycle treatment in the semi-solidified solution for 3 times, and a silk fibroin / agarose coating was prepared on the surface of the PBS / PLA coating, that is, a maltitol rod-PBS / PLA coating-silk fibroin / agarose coating was obtained.

[0052] 3) Dissolve PBS / PLA with a weight fraction of 33.39% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well to a concentration of 5 mg / mL. Then, the maltitol rod-PBS / PLA coating-silk fibroin / agarose coating is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on its surface, thereby obtaining an artificial nerve guide catheter with maltitol rod and porogen.

[0053] 4) soaking in an aqueous solution to remove the maltitol rod and the porogen, thereby obtaining a pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter.

[0054] Example 4

[0055] This embodiment provides a pressure-resistant, slow-release artificial nerve guide catheter, and the preparation method thereof is specifically as follows:

[0056] 1) Dissolve PBS / PLA with a weight fraction of 33.23% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on the surface of the maltitol rod, thereby obtaining a maltitol rod-PBS / PLA coating;

[0057] 2) 4.11% silk fibroin by weight, 5.48% agarose by weight and 0.91% ciprofloxacin by weight were dissolved in 80°C water / glycerol (volume ratio = 9:1, glycerol weight fraction 23.05%), stirred at constant temperature to fully dissolve, and then cooled to a semi-solidified state. The above maltitol rod-PBS / PLA coating was subjected to 5 cycles of leaching-room temperature curing in the semi-solidified solution to prepare a silk fibroin / agarose coating on the surface of the PBS / PLA coating, that is, a maltitol rod-PBS / PLA coating-silk fibroin / agarose coating was obtained;

[0058] 3) Dissolve PBS / PLA with a weight fraction of 33.23% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod-PBS / PLA coating-silk fibroin / agarose coating is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on its surface, thereby obtaining an artificial nerve guide catheter with maltitol rod and porogen;

[0059] 4) soaking in an aqueous solution to remove the maltitol rod and the porogen, thereby obtaining a pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter.

[0060] Example 5

[0061] This embodiment provides a pressure-resistant, slow-release artificial nerve guide catheter, and the preparation method thereof is specifically as follows:

[0062] 1) Dissolve PBS / PLA with a weight fraction of 32.93% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on the surface of the maltitol rod, thereby obtaining a maltitol rod-PBS / PLA coating;

[0063] 2) Dissolve 5.34% silk fibroin by weight, 5.48% agarose by weight, and 0.27% ciprofloxacin by weight in 80°C water / glycerol (volume ratio = 9:1, glycerol weight fraction is 23.05%), stir at constant temperature to fully dissolve, and then cool to a semi-solid state. The above-mentioned maltitol rod-PBS / PLA coating is subjected to 3 cycles of leaching-room temperature curing in the semi-solid solution to prepare a silk fibroin / agarose coating on the surface of the PBS / PLA coating, that is, to obtain a maltitol rod-PBS / PLA coating-silk fibroin / agarose coating;

[0064] 3) Dissolve PBS / PLA with a weight fraction of 32.93% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod-PBS / PLA coating-silk fibroin / agarose coating is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on its surface, thereby obtaining an artificial nerve guide catheter with maltitol rod and porogen;

[0065] 4) soaking in an aqueous solution to remove the maltitol rod and the porogen, thereby obtaining a pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter.

[0066] Example 6

[0067] This embodiment provides a pressure-resistant, slow-release artificial nerve guide catheter, and the preparation method thereof is specifically as follows:

[0068] 1) Dissolve PBS / PLA with a weight fraction of 32.77% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on the surface of the maltitol rod, thereby obtaining a maltitol rod-PBS / PLA coating;

[0069] 2) Dissolve 5.34% silk fibroin by weight, 5.48% agarose by weight, and 0.59% ciprofloxacin by weight in 80°C water / glycerol (volume ratio = 9:1, glycerol weight fraction is 23.05%), stir at constant temperature to fully dissolve, and then cool to a semi-solid state. The above-mentioned maltitol rod-PBS / PLA coating is subjected to a leaching-room temperature curing cycle for 3 times in the semi-solidified solution to prepare a silk fibroin / agarose coating on the surface of the PBS / PLA coating, that is, to obtain a maltitol rod-PBS / PLA coating-silk fibroin / agarose coating;

[0070] 3) Dissolve PBS / PLA with a weight fraction of 32.77% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod-PBS / PLA coating-silk fibroin / agarose coating is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on its surface, thereby obtaining an artificial nerve guide catheter with maltitol rod and porogen;

[0071] 4) soaking in an aqueous solution to remove the maltitol rod and the porogen, thereby obtaining a pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter.

[0072] Example 7

[0073] This embodiment provides a pressure-resistant, slow-release artificial nerve guide catheter, and the preparation method thereof is specifically as follows:

[0074] 1) Dissolve PBS / PLA with a weight fraction of 32.61% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on the surface of the maltitol rod, thereby obtaining a maltitol rod-PBS / PLA coating;

[0075] 2) Dissolve 5.34% silk fibroin by weight, 5.48% agarose by weight, and 0.91% ciprofloxacin by weight in 80°C water / glycerol (volume ratio = 9:1, glycerol weight fraction is 23.05%), stir at constant temperature to fully dissolve, and then cool to a semi-solid state. The maltitol rod-PBS / PLA coating is subjected to a leaching-room temperature curing cycle for 3 times in the semi-solidified solution to prepare a silk fibroin / agarose coating on the surface of the PBS / PLA coating, that is, to obtain a maltitol rod-PBS / PLA coating-silk fibroin / agarose coating;

[0076] 3) Dissolve PBS / PLA with a weight fraction of 32.61% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod-PBS / PLA coating-silk fibroin / agarose coating is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on its surface, thereby obtaining an artificial nerve guide catheter with maltitol rod and porogen;

[0077] 4) soaking in an aqueous solution to remove the maltitol rod and the porogen, thereby obtaining a pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter.

[0078] Example 8

[0079] This embodiment provides a pressure-resistant, slow-release artificial nerve guide catheter, and the preparation method thereof is specifically as follows:

[0080] 1) Dissolve PBS / PLA with a weight fraction of 31.49% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on the surface of the maltitol rod, thereby obtaining a maltitol rod-PBS / PLA coating;

[0081] 2) 8.22% silk fibroin by weight, 5.48% agarose by weight and 0.27% ciprofloxacin by weight were dissolved in 80°C water / glycerol (volume ratio = 9:1, glycerol weight fraction 23.05%), stirred at constant temperature to fully dissolve, and then cooled to a semi-solidified state. The above maltitol rod-PBS / PLA coating was subjected to leaching-room temperature curing cycle treatment in the semi-solidified solution for 3 times, and a silk fibroin / agarose coating was prepared on the surface of the PBS / PLA coating, that is, a maltitol rod-PBS / PLA coating-silk fibroin / agarose coating was obtained;

[0082] 3) Dissolve PBS / PLA with a weight fraction of 31.49% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod-PBS / PLA coating-silk fibroin / agarose coating is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on its surface, thereby obtaining an artificial nerve guide catheter with maltitol rod and porogen;

[0083] 4) soaking in an aqueous solution to remove the maltitol rod and the porogen, thereby obtaining a pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter.

[0084] Example 9

[0085] This embodiment provides a pressure-resistant, slow-release artificial nerve guide catheter, and the preparation method thereof is specifically as follows:

[0086] 1) Dissolve PBS / PLA with a weight fraction of 31.33% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on the surface of the maltitol rod, thereby obtaining a maltitol rod-PBS / PLA coating;

[0087] 2) 8.22% silk fibroin by weight, 5.48% agarose by weight and 0.59% ciprofloxacin by weight were dissolved in 80°C water / glycerol (volume ratio = 9:1, glycerol weight fraction 23.05%), stirred at constant temperature to fully dissolve, and then cooled to a semi-solidified state. The above maltitol rod-PBS / PLA coating was subjected to leaching-room temperature curing cycle treatment in the semi-solidified solution for 3 times, and a silk fibroin / agarose coating was prepared on the surface of the PBS / PLA coating, that is, a maltitol rod-PBS / PLA coating-silk fibroin / agarose coating was obtained;

[0088] 3) Dissolve PBS / PLA with a weight fraction of 31.33% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod-PBS / PLA coating-silk fibroin / agarose coating is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on its surface, thereby obtaining an artificial nerve guide catheter with maltitol rod and porogen;

[0089] 4) soaking in an aqueous solution to remove the maltitol rod and the porogen, thereby obtaining a pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter.

[0090] Example 10

[0091] This embodiment provides a pressure-resistant, slow-release artificial nerve guide catheter, and the preparation method thereof is specifically as follows:

[0092] 1) Dissolve PBS / PLA with a weight fraction of 31.17% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on the surface of the maltitol rod, thereby obtaining a maltitol rod-PBS / PLA coating;

[0093] 2) 8.22% silk fibroin by weight, 5.48% agarose by weight and 0.91% ciprofloxacin by weight were dissolved in 80°C water / glycerol (volume ratio = 9:1, glycerol weight fraction 23.05%), stirred at constant temperature to fully dissolve, and then cooled to a semi-solidified state. The maltitol rod-PBS / PLA coating was subjected to a leaching-room temperature curing cycle for 3 times in the semi-solidified solution to prepare a silk fibroin / agarose coating on the surface of the PBS / PLA coating, that is, a maltitol rod-PBS / PLA coating-silk fibroin / agarose coating was obtained;

[0094] 3) Dissolve PBS / PLA with a weight fraction of 31.17% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod-PBS / PLA coating-silk fibroin / agarose coating is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on its surface, thereby obtaining an artificial nerve guide catheter with maltitol rod and porogen;

[0095] 4) soaking in an aqueous solution to remove the maltitol rod and the porogen, thereby obtaining a pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter.

[0096] Embodiment 11

[0097] This embodiment provides a pressure-resistant, slow-release artificial nerve guide catheter, and the preparation method thereof is specifically as follows:

[0098] 1) Dissolve PBS / PLA with a weight fraction of 31.63% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on the surface of the maltitol rod, thereby obtaining a maltitol rod-PBS / PLA coating;

[0099] 2) 8.22% silk fibroin by weight and 5.48% agarose by weight were dissolved in 80°C water / glycerol (volume ratio = 9:1, glycerol weight fraction 23.05%), stirred at constant temperature to fully dissolve, and then cooled to a semi-solidified state. The above maltitol rod-PBS / PLA coating was subjected to leaching-room temperature curing cycle treatment in the semi-solidified solution for 3 times, and a silk fibroin / agarose coating was prepared on the surface of the PBS / PLA coating, that is, a maltitol rod-PBS / PLA coating-silk fibroin / agarose coating was obtained;

[0100] 3) Dissolve PBS / PLA with a weight fraction of 31.63% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod-PBS / PLA coating-silk fibroin / agarose coating is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on its surface, thereby obtaining an artificial nerve guide catheter with maltitol rod and porogen;

[0101] 4) soaking in an aqueous solution to remove the maltitol rod and the porogen, thereby obtaining a pressure-resistant, drug-free PBS / PLA-silk fibroin / agarose artificial nerve guide catheter.

[0102] Example 12

[0103] This embodiment provides a pressure-resistant, slow-release artificial nerve guide catheter, and the preparation method thereof is specifically as follows:

[0104] 1) Dissolve PBS / PLA with a weight fraction of 33.07% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on the surface of the maltitol rod, thereby obtaining a maltitol rod-PBS / PLA coating;

[0105] 2) Dissolve 5.34% silk fibroin by weight and 5.48% agarose by weight in 80°C water / glycerol (volume ratio = 9:1, glycerol weight fraction is 23.05%), stir at constant temperature to fully dissolve, and then cool to a semi-solid state. The above maltitol rod-PBS / PLA coating is subjected to 3 cycles of leaching-room temperature curing in the semi-solid solution to prepare a silk fibroin / agarose coating on the surface of the PBS / PLA coating, that is, to obtain a maltitol rod-PBS / PLA coating-silk fibroin / agarose coating;

[0106] 3) Dissolve PBS / PLA with a weight fraction of 33.07% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod-PBS / PLA coating-silk fibroin / agarose coating is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on its surface, thereby obtaining an artificial nerve guide catheter with maltitol rod and porogen;

[0107] 4) soaking in an aqueous solution to remove the maltitol rod and the porogen, thereby obtaining a pressure-resistant, drug-free PBS / PLA-silk fibroin / agarose artificial nerve guide catheter.

[0108] Example 13

[0109] This embodiment provides a pressure-resistant, slow-release artificial nerve guide catheter, and the preparation method thereof is specifically as follows:

[0110] 1) Dissolve PBS / PLA with a weight fraction of 33.68% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 5 mg / mL. Then, the maltitol rod is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on the surface of the maltitol rod, thereby obtaining a maltitol rod-PBS / PLA coating;

[0111] 2) Dissolve 4.11% silk fibroin by weight and 5.48% agarose by weight in 80°C water / glycerol (volume ratio = 9:1, glycerol weight fraction is 23.05%), stir at constant temperature to fully dissolve, and then cool to a semi-solidified state. The above maltitol rod-PBS / PLA coating is subjected to 3 cycles of leaching-room temperature curing in the semi-solidified solution to prepare a silk fibroin / agarose coating on the surface of the PBS / PLA coating, that is, to obtain a maltitol rod-PBS / PLA coating-silk fibroin / agarose coating;

[0112] 3) Dissolve PBS / PLA with a weight fraction of 33.68% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 10 mg / mL. Then, the maltitol rod-PBS / PLA coating-silk fibroin / agarose coating is subjected to 5 extraction-drying cycles in the above chloroform solution to form a PBS / PLA coating on its surface, thereby obtaining an artificial nerve guide catheter with maltitol rod and porogen;

[0113] 4) soaking in an aqueous solution to remove the maltitol rod and the porogen, thereby obtaining a pressure-resistant, drug-free PBS / PLA-silk fibroin / agarose artificial nerve guide catheter.

[0114] Embodiment 14

[0115] This embodiment provides a pressure-resistant, slow-release artificial nerve guide catheter, and the preparation method thereof is specifically as follows:

[0116] 1) Dissolve PBS / PLA with a weight fraction of 31.17% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 10 mg / mL. Then, the maltitol rod is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on the surface of the maltitol rod, thereby obtaining a maltitol rod-PBS / PLA coating;

[0117] 2) 8.22% silk fibroin by weight, 5.48% agarose by weight and 0.91% ciprofloxacin by weight were dissolved in 80°C water / glycerol (volume ratio = 9:1, glycerol weight fraction 23.05%), stirred at constant temperature to fully dissolve, and then cooled to a semi-solidified state. The maltitol rod-PBS / PLA coating was subjected to a leaching-room temperature curing cycle for 3 times in the semi-solidified solution to prepare a silk fibroin / agarose coating on the surface of the PBS / PLA coating, that is, a maltitol rod-PBS / PLA coating-silk fibroin / agarose coating was obtained;

[0118] 3) Dissolve PBS / PLA with a weight fraction of 31.17% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well to a concentration of 10 mg / mL. Then, the maltitol rod-PBS / PLA coating-silk fibroin / agarose coating is subjected to 5 extraction-drying cycles in the above chloroform solution to form a PBS / PLA coating on its surface, thereby obtaining an artificial nerve guide catheter with a maltitol rod and a porogen.

[0119] 4) soaking in an aqueous solution to remove the maltitol rod and the porogen, thereby obtaining a pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter.

[0120] Embodiment 15

[0121] This embodiment provides a pressure-resistant, slow-release artificial nerve guide catheter, and the preparation method thereof is specifically as follows:

[0122] 1) Dissolve PBS / PLA with a weight fraction of 31.49% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 20 mg / mL. Then, the maltitol rod is subjected to 5 times of leaching-drying cycle in the above chloroform solution to form a PBS / PLA coating on the surface of the maltitol rod, thereby obtaining a maltitol rod-PBS / PLA coating;

[0123] 2) 8.22% silk fibroin by weight, 5.48% agarose by weight and 0.27% ciprofloxacin by weight were dissolved in 80°C water / glycerol (volume ratio = 9:1, glycerol weight fraction 23.05%), stirred at constant temperature to fully dissolve, and then cooled to a semi-solidified state. The above maltitol rod-PBS / PLA coating was subjected to leaching-room temperature curing cycle treatment in the semi-solidified solution for 3 times, and a silk fibroin / agarose coating was prepared on the surface of the PBS / PLA coating, that is, a maltitol rod-PBS / PLA coating-silk fibroin / agarose coating was obtained;

[0124] 3) Dissolve PBS / PLA with a weight fraction of 31.49% in chloroform solution. Add ammonium bicarbonate porogen and stir to mix well. The concentration is 20 mg / mL. Then, the maltitol rod-PBS / PLA coating-silk fibroin / agarose coating is subjected to 5 extraction-drying cycles in the above chloroform solution to form a PBS / PLA coating on its surface, thereby obtaining an artificial nerve guide catheter with maltitol rod and porogen;

[0125] 4) soaking in an aqueous solution to remove the maltitol rod and the porogen, thereby obtaining a pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter.

[0126] Test case performance characterization

[0127] 1. Physical and molecular structure characterization: Figure 1 As shown in the stereomicroscope photograph of the cross section of the pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter prepared in Example 10 in Figure A, the artificial nerve guide catheter provided by the present invention consists of a three-layer structure.

[0128] From the infrared spectra of each layer (Figure B), it can be seen that in the infrared spectrum of the middle layer of the pressure-resistant and sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter prepared in Example 10: -1 、924cm -1 and 1035cm -1 The peak at 1635 cm can be attributed to CO in agarose (axial deformation). -1 、1516cm -1 and 1236cm -1 The peaks at 3367 cm-1 correspond to amide I, amide II, and amide III of silk protein. The strong absorption peak of -OH of glycerol appears at 3367 cm-1. -1 The characteristic peak of ciprofloxacin appears at 1402 cm -1 (stretching vibration of the protons of the amine group in the piperazine moiety) and 1300 cm -1 The presence of these characteristic peaks proves that the middle layer of the artificial nerve guide catheter provided by the present invention is composed of silk fibroin, agarose, glycerol and ciprofloxacin. In the infrared spectrum of the inner and outer layers of the pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter: 1738cm -1 Corresponding to the carboxylic acid group of PLA, at the same time, 3427 cm -1 The characteristic absorption peak of hydroxyl group appeared. The carbon-oxygen bond bending vibration of PBS appeared at 1450-1460cm -1 The existence of these characteristic peaks proves that the inner layer and the outer layer of the artificial nerve guiding catheter provided by the present invention are composed of PBS / PLA.

[0129] 2. Characterization of inner and outer pore structures: Figure 2 Scanning electron microscope photos show that the pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter prepared by using 5 mg / mL porogen in Example 8 consists of a three-layer structure (Figure A). The middle layer has an extremely high pore density with a pore size of tens to tens of microns (Figure B). The inner layer (Figure C) and the outer layer (Figure D) have a pore structure with a size of about 200 nm on the surface and a lower pore density. The pore density of the outer layer is higher than that of the inner layer.

[0130] In Example 15, the inner layer (Figure E) and outer layer (Figure F) of the pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter prepared using 20 mg / mL porogen have a pore structure of about 200 nm in size, and the pore density is significantly increased.

[0131] 3. Characterization Methods

[0132] The tensile and bending properties of the samples were measured at room temperature using a universal mechanical analyzer. For the tensile test, the length and outer diameter of the nerve guide catheter were 15 mm and 3.5 mm, respectively. The clamp distance and crosshead movement speed were 10 mm and 20 mm / min, respectively. For the bending properties, the standard three-point bending test method was used, and the corresponding data were 30 mm, 3.5 mm, 25 mm, and 20 mm / min, respectively.

[0133] from Figure 3 As can be seen from Figure A, the elastic strength and elastic modulus of the compression-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter prepared in Example 4 reached 0.226±0.025MPa and 2.364±0.335MPa, and the flexural strength and flexural modulus reached 0.702±0.248MPa and 3.872±2.468MPa.

[0134] The elastic properties of the compression-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter prepared in Example 10 were significantly enhanced, and its elastic strength and elastic modulus reached 0.989±0.118MPa and 7.256±0.757MPa; however, the bending properties were reduced, and its bending strength and bending modulus reached 0.175±0.041MPa and 0.3±0.099MPa.

[0135] As can be seen from Figure B, the pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter prepared in Example 10 did not undergo significant changes in pressure during 15 cycles of continuous compression and relaxation, demonstrating that the catheter still maintained good shape recovery performance.

[0136] 4. Characterization Methods

[0137] The release kinetics curve of ciprofloxacin in the sample was determined in a phosphate buffer containing 0.1U / mL protease XIV. Specifically, prepare 20mL of phosphate buffer containing 0.1U / mL protease XIV at a constant temperature of 37°C and put it into a dialysis bag (molecular retention is 3500D), and prepare 400mL of phosphate buffer at a constant temperature of 37°C and put it into a beaker. Place a sample with a length of 10mm in the solution of the above dialysis bag. Then, place the dialysis bag in the above beaker containing phosphate buffer. Stir gently at 37°C, and at different time points, take out 0.4mL of phosphate buffer for testing from the beaker (add the same volume of fresh phosphate buffer at the same time). Use a spectrophotometer to measure the light absorption value of the test solution at a wavelength of 271nm. Using a standard ciprofloxacin solution, draw a ciprofloxacin light absorption-content standard curve, calculate the ciprofloxacin concentration in the test solution, and after conversion, you can obtain the ciprofloxacin release kinetics in the sample.

[0138] from Figure 4 The drug release kinetics curves of the pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheters prepared in Examples 4 and 10 show that the artificial nerve guide catheters can slowly release ciprofloxacin, and the in vitro release amount reaches 4.3-4.9 μg / mL in 20 days. The difference in silk fibroin content in the artificial nerve guide catheters has no significant effect on the release of ciprofloxacin.

[0139] 5. From Figure 5 It can be seen from the results of the inhibition zone experiment on Escherichia coli that there is no inhibition zone around the pressure-resistant, drug-free PBS / PLA-silk fibroin / agarose artificial nerve guide catheter (control sample) prepared in Example 11, which proves that the pressure-resistant, drug-free PBS / PLA-silk fibroin / agarose artificial nerve guide catheter (control sample) does not have antibacterial activity. Inhibition zones can be seen around the pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheters prepared in Examples 8 and 10, and the inhibition zone corresponding to Example 10 is significantly larger than the inhibition zone corresponding to Example 8, which proves that the pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter has antibacterial activity against E. coli, and the antibacterial activity increases with the increase of ciprofloxacin content. From the results of the inhibition zone experiment on Staphylococcus aureus in Figure B, it can be seen that the results are similar to those of Escherichia coli, but the size of the inhibition zone is larger, proving that the pressure-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter has antibacterial properties against both Staphylococcus aureus and Escherichia coli, but the antibacterial performance against Staphylococcus aureus is stronger than that against Escherichia coli.

[0140] 6. Repair of 10 mm sciatic nerve defect in New Zealand white rabbits using the compression-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter prepared in Example 10:

[0141] from Figure 6 As can be seen from Figure A, the compression-resistant, sustained-release PBS / PLA-silk fibroin / agarose artificial nerve guide catheter prepared by Example 10 can connect broken nerves together through end-to-end anastomosis. As can be seen from Figure B, electromyographic signals can be detected 16 weeks after the nerve guide catheter is implanted, proving that nerve regeneration is successful. As can be seen from the HE staining image in Figure C, there are regenerated nerves in the nerve guide catheter cavity 12 weeks after the nerve guide catheter is implanted. As can be seen from the HE staining image in Figure C, S100 fluorescent immunohistochemistry analysis 12 weeks after the nerve guide catheter is implanted shows that Schwann cells are detected in the nerve guide catheter, further proving the generation of regenerated nerves.

[0142] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pressure-resistant, slow-release artificial nerve guide catheter, characterized in that: From the inside to the outside, they are the inner layer of porous structure polymer material, the gel material layer, and the outer layer of porous structure polymer material.

2. The pressure-resistant, slow-release artificial nerve guide catheter according to claim 1, characterized in that: The porous structure polymer material is a PBS / PLA composite material.

3. The pressure-resistant, slow-release artificial nerve guide catheter according to claim 2, characterized in that: The gel material is a composite material of silk fibroin and agar.

4. The pressure-resistant, slow-release artificial nerve guide catheter according to claim 3, characterized in that: The gel material layer is also loaded with antibacterial active ingredients. Optionally, the antibacterial active ingredient is ciprofloxacin.

5. The pressure-resistant, slow-release artificial nerve guide catheter according to claim 4, characterized in that: The porous structure polymer material is prepared by using a PBS / PLA organic solvent solution containing a porogen; Optionally, the PBS / PLA organic solvent solution containing the porogen is a 31.17 wt % to 33.55 wt % PBS / PLA chloroform solution containing 5 to 20 mg / mL of the porogen.

6. The pressure-resistant, slow-release artificial nerve guide catheter according to claim 5, characterized in that: The gel material layer is prepared from a water / glycerol composite solvent solution of silk fibroin, agar, and / or ciprofloxacin. Optionally, the mass ratio of silk fibroin to agarose in the water / glycerol composite solvent solution of silk fibroin, agar, and / or ciprofloxacin is 0.75-1.5: the water / glycerol composite solvent is a composite solvent with a volume ratio of water to glycerol of 9:1; Optionally, the silk fibroin mass percentage is 4.11wt% to 8.22wt%, the agarose mass percentage is 5.48wt%, and the glycerol mass percentage is 23.05wt%.

7. A method for preparing the pressure-resistant, slow-release artificial nerve guide catheter as claimed in claim 6, characterized in that: The steps include: 1) Prepare a water-soluble mandrel; 2) preparing a PBS / PLA organic solvent solution containing a porogen, and preparing an inner PBS / PLA coating on the surface of the mandrel by a leaching-drying cycle treatment method; 3) preparing a water / glycerol composite solvent solution of silk fibroin, agar, and / or ciprofloxacin, cooling the solution to a semi-solid state, and further preparing a drug-loaded or non-drug-loaded silk fibroin / agarose coating on the surface of the PBS / PLA coating prepared in step 2) by an extraction-room temperature solidification cycle treatment method; 4) preparing a PBS / PLA organic solvent solution containing a porogen, and preparing an outer PBS / PLA coating on the surface of the silk fibroin / agarose coating prepared in step 3) by using an extraction-drying cycle treatment method; 5) Soaking the semi-finished product prepared in step 4) in an aqueous solution to remove the maltitol rod and the pore-forming agent, thereby obtaining the pressure-resistant, sustained-release artificial nerve guide catheter.

8. The method for preparing the pressure-resistant, sustained-release artificial nerve guide catheter according to claim 7, characterized in that: The specific method for preparing the water-soluble core rod in step 1) is to use 3D printing technology to print out maltitol rods using maltitol as raw material.

9. The method for preparing the pressure-resistant and sustained-release artificial nerve guiding catheter according to claim 8, characterized in that: The specific method of preparing the PBS / PLA organic solvent solution containing the porogen in step 2) and step 4) is to prepare a chloroform solution of the PBS / PLA mixture and add the ammonium bicarbonate porogen; The specific method for preparing the water / glycerol composite solvent solution of silk fibroin, agar, and / or ciprofloxacin in step 3) is to dissolve silk fibroin, agarose and / or ciprofloxacin in 80°C water / glycerol and stir at constant temperature to fully dissolve.

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