Polycaprolactone stent with multi-layer pore structure as well as preparation method and application of polycaprolactone stent

Abstract

The invention belongs to the technical field of medical stents, and particularly relates to a polycaprolactone stent with a multi-layer pore structure as well as a preparation method and application of the polycaprolactone stent. The polycaprolactone stent comprises a polycaprolactone stent body and ZIF8 attached to the surface of the polycaprolactone stent body, a base material of the polycaprolactone stent body is polycaprolactone, micron pore structures are arranged between fibers of the polycaprolactone and on the surfaces of the fibers, and the size of the micron pore structures between the fibers is larger than that of the micron pore structures on the surfaces of the fibers. The polycaprolactone stent body is provided with macroscopic macropores and microscopic pores, so that the specific surface area and the surface roughness of the stent body can be improved, the degradation effect of the polycaprolactone stent body is further improved, and cell adhesion can be promoted; besides, the large specific surface area can provide more sites for ZIF8 deposition, the bioactivity of the polycaprolactone scaffold is improved, the polycaprolactone scaffold is suitable for regeneration and repair of bone defects, and the polycaprolactone scaffold has good mechanical strength, biocompatibility and antibacterial property.

Description

Technical Field

[0001] The invention belongs to the technical field of medical stents, and particularly relates to a polycaprolactone stent with a multi-level pore structure and a preparation method and application thereof. Background Art

[0002] The ideal bone tissue engineering scaffold should have an interconnected porous network structure, providing interconnected channels for cell migration, ion transport, and cell-to-cell interaction. Conventional technologies include foaming, sacrificial template method, freeze drying and casting. 3D printing, also known as additive manufacturing technology, has penetrated into all walks of life due to its significant advantages in high precision, personalized manufacturing and complex shape construction. 3D printing obtains a three-dimensional entity by stacking raw materials layer by layer on a plane. Currently, the more mature 3D printing technologies include photopolymerization 3D printing, fused deposition 3D printing, inkjet 3D printing and laser sintering 3D printing. Among them, the basic principle of fused deposition 3D printing technology is that the base material is heated in the nozzle to a temperature slightly higher than its melting point. Under the action of pressure, the base material is extruded from the nozzle into filaments, and the nozzle moves in three dimensions under computer control, thereby stacking the base material filaments into shape. Compared with other scaffold preparation technologies, 3D printing technology can freely design the porosity and shape of the scaffold, which can well meet the needs of bone tissue engineering.

[0003] Polycaprolactone (PCL) is a commonly used material for 3D printing. It is widely used in the medical field because of its good biocompatibility, non-toxicity, degradability, and ability to induce tissue regeneration and provide a place for cell adhesion, proliferation and differentiation. However, the hydrophobicity of PCL will affect the adhesion of the scaffold, and its mechanical structure will be significantly damaged after degradation.

[0004] At present, there are many studies on polycaprolactone-based scaffolds, but the obtained polycaprolactone-based scaffolds or ZIF8 surface modified materials generally have the following problems: 1) insufficient osteogenic activity of the material; 2) the scaffolds degrade too slowly; 3) the preparation process is complicated and difficult to industrialize.

[0005] Therefore, it is of great significance to provide a polycaprolactone scaffold with good osteogenic activity and degradability, which can promote the repair and regeneration of bone tissue defects. Summary of the invention

[0006] The present invention aims to solve one or more technical problems existing in the above-mentioned prior art and at least provide a beneficial choice. Specifically, the present invention provides a polycaprolactone scaffold, which has good osteogenic activity and degradability and can promote the repair and regeneration of bone tissue defects.

[0007] The inventive concept of the present invention is as follows: the polycaprolactone scaffold of the present invention comprises a polycaprolactone scaffold body, and a zeolite imidazolate framework material (ZIF8) attached to the surface of the polycaprolactone scaffold body; the matrix material of the polycaprolactone scaffold body is polycaprolactone, and the fibers and the fiber surface of the polycaprolactone both have a micron pore structure; the size of the micron pore structure between the fibers of the polycaprolactone is larger than the size of the micron pore structure on the fiber surface of the polycaprolactone. The fibers and the fiber surface of the polycaprolactone of the present invention have a micron pore structure, so that the polycaprolactone scaffold body simultaneously obtains macroscopic macropores (gaps between fibers) and microscopic pores (holes on the fiber surface), significantly improving the specific surface area of ​​the polycaprolactone scaffold body, promoting the degradation of the polycaprolactone scaffold body, making it more suitable for the regeneration and repair of bone defects, and at the same time, the macroscopic macropores and microscopic pores can also significantly improve the surface roughness of the polycaprolactone scaffold body, so as to achieve the purpose of promoting cell adhesion of the polycaprolactone scaffold. In addition, the deposition and modification of ZIF8 on the surface of the polycaprolactone scaffold can give the polycaprolactone scaffold biological activity and induce osteogenic differentiation of stem cells. The large specific surface area of ​​the polycaprolactone scaffold can provide more sites for the deposition of ZIF8, which is beneficial to enhance the biological activity of the polycaprolactone scaffold and is suitable for the regeneration and repair of bone defects.

[0008] Therefore, the first aspect of the present invention provides a polycaprolactone scaffold having a multi-level pore structure.

[0009] Specifically, the polycaprolactone stent with a multi-level pore structure includes a polycaprolactone stent body and a zeolite imidazolate framework material attached to the surface of the polycaprolactone stent body;

[0010] The matrix material of the polycaprolactone stent body is polycaprolactone, and the fibers and the fiber surfaces of the polycaprolactone have micron pore structures; the size of the micron pore structure between the polycaprolactone fibers is larger than the size of the micron pore structure on the fiber surfaces of the polycaprolactone.

[0011] Preferably, the polycaprolactone stent body is a 3D printed stent.

[0012] Preferably, the molecular weight of the polycaprolactone is 27,000-65,000 Daltons; further preferably, the molecular weight of the polycaprolactone is 30,000-60,000 Daltons.

[0013] Preferably, the size of the micron pore structure between the fibers of polycaprolactone is 200-650 μm; further preferably, the size of the micron pore structure between the fibers of polycaprolactone is 200-600 μm.

[0014] Preferably, the size of the micron pore structure on the surface of the polycaprolactone fiber is 0.9-110 μm; further preferably, the size of the micron pore structure on the surface of the polycaprolactone fiber is 1-100 μm.

[0015] The second aspect of the present invention provides a method for preparing the polycaprolactone scaffold with a multi-level pore structure according to the first aspect of the present invention.

[0016] Specifically, the method for preparing the polycaprolactone scaffold with a multi-level pore structure comprises the following steps:

[0017] (1) mixing polycaprolactone, gluconolactone and a solvent to obtain a gluconolactone / polycaprolactone solution;

[0018] (2) preparing the gluconolactone / polycaprolactone solution obtained in step (1) into a gluconolactone / polycaprolactone film, and then printing it to obtain a gluconolactone / polycaprolactone scaffold;

[0019] (3) mixing the gluconolactone / polycaprolactone scaffold obtained in step (2) with an aqueous solution, and shaking to obtain a polycaprolactone scaffold body; and mixing the polycaprolactone scaffold body, a 2-methylimidazole aqueous solution, and a zinc nitrate aqueous solution, and reacting them to obtain the polycaprolactone scaffold.

[0020] Preferably, in step (1), the mass ratio of gluconolactone to polycaprolactone is (0.45-3.3):1; further preferably, the mass ratio of gluconolactone to polycaprolactone is (0.5-3):1.

[0021] Preferably, in step (1), the mass volume ratio of the polycaprolactone to the solvent is (0.35-4.5) g:10 mL; further preferably, the mass volume ratio of the polycaprolactone to the solvent is (0.4-4) g:10 mL.

[0022] Preferably, in step (1), the solvent comprises dichloromethane.

[0023] Preferably, in step (1), the mixing is carried out by stirring, the stirring speed is 270-650 rpm, and the stirring time is 22-100 h; further preferably, the stirring speed is 300-600 rpm, and the stirring time is 24-96 h.

[0024] Preferably, in step (2), the gluconolactone / polycaprolactone solution is placed on a substrate and allowed to stand to obtain a gluconolactone / polycaprolactone film.

[0025] Preferably, the substrate comprises a glass culture dish.

[0026] Preferably, the standing still is placing in a ventilated place for 22-80 hours; further preferably, the standing still is placing in a ventilated place for 24-72 hours.

[0027] Preferably, in step (2), the printing process is to first place the gluconolactone / polycaprolactone film into a printing cartridge, match the needle, set the printing parameters and heating temperature, and then print layer by layer to obtain the gluconolactone / polycaprolactone scaffold.

[0028] Preferably, the needle tip diameter is 0.16-0.45 mm; further preferably, the needle tip diameter is 0.15-0.4 mm.

[0029] Preferably, the temperature of the barrel is 50-95°C; further preferably, the temperature of the barrel is 55-90°C.

[0030] Preferably, the temperature of the needle is 55-95°C; further preferably, the temperature of the needle is 60-95°C.

[0031] Preferably, the fiber spacing of the polycaprolactone fibers in the polycaprolactone stent body is (0.70-0.90) mm×(0.70-0.90) mm; further preferably, the fiber spacing of the polycaprolactone fibers in the polycaprolactone stent body is (0.75-0.85) mm×(0.75-0.85) mm; further preferably, the fiber spacing of the polycaprolactone fibers in the polycaprolactone stent body is 0.8 mm×0.8 mm.

[0032] Preferably, the fiber orientation of the polycaprolactone fibers in the polycaprolactone film is 0-90°.

[0033] Preferably, the printing speed is 4.5-16.5 mm / s; further preferably, the printing speed is 5-15 mm / s.

[0034] Preferably, the air pressure during printing is 320-440 kPa; further preferably, the air pressure during printing is 350-400 kPa.

[0035] Preferably, the support layer height during printing is 65-95% of the needle diameter; further preferably, the support layer height during printing is 70-90% of the needle diameter.

[0036] Preferably, in step (3), the mass volume ratio of the gluconolactone / polycaprolactone scaffold to the aqueous solution is 1 mg:(2.2-16.5) mL; further preferably, the mass volume ratio of the gluconolactone / polycaprolactone scaffold to the aqueous solution is 1 mg:(2.5-15) mL.

[0037] Preferably, in step (3), the oscillation temperature is room temperature.

[0038] Preferably, in step (3), the shaking time is 22-52 hours; further preferably, in step (3), the shaking time is 24-48 hours.

[0039] Specifically, after the shaking, a polycaprolactone stent body with a multi-level pore structure is obtained.

[0040] Preferably, in step (3), the concentration of 2-methylimidazole in the 2-methylimidazole aqueous solution is 27-65 mg / mL; further preferably, the concentration of 2-methylimidazole in the 2-methylimidazole aqueous solution is 30-60 mg / mL.

[0041] Preferably, in step (3), the concentration of zinc nitrate in the aqueous zinc nitrate solution is 5.5-26 mg / mL; further preferably, in step (3), the concentration of zinc nitrate in the aqueous zinc nitrate solution is 6-24 mg / mL.

[0042] Preferably, in step (3), the volume ratio of the 2-methylimidazole aqueous solution to the zinc nitrate aqueous solution is (3.5-11):1; further preferably, in step (3), the volume ratio of the 2-methylimidazole aqueous solution to the zinc nitrate aqueous solution is (4-10):1.

[0043] Preferably, in step (3), the reaction further includes a cleaning and drying process.

[0044] The third aspect of the present invention provides a use of the polycaprolactone scaffold described in the first aspect of the present invention in preparing a product for repairing and regenerating bone tissue defects.

[0045] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0046] (1) The matrix material of the polycaprolactone scaffold body of the present invention is polycaprolactone, and the fibers and fiber surfaces of the polycaprolactone have a microporous structure, and the size of the microporous structure between the fibers is larger than the size of the microporous structure on the fiber surface, so that the polycaprolactone scaffold body simultaneously obtains macroscopic macropores (gaps between fibers) and microscopic pores (holes on the fiber surface), significantly improving the specific surface area of ​​the polycaprolactone scaffold body, which can improve the degradation effect of the polycaprolactone scaffold body, making it more suitable for the regeneration and repair of bone defects, and at the same time, the macroscopic macropores and microscopic pores can also significantly improve the surface roughness of the polycaprolactone scaffold body, so as to achieve the purpose of significantly promoting the adhesion of polycaprolactone scaffold cells. In addition, ZIF8 is deposited and modified on the surface of the polycaprolactone scaffold body, which can give the polycaprolactone scaffold biological activity, can significantly induce osteogenic differentiation of stem cells, and the large specific surface area of ​​the polycaprolactone scaffold body can provide more sites for the deposition of ZIF8, which is conducive to improving the biological activity of the polycaprolactone scaffold, and is suitable for the regeneration and repair of bone defects.

[0047] (2) The ZIF8 on the surface of the polycaprolactone stent body of the present invention will accelerate degradation under acidic microenvironments such as bacterial infection, thereby producing antibacterial properties.

[0048] (3) The preparation method of the present invention has a simple process, low requirements on equipment, and the raw materials are readily available from industrial sources, low cost, and easy to realize industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a tensile strength diagram of the polycaprolactone stents of Examples 1-4 and Comparative Examples 1-4 of the present invention;

[0050] Figure 2 The alkaline phosphatase activity diagram of the polycaprolactone scaffolds of Examples 1-4 of the present invention and Comparative Examples 1-4. DETAILED DESCRIPTION

[0051] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0052] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0053] Example 1

[0054] A method for preparing a polycaprolactone scaffold with a multi-level pore structure comprises the following steps:

[0055] (1) 10 g of polycaprolactone (molecular weight 30,000 Daltons) was dissolved in 25 mL of dichloromethane, and 5 g of gluconolactone was added, and mechanical stirring was performed at 300 rpm for 24 h to obtain a gluconolactone / polycaprolactone solution;

[0056] (2) pouring the gluconolactone / polycaprolactone solution obtained in step (1) onto a glass culture dish and placing it under ventilation for 48 hours to obtain a gluconolactone / polycaprolactone film; placing the gluconolactone / polycaprolactone film into a printing barrel, with a needle diameter of 0.2 mm, a barrel temperature of 90° C., a needle temperature of 95° C., a fiber spacing of 0.8 mm×0.8 mm, a printing rate of 10 mm / s, a fiber orientation of “0-90°”, an air pressure of 350 kPa, and a scaffold layer height of 75% of the needle diameter, and printing layer by layer to obtain a gluconolactone / polycaprolactone scaffold;

[0057] (3) 20 mg of the gluconolactone / polycaprolactone scaffold obtained in step (2) was immersed in 50 mL of aqueous solution and shaken at room temperature for 24 hours to obtain a polycaprolactone scaffold body with a multi-level porous structure; the polycaprolactone scaffold body with a multi-level porous structure was added to 100 mL of a 30 mg / mL 2-methylimidazole aqueous solution, and 25 mL of a 6 mg / mL zinc nitrate aqueous solution was added dropwise during stirring. After the reaction, the mixture was washed and freeze-dried to obtain a polycaprolactone scaffold with a multi-level porous structure.

[0058] Example 2

[0059] A method for preparing a polycaprolactone scaffold with a multi-level pore structure comprises the following steps:

[0060] (1) Dissolve 10 g of polycaprolactone (molecular weight 40,000 Daltons) in 100 mL of dichloromethane, add 10 g of gluconolactone, and stir magnetically at 600 rpm for 72 h to obtain a gluconolactone / polycaprolactone solution;

[0061] (2) pouring the gluconolactone / polycaprolactone solution obtained in step (1) onto a glass culture dish and placing it under ventilation for 72 hours to obtain a gluconolactone / polycaprolactone film; placing the gluconolactone / polycaprolactone film into a printing barrel, with a needle diameter of 0.25 mm, a barrel temperature of 65° C., a needle temperature of 70° C., a fiber spacing of 0.8 mm×0.8 mm, a printing rate of 10 mm / s, a fiber orientation of “0-90°”, an air pressure of 380 kPa, and a scaffold layer height of 80% of the needle diameter, and printing layer by layer to obtain a gluconolactone / polycaprolactone scaffold;

[0062] (3) 20 mg of the gluconolactone / polycaprolactone scaffold obtained in step (2) was immersed in 200 mL of aqueous solution and shaken at room temperature for 48 hours to obtain a polycaprolactone scaffold body with a multi-level pore structure; the polycaprolactone scaffold body with a multi-level pore structure was added to 60 mL of a 50 mg / mL 2-methylimidazole aqueous solution, and 6 mL of a 24 mg / mL zinc nitrate aqueous solution was added dropwise during stirring. After the reaction, the mixture was washed and freeze-dried to obtain a polycaprolactone scaffold with a multi-level pore structure.

[0063] Example 3

[0064] A method for preparing a polycaprolactone scaffold with a multi-level pore structure comprises the following steps:

[0065] (1) Dissolve 10 g of polycaprolactone (molecular weight 60,000 Daltons) in 150 mL of dichloromethane, add 10 g of gluconolactone, and stir magnetically at 500 rpm for 96 h to obtain a gluconolactone / polycaprolactone solution;

[0066] (2) pouring the gluconolactone / polycaprolactone solution obtained in step (1) onto a glass culture dish and placing it under ventilation for 24 hours to obtain a gluconolactone / polycaprolactone film; placing the gluconolactone / polycaprolactone film into a printing barrel, with a needle diameter of 0.15 mm, a barrel temperature of 55° C., a needle temperature of 65° C., a fiber spacing of 0.8 mm×0.8 mm, a printing rate of 5 mm / s, a fiber orientation of “0-90°”, an air pressure of 350 kPa, and a scaffold layer height of 90% of the needle diameter, and printing layer by layer to obtain a gluconolactone / polycaprolactone scaffold;

[0067] (3) 20 mg of the gluconolactone / polycaprolactone scaffold obtained in step (2) was immersed in 300 mL of aqueous solution and shaken at room temperature for 48 hours to obtain a polycaprolactone scaffold body with a multi-level porous structure; the polycaprolactone scaffold body with a multi-level porous structure was added to 50 mL of a 60 mg / mL 2-methylimidazole aqueous solution, and 8 mL of an 18 mg / mL zinc nitrate aqueous solution was added dropwise during stirring. After the reaction, the mixture was washed and freeze-dried to obtain a polycaprolactone scaffold with a multi-level porous structure.

[0068] Example 4

[0069] A method for preparing a polycaprolactone scaffold with a multi-level pore structure comprises the following steps:

[0070] (1) Dissolve 10 g of polycaprolactone (molecular weight 40,000 Daltons) in 250 mL of dichloromethane, add 20 g of gluconolactone, and stir mechanically at 500 rpm for 48 h to obtain a gluconolactone / polycaprolactone solution;

[0071] (2) pouring the gluconolactone / polycaprolactone solution obtained in step (1) onto a glass culture dish and placing it under ventilation for 48 hours to obtain a gluconolactone / polycaprolactone film; placing the gluconolactone / polycaprolactone film into a printing barrel, with a needle diameter of 0.4 mm, a barrel temperature of 80° C., a needle temperature of 90° C., a fiber spacing of 0.8 mm×0.8 mm, a printing rate of 15 mm / s, a fiber orientation of “0-90°”, an air pressure of 400 kPa, and a scaffold layer height of 70% of the needle diameter, and printing layer by layer to obtain a gluconolactone / polycaprolactone scaffold;

[0072] (3) Soaking 20 mg of the gluconolactone / polycaprolactone scaffold obtained in step (2) in 200 mL of aqueous solution and shaking at room temperature for 48 hours to obtain a polycaprolactone scaffold body with a multi-level pore structure; adding the polycaprolactone scaffold body with a multi-level pore structure to 70 mL of a 45 mg / mL 2-methylimidazole aqueous solution, adding 8 mL of a 20 mg / mL zinc nitrate aqueous solution dropwise while stirring, washing after the reaction, and freeze-drying to obtain a polycaprolactone scaffold with a multi-level pore structure.

[0073] Comparative Example 1

[0074] The difference between Comparative Example 1 and Example 2 is that no gluconolactone is added in step (1) of Comparative Example 1.

[0075] Specifically, the preparation method of the polycaprolactone stent in Comparative Example 1 comprises the following steps:

[0076] (1) Dissolving 10 g of polycaprolactone (molecular weight: 40,000 Daltons) in 100 mL of dichloromethane to obtain a polycaprolactone solution;

[0077] (2) pouring the polycaprolactone solution obtained in step (1) onto a glass culture dish and placing it under ventilation for 72 hours to obtain a polycaprolactone film; placing the polycaprolactone film into a printing barrel, with a needle diameter of 0.25 mm, a barrel temperature of 65° C., a needle temperature of 70° C., a fiber spacing of 0.8 mm×0.8 mm, a printing rate of 10 mm / s, a fiber orientation of “0-90°”, an air pressure of 380 kPa, and a stent layer height of 80% of the needle diameter, printing layer by layer to obtain a polycaprolactone stent body;

[0078] (3) The polycaprolactone stent obtained in step (2) was added to 70 mL of a 45 mg / mL 2-methylimidazole aqueous solution, and 8 mL of a 20 mg / mL zinc nitrate aqueous solution was added dropwise while stirring. After the reaction, the mixture was washed and freeze-dried to obtain a polycaprolactone stent.

[0079] Comparative Example 2

[0080] The only difference between Comparative Example 2 and Example 2 is that ZIF8 is not attached to the surface of the polycaprolactone stent body in Comparative Example 2, that is, in step (3) of Comparative Example 2, the polycaprolactone stent body with a multi-level pore structure is not added to the 2-methylimidazole aqueous solution, and the zinc nitrate aqueous solution is not added dropwise during stirring. The rest is the same as Example 2.

[0081] The step (3) of Comparative Example 2 is specifically as follows:

[0082] 20 mg of the gluconolactone / polycaprolactone scaffold obtained in step (2) was immersed in 200 mL of aqueous solution and shaken at room temperature for 48 hours to obtain a polycaprolactone scaffold.

[0083] Comparative Example 3

[0084] The difference between Comparative Example 3 and Example 2 is that the fiber surface of the polycaprolactone in Comparative Example 3 does not have a microporous structure, and ZIF8 is not attached to the surface of the polycaprolactone scaffold body, that is, gluconolactone is not added in step (1) of Comparative Example 3, and the polycaprolactone scaffold body is not added to the 2-methylimidazole aqueous solution in step (3), and the zinc nitrate aqueous solution is not added dropwise during stirring. The rest is the same as Example 2.

[0085] Comparative Example 3 The preparation method of the polycaprolactone scaffold comprises the following steps:

[0086] (1) dissolving 10 g of polycaprolactone (molecular weight 40,000 Daltons) in 100 mL of dichloromethane to obtain a polycaprolactone solution;

[0087] (2) pouring the polycaprolactone solution obtained in step (1) onto a glass culture dish and placing it under ventilation for 72 hours to obtain a polycaprolactone film; placing the polycaprolactone film into a printing barrel, with a needle diameter of 0.25 mm, a barrel temperature of 65° C., a needle temperature of 70° C., a fiber spacing of 0.8 mm×0.8 mm, a printing rate of 10 mm / s, a fiber orientation of “0-90°”, an air pressure of 380 kPa, and a support layer height of 80% of the needle diameter, and printing layer by layer to obtain a polycaprolactone support body;

[0088] (3) Soak 20 mg of the polycaprolactone stent obtained in step (2) in 200 mL of aqueous solution and shake it at room temperature for 48 hours to obtain a polycaprolactone stent.

[0089] Comparative Example 4

[0090] The difference between Comparative Example 4 and Example 2 is that in Comparative Example 4, the surface of the polycaprolactone stent body is not attached with ZIF8, but with chitosan, that is, in step (3) of Comparative Example 4, the polycaprolactone stent body is not added to the 2-methylimidazole aqueous solution, nor is the zinc nitrate aqueous solution added dropwise during stirring, but is immersed in the chitosan solution, and the rest is the same as in Example 2.

[0091] Comparative Example 4 A method for preparing a polycaprolactone stent comprises the following steps:

[0092] (1) dissolving 10 g of polycaprolactone (molecular weight 40,000 Daltons) in 100 mL of dichloromethane to obtain a polycaprolactone solution;

[0093] (2) pouring the polycaprolactone solution obtained in step (1) onto a glass culture dish and placing it under ventilation for 72 hours to obtain a polycaprolactone film; placing the polycaprolactone film into a printing barrel, with a needle diameter of 0.25 mm, a barrel temperature of 65° C., a needle temperature of 70° C., a fiber spacing of 0.8 mm×0.8 mm, a printing rate of 10 mm / s, a fiber orientation of “0-90°”, an air pressure of 380 kPa, and a support layer height of 80% of the needle diameter, and printing layer by layer to obtain a polycaprolactone support body;

[0094] (3) Soak 20 mg of the polycaprolactone scaffold obtained in step (2) in 200 mL of an aqueous solution containing 2% chitosan and 2% acetic acid, and shake at room temperature for 48 hours to obtain a chitosan-modified polycaprolactone scaffold.

[0095] Performance Testing

[0096] 1. Tensile strength test

[0097] After drying, the polycaprolactone stents of Examples 1-4 and Comparative Examples 1-4 were placed in an INSTON test machine (34sc -5 ) with a compression speed of 1 mm / min, and mechanical properties tests were performed. The tensile strength test results are shown in Figure 1 As shown. Figure 1 It can be seen that compared with the polycaprolactone scaffold without a multi-level pore structure (Comparative Examples 1 and 3), the compressive strength of the polycaprolactone scaffold with a multi-level pore structure (Examples 1-4, Comparative Examples 2 and 4) is lower. When the polycaprolactone scaffold has a multi-level pore structure, its mechanical strength is more suitable for bone repair because it is closer to the strength of bone tissue.

[0098] 2. In vitro cytotoxicity evaluation

[0099] L929 mouse fibroblasts that had been cultured for 24 hours and were growing vigorously were digested and prepared into a density of 1.0×10 5 / mL, inoculated in a 96-well plate, 100 μL per well; after the cells grow into a monolayer, remove the original culture medium, and add 100 μL of the test sample (polycaprolactone scaffold of Example 1-4, Comparative Example 1-4) extract, blank control solution, positive control solution and negative control solution, respectively, with 6 replicates in each group, wherein the blank control solution is a complete medium containing 3 g / L phenol; the positive control solution is an extract containing 0.3 g / mL of high-density polyethylene in a complete medium at 37±1°C for 24 h. After the sample addition was completed, the 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours; after 24 hours of incubation, the original culture medium was aspirated, 50 μL of MTT (1 mg / mL) was added to each well, and the culture was continued for 2 hours. After the end, the supernatant was aspirated, and 100 μL of 99.5% pure isopropanol was added to dissolve the crystals; the absorbance value was measured on an ELISA instrument with 570 nm as the main absorption wavelength and 650 nm as the reference wavelength to calculate the cell survival rate.

[0100] The cell viability was calculated according to the following formula: Cell viability (%) = 100 × OD 570e / OD 570b .

[0101] OD 570e OD is the average optical density of the test sample or control sample extract; 570b is the average optical density of the blank.

[0102] The in vitro cell survival rates of the polycaprolactone scaffolds of Examples 1-4 and Comparative Examples 1-4 are shown in Table 1.

[0103] Table 1: In vitro cell survival rates of polycaprolactone scaffolds of Examples 1-4 and Comparative Examples 1-4

[0104]

[0105] It can be seen from Table 1 that the polycaprolactone scaffold prepared in the present invention has no cytotoxicity.

[0106] 3. Hemolytic performance test

[0107] According to the amount of blood used in the test, blood is collected from the heart of a healthy rabbit, such as 10 mL of blood, and 0.5 mL of potassium oxalate solution with a mass concentration of 20 g / L is added to prepare fresh anticoagulated rabbit blood; 8 mL of fresh anticoagulated rabbit blood is taken and diluted with 10 mL of sodium chloride injection with a mass concentration of 9 g / L. The test sample group (Example 1-4, Comparative Example 1-4) adds the test sample according to the extraction ratio (0.2 g / mL), and then adds 10 mL of sodium chloride injection; 10 mL of sodium chloride injection is added to each tube of the negative control group; 10 mL of distilled water is added to each tube of the positive control group. Three tubes are operated in parallel in each group. After all the test tubes are placed in a constant temperature water bath at (37 ± 1) ° C for 30 minutes, 0.2 mL of diluted rabbit blood is added to each test tube, gently mixed, and placed in a (37 ± 1) ° C water bath for 60 minutes. Pour out the liquid in the tube and centrifuge at 800g for 5 minutes. Pipette the supernatant into a cuvette and measure the absorbance at a wavelength of 545nm using a spectrophotometer. The absorbance of the test group and the control group is the average of the three tubes. The absorbance of the negative control tube should not be greater than 0.03; the absorbance of the positive control tube should be 0.8±0.3, otherwise the test should be repeated. Calculate according to the following formula:

[0108] Hemolysis rate (%) = (absorbance of the test sample group - absorbance of the negative control group) / (absorbance of the positive control group - absorbance of the negative control group) × 100%.

[0109] The hemolysis rate results of the polycaprolactone stents of Examples 1-4 and Comparative Examples 1-4 are shown in Table 2.

[0110] Table 2: Hemolysis rate of polycaprolactone stents in Examples 1-4 and Comparative Examples 1-4

[0111]

[0112] It can be seen from Table 2 that the hemolysis rate of the polycaprolactone stent prepared by the method of the present invention is less than 5%, and there is no hemolysis risk.

[0113] 4. In vitro osteogenic differentiation test of pre-osteoblasts

[0114] The polycaprolactone scaffolds prepared in Examples 1-4 and Comparative Examples 1-4 were irradiated and sterilized, immersed in DMEM basal culture medium at a concentration of 10 mg / mL, and extracted at 120 rpm in a 37°C shaker for 24 h. After the extraction, the mixture of the polycaprolactone scaffold and the culture medium was centrifuged at 1000 rpm, and the supernatant was collected, which was the extract. The collected extracts were diluted 2 times with the corresponding DMEM culture medium, and finally 10% fetal bovine serum was added to obtain a conditioned medium.

[0115] Mouse embryonic osteoblast precursor cells (MC3T3-E1 cells) were cultured at 1×10 5The cells were inoculated in a 24-well plate at a density of 100 cells / mL. After 24 hours of adherent culture, the conditioned medium was replaced and cultured in an incubator at 37°C and 5% carbon dioxide atmosphere. The culture medium was replaced every 2-3 days. After 7 days of culture, the osteogenic differentiation performance of MC3T3-E1 cells was detected by the alkaline phosphatase secreted by them and measured by the pNPP method. The specific steps are as follows: MC3T3-E1 cells were washed with PBS solution and immersed in a PBS solution containing 0.1 mol / L glycine, 1 mmol / L magnesium chloride and 0.05% Triton X-100 (octoxynol); after the cells were dissolved, the dissolved solution was evenly mixed with disodium p-nitrophenyl phosphate, and the mixed solution was placed at 37°C for 30 minutes; then, the mixed solution was added dropwise to a 96-well plate, and the absorbance value of each well at a wavelength of 405 nm was measured by an ELISA reader.

[0116] Definition of alkaline phosphatase activity unit: In diethanolamine (DEA) buffer at pH 9.8, at 37°C, the amount of alkaline phosphatase required to hydrolyze para-nitrophenyl phosphate chromogenic substrate to produce 1 micromole of p-nitrophenol per minute is defined as one enzyme activity unit, also known as one DEA enzyme activity unit. In glycine buffer at pH 9.6, at 25°C, the amount of alkaline phosphatase required to hydrolyze para-nitrophenyl phosphate chromogenic substrate to produce 1 micromole of p-nitrophenol per minute is defined as one enzyme activity unit, also known as one glycine enzyme activity unit. One glycine enzyme activity unit is approximately equivalent to 3 DEA enzyme activity units. Based on the definition of enzyme activity, the alkaline phosphatase activity in the sample is calculated. The alkaline phosphatase activity of the polycaprolactone scaffolds of Examples 1-4 and Comparative Examples 1-4 is shown in the figure. Figure 2 shown.

[0117] Depend on Figure 2 It can be seen that the scaffolds in Examples 1-4 of the present invention have a good effect of inducing cells to secrete alkaline phosphatase. In Comparative Example 1, without the use of gluconolactone, the polycaprolactone scaffold without a multi-level pore structure has a cell activity worse than that of the polycaprolactone scaffold with a multi-level pore structure, and therefore also affects the cell secretion of alkaline phosphatase. Comparative Example 2 does not contain a ZIF8 layer with good biological activity, and the alkaline phosphatase activity is significantly reduced compared with Example 2. Comparative Example 3 does not contain a ZIF8 layer with good biological activity, nor does it have a multi-level pore structure, and its alkaline phosphatase activity is the lowest. Comparative Example 4 uses a chitosan coating to replace the ZIF8 coating. Although the effect is better than that of Comparative Example 3, the alkaline phosphatase activity is still significantly worse than that of Example 2.

[0118] 6. Antibacterial performance test

[0119] Take fresh slant cultures of Staphylococcus aureus and Escherichia coli, count the live bacteria in the bacterial suspension, and prepare a 5×10-60 suspension with 0.03 mol / L PBS (pH=7.2-7.4) containing 1% peptone. 5 -10×10 6 cfu / mL of bacterial suspension. Place the polycaprolactone stent samples of Examples 1-4 and Comparative Examples 1-4 in sterile dishes, add 50 μL of bacterial suspension on each sample, record the time of adding bacteria to each tube, inoculate the blood plate 60 minutes after adding bacteria, and place the sample in a 5 mL nutrient broth tube. Culture the blood plates and broth tubes inoculated with bacteria at 37°C for 48 hours, observe the preliminary results, and continue to culture in sterile growth tubes until the 28th day. If the broth tube is turbid and there is bacterial growth on the blood plate, it is recorded as positive, indicated by (+); if it is still clear on the 35th day, it is considered as sterile growth, indicated by (–).

[0120] The antibacterial performance test results of the polycaprolactone stents of Examples 1-4 and Comparative Examples 1-4 are shown in Table 3.

[0121] Table 3: Antibacterial performance test results of polycaprolactone stents in Examples 1-4 and Comparative Examples 1-4

[0122]

[0123] It can be seen from Table 3 that the polycaprolactone stents prepared in Examples 1-4 of the present invention all have good long-term antibacterial effects and can grow aseptically for 35 days. Comparative Examples 2-4 do not contain ZIF8 and have no antibacterial effects.

[0124] In summary, the polycaprolactone scaffold body of the present invention simultaneously obtains macroscopic macropores (gaps between fibers) and microscopic pores (holes on the fiber surface), significantly improving the specific surface area of ​​the polycaprolactone scaffold body, and can improve the effect of polycaprolactone scaffold body degradation, making it more suitable for regeneration and repair of bone defects, while macroscopic macropores and microscopic pores can also significantly improve the surface roughness of the polycaprolactone scaffold body, and achieve the purpose of significantly promoting the adhesion of polycaprolactone scaffold cells. In addition, ZIF8 is deposited and modified on the surface of the polycaprolactone scaffold body, which can give the polycaprolactone scaffold biological activity, can significantly induce osteogenic differentiation of stem cells, and the large specific surface area of ​​the polycaprolactone scaffold body can provide more sites for the deposition of ZIF8, which is conducive to improving the biological activity of the polycaprolactone scaffold, suitable for regeneration and repair of bone defects, and has good mechanical strength, biocompatibility, and antibacterial properties.

[0125] The above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A polycaprolactone stent, characterized in that: It includes a polycaprolactone stent body and a zeolite imidazolate framework material attached to the surface of the polycaprolactone stent body; The matrix material of the polycaprolactone stent body is polycaprolactone, and the fibers and the fiber surfaces of the polycaprolactone have micron pore structures, and the size of the micron pore structures between the polycaprolactone fibers is larger than the size of the micron pore structures on the fiber surfaces of the polycaprolactone.

2. The polycaprolactone stent according to claim 1, characterized in that: The polycaprolactone stent body is a 3D printed stent.

3. The polycaprolactone stent according to claim 1, characterized in that: The molecular weight of the polycaprolactone is 27,000-65,000 Daltons.

4. The polycaprolactone stent according to claim 1, characterized in that: The size of the microporous structure between the fibers of the polycaprolactone is 200-650 μm.

5. The polycaprolactone stent according to claim 1, characterized in that: The size of the micropore structure on the surface of the polycaprolactone fiber is 0.9-110 μm.

6. The method for preparing the polycaprolactone stent according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) mixing polycaprolactone, gluconolactone and a solvent to obtain a gluconolactone / polycaprolactone solution; (2) preparing the gluconolactone / polycaprolactone solution obtained in step (1) into a gluconolactone / polycaprolactone film, and then printing it to obtain a gluconolactone / polycaprolactone scaffold; (3) mixing the gluconolactone / polycaprolactone scaffold obtained in step (2) with an aqueous solution, and shaking to obtain a polycaprolactone scaffold body; and mixing the polycaprolactone scaffold body, a 2-methylimidazole aqueous solution, and a zinc nitrate aqueous solution, and reacting them to obtain the polycaprolactone scaffold.

7. The preparation method according to claim 6, characterized in that: In step (1), the mass ratio of gluconolactone to polycaprolactone is (0.45-3.3):1; and / or the mass volume ratio of polycaprolactone to solvent is (0.35-4.5) g:10 mL; and / or the mixing is carried out by stirring, the stirring speed is 270-650 rpm, and the stirring time is 22-100 h.

8. The preparation method according to claim 6, characterized in that: In step (2), the gluconolactone / polycaprolactone solution is placed on a substrate and allowed to stand to obtain the gluconolactone / polycaprolactone film.

9. The preparation method according to claim 6, characterized in that: In step (3), the mass volume ratio of the gluconolactone / polycaprolactone scaffold to the aqueous solution is 1 mg:(2.2-16.5) mL; and / or the concentration of 2-methylimidazole in the 2-methylimidazole aqueous solution is 27-65 mg / mL; and / or the concentration of zinc nitrate in the zinc nitrate aqueous solution is 5.5-26 mg / mL; and / or the volume ratio of the 2-methylimidazole aqueous solution to the zinc nitrate aqueous solution is (3.5-11):

1.

10. Use of the polycaprolactone scaffold according to any one of claims 1 to 5 in preparing products for repairing and regenerating bone tissue defects.

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