Modified polycaprolactone composite scaffold and preparation method and application thereof

Abstract

The application discloses a modified polycaprolactone composite scaffold and a preparation method and application thereof, and the polycaprolactone composite scaffold has a porous structure, wherein the pores on the surface and inside are interconnected, and the pore diameter is gradually increased from the surface to the inside; the surface of the polycaprolactone composite scaffold is loaded with metal organic framework materials loaded with antibacterial drugs inside. The modified polycaprolactone composite scaffold of the application has macro-pores (fiber gaps) and micro-pores (fiber surface holes) at the same time, can not only improve the surface roughness of the scaffold, achieve the purpose of significantly promoting cell adhesion of the scaffold, but also can significantly improve the specific surface area of the scaffold, accelerate the degradation of polycaprolactone and release more acidic degradation products, thereby accelerating the degradation of polydopamine and accelerating the release of the drugs embedded inside, and the problem that the polydopamine is slow in degradation in vivo and in vitro is solved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical materials, and particularly relates to a modified polycaprolactone composite material stent and a preparation method and application thereof. BACKGROUND

[0002] In recent years, with the development of bone tissue engineering technology, the use of tissue engineering prepared prosthesis shows good cartilage repair effect, and it is hoped that tissue engineering technology can be used to treat growth plate injury. Cartilage tissue engineering generally includes a scaffold and active substances. An ideal bone tissue engineering scaffold should have an interconnected porous network structure to provide interconnected channels for cell migration, ion transport, and cell-cell interaction. Conventional techniques include foaming method, sacrificial template method, freeze-drying method and casting method, etc. 3D printing, also known as additive manufacturing technology, has unique advantages in high precision, personalized manufacturing and complex shape construction, and has penetrated into various industries, leading to innovation and triggering a global manufacturing revolution. 3D printing obtains a three-dimensional entity by layering materials on a plane, and 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, which has good biocompatibility, non-toxicity, degradability, and can induce tissue regeneration, provide a place for cell adhesion, proliferation and differentiation, and is widely used in the field of medical science. However, the hydrophobicity of PCL will affect the adhesion of the formed scaffold, and the mechanical structure is obviously destroyed after degradation. By introducing artificial materials through surface modification, physical doping, chemical grafting and other methods, the characteristics of polycaprolactone that are not possessed can be supplemented, and by adjusting the mixed components, the respective advantages of the components can be maximized, and thus the ideal parameter requirements of bone repair materials can be met. In addition, polydopamine microspheres have a certain drug loading capacity and can be used as a drug carrier, which can be used in bone tissue engineering technology.

[0004] The existing technologies of bone tissue engineering scaffolds include the following: Document

Xu Yingke. 3D printing of polydopamine microspheres loaded with poly (caprolactone) / β-tricalcium phosphate scaffold characterization and biocompatibility research[D]. Hainan Medical College, 2023. DOI: 10.27952 / d.cnki.ghnyx.2023.000017.

Dong Bo. Research on the repair effect of 3D printed composite scaffold loaded with FK-16 on infected bone defects[D]. Jilin University, 2024. DOI: 10.27162 / d.cnki.gjlin.2024.005386.

[0005] In order to overcome the problems of the above existing technologies, one of the purposes of the present application is to provide a modified poly (caprolactone) composite scaffold. The second purpose of the present application is to provide a preparation method of the modified poly (caprolactone) composite scaffold. The third purpose of the present application is to provide an application of the modified poly (caprolactone) composite scaffold. The gradient pore structure scaffold of the present application not only has good porosity and biocompatibility, antibacterial performance, and induction of stem cell osteogenic differentiation, but especially can accelerate the release of antibacterial drugs under infection conditions, and start the slow release of the internally encapsulated drugs for promoting tissue regeneration and repair. It is suitable for the repair and regeneration of bone tissue defects under infection.

[0006] In order to achieve the above purposes, the technical scheme adopted by the present application is as follows:

[0007] The first aspect of the present application provides a modified poly (caprolactone) composite scaffold, comprising a poly (caprolactone) scaffold matrix, the surface of the poly (caprolactone) scaffold matrix being loaded with a metal organic framework material, the metal organic framework material internally loading an antibacterial drug; the surface and interior of the poly (caprolactone) scaffold matrix being distributed with intercommunicating pores, the pores gradually increasing from the surface to the interior of the poly (caprolactone) scaffold matrix; the poly (caprolactone) scaffold matrix being distributed with drug loaded materials, the drug loaded materials comprising mesoporous ceramics, the mesoporous ceramics internally filling a tissue regeneration and repair drug, the surface of the mesoporous ceramics being coated with polydopamine.

[0008] The scaffold pores in the present application gradually increase from the surface of the polycaprolactone scaffold matrix to the inside, forming a gradient pore and a multi-level pore structure, wherein the internal pore of the polycaprolactone scaffold matrix refers to the pore formed by the stacking of fibers (macroscopic hole, several hundred microns), and the external pore of the polycaprolactone scaffold matrix refers to the pore formed on the fiber due to the pore-forming technology (microscopic hole, several to several tens of microns).

[0009] Preferably, the surface of the polycaprolactone composite has pores with a size of 1-100 μm.

[0010] Preferably, the mesoporous ceramic is at least one selected from mesoporous silica, mesoporous calcium silicate, mesoporous magnesium silicate, mesoporous zinc silicate, mesoporous strontium silicate, mesoporous bioglass, mesoporous hydroxyapatite.

[0011] Preferably, the mesoporous ceramic is a particle with a particle size of 30-1500 nm.

[0012] More preferably, the specific surface area of the mesoporous ceramic is 100-2000 m 2 / g, and the average pore size is 2-50 nm.

[0013] Preferably, the metal-organic framework material is a zeolitic imidazolate framework material self-assembled by coordination of zinc ions and 2-methyl imidazole.

[0014] Preferably, the drug for promoting tissue regeneration and repair is at least one selected from bone morphogenetic protein-2, bone morphogenetic protein-7, vascular endothelial cell factor, platelet-derived factor, curcumin, melatonin, alendronate sodium, naringin, resveratrol, dexamethasone, gentamicin sulfate, epidermal cell growth factor, fibroblast growth factor, keratinocyte growth factor, nerve growth factor, berberine hydrochloride, gentamicin, transforming growth factor, insulin-like growth factor, parathyroid hormone, growth hormone, interleukin, alendronate sodium, salmon calcitonin, zoledronate sodium, ibandronate sodium, strontium ranelate, vitamin D.

[0015] Preferably, the antibacterial drug is at least one selected from ertapenem, imipenem, meropenem, piperacillin / tazobactam, amikacin, colistin, polymyxin B, amikacin, linezolid, vancomycin, tetracycline, tigecycline, fluorouracil, amphotericin B, caspofungin, voriconazole, triclosan, chlorhexidine, penicillin, roxithromycin, clarithromycin, azithromycin, tobramycin, etimicin, netilmicin, tetracycline, chloramphenicol, ciprofloxacin, levofloxacin, moxifloxacin, metronidazole, ornidazole, tinidazole, itraconazole, mycostatin, griseofulvin.

[0016] Preferably, the molecular weight of the polycaprolactone is 3-100,000 daltons.

[0017] The second aspect of the present application provides a preparation method of the modified polycaprolactone composite scaffold of the first aspect, comprising the following steps:

[0018] S1, mixing the drug-loaded material, gluconolactone and polycaprolactone in a solvent to obtain a mixed solution, transferring the mixed solution to a mold and placing it for a period of time to obtain a mixed material film; using the mixed material film as a printing material, 3D printing to obtain a scaffold with a gradient pore structure; after printing, immersing the scaffold with a gradient pore structure in water to remove the gluconolactone and residual solvent;

[0019] S2, reacting the scaffold with a gradient pore structure, an antibacterial drug, 2-methylimidazole and a zinc source in water to obtain the modified polycaprolactone composite scaffold.

[0020] Preferably, the preparation method of the drug-loaded material comprises the following steps: immersing the mesoporous ceramic in a solution containing a tissue regeneration and repair promoting drug to obtain mesoporous ceramic encapsulating the tissue regeneration and repair promoting drug; immersing the mesoporous bioceramic encapsulating the tissue regeneration and repair promoting drug in a solution containing dopamine to obtain the drug-loaded material.

[0021] More preferably, the solvent of the solution containing the tissue regeneration and repair promoting drug is water.

[0022] More preferably, the mass ratio of the mesoporous ceramic, the tissue regeneration and repair promoting drug and dopamine is (50-1000):(0.005-10):100.

[0023] Preferably, the mass ratio of the drug-loaded material and polycaprolactone is (0.005-0.05):1.

[0024] Preferably, the mass ratio of the gluconolactone and polycaprolactone is (0.5-3):1.

[0025] Preferably, the solvent is dichloromethane.

[0026] Preferably, the process conditions of the 3D printing meet at least one of the following:

[0027] a) the 3D printing uses a printing needle with a diameter of 0.15-0.4 mm;

[0028] b) the printing and heating temperature: the barrel is 60-80℃, and the needle is 60-90℃;

[0029] c) the fiber spacing is 0.8mm×0.8mm; the printing rate is 8-17mm / s;

[0030] d) the air pressure is 320-400Kpa;

[0031] e) the scaffold layer height is 75-95% of the needle diameter.

[0032] f) the fiber orientation is "0-90°".

[0033] The fiber spacing refers to the distance between the adjacent fiber axes in the transverse and longitudinal directions. The fiber orientation refers to the angle between the two layers of fibers. The scaffold layer height refers to the layer thickness of each layer printed.

[0034] Preferably, in step S1, the mixing time is 30-100h.

[0035] More preferably, the mixing is carried out under stirring conditions, wherein the stirring rate is 100-1000rpm.

[0036] Preferably, the placing time is 50-100h.

[0037] Preferably, the film forming conditions of the mixed material film are volatilization film forming.

[0038] Preferably, the mass ratio of the 2-methylimidazole, the antibacterial drug and the zinc source is (18-23):(4-10):1.

[0039] Preferably, the zinc source is zinc nitrate.

[0040] Preferably, step S2 specifically comprises the following steps: adding the scaffold with gradient pore structure into the aqueous solution containing the antibacterial drug and the 2-methylimidazole for dispersion, then slowly adding the zinc source for reaction, and after the reaction, washing and freeze-drying to obtain the modified polycaprolactone composite scaffold.

[0041] The third aspect of the present application provides the use of the modified polycaprolactone composite scaffold of the first aspect in the preparation of a product for promoting bone tissue repair and regeneration.

[0042] The present application has the following beneficial effects:

[0043] The present application provides a modified polycaprolactone composite scaffold, comprising a polycaprolactone composite scaffold; the polycaprolactone composite scaffold has a porous structure, wherein the pores on the surface and inside are interconnected, and the pore size gradually increases from the surface to the inside, so that the printed scaffold simultaneously obtains macroscopic large pores (fiber gaps) and microscopic small pores (fiber surface holes), which not only can improve the surface roughness of the scaffold to achieve the purpose of significantly promoting cell adhesion of the scaffold, but also can significantly increase the specific surface area of the scaffold, so that the polycaprolactone degrades faster and releases more acidic degradation products, thereby accelerating the degradation of polydopamine and accelerating the release of the drugs embedded inside, solving the problem of slow degradation of polydopamine in vivo and in vitro.

[0044] The specific beneficial effects are as follows:

[0045] (1) The surface of the modified polycaprolactone composite scaffold of the application also carries a metal organic framework material loaded with antibacterial drugs, which not only enhances the antibacterial performance of the gradient pore structure polycaprolactone scaffold, but also enables slow release of the internal tissue regeneration and repair promoting drugs. In the case of bone infection, the lower pH in the local microenvironment will significantly accelerate the degradation of the metal organic framework material, and the antibacterial drugs loaded therein will be released faster, achieving better antibacterial and bone regeneration effects. In addition, the polycaprolactone composite scaffold of the application has micro-pores, providing more sites for the deposition of drug-loaded metal organic framework materials, which is beneficial to further improve the biological activity of the scaffold.

[0046] (2) The modified polycaprolactone composite scaffold of the application uses drug-loaded mesoporous ceramics as a scaffold forming material, which can enhance the mechanical strength of the scaffold and the drug-loaded material, making it suitable for bone repair. The modified polycaprolactone composite scaffold of the application further disperses the tissue regeneration and repair promoting drugs in the interior of polycaprolactone, which can achieve better therapeutic effect compared to the conventional use of polydopamine coating to adsorb drugs.

[0047] (3) The application provides a preparation method of the above-mentioned modified polycaprolactone composite scaffold, which is simple and feasible. The use of gluconolactone as a pore-forming agent enables the scaffold to have a gradient pore structure, thereby enabling the printed scaffold to have both macro-pores (inter-fiber gaps) and micro-pores (fiber surface holes), and the gluconolactone is easily soluble in water and easy to eliminate after reaction.

[0048] (4) The modified polycaprolactone composite scaffold of the application has great application potential in the preparation of products for promoting bone tissue repair and regeneration. The surface modified gradient pore structure scaffold has good porosity and biocompatibility, antibacterial performance, and the characteristics of inducing osteogenic differentiation of stem cells. In the case of infection, the metal organic framework material can accelerate the release of antibacterial drugs, and then start the slow release of the internal encapsulated tissue regeneration and repair promoting drugs, so it is suitable for the repair and regeneration of bone tissue defects under infection. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 In vitro release performance of tissue regeneration and repair promoting drugs of the materials prepared in the examples and comparative examples;

[0050] Figure 2 In vitro release performance of tissue regeneration and repair promoting drugs of the materials prepared in the examples and comparative examples;

[0051] Figure 3 In vitro antibacterial drug release performance of the materials prepared in the examples and comparative examples;

[0052] Figure 4In vitro antibacterial drug release performance of the material prepared for Example 1 and the comparative example;

[0053] Figure 5 Alkaline phosphatase activity of the stent prepared for the example and the comparative example. DETAILED DESCRIPTION

[0054] The present application will be further described in details by specific examples. The raw materials used in the following examples, if not specifically stated, can be obtained from conventional commercial channels or prepared and isolated by simple synthesis; the processes used, if not specifically stated, are conventional processes in the art.

[0055] Example 1

[0056] This example provides a modified polycaprolactone composite stent, and the preparation method is as follows:

[0057] S1, 1000 mg of mesoporous zinc silicate (average particle size between 300 nm and 800 nm, specific surface area between 400 m 2 / g and 900 m 2 / g, average pore size between 30 nm and 50 nm) is dispersed in 100 mL of an aqueous solution containing 0.2 mg of bone morphogenetic protein-2, stirred at 200 rpm for 18 h, and freeze-dried to obtain a mesoporous material loaded with a therapeutic agent; 300 mg of the mesoporous material loaded with the therapeutic agent is dispersed in 100 mL of an aqueous solution of 2 mg / mL dopamine (pH = 8.5) at 0°C, stirred at 200 rpm for 10 min, and freeze-dried to obtain polydopamine-coated drug-loaded mesoporous material.

[0058] S2, 10 g of polycaprolactone (molecular weight: 40,000 daltons) is dissolved in 100 mL of dichloromethane, and then 10 g of gluconolactone and 150 mg of drug-loaded mesoporous ceramic@polydopamine particles are added, and magnetic stirring is performed at 300 rpm for 72 h to obtain a gluconolactone / drug-loaded mesoporous ceramic@polydopamine particle / polycaprolactone solution. The solution is poured onto a glass culture dish, and placed in a ventilated state for 72 h to obtain a gluconolactone / drug-loaded mesoporous ceramic@polydopamine particle / polycaprolactone film.

[0059] S3, the film is put into a printing cartridge, the needle diameter is 0.25mm, the cartridge temperature is 60℃, the needle temperature is 65℃, the fiber spacing is 0.8mm*0.8mm, the printing rate is 10mm / s, the fiber direction is "0-90°", the air pressure is 380Kpa, and the support layer height is 80% of the needle diameter. Gluconolactone / drug-loaded mesoporous ceramic@polydopamine particle / polycaprolactone scaffold is printed layer by layer. 20mg of gluconolactone / drug-loaded mesoporous ceramic@polydopamine particle / polycaprolactone scaffold is soaked in 10mL aqueous solution, ultrasonic is obtained at room temperature for 0.5h, and gradient pore structure drug-loaded mesoporous ceramic@polydopamine particle / polycaprolactone scaffold is obtained.

[0060] S4, the gradient pore structure scaffold is added to 12mL of 41mg / mL 2-methylimidazole aqueous solution (containing 15mg / mL vancomycin), 2mL of 12mg / mL zinc nitrate aqueous solution is added dropwise under stirring, and after reaction, cleaning, freeze-drying, gradient pore structure, drug-loaded ZIF8 surface modified drug-loaded mesoporous ceramic@polydopamine / polycaprolactone scaffold is obtained.

[0061] The modified polycaprolactone composite scaffold prepared in Example 1 is a porous structure, and the scaffold pores gradually increase from the surface to the inside of the polycaprolactone scaffold matrix, wherein the inside pores of the polycaprolactone scaffold matrix refer to the pores formed by the stacking of fibers (macroscopic pores, several hundred microns), and the outside pores of the polycaprolactone scaffold matrix refer to the pores formed on the fibers due to the poring technology (microscopic pores, several to several tens of microns).

[0062] Example 2

[0063] The present embodiment provides a modified polycaprolactone composite scaffold, and a preparation method thereof is as follows:

[0064] S1, 1000mg of mesoporous bioglass (average particle size between 400nm and 600nm, specific surface area between 100m 2 / g and 700m 2 / g, average pore size between 10nm and 30nm) is dispersed in 100mL of water solution containing 100mg of dexamethasone at 37℃, 500rpm stirring for 4h, and freeze-drying to obtain a therapeutic agent-loaded mesoporous material; 900mg of the therapeutic agent-loaded mesoporous material is dispersed in 100mL of 3mg / mL dopamine aqueous solution (pH=8.4) at 37℃, 100rpm stirring for 20min, and freeze-drying to obtain a polydopamine-coated drug-loaded mesoporous material.

[0065] S2, 10 g of polycaprolactone (molecular weight: 40,000 daltons) was dissolved in 20 mL of dichloromethane, 20 g of gluconolactone and 50 mg of drug-loaded mesoporous ceramic @ polydopamine particles were added, and magnetic stirring was performed at 600 rpm for 60 h to obtain a gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone solution. The solution was poured onto a glass culture dish and placed in a ventilated state for 48 h to obtain a gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone film.

[0066] S3, the film was placed in a printing cartridge, the needle diameter was 0.3 mm, the cartridge temperature was 70°C, the needle temperature was 75°C, the fiber spacing was 0.8 mm x 0.8 mm, the printing rate was 8 mm / s, the fiber direction was "0-90°", the air pressure was 320 Kpa, and the support layer height was 85% of the needle diameter. The gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold was printed layer by layer.

[0067] S4, 20 mg of the gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold was soaked in 25 mL of an aqueous solution, ultrasonicated at room temperature for 0.4 h to obtain a gradient pore structure drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold. The gradient pore structure scaffold was added to 13 mL of a 38 mg / mL aqueous solution of 2-methylimidazole (containing 18 mg / mL chloramphenicol), 1 mL of a 24 mg / mL aqueous solution of zinc nitrate was added dropwise under stirring, and after reaction, the gradient pore structure, drug-loaded ZIF8 surface modified drug-loaded mesoporous ceramic @ polydopamine / polycaprolactone scaffold was obtained after washing, freezing and drying.

[0068] Example 3

[0069] The present embodiment provides a modified polycaprolactone composite scaffold, and the preparation method is as follows:

[0070] S1, at 20°C, 500 mg of mesoporous silica (average particle size between 30 nm and 600 nm, specific surface area between 500 m 2 / g and 2000 m 2 / g, average pore size between 2 nm and 6 nm) was dispersed in 50 mL of an aqueous solution containing 30 mg of curcumin, stirred at 100 rpm for 24 h, and freeze-dried to obtain a therapeutic agent-loaded mesoporous material; at 30°C, 150 mg of the therapeutic agent-loaded mesoporous material was dispersed in 100 mL of a 1 mg / mL aqueous solution of dopamine (pH = 8), stirred at 400 rpm for 15 h, and freeze-dried to obtain a polydopamine-coated drug-loaded mesoporous material.

[0071] S2, 10 g of polycaprolactone (molecular weight: 100,000 Dalton) was dissolved in 125 mL of dichloromethane, 30 g of gluconolactone and 110 mg of drug-loaded mesoporous ceramic @ polydopamine particles were added, and mechanical stirring was carried out at 800 rpm for 36 h to obtain a gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone solution. The solution was poured onto a glass culture dish and placed in a ventilated state for 48 h to obtain a gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone film.

[0072] S3, the film was placed in a printing cartridge, the needle diameter was 0.15 mm, the cartridge temperature was 80°C, the needle temperature was 60°C, the fiber spacing was 0.8 mm x 0.8 mm, the printing rate was 12 mm / s, the fiber direction was "0-90°", the air pressure was 400 Kpa, and the scaffold layer height was 95% of the needle diameter. The gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold was printed layer by layer. 20 mg of the gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold was soaked in 50 mL of an aqueous solution, ultrasonicated at room temperature for 1 hour, and a gradient pore structure drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold was obtained.

[0073] S4, the gradient pore structure scaffold was added to 12 mL of a 34 mg / mL aqueous solution of 2-methylimidazole (containing 25 mg / mL of chlorhexidine), 3 mL of a 7 mg / mL aqueous solution of zinc nitrate was added dropwise under stirring, and after reaction, the product was washed, freeze-dried, and a gradient pore structure, drug-loaded ZIF8 surface-modified drug-loaded mesoporous ceramic @ polydopamine / polycaprolactone scaffold was obtained.

[0074] Example 4

[0075] The present embodiment provides a modified polycaprolactone composite scaffold, and the preparation method is as follows:

[0076] S1, 1000 mg of mesoporous hydroxyapatite (average particle size between 1000 nm and 1500 nm, specific surface area between 100 m 2 / g and 500 m 2 / g, average pore size between 2 nm and 10 nm) was dispersed in 100 mL of an aqueous solution containing 80 mg of resveratrol, stirred at 400 rpm for 8 h, and freeze-dried to obtain a therapeutic agent-loaded mesoporous material; 600 mg of the therapeutic agent-loaded mesoporous material was dispersed in 100 mL of a 1.5 mg / mL aqueous solution of dopamine (pH = 9) at 20°C, stirred at 600 rpm for 2 h, and freeze-dried to obtain a polydopamine-coated drug-loaded mesoporous material.

[0077] S2, 10 g of polycaprolactone (molecular weight: 60,000 Dalton) was dissolved in 50 mL of dichloromethane, 5 g of gluconolactone and 80 mg of drug-loaded mesoporous ceramic @ polydopamine particles were added, and mechanical stirring was carried out at 400 rpm for 54 h to obtain a gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone solution. The solution was poured onto a glass culture dish and placed in a ventilated state for 60 h to obtain a gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone film.

[0078] S3, the film was placed in a printing cartridge, the needle diameter was 0.4 mm, the cartridge temperature was 75°C, the needle temperature was 90°C, the fiber spacing was 0.8 mm x 0.8 mm, the printing rate was 17 mm / s, the fiber direction was "0-90°", the air pressure was 360 Kpa, and the scaffold layer height was 75% of the needle diameter. The gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold was printed layer by layer. 20 mg of the gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold was soaked in 4 mL of an aqueous solution, ultrasonicated at room temperature for 0.5 h, and a gradient pore structure drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold was obtained.

[0079] S4, the gradient pore structure scaffold was added to 16 mL of a 53 mg / mL aqueous solution of 2-methylimidazole (containing 10 mg / mL of penicillin), 4 mL of a 10 mg / mL aqueous solution of zinc nitrate was added dropwise under stirring, and after reaction, the product was washed, freeze-dried, and a gradient pore structure, drug-loaded ZIF8 surface-modified drug-loaded mesoporous ceramic @ polydopamine / polycaprolactone scaffold was obtained.

[0080] Comparative Example 1

[0081] This comparative example provides a preparation method of a ZIF8 surface-modified polycaprolactone scaffold, which is substantially the same as that of Example 1, except that gluconolactone is not used, and the preparation method is as follows:

[0082] S1, 1000 mg of mesoporous zinc silicate (average particle size between 300 nm and 800 nm, specific surface area between 400 m 2 / g and 900 m 2 / g, average pore size between 30 nm and 50 nm) was dispersed in 100 mL of an aqueous solution containing 0.2 mg of bone morphogenetic protein-2, stirred at 200 rpm for 18 h, and freeze-dried to obtain a therapeutic agent-loaded mesoporous material; 300 mg of the therapeutic agent-loaded mesoporous material was dispersed in 100 mL of a 2 mg / mL aqueous solution of dopamine (pH = 8.5) at 0°C, stirred at 200 rpm for 10 min, and freeze-dried to obtain a polydopamine-coated drug-loaded mesoporous material.

[0083] S2, 10 g of polycaprolactone (molecular weight: 40,000 daltons) was dissolved in 100 mL of dichloromethane, and 150 mg of drug-loaded mesoporous ceramic @ polydopamine particles were added, and stirred at 300 rpm for 72 h to obtain a drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone solution. Pour the solution onto a glass culture dish and place it in a ventilated state for 72 h to obtain a drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone film.

[0084] S3, the film was placed in a printing cartridge, the needle diameter was 0.25 mm, the cartridge temperature was 60°C, the needle temperature was 65°C, the fiber spacing was 0.8 mm x 0.8 mm, the printing rate was 10 mm / s, the fiber direction was "0-90°", the air pressure was 380 Kpa, and the support layer height was 80% of the needle diameter. Layered printing of drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffolds. 20 mg of drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffolds were soaked in 10 mL of an aqueous solution, and ultrasonic treatment was performed at room temperature for 0.5 hours to obtain drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffolds.

[0085] S4, the scaffold was added to 12 mL of a 41 mg / mL aqueous solution of 2-methylimidazole (containing 15 mg / mL of vancomycin), and 2 mL of a 12 mg / mL aqueous solution of zinc nitrate was added dropwise under stirring. After the reaction, the product was washed, freeze-dried, and a drug-loaded ZIF8 surface-modified drug-loaded mesoporous ceramic @ polydopamine / polycaprolactone scaffold was obtained.

[0086] The modified polycaprolactone composite scaffold prepared in Comparative Example 1 had a relatively smooth surface, and occasionally had some micrometer to tens of micrometer holes.

[0087] Comparative Example 2

[0088] This comparative example provides a method for preparing a gradient pore structure polycaprolactone scaffold, which is substantially the same as Example 1, except that ZIF8 surface modification is not performed, and the preparation method is as follows:

[0089] S1, 1000 mg of mesoporous zinc silicate (average particle size between 300 nm and 800 nm, specific surface area between 400 m 2 / g and 900 m 2 / g, average pore size between 30 nm and 50 nm) was dispersed in 100 mL of an aqueous solution containing 0.2 mg of bone morphogenetic protein-2, and stirred at 200 rpm for 18 h. After freeze-drying, a therapeutic agent-loaded mesoporous material was obtained; 300 mg of the therapeutic agent-loaded mesoporous material was dispersed in 100 mL of a 2 mg / mL dopamine aqueous solution (pH = 8.5) at 0°C, and stirred at 200 rpm for 10 min. After freeze-drying, a polydopamine-coated drug-loaded mesoporous material was obtained.

[0090] S2, 10 g of polycaprolactone (molecular weight: 40,000 Dalton) was dissolved in 100 mL of dichloromethane, and 10 g of gluconolactone and 150 mg of drug-loaded mesoporous ceramic @ polydopamine particles were added, and stirred at 300 rpm for 72 h to obtain a gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone solution. The solution was poured onto a glass culture dish and placed in a ventilated state for 72 h to obtain a gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone film.

[0091] S3, the film was placed in a printing cartridge, the needle diameter was 0.25 mm, the cartridge temperature was 60°C, the needle temperature was 65°C, the fiber spacing was 0.8 mm x 0.8 mm, the printing rate was 10 mm / s, the fiber direction was "0-90°", the air pressure was 380 Kpa, and the support layer height was 80% of the needle diameter. The gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold was printed layer by layer. 20 mg of the gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold was soaked in 10 mL of an aqueous solution, and ultrasonic treatment was performed at room temperature for 0.5 h to obtain a gradient pore structure drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold.

[0092] S4, the gradient pore structure scaffold was added to 12 mL of an aqueous solution containing 15 mg / mL of vancomycin and the like, washed and freeze-dried to obtain a gradient pore structure, surface drug-loaded drug-loaded mesoporous ceramic @ polydopamine / polycaprolactone scaffold.

[0093] Comparative Example 3

[0094] This comparative example provides a method for preparing a polycaprolactone scaffold, which is substantially the same as Example 1, except that no drug is loaded in ZIF8, and the preparation method is as follows:

[0095] S1, 1000 mg of mesoporous zinc silicate (average particle size between 300 nm and 800 nm, specific surface area between 400 m 2 / g and 900 m 2 / g, average pore size between 30 nm and 50 nm) was dispersed in 100 mL of an aqueous solution containing 0.2 mg of bone morphogenetic protein-2, stirred at 200 rpm for 18 h, and freeze-dried to obtain a therapeutic agent-loaded mesoporous material; 300 mg of the therapeutic agent-loaded mesoporous material was dispersed in 100 mL of a 2 mg / mL dopamine aqueous solution (pH = 8.5) at 0°C, stirred at 200 rpm for 10 min, and freeze-dried to obtain a drug-loaded mesoporous material coated with polydopamine.

[0096] S2, 10 g of polycaprolactone (molecular weight: 40,000 daltons) was dissolved in 100 mL of dichloromethane, 10 g of gluconolactone and 150 mg of drug-loaded mesoporous ceramic @ polydopamine particles were added, and magnetic stirring was carried out at 300 rpm for 72 h to obtain a gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone solution. The solution was poured onto a glass culture dish and placed in a ventilated state for 72 h to obtain a gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone film.

[0097] S3, the film was placed in a printing cartridge, the needle diameter was 0.25 mm, the cartridge temperature was 60°C, the needle temperature was 65°C, the fiber spacing was 0.8 mm x 0.8 mm, the printing rate was 10 mm / s, the fiber direction was "0-90°", the air pressure was 380 Kpa, and the scaffold layer height was 80% of the needle diameter. The gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold was printed layer by layer. 20 mg of the gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold was soaked in 10 mL of an aqueous solution, and ultrasonic treatment was carried out at room temperature for 0.5 h to obtain a gradient pore structure drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold.

[0098] S4, the gradient pore structure scaffold was added to 12 mL of a 41 mg / mL aqueous solution of 2-methylimidazole, 2 mL of a 12 mg / mL aqueous solution of zinc nitrate was added dropwise under stirring, and after reaction, the product was washed, freeze-dried to obtain a gradient pore structure, ZIF8 surface modified drug-loaded mesoporous ceramic @ polydopamine / polycaprolactone scaffold.

[0099] Comparative Example 4

[0100] This comparative example provides a preparation method of a polycaprolactone scaffold, which is substantially the same as that of Example 1, except that it does not contain drug-loaded ZIF8, and the preparation method is as follows:

[0101] S1, 1000 mg of mesoporous zinc silicate (average particle size between 300 nm and 800 nm, specific surface area between 400 m 2 / g and 900 m 2 / g, average pore size between 30 nm and 50 nm) was dispersed in 100 mL of an aqueous solution containing 0.2 mg of bone morphogenetic protein-2, and stirring was carried out at 200 rpm for 18 h. After freeze-drying, a therapeutic agent-loaded mesoporous material was obtained; 300 mg of the therapeutic agent-loaded mesoporous material was dispersed in 100 mL of a 2 mg / mL aqueous solution of dopamine (pH = 8.5) at 0°C, and stirring was carried out at 200 rpm for 10 min. After freeze-drying, a polydopamine-coated drug-loaded mesoporous material was obtained.

[0102] S2, 10 g of polycaprolactone (molecular weight: 40,000 daltons) was dissolved in 100 mL of dichloromethane, 10 g of gluconolactone and 150 mg of drug-loaded mesoporous ceramic @ polydopamine particles were added, and magnetic stirring was carried out at 300 rpm for 72 h to obtain a gluconolactone / mesoporous ceramic @ polydopamine particle / polycaprolactone solution. The solution was poured onto a glass culture dish and placed in a ventilated state for 72 h to obtain a gluconolactone / mesoporous ceramic @ polydopamine particle / polycaprolactone film.

[0103] S3, the film was placed in a printing cartridge, the needle diameter was 0.25 mm, the cartridge temperature was 60°C, the needle temperature was 65°C, the fiber spacing was 0.8 mm x 0.8 mm, the printing rate was 10 mm / s, the fiber direction was "0-90°", the air pressure was 380 Kpa, and the scaffold layer height was 80% of the needle diameter. The gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold was printed layer by layer. 20 mg of the gluconolactone / drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold was soaked in 10 mL of an aqueous solution, and ultrasonic treatment was carried out at room temperature for 0.5 h to obtain a gradient pore structure drug-loaded mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold.

[0104] Comparative Example 5

[0105] This comparative example provides a preparation method of a polycaprolactone scaffold, which is substantially the same as that of Example 1, except that the bioactive ceramic @ polydopamine particles are not drug-loaded, and the preparation method is as follows:

[0106] S1, 1000 mg of mesoporous zinc silicate (average particle size between 300 nm and 800 nm, specific surface area between 400 m 2 / g and 900 m 2 / g, average pore size between 30 nm and 50 nm) was dispersed in 100 mL of an aqueous solution at 0°C, stirred at 200 rpm for 18 h, and freeze-dried. 300 mg of the loaded mesoporous material was dispersed in 100 mL of a 2 mg / mL dopamine aqueous solution (pH = 8.5) at 0°C, stirred at 200 rpm for 10 min, and freeze-dried to obtain polydopamine-coated mesoporous material.

[0107] S2, 10 g of polycaprolactone (molecular weight: 40,000 daltons) was dissolved in 100 mL of dichloromethane, 10 g of gluconolactone and 150 mg of drug-loaded mesoporous ceramic @ polydopamine particles were added, and magnetic stirring was carried out at 300 rpm for 72 h to obtain a gluconolactone / mesoporous ceramic @ polydopamine particle / polycaprolactone solution. The solution was poured onto a glass culture dish and placed in a ventilated state for 72 h to obtain a gluconolactone / mesoporous ceramic @ polydopamine particle / polycaprolactone film.

[0108] S3, the film was put into a printing cartridge, the needle diameter was 0.25 mm, the cartridge temperature was 60°C, the needle temperature was 65°C, the fiber spacing was 0.8 mm x 0.8 mm, the printing rate was 10 mm / s, the fiber direction was "0-90°", the air pressure was 380 Kpa, and the support layer height was 80% of the needle diameter. The glucose lactone / mesoporous ceramic@polydopamine particle / polycaprolactone scaffold was printed layer by layer. 20 mg of the glucose lactone / mesoporous ceramic@polydopamine particle / polycaprolactone scaffold was soaked in 10 mL of an aqueous solution, ultrasonicated at room temperature for 0.5 hours, and a gradient pore structure mesoporous ceramic@polydopamine particle / polycaprolactone scaffold was obtained.

[0109] S4, the gradient pore structure scaffold was added to 12 mL of a 41 mg / mL aqueous solution of 2-methylimidazole (containing 15 mg / mL of vancomycin), 2 mL of a 12 mg / mL aqueous solution of zinc nitrate was added dropwise under stirring, and after reaction, the gradient pore structure, drug-loaded ZIF8 surface modified mesoporous ceramic@polydopamine / polycaprolactone scaffold was obtained after washing, freeze-drying.

[0110] Comparative Example 6

[0111] This comparative example provides a preparation method of a polycaprolactone scaffold, which is substantially the same as that of Example 1, except that the drug-loaded polydopamine particles do not contain biologically active ceramic, and the preparation method is as follows:

[0112] S1, 0.2 mg of bone morphogenetic protein-2 was dissolved in 1000 mL of a 2 mg / mL aqueous solution of dopamine (pH = 8.5) at 0°C, stirred at 200 rpm for 10 min, and after freeze-drying, drug-loaded polydopamine particles were obtained.

[0113] S2, 10 g of polycaprolactone (molecular weight: 40,000 daltons) was dissolved in 100 mL of dichloromethane, 10 g of gluconolactone and 150 mg of drug-loaded polydopamine were added, and magnetic stirring was performed at 300 rpm for 72 h to obtain a gluconolactone / drug-loaded polydopamine / polycaprolactone solution. The solution was poured onto a glass culture dish and placed in a ventilated state for 72 h to obtain a gluconolactone / drug-loaded polydopamine / polycaprolactone film.

[0114] S3, the film was put into a printing cartridge, the needle diameter was 0.25 mm, the cartridge temperature was 60°C, the needle temperature was 65°C, the fiber spacing was 0.8 mm x 0.8 mm, the printing rate was 10 mm / s, the fiber direction was "0-90°", the air pressure was 380 Kpa, and the support layer height was 80% of the needle diameter. The glucose lactone / drug-loaded polydopamine / polycaprolactone scaffold was printed layer by layer. 20 mg of the glucose lactone / drug-loaded polydopamine / polycaprolactone scaffold was soaked in 10 mL of an aqueous solution, ultrasonicated at room temperature for 0.5 hours, and a gradient pore structure drug-loaded polydopamine / polycaprolactone scaffold was obtained.

[0115] S4, the gradient pore structure scaffold was added to 12 mL of a 41 mg / mL aqueous solution of 2-methylimidazole (containing 15 mg / mL of vancomycin), 2 mL of a 12 mg / mL aqueous solution of zinc nitrate was added dropwise under stirring, and after reaction, the gradient pore structure drug-loaded ZIF8 surface modified drug-loaded polydopamine / polycaprolactone scaffold was obtained after washing, freeze-drying.

[0116] Comparative Example 7

[0117] This comparative example provides a preparation method of a polycaprolactone scaffold, which is substantially the same as that of Example 1, except that it does not contain polydopamine, and the preparation method is as follows:

[0118] S1, 1000 mg of mesoporous zinc silicate (average particle size between 300 nm and 800 nm, specific surface area between 400 m 2 / g and 900 m 2 / g, average pore size between 30 nm and 50 nm) was dispersed in 100 mL of an aqueous solution containing 0.2 mg of bone morphogenetic protein-2, stirred at 200 rpm for 18 h, and freeze-dried to obtain a drug-loaded mesoporous ceramic.

[0119] S2, 10 g of polycaprolactone (molecular weight: 40,000 daltons) was dissolved in 100 mL of dichloromethane, 10 g of glucose lactone and 150 mg of drug-loaded mesoporous ceramic particles were added, and magnetic stirring was performed at 300 rpm for 72 h to obtain a glucose lactone / drug-loaded mesoporous ceramic particle / polycaprolactone solution. The solution was poured onto a glass culture dish and placed in a ventilated state for 72 h to obtain a glucose lactone / drug-loaded mesoporous ceramic particle / polycaprolactone film.

[0120] S3, the film is put into the printing cartridge, the needle diameter is 0.25mm, the cartridge temperature is 60℃, the needle temperature is 65℃, the fiber spacing is 0.8mm*0.8mm, the printing rate is 10mm / s, the fiber direction is "0-90°", the air pressure is 380Kpa, and the scaffold layer height is 80% of the needle diameter. The glucose lactone / drug-loaded mesoporous ceramic particle / polycaprolactone scaffold is printed layer by layer. 20mg of the glucose lactone / drug-loaded mesoporous ceramic particle / polycaprolactone scaffold is soaked in 10mL of an aqueous solution, ultrasonic treatment is carried out at room temperature for 0.5h, and a gradient pore structure drug-loaded mesoporous ceramic particle / polycaprolactone scaffold is obtained.

[0121] S4, the gradient pore structure scaffold is added to 12mL of a 41mg / mL aqueous 2-methylimidazole solution (containing 15mg / mL of vancomycin), 2mL of a 12mg / mL aqueous zinc nitrate solution is added dropwise under stirring, and after reaction, the gradient pore structure drug-loaded ZIF8 surface modified drug-loaded mesoporous ceramic / polycaprolactone scaffold is obtained after washing and freeze-drying.

[0122] Comparative Example 8

[0123] The present comparative example provides a preparation method of a polycaprolactone scaffold, which is substantially the same as that of Example 1, except that the drug-loaded mesoporous ceramic@polydopamine particle is not contained, and the preparation method is specifically as follows:

[0124] S1, 10g of polycaprolactone (molecular weight: 40,000 daltons) is dissolved in 100mL of dichloromethane, 10g of glucose lactone is added, magnetic stirring is carried out at 300rpm for 72h, and a glucose lactone / polycaprolactone solution is obtained. The solution is poured on a glass culture dish, and placed under ventilation for 72h to obtain a glucose lactone / polycaprolactone film.

[0125] S2, the film is put into the printing cartridge, the needle diameter is 0.25mm, the cartridge temperature is 60℃, the needle temperature is 65℃, the fiber spacing is 0.8mm*0.8mm, the printing rate is 10mm / s, the fiber direction is "0-90°", the air pressure is 380Kpa, and the scaffold layer height is 80% of the needle diameter. The glucose lactone / polycaprolactone scaffold is printed layer by layer. 20mg of the glucose lactone / polycaprolactone scaffold is soaked in 10mL of an aqueous solution, ultrasonic treatment is carried out at room temperature for 0.5h, and a gradient pore structure polycaprolactone scaffold is obtained.

[0126] S4, the gradient pore structure scaffold is added to 12mL of a 41mg / mL aqueous 2-methylimidazole solution (containing 15mg / mL of vancomycin), 2mL of a 12mg / mL aqueous zinc nitrate solution is added dropwise under stirring, and after reaction, the gradient pore structure drug-loaded ZIF8 surface modified polycaprolactone scaffold is obtained after washing and freeze-drying.

[0127] Comparative Example 9

[0128] The present comparative example provides a preparation method of a polycaprolactone scaffold, which is substantially the same as that of Example 1, except that it does not contain drug-loaded mesoporous ceramic polydopamine particles, gluconolactone and drug-loaded ZIF8, and the preparation method is as follows:

[0129] 10 g of polycaprolactone (molecular weight: 40,000 daltons) was placed in a printing cartridge, the needle diameter was 0.25 mm, the cartridge temperature was 60°C, the needle temperature was 65°C, the fiber spacing was 0.8 mm x 0.8 mm, the printing rate was 10 mm / s, the fiber direction was "0-90°", the air pressure was 380 Kpa, and the scaffold layer height was 80% of the needle diameter. The gluconolactone / polycaprolactone scaffold was printed layer by layer. 20 mg of the gluconolactone / polycaprolactone scaffold was soaked in 10 mL of an aqueous solution, and ultrasonicated at room temperature for 0.5 hours to obtain a polycaprolactone scaffold.

[0130] Experimental analysis

[0131] The scaffold materials prepared in the examples and comparative examples were evaluated for the following properties, and the results are shown in Table 1-3. Figures 1-2 and Table 1-3.

[0132] 1. In vitro cytotoxicity evaluation

[0133] The prepared gel was evaluated and scored according to the requirements of GB / T 16886.5.

[0134] The L929 mouse fibroblasts that had grown vigorously for 24 h were digested and prepared into a density of 1.0 x 10 5 / mL, 100 μL per well, and inoculated into a 96-well plate. After the cells grew into a monolayer, the original culture solution was removed, and 100 μL of test sample extract, blank control, positive control and negative control were added, respectively, 6 replicates per group. After the addition was completed, the 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours.

[0135] After 24 h of culture, the original culture solution was aspirated, 50 μL of MTT (1 mg / mL) was added per well, and the culture was continued for 2 hours. After the end, the supernatant was aspirated, and 100 μL of 99.5% pure isopropyl alcohol was added to dissolve the crystals. The absorbance value was measured on an enzyme marker instrument with 570 nm as the main absorption wavelength and 650 nm as the reference wavelength, and the cell survival rate was calculated.

[0136] The cell survival rate was calculated according to the following formula: Cell survival rate (%) = 100 x OD 570e / OD570 b

[0137] OD 570eOD is the average value of the optical density of the test sample or control sample leaching solution.

[0138] OD 570b OD is the average value of the optical density of the blank.

[0139] The experimental results are shown in Table 1 below:

[0140] Table 1 In vitro cell survival rate of the stents prepared in the examples and comparative examples

[0141]

[0142] 2. Hemolysis test

[0143] According to the amount of blood for testing, blood is collected from the heart of a healthy rabbit. For example, 10 mL of blood is collected, 0.5 mL of a 20 g / L potassium oxalate solution is added, and fresh anticoagulated rabbit blood is prepared. 8 mL of fresh anticoagulated rabbit blood is diluted with 10 mL of a 9 g / L sodium chloride injection. The test product group is added with the test product according to the leaching ratio, and then 10 mL of sodium chloride injection is added. The negative control group is added with 10 mL of sodium chloride per tube. The positive control group is added with 10 mL of distilled water per tube. Each group is operated in parallel for 3 tubes. After all the test tubes are placed in a constant temperature water bath at (37±1) °C for 30 min, 0.2 mL of diluted rabbit blood is added to each test tube, mixed gently, and then placed in a (37±1) °C water bath for continuous incubation for 60 min. The liquid in the tube is poured out and centrifuged at 800 g for 5 min. The supernatant is transferred to a cuvette, and the absorbance is measured at a wavelength of 545 nm using a spectrophotometer. The absorbance of the test product group and the control group is the average value of 3 tubes. The absorbance of the negative control tube should be not more than 0.03; the absorbance of the positive control tube should be 0.8±0.3, otherwise the test should be repeated. The hemolysis rate is calculated according to the following formula: hemolysis rate = (absorbance of the test product group - absorbance of the negative control group) / (absorbance of the positive control group - absorbance of the negative control group) x 100%, and the results are shown in Table 2:

[0144] Table 2 Hemolysis rate of the materials prepared in the examples and comparative examples

[0145]

[0146] From the evaluation results of the hemolysis rate of the examples and comparative examples (Table 2), it can be seen that the hemolysis rate of the hydrogel prepared by the method of the present application is less than 5%, and there is no risk of hemolysis.

[0147] 3. In vitro drug release performance test

[0148] The in vitro drug release performance evaluation method is as follows: At 37℃ and 60 rpm, 500 mg of the product is immersed in 200 mL of PBS (pH = 7.4) in a constant temperature shaker. The test solution is collected periodically, and an equal amount of PBS is added. The content of the therapeutic agent in the collected test solution is determined by high performance liquid chromatography (HPLC), and compared with the total load of the therapeutic agent in the product to calculate the cumulative release rate of the therapeutic agent.

[0149] Results of in vitro drug release performance testing for promoting tissue regeneration and repair are shown below. Figure 1 and 2 The scaffolds prepared in Examples 1-4 all exhibited release periods exceeding 5 weeks for various drugs promoting tissue regeneration and repair. Comparative Example 1, which did not use gluconolactone, had scaffold fibers lacking a porous structure, resulting in a significantly lower drug release rate compared to Example 1. Comparative Examples 2 and 4, without ZIF8 surface modification, showed significantly increased burst release and a significantly faster release rate compared to Example 1, with near-complete release within 5 weeks. The drug-loaded polydopamine particles in Comparative Example 6 did not contain bioactive ceramics, thus having little impact on drug release, and their release curves were similar to those of Example 1. Comparative Example 7, which did not contain polydopamine, showed significantly increased burst release and a significantly faster release rate compared to Example 1, with near-complete release within 4 weeks.

[0150] Results of in vitro antibacterial drug release performance testing are shown below Figure 3 and 4 The scaffolds prepared in Examples 1-4 all had release periods exceeding 3 weeks for various antibacterial drugs. Comparative Example 1 did not use gluconolactone, and its scaffold fibers did not contain a porous structure; its antibacterial drug release rate was close to that of Example 1. Compared with Example 1, the modifications in Comparative Examples 5-8 had little effect on the release of antibacterial drugs embedded on the scaffold surface, and the release curves were all close to those of Example 1. Comparative Example 2 did not contain a ZIF8 layer, and the antibacterial drugs were directly adsorbed onto the scaffold surface. Compared with Example 1, the burst release was significantly increased, and the release rate was significantly faster, with the drugs being basically released within 1 week.

[0151] 4. Detection of osteogenic differentiation performance of pre-osteoblasts induced in vitro

[0152] The composite materials prepared in Examples 1-5 and Comparative Examples 1-3 were irradiated and sterilized, then immersed in DMEM basal medium at a concentration of 10 mg / mL and extracted at 120 rpm for 24 h in a shaker at 37°C. After extraction, the mixture of composite material and medium was centrifuged at 1000 rpm and the supernatant was collected. The collected extracts were diluted 2-fold with the corresponding DMEM medium, and finally 10% fetal bovine serum was added to obtain conditioned medium.

[0153] MC3T3-E1 cells were distributed at a ratio of 1×10⁻⁶ per well. 5The individual density was inoculated in 24-well plates, and after 24 h of adherent culture, the conditioned medium was replaced, and the culture was carried out in an incubator at 37°C and in a 5% carbon dioxide atmosphere. The medium was replaced every 2-3 days, and after 7 days of culture, the osteogenic differentiation performance of the MC3T3-E1 cells was detected by the alkaline phosphatase secreted by the cells, and the pNPP method was used for determination. The specific steps are as follows: after the cells were washed with PBS solution, they were immersed in a PBS solution containing 0.1 mol / L glycine, 1 mmol / L magnesium chloride, and 0.05% triton X-100 (octylphenoxypolyethoxyethanol). After the cells were lysed, the lysate was uniformly mixed with para-nitrophenyl phosphate disodium salt, and the mixture was placed at 37°C for 30 min. Subsequently, the mixture was added dropwise to a 96-well plate, and the absorbance value of each well at 405 nm was determined by an enzyme-labeled instrument.

[0154] Definition of alkaline phosphatase activity unit: the amount of alkaline phosphatase required to hydrolyze 1 micromole of para-nitrophenyl phosphate chromogenic substrate to produce para-nitrophenol per minute in a diethanolamine (DEA) buffer at pH 9.8 and 37°C is defined as one enzyme activity unit, also known as one DEA enzyme activity unit. The amount of alkaline phosphatase required to hydrolyze 1 micromole of para-nitrophenyl phosphate chromogenic substrate to produce para-nitrophenol per minute in a glycine buffer at pH 9.6 and 25°C 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. According to the definition of enzyme activity, the alkaline phosphatase activity in the sample was calculated and plotted, respectively, as shown in Figure 3

[0155] Figure 5 ​​It can be seen that the scaffolds in Examples 1-4 have good effects of inducing cells to secrete alkaline phosphatase, but Comparative Example 1 does not have a gradient pore structure without using gluconolactone, and the cell activity is not as good as that of the scaffold with a gradient pore structure, thus affecting the secretion of alkaline phosphatase by the cells, and the alkaline phosphatase activity is slightly lower than that of Example 1. Comparative Example 3 does not load antibacterial drugs, and the alkaline phosphatase activity is close to that of Example 1. The drug-loaded polydopamine particles in Comparative Example 6 do not contain bioactive ceramics, thus affecting the secretion of alkaline phosphatase by the cells, and the alkaline phosphatase activity is also slightly lower than that of Example 1. Comparative Examples 2 and 4 do not contain ZIF8 layers with good bioactivity, and the alkaline phosphatase activity is significantly lower than that of Example 1. Comparative Example 7 does not contain polydopamine with osteogenesis activity, but the release of the drug is significantly accelerated, thus affecting the secretion of alkaline phosphatase by the cells, and the alkaline phosphatase activity is also significantly lower than that of Example 1. The bioactive ceramic / polydopamine particles in Comparative Example 5 do not load drugs, and the alkaline phosphatase activity is also significantly lower than that of Example 1. Comparative Example 8 does not contain drug-loaded bioactive ceramic / polydopamine particles, and the alkaline phosphatase activity is also significantly lower than that of Example 1, and the alkaline phosphatase activity is lower than that of Comparative Example 5. Comparative Example 9 does not contain ZIF8 layers with good bioactivity, does not have a gradient pore structure, does not load mesoporous ceramic / polydopamine particles and antibacterial drugs, and the alkaline phosphatase activity is the lowest.

[0156] 5. Detection of antibacterial performance of the scaffold

[0157] Fresh slant cultures of Staphylococcus aureus and Escherichia coli were taken, and the viable cell count was performed on the bacterial liquid, and the bacterial suspension with a bacterial content of 5x10 5 ~ 10x10 6 cfu / mL was prepared by using a diluent (0.03 mol / L PBS (pH = 7.2-7.4) containing 1% peptone). The samples were placed in sterile petri dishes, 50 μL of the bacterial suspension was added to each sample, the time of adding the bacteria to each tube was recorded, and blood plates were inoculated 60 min after the bacteria were added. At the same time, the samples were placed in 5 mL nutrient broth tubes. The blood plates inoculated with bacteria and the broth tubes were incubated at 37°C for 48 h, and the preliminary results were observed. The sterile growth tubes were continuously cultured until the 28th day. If the broth tube was turbid and bacterial growth was observed on the blood plate, it was recorded as positive, and was represented by (+). If it was still clear on the 35th day, it was considered as sterile growth, and was represented by (-).

[0158] Table 3. Antibacterial effects of the scaffolds prepared in Examples and Comparative Examples

[0159]

[0160]

[0161] As shown in Table 3, the composite stents prepared by the examples 1-4 and the comparative examples 1, 5-8 of the present application all have good long-acting antibacterial effect, and are sterile for 35 days. The comparative examples 2, 3, 4 and 9 do not contain ZIF8 or antibacterial drugs, and do not have antibacterial effect.

[0162] The above describes the preferred embodiments of the present application. It should be noted that, for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements are also considered to be within the scope of protection of the present application.

Claims

1. A modified polycaprolactone composite scaffold, characterized in that, The poly-caprolactone support base is internally loaded with a metal organic framework material, and the surface and interior of the poly-caprolactone support base are distributed with interconnected pores which gradually increase from the surface to the interior of the poly-caprolactone support base; the poly-caprolactone support base is distributed with drug-loaded materials, and the drug-loaded materials comprise mesoporous ceramics, the interior of the mesoporous ceramics is filled with tissue regeneration and repair drugs, and the surface of the mesoporous ceramics is coated with polydopamine; The mesoporous ceramics are at least one of mesoporous silica, mesoporous calcium silicate, mesoporous magnesium silicate, mesoporous zinc silicate, mesoporous strontium silicate, mesoporous bioglass and mesoporous hydroxyapatite; the mesoporous ceramics are particles with a particle size of 30-1500 nm; The preparation method of the modified poly-caprolactone composite support comprises the following steps: S1, mixing the drug-loaded material, gluconolactone and poly-caprolactone in a solvent to obtain a mixed solution, transferring the mixed solution to a mold and placing it for a period of time to obtain a mixed material film; using the mixed material film as a printing material, 3D printing to obtain a support with a gradient pore structure; After printing is completed, the support with the gradient pore structure is soaked in water to remove the gluconolactone and residual solvents; S2, reacting the support with the gradient pore structure, an antibacterial drug, 2-methylimidazole and a zinc source in water to prepare the modified poly-caprolactone composite support; The preparation method of the drug-loaded material comprises the following steps: immersing the mesoporous ceramics in a solution containing a tissue regeneration and repair drug to obtain mesoporous ceramics encapsulating the tissue regeneration and repair drug; immersing the mesoporous bioceramics encapsulating the tissue regeneration and repair drug in a solution containing dopamine to obtain the drug-loaded material.

2. The modified polycaprolactone composite scaffold of claim 1, wherein, The surface of the poly-caprolactone composite material has pores with a size of 1-100 μm.

3. The method of producing a modified polycaprolactone composite scaffold according to any one of claims 1-2, characterized in that, The preparation method comprises the following steps: S1, mixing the drug-loaded material, gluconolactone and poly-caprolactone in a solvent to obtain a mixed solution, transferring the mixed solution to a mold and placing it for a period of time to obtain a mixed material film; using the mixed material film as a printing material, 3D printing to obtain a support with a gradient pore structure; After printing is completed, the support with the gradient pore structure is soaked in water to remove the gluconolactone and residual solvents; S2, reacting the support with the gradient pore structure, an antibacterial drug, 2-methylimidazole and a zinc source in water to prepare the modified poly-caprolactone composite support.

4. The method for preparing the modified polycaprolactone composite scaffold according to claim 3, characterized in that, The mass ratio of the drug-loaded material to poly-caprolactone is (0.005-0.05):1; And / or, the mass ratio of the gluconolactone to poly-caprolactone is (0.5-3):

1.

5. The method for preparing the modified polycaprolactone composite scaffold according to claim 3, characterized in that, The process conditions of the 3D printing meet at least one of the following conditions: a) the 3D printing uses a printing needle with a diameter of 0.15-0.4 mm; b) printing and heating temperature: barrel 60-80℃, needle 60-90℃; c) fiber spacing 0.8mm×0.8mm; printing rate 8-17mm / s; d) air pressure 320-400Kpa; e) support layer height is 75-95% of the needle diameter.

6. The method for preparing the modified polycaprolactone composite scaffold according to claim 3, characterized in that, The mixing time in step S1 is 30-100 h; And / or, the placing time is 50-100 h.

7. The method for preparing the modified polycaprolactone composite scaffold according to claim 3, characterized in that, The mass ratio of the 2-methyl imidazole, the antibacterial drug and the zinc source is (18-23):(4-10):

1. And / or, the zinc source is zinc nitrate.

8. Use of the modified polycaprolactone composite scaffold according to any one of claims 1-2 in the preparation of a product for promoting bone tissue repair and regeneration.

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