A hydrogel-filled polycaprolactone composite scaffold and a preparation method and application thereof

Abstract

The application discloses a kind of hydrogel filled polycaprolactone composite material scaffolds and its preparation method and application, the polycaprolactone composite material scaffold has porous structure, wherein the surface and internal pore are interconnected, and the aperture from surface to inside is gradiently larger;The pore of the polycaprolactone composite material scaffold is filled with drug-loaded gel composite material.The hydrogel filled polycaprolactone composite material scaffold of the application prints scaffold and has macroscopic macropore and microscopic small hole simultaneously, not only can improve the surface roughness of scaffold, reach the purpose of significantly promoting scaffold cell adhesion, also can significantly improve the specific surface area of scaffold, make polycaprolactone accelerate degradation and release more acidic degradation product, solve the problem that polydopamine is slow in degradation in vitro and in vivo.The application fills good hydrogel in the pore of printing scaffold, can further regulate the hydrophilicity and hydrophobicity of material while maintaining the mechanical strength of material, so that it is more suitable for the regeneration and repair of bone defect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical materials, and particularly relates to a hydrogel-filled polycaprolactone composite scaffold 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 to prepare prostheses has shown 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 raw 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 3D printing material, which has good biocompatibility, non-toxicity, and biodegradability, 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 can be supplemented, and by adjusting the mixed components, the respective advantages of the components can be maximized, thereby meeting the ideal parameter requirements of bone repair materials. In addition, polydopamine microspheres have a certain drug loading capacity and can be used as a drug carrier for bone tissue engineering technology.

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

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 hydrogel-filled poly (caprolactone) composite scaffold. The second purpose of the present application is to provide a preparation method of the above hydrogel-filled poly (caprolactone) composite scaffold. The third purpose of the present application is to provide an application of the above hydrogel-filled 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 also can accelerate the release of antibacterial drugs in the case of infection, and start the slow release of the internally encapsulated drugs that promote tissue regeneration and repair. It is suitable for the repair and regeneration of infected bone tissue defects.

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

[0007] The first aspect of the present application provides a hydrogel-filled polycaprolactone composite scaffold, comprising a polycaprolactone scaffold matrix; the surface and interior of the polycaprolactone scaffold matrix are distributed with interconnected pores, the pores gradually increase from the surface to the interior of the polycaprolactone scaffold matrix; the polycaprolactone scaffold matrix is distributed with a drug-loaded material, the drug-loaded material comprises mesoporous ceramics, the mesoporous ceramics are filled with tissue regeneration and repair drugs inside, and the surface of the mesoporous ceramics is coated with polydopamine; the pores of the polycaprolactone scaffold matrix are filled with a gel composite material; the gel composite material comprises a hydrogel and a metal organic framework material wrapped in the hydrogel; the metal organic framework material is loaded with an antibacterial drug inside.

[0008] The pores of the scaffold in the present application gradually increase from the surface to the interior of the polycaprolactone scaffold matrix, forming a gradient pore and a multi-level pore structure, and the porous structure is filled with a gel composite material, wherein the interior pores of the polycaprolactone scaffold matrix refer to the pores formed by the stacking of fibers (macroscopic pores, several hundred microns), and the exterior pores of the polycaprolactone scaffold matrix refer to the pores formed on the fibers due to the pore-forming technology (microscopic pores, several to tens of microns).

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

[0010] Preferably, the mesoporous ceramics are selected from at least one of mesoporous silica, mesoporous calcium silicate, mesoporous magnesium silicate, mesoporous zinc silicate, mesoporous strontium silicate, mesoporous bioglass, and mesoporous hydroxyapatite.

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

[0012] Preferably, the specific surface area of the mesoporous ceramics 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 from zinc ions and 2-methyl imidazole.

[0014] Preferably, the tissue regeneration and repair drug is selected from at least one of 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, and vitamin D.

[0015] Preferably, the antibacterial drug is at least one of 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, nystatin, and griseofulvin.

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

[0017] Preferably, the raw material matrix of the hydrogel is at least one of methylacrylated hyaluronic acid, methylacrylated silk fibroin, methylacrylated chondroitin sulfate, methylacrylated chitosan, methylacrylated carboxymethyl chitosan, methylacrylated gelatin, methylacrylated sodium alginate, hyaluronic acid, silk fibroin, chondroitin sulfate, chitosan, carboxymethyl chitosan, gelatin, and sodium alginate.

[0018] The second aspect of the present application provides a preparation method of the hydrogel-filled polycaprolactone composite scaffold of the first aspect, comprising the following steps:

[0019] 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 is completed, immersing the scaffold with the gradient pore structure in water to remove the gluconolactone and residual solvent;

[0020] S2, reacting an antibacterial drug, 2-methylimidazole and a zinc source in water to obtain the metal-organic framework material, mixing the metal-organic framework material, the hydrogel matrix, the crosslinking agent and the scaffold with the gradient pore structure, and performing photocuring to prepare the hydrogel-filled polycaprolactone composite scaffold.

[0021] Preferably, the preparation method of the drug-loaded material comprises the following steps: immersing mesoporous ceramics in a solution containing a tissue regeneration and repair promoting drug to obtain mesoporous ceramics encapsulating the tissue regeneration and repair promoting drug; immersing the mesoporous bioceramics encapsulating the tissue regeneration and repair promoting drug in a solution containing dopamine to obtain the drug-loaded material.

[0022] More preferably, the solvent of the solution containing the tissue regeneration and repair promoting drug is water.

[0023] More preferably, the mass ratio of the mesoporous ceramic, the promoting regenerative repair drug and dopamine is (50-1000):(0.005-10):100.

[0024] Preferably, the mass ratio of the drug-loaded material and polycaprolactone is (0.005-0.05):1.

[0025] Preferably, the mass ratio of the gluconolactone and polycaprolactone is (0.5-3):1.

[0026] Preferably, the solvent is dichloromethane.

[0027] Preferably, the process conditions of the 3D printing meet at least one of the following:

[0028] a) The 3D printing uses a printing needle with a diameter of 0.15-0.4 mm;

[0029] b) The printing and heating temperature: barrel 60-80℃, needle 60-90℃;

[0030] c) The fiber spacing is 0.8mm×0.8mm; the printing speed is 8-17mm / s;

[0031] d) The air pressure is 320-400Kpa;

[0032] e) The scaffold layer height is 75-95% of the needle diameter;

[0033] f) The fiber direction is "0-90°".

[0034] The fiber spacing refers to the distance between the central axes of adjacent fibers in the horizontal and vertical directions. The fiber direction is the angle between the two layers of fibers. The scaffold layer height refers to the layer thickness of each layer printed.

[0035] Preferably, in step S1, the mixing time is 50-100h.

[0036] More preferably, the mixing is carried out under stirring conditions, wherein the stirring rate is 100-1000rpm.

[0037] Preferably, the placement time is 50-100h.

[0038] Preferably, the film-forming conditions of the mixed material film are volatilization film-forming.

[0039] Preferably, the mass ratio of the 2-methylimidazole, the antibacterial drug and the zinc source is (18-23):(4-10):1.

[0040] Preferably, the zinc source is zinc nitrate.

[0041] Preferably, the hydrogel matrix is selected from at least one of methacrylated hyaluronic acid, methacrylated fibroin, methacrylated chondroitin sulfate, methacrylated chitosan, methacrylated carboxymethyl chitosan, methacrylated gelatin, methacrylated sodium alginate, hyaluronic acid, fibroin, chondroitin sulfate, chitosan, carboxymethyl chitosan, gelatin, and sodium alginate.

[0042] Preferably, the step S2 specifically comprises the following steps: slowly adding a zinc source into an aqueous solution containing an antibacterial drug and 2-methylimidazole to react, to obtain the metal organic framework material; mixing the metal organic framework material, the hydrogel matrix, and a crosslinking agent in a buffer solution to obtain a hydrogel matrix mixed solution; and adding the scaffold with a gradient pore structure into the hydrogel matrix mixed solution to perform photocuring, to obtain the hydrogel-filled polycaprolactone composite scaffold.

[0043] More preferably, the mass-to-volume ratio of the scaffold with a gradient pore structure to the hydrogel matrix mixed solution is (0.5-1.5) g:10 mL.

[0044] More preferably, the photocuring time is 30-120 s.

[0045] More preferably, the photoinitiator is phenyl-2,4,6-trimethylbenzoyl lithium phosphite.

[0046] More preferably, the buffer solution is a PBS solution.

[0047] More preferably, the photocuring is performed using blue light.

[0048] The third aspect of the present application provides an application of the hydrogel-filled polycaprolactone composite scaffold of the first aspect in the preparation of a product for promoting bone tissue repair and regeneration.

[0049] The present application has the following beneficial effects:

[0050] The present application provides a hydrogel-filled polycaprolactone composite scaffold, comprising a polycaprolactone composite scaffold; the polycaprolactone composite scaffold has a porous structure, in which 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 (inter-fiber gaps) and microscopic small pores (pore holes on the surface of the fiber), 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 as to 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 solving the problem of slow degradation of polydopamine in vivo and in vitro.

[0051] The specific beneficial effects are as follows:

[0052] (1) The hydrogel-filled polycaprolactone composite scaffold of the present application is filled with a gel composite, which comprises a hydrogel and a metal organic framework material wrapped in the hydrogel, and the metal organic framework material can enhance the antibacterial performance of the gradient pore structure polycaprolactone scaffold; in the case of bone infection, the pH in the local microenvironment is lower, which will significantly accelerate the degradation of the metal organic framework material, and can accelerate the release of the antibacterial drugs loaded therein, and synergistically achieve better antibacterial and bone regeneration effects. In addition, the present application fills the pores of the printed scaffold with a hydrogel with good hydrophilicity, which can further regulate the hydrophilicity of the material while maintaining the mechanical strength of the material, making it more suitable for the regeneration and repair of bone defects.

[0053] (2) The hydrogel-filled polycaprolactone composite scaffold of the present application uses drug-loaded mesoporous ceramics as a scaffold forming material, which is further dispersed in the interior of polycaprolactone, not only enhancing the mechanical strength of the scaffold and the drug-loaded material, making it suitable for bone repair, but also having a controlled release drug effect.

[0054] (3) The present application provides a preparation method of the above-mentioned hydrogel-filled polycaprolactone composite scaffold, which is simple and feasible. The use of gluconolactone as a pore-forming agent can make the scaffold have a gradient pore structure, so that the printed scaffold can simultaneously obtain macroscopic macropores (inter-fiber gaps) and microscopic micropores (fiber surface pores), and gluconolactone is easily soluble in water and easy to eliminate after reaction.

[0055] (4) The hydrogel-filled polycaprolactone composite scaffold of the present 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 stem cell osteogenic differentiation. 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 internally 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

[0056] Figure 1 In vitro release performance of tissue regeneration and repair promoting drugs of the materials prepared in the examples and comparative examples;

[0057] Figure 2 In vitro release performance of tissue regeneration and repair promoting drugs of the materials prepared in the examples and comparative examples;

[0058] Figure 3 In vitro antibacterial drug release performance of the materials prepared in the examples and comparative examples;

[0059] Figure 4In vitro antibacterial drug release performance of the material prepared in Example 1 and the comparative example;

[0060] Figure 5 Alkaline phosphatase activity of the scaffold prepared in the example and the comparative example. DETAILED DESCRIPTION

[0061] 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.

[0062] Example 1

[0063] This example provides a hydrogel-filled polycaprolactone composite scaffold, and the preparation method is as follows:

[0064] S1, 1000 mg of mesoporous bioglass (average particle size between 200-1000 nm, specific surface area between 300-900 m 2 / g, average pore size between 10 nm-45 nm) was dispersed in 100 mL aqueous solution (containing 50 mg of curcumin) at 25°C, stirred at 300 rpm for 12 h, and freeze-dried; 500 mg of the loaded mesoporous material was dispersed in 100 mL of 2 mg / mL dopamine aqueous solution (pH=8.4) at 20°C, stirred at 200 rpm for 16 min, and freeze-dried to obtain polydopamine-coated mesoporous material.

[0065] S2, 10 g of polycaprolactone (molecular weight: 40,000 daltons) was dissolved in 100 mL of dichloromethane, and then 12 g of gluconolactone and 1000 mg of mesoporous ceramic@polydopamine particles were added, and magnetic stirring was carried out at 500 rpm for 50 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.

[0066] S3, the film was placed in a printing cartridge, the needle diameter was 0.2 mm, the cartridge temperature was 65°C, the needle temperature was 70°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 350 Kpa, and the scaffold layer height was 80% of the needle diameter. The gluconolactone / mesoporous ceramic@polydopamine particle / polycaprolactone scaffold was printed layer by layer. 20 mg of the gluconolactone / mesoporous ceramic@polydopamine particle / polycaprolactone scaffold was soaked in 10 mL of aqueous solution, ultrasonicated at room temperature for 0.5 h, and a gradient pore structure mesoporous ceramic@polydopamine particle / polycaprolactone scaffold was obtained.

[0067] S4, 20 mg of vancomycin was added to 20 mL of 27 mg / mL aqueous 2-methylimidazole solution, 4 mL of 6.5 mg / mL aqueous zinc nitrate solution was added dropwise under stirring, and after reaction, the product was washed and freeze-dried to obtain drug-loaded ZIF8. 250 mg of methacrylated sodium alginate was dissolved in 10 mL of PBS solution containing 0.2% phenyl-2,4,6-trimethylbenzoyl lithium phosphite, and 200 mg of drug-loaded ZIF8 was added to obtain a drug-loaded ZIF8-containing hydrogel solution. 60 mg of bioactive ceramic / polycaprolactone scaffold was placed in the drug-loaded ZIF8-containing hydrogel solution, removed after dispersion for 2 min, and cured by blue light irradiation for 60 s. The scaffold was frozen in a -20°C refrigerator and then freeze-dried in a freeze dryer to obtain a drug-loaded mesoporous ceramic@polydopamine / polycaprolactone scaffold with a drug-loaded ZIF8 hydrogel filled gradient pore structure.

[0068] The modified polycaprolactone composite scaffold prepared in Example 1 has a porous structure, and the porous structure is filled with a gel composite. The pores of the scaffold gradually increase from the surface of the polycaprolactone scaffold matrix to the inside. The inside pores of the polycaprolactone scaffold matrix refer to the pores formed by the stacking of fibers (macroscopic pores, hundreds of microns), and the outside pores of the polycaprolactone scaffold matrix refer to the pores formed on the fibers due to the poration technology (microscopic pores, several to tens of microns).

[0069] Example 2

[0070] This example provides a hydrogel-filled polycaprolactone composite scaffold, and the preparation method is as follows:

[0071] S1, 1000 mg of mesoporous silica (average particle size between 30-600 nm, specific surface area between 500-1800 m 2 / g, average pore size between 2 nm-10 nm) was dispersed in 100 mL of aqueous solution (containing 0.3 mg of bone morphogenetic protein-7) at 0°C, stirred at 400 rpm for 8 h, and freeze-dried. 700 mg of mesoporous material was dispersed in 100 mL of 2.8 mg / mL dopamine aqueous solution (pH=8.2) at 0°C, stirred at 300 rpm for 12 min, and freeze-dried to obtain polydopamine-coated mesoporous material.

[0072] S2, 10 g of polycaprolactone (molecular weight: 60,000 daltons) was dissolved in 150 mL of dichloromethane, and 16 g of gluconolactone and 800 mg of mesoporous ceramic@polydopamine particles were added, and the mixture was stirred at 400 rpm for 40 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 60 h to obtain a gluconolactone / mesoporous ceramic@polydopamine particle / polycaprolactone film.

[0073] S3, the film is put into a printing cartridge, the needle diameter is 0.3mm, the cartridge temperature is 70℃, the needle temperature is 75℃, the fiber spacing is 0.8mm*0.8mm, the printing rate is 7mm / s, the fiber direction is "0-90°", the air pressure is 340Kpa, and the support layer height is 85% of the needle diameter. The glucose lactone / mesoporous ceramic@polydopamine particle / polycaprolactone support is printed layer by layer. 20mg of glucose lactone / mesoporous ceramic@polydopamine particle / polycaprolactone support is soaked in 25mL aqueous solution, ultrasonic is obtained at room temperature for 0.75h, and a gradient pore structure mesoporous ceramic@polydopamine particle / polycaprolactone support is obtained.

[0074] S4, 16mg of gentamicin is added to 20mL of 38mg / mL 2-methylimidazole aqueous solution, 2mL of 18mg / mL zinc nitrate aqueous solution is added dropwise under stirring, and after reaction, washing, freeze-drying, drug-loaded ZIF8 is obtained. 200mg of methacrylated hyaluronic acid is dissolved in 10mL of PBS solution containing 0.3% phenyl-2,4,6-trimethylbenzoyl lithium phosphite, and then 300mg of drug-loaded ZIF8 is added to obtain a drug-loaded ZIF8-containing hydrogel solution. 100mg of bioactive ceramic / polycaprolactone support is placed in the drug-loaded ZIF8-containing hydrogel solution, dispersed for 6min, taken out, and cured by blue light irradiation for 50s. The support is placed in a-20℃ refrigerator and freeze-dried in a freeze dryer to obtain a drug-loaded ZIF8 hydrogel filled gradient pore structure drug-loaded mesoporous ceramic / polydopamine / polycaprolactone support.

[0075] Example 3

[0076] The present embodiment provides a hydrogel-filled polycaprolactone composite support, and the preparation method is as follows:

[0077] S1, at 15℃, 1000mg of mesoporous calcium silicate (average particle size between 1000-1200nm, specific surface area between 200-900m 2 / g, average pore size between 5nm-15nm) is dispersed in 100mL of aqueous solution (containing 40mg of alendronate sodium), 600rpm stirring for 6h, freeze-drying; at 40℃, 400mg of loaded mesoporous material is dispersed in 100mL of 1.6mg / mL dopamine aqueous solution (pH=8.6), 600rpm stirring for 2min, freeze-drying to obtain polydopamine-coated mesoporous material.

[0078] S2, 10 g polycaprolactone (molecular weight: 80,000 daltons) was dissolved in 250 mL dichloromethane, 24 g gluconolactone and 2200 mg mesoporous ceramic @ polydopamine particles were added, mechanical stirring at 600 rpm for 36 h, to obtain a gluconolactone / mesoporous ceramic @ polydopamine particle / polycaprolactone solution. Pour the solution on a glass culture dish, place in a ventilated state for 96 h, to obtain a gluconolactone / mesoporous ceramic @ polydopamine particle / polycaprolactone film.

[0079] S3, the film was placed in a printing cartridge, the needle diameter was 0.45 mm, the cartridge temperature was 85°C, the needle temperature was 90°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 400 Kpa, and the scaffold layer height was 95% of the needle diameter. The gluconolactone / mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold was printed layer by layer. 20 mg of gluconolactone / mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold was soaked in 50 mL aqueous solution, ultrasonic for 1 hour at room temperature, to obtain a gradient pore structure mesoporous ceramic @ polydopamine particle / polycaprolactone scaffold.

[0080] S4, 40 mg of triclosan was added to 20 mL of 33 mg / mL 2-methylimidazole aqueous solution, 3 mL of 10 mg / mL zinc nitrate aqueous solution was added dropwise under stirring, and after reaction, it was washed, freeze-dried to obtain drug-loaded ZIF8. 100 mg of methacrylated carboxymethyl gelatin was dissolved in 10 mL of PBS solution containing 0.15% phenyl-2,4,6-trimethylbenzoyl lithium phosphite, and 200 mg of drug-loaded ZIF8 was added to obtain a drug-loaded ZIF8-containing hydrogel solution. 150 mg of bioactive ceramic / polycaprolactone scaffold was placed in the drug-loaded ZIF8-containing hydrogel solution, dispersed for 10 min, then taken out and cured by blue light irradiation for 120 s. The scaffold was placed in a -20°C refrigerator and freeze-dried in a freeze dryer to obtain a drug-loaded ZIF8 hydrogel-filled gradient pore structure drug-loaded mesoporous ceramic @ polydopamine / polycaprolactone scaffold.

[0081] Example 4

[0082] The present embodiment provides a hydrogel-filled polycaprolactone composite scaffold, and the preparation method is as follows:

[0083] S1, at 15°C, 1000 mg of mesoporous calcium silicate (average particle size between 1000-1200 nm, specific surface area between 200-900 m 2S1, 10 g of mesoporous ceramic particles (average pore size between 2 nm and 10 nm, average pore size between 5 nm and 15 nm) were dispersed in 100 mL of an aqueous solution containing 40 mg of alendronate sodium, stirred at 600 rpm for 6 h, and freeze-dried; 400 mg of the loaded mesoporous material was dispersed in 100 mL of an aqueous solution of 1.6 mg / mL dopamine (pH = 8.6), stirred at 600 rpm for 2 min, and freeze-dried to obtain the polydopamine-coated mesoporous material.

[0084] S2, 10 g of polycaprolactone (molecular weight: 80,000 daltons) was dissolved in 250 mL of dichloromethane, and 24 g of gluconolactone and 2200 mg of mesoporous ceramic@polydopamine particles were added, and mechanical stirring was performed at 600 rpm for 36 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 96 h to obtain a gluconolactone / mesoporous ceramic@polydopamine particle / polycaprolactone film.

[0085] S3, the film was placed in a printing cartridge, the needle diameter was 0.45 mm, the cartridge temperature was 85°C, the needle temperature was 90°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 400 Kpa, and the support layer height was 95% of the needle diameter, and the gluconolactone / mesoporous ceramic@polydopamine particle / polycaprolactone scaffold was printed layer by layer. 20 mg of the gluconolactone / 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 mesoporous ceramic@polydopamine particle / polycaprolactone scaffold was obtained.

[0086] S4, 40 mg of triclosan was added to 20 mL of a 33 mg / mL aqueous solution of 2-methylimidazole, 3 mL of a 10 mg / mL aqueous solution of zinc nitrate was added dropwise under stirring, and after the reaction, the product was washed and freeze-dried to obtain drug-loaded ZIF8. 100 mg of methacrylated carboxymethyl gelatin was dissolved in 10 mL of a PBS solution containing 0.15% phenyl-2,4,6-trimethylbenzoyl lithium phosphite, and 200 mg of drug-loaded ZIF8 was added to obtain a drug-loaded ZIF8-containing hydrogel solution. 150 mg of the bioactive ceramic / polycaprolactone scaffold was placed in the drug-loaded ZIF8-containing hydrogel solution, dispersed for 10 min, and then taken out and cured by blue light irradiation for 120 s. The scaffold was frozen in a -20°C refrigerator and then freeze-dried in a freeze dryer to obtain a drug-loaded ZIF8 hydrogel-filled gradient-pore-structure drug-loaded mesoporous ceramic@polydopamine / polycaprolactone scaffold.

[0087] Comparative Example 1

[0088] The 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 no gluconolactone is used, and the preparation method is specifically as follows:

[0089] At 25°C, 1000 mg of mesoporous bioglass (average particle size between 200-1000 nm, specific surface area between 300-900 m 2 / g, average pore size between 10 nm-45 nm) is dispersed in 100 mL of an aqueous solution containing 50 mg of curcumin, stirred at 300 rpm for 12 h, and freeze-dried; at 20°C, 500 mg of the loaded mesoporous material is dispersed in 100 mL of an aqueous solution of 2 mg / mL dopamine (pH = 8.4), stirred at 200 rpm for 16 min, and freeze-dried to obtain a polydopamine-coated mesoporous material. 10 g of polycaprolactone (molecular weight: 40,000 daltons) is dissolved in 100 mL of dichloromethane, and then 1000 mg of the mesoporous ceramic@polydopamine particle is added, and magnetic stirring is performed at 500 rpm for 50 h to obtain a 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 mesoporous ceramic@polydopamine particle / polycaprolactone film. The film is placed in a printing cartridge, the needle diameter is 0.2 mm, the cartridge temperature is 65°C, the needle temperature is 70°C, the fiber spacing is 0.8 mm x 0.8 mm, the printing rate is 10 mm / s, the fiber direction is “0-90°”, the air pressure is 350 Kpa, and the scaffold layer height is 80% of the needle diameter, and the mesoporous ceramic@polydopamine particle / polycaprolactone scaffold is printed layer by layer. 20 mg of the mesoporous ceramic@polydopamine particle / polycaprolactone scaffold is soaked in 10 mL of an aqueous solution, ultrasonicated at room temperature for 0.5 h, and a mesoporous ceramic@polydopamine particle / polycaprolactone scaffold is obtained. 20 mg of vancomycin is added to 20 mL of a 27 mg / mL aqueous solution of 2-methylimidazole, 4 mL of a 6.5 mg / mL aqueous solution of zinc nitrate is added dropwise under stirring, and after reaction, washing and freeze-drying, a drug-loaded ZIF8 is obtained. 250 mg of methacrylated sodium alginate is dissolved in 10 mL of a PBS solution containing 0.2% phenyl-2,4,6-trimethylbenzoyl lithium phosphite, and then 200 mg of the drug-loaded ZIF8 is added to obtain a drug-loaded ZIF8-containing hydrogel solution. 60 mg of the bioactive ceramic / polycaprolactone scaffold is placed in the drug-loaded ZIF8-containing hydrogel solution, dispersed for 2 min, taken out, and cured by blue light irradiation for 60 s. The scaffold is frozen in a -20°C refrigerator, and then freeze-dried in a freeze dryer to obtain a drug-loaded ZIF8 hydrogel-filled drug-loaded mesoporous ceramic@polydopamine / polycaprolactone scaffold.

[0090] The modified polycaprolactone composite scaffold prepared in Comparative Example 1 has a relatively smooth surface, and occasionally has some micrometer to tens of micrometer holes.

[0091] Comparative Example 2

[0092] This comparative example provides a method for preparing a gradient-pore-structure polycaprolactone scaffold, which is substantially the same as that of Example 1, except that the ZIF8 surface is not modified, and the preparation method is as follows:

[0093] At 25°C, 1000 mg of mesoporous bioglass (average particle size between 200-1000 nm, specific surface area between 300-900 m 2 / g, average pore size between 10 nm-45 nm) is dispersed in 100 mL of an aqueous solution containing 50 mg of curcumin, stirred at 300 rpm for 12 h, and freeze-dried; at 20°C, 500 mg of the loaded mesoporous material is dispersed in 100 mL of an aqueous solution of 2 mg / mL dopamine (pH = 8.4), stirred at 200 rpm for 16 min, and freeze-dried to obtain polydopamine-coated mesoporous material. 10 g of polycaprolactone (molecular weight: 40,000 daltons) is dissolved in 100 mL of dichloromethane, and then 12 g of gluconolactone and 1000 mg of mesoporous ceramic@polydopamine particles are added, and magnetic stirring is performed at 500 rpm for 50 h to obtain a gluconolactone / 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 / mesoporous ceramic@polydopamine particle / polycaprolactone film. The film is placed in a printing cartridge, the needle diameter is 0.2 mm, the cartridge temperature is 65°C, the needle temperature is 70°C, the fiber spacing is 0.8 mm x 0.8 mm, the printing rate is 10 mm / s, the fiber direction is “0-90°”, the air pressure is 350 Kpa, and the scaffold layer height is 80% of the needle diameter, and the gluconolactone / mesoporous ceramic@polydopamine particle / polycaprolactone scaffold is printed layer by layer. 20 mg of the gluconolactone / mesoporous ceramic@polydopamine particle / polycaprolactone scaffold is soaked in 10 mL of an aqueous solution, ultrasonicated at room temperature for 0.5 h, and a gradient-pore-structure mesoporous ceramic@polydopamine particle / polycaprolactone scaffold is obtained. 60 mg of the bioactive ceramic / polycaprolactone scaffold is placed in an aqueous hydrogel solution containing 20 mg of vancomycin, dispersed for 2 min, and then removed, and irradiated with blue light for 60 s to solidify. The scaffold is frozen in a -20°C refrigerator, and then freeze-dried in a freeze dryer to obtain a gradient-pore-structure drug-loaded mesoporous ceramic@polydopamine / polycaprolactone scaffold filled with drug-loaded hydrogel.

[0094] Comparative Example 3

[0095] This comparative example provides a method for preparing a polycaprolactone scaffold, which is substantially the same as that of Example 1, except that no drug is loaded in the ZIF8, and the preparation method is as follows:

[0096] At 25 °C, 1000 mg mesoporous bioglass (average particle size between 200-1000 nm, specific surface area between 300-900 m 2 / g between 10 nm and 45 nm) was dispersed in 100 mL aqueous solution (containing 50 mg curcumin), stirred at 300 rpm for 12 h, and freeze-dried; 500 mg of the loaded mesoporous material was dispersed in 100 mL of 2 mg / mL dopamine aqueous solution (pH = 8.4), stirred at 200 rpm for 16 min, and freeze-dried to obtain polydopamine-coated mesoporous material. 10 g of polycaprolactone (molecular weight: 40,000 Dalton) was dissolved in 100 mL dichloromethane, and then 12 g of gluconolactone and 1000 mg of mesoporous ceramic@polydopamine particles were added, and the mixture was stirred at 500 rpm for 50 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. The film was placed in a printing cartridge, and a scaffold of gluconolactone / mesoporous ceramic@polydopamine particle / polycaprolactone was printed layer by layer with a needle diameter of 0.2 mm, a cartridge temperature of 65 °C, a needle temperature of 70 °C, a fiber spacing of 0.8 mm x 0.8 mm, a printing rate of 10 mm / s, a fiber direction of “0-90°”, an air pressure of 350 Kpa, and a scaffold layer height of 80% of the needle diameter. 20 mg of the gluconolactone / mesoporous ceramic@polydopamine particle / polycaprolactone scaffold was soaked in 10 mL of an aqueous solution, and a gradient-pore-structure mesoporous ceramic@polydopamine particle / polycaprolactone scaffold was obtained by ultrasonicating at room temperature for 0.5 h. In 20 mL of a 27 mg / mL aqueous solution of 2-methylimidazole, 4 mL of a 6.5 mg / mL aqueous solution of zinc nitrate was added dropwise with stirring, and after the reaction, the ZIF8 was obtained by washing and freeze-drying. 250 mg of methacrylated sodium alginate was dissolved in 10 mL of a PBS solution containing 0.2% phenyl-2,4,6-trimethylbenzoyl lithium phosphite, and then 200 mg of ZIF8 was added to obtain a ZIF8-containing hydrogel solution. 60 mg of the bioactive ceramic / polycaprolactone scaffold was placed in the ZIF8-containing hydrogel solution, dispersed for 2 min, removed, and cured by blue light irradiation for 60 s. The scaffold was frozen in a -20 °C refrigerator and then freeze-dried in a freeze dryer to obtain a ZIF8-containing hydrogel-filled gradient-pore-structure drug-loaded mesoporous ceramic@polydopamine / polycaprolactone scaffold.

[0097] Comparative Example 4

[0098] This comparative example provides a method for preparing 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:

[0099] At 25℃, 1000 mg of mesoporous bioglass (average particle size between 200 and 1000 nm, specific surface area between 300 and 900 m²) was added. 2 The polydopamine-coated mesoporous material (with an average pore size between 10 nm and 45 nm) was dispersed in 100 mL of aqueous solution (containing 50 mg curcumin), stirred at 300 rpm for 12 h, and then freeze-dried. At 20 °C, 500 mg of the loaded mesoporous material was dispersed in 100 mL of an aqueous solution of 2 mg / mL dopamine (pH = 8.4), stirred at 200 rpm for 16 min, and then freeze-dried to obtain polydopamine-coated mesoporous material. 10 g of polycaprolactone (molecular weight: 40,000 Daltons) was dissolved in 100 mL of dichloromethane, and then 12 g of gluconolactone and 1000 mg of mesoporous ceramic@polydopamine particles were added. The mixture was magnetically stirred at 500 rpm for 50 h to obtain a gluconolactone / mesoporous ceramic@polydopamine particle / polycaprolactone solution. This solution was poured onto a glass petri dish and placed under aeration for 72 h to obtain a gluconolactone / mesoporous ceramic@polydopamine particle / polycaprolactone film. The film was placed in a printing barrel with a needle diameter of 0.2 mm, barrel temperature of 65℃, needle temperature of 70℃, fiber spacing of 0.8 mm × 0.8 mm, printing speed of 10 mm / s, fiber orientation of "0-90°", air pressure of 350 kPa, and scaffold layer height of 80% of needle diameter. Gluconolactone / mesoporous ceramic@polydopamine particles / polycaprolactone scaffold was printed layer by layer. 20 mg of gluconolactone / mesoporous ceramic@polydopamine particles / polycaprolactone scaffold was immersed in 10 mL of aqueous solution and sonicated at room temperature for 0.5 hours to obtain a gradient-pore structure mesoporous ceramic@polydopamine particles / polycaprolactone scaffold. 250 mg of sodium methacrylamide alginate was dissolved in 10 mL of PBS solution containing 0.2% phenyl-2,4,6-trimethylbenzoyl lithium phosphite to obtain a hydrogel solution. 60 mg of bioactive ceramic / polycaprolactone scaffold was placed in the hydrogel solution, dispersed for 2 min, and then removed and cured by blue light irradiation for 60 s. The scaffold was then frozen in a -20°C freezer and freeze-dried to obtain a hydrogel-filled gradient pore structure drug-loaded mesoporous ceramic@polydopamine / polycaprolactone scaffold.

[0100] Comparative Example 5

[0101] This comparative example provides a method for preparing a polycaprolactone scaffold, which is largely the same as that in Example 1, except that the bioactive ceramic@polydopamine particles do not contain drug loading. The specific preparation method is as follows:

[0102] At 25℃, 1000 mg of mesoporous bioglass (average particle size between 200 and 1000 nm, specific surface area between 300 and 900 m²) was added. 2The mesoporous ceramic@polydopamine particles were prepared as follows: 1 g of mesoporous ceramic particles (pore size: 2-50 nm, average pore size: 10-45 nm) were dispersed in 100 mL of an aqueous solution of dopamine (2 mg / mL, pH = 8.4) and stirred at 200 rpm for 16 min, and then freeze-dried to obtain polydopamine-coated mesoporous ceramic particles. 10 g of polycaprolactone (molecular weight: 40,000) was dissolved in 100 mL of dichloromethane, and then 12 g of gluconolactone and 1000 mg of the mesoporous ceramic@polydopamine particles were added, and the mixture was stirred at 500 rpm for 50 h to obtain a solution of the gluconolactone / mesoporous ceramic@polydopamine particle / polycaprolactone. The solution was poured onto a glass dish and left to stand in a ventilated state for 72 h to obtain a film of the gluconolactone / mesoporous ceramic@polydopamine particle / polycaprolactone. The film was placed in a printing cartridge, and a scaffold of the gluconolactone / mesoporous ceramic@polydopamine particle / polycaprolactone was printed layer by layer with a needle diameter of 0.2 mm, a cartridge temperature of 65°C, a needle temperature of 70°C, a fiber spacing of 0.8 mm x 0.8 mm, a printing rate of 10 mm / s, a fiber direction of "0-90°", an air pressure of 350 Kpa, and a scaffold layer height of 80% of the needle diameter. 20 mg of the scaffold of the gluconolactone / mesoporous ceramic@polydopamine particle / polycaprolactone was soaked in 10 mL of an aqueous solution, and an ultrasonic treatment was performed at room temperature for 0.5 h to obtain a scaffold of the gradient-pore-structure mesoporous ceramic@polydopamine particle / polycaprolactone. 20 mg of vancomycin was added to 20 mL of an aqueous solution of 2-methylimidazole (27 mg / mL), and 4 mL of an aqueous solution of zinc nitrate (6.5 mg / mL) was added dropwise under stirring. After the reaction, the product was washed and freeze-dried to obtain a drug-loaded ZIF8. 250 mg of methacrylated sodium alginate was dissolved in 10 mL of a PBS solution containing 0.2% lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and then 200 mg of the drug-loaded ZIF8 was added to obtain a hydrogel solution containing the drug-loaded ZIF8. 60 mg of the bioactive ceramic / polycaprolactone scaffold was placed in the hydrogel solution containing the drug-loaded ZIF8, dispersed for 2 min, taken out, and cured by irradiation with blue light for 60 s. The scaffold was frozen in a refrigerator at -20°C, and then freeze-dried in a freeze dryer to obtain a scaffold of the gradient-pore-structure mesoporous ceramic@polydopamine / polycaprolactone filled with the drug-loaded ZIF8 hydrogel.

[0103] Comparative Example 6

[0104] This comparative example provides a method for preparing a polycaprolactone scaffold, which is substantially the same as that of Example 1, except that the drug-loaded polydopamine particles do not contain bioactive ceramic, and the preparation method is as follows:

[0105] At 20℃, 50mg curcumin was dispersed in 100mL 2mg / mL dopamine aqueous solution (pH=8.4) with 200rpm stirring for 16min, and drug-loaded polydopamine particles were obtained after freeze-drying. 10g polycaprolactone (molecular weight: 40,000 Dalton) was dissolved in 100mL dichloromethane, and then 12g gluconolactone and 1000mg drug-loaded polydopamine particles were added, and the solution was stirred at 500rpm for 50h to obtain a gluconolactone / drug-loaded polydopamine particle / polycaprolactone solution. The solution was poured onto a glass dish and placed in a ventilated state for 72h to obtain a gluconolactone / drug-loaded polydopamine particle / polycaprolactone film. The film was placed in a printing cartridge, and a 0.2mm diameter needle, a cartridge temperature of 65℃, a needle temperature of 70℃, a fiber spacing of 0.8mm x 0.8mm, a printing rate of 10mm / s, a fiber direction of "0-90°", an air pressure of 350Kpa, and a scaffold layer height of 80% of the needle diameter were used to layer-by-layer print a gluconolactone / drug-loaded polydopamine particle / polycaprolactone scaffold. 20mg of the gluconolactone / drug-loaded polydopamine particle / polycaprolactone scaffold was soaked in 10mL of an aqueous solution, and an ultrasonic wave was applied for 0.5h at room temperature to obtain a gradient-pore-structured drug-loaded polydopamine particle / polycaprolactone scaffold. 20mg of vancomycin was added to 20mL of a 27mg / mL aqueous solution of 2-methylimidazole, and 4mL of a 6.5mg / mL aqueous solution of zinc nitrate was added dropwise under stirring. After the reaction, the product was washed and freeze-dried to obtain drug-loaded ZIF8. 250mg of methacrylated sodium alginate was dissolved in 10mL of a PBS solution containing 0.2% phenyl-2,4,6-trimethylbenzoyl lithium phosphite, and 200mg of drug-loaded ZIF8 was added to obtain a drug-loaded ZIF8-containing hydrogel solution. 60mg of the gradient-pore-structured drug-loaded polydopamine particle / polycaprolactone scaffold was placed in the drug-loaded ZIF8-containing hydrogel solution, dispersed for 2min, and then removed and cured by blue light irradiation for 60s. The scaffold was frozen in a -20℃ refrigerator and then freeze-dried to obtain a gradient-pore-structured drug-loaded polydopamine particle / polycaprolactone scaffold filled with drug-loaded ZIF8 hydrogel.

[0106] Comparative Example 7

[0107] This comparative example provides a method for preparing 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:

[0108] At 25℃, 1000mg mesoporous bioglass (average particle size between 200-1000nm, specific surface area between 300-900m 2The drug-loaded mesoporous ceramic was obtained by dispersing 10 g of the drug-loaded mesoporous ceramic (average pore size between 2 nm and 50 nm, average pore size between 10 nm and 45 nm) in 100 mL of an aqueous solution (containing 50 mg of curcumin) and stirring at 300 rpm for 12 h, and then freeze-drying. 10 g of polycaprolactone (molecular weight: 40,000 daltons) was dissolved in 100 mL of dichloromethane, and then 12 g of gluconolactone and 1000 mg of the drug-loaded mesoporous ceramic were added, and the solution was stirred at 500 rpm for 50 h by magnetic stirring to obtain a gluconolactone / drug-loaded mesoporous ceramic / polycaprolactone solution. The solution was poured onto a glass culture dish, and left to stand in a ventilated state for 72 h to obtain a gluconolactone / drug-loaded mesoporous ceramic / polycaprolactone film. The film was placed in a printing cartridge, and a fiber scaffold was printed layer by layer by using a needle with a diameter of 0.2 mm, a cartridge temperature of 65°C, a needle temperature of 70°C, a fiber spacing of 0.8 mm x 0.8 mm, a printing rate of 10 mm / s, a fiber direction of “0-90°”, an air pressure of 350 Kpa, and a scaffold layer height of 80% of the diameter of the needle. 20 mg of the gluconolactone / drug-loaded mesoporous ceramic / polycaprolactone scaffold was soaked in 10 mL of an aqueous solution, and an ultrasonic wave was applied thereto at room temperature for 0.5 h to obtain a gradient-pore-structure drug-loaded mesoporous ceramic / polycaprolactone scaffold. 20 mg of vancomycin was added to 20 mL of a 27 mg / mL aqueous solution of 2-methylimidazole, and 4 mL of a 6.5 mg / mL aqueous solution of zinc nitrate was added dropwise under stirring, and then the reaction product was washed and freeze-dried to obtain a drug-loaded ZIF8. 250 mg of methacrylated sodium alginate was dissolved in 10 mL of a PBS solution containing 0.2% of lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and then 200 mg of the drug-loaded ZIF8 was added to obtain a drug-loaded ZIF8-containing hydrogel solution. 60 mg of the bioactive ceramic / polycaprolactone scaffold was placed in the drug-loaded ZIF8-containing hydrogel solution, and dispersed for 2 min, and then taken out and cured by irradiation with blue light for 60 s. The scaffold was frozen in a refrigerator at -20°C, and then freeze-dried to obtain a gradient-pore-structure drug-loaded mesoporous ceramic / polycaprolactone scaffold filled with drug-loaded ZIF8 hydrogel.

[0109] Comparative Example 8

[0110] This comparative example provides a method for preparing a polycaprolactone scaffold, which is substantially the same as that of Example 1, except that the drug-loaded mesoporous ceramic@polydopamine particles are not contained, and the preparation method is as follows:

[0111] The 10 g polycaprolactone (molecular weight: 40,000 Dalton) was dissolved in 100 mL dichloromethane, 12 g of gluconolactone was added, and the solution was stirred at 500 rpm for 50 h to obtain a gluconolactone / polycaprolactone solution. The solution was poured onto a glass culture dish and placed in a ventilated state for 72 h to obtain a gluconolactone / polycaprolactone film. The film was placed in a printing cartridge, the needle diameter was 0.2 mm, the cartridge temperature was 65°C, the needle temperature was 70°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 350 Kpa, and the support 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 ultrasonic treatment was performed at room temperature for 0.5 h to obtain a gradient pore structure polycaprolactone scaffold. 20 mg of vancomycin was added to 20 mL of a 27 mg / mL aqueous solution of 2-methylimidazole, 4 mL of a 6.5 mg / mL aqueous solution of zinc nitrate was added dropwise under stirring, and after reaction, the product was washed, freeze-dried to obtain drug-loaded ZIF8. 250 mg of methacrylated sodium alginate was dissolved in 10 mL of a PBS solution containing 0.2% phenyl-2,4,6-trimethylbenzoyl lithium phosphite, and 200 mg of drug-loaded ZIF8 was added to obtain a drug-loaded ZIF8-containing hydrogel solution. 60 mg of the gradient pore structure polycaprolactone scaffold was placed in the drug-loaded ZIF8-containing hydrogel solution, and after dispersion for 2 min, it was taken out and cured by blue light irradiation for 60 s. The scaffold was frozen in a -20°C refrigerator and then freeze-dried in a freeze dryer to obtain a drug-loaded ZIF8-containing hydrogel filled gradient pore structure polycaprolactone scaffold.

[0112] Comparative Example 9

[0113] This comparative example provides a method for preparing a polycaprolactone scaffold, which is substantially the same as 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:

[0114] Put 10 g polycaprolactone (molecular weight: 40,000 Dalton) into a printing cartridge, the needle diameter is 0.2 mm, the cartridge temperature is 65℃, the needle temperature is 70℃, the fiber spacing is 0.8 mm x 0.8 mm, the printing rate is 10 mm / s, the fiber direction is "0-90°", the air pressure is 350 Kpa, and the support layer height is 80% of the needle diameter. The polycaprolactone scaffold is printed layer by layer. Soak 20 mg of the polycaprolactone scaffold in 10 mL of an aqueous solution, and ultrasonicate at room temperature for 0.5 hours to obtain a gradient pore structure polycaprolactone scaffold. Dissolve 250 mg of methacrylated sodium alginate in 10 mL of a PBS solution containing 0.2% phenyl-2,4,6-trimethylbenzoyl lithium phosphite to obtain a hydrogel solution. Place 60 mg of the gradient pore structure polycaprolactone scaffold in the hydrogel solution, remove it after 2 minutes of dispersion, and irradiate it with blue light for 60 seconds to solidify. Place the scaffold in a -20℃ refrigerator to freeze, and then freeze-dry it in a freeze dryer to obtain a hydrogel-filled polycaprolactone scaffold.

[0115] Experimental analysis

[0116] The scaffold materials prepared in the examples and comparative examples were evaluated for the following properties, and the results are shown in Figures 1-2 and Tables 1-3.

[0117] 1. In vitro cytotoxicity evaluation

[0118] The prepared gel was evaluated and scored according to the requirements of GB / T 16886.5.

[0119] The L929 mouse fibroblasts that had been cultured for 24 hours and were growing vigorously were digested and prepared into a solution with a density of 1.0 x 10 5 cells / mL, and 100 μL was inoculated into each well of a 96-well plate. After the cells formed a monolayer, the original culture solution was removed, and 100 μL of a test sample extract, a blank control, a positive control, and a negative control were added to each group, with 6 replicate wells. After the addition was complete, the 96-well plate was placed in a 37℃, 5% CO2 incubator for 24 hours.

[0120] After 24 hours of culture, the original culture solution 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 of the culture, 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 at a main absorption wavelength of 570 nm and a reference wavelength of 650 nm, and the cell survival rate was calculated.

[0121] The cell survival rate was calculated according to the following formula: cell survival rate (%) = 100 x OD 570e / OD570 b

[0122] OD 570eOD is the average value of the optical density of the test sample or control sample leaching solution.

[0123] OD 570b OD is the average value of the optical density of the blank.

[0124] The experimental results are shown in Table 1 below:

[0125] Table 1 In vitro cell survival rate of the stents prepared in the examples and comparative examples

[0126]

[0127] 2. Hemolysis test

[0128] 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 test product group - absorbance of negative control group) / (absorbance of positive control group - absorbance of negative control group) x 100%, and the results are shown in Table 2:

[0129] Table 2 Hemolysis rate of the materials prepared in the examples and comparative examples

[0130]

[0131] 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.

[0132] 3. In vitro drug release performance test

[0133] The in vitro drug release performance evaluation method is as follows: 500 mg of the product is immersed in 200 mL of PBS (pH = 7.4) in a constant temperature shaker at 37°C and 60 rpm, the test solution is collected regularly, and an equal amount of PBS is supplemented, the content of the therapeutic agent in the collected test solution is determined by high performance liquid chromatography (HPLC), compared with the total amount of the therapeutic agent loaded in the product, and the cumulative release rate of the therapeutic agent is calculated.

[0134] The in vitro drug release performance detection results of the tissue regeneration and repair promoting drugs are shown in Table 1. Figure 1 and 2 The release period of the scaffolds prepared in Examples 1-4 to various tissue regeneration and repair promoting drugs is more than 5 weeks. The scaffold fibers of Comparative Example 1 do not contain a porous structure, and the drug release rate is significantly lower than that of Example 1, and the release period is significantly longer; Comparative Examples 2 and 4 do not contain ZIF8, and the drug-loaded polydopamine particles in Comparative Example 6 do not contain bioactive ceramics, which have little effect on the release of the drug, and the release curves are close to those of Example 1. The burst release in Comparative Example 7 is significantly increased compared with Example 1, and the release rate is significantly increased, and it is basically released at 5 weeks.

[0135] The in vitro drug release performance detection results of the antibacterial drugs are shown in Table 2. Figure 3 and 4 The release period of the scaffolds prepared in Examples 1-4 to various antibacterial drugs is more than 3 weeks. The scaffold fibers of Comparative Example 1 do not contain a porous structure, and the antibacterial drug release rate is close to that of Example 1; compared with Example 1, the changes of Comparative Examples 5-8 have little effect on the release of the antibacterial drug embedded on the surface of the scaffold, and the release curves are also close to those of Example 1; the antibacterial drug in Comparative Example 2 is not wrapped by ZIF8, but is directly dispersed in the hydrogel, and compared with Example 1, the burst release is significantly increased, and the release rate is significantly increased, and it is basically released at 2 weeks.

[0136] 4. In vitro induction of pre-osteoblast osteogenic differentiation performance detection

[0137] The composite materials prepared in Examples 1-5 and Comparative Examples 1-3 are sterilized by irradiation and then soaked in DMEM basic culture medium at a concentration of 10 mg / mL, and then put into a 37°C shaker at 120 rpm for 24 h of extraction. After the extraction is completed, the mixture of the composite material and the culture medium is centrifuged at 1000 rpm, and the supernatant is collected. The collected extraction solution is diluted 2 times with the corresponding DMEM culture medium, and finally 10% fetal bovine serum is added to obtain the conditioned medium.

[0138] The MC3T3-E1 cells are inoculated at 1×10 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.

[0139] 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

[0140] 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 particles 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 with good bioactivity, nor does it have gradient pore structure, drug-loaded mesoporous ceramic / polydopamine particles, and antibacterial drugs, and the alkaline phosphatase activity is the lowest.

[0141] 5. Detection of antibacterial performance of the scaffold

[0142] 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 5×10 5 ~ 10×10 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 cultured at 37°C for 48 h, and the preliminary results were observed. The sterile growth tubes were continuously cultured to the 28th day. If the broth tube was turbid and bacterial growth was observed on the blood plate, it was recorded as positive, indicated by (+); if it was still clear on the 35th day, it was considered as sterile growth, indicated by (-).

[0143] Table 3. Antibacterial effects of the scaffolds prepared in Examples and Comparative Examples

[0144]

[0145] As shown in Table 3, the composite stents prepared by the examples 1-4 and the comparative examples 1, 5-8 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.

[0146] 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, which are also considered within the scope of protection of the present application.

Claims

1. A hydrogel-filled polycaprolactone composite scaffold, characterized in that, The scaffold includes a polycaprolactone (PCL) scaffold matrix; the PCL scaffold matrix has interconnected pores distributed on its surface and interior, the pores gradually increasing in size from the surface to the interior; a drug-loaded material is distributed within the PCL scaffold matrix, the drug-loaded material including mesoporous ceramic, the mesoporous ceramic being filled with tissue regeneration and repair drugs, and the surface of the mesoporous ceramic being coated with polydopamine; the pores of the PCL scaffold matrix are filled with a gel composite material; the gel composite material includes a hydrogel and a metal-organic framework (MOF) material encapsulated within the hydrogel; the MOF material is loaded with an antibacterial drug; The mesoporous ceramic is selected from at least one of mesoporous silicon, mesoporous calcium silicate, mesoporous magnesium silicate, mesoporous zinc silicate, mesoporous strontium silicate, mesoporous bioglass, and mesoporous hydroxyapatite. The raw material matrix of the hydrogel is selected from at least one of the following: methacrylamide hyaluronic acid, methacrylamide silk fibroin, methacrylamide chondroitin sulfate, methacrylamide chitosan, methacrylamide carboxymethyl chitosan, methacrylamide gelatin, methacrylamide sodium alginate, hyaluronic acid, silk fibroin, chondroitin sulfate, chitosan, carboxymethyl chitosan, gelatin, and sodium alginate. The method for preparing the hydrogel-filled polycaprolactone composite scaffold includes the following steps: S1. The drug-loaded material, gluconolactone and polycaprolactone are mixed in a solvent to obtain a mixed solution, which is then transferred to a mold and left for a period of time to obtain a mixed material film; the mixed material film is used as a printing material to 3D print a scaffold with a gradient hole structure. After printing, the support with a gradient pore structure is immersed in water to remove gluconolactone and residual solvent. S2. The antibacterial drug, 2-methylimidazole and zinc source are reacted in water to obtain the metal-organic framework material. The metal-organic framework material, hydrogel matrix, crosslinking agent and the scaffold with gradient pore structure are mixed and photocured to obtain the hydrogel-filled polycaprolactone composite material scaffold. The preparation method of the drug-loaded material includes the following steps: immersing mesoporous ceramic in a solution containing a drug that promotes tissue regeneration and repair to obtain mesoporous ceramic encapsulated with the drug; immersing the mesoporous bioceramic encapsulated with the drug in a solution containing dopamine to obtain the drug-loaded material.

2. The hydrogel-filled polycaprolactone composite scaffold according to claim 1, characterized in that, The surface of the polycaprolactone composite material has pores with a size of 1-100 μm.

3. The method for preparing the hydrogel-filled polycaprolactone composite scaffold according to any one of claims 1-2, characterized in that, Includes the following steps: S1. The drug-loaded material, gluconolactone and polycaprolactone are mixed in a solvent to obtain a mixed solution, which is then transferred to a mold and left for a period of time to obtain a mixed material film; the mixed material film is used as a printing material to 3D print a scaffold with a gradient hole structure. After printing, the support with a gradient pore structure is immersed in water to remove gluconolactone and residual solvent. S2. The antibacterial drug, 2-methylimidazole and zinc source are reacted in water to obtain the metal-organic framework material. The metal-organic framework material, hydrogel matrix, crosslinking agent and the scaffold with gradient pore structure are mixed and photocured to obtain the hydrogel-filled polycaprolactone composite material scaffold.

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

1.

5. The method for preparing the hydrogel-filled polycaprolactone composite scaffold according to claim 3, characterized in that, The 3D printing process conditions satisfy at least one of the following: a) 3D printing uses printing needles with a diameter of 0.15~0.4mm; b) Printing and heating temperatures: barrel 60~80℃, needle tip 60~90℃; c) Fiber spacing 0.8mm × 0.8mm; printing speed 8~17mm / s; d) The air pressure is 320-400 kPa; e) The height of the stent layer is 75-95% of the needle diameter.

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

7. The method for preparing the hydrogel-filled polycaprolactone composite scaffold according to claim 3, characterized in that, The mass ratio of the 2-methylimidazole, the antibacterial drug, and the zinc source is (18-23):(4-10):1; And / or, the zinc source is zinc nitrate.

8. The use of the hydrogel-filled polycaprolactone composite scaffold according to any one of claims 1-2 in the preparation of products that promote bone tissue repair and regeneration.

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