A drug-loaded polydopamine hydrogel filled polycaprolactone scaffold, and a preparation method and application thereof

Abstract

The present application belongs to the technical field of medical stents, and particularly relates to a drug-loaded polydopamine hydrogel filled polycaprolactone stent and a preparation method and application thereof. The polycaprolactone stent comprises a polycaprolactone stent body, and drug-loaded mesoporous ceramics and zeolite imidazolate framework material embedded in the polycaprolactone stent body; the drug-loaded mesoporous ceramics are embedded in the zeolite imidazolate framework material; the matrix material of the polycaprolactone stent body is polycaprolactone, the micropore structure between fibers and the surface of the fibers of the polycaprolactone has a micropore structure, and the size of the micropore structure between the fibers is greater than that of the surface of the fibers; the micropore structure is filled with drug-loaded polydopamine hydrogel. The polycaprolactone stent has good porosity, biocompatibility and antibacterial property, can induce stem cell osteogenic differentiation, can accelerate the release of antibacterial drugs in the case of infection, and the internally encapsulated drugs for promoting tissue regeneration and repair can be slowly released, and is suitable for the repair and regeneration of infected bone tissue defects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical stents, and particularly relates to a drug-loaded polydopamine hydrogel-filled polycaprolactone stent and a preparation method and application thereof. BACKGROUND

[0002] An ideal bone tissue engineering scaffold should have a porous network structure for cell migration, ion transport, and cell-cell interaction. Conventional techniques include foaming, sacrificial template method, freeze-drying method, and casting method, etc. 3D printing, also known as additive manufacturing technology, has penetrated into various industries due to its high precision, personalized manufacturing, and significant advantages in complex shape construction. 3D printing obtains a three-dimensional entity by layer-by-layer accumulation of raw materials on a plane. Compared with other scaffold preparation techniques, 3D printing technology can freely design the porosity and shape of the scaffold, and 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, biodegradability, and can induce tissue regeneration, and is widely used in the field of medical science. However, the hydrophobicity of PCL affects the adhesion of the scaffold, and the mechanical structure is significantly destroyed after degradation.

[0004] At present, there are many studies on polycaprolactone-based scaffolds, but the prepared polycaprolactone-based scaffolds, polydopamine-containing drug-loaded particles, and drug-loaded ZIF8 generally have the following problems: 1) polydopamine degrades slowly, and it is difficult to effectively release enough drugs to achieve antibacterial and osteogenic effects; 2) the surface of the polycaprolactone scaffold is too hydrophobic, which is not conducive to cell adhesion and proliferation; 3) the components of the material are relatively single, and it is difficult to achieve good antibacterial and bone regeneration effects; 4) the preparation process is complex and difficult to industrialize.

[0005] Therefore, it is of great significance to provide a polycaprolactone scaffold with good osteogenic activity, drug release performance, cell adhesion, and mechanical strength. SUMMARY

[0006] The present application aims to solve one or more technical problems in the prior art, and at least provide a beneficial alternative. Specifically, the present application provides a polycaprolactone scaffold with good osteogenic activity, drug release performance, cell adhesion, and mechanical strength.

[0007] The inventive concept of this invention is as follows: The polycaprolactone scaffold of this invention comprises a polycaprolactone scaffold body and a drug-loaded mesoporous ceramic and a zeolite imidazole ester framework material embedded within the polycaprolactone scaffold body; the drug-loaded mesoporous ceramic is embedded in the zeolite imidazole ester framework material; the matrix material of the polycaprolactone scaffold body is polycaprolactone, and both the fibers and the surface of the polycaprolactone fibers have micron-sized pore structures, with the size of the micron-sized pore structures between the polycaprolactone fibers being larger than the size of the micron-sized pore structures on the surface of the polycaprolactone fibers; the micron-sized pore structures are filled with hydrogel; and the hydrogel contains drug-loaded polydopamine. The polycaprolactone scaffold body of this invention has macropores (between fibers) and micropores (pores on the fiber surface), which can significantly increase the specific surface area of ​​the polycaprolactone scaffold body, accelerate the degradation of polycaprolactone and release more acidic degradation products, thereby accelerating the degradation of polydopamine and accelerating the release of the internally embedded antibacterial drug. Simultaneously, macroscopic macropores and microscopic micropores can increase the surface roughness of the polycaprolactone scaffold, thereby improving its cell adhesion. Drug-loaded mesoporous ceramics embedded in a zeolite imidazole ester framework can delay drug release, achieving a long-lasting controlled-release effect, improving antibacterial properties, significantly inducing osteogenic differentiation of stem cells, and enhancing the bioactivity and mechanical strength of the drug-loaded particles. Furthermore, the hydrogel and drug-loaded polydopamine filling the scaffold pores can improve the scaffold's mechanical strength and bioactivity, endowing it with antibacterial properties and drug controlled-release capabilities, allowing for better therapeutic effects through controlled-release drugs.

[0008] Therefore, a first aspect of the present invention provides a polycaprolactone scaffold filled with drug-loaded polydopamine hydrogel.

[0009] Specifically, the drug-loaded polydopamine hydrogel-filled polycaprolactone scaffold includes a polycaprolactone scaffold body and a drug-loaded mesoporous ceramic and zeolite imidazole ester skeleton material embedded in the polycaprolactone scaffold body.

[0010] The drug-loaded mesoporous ceramic is embedded in the zeolite imidazole ester framework material;

[0011] The matrix material of the polycaprolactone scaffold body is polycaprolactone fiber. The polycaprolactone fibers and the surface of the polycaprolactone fibers have micron-sized pore structures. The size of the micron-sized pore structure between the polycaprolactone fibers is larger than the size of the micron-sized pore structure on the surface of the polycaprolactone fibers.

[0012] The microporous structure is filled with hydrogel; the hydrogel contains drug-loaded polydopamine.

[0013] Preferably, the polycaprolactone scaffold body is a 3D printed scaffold.

[0014] Preferably, the molecular weight of the polycaprolactone is 27-110 kiloDaltons; further preferably, the molecular weight of the polycaprolactone is 3-10 kiloDaltons.

[0015] Preferably, the size of the micropore structure between the fibers of the polycaprolactone is 200-650 μm; further preferably, the size of the micropore structure between the fibers of the polycaprolactone is 200-600 μm.

[0016] Preferably, the size of the micropore structure on the surface of the fibers of the polycaprolactone is 0.9-110 μm; further preferably, the size of the micropore structure on the surface of the fibers of the polycaprolactone is 1-100 μm.

[0017] Preferably, the mesoporous ceramic in the drug-loaded mesoporous ceramic comprises at least one of mesoporous silica, mesoporous calcium silicate, mesoporous magnesium silicate, mesoporous zinc silicate, mesoporous strontium silicate, mesoporous bioglass, mesoporous hydroxyapatite.

[0018] Preferably, the particle size of the mesoporous ceramic in the drug-loaded mesoporous ceramic is 45-1100 nm; further preferably, the particle size of the mesoporous ceramic in the drug-loaded mesoporous ceramic is 50-1000 nm.

[0019] Preferably, the specific surface area of the mesoporous ceramic in the drug-loaded mesoporous ceramic is 140-2000 m 2 / g; further preferably, the specific surface area of the mesoporous ceramic in the drug-loaded mesoporous ceramic is 150-1800 m 2 / g.

[0020] Preferably, the average pore size of the mesoporous ceramic in the drug-loaded mesoporous ceramic is 2-45 nm; further preferably, the average pore size of the mesoporous ceramic in the drug-loaded mesoporous ceramic is 2-40 nm.

[0021] Preferably, the drug in the drug-loaded mesoporous ceramic comprises a tissue regeneration and repair promoting drug.

[0022] Further preferably, the tissue regeneration and repair promoting drug comprises at least one of bone morphogenetic protein-2, bone morphogenetic protein-7, vascular endothelial cell factor, platelet-derived factor, epidermal cell growth factor, fibroblast growth factor, keratinocyte growth factor, nerve growth factor, transforming growth factor, insulin-like growth factor, parathyroid hormone, growth hormone, interleukin, curcumin, melatonin, alendronate sodium, zoledronate sodium, ibandronate sodium, naringin, resveratrol, dexamethasone, gentamicin sulfate, berberine hydrochloride, gentamicin, salmon calcitonin, strontium ranelate, vitamin D.

[0023] Preferably, the hydrogel comprises 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, sodium alginate.

[0024] Preferably, the drug in the drug-loaded polydopamine comprises an antibacterial drug.

[0025] Further preferably, the antibacterial drug comprises 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, gentamicin, netilmicin, tetracycline, chloramphenicol, ciprofloxacin, levofloxacin, moxifloxacin, metronidazole, ornidazole, tinidazole, itraconazole, mycetin, griseofulvin.

[0026] The second aspect of the present application provides a preparation method of the drug-loaded polydopamine hydrogel filled polycaprolactone scaffold of the first aspect of the present application.

[0027] Specifically, the preparation method of the drug-loaded polydopamine hydrogel filled polycaprolactone scaffold comprises the following steps:

[0028] (1) mixing mesoporous ceramics and a drug-containing aqueous solution to obtain drug-loaded mesoporous ceramics; mixing the drug-loaded mesoporous ceramics and a 2-methyl imidazole aqueous solution, then adding a zinc source aqueous solution, and reacting to obtain a material of zeolite imidazolate framework embedded drug-loaded mesoporous ceramics;

[0029] (2) mixing polycaprolactone, a solvent, gluconolactone, and the material of zeolite imidazolate framework embedded drug-loaded mesoporous ceramics obtained in step (1) to obtain a mixed solution, preparing the mixed solution into a film, then printing to obtain a gluconolactone / drug-loaded mesoporous ceramics@zeolite imidazolate framework / polycaprolactone scaffold, and placing it in an aqueous solution, and ultrasonic treatment to obtain a drug-loaded mesoporous ceramic@zeolite imidazolate framework / polycaprolactone scaffold;

[0030] (3) adding drug-loaded polydopamine to a hydrogel solution containing a photoinitiator to obtain a drug-loaded polydopamine-containing hydrogel solution; then placing the drug-loaded mesoporous ceramic@zeolite imidazolate framework / polycaprolactone scaffold obtained in step (2) into the drug-loaded polydopamine-containing hydrogel solution, then taking it out, and solidifying to obtain the drug-loaded polydopamine hydrogel filled polycaprolactone scaffold.

[0031] Preferably, in step (1), the mass ratio of the drug to the mesoporous ceramic is (0.0005-0.055):1; further preferably, the mass ratio of the drug to the mesoporous ceramic is (0.0005-0.05):1.

[0032] Preferably, in step (1), the mixing of the mesoporous ceramic and the drug-containing aqueous solution is performed by stirring, the stirring speed is 270-900 rpm, the stirring time is 3.5-20 h, and the stirring temperature is 0-37℃; further preferably, the stirring speed is 300-800 rpm, the stirring time is 4-18 h, and the stirring temperature is 0-37℃.

[0033] Preferably, in step (1), after mixing the mesoporous ceramic and the drug-containing aqueous solution, drying is performed to obtain the drug-loaded mesoporous ceramic.

[0034] Preferably, in step (1), the concentrations of 2-methylimidazole and the drug-loaded mesoporous ceramic in the 2-methylimidazole aqueous solution are 17-58 mg / mL and 7-20 mg / mL, respectively; further preferably, the concentrations of 2-methylimidazole and the drug-loaded mesoporous ceramic in the 2-methylimidazole aqueous solution are 19-53 mg / mL and 8-18 mg / mL, respectively.

[0035] Preferably, in step (1), the zinc source aqueous solution comprises a zinc nitrate aqueous solution; the concentration of zinc nitrate in the zinc nitrate aqueous solution is 5.5-33 mg / mL; further preferably, in step (1), the concentration of zinc nitrate in the zinc nitrate aqueous solution is 6-30 mg / mL.

[0036] Preferably, in step (1), the volume ratio of the 2-methylimidazole aqueous solution to the zinc source aqueous solution is (5.5-13):1; further preferably, in step (1), the volume ratio of the 2-methylimidazole aqueous solution to the zinc source aqueous solution is (6-12):1.

[0037] Preferably, in step (1), after the reaction, a washing and drying process is further included.

[0038] Preferably, in step (2), the mass ratio of the glucose lactone, the material for embedding the drug-loaded mesoporous ceramic in the zeolitic imidazolate framework, and the polycaprolactone is (0.45-3.3):(0.05-0.33):1; further preferably, in step (2), the mass ratio of the glucose lactone, the material for embedding the drug-loaded mesoporous ceramic in the zeolitic imidazolate framework, and the polycaprolactone is (0.5-3):(0.05-0.3):1.

[0039] Preferably, in step (2), the mass-volume ratio of the polycaprolactone to the solvent is (0.45-9) g: 10 mL; further preferably, in step (2), the mass-volume ratio of the polycaprolactone to the solvent is (0.5-8) g: 10 mL.

[0040] Preferably, in step (2), the solvent comprises dichloromethane.

[0041] Preferably, in step (2), the mixing is performed by stirring, the stirring speed is 270-900 rpm, and the stirring time is 22-80 h; further preferably, the stirring speed is 300-800 rpm, and the stirring time is 24-72 h.

[0042] Preferably, the stirring comprises any one of mechanical stirring and magnetic stirring.

[0043] Specifically, in step (2), the mixed solution is a lactobionate / drug-loaded mesoporous ceramic@zeolitic imidazolate framework / polycaprolactone solution.

[0044] Preferably, in step (2), the process of preparing the mixed solution into a film is placing the mixed solution on a plane and standing to obtain.

[0045] Preferably, the standing is placing in a ventilated place, and the standing time is 44-80 h; further preferably, the standing time is 48-72 h.

[0046] Specifically, in step (2), the film is a lactobionate / drug-loaded mesoporous ceramic@zeolitic imidazolate framework / polycaprolactone film.

[0047] Preferably, in step (2), the printing is 3D printing.

[0048] Preferably, in step (2), the process of printing is placing the lactobionate / drug-loaded mesoporous ceramic@zeolitic imidazolate framework / polycaprolactone film into a printing cartridge, matching a needle, setting printing parameters and heating temperature, and then printing layer by layer to obtain the lactobionate / drug-loaded mesoporous ceramic@zeolitic imidazolate framework / polycaprolactone scaffold.

[0049] Preferably, the diameter of the needle is 0.18-0.45 mm; further preferably, the diameter of the needle is 0.2-0.5 mm.

[0050] Preferably, the temperature of the cartridge is 55-95 ℃; further preferably, the temperature of the cartridge is 60-90 ℃.

[0051] Preferably, the temperature of the needle is 60-95℃; further preferably, the temperature of the needle is 65-95℃.

[0052] Preferably, the fiber spacing of the polycaprolactone fibers in the polycaprolactone scaffold body is (0.70-0.90) mm x (0.70-0.90) mm; further preferably, the fiber spacing of the polycaprolactone fibers in the polycaprolactone scaffold body is (0.75-0.85) mm x (0.75-0.85) mm; further preferably, the fiber spacing of the polycaprolactone fibers in the polycaprolactone scaffold body is 0.8 mm x 0.8 mm.

[0053] Preferably, the fiber orientation of the polycaprolactone fibers in the polycaprolactone scaffold body is 0-90°.

[0054] Preferably, the printing rate is 7-16.5 mm / s; further preferably, the printing rate is 8-15 mm / s.

[0055] Preferably, the air pressure during printing is 300-460 kPa; further preferably, the air pressure during printing is 320-420 kPa.

[0056] Preferably, the scaffold layer height during printing is 70-95% of the needle diameter; further preferably, the scaffold layer height during printing is 75-90% of the needle diameter.

[0057] Preferably, in step (2), the mass-volume ratio of the glucose lactone / drug-loaded mesoporous ceramic@zeolitic imidazolate framework / polycaprolactone scaffold to the aqueous solution is 1 mg:(0.27-4.5) mL; further preferably, the mass-volume ratio of the glucose lactone / drug-loaded mesoporous ceramic@zeolitic imidazolate framework / polycaprolactone scaffold to the aqueous solution is 1 mg:(0.3-4) mL.

[0058] Preferably, in step (2), the ultrasonic treatment time is 0.35-1.1 h; further preferably, the ultrasonic treatment time is 0.4-1 h.

[0059] Preferably, in step (2), the ultrasonic treatment temperature is room temperature.

[0060] Specifically, the drug-loaded mesoporous ceramic@zeolitic imidazolate framework / polycaprolactone scaffold obtained in step (2) is a drug-loaded mesoporous ceramic@zeolitic imidazolate framework / polycaprolactone scaffold with a multi-level pore structure, i.e., the fibers of polycaprolactone and the surface of the fibers of polycaprolactone both have micropore structures.

[0061] Preferably, in step (3), the preparation process of the drug-loaded polydopamine is as follows: mixing the drug and the aqueous solution of dopamine, and stirring to obtain the drug-loaded polydopamine; the concentration of dopamine in the aqueous solution of dopamine is 1.5-4.5 mg / mL; the mass-volume ratio of the drug to the aqueous solution of dopamine is (9-33) mg: 10 mL; further preferably, the concentration of dopamine in the aqueous solution of dopamine is 1.6-4 mg / mL; and the mass-volume ratio of the drug to the aqueous solution of dopamine is (10-30) mg: 10 mL.

[0062] Preferably, the stirring speed is 270-900 rpm, and the stirring time is 3.5-13 h; further preferably, the stirring speed is 300-800 rpm, and the stirring time is 4-12 h.

[0063] Preferably, the pH value of the aqueous solution of dopamine is 7.5-10; further preferably, the pH value of the aqueous solution of dopamine is 8.2-9.2.

[0064] Preferably, in step (3), the concentration of the hydrogel in the hydrogel solution is 1.4-3.3%; further preferably, in step (3), the concentration of the hydrogel in the hydrogel solution is 1.5-3%.

[0065] Preferably, in step (3), the hydrogel is dissolved in the PBS solution containing the photoinitiator to obtain the hydrogel solution.

[0066] Preferably, in step (3), the mass-volume ratio of the drug-loaded polydopamine to the hydrogel solution is (11-52) mg: 1 mL; further preferably, in step (3), the mass-volume ratio of the drug-loaded polydopamine to the hydrogel solution is (12-48) mg: 1 mL.

[0067] Preferably, in step (3), the photoinitiator comprises phenyl-2,4,6-trimethylbenzoyl lithium phosphite.

[0068] Preferably, the concentration of the photoinitiator in the PBS solution is 0.22-0.45%; further preferably, the concentration of the photoinitiator in the PBS solution is 0.25-0.4%.

[0069] Preferably, in step (3), the mass-volume ratio of the drug-loaded mesoporous ceramic@zeolitic imidazolate framework / poly-caprolactone scaffold to the hydrogel solution containing the drug-loaded polydopamine is (0.04-0.18) g: 10 mL; further preferably, the mass-volume ratio of the drug-loaded mesoporous ceramic@zeolitic imidazolate framework / poly-caprolactone scaffold to the hydrogel solution containing the drug-loaded polydopamine is (0.04-0.16) g: 10 mL.

[0070] Preferably, in step (3), the time of the implantation is 2.5-13 min; further preferably, the time of the implantation is 3-12 min.

[0071] Preferably, in step (3), the time of the solidification is 27-130 s; further preferably, the time of the solidification is 30-120 s.

[0072] Preferably, in step (3), the solidification further comprises the processes of freezing and freeze-drying.

[0073] The third aspect of the present application provides a use of the drug-loaded polydopamine hydrogel filled polycaprolactone scaffold of the first aspect of the present application in the preparation of a bone tissue defect repair and regeneration product.

[0074] Compared with the prior art, the technical scheme provided by the present application has the following beneficial effects:

[0075] (1) The present application can delay the release of the drug for promoting tissue regeneration and repair by encapsulating the drug-loaded mesoporous bioceramics in the zeolitic imidazolate framework material (ZIF8), achieve the effect of long-acting controlled release, endow the polycaprolactone scaffold with good antibacterial performance, and significantly induce the osteogenic differentiation of stem cells, making it more suitable for the regeneration and repair of bone defects. In addition, the pore structure in the polycaprolactone fibers reduces the mechanical strength of the polycaprolactone scaffold, and the bioactive ceramic in the drug-loaded mesoporous ceramic@ZIF8 particles filled in the fibers can enhance the bioactivity and mechanical strength of the drug-loaded mesoporous ceramic@ZIF8, thereby maintaining the bioactivity and mechanical strength of the polycaprolactone scaffold, making it suitable for bone repair.

[0076] (2) In the case of bone infection, the pH in the local microenvironment is lower, which will significantly accelerate the degradation of ZIF8 and accelerate the release of the drug for promoting tissue regeneration and repair, thereby achieving better antibacterial and bone regeneration effects. In addition, the present application can accelerate the degradation of polycaprolactone and accelerate the release of the drug under the assistance of ultrasound, thereby achieving better antibacterial and bone regeneration effects.

[0077] (3) Polydopamine degrades very slowly in vivo and in vitro, and the present application makes the printed scaffold obtain macroscopic large pores (fiber gaps) and microscopic small pores (fiber surface pores) by pore-forming treatment of the polycaprolactone fibers of the printed scaffold, significantly increases the specific surface area of the scaffold, accelerates the degradation of polycaprolactone and releases more acidic degradation products, thereby accelerating the degradation of polydopamine and accelerating the release of the antibacterial drug embedded therein.

[0078] (4) The surface of polycaprolactone is too hydrophobic to be suitable for cell adhesion, and the polycaprolactone fibers of the printed scaffold are subjected to pore-forming treatment in the application, so that the printed scaffold has macroscopic large pores (fiber gaps) and microscopic small pores (fiber surface pores) at the same time, and the surface roughness of the scaffold is significantly improved, which can improve the cell adhesion effect of the scaffold.

[0079] (5) The hydrogel and drug-loaded polydopamine particles are filled in the pores of the 3D printed scaffold in the application, compared with the hydrogel filled alone, the mechanical strength is greatly increased, and the scaffold is endowed with antibacterial properties and improved biological activity. At the same time, the hydrogel with good hydrophilicity filled in the pores can further regulate the hydrophilic and hydrophobic properties of the material while maintaining the mechanical strength of the material, making it more suitable for bone defect regeneration and repair.

[0080] (6) The printed scaffold is partially filled with neutral-soluble methacrylated hydrogel in the application, which can avoid the problem that the acidic microenvironment of the scaffold is not conducive to cell survival. In addition, when the hydrogel is sodium alginate or methacrylated sodium alginate, the divalent metal ions such as calcium ions and zinc ions released by the degradation of the bioactive ceramic and ZIF8 can crosslink the sodium alginate or methacrylated sodium alginate, thereby maintaining the mechanical strength of the hydrogel.

[0081] (7) The polydopamine of the application loads drugs by embedding, forms drug-loaded particles, and then disperses in the interior of the hydrogel and in the pores of the scaffold, which makes the polycaprolactone scaffold have a better slow-release drug effect, while the conventional method is to use the adhesive effect of the polydopamine coating to adsorb and load drugs.

[0082] (8) The preparation method of the application has simple process, low requirement for equipment, easily available raw materials, low cost, and is easy to realize industrialization. BRIEF DESCRIPTION OF DRAWINGS

[0083] Figure 1 The drug release curve diagram of the polycaprolactone scaffold of the application examples 1-4, comparative examples 1-4, 6-7, 10 in promoting tissue regeneration and repair in vitro;

[0084] Figure 2 The drug release curve diagram of the polycaprolactone scaffold of the application examples 1, comparative examples 1-4, 6-7, 10 in promoting tissue regeneration and repair in vitro;

[0085] Figure 3 The drug release curve diagram of the polycaprolactone scaffold of the application examples 1-4, comparative examples 1-2, 5-8, 10 in promoting tissue regeneration and repair in vitro;

[0086] Figure 4 The drug release curve diagram of the polycaprolactone scaffold of the application examples 1, comparative examples 1-2, 5-8, 10 in promoting tissue regeneration and repair in vitro;

[0087] Figure 5 Figure 1 is a graph showing the alkaline phosphatase activity of polycaprolactone scaffolds of Examples 1-4 and Comparative Examples 1-10 of the present application. DETAILED DESCRIPTION

[0088] In order to make the skilled in the art more clearly understand the technical solutions described in the present application, the following examples are listed for illustration. It should be noted that the following examples do not constitute a limitation on the scope of protection required by the present application.

[0089] The raw materials, reagents or devices used in the following examples, if not specifically stated, can be obtained from conventional commercial channels, or can be obtained by existing known methods.

[0090] Example 1

[0091] A method for preparing a drug-loaded polydopamine hydrogel-filled polycaprolactone scaffold, comprising the following steps:

[0092] (1) 100 mg of mesoporous calcium silicate (average particle size between 300-1000 nm, specific surface area between 300-900 m 2 / g, average pore size between 2-30 nm) is dispersed in 10 mL of an aqueous solution containing 0.05 mg of bone morphogenetic protein-2, stirred at a speed of 300 rpm for 18 h, and freeze-dried to obtain drug-loaded mesoporous ceramic; 300 mg of drug-loaded mesoporous ceramic is added to 24 mL of a 35 mg / mL aqueous solution of 2-methylimidazole, and 3 mL of a 14 mg / mL aqueous solution of zinc nitrate is added dropwise during stirring, and after reaction, the drug-loaded mesoporous ceramic@ZIF8 particles are obtained after washing and freeze-drying;

[0093] (2) 10 g polycaprolactone (molecular weight of 40,000 Dalton) was dissolved in 50 mL dichloromethane, 10 g gluconolactone and 1.5 g drug-loaded mesoporous ceramic@ZIF8 particles obtained in step (1) were added, and the mixture was stirred at a speed of 500 rpm for 72 h to obtain a gluconolactone / drug-loaded mesoporous ceramic@ZIF8 / 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@ZIF8 / polycaprolactone film; the film was placed in a printing cartridge, the needle diameter was 0.25 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 10 mm / s, the fiber direction was "0-90°", the air pressure was 360 kPa, and the scaffold layer height was 80% of the needle diameter, and the scaffold was printed layer by layer to obtain a gluconolactone / drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold; 50 mg of the gluconolactone / drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold was soaked in 50 mL of an aqueous solution, and ultrasonic treatment was performed at room temperature for 0.5 h to obtain a drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold with a multi-level pore structure;

[0094] (3) 20 mg of gentamicin was dissolved in 10 mL of a 2 mg / mL dopamine aqueous solution (pH = 8.4), stirred at a speed of 300 rpm for 12 min, and then freeze-dried to obtain drug-loaded polydopamine; 180 mg of methacrylated sodium alginate was dissolved in 10 mL of a PBS solution containing 0.3% phenyl-2,4,6-trimethylbenzoyl lithium phosphite, and 240 mg of drug-loaded polydopamine was added to obtain a drug-loaded polydopamine-containing hydrogel solution; 100 mg of the drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold with a multi-level pore structure obtained in step (2) was placed in the drug-loaded polydopamine-containing hydrogel solution, dispersed for 6 min, removed, irradiated with blue light for 90 s to solidify, then placed in a -20°C refrigerator to freeze, and freeze-dried in a freeze dryer to obtain a drug-loaded polydopamine-containing hydrogel-filled polycaprolactone scaffold with a multi-level pore structure drug-loaded mesoporous ceramic@ZIF8.

[0095] Example 2

[0096] A method for preparing a drug-loaded polydopamine hydrogel-filled polycaprolactone scaffold, comprising the following steps:

[0097] (1) At 15°C, 100 mg of mesoporous hydroxyapatite (average particle size between 100-600 nm, specific surface area between 150-500 m 2The drug-loaded mesoporous ceramic@ZIF8 particles were obtained by dispersing 280 mg of the drug-loaded mesoporous ceramic in 24 mL of a 27 mg / mL aqueous solution of 2-methylimidazole, adding 3 mL of a 10 mg / mL aqueous solution of zinc nitrate dropwise during stirring, and then cleaning and freeze-drying after the reaction;

[0098] (2) 10 g of polycaprolactone (molecular weight: 100,000 daltons) was dissolved in 200 mL of dichloromethane, 30 g of gluconolactone and 3 g of the drug-loaded mesoporous ceramic@ZIF8 particles obtained in step (1) were added, and the mixture was stirred at 800 rpm for 60 h to obtain a gluconolactone / drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone solution. The solution was poured onto a glass culture dish and left to stand in a ventilated state for 48 h to obtain a gluconolactone / drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone film. The film was placed in a printing cartridge, and a polycaprolactone scaffold with a multi-level pore structure filled with drug-loaded mesoporous ceramic@ZIF8 was printed layer by layer with a needle diameter of 0.5 mm, a cartridge temperature of 90°C, a needle temperature of 95°C, a fiber spacing of 0.8 mm x 0.8 mm, a printing rate of 8 mm / s, a fiber direction of “0-90°”, an air pressure of 320 kPa, and a scaffold layer height of 90% of the needle diameter.

[0099] (3) 30 mg of vancomycin was dissolved in 10 mL of a 4 mg / mL aqueous solution of dopamine (pH = 8.2) and stirred at 800 rpm for 4 min. After freeze-drying, drug-loaded polydopamine was obtained. 150 mg of methacrylated silk fibroin was dissolved in 10 mL of a PBS solution containing 0.35% phenyl-2,4,6-trimethylbenzoyl lithium phosphite, and 480 mg of drug-loaded polydopamine was added to obtain a drug-loaded polydopamine-containing hydrogel solution. 160 mg of the multi-level pore structure drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold obtained in step (2) was placed in the drug-loaded polydopamine-containing hydrogel solution, dispersed for 9 min, removed, irradiated with blue light for 60 s to solidify, then placed in a -20°C refrigerator to freeze, and freeze-dried in a freeze dryer to obtain a drug-loaded polydopamine-containing hydrogel-filled multi-level pore structure drug-loaded mesoporous ceramic@ZIF8 polycaprolactone scaffold.

[0100] Example 3

[0101] A method for preparing a drug-loaded polydopamine hydrogel-filled polycaprolactone scaffold, comprising the following steps:

[0102] (1) 100 mg mesoporous bioglass (average particle size between 300-900 nm, specific surface area between 200-1000 m 2 / g, average pore size between 2-40 nm) was dispersed in 10 mL of an aqueous solution containing 50 mg of alendronate sodium at 37°C, stirred at a speed of 800 rpm for 4 h, and freeze-dried to obtain drug-loaded mesoporous ceramics; 192 mg of drug-loaded mesoporous ceramics was added to 24 mL of an aqueous solution of 2-methylimidazole at 19 mg / mL, and 4 mL of an aqueous solution of zinc nitrate at 6 mg / mL was added dropwise during stirring, and after the reaction, the drug-loaded mesoporous ceramics@ZIF8 particles were washed and freeze-dried to obtain drug-loaded mesoporous ceramics@ZIF8 particles;

[0103] (2) 10 g of polycaprolactone (molecular weight: 60,000 daltons) was dissolved in 100 mL of dichloromethane, and then 20 g of gluconolactone and 2 g of drug-loaded mesoporous ceramics@ZIF8 particles obtained in step (1) were added, and mechanical stirring was performed at a speed of 400 rpm for 48 h to obtain a gluconolactone / drug-loaded mesoporous ceramics@ZIF8 / polycaprolactone solution; the solution was poured onto a glass culture dish, and left to stand in a ventilated state for 60 h to obtain a gluconolactone / drug-loaded mesoporous ceramics@ZIF8 / polycaprolactone film; the film was placed in a printing cartridge, and a needle diameter of 0.35 mm, a cartridge temperature of 80°C, a needle temperature of 85°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 340 kPa, and a scaffold layer height of 85% of the needle diameter were used to perform layer-by-layer printing to obtain a gluconolactone / drug-loaded mesoporous ceramics@ZIF8 / polycaprolactone scaffold; 50 mg of the gluconolactone / drug-loaded mesoporous ceramics@ZIF8 / polycaprolactone scaffold was soaked in 100 mL of an aqueous solution, and a multi-level porous drug-loaded mesoporous ceramics@ZIF8 / polycaprolactone scaffold was obtained after ultrasonic treatment for 0.4 hours at room temperature;

[0104] (3) 10 mg of metronidazole was dissolved in 10 mL of an aqueous solution of dopamine at 3 mg / mL (pH = 9), and stirred at a speed of 400 rpm for 9 min, and then freeze-dried to obtain drug-loaded polydopamine; 200 mg of methacrylated carboxymethyl chitosan was dissolved in 10 mL of a PBS solution containing 0.4% phenyl-2,4,6-trimethylbenzoyl lithium phosphite, and then 360 mg of drug-loaded polydopamine was added to obtain a drug-loaded polydopamine-containing hydrogel solution; 120 mg of the multi-level porous drug-loaded mesoporous ceramics@ZIF8 / polycaprolactone scaffold obtained in step (2) was placed in the drug-loaded polydopamine-containing hydrogel solution, dispersed for 3 min, removed, and cured by blue light irradiation for 30 s, and then placed in a -20°C refrigerator for freezing, and freeze-dried in a freeze dryer to obtain a drug-loaded polydopamine-containing hydrogel-filled multi-level porous drug-loaded mesoporous ceramics@ZIF8 polycaprolactone scaffold.

[0105] Example 4

[0106] A method for preparing a drug-loaded polydopamine hydrogel filled polycaprolactone scaffold, comprising the following steps:

[0107] (1) At 25°C, 100 mg of mesoporous silica (average particle size between 50-500 nm, specific surface area between 500-1800 m 2 / g, average pore size between 2-20 nm) is dispersed in 10 mL of an aqueous solution containing 25 mg of curcumin, stirred at a speed of 600 rpm for 8 h, and freeze-dried to obtain drug-loaded mesoporous ceramic; 432 mg of drug-loaded mesoporous ceramic is added to 24 mL of a 53 mg / mL aqueous solution of 2-methylimidazole, and 2 mL of a 30 mg / mL aqueous solution of zinc nitrate is added dropwise during stirring, and after reaction, the drug-loaded mesoporous ceramic@ZIF8 particles are obtained after washing and freeze-drying;

[0108] (2) 10 g of polycaprolactone (molecular weight 30,000 daltons) is dissolved in 12.5 mL of dichloromethane, and then 5 g of gluconolactone and 0.5 g of drug-loaded mesoporous ceramic@ZIF8 particles obtained in step (1) are added, and mechanical stirring is carried out at a speed of 300 rpm for 24 h to obtain a gluconolactone / drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone solution; the solution is poured onto a glass culture dish and placed in a ventilated state for 64 h to obtain a gluconolactone / drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone film; the film is placed in a printing cartridge, the needle diameter is 0.2 mm, the cartridge temperature is 60°C, the needle temperature is 65°C, the fiber spacing is 0.8 mm x 0.8 mm, the printing rate is 15 mm / s, the fiber direction is “0-90°”, the air pressure is 420 kPa, and the scaffold layer height is 75% of the needle diameter, and the scaffold is printed layer by layer to obtain a gluconolactone / drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold; 50 mg of the gluconolactone / drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold is soaked in 15 mL of an aqueous solution, and ultrasonic treatment is carried out at room temperature for 1 hour to obtain a drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold with a multi-level pore structure;

[0109] (3) 18 mg of penicillin was dissolved in 10 mL of 1.6 mg / mL dopamine aqueous solution (pH = 9.2) and stirred at 600 rpm for 6 min, and then drug-loaded polydopamine was obtained by freeze-drying. 300 mg of methacrylated carboxymethyl gelatin was dissolved in 10 mL of PBS solution containing 0.25% lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and then 120 mg of drug-loaded polydopamine was added to obtain a drug-loaded polydopamine-containing hydrogel solution. 40 mg of the drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold with a multi-level pore structure obtained in step (2) was placed in the drug-loaded polydopamine-containing hydrogel solution, and after being dispersed for 12 min, it was taken out, irradiated with blue light for 120 s for curing, then placed in a-20°C refrigerator for freezing, and then freeze-dried in a freeze dryer to obtain a polycaprolactone scaffold filled with drug-loaded polydopamine hydrogel and drug-loaded mesoporous ceramic@ZIF8 with a multi-level pore structure.

[0110] Comparative Example 1

[0111] The difference between Comparative Example 1 and Example 1 is only that no gluconolactone is added in step (2) of Comparative Example 1, that is, the drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold obtained in step (2) of Comparative Example 1 does not have a pore structure, and the others are the same as in Example 1.

[0112] Specifically, step (2) of Comparative Example 1 is as follows:

[0113] 10 g of polycaprolactone (molecular weight: 40,000 daltons) was dissolved in 50 mL of dichloromethane, and then 1.5 g of drug-loaded mesoporous ceramic@ZIF8 particles were added and magnetically stirred at 500 rpm for 72 h to obtain a drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone solution. The solution was poured onto a glass culture dish and placed in a ventilated state for 72 h to obtain a drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone film. The film was placed in a printing cartridge, the needle diameter was 0.25 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 10 mm / s, the fiber direction was “0-90°”, the air pressure was 360 kPa, and the scaffold layer height was 80% of the needle diameter. The drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold was printed layer by layer. 50 mg of the drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold was soaked in 50 mL of an aqueous solution, ultrasonicated at room temperature for 0.5 hour, and then the drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold was obtained.

[0114] Comparative Example 2

[0115] The difference between Comparative Example 2 and Example 1 is only that no polydopamine is added in step (3) of Comparative Example 2, and the others are the same as in Example 1.

[0116] Specifically, step (3) of Comparative Example 2 is as follows:

[0117] 180 mg of methacrylated sodium alginate was dissolved in 10 mL of PBS solution containing 0.3% phenyl-2,4,6-trimethylbenzoyl lithium phosphite, and 20 mg of gentamicin was added to obtain a drug-loaded hydrogel solution; 100 mg of a drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold with a multi-level pore structure was placed in the drug-loaded hydrogel, and after 6 min of dispersion, it was taken out, irradiated with blue light for 90 s for curing, then placed in a-20℃ refrigerator for freezing, and freeze-dried in a freeze dryer to obtain a polycaprolactone scaffold with a multi-level pore structure filled with drug-loaded mesoporous ceramic@ZIF8 of drug-loaded hydrogel.

[0118] Comparative Example 3

[0119] The difference between Comparative Example 3 and Example 1 is that no antibacterial drug is added in step (3) of Comparative Example 3, that is, the polydopamine is not drug-loaded, and the others are the same as in Example 1.

[0120] Specifically, step (3) of Comparative Example 3 is as follows:

[0121] 180 mg of methacrylated sodium alginate was dissolved in 10 mL of PBS solution containing 0.3% phenyl-2,4,6-trimethylbenzoyl lithium phosphite, and 240 mg of polydopamine was added to obtain a polydopamine-containing hydrogel solution; 100 mg of a drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold with a multi-level pore structure was placed in the polydopamine-containing hydrogel solution, and after 6 min of dispersion, it was taken out, irradiated with blue light for 90 s for curing, then placed in a-20℃ refrigerator for freezing, and freeze-dried in a freeze dryer to obtain a polycaprolactone scaffold with a multi-level pore structure filled with drug-loaded mesoporous ceramic@ZIF8 of drug-loaded hydrogel.

[0122] Comparative Example 4

[0123] The difference between Comparative Example 4 and Example 1 is that Comparative Example 4 does not contain drug-loaded polydopamine particles, that is, no drug is added in step (3), and no polydopamine is added, and the others are the same as in Example 1.

[0124] Specifically, step (3) of Comparative Example 4 is as follows:

[0125] 180 mg of methacrylated sodium alginate was dissolved in 10 mL of PBS solution containing 0.3% phenyl-2,4,6-trimethylbenzoyl lithium phosphite to obtain a hydrogel solution; 100 mg of a drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold with a multi-level pore structure was placed in the hydrogel solution, and after 6 min of dispersion, it was taken out, irradiated with blue light for 90 s for curing, then placed in a-20℃ refrigerator for freezing, and freeze-dried in a freeze dryer to obtain a polycaprolactone scaffold with a multi-level pore structure filled with drug-loaded mesoporous ceramic@ZIF8 of drug-loaded hydrogel.

[0126] Comparative Example 5

[0127] The difference between Comparative Example 5 and Example 1 is that no drug is loaded in the mesoporous ceramic@ZIF8 particles of Comparative Example 5, i.e. no mesoporous ceramic is added in step (1), and the rest is the same as Example 1.

[0128] Specifically, step (1) of Comparative Example 5 is as follows:

[0129] 300 mg of mesoporous calcium silicate (average particle size between 300-1000 nm, specific surface area between 300-900 m 2 / g, average pore size between 2-30 nm) is added to 24 mL of 35 mg / mL aqueous 2-methylimidazole solution, 3 mL of 14 mg / mL aqueous zinc nitrate solution is added dropwise during stirring, and mesoporous ceramic@ZIF8 particles are obtained after washing, freeze-drying and reaction.

[0130] Comparative Example 6

[0131] The difference between Comparative Example 6 and Example 1 is that no mesoporous ceramic is contained in the drug-loaded ZIF8 particles of Comparative Example 6, i.e. no mesoporous ceramic is added in step (1), and the rest is the same as Example 1.

[0132] Specifically, step (1) of Comparative Example 6 is as follows:

[0133] 0.05 mg of bone morphogenetic protein-2 is added to 24 mL of 35 mg / mL aqueous 2-methylimidazole solution, 3 mL of 14 mg / mL aqueous zinc nitrate solution is added dropwise during stirring, and drug-loaded ZIF8 particles are obtained after washing, freeze-drying and reaction.

[0134] Comparative Example 7

[0135] The difference between Comparative Example 7 and Example 1 is that no ZIF8 is contained in Comparative Example 7, i.e. no ZIF8 is added in step (1), and the rest is the same as Example 1.

[0136] Specifically, step (1) of Comparative Example 7 is as follows:

[0137] 100 mg of mesoporous calcium silicate (average particle size between 300-1000 nm, specific surface area between 300-900 m 2 / g, average pore size between 2-30 nm) is dispersed in 10 mL of aqueous solution containing 0.05 mg of bone morphogenetic protein-2 at 0°C, stirred at a speed of 300 rpm for 18 h, and freeze-dried to obtain drug-loaded mesoporous ceramic.

[0138] Comparative Example 8

[0139] The difference between Comparative Example 8 and Example 1 is that Comparative Example 8 does not contain drug-loaded mesoporous ceramic@ZIF8 particles, i.e., step (1) is not performed, and the others are the same as Example 1.

[0140] Comparative Example 9

[0141] The difference between Comparative Example 9 and Example 1 is that Comparative Example 9 does not contain drug-loaded mesoporous ceramic@ZIF8 particles, gluconolactone, and drug-loaded polydopamine, and the others are the same as Example 1.

[0142] Specifically, the preparation method of the polycaprolactone scaffold of Comparative Example 9 comprises the following steps:

[0143] 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 70°C, the needle temperature was 75°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 360 kPa, and the scaffold layer height was 80% of the needle diameter. Layer by layer printing was performed to obtain a scaffold. 50 mg of the polycaprolactone scaffold was soaked in 50 mL of an aqueous solution, ultrasonicated at room temperature for 0.5 hours to obtain a polycaprolactone scaffold;

[0144] 180 mg of methacrylated sodium alginate was dissolved in 10 mL of a PBS solution containing 0.3% phenyl-2,4,6-trimethylbenzoyl lithium phosphite to obtain a hydrogel solution; 100 mg of the polycaprolactone scaffold was placed in the hydrogel solution, dispersed for 6 min, removed, irradiated with blue light for 90 s to solidify, then placed in a -20°C refrigerator to freeze, and then freeze-dried in a freeze dryer to obtain a hydrogel-filled polycaprolactone scaffold.

[0145] Comparative Example 10

[0146] The difference between Comparative Example 10 and Example 1 is that Comparative Example 10 forms a drug-loaded polydopamine coating on the surface of the polycaprolactone scaffold, and then fills the hydrogel, i.e., steps (1) and (2) are the same as Example 1.

[0147] Steps (2) and (3) are as follows:

[0148] (2) 10 g polycaprolactone (molecular weight 40,000 Dalton) was dissolved in 50 mL dichloromethane, 10 g gluconolactone and 1.5 g drug-loaded mesoporous ceramic@ZIF8 particles obtained in step (1) were added, and the mixture was stirred at a speed of 500 rpm for 72 h to obtain a gluconolactone / drug-loaded mesoporous ceramic@ZIF8 / 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@ZIF8 / polycaprolactone film; the film was placed in a printing cartridge, the needle diameter was 0.25 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 10 mm / s, the fiber direction was "0-90°", the air pressure was 360 kPa, and the scaffold layer height was 80% of the needle diameter, and the scaffold was printed layer by layer to obtain a gluconolactone / drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold; 50 mg of the gluconolactone / drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold was soaked in 50 mL of an aqueous solution, and ultrasonic treatment was performed at room temperature for 0.5 h to obtain a drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold with a multi-level pore structure;

[0149] (3) 20 mg gentamicin was dissolved in 10 mL of a 2 mg / mL dopamine aqueous solution (pH = 8.4), and a drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold with a multi-level pore structure was added, and the mixture was stirred at a speed of 300 rpm for 12 min. After freeze-drying, a drug-loaded polydopamine-coated drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold with a multi-level pore structure was obtained; 180 mg of methacrylated sodium alginate was dissolved in 10 mL of a PBS solution containing 0.3% phenyl-2,4,6-trimethylbenzoyl lithium phosphite, and 100 mg of the drug-loaded polydopamine-coated drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold with a multi-level pore structure was added to the hydrogel solution, dispersed for 6 min, removed, irradiated with blue light for 90 s to solidify, then placed in a -20°C refrigerator to freeze, and freeze-dried in a freeze dryer to obtain a hydrogel-filled drug-loaded polydopamine-coated drug-loaded mesoporous ceramic@ZIF8 / polycaprolactone scaffold with a multi-level pore structure.

[0150] Performance test

[0151] 1. In vitro cytotoxicity evaluation

[0152] After the L929 mouse fibroblasts which had been cultured for 24 h and grew vigorously were digested, a solution with a density of 1.0 x 10 5The cells were inoculated in 96-well plates at 1×104 / mL, 100 μL per well. After the cells grew into a monolayer, the original culture solution was removed, and 100 μL of the test sample (poly (ε-caprolactone) scaffold of Examples 1-4 and Comparative Examples 1-10) leaching solution, blank control solution, positive control solution and negative control solution were added, respectively, 6 replicates in each group. The blank control solution was without any addition; the positive control solution was complete culture medium containing 3 g / L phenol; the positive control solution was leaching solution of high-density polyethylene containing 0.3 g / mL leached in complete culture medium at 37±1°C for 24 h; after the addition was completed, the 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 h; 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 h; 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 at 570 nm as the main absorption wavelength and 650 nm as the reference wavelength, and the cell survival rate was calculated.

[0153] The cell survival rate was calculated according to the following formula: cell survival rate (%) = 100×OD 570e / OD 570b ;

[0154] OD 570e is the average value of the optical density of the test sample or control sample leaching solution;

[0155] OD 570b is the average value of the blank optical density.

[0156] The in vitro cell survival rates of the poly (ε-caprolactone) scaffolds of the control group, Examples 1-4 and Comparative Examples 1-10 are shown in Table 1.

[0157] Table 1: In vitro cell survival rates of the poly (ε-caprolactone) scaffolds of the control group, Examples 1-4 and Comparative Examples 1-10

[0158]

[0159] As can be seen from Table 1, the poly (ε-caprolactone) scaffold prepared in the application has no cytotoxicity.

[0160] 2. Hemolysis performance test

[0161] According to the blood volume for test, blood is collected from the heart of healthy rabbits, for example, 10 mL of blood is collected, 0.5 mL of 20 g / L potassium oxalate solution is added, and fresh anticoagulant rabbit blood is prepared; 8 mL of fresh anticoagulant rabbit blood is diluted with 10 mL of 9 g / L sodium chloride injection. The test sample group (Examples 1-4 and Comparative Examples 1-10) is added with the test sample according to the extraction ratio (0.2 g / mL), and then 10 mL of sodium chloride injection is added; the negative control group is added with 10 mL of sodium chloride injection per tube; and 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) ℃ for 30 min, 0.2 mL of diluted rabbit blood is added to each test tube, mixed gently, and then placed in a water bath at 37±1 ℃ for continuous incubation for 60 min. The liquid in each test tube is poured out and centrifuged at 800 g for 5 min, and the supernatant is transferred into a cuvette, and the absorbance is measured at a wavelength of 545 nm by using a spectrophotometer. The absorbance of the test sample 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; and the absorbance of the positive control tube should be 0.8±0.3, otherwise the test should be repeated.

[0162] The hemolysis rate is calculated according to the following formula:

[0163] Hemolysis rate (%) = (absorbance of the test sample group - absorbance of the negative control group) / (absorbance of the positive control group - absorbance of the negative control group) x 100%.

[0164] The hemolysis rate of the polycaprolactone stents in Examples 1-4 and Comparative Examples 1-10 is shown in Table 2.

[0165] Table 2: Hemolysis rate of polycaprolactone stents in Examples 1-4 and Comparative Examples 1-10

[0166]

[0167]

[0168] As can be seen from Table 2, the hemolysis rate of the polycaprolactone stents prepared by the method of the present application is less than 5%, and there is no risk of hemolysis.

[0169] 3. In vitro drug release performance test

[0170] The in vitro drug release performance evaluation method is as follows: 500 mg of the polycaprolactone stent is immersed in 200 mL of PBS (pH = 7.4) at 37 ℃ and 60 rpm in a constant-temperature shaker, the test solution is collected regularly, and an equal amount of PBS is supplemented. The collected test solution is determined for the content of the drug by using high performance liquid chromatography (HPLC), compared with the total amount of the drug loaded in the product, and the cumulative release rate of the drug is calculated.

[0171] The in vitro drug release curves for promoting tissue regeneration and repair using polycaprolactone scaffolds in Examples 1-4, Comparative Examples 1-4, 6-7, and 10 are shown below. Figure 1 As shown; the in vitro drug release curves for promoting tissue regeneration and repair using polycaprolactone scaffolds in Examples 1, 1-4, 6-7, and 10 are shown in the figure. Figure 2 As shown.

[0172] The in vitro antibacterial drug release curves of polycaprolactone scaffolds in Examples 1-4 and Comparative Examples 1-2, 5-8, and 10 are shown below. Figure 3 As shown; the in vitro antibacterial drug release curves of polycaprolactone scaffolds in Examples 1, 1-2, 5-8, and 10 are as follows. Figure 4 As shown.

[0173] Depend on Figures 1-2 It can be seen that the polycaprolactone scaffolds in Examples 1-4 all had release periods exceeding 5 weeks for various tissue regeneration and repair-promoting drugs. Comparative Example 1 did not use gluconolactone, and its scaffold fibers lacked a porous structure, resulting in a significantly lower release rate and a significantly longer release period for the tissue regeneration and repair-promoting drugs compared to Example 1. Comparative Example 3 did not contain antibacterial drugs, having almost no effect on the drug release curve. Comparative Examples 2 and 4 did not contain polydopamine, and the drug-loaded ZIF8 particles in Comparative Example 6 did not contain bioactive ceramics, having little impact on drug release; their release curves were similar to those of Example 1. In Comparative Example 7, the drug-loaded mesoporous ceramic particles were not encapsulated with ZIF8, resulting in a significantly increased burst release and a significantly faster release rate compared to Example 1, with a cumulative release rate approaching 90% at 5 weeks. Comparative Example 10, with a PDA coating, had a certain delaying effect on drug release, with a drug release rate between that of Example 1 and Comparative Example 1.

[0174] Depend on Figures 3-4 It can be seen that the release periods of various antibacterial drugs by the polycaprolactone scaffolds in Examples 1-4 all exceeded 3 weeks. Compared with Example 1, Comparative Example 1 did not use gluconolactone, and its scaffold fibers did not contain a porous structure, resulting in a slightly lower antibacterial drug release rate than that of Example 1. Comparative Examples 5-8 had little impact on the release of antibacterial drugs embedded on the scaffold surface, and their release curves were all close to those of Example 1. In Comparative Example 2, the antibacterial drug was not encapsulated by polydopamine and was directly dispersed in the hydrogel, resulting in a significantly increased burst release and a significantly faster release rate compared to Example 1. Comparative Example 10 formed a drug-loaded polydopamine coating on the surface of the polycaprolactone scaffold and then filled it with hydrogel, making the release rate of Comparative Example 10 significantly faster than that of Example 1.

[0175] 4. Detection of osteogenic differentiation performance of pre-osteoblasts induced in vitro

[0176] The polycaprolactone scaffolds prepared in Examples 1-4 and Comparative Examples 1-10 were sterilized by irradiation, soaked in DMEM basic medium at a concentration of 10 mg / mL, and then placed in a 37°C shaker at a rotation speed of 120 rpm for 24 h. After the extraction was completed, the polycaprolactone scaffold and medium mixture was centrifuged at a rotation speed of 1000 rpm, and the supernatant, i.e., the extract, was collected. The collected extract was diluted 2-fold with the corresponding DMEM medium, and finally 10% fetal bovine serum was added to obtain the conditioned medium.

[0177] Mouse embryonic osteogenic precursor cells (MC3T3-E1 cells) were seeded in a 24-well plate at a density of 1 x 10 5 cells per well, and adherent culture was performed for 24 h. The conditioned medium was then replaced, and the plate was incubated in a 37°C incubator under 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 measuring the alkaline phosphatase secreted by the cells. The pNPP method was used for the measurement, and the specific steps were as follows: after the MC3T3-E1 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 disodium para-nitrophenyl phosphate, 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 of each well at a wavelength of 405 nm was measured using an enzyme-labeled instrument.

[0178] Definition of alkaline phosphatase activity unit: the amount of alkaline phosphatase required to hydrolyze 1 micromole of para-nitrophenol (p-nitrophenol) from the para-nitrophenyl phosphate chromogenic substrate per minute in a diethanolamine (DEA) buffer at pH 9.8 and at 37°C is defined as one enzyme activity unit (mmol / min / mg), also referred to as one DEA enzyme activity unit. The amount of alkaline phosphatase required to hydrolyze 1 micromole of para-nitrophenol from the para-nitrophenyl phosphate chromogenic substrate per minute in a glycine buffer at pH 9.6 and at 25°C is defined as one enzyme activity unit, also referred to as one glycine (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 the alkaline phosphatase activity of the polycaprolactone scaffolds of Examples 1-4 and Comparative Examples 1-10 is shown in the graph of Figure 5

[0179] The polycaprolactone scaffolds prepared in Examples 1-4 and Comparative Examples 1-10 were sterilized by irradiation, soaked in DMEM basic medium at a concentration of 10 mg / mL, and then placed in a 37°C shaker at a rotation speed of 120 rpm for 24 h. After the extraction was completed, the polycaprolactone scaffold and medium mixture was centrifuged at a rotation speed of 1000 rpm, and the supernatant, i.e., the extract, was collected. The collected extract was diluted 2-fold with the corresponding DMEM medium, and finally 10% fetal bovine serum was added to obtain the conditioned medium. Figure 5 ​It can be seen that the polycaprolactone scaffolds of the embodiments 1-4 of the present application have good effect of inducing cells to secrete alkaline phosphatase. The comparative example 1 does not use gluconolactone, and the polycaprolactone scaffold does not have a multi-level pore structure, and the cell activity is lower than that of the scaffold with a multi-level pore structure, thus affecting the secretion of alkaline phosphatase by the cells, and the alkaline phosphatase activity is slightly lower than that of the embodiment 1. The comparative example 3 does not load antibacterial drugs, and the alkaline phosphatase activity is close to that of the embodiment 1. The comparative examples 2 and 4 do not contain polydopamine with good biological activity, and the alkaline phosphatase activity is significantly lower than that of the embodiment 1. The drug-loaded ZIF8 particles in the comparative example 6 do not contain biologically active ceramics, thus affecting the secretion of alkaline phosphatase by the cells, and the alkaline phosphatase activity is also significantly lower than that of the embodiment 1. The comparative example 7 does not contain ZIF8 with osteogenesis-promoting activity, and the release of the drugs is significantly accelerated, thus affecting the secretion of alkaline phosphatase by the cells, and the alkaline phosphatase activity is significantly lower than that of the embodiment 1. The biologically active ceramic / ZIF8 particles in the comparative example 5 do not load drugs, and the alkaline phosphatase activity is significantly lower than that of the embodiment 1. The comparative example 8 does not contain drug-loaded biologically active ceramic / ZIF8 particles, and the alkaline phosphatase activity is also significantly lower than that of the embodiment 1, and the alkaline phosphatase activity is lower than that of the comparative example 5. The comparative example 9 does not contain polydopamine with good biological activity, does not have a multi-level pore structure, does not load mesoporous ceramic@ZIF8 particles and antibacterial drugs, and the alkaline phosphatase activity is the lowest. The comparative example 10 is changed to form a polydopamine coating, and the alkaline phosphatase activity is between that of the comparative example 2 and the embodiment 1.

[0180] 5. Anti-bacterial performance test

[0181] Fresh cultures of Staphylococcus aureus and Escherichia coli were taken, and the viable bacterial count was determined, and the bacterial suspensions with a bacterial content of 5x10 5 -10x10 6 cfu / mL were prepared using 1% proteose peptone in 0.03 mol / L PBS (pH = 7.2-7.4). The polycaprolactone scaffold samples of the embodiments 1-4 and the comparative examples 1-10 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, and 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, the preliminary results were observed, and 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 indicated as (+); if it was still clear on the 35th day, it was considered as sterile growth, and indicated as (-).

[0182] The results of the anti-bacterial performance test of the polycaprolactone scaffolds of the embodiments 1-4 and the comparative examples 1-10 are shown in Table 3.

[0183] Table 3: Results of antibacterial performance test of polycaprolactone scaffolds of examples 1-4 and comparative examples 1-10

[0184]

[0185] As can be seen from Table 3, the polycaprolactone scaffolds prepared in examples 1-4 of the present application all have good long-acting antibacterial effect, and no bacteria grow for 35 days. Comparative example 9 does not contain ZIF8 and does not contain antibacterial drugs, and does not have antibacterial effect.

[0186] In summary, the polycaprolactone scaffold body of the present application has both macroscopic large pores (inter-fiber gaps) and microscopic small pores (fiber surface holes), which significantly increases the specific surface area of the polycaprolactone scaffold body, accelerates the degradation of polycaprolactone and releases more acidic degradation products, thereby accelerating the degradation of polydopamine and accelerating the release of the antibacterial drugs embedded therein. At the same time, the macroscopic large pores and the microscopic small pores can also significantly increase the surface roughness of the polycaprolactone scaffold body, achieving the purpose of promoting cell adhesion of the polycaprolactone scaffold. The drug-loaded mesoporous bioceramics is embedded in ZIF8, which can delay the release of the drug, achieve the effect of long-acting controlled release, improve the antibacterial performance, and induce osteogenic differentiation of stem cells, which is suitable for repair and regeneration of infected bone tissue defects, and the bioactive ceramic can enhance the bioactivity and mechanical strength of the drug-loaded particles. In addition, the hydrogel and drug-loaded polydopamine particles filled in the pores of the scaffold can improve the mechanical strength and bioactivity of the scaffold, and endow the scaffold with antibacterial performance.

[0187] The above examples are only used to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A polycaprolactone scaffold, characterized in that, The poly-caprolactone scaffold body and the drug-loaded mesoporous ceramic and zeolitic imidazolate framework embedded in the poly-caprolactone scaffold body; The drug-loaded mesoporous ceramic is embedded in the zeolitic imidazolate framework; The base material of the poly-caprolactone scaffold body is poly-caprolactone, the microporous structure is formed between and on the surface of the fibers of the poly-caprolactone, the size of the microporous structure between the fibers of the poly-caprolactone is larger than the size of the microporous structure on the surface of the fibers of the poly-caprolactone; The microporous structure is filled with a hydrogel, and the drug-loaded polydopamine is embedded in the hydrogel; The preparation method of the poly-caprolactone scaffold comprises the following steps: (1) mixing mesoporous ceramic and a drug-containing aqueous solution, drying to obtain drug-loaded mesoporous ceramic, mixing the drug-loaded mesoporous ceramic with an aqueous solution of 2-methyl imidazole, then adding an aqueous solution of zinc source, and reacting to obtain a material of zeolitic imidazolate framework embedded drug-loaded mesoporous ceramic; (2) mixing poly-caprolactone, a solvent, gluconolactone and the material of zeolitic imidazolate framework embedded drug-loaded mesoporous ceramic obtained in step (1) to obtain a mixed solution, preparing the mixed solution into a film, then printing to obtain a gluconolactone / drug-loaded mesoporous ceramic@zeolitic imidazolate framework / poly-caprolactone scaffold, and placing it in an aqueous solution, and ultrasonic treatment to obtain a drug-loaded mesoporous ceramic@zeolitic imidazolate framework / poly-caprolactone scaffold; (3) adding drug-loaded polydopamine to a hydrogel solution containing a photoinitiator to obtain a hydrogel solution containing drug-loaded polydopamine, then placing the drug-loaded mesoporous ceramic@zeolitic imidazolate framework / poly-caprolactone scaffold obtained in step (2) into the hydrogel solution containing drug-loaded polydopamine, then taking it out, and solidifying to obtain the poly-caprolactone scaffold.

2. The polycaprolactone scaffold of claim 1, wherein, The poly-caprolactone scaffold body is a 3D printed scaffold; and / or the molecular weight of the poly-caprolactone is 27-110 kilodaltons.

3. The polycaprolactone scaffold of claim 1, wherein, The size of the microporous structure between the fibers of the poly-caprolactone is 200-650 microns; and / or the size of the microporous structure on the surface of the fibers of the poly-caprolactone is 0.9-110 microns.

4. The polycaprolactone scaffold of claim 1, wherein, The mesoporous ceramic in the drug-loaded mesoporous ceramic comprises at least one of mesoporous silica, mesoporous calcium silicate, mesoporous magnesium silicate, mesoporous zinc silicate, mesoporous strontium silicate, mesoporous bioglass, and mesoporous hydroxyapatite; and / or, the particle size of the mesoporous ceramic in the drug-loaded mesoporous ceramic is 45-1100 nm; and / or, the specific surface area of the mesoporous ceramic in the drug-loaded mesoporous ceramic is 140-2000 m 2 / g; and / or, the average pore size of the mesoporous ceramic in the drug-loaded mesoporous ceramic is 2-45 nm.

5. The polycaprolactone scaffold of claim 1, wherein, The drug in the drug-loaded mesoporous ceramic includes a drug for promoting tissue regeneration and repair; and / or the hydrogel includes at least one of methacrylated hyaluronic acid, methacrylated silk fibroin, methacrylated chondroitin sulfate, methacrylated chitosan, methacrylated carboxymethyl chitosan, methacrylated gelatin, methacrylated sodium alginate, hyaluronic acid, silk fibroin, chondroitin sulfate, chitosan, carboxymethyl chitosan, gelatin, and sodium alginate; and / or the drug in the drug-loaded polydopamine includes an antibacterial drug.

6. The polycaprolactone scaffold of claim 1, wherein, In step (1), the mass ratio of the drug to the mesoporous ceramic is (0.0005-0.055):1; and / or, the concentrations of 2-methylimidazole and the drug-loaded mesoporous ceramic in the 2-methylimidazole aqueous solution are 17-58 mg / mL and 7-20 mg / mL, respectively; and / or, the zinc source aqueous solution comprises a zinc nitrate aqueous solution; the concentration of zinc nitrate in the zinc nitrate aqueous solution is 5.5-33 mg / mL; and / or, the volume ratio of the 2-methylimidazole aqueous solution to the zinc source aqueous solution is (5.5-13):

1.

7. The polycaprolactone scaffold of claim 1, wherein, In step (2), the mass ratio of the glucose lactone, the material for embedding the drug-loaded mesoporous ceramic in the zeolite imidazolate framework, and the polycaprolactone is (0.45-3.3):(0.05-0.33):1; and / or, the mass-volume ratio of the polycaprolactone to the solvent is (0.45-9) g:10 mL; and / or, the mass-volume ratio of the glucose lactone / drug-loaded mesoporous ceramic@zeolite imidazolate framework / polycaprolactone scaffold to the aqueous solution is 1 mg:(0.27-4.5) mL.

8. The polycaprolactone scaffold of claim 1, wherein, In step (3), the preparation process of the drug-loaded polydopamine is mixing the drug and a dopamine aqueous solution, and stirring to obtain the drug-loaded polydopamine; the concentration of dopamine in the dopamine aqueous solution is 1.5-4.5 mg / mL; the mass-volume ratio of the drug to the dopamine aqueous solution is (9-33) mg:10 mL; and / or, the concentration of the hydrogel in the hydrogel solution is 1.4-3.3%; and / or, the mass-volume ratio of the drug-loaded polydopamine to the hydrogel solution is (11-52) mg:1 mL; and / or, the mass-volume ratio of the drug-loaded mesoporous ceramic@zeolite imidazolate framework / polycaprolactone scaffold to the hydrogel solution containing the drug-loaded polydopamine is (0.04-0.18) g:10 mL; and / or, the implantation time is 2.5-13 min.

9. Use of the polycaprolactone scaffold of any one of claims 1-8 in the preparation of a product for repairing and regenerating bone tissue defects.

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