A modified polylactic acid-glycolic acid copolymer composite scaffold and a preparation method and application thereof

Abstract

The application discloses a modified polylactic acid-glycolic acid copolymer composite scaffold and a preparation method and application thereof. The polylactic acid-glycolic acid copolymer composite scaffold has a porous structure, wherein the pores on the surface and inside are interconnected, and the pore diameter gradually increases from the surface to the inside; and the polylactic acid-glycolic acid copolymer composite scaffold is loaded with a metal organic framework material on the surface. The modified polylactic acid-glycolic acid copolymer composite scaffold has macro-pores and micro-pores at the same time, which can not only improve the surface roughness of the scaffold, so as to significantly promote cell adhesion of the scaffold, but also significantly improve the specific surface area of the scaffold, so that the polylactic acid-glycolic acid copolymer is accelerated to degrade and release more acidic degradation products, thereby accelerating the degradation of polydopamine and accelerating the release of the drugs embedded in the polydopamine, and the problem that the polydopamine is slow in degradation in vivo and in vitro is solved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical materials, and particularly relates to a modified polylactic acid-glycolic acid copolymer 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 innovation and triggering global manufacturing revolution. 3D printing obtains a three-dimensional entity by layer-by-layer accumulation of 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] Polylactic acid-glycolic acid copolymer is a synthetic copolymer of polylactic acid and polyglycolic acid, which can be dissolved in a wide range of solvents and can be easily controlled to the desired size and shape, has good biocompatibility and mechanical strength. The 3D printed polylactic acid-glycolic acid copolymer scaffold is prepared by melt deposition method at a melting temperature of 197℃. However, it is found in the research process that polylactic acid-glycolic acid copolymer has the disadvantages of acid degradation and lack of cell binding sites, and needs to be modified by combining other materials. By loading drugs, surface modification, physical doping, chemical grafting and other ways to introduce artificial materials, the characteristics of polylactic acid-glycolic acid copolymer can be supplemented, and by adjusting the mixed components, the respective advantages of the components can be maximized, and the ideal parameter requirements of bone repair materials can be met. In addition, polydopamine microspheres have a certain drug loading capacity and can be used as drug carriers 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 Poly (Caprolactone) / β-Tricalcium Phosphate Scaffolds 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 Scaffolds Loaded with FK-16 on Infectious Bone Defects[D]. Jilin University, 2024. DOI:10.27162 / d.cnki.gjlin.2024.005386.

[0005] In order to overcome the problems of the above existing technologies, one of the purposes of the present application is to provide a modified polylactic acid-glycolic acid copolymer composite material scaffold. The second purpose of the present application is to provide a preparation method of the modified polylactic acid-glycolic acid copolymer composite material scaffold. The third purpose of the present application is to provide an application of the modified polylactic acid-glycolic acid copolymer composite material 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 bone tissue defects under infection.

[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 modified polylactic acid-glycolic acid copolymer composite scaffold, comprising a polylactic acid-glycolic acid copolymer matrix, a metal organic framework material loaded on the surface of the polylactic acid-glycolic acid copolymer scaffold matrix, and an antibacterial drug loaded in the metal organic framework material; the polylactic acid-glycolic acid copolymer scaffold matrix is distributed with interconnected pores, the pores gradually increase from the surface to the inside of the polylactic acid-glycolic acid copolymer scaffold matrix; the polylactic acid-glycolic acid copolymer scaffold matrix is distributed with drug-loaded materials, the drug-loaded materials comprise a ceramic material and a tissue regeneration and repair promoting drug, and the ceramic material and the tissue regeneration and repair promoting drug are coated with polydopamine on the surface.

[0008] The pores of the scaffold in the present application gradually increase from the surface to the inside of the polylactic acid-glycolic acid copolymer scaffold matrix, forming a gradient pore and a multi-level pore structure, wherein the internal pores of the polylactic acid-glycolic acid copolymer scaffold matrix refer to the pores formed by the stacking of fibers (macroscopic pores, several hundred microns), and the external pores of the polylactic acid-glycolic acid copolymer 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 polylactic acid-glycolic acid copolymer composite has pores with a size of 1-100 μm.

[0010] Preferably, the ceramic material is selected from at least one of hydroxyapatite, bioglass, calcium silicate, calcium carbonate, calcium sulfate, tricalcium phosphate, octacalcium phosphate, and amorphous calcium phosphate.

[0011] Preferably, the metal organic framework material is a zeolitic imidazolate framework material self-assembled by coordination of zinc ions and 2-methyl imidazole.

[0012] Preferably, the tissue regeneration and repair promoting 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.

[0013] Preferably, the molecular weight of the polylactic acid-glycolic acid copolymer is 1-100,000 Daltons.

[0014] The second aspect of the present application provides a preparation method of the modified polylactic acid-glycolic acid copolymer composite scaffold of the first aspect, comprising the following steps:

[0015] S1, mixing the drug-loaded material, gluconolactone and polylactic acid-glycolic acid copolymer in a solvent to obtain a mixed solution, transferring the mixed solution to a mold and placing it for a period of time to obtain a mixed material film; using the mixed material film as a printing material, 3D printing to obtain a scaffold with a gradient pore structure; after printing, immersing the scaffold with the gradient pore structure in water to remove the gluconolactone and residual solvent;

[0016] S2, reacting the scaffold with the gradient pore structure, 2-methylimidazole and a zinc source in water to obtain the modified polylactic acid-glycolic acid copolymer composite scaffold.

[0017] Preferably, the preparation method of the drug-loaded material comprises the following steps: dispersing a tissue regeneration and repair promoting drug and a ceramic material in a dopamine-containing aqueous solution, and mixing and reacting to obtain the drug-loaded material.

[0018] More preferably, the mass ratio of the ceramic material, the regeneration and repair promoting drug and dopamine is (50-1000):(0.005-10):100.

[0019] Preferably, the mass ratio of the drug-loaded material and the polylactic acid-glycolic acid copolymer is (0.005-0.05):1.

[0020] Preferably, the mass ratio of the gluconolactone and the polylactic acid-glycolic acid copolymer is (0.5-3):1.

[0021] Preferably, the solvent is dichloromethane.

[0022] Preferably, the process conditions of the 3D printing satisfy at least one of the following:

[0023] a) the 3D printing uses a printing needle with a diameter of 0.15-0.4 mm;

[0024] b) the printing and heating temperature: the barrel is 60-80℃, and the needle is 60-90℃;

[0025] c) the fiber spacing is 0.8mm×0.8mm; the printing rate is 8-17mm / s;

[0026] d) the air pressure is 320-400Kpa;

[0027] e) the scaffold layer height is 75-95% of the needle diameter;

[0028] f) the fiber direction is "0-90°".

[0029] 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 included angle between the two layers of fibers. The scaffold layer height refers to the layer thickness of each layer printed.

[0030] Preferably, in step S1, the mixing time is 30-100h.

[0031] More preferably, the mixing is carried out under stirring conditions, wherein the stirring rate is 100-1000rpm.

[0032] Preferably, the placing time is 40-100h.

[0033] Preferably, the film forming condition of the mixed material film is volatilization film forming.

[0034] Preferably, the mass ratio of the 2-methyl imidazole and the zinc source is (18-60):1.

[0035] Preferably, the zinc source is zinc nitrate.

[0036] Preferably, the step S2 specifically comprises the following steps: adding the scaffold with gradient pore structure into the 2-methyl imidazole-containing aqueous solution for dispersion, then slowly adding the zinc source for reaction, and after the reaction, washing and freeze-drying to obtain the modified polylactic acid-glycolic acid copolymer composite scaffold.

[0037] The third aspect of the present application provides an application of the modified polylactic acid-glycolic acid copolymer composite scaffold of the first aspect in preparing a product for promoting bone tissue repair and regeneration.

[0038] The present application has the following beneficial effects:

[0039] The present application provides a modified polylactic acid-glycolic acid copolymer composite scaffold, comprising a polylactic acid-glycolic acid copolymer composite scaffold; the polylactic acid-glycolic acid copolymer composite scaffold has a porous structure, wherein the pores on the surface and inside are interconnected, and the pore size gradually increases from the surface to the inside, so that the printed scaffold obtains macro-pores (fiber gaps) and micro-pores (fiber surface holes) at the same time, which not only can improve the surface roughness of the scaffold, achieve the purpose of significantly promoting the cell adhesion of the scaffold, but also can significantly improve the specific surface area of the scaffold, accelerate the degradation of the polylactic acid-glycolic acid copolymer and release more acidic degradation products, thereby accelerating the degradation of the polydopamine and accelerating the release of the drugs embedded inside, solving the problem of slow degradation of the polydopamine in vivo and in vitro.

[0040] The specific beneficial effects are as follows:

[0041] (1) The surface of the modified polylactic acid-glycolic acid copolymer composite scaffold of the application also carries a metal organic framework material loaded with drugs, which not only enhances the antibacterial performance of the gradient pore structure polylactic acid-glycolic acid copolymer scaffold, but also significantly induces osteogenic differentiation of stem cells, making it more suitable for the regeneration and repair of bone defects. In addition, the polylactic acid-glycolic acid copolymer composite scaffold of the application has micro-pores, providing more sites for the deposition of drug-loaded metal organic framework materials, which is conducive to further improving the biological activity of the scaffold.

[0042] (2) The modified polylactic acid-glycolic acid copolymer composite scaffold of the application uses ceramic material as a scaffold forming material, which can enhance the mechanical strength of the scaffold and the drug-loaded material, making it suitable for bone repair. The modified polylactic acid-glycolic acid copolymer composite scaffold of the application further disperses the tissue regeneration and repair drugs in the interior of the polylactic acid-glycolic acid copolymer, which can achieve better therapeutic effect compared to the conventional use of polydopamine coating adhesion to adsorb drug-loaded drugs.

[0043] (3) The application provides a preparation method of the above-mentioned modified polylactic acid-glycolic acid copolymer 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 have both macro-pores (fiber gaps) and micro-pores (fiber surface pores), and gluconolactone is easily soluble in water and easy to eliminate after reaction.

[0044] (4) The modified polylactic acid-glycolic acid copolymer composite scaffold of the application has great application potential in the preparation of products for promoting bone tissue repair and regeneration. The surface modified gradient pore structure scaffold has good porosity, biocompatibility, antibacterial performance, and osteogenic differentiation of stem cells. BRIEF DESCRIPTION OF DRAWINGS

[0045] Fig. 1 In Vitro Release Performance of Tissue Regeneration and Repair Drugs of the Materials Prepared in Examples and Comparative Examples;

[0046] Fig. 2 Alkaline Phosphatase Activity of the Scaffolds Prepared in Examples and Comparative Examples. DETAILED DESCRIPTION

[0047] The content of the application will be further described in detail through specific examples. In the following examples, the raw materials used, unless otherwise specified, can be obtained from conventional commercial channels or prepared and isolated by simple synthesis; the processes used, unless otherwise specified, all use conventional processes in the art.

[0048] Example 1

[0049] The embodiment provides a modified polylactic acid-glycolic acid copolymer composite scaffold, and a preparation method thereof.

[0050] S1, 0.01 mg of bone morphogenetic protein-2 and 300 mg of bioglass are dissolved / dispersed in 100 ml of weakly alkaline 1.5 mg / ml dopamine aqueous solution (pH = 8.4), and 400 rpm stirring is carried out for 8 h to obtain drug-loaded biologically active ceramic @ polydopamine particles.

[0051] S2, 10 g of polylactic acid-glycolic acid copolymer (molecular weight: 30,000 daltons) is dissolved in 50 ml of dichloromethane, 10 g of gluconolactone and 100 mg of drug-loaded biologically active ceramic @ polydopamine particles are added, and mechanical stirring is carried out at 500 rpm for 48 h to obtain a gluconolactone / drug-loaded biologically active ceramic @ polydopamine particle / polylactic acid-glycolic acid copolymer solution. The solution is poured on a glass culture dish, and placed in a ventilated state for 72 h to obtain a gluconolactone / drug-loaded biologically active ceramic @ polydopamine particle / polylactic acid-glycolic acid copolymer film.

[0052] S3, the film is placed into a printing cartridge, a needle diameter is 0.25 mm, a cartridge temperature is 85 DEG C, a needle temperature is 90 DEG C, a fiber spacing is 0.8 mm*0.8 mm, a printing rate is 12 mm / s, a fiber direction is '0-90 DEG ', an air pressure is 320 Kpa, and a scaffold layer height is 85% of the needle diameter, and the gluconolactone / drug-loaded biologically active ceramic @ polydopamine particle / polylactic acid-glycolic acid copolymer scaffold is printed layer by layer. 30 mg of the gluconolactone / drug-loaded biologically active ceramic @ polydopamine particle / polylactic acid-glycolic acid copolymer scaffold is soaked in 25 ml of an aqueous solution, ultrasonic treatment is carried out at room temperature for 0.5 h, and a gradient pore structure drug-loaded biologically active ceramic @ polydopamine particle / polylactic acid-glycolic acid copolymer scaffold is obtained.

[0053] S4, the gradient pore structure scaffold is added into 28 ml of 12 mg / ml 2-methylimidazole aqueous solution, 4 ml of 4 mg / ml zinc nitrate aqueous solution is added dropwise under stirring, and after reaction, the gradient pore structure, ZIF8 surface modified drug-loaded biologically active ceramic @ polydopamine / polylactic acid-glycolic acid copolymer scaffold is obtained after cleaning and freeze-drying.

[0054] Embodiment 2

[0055] The embodiment provides a modified polylactic acid-glycolic acid copolymer composite scaffold, and a preparation method thereof.

[0056] S1, 10 mg gentamicin sulfate and 400 mg hydroxyapatite were dissolved / dispersed in 100 ml of weakly basic 1 mg / ml dopamine aqueous solution (pH = 8.6), and the reaction was stirred at 600 rpm for 6 h to obtain drug-loaded bioactive ceramic@polydopamine particles.

[0057] S2, 10 g of polylactic acid-glycolic acid copolymer (molecular weight: 30,000 daltons) was dissolved in 20 ml of dichloromethane, and then 20 g of gluconolactone and 150 mg of drug-loaded bioactive ceramic@polydopamine particles were added, and the mixture was mechanically stirred at 600 rpm for 48 h to obtain a gluconolactone / drug-loaded bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer solution. The solution was poured onto a glass culture dish and placed in a ventilated state for 60 h to obtain a gluconolactone / drug-loaded bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer film.

[0058] S3, the film was placed in a printing cartridge, the needle diameter was 0.1 mm, the cartridge temperature was 95°C, the needle temperature was 85°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 support layer height was 80% of the needle diameter. The gluconolactone / drug-loaded bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer scaffold was printed layer by layer. 30 mg of the gluconolactone / drug-loaded bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer scaffold was soaked in 30 ml of an aqueous solution, and an ultrasonic wave was applied for 0.6 hours at room temperature to obtain a gradient pore structure drug-loaded bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer scaffold.

[0059] S4, the gradient pore structure scaffold was added to 29 ml of an aqueous solution of 18 mg / ml 2-methylimidazole, and 3 ml of an aqueous solution of 3 mg / ml zinc nitrate was added dropwise under stirring. After the reaction, the product was washed, freeze-dried, and a gradient pore structure, ZIF8 surface modified drug-loaded bioactive ceramic@polydopamine / polylactic acid-glycolic acid copolymer scaffold was obtained.

[0060] Example 3

[0061] The present example provides a modified polylactic acid-glycolic acid copolymer composite scaffold, and a preparation method thereof is as follows:

[0062] S1, 5 mg of curcumin and 100 mg of octacalcium phosphate were dissolved / dispersed in 100 ml of weakly basic 2 mg / ml dopamine aqueous solution (pH = 8), and the reaction was stirred at 200 rpm for 12 h to obtain drug-loaded bioactive ceramic@polydopamine particles.

[0063] S2, 10 g of polylactic acid-glycolic acid copolymer (molecular weight: 100,000 daltons) was dissolved in 100 ml of dichloromethane, 5 g of gluconolactone and 50 mg of drug-loaded bioactive ceramic @ polydopamine particles were added, and magnetic stirring was carried out at 300 rpm for 36 h to obtain a gluconolactone / drug-loaded bioactive ceramic @ polydopamine particle / polylactic acid-glycolic acid copolymer solution. The solution was poured onto a glass culture dish and placed in a ventilated state for 48 h to obtain a gluconolactone / drug-loaded bioactive ceramic @ polydopamine particle / polylactic acid-glycolic acid copolymer film.

[0064] S3, the film was placed in a printing cartridge, the needle diameter was 0.35 mm, the cartridge temperature was 110°C, the needle temperature was 105°C, the fiber spacing was 0.8 mm x 0.8 mm, the printing rate was 5 mm / s, the fiber direction was "0-90°", the air pressure was 300 Kpa, and the support layer height was 75% of the needle diameter. The gluconolactone / drug-loaded bioactive ceramic @ polydopamine particle / polylactic acid-glycolic acid copolymer scaffold was printed layer by layer. 30 mg of the gluconolactone / drug-loaded bioactive ceramic @ polydopamine particle / polylactic acid-glycolic acid copolymer scaffold was soaked in 9 ml of an aqueous solution, and ultrasonic treatment was carried out at room temperature for 1 h to obtain a gradient pore structure drug-loaded bioactive ceramic @ polydopamine particle / polylactic acid-glycolic acid copolymer scaffold.

[0065] S4, the gradient pore structure scaffold was added to 36 ml of a 9 mg / ml aqueous solution of 2-methylimidazole, 3 ml of a 5 mg / ml aqueous solution of zinc nitrate was added dropwise under stirring, and after reaction, the product was washed, freeze-dried to obtain a gradient pore structure, ZIF8 surface modified drug-loaded bioactive ceramic @ polydopamine / polylactic acid-glycolic acid copolymer scaffold.

[0066] Example 4

[0067] The present example provides a modified polylactic acid-glycolic acid copolymer composite scaffold, and the preparation method is as follows:

[0068] S1, 4 mg of dexamethasone and 800 mg of calcium carbonate were dissolved / dispersed in 100 ml of weakly basic 0.8 mg / ml dopamine aqueous solution (pH = 9), and stirring was carried out at 800 rpm for 4 h to obtain drug-loaded bioactive ceramic @ polydopamine particles.

[0069] S2, 10 g of polylactic acid-glycolic acid copolymer (molecular weight: 10,000 daltons) was dissolved in 200 ml of dichloromethane, 30 g of gluconolactone and 200 mg of drug-loaded bioactive ceramic @ polydopamine particles were added, and magnetic stirring was performed at 1000 rpm for 72 h to obtain a gluconolactone / drug-loaded bioactive ceramic @ polydopamine particle / polylactic acid-glycolic acid copolymer solution. The solution was poured onto a glass culture dish and placed in a ventilated state for 60 h to obtain a gluconolactone / drug-loaded bioactive ceramic @ polydopamine particle / polylactic acid-glycolic acid copolymer film.

[0070] S3, the film was placed in a printing cartridge, the needle diameter was 0.45 mm, the cartridge temperature was 90°C, the needle temperature was 80°C, the fiber spacing was 0.8 mm x 0.8 mm, the printing rate was 15 mm / s, the fiber direction was "0-90°", the air pressure was 380 Kpa, and the support layer height was 90% of the needle diameter. The gluconolactone / drug-loaded bioactive ceramic @ polydopamine particle / polylactic acid-glycolic acid copolymer scaffold was printed layer by layer. 30 mg of the gluconolactone / drug-loaded bioactive ceramic @ polydopamine particle / polylactic acid-glycolic acid copolymer scaffold was soaked in 60 ml of an aqueous solution, and ultrasonic treatment was performed at room temperature for 0.8 hours to obtain a gradient pore structure drug-loaded bioactive ceramic @ polydopamine particle / polylactic acid-glycolic acid copolymer scaffold.

[0071] S4, the gradient pore structure scaffold was added to 22 ml of a 24 mg / ml aqueous solution of 2-methylimidazole, and 2 ml of a 13 mg / ml aqueous solution of zinc nitrate was added dropwise under stirring. After reaction, washing, and freeze-drying, a gradient pore structure, ZIF8 surface modified drug-loaded bioactive ceramic @ polydopamine / polylactic acid-glycolic acid copolymer scaffold was obtained.

[0072] Comparative Example 1

[0073] This comparative example provides a preparation method of a ZIF8 surface modified polylactic acid-glycolic acid copolymer scaffold, which is substantially the same as Example 1, except that gluconolactone is not used, and the preparation method is as follows:

[0074] S1, 0.01 mg of bone morphogenetic protein-2 and 300 mg of bioglass were dissolved / dispersed in 100 ml of weakly basic 1.5 mg / ml dopamine aqueous solution (pH = 8.4), and stirring was performed at 400 rpm for 8 h to obtain drug-loaded bioactive ceramic @ polydopamine particles.

[0075] S2, 10 g of polylactic acid-glycolic acid copolymer (molecular weight: 30,000 daltons) was dissolved in 50 ml of dichloromethane, and 100 mg of drug-loaded bioactive ceramic@polydopamine particles were added, and mechanical stirring was carried out at 500 rpm for 48 h to obtain a drug-loaded bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer solution. The solution was poured onto a glass culture dish and placed in a ventilated state for 72 h to obtain a drug-loaded bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer film.

[0076] S3, the film was placed in a printing cartridge, the needle diameter was 0.25 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 12 mm / s, the fiber direction was "0-90°", the air pressure was 320 Kpa, and the support layer height was 85% of the needle diameter. The drug-loaded bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer scaffold was printed layer by layer. 30 mg of drug-loaded bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer scaffold was soaked in 25 ml of aqueous solution, and ultrasonic treatment was carried out at room temperature for 0.5 h to obtain a drug-loaded bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer scaffold.

[0077] S4, the gradient pore structure scaffold was added to 28 ml of 12 mg / ml 2-methyl imidazole aqueous solution, and 4 ml of 4 mg / ml zinc nitrate aqueous solution was added dropwise under stirring. After reaction, washing, freeze-drying, a ZIF8 surface modified drug-loaded bioactive ceramic@polydopamine / polylactic acid-glycolic acid copolymer scaffold was obtained.

[0078] The modified polycaprolactone composite scaffold prepared in Comparative Example 1 has a relatively smooth surface, and occasionally has some micropores or nanopores.

[0079] Comparative Example 2

[0080] This comparative example provides a method for preparing a gradient pore structure polylactic acid-glycolic acid copolymer scaffold, which is substantially the same as Example 1, except that ZIF8 surface modification is not performed, and the preparation method is as follows:

[0081] S1, 0.01 mg of bone morphogenetic protein-2 and 300 mg of bioglass were dissolved / dispersed in 100 ml of weakly basic 1.5 mg / ml dopamine aqueous solution (pH = 8.4), and stirring was carried out at 400 rpm for 8 h to obtain drug-loaded bioactive ceramic@polydopamine particles.

[0082] S2, 10 g of polylactic acid-glycolic acid copolymer (molecular weight: 30,000 daltons) was dissolved in 50 ml of dichloromethane, 10 g of gluconolactone and 100 mg of drug-loaded bioactive ceramic@polydopamine particles were added, and mechanical stirring was carried out at 500 rpm for 48 h to obtain a gluconolactone / drug-loaded bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer 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 bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer film.

[0083] S3, the film was placed in a printing cartridge, the needle diameter was 0.25 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 12 mm / s, the fiber direction was "0-90°", the air pressure was 320 Kpa, and the scaffold layer height was 85% of the needle diameter. The gluconolactone / drug-loaded bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer scaffold was printed layer by layer. 30 mg of the gluconolactone / drug-loaded bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer scaffold was soaked in 25 ml of an aqueous solution, and ultrasonic treatment was carried out at room temperature for 0.5 h to obtain a gradient pore structure drug-loaded bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer scaffold.

[0084] Comparative Example 3

[0085] This comparative example provides a preparation method of a polylactic acid-glycolic acid copolymer scaffold, which is substantially the same as that of Example 1, except that the bioactive ceramic@polydopamine particles are not drug-loaded, and the preparation method is as follows:

[0086] S1, 300 mg of bioglass was dissolved / dispersed in 100 ml of weakly basic 1.5 mg / ml dopamine aqueous solution (pH = 8.4), and stirring was carried out at 400 rpm for 8 h to obtain bioactive ceramic@polydopamine particles.

[0087] S2, 10 g of polylactic acid-glycolic acid copolymer (molecular weight: 30,000 daltons) was dissolved in 50 ml of dichloromethane, 10 g of gluconolactone and 100 mg of drug-loaded bioactive ceramic@polydopamine particles were added, and mechanical stirring was carried out at 500 rpm for 48 h to obtain a gluconolactone / drug-loaded bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer 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 bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer film.

[0088] S3, the film was put into the printing cartridge, the needle diameter was 0.25 mm, the cartridge temperature was 85 ℃, the needle temperature was 90 ℃, the fiber spacing was 0.8 mm x 0.8 mm, the printing rate was 12 mm / s, the fiber direction was "0-90°", the air pressure was 320 Kpa, and the scaffold layer height was 85% of the needle diameter. Gluconolactone / bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer scaffold was printed layer by layer. 30 mg of the gluconolactone / bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer scaffold was soaked in 25 ml of an aqueous solution, ultrasonicated at room temperature for 0.5 h, and a gradient pore structure bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer scaffold was obtained.

[0089] S4, the gradient pore structure scaffold was added to 28 ml of a 12 mg / ml aqueous solution of 2-methylimidazole, 4 ml of a 4 mg / ml aqueous solution of zinc nitrate was added dropwise under stirring, and after reaction, the gradient pore structure, ZIF8 surface modified bioactive ceramic@polydopamine / polylactic acid-glycolic acid copolymer scaffold was obtained after washing, freeze-drying.

[0090] Comparative Example 4

[0091] The present comparative example provides a preparation method of a polylactic acid-glycolic acid copolymer scaffold, which is substantially the same as that of Example 1, except that the drug-loaded polydopamine particles do not contain bioactive ceramics, and the preparation method is specifically as follows:

[0092] S1, 0.01 mg of bone morphogenetic protein-2 was dissolved in 100 ml of weakly alkaline 1.5 mg / ml dopamine aqueous solution (pH = 8.4), and the reaction was stirred at 400 rpm for 8 h to obtain drug-loaded polydopamine particles.

[0093] S2, 10 g of polylactic acid-glycolic acid copolymer (molecular weight: 30,000 daltons) was dissolved in 50 ml of dichloromethane, 10 g of gluconolactone and 100 mg of drug-loaded polydopamine particles were added, and mechanical stirring was carried out at 500 rpm for 48 h to obtain a gluconolactone / drug-loaded polydopamine particle / polylactic acid-glycolic acid copolymer solution. The solution was poured onto a glass culture dish, and placed under ventilation for 72 h to obtain a gluconolactone / drug-loaded polydopamine particle / polylactic acid-glycolic acid copolymer film.

[0094] S3, the film was put into the printing cartridge, the needle diameter was 0.25 mm, the cartridge temperature was 85 ℃, the needle temperature was 90 ℃, the fiber spacing was 0.8 mm x 0.8 mm, the printing rate was 12 mm / s, the fiber direction was "0-90°", the air pressure was 320 Kpa, and the scaffold layer height was 85% of the needle diameter. Gluconolactone / bioactive ceramic@polydopamine particle / polylactic acid-glycolic acid copolymer scaffold was printed layer by layer.

[0095] S4, the gradient pore structure scaffold was added to 28 ml of 12 mg / ml 2-methylimidazole aqueous solution, 4 ml of 4 mg / ml zinc nitrate aqueous solution was added dropwise under stirring, and after reaction, the scaffold was washed, freeze-dried, and a gradient pore structure, ZIF8 surface modified polydopamine / polylactic acid-glycolic acid copolymer scaffold was obtained.

[0096] Comparative Example 5

[0097] The present comparative example provides a preparation method of a polylactic acid-glycolic acid copolymer scaffold, which is substantially the same as that of Example 1, except that it does not contain drug-loaded biologically active ceramic / polydopamine particles, and the preparation method is as follows:

[0098] S1, 10 g of polylactic acid-glycolic acid copolymer (molecular weight: 30,000 daltons) was dissolved in 50 ml of dichloromethane, and then 10 g of gluconolactone was added, and mechanical stirring was carried out at 500 rpm for 48 h to obtain a gluconolactone / polylactic acid-glycolic acid copolymer solution. The solution was poured onto a glass culture dish and placed in a ventilated state for 72 h to obtain a gluconolactone / polylactic acid-glycolic acid copolymer film.

[0099] S2, the film was placed in a printing cartridge, the needle diameter was 0.25 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 12 mm / s, the fiber direction was "0-90°", the air pressure was 320 Kpa, and the scaffold layer height was 85% of the needle diameter. The gluconolactone / polylactic acid-glycolic acid copolymer scaffold was printed layer by layer. 30 mg of the gluconolactone / polylactic acid-glycolic acid copolymer scaffold was soaked in 25 ml of aqueous solution, and ultrasonic treatment was carried out at room temperature for 0.5 h to obtain a gradient pore structure polylactic acid-glycolic acid copolymer scaffold.

[0100] S3, the gradient pore structure scaffold was added to 28 ml of 12 mg / ml 2-methylimidazole aqueous solution, 4 ml of 4 mg / ml zinc nitrate aqueous solution was added dropwise under stirring, and after reaction, the scaffold was washed, freeze-dried, and a gradient pore structure, ZIF8 surface modified polylactic acid-glycolic acid copolymer scaffold was obtained.

[0101] Comparative Example 6

[0102] The present comparative example provides a preparation method of a polylactic acid-glycolic acid copolymer scaffold, which is substantially the same as that of Example 1, except that it does not contain drug-loaded biologically active ceramic / polydopamine particles, gluconolactone, and ZIF8, and the preparation method is as follows:

[0103] The 10 g of polylactic acid-glycolic acid copolymer (molecular weight: 30,000 Dalton) was put into a printing cartridge, the needle diameter was 0.25 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 12 mm / s, the fiber direction was "0-90°", the air pressure was 320 Kpa, and the scaffold layer height was 85% of the needle diameter. The polylactic acid-glycolic acid copolymer scaffold was printed layer by layer. The 30 mg of polylactic acid-glycolic acid copolymer scaffold was soaked in 25 ml of aqueous solution, and ultrasonic was performed at room temperature for 0.5 hours to obtain the polylactic acid-glycolic acid copolymer scaffold.

[0104] Experimental analysis

[0105] The scaffold materials prepared in the examples and comparative examples were evaluated for the following properties, and the results are shown in Figs. 1-2 and Tables 1-3.

[0106] 1. In vitro cytotoxicity evaluation

[0107] The prepared gel was evaluated and scored according to the requirements of GB / T 16886.5.

[0108] The L929 mouse fibroblasts that had grown vigorously for 24 h were digested and prepared into a density of 1.0 x 10 5 / mL, 100 μL of which was inoculated into a 96-well plate. After the cells grew into a monolayer, the original culture solution was removed, and 100 μL of test sample extract, blank control, positive control, and negative control were added, respectively, with 6 replicates in each group. After the addition was completed, the 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours.

[0109] After 24 h 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, 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 with 570 nm as the main absorption wavelength and 650 nm as the reference wavelength, and the cell survival rate was calculated.

[0110] The cell survival rate was calculated according to the following formula: Cell survival rate (%) = 100 x OD 570e / OD570 b OD 570e is the average optical density of the test sample or control sample extract.

[0111] OD 570b is the average blank optical density.

[0112] The experimental results are shown in Table 1 below:

[0113] Table 1 In vitro cell survival rate of the scaffolds prepared in the examples and comparative examples

[0114]

[0115] The results of the in vitro cytotoxicity evaluation of the examples and the comparative examples (Table 1) show that the composite gels prepared by the method of the present application have no cytotoxicity.

[0116] 2. Hemolysis test

[0117] The blood was collected from the heart of healthy rabbits according to the amount of blood for the test. For example, 10 mL of blood was collected, 0.5 mL of 20 g / L potassium oxalate solution was added, and fresh anticoagulated rabbit blood was prepared. 8 mL of fresh anticoagulated rabbit blood was diluted with 10 mL of 9 g / L sodium chloride injection. The test product group was added with the test product according to the extraction ratio, and then 10 mL of sodium chloride injection was added. The negative control group was added with 10 mL of sodium chloride per tube. The positive control group was added with 10 mL of distilled water per tube. Each group was operated in parallel for 3 tubes. After all the test tubes were placed in a constant temperature water bath at (37±1) °C for 30 min, 0.2 mL of diluted rabbit blood was 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 was poured out and centrifuged at 800 g for 5 min. The supernatant was transferred to a cuvette, and the absorbance was measured at a wavelength of 545 nm by a spectrophotometer. The absorbance of the test product group and the control group was the average 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 was calculated according to the following formula: hemolysis rate = (absorbance of the test product group - absorbance of the negative control group) / (absorbance of the positive control group - absorbance of the negative control group) x 100%, and the results are shown in Table 2:

[0118] Table 2 Hemolysis rate of the materials prepared in the examples and the comparative examples

[0119]

[0120] The results of the hemolysis rate evaluation of the examples and the comparative examples (Table 2) show that the hydrogels prepared by the method of the present application have a hemolysis rate of less than 5%, and have no hemolysis risk.

[0121] 3. In vitro drug release performance test

[0122] 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) at 37 °C and 60 rpm in a constant temperature shaker, 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.

[0123] The results of the in vitro drug release performance test are shown in Table 3:Fig. 1 The stents prepared in Examples 1-4 all have a drug release period of more than 5 weeks. Comparative Example 1 does not use gluconolactone, and the stent fibers thereof do not contain a porous structure, and the drug release rate thereof is lower than that of Example 1; Comparative Example 2 does not undergo ZIF8 surface modification, and the burst release is significantly increased and the release rate is significantly increased, and the drug is basically released at 4 weeks. The drug-loaded polydopamine particles in Comparative Example 4 do not contain bioactive ceramics, and the drug release is close to that of Example 1.

[0124] 4. Detection of osteogenic differentiation performance of preosteoblasts in vitro

[0125] The composite materials prepared in Examples 1-5 and Comparative Examples 1-3 were sterilized by irradiation and then soaked in DMEM basic culture medium at a concentration of 10 mg / mL, and then placed in a 37°C shaker at 120 rpm for 24 h. After the extraction was completed, the composite material and the culture medium mixture were centrifuged at 1000 rpm, and the supernatant was collected. The collected extraction solution was diluted 2 times with the corresponding DMEM culture medium, and finally 10% fetal bovine serum was added to obtain the conditioned medium.

[0126] MC3T3-E1 cells were seeded in a 24-well plate at a density of 1×10 5 The cells were washed with PBS solution, and then 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 p-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 measured by an enzyme-labeled instrument.

[0127] Definition of alkaline phosphatase activity unit: The amount of alkaline phosphatase required to hydrolyze 1 micromole of p-nitrophenol from para-nitrophenyl phosphate chromogenic substrate per minute in diethanolamine (DEA) buffer at pH 9.8 and 37°C is defined as one enzyme activity unit, also referred to as one DEA enzyme activity unit. The amount of alkaline phosphatase required to hydrolyze 1 micromole of p-nitrophenol from para-nitrophenyl phosphate chromogenic substrate per minute in glycine buffer at pH 9.6 and 25°C is defined as one enzyme activity unit, also referred to as one Glycine enzyme activity unit. One Glycine enzyme activity unit is approximately equivalent to 3 DEA enzyme activity units. The alkaline phosphatase activity in the samples was calculated according to the definition of enzyme activity and plotted respectively.

[0128] By Fig. 2 It can be seen that the scaffolds in Examples 1-4 of the present application all 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 its cell activity is not as good as that 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. The drug-loaded polydopamine particles in Comparative Example 5 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 Example 2 does not contain a ZIF8 layer with good bioactivity, and the alkaline phosphatase activity is significantly lower than that of Example 1. The bioactive ceramic / polydopamine particles in Comparative Example 4 do not load drugs, and the alkaline phosphatase activity is also significantly lower than that of Example 1. The drug-free bioactive ceramic / polydopamine particles in Comparative Example 4 are also significantly lower than that of Example 1, and the alkaline phosphatase activity is lower than that of Comparative Example 4. Comparative Example 6 does not contain a ZIF8 layer with good bioactivity, does not have a gradient pore structure, and does not have drug-loaded bioactive ceramic / polydopamine particles, and the alkaline phosphatase activity is the lowest.

[0129] 5. Detection of antibacterial performance of the scaffold

[0130] Fresh cultures of Staphylococcus aureus and Escherichia coli were taken, and the viable bacterial count was performed on the bacterial solution, and the diluent (0.03 mol / L PBS (pH = 7.2-7.4) containing 1% proteose peptone) was used to prepare bacterial solutions with a bacterial content of 5×10 5 ~ 10×10 6The samples were placed in sterile flat dishes, and 50 μL of the bacterial suspension was added to each sample. The time of bacterial addition was recorded for each tube, and blood plates were inoculated 60 min after the bacterial addition. The samples were also placed in 5 mL nutrient broth tubes. The blood plates and broth tubes inoculated with bacteria were incubated at 37°C for 48 h, and the preliminary results were observed. The samples were incubated in sterile growth tubes until the 28th day. If the broth tubes were turbid and the blood plates had bacterial growth, the result was positive, indicated by (+). If the broth tubes were clear on the 35th day, the result was considered to be sterile growth, indicated by (-).

[0131] Table 3. Antimicrobial effect of the stents prepared in the examples and comparative examples

[0132]

[0133] As shown in Table 3, the composite stents prepared in Examples 1-4 and Comparative Examples 1, 3, 4, and 5 have good long-term antimicrobial effect and are sterile on the 35th day. Comparative Examples 2 and 6 do not contain ZIF8 and do not have antimicrobial effect.

[0134] The above description is a preferred embodiment of the present application. It should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application. These improvements and refinements are also considered to be within the scope of the present application.

Claims

1. A modified polylactic-co-glycolic acid composite scaffold, characterized in that, The poly-lactic-glycolic acid copolymer scaffold substrate has a surface loaded with a metal organic framework material, and the metal organic framework material has an antibacterial drug loaded inside; the surface and the interior of the poly-lactic-glycolic acid copolymer scaffold substrate are distributed with interconnected pores, the pores gradually increase from the surface to the interior of the poly-lactic-glycolic acid copolymer scaffold substrate; the poly-lactic-glycolic acid copolymer scaffold substrate is distributed with a drug-loaded material, the drug-loaded material includes a ceramic material and a tissue regeneration and repair promoting drug, and the ceramic material and the tissue regeneration and repair promoting drug are coated with polydopamine on the surface; The ceramic material is at least one selected from hydroxyapatite, bioglass, calcium silicate, calcium carbonate, calcium sulfate, tricalcium phosphate, octacalcium phosphate, and amorphous calcium phosphate; The preparation method of the modified poly-lactic-glycolic acid copolymer composite scaffold includes the following steps: S1, the drug-loaded material, gluconolactone and poly-lactic-glycolic acid copolymer are mixed in a solvent to obtain a mixed solution, which is transferred to a mold and placed for a period of time to obtain a mixed material film; the mixed material film is used as a printing material, and a scaffold with a gradient pore structure is obtained by 3D printing; After printing, the scaffold with a gradient pore structure is soaked in water to remove gluconolactone and residual solvent; S2, the scaffold with a gradient pore structure, 2-methylimidazole and a zinc source are reacted in water to obtain the modified poly-lactic-glycolic acid copolymer composite scaffold; The preparation method of the drug-loaded material includes the following steps: dispersing the tissue regeneration and repair promoting drug and the ceramic material in a dopamine-containing aqueous solution, and mixing and reacting to obtain the drug-loaded material; The mass ratio of the drug-loaded material to poly-lactic-glycolic acid copolymer is (0.005-0.05):1; the mass ratio of gluconolactone to poly-lactic-glycolic acid copolymer is (0.5-3):

1.

2. The modified polylactic-co-glycolic acid composite scaffold of claim 1, wherein, The surface of the poly-lactic-glycolic acid copolymer composite material has pores with a size of 1-100 μm.

3. The method of producing a modified polylactic-co-glycolic acid composite scaffold according to any one of claims 1-2, characterized in that, The preparation method includes the following steps: S1, the drug-loaded material, gluconolactone and poly-lactic-glycolic acid copolymer are mixed in a solvent to obtain a mixed solution, which is transferred to a mold and placed for a period of time to obtain a mixed material film; the mixed material film is used as a printing material, and a scaffold with a gradient pore structure is obtained by 3D printing; After printing, the scaffold with a gradient pore structure is soaked in water to remove gluconolactone and residual solvent; S2, the scaffold with a gradient pore structure, 2-methylimidazole and a zinc source are reacted in water to obtain the modified poly-lactic-glycolic acid copolymer composite scaffold.

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

5. The method for preparing the modified polylactic acid-glycolic acid copolymer composite scaffold according to claim 3, characterized in that, The mixing time in step S1 is 30-100 h; And / or, the placing time is 40-100 h.

6. The method for preparing the modified polylactic acid-glycolic acid copolymer composite scaffold according to claim 3, characterized in that, The mass ratio of the 2-methyl imidazole and the zinc source is (18-60):

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

7. Use of the modified polylactic-glycolic acid copolymer composite scaffold according to any one of claims 1-2 in the preparation of a product for promoting bone tissue repair and regeneration.

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