A porous scaffold for repairing bone defects loaded with black phosphorus capable of eliminating reactive oxygen species and releasing drugs and a preparation method thereof

Abstract

The present application relates to a kind of porous scaffold for repairing bone defect loaded with black phosphorus active oxygen elimination and slow-release drug and its preparation method;The scaffold includes the amino cation polymer grafted with the phenylboronic acid (PBA) and cyclodextrin (CD) that can eliminate active oxygen, black phosphorus nanosheet (BP) and scaffold material poly (lactic acid-glycolic acid) (PLGA).The porous scaffold loads a large number of black phosphorus nanosheet that can be hydrolyzed into bone repair nutrients, and the porous structure provides sufficient space for cell migration and growth, realizes the good repair of bone.Active oxygen elimination ability, can eliminate active oxygen in diabetic microenvironment.In addition, cyclodextrin can load a variety of drugs, such as osteogenic differentiation induction drug, and realize the slow release of drug.The porous scaffold has good biocompatibility, and the degradation product is harmless to human body, can be used for the tissue repair of bone defect, can be widely applied in the field of biological medicine and tissue engineering, and has been proved to have good repair ability in the diabetic rat skull defect model.

Description

Technical Field

[0001] This invention relates to a porous scaffold for bone defect repair loaded with black phosphorus that can eliminate reactive oxygen species and release drugs in a sustained manner, and its preparation method. The scaffold can be used in bone defect repair, especially in the repair of diabetic bone defects, and belongs to the field of biomedical materials technology. Background Technology

[0002] Diabetes mellitus is a chronic metabolic disease characterized by disordered insulin and glucose metabolism, posing a serious threat to human health and quality of life worldwide. As a systemic disease, studies have shown that a high-glucose environment inhibits osteoblast differentiation and growth of osteoblasts and bone marrow mesenchymal stem cells. Glycolysis is the basic metabolic pathway of glucose catabolism, but in a high-glucose microenvironment, excess glucose may activate other metabolic pathways, such as the polyol pathway, the protein kinase C signaling pathway, and the formation of advanced glycation end products (AGEs), leading to excessive ROS production, inducing oxidative stress, and the secretion of inflammatory cytokines. Therefore, diabetes increases the risk of fractures, the incidence of nonunion and delayed healing, and hinders bone remodeling.

[0003] Natural bone is a highly vascularized tissue, relying on the vascular system to supply blood and nutrients to maintain its integrity. Due to the complexity of the bone microenvironment, complete bone regeneration remains a significant challenge. Clinically, methods such as autologous bone grafting, allogeneic bone transplantation, and the Ilizarov technique are commonly used, but these face challenges such as infection and limited bone sources. In bone tissue engineering, inorganic materials such as phosphates and silicates are widely used. However, in areas with large bone defects, especially when other diseases (such as diabetes and osteoporosis) are present, the repair effect of these materials alone is insufficient. Phosphorus is an important element in human bone composition, and black phosphorus nanosheets (BP) exhibit excellent biodegradability and biocompatibility in vivo due to their high reactivity with oxygen and water. Their biodegradation products are non-toxic phosphate ions or phosphates, which can capture calcium in the body. 2+ It forms calcium phosphate deposits and serves as a resource for bone tissue mineralization. Therefore, black phosphorus is an attractive biomaterial for future repair of bone tissue in the body.

[0004] This invention prepares two-dimensional black phosphorus nanosheets with surface functionalized phenylboronic acid and drug-loaded cyclodextrin to eliminate reactive oxygen species (ROS), and adsorbs them onto a porous PLGA scaffold. This porous scaffold exhibits excellent biocompatibility and degradability. Its porous structure provides sufficient space for cell migration and growth, continuously releasing phosphorus ions to promote osteoblast activity and osteogenic differentiation of mesenchymal stem cells, thus promoting bone regeneration. Furthermore, this scaffold can eliminate excess ROS in the diabetic microenvironment, facilitating bone repair. The scaffold can load various drugs, such as osteogenic differentiation-inducing drugs, achieving sustained drug release. Therefore, this reactive oxygen species-eliminating scaffold loaded with the novel material black phosphorus nanosheets provides a new perspective for bone tissue engineering. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a porous scaffold loaded with black phosphorus nanosheets that can eliminate reactive oxygen species and release drugs in a sustained manner.

[0006] Another objective of this invention is to provide the application of the aforementioned porous scaffold loaded with black phosphorus nanosheets that can eliminate reactive oxygen species and release drugs sustainably in bone defect repair, especially in the repair of diabetic bone defects. This scaffold exhibits good biocompatibility and degradability, provides space for cell migration and growth, can eliminate excessive reactive oxygen species in diabetes, sustain drug release, and continuously provide nutrients for bone repair, thereby achieving the repair of diabetic bone defects.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] A porous scaffold for bone defect repair loaded with black phosphorus that can eliminate reactive oxygen species and release drugs sustainably is composed of a porous PLGA scaffold, functionalized black phosphorus nanosheets, and a functionalized amino-cationic polymer composited with the nanosheets. The functionalized black phosphorus nanosheets and the functionalized amino-cationic polymer fill the pores and surface of the porous PLGA scaffold, and the functionalized amino-cationic polymer carries the drug.

[0009] The molecular weight of the PLGA is 100-300 kDa.

[0010] The amino-cationic polymer has a molecular weight of 3.5–10 kDa.

[0011] The porous PLGA scaffold is prepared by either a pore-forming agent leaching method or a phase separation method.

[0012] The black phosphorus nanosheets were prepared by ultrasonic exfoliation.

[0013] The mass ratio of the porous PLGA scaffold, the functionalized black phosphorus nanosheets, and the functionalized amino cationic polymer is typically (1-2):(0.05-0.2):(0.5-2).

[0014] The above-described method for preparing a porous scaffold for bone defect repair loaded with black phosphorus that can eliminate reactive oxygen species and release drugs sustainably includes the following steps:

[0015] (1) Fabrication of porous PLGA scaffolds, and cutting them into suitable shapes and sizes using a scalpel and dental drill:

[0016] (2) Preparation of functionalized black phosphorus nanosheets;

[0017] (3) Preparation of functionalized amino cationic polymers, namely amino cationic polymers grafted with cyclodextrin and phenylboronic acid.

[0018] (4) The composite of functionalized amino cationic polymer and functionalized black phosphorus nanosheets and drug loading, the resulting surface-functionalized black phosphorus nanosheets are composited with porous PLGA scaffold, and finally freeze-dried to obtain the scaffold.

[0019] Furthermore, the method for preparing the porous PLGA support in step (1) is as follows:

[0020] S1: Gelatin particles are screened using molecular sieves, filled into a cylindrical mold, and uniformly permeated with an 85% ethanol solution (usually by mass percentage). The mold filled with gelatin particles is dried in a 37°C oven, and the mold is removed to obtain a gelatin template.

[0021] S2: Dissolve the PLGA in an organic solvent with a low melting point to obtain a PLGA solution;

[0022] S3: Immerse the gelatin template in a PLGA solution and introduce the solution into the scaffold through repeated negative pressure-decompression. Freeze the template containing the PLGA organic solution and then freeze-dry it at -20°C. Immerse the dried PLGA / gelatin scaffold in warm water to dissolve and remove the gelatin, then freeze-dry to obtain a porous PLGA scaffold.

[0023] S4: Use a scalpel and dental drill to cut to the appropriate shape and size.

[0024] The gelatin particles in step S1 are 50-500 μm in size; the cylindrical mold has a diameter of 5-15 mm; and the drying time in an oven at 37°C is 12-36 hours.

[0025] The organic solution mentioned in step S2 is dioxane; the PLGA concentration is 8-20% (w / v);

[0026] Step S3 involves 3 to 6 cycles of negative pressure-decompression; freezing temperature of -20 to -80°C; freezing time of 8 to 24 hours; freeze-drying time of 12 to 24 hours; and immersion in 37°C water for 3 to 10 days.

[0027] The porous PLGA support described in step S3 has a pore size of 50-500 μm and good pore connectivity.

[0028] Step S4: Cut the PLGA porous bracket into cylinders with a height of 0.5-8mm and a diameter of 3-15mm.

[0029] Furthermore, the preparation method of the functionalized black phosphorus nanosheets in step (2) includes:

[0030] S1: Dissolve tannic acid in an organic solution;

[0031] S2: Place the black phosphorus crystals in an organic solution of tannic acid and use an ultrasonic breaker to perform ultrasonic peeling under ice bath conditions;

[0032] S3: Large pieces of black phosphorus are removed by low-speed centrifugation, and black phosphorus nanosheets are obtained by high-speed centrifugation. The water washing and centrifugation steps are repeated, and finally the black phosphorus nanosheets are dispersed in deionized water.

[0033] The organic solution mentioned in step S1 is N-methylpyrrolidone, and the tannic acid concentration is 0.5-2 mg / mL;

[0034] In step S2, the mass ratio of black phosphorus crystals to solution is 1–2 mg / mL, the ultrasonic power is 20–35%, and the ultrasonic time is 8–16 hours.

[0035] In step S3, the low-speed centrifugation speed is 2500-3500 rpm and the low-speed centrifugation time is 10-20 minutes; the high-speed centrifugation speed is 9000-12000 rpm and the high-speed centrifugation time is 15-25 minutes; the water washing is repeated 1-2 times; after washing, the centrifugation speed is 9000-12000 rpm and the centrifugation time is 15-25 minutes; the precipitate is uniformly dispersed in deionized water at 1-3 mg / mL.

[0036] The black phosphorus nanosheets described in step S3 are 300–700 nm in size and 2–30 nm in thickness.

[0037] Further, the preparation method of the amino cationic polymer grafted with cyclodextrin and phenylboronic acid in step (3) is as follows:

[0038] S1: Dissolve the amino-cationic polymer in an aqueous solution, and dissolve the cyclodextrin (CD) in the amino-cationic polymer aqueous solution;

[0039] S2: Add acidic solution to adjust the pH of the solution;

[0040] S3: Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and carry out the EDC / NHS reaction while maintaining the pH of the solution constant;

[0041] S4: Dialyze the obtained solution using a dialysis bag and freeze-dry to obtain the product;

[0042] S5: Replace the amino cationic polymer with the product of S4, replace the cyclodextrin with 4-carboxy-2-fluoro-phenylboronic acid (PBA), and graft 4-carboxy-2-fluoro-phenylboronic acid (PBA) onto the amino cationic polymer using the same method as S1-S4.

[0043] The amino-cationic polymer mentioned in step S1 is at least one of hyperbranched polylysine, polylysine, polyetherimide, and chitin.

[0044] The aqueous solution mentioned in step S1 is a PBS solution, the concentration of the amino cationic polymer is 1-6 mg / mL, and the molar ratio of the amino cationic polymer to cyclodextrin is 1:5-1:12.

[0045] The acidic solution mentioned in step S2 is concentrated hydrochloric acid, and the pH is adjusted to pH=6;

[0046] In step S3, the molar ratio of EDC to cyclodextrin is 20-25, the molar ratio of NHS to cyclodextrin is 10-12, the reaction temperature is 37°C, and the reaction time is 12-16 hours.

[0047] The dialysis bag size in step S4 is 3500 molecular weight, and the dialysis time is 5 to 10 days.

[0048] Furthermore, the preparation method of the PLGA porous scaffold in step (4) which combines functionalized amino cationic polymer, functionalized black phosphorus, and drug is as follows:

[0049] S1: Dissolve the functionalized amino cationic polymer obtained in step (3) in an aqueous solution and resuspend the black phosphorus nanosheets obtained in step (2);

[0050] S2: Disperse the mixed solution from step S1 using ultrasound, stir, and then let it stand at 4°C. Centrifuge the solution after standing, and redisperse the resulting precipitate in deionized water.

[0051] S3: Dissolve the drug in a volatile organic solution, add it dropwise slowly to the solution obtained in step S2 under ultrasonication, stir at room temperature with the container open, seal the container, and shake it in a shaker.

[0052] S4: Immerse the dried PLGA scaffold obtained in step (1) in the solution of step S3, shake slowly, remove and freeze dry to obtain the composite scaffold.

[0053] The concentration of the functionalized amino cationic polymer in step S1 is 2-10 mg / mL, and the concentration of black phosphorus nanosheets is 1-3 mg / mL;

[0054] In step S2, the ultrasonic dispersion time is 10-30 minutes, the stirring time is 2-6 hours, the standing time at 4°C is 4-12 hours, the centrifugation speed is 8000-12000 rpm, the centrifugation time is 10-15 minutes, and the concentration of black phosphorus nanosheets redispersed in water is 1-3 mg / mL.

[0055] The organic solution mentioned in step S3 can be an ethanol solution, the drug is at least one of simvastatin, lovastatin, alendronate sodium, and risedronate sodium, the drug concentration is 2.5-5 mg / mL, the open stirring time is 4-8 hours, the shaker temperature is 37°C, the shaker speed is 50-120 rpm, and the shaker incubation time is 12-16 hours.

[0056] The incubation time in step S4 is 0.5 to 4 hours.

[0057] The aforementioned porous scaffold for bone defect repair that supports black phosphorus nanosheets and can eliminate reactive oxygen species and release drugs has its reactive oxygen species elimination ability derived from phenylboronic acid grafted onto an amino cationic polymer and tannic acid on the surface of black phosphorus.

[0058] The aforementioned porous scaffold for bone defect repair, which is loaded with black phosphorus nanosheets and can eliminate reactive oxygen species to release drugs, has a drug release capacity derived from cyclodextrin grafted onto an amino cationic polymer.

[0059] The drug loading rate of the porous scaffold for bone defect repair with black phosphorus nanosheets loaded with active oxygen-eliminating sustained-release drugs is 0.5-8%.

[0060] The aforementioned porous scaffold for bone defect repair, loaded with black phosphorus nanosheets and capable of eliminating reactive oxygen species and releasing drugs, is suitable for various bone defect diseases.

[0061] The application principle of the porous scaffold for bone defect repair, loaded with black phosphorus nanosheets and capable of eliminating reactive oxygen species and releasing drugs, is as follows: the interconnected porous structure of the scaffold provides ample space for cell migration and growth; the black phosphorus nanosheets continuously hydrolyze into phosphates, providing nutrients for osteogenic formation; and the tannic acid and grafted phenylboronic acid on the surface of the black phosphorus can eliminate excessive reactive oxygen species in the diabetic microenvironment. It can load and release various drugs, such as osteogenic differentiation drugs, promoting osteoblast activity and osteogenic differentiation of mesenchymal stem cells, thus promoting bone regeneration and bone integration.

[0062] Compared with existing materials for repairing bone defects in diabetic patients, the porous scaffold for bone defect repair prepared in this invention, which is loaded with black phosphorus nanosheets and can eliminate reactive oxygen species to release drugs, has the following advantages:

[0063] 1) In this invention, black phosphorus two-dimensional nanosheet material is selected, which has good biocompatibility and degradability, and the biodegradation products are non-toxic phosphate ions or phosphates, which react with Ca... 2+It forms calcium phosphate deposits, providing a continuous and sufficient supply of nutrients for bone tissue mineralization, and significantly accelerating the bone repair process.

[0064] 2) In this invention, in view of the fact that excessive secretion of reactive oxygen species in the diabetic microenvironment can hinder the repair process, tannic acid and amino cationic polymers grafted with phenylboronic acid are selected. These can quickly consume reactive oxygen species in the surrounding environment, regulate the inflammatory microenvironment, regulate the production of cytokines and the polarization of macrophages, and have a positive effect on bone repair.

[0065] 3) In this invention, by grafting cyclodextrin onto HBPL and then adsorbing it onto the surface of black phosphorus with positive and negative charges, a variety of drugs can be efficiently loaded, such as osteogenic differentiation drugs and drugs that inhibit bone resorption, and the drugs can be released slowly at the disease site, continuously promoting osteogenic effects during the long process required for bone repair; at the same time, black phosphorus can provide nutrients to synergistically promote bone repair. Attached Figure Description

[0066] Figure 1 Illustration of the preparation of a porous scaffold for bone defect repair with drugs that can be sustained-released by eliminating reactive oxygen species and loaded with black phosphorus nanosheets;

[0067] Figure 2 The structural formula and NMR image of hyperbranched polylysine (HBPL) grafted with cyclodextrin and phenylboronic acid;

[0068] Figure 3 The phosphate hydrolysis release curve of the porous scaffold for bone defect repair prepared in Example 1 with black phosphorus nanosheets in aqueous solution.

[0069] Figure 4 The ALP content of the porous scaffold for bone defect repair prepared with black phosphorus nanosheets in Example 1 was determined after 14 days of in vitro induction of MC3T3-E1 osteoblasts.

[0070] Figure 5 H&E and Masson trichrome staining of tissue sections of the porous scaffold for bone defect repair prepared with black phosphorus nanosheets in Example 1 at 8 and 12 weeks of skull defect repair in diabetic rats. Detailed Implementation

[0071] The technical solutions of the present invention are further illustrated below with reference to embodiments, but these embodiments are not intended to limit the present invention.

[0072] Example 1:

[0073] A PLGA porous scaffold with surface-adsorbed functionalized black phosphorus nanosheets was prepared. Tannins and phenylboronic acid on the black phosphorus surface possess anti-inflammatory and antioxidant capabilities. Cyclodextrin on the surface was used to load osteogenic differentiation drugs, achieving sustained drug release (e.g., ...). Figure 1 As shown in the figure, its specific preparation process is as follows:

[0074] (1) Fabrication of PLGA porous scaffold

[0075] Gelatin particles of 280–450 μm size were screened using molecular sieves and filled into a 7 mm diameter mold, which was then uniformly permeated with an 85% ethanol solution. The mold filled with gelatin particles was placed in a 37°C oven for 24 hours and then removed. PLGA with a molecular weight of 122 kDa was dissolved in a dioxane solution at a concentration of 12% (w / v). The gelatin template removed from the mold was immersed in the PLGA solution, and the solution was introduced into the scaffold through repeated negative pressure-depressurization four times. The scaffold was placed at -20°C for 16 hours and then freeze-dried at -20°C for 12 hours. The dried PLGA / gelatin scaffold was then immersed in water at 37°C for 5 days and freeze-dried for 16 hours to obtain a porous PLGA scaffold.

[0076] (2) Preparation of black phosphorus nanosheets

[0077] Tannic acid was dissolved in N-methylpyrrolidone at a concentration of 1 mg / mL. 30 mg of black phosphorus crystals were added to 20 mL of the tannic acid / N-methylpyrrolidone solution, and the mixture was sonicated at 25% power for 12 hours in an ice bath. The mixture was centrifuged at 3000 rpm for 10 minutes, and the supernatant was centrifuged at 10000 rpm for 15 minutes. The precipitate was washed with deionized water and centrifuged at 10000 rpm for 15 minutes, and this process was repeated once.

[0078] (3) Synthesis of hyperbranched polylysine grafted with cyclodextrin and phenylboronic acid

[0079] HBPL was dissolved in PBS solution at 2 mg / mL. Cyclodextrin was added and dissolved at a molar ratio of HBPL:cyclodextrin = 1:10. Concentrated hydrochloric acid was added to adjust the pH of the solution to 6. 20 equivalents of EDC and 10 equivalents of NHS were added sequentially. After reacting at 37°C for 12 hours while maintaining pH = 6, the resulting solution was dialyzed for 8 days using a 3500 dialysis bag and then freeze-dried to obtain HBPL-CD. 4-Carboxy-2-fluorophenylboronic acid (PBA) was grafted onto HBPL-CD using the same method to obtain HBPL-CD-PBA. The grafting rate of cyclodextrin was calculated to be approximately 45% and that of phenylboronic acid to be approximately 20% (e.g., ...). Figure 2 ).

[0080] (4) Dissolve the HBPL-CD-PBA obtained in step (3) in water at a concentration of 5 mg / mL and resuspend the black phosphorus nanosheets to a concentration of 2 mg / mL. Disperse the mixture ultrasonically for 15 min, stir for 2 hours, and allow it to stand at 4°C for 4 hours. Centrifuge at 8000 rpm for 10 minutes to redisperse the resulting precipitate in deionized water to achieve a black phosphorus concentration of 2 mg / mL. Add 2.5 mg / mL simvastatin / ethanol solution dropwise to the HBPL@black phosphorus solution under ultrasonication. Stir for 6 hours with an open container, then shake at 37°C and 100 rpm for 12 hours. Cut the obtained PLGA-dried scaffold into cylinders with a height of 1 mm and a diameter of 5 mm using a scalpel and dental drill. Immerse the cylinders in the above solution, shake slowly for 2 hours, and then freeze-dry to obtain a porous scaffold adsorbed with surface-functionalized black phosphorus nanosheets (referred to as a black phosphorus scaffold). The size of the black phosphorus nanosheets, measured by transmission electron microscopy, was approximately 600 nm. Dynamic light scattering (DLS) analysis revealed a change in particle potential from -18 eV to 30 eV before and after adsorption of positively charged HBPL. Scanning electron microscopy showed that black phosphorus uniformly covered the surface and pores of the porous scaffold, with a drug loading of approximately 60 μg. The sustained-release curve of phosphate from the black phosphorus scaffold in aqueous solution is shown below. Figure 3 .

[0081] To verify the osteogenic induction effect of black phosphorus scaffolds and blank PLGA scaffolds on MC3T3-E1 cells, intracellular alkaline phosphatase (ALP) levels were measured at 7 and 14 days. Figure 4 Black phosphorus scaffolds significantly improved ALP activity compared to PLGA scaffolds, demonstrating their osteogenic differentiation induction ability. A 5mm diameter, 1mm thick scaffold was implanted into skull defects in diabetic rats. Stools were harvested at 8 and 12 weeks and stained with H&E and Masson trichrome (e.g., ...). Figure 5 Compared to blank PLGA scaffolds, black phosphorus scaffolds exhibit more bone ingrowth and collagen deposition on both the surface and interior, and demonstrate good integration with surrounding tissues.

[0082] Example 2:

[0083] (1) Fabrication of PLGA porous scaffold

[0084] Gelatin particles of 50–300 μm size were screened using molecular sieves and filled into a 7 mm diameter mold, which was then uniformly permeated with an 85% ethanol solution. The mold filled with gelatin particles was placed in a 37°C oven for 24 hours and then removed. PLGA with a molecular weight of 122 kDa was dissolved in a 10% (w / v) dioxane solution. The gelatin template removed from the mold was immersed in the PLGA solution, and the solution was introduced into the scaffold through repeated negative pressure-decompression cycles (5 times). The scaffold was placed at -20°C for 16 hours and then freeze-dried at -20°C for 12 hours. The dried PLGA / gelatin scaffold was then immersed in water at 37°C for 7 days and freeze-dried for 16 hours to obtain a porous PLGA scaffold.

[0085] (2) Preparation of black phosphorus nanosheets

[0086] Tannic acid was dissolved in N-methylpyrrolidone at a concentration of 1.5 mg / mL. 30 mg of black phosphorus crystals were added to 20 mL of the tannic acid / N-methylpyrrolidone solution, and the mixture was sonicated at 30% power for 16 hours in an ice bath. The mixture was centrifuged at 3000 rpm for 10 minutes, and the supernatant was centrifuged at 12000 rpm for 15 minutes. The precipitate was washed with deionized water and centrifuged at 12000 rpm for 15 minutes, and this process was repeated once.

[0087] (3) Synthesis of hyperbranched polylysine grafted with cyclodextrin and phenylboronic acid

[0088] HBPL was dissolved in PBS at a concentration of 2 mg / mL. Cyclodextrin was added and dissolved at a molar ratio of 1:12. Concentrated hydrochloric acid was added to adjust the pH of the solution to 6. 20 equivalents of EDC and 10 equivalents of NHS were added sequentially. The reaction was carried out at 37°C for 14 hours while maintaining pH 6. The resulting solution was then dialyzed for 8 days using a 3500 dialysis bag and freeze-dried to obtain HBPL-CD. 4-Carboxy-2-fluorophenylboronic acid (PBA) was grafted onto HBPL-CD using the same method to obtain HBPL-CD-PBA.

[0089] (4) The HBPL-CD-PBA obtained in step (3) was dissolved in water at a concentration of 3 mg / mL, and the black phosphorus nanosheets were resuspended, with a black phosphorus nanosheet concentration of 1 mg / mL. The mixture was ultrasonically dispersed for 10 min, stirred for 2 hours, and allowed to stand at 4°C for 8 hours. After centrifugation at 12000 rpm for 15 minutes, the resulting precipitate was redispersed in deionized water to achieve a black phosphorus concentration of 1 mg / mL. A 1 mg / mL alendronate sodium / ethanol solution was added dropwise to the HBPL@black phosphorus solution under ultrasonication. After stirring with an open container for 4 hours, the mixture was shaken at 37°C and 100 rpm for 12 hours. The obtained PLGA dried scaffold was cut into cylinders with a height of 2 mm and a diameter of 4 mm using a scalpel and dental drill, and immersed in the above solution. After shaking slowly for 3 hours, the cylinders were freeze-dried to obtain a porous scaffold adsorbed with surface-functionalized black phosphorus nanosheets. The size of the black phosphorus nanosheets was measured to be approximately 450 nm by transmission electron microscopy.

Claims

1. A porous scaffold for bone defect repair loaded with black phosphorus that can eliminate reactive oxygen species and release drugs sustainably, characterized in that, The scaffold is composed of a porous PLGA scaffold, functionalized black phosphorus nanosheets, and a functionalized amino-cationic polymer composited with the nanosheets. The functionalized black phosphorus nanosheets and the functionalized amino-cationic polymer fill the pores and surface of the porous PLGA scaffold, and the functionalized amino-cationic polymer carries a drug. The functionalized black phosphorus nanosheets are prepared by ultrasonic exfoliation, and the preparation method includes: (1) Dissolve tannic acid in N-methylpyrrolidone at a concentration of 0.5–2 mg / mL; (2) Place the black phosphorus crystals in a tannic acid / N-methylpyrrolidone solution and use an ultrasonic disruptor under an ice bath at 20-35% power for 8-16 hours. (3) Centrifuge the obtained mixture at 2500-3500 rpm for 10-20 minutes, and centrifuge the obtained supernatant at 9000-12000 rpm for 15-25 minutes; wash the precipitate in deionized water and centrifuge at 9000-12000 rpm for 15-25 minutes, repeating 1-2 times; (4) The precipitate was uniformly dispersed in deionized water at a concentration of 1–3 mg / mL; The preparation method of the functionalized amino cationic polymer is as follows: (1) Dissolve the amino cationic polymer in PBS solution at a concentration of 1–4 mg / mL, and add cyclodextrin at a molar ratio of amino cationic polymer to cyclodextrin of 1:5–1:12 and dissolve. (2) Add concentrated hydrochloric acid to adjust the pH of the solution to 6; (3) Add 20-25 equivalents of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 10-12 equivalents of N-hydroxysuccinimide (NHS) sequentially, maintain pH=6 at 37°C and react for 12-16 hours; (4) After dialyzing the obtained solution with a 3500 Da dialysis bag for 5 to 10 days, freeze-dry to obtain the product; (5) Replace the amino cationic polymer with the product obtained in step (4), replace the cyclodextrin with 4-carboxy-2-fluoro-phenylboronic acid (PBA), and repeat steps (1) to (4) to obtain the functionalized amino cationic polymer.

2. The porous scaffold for bone defect repair loaded with black phosphorus to eliminate reactive oxygen species and release drugs under sustained release, as described in claim 1, is characterized in that... The porous PLGA support has pores with a diameter of 50 to 500 μm running through it.

3. The porous scaffold for bone defect repair loaded with black phosphorus to eliminate reactive oxygen species and sustain-release drugs according to claim 1, characterized in that, The porous PLGA scaffold is prepared by pore-forming agent leaching or phase separation.

4. The porous scaffold for bone defect repair loaded with black phosphorus to eliminate reactive oxygen species and release drugs under sustained release, as described in claim 1, is characterized in that... The method for preparing the porous PLGA support is as follows: (1) Use molecular sieves to screen gelatin particles with a size of 50-500 μm; (2) Fill the gelatin particles into a mold with a diameter of 5-15 mm and allow them to be evenly permeated with 85% ethanol solution; (3) Place the mold filled with gelatin particles in a 37°C oven for 12 to 36 hours; (4) PLGA with a molecular weight of 100-300 kDa is dissolved in dioxane solution at a concentration of 8-20% (W / V); (5) Immerse the gelatin template that has been removed from the mold in PLGA solution, and introduce the solution into the interior of the support by repeatedly applying negative pressure and releasing pressure 3 to 6 times. Place the support at -20 to -80°C for 8 to 24 hours. (6) Freeze-dry at -20°C for 12 to 24 hours; (7) The dried PLGA / gelatin stent obtained in step (6) is soaked in water at 37°C for 3 to 10 days and then freeze-dried for 12 to 24 hours to obtain a porous PLGA stent.

5. The porous scaffold for bone defect repair loaded with black phosphorus to eliminate reactive oxygen species and release drugs under sustained release, as described in claim 1, is characterized in that... The black phosphorus nanosheets are 300–700 nm in size and 2–30 nm in thickness.

6. The porous scaffold for bone defect repair loaded with black phosphorus to eliminate reactive oxygen species and release drugs under sustained release, as described in claim 1, is characterized in that... The amino-cationic polymer is at least one of hyperbranched polylysine, polylysine, polyetherimide, and chitin.

7. The method for preparing a porous scaffold for bone defect repair loaded with black phosphorus that can eliminate reactive oxygen species and release drugs under sustained conditions, as described in claim 1, is characterized in that... The method for preparing the porous scaffold for bone defect repair includes: (1) The amino cationic polymer grafted with cyclodextrin and phenylboronic acid was dissolved in water at a concentration of 2-10 mg / mL and black phosphorus nanosheets were resuspended, with a black phosphorus nanosheet concentration of 1-3 mg / mL. (2) Disperse the mixed solution in step (1) ultrasonically for 10 to 30 minutes, stir for 2 to 6 hours, and then let it stand at 4°C for 4 to 12 hours; (3) Centrifuge the solution in step (2) at 8000-12000 rpm for 10-15 minutes, and redisperse the resulting precipitate in deionized water so that the concentration of black phosphorus nanosheets is 1-3 mg / mL; (4) Add the 2.5-5 mg / mL drug ethanol solution dropwise to the solution obtained in step (3) under sonication, stir with the open end for 4-8 hours, and then shake at 37°C for 50-120 rpm for 12-16 hours; (5) Cut the obtained PLGA drying bracket to the required size; (6) Immerse the dried PLGA scaffold in the solution of step (4), shake slowly for 0.5 to 4 hours, and freeze dry to obtain a porous scaffold for bone defect repair loaded with black phosphorus to eliminate reactive oxygen species and release drugs in a sustained manner.

8. The method for preparing a porous scaffold for bone defect repair loaded with black phosphorus to eliminate reactive oxygen species and release drugs under sustained release, as described in claim 7, is characterized in that... The drug loaded on the porous scaffold for bone defect repair is at least one of simvastatin, lovastatin, alendronate sodium, and risedronate sodium.

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