pH-responsive metal-polyphenol-modified 3d-printed bone repair scaffold and preparation method thereof

Abstract

The application discloses a pH-responsive metal-polyphenol modified 3D printing bone repair scaffold and a preparation method thereof, and comprises the following steps: S1, dissolving PLGA in dichloromethane, adding β-TCP nanoparticles, and stirring to form a uniform suspension; S2, dissolving tannic acid in a Tris-HCl buffer solution with a pH value of 8.5 to obtain a TA solution, mixing the TA solution with an equal volume of a ZnCl2 solution, centrifuging and washing three times, and freeze-drying to obtain Zn-TA-MPN nanoparticles; S3, dispersing the Zn-TA-MPN nanoparticles in dichloromethane, mixing the Zn-TA-MPN nanoparticles with the PLGA / β-TCP suspension to form a bio-printing ink containing the Zn-TA-MPN nanoparticles; S4, preparing a scaffold through a 3D printing technology, obtaining a Zn-TA / PLGA / β-TCP scaffold after freeze-drying, then immersing the scaffold in a TA solution, washing the scaffold with PBS, then immersing the scaffold in an AgNO3 solution, forming an Ag-TA-MPN coating on the surface of the scaffold, and obtaining a Zn-TA / PLGA / β-TCP@Ag-TA scaffold. Thus, the scaffold can be used for repairing an infectious bone defect, the pH-responsive release of antibacterial and osteogenic components is realized, and the synergistic treatment of infection control and bone regeneration is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of materials, in particular to a pH-responsive metal-polyphenol modified 3D-printed bone repair scaffold and a preparation method thereof. BACKGROUND

[0002] The treatment of infected bone defects requires simultaneous control of infection and promotion of bone regeneration, but existing scaffolds are difficult to achieve dynamic balance of the two. Traditional antibacterial materials (such as antibiotics) are prone to cause drug resistance, and lack the function of promoting bone regeneration; and a single osteogenic scaffold is easily colonized by bacteria in an infected environment, leading to repair failure.

[0003] Although the existing 3D-printed PLGA / β-TCP composite scaffold has biodegradability and bone conductivity, it lacks antibacterial ability; metal ions (such as Ag + , Zn 2+ ) have antibacterial or bone regeneration-promoting effects, but their release is uncontrollable when directly doped into the scaffold, which may cause cytotoxicity. Tannic acid (TA) as a natural polyphenol has certain antibacterial and anti-inflammatory effects, and can form a multifunctional metal-polyphenol nanonetwork (MPN) by coordination with metal ions. Metal-polyphenol networks are of concern due to their pH responsiveness, multifunctionality and simple synthesis characteristics. Some studies have used MPN coated with TA and Ag + for antibacterial modification of medical device surfaces, but the coating is limited to static antibacterial applications and cannot meet the dynamic needs of antibacterial infection and bone formation during bone repair. Some studies have achieved pH-responsive drug release by loading drugs in MPN, but they have not combined this with the biomimetic structure design of bone scaffolds. In addition, existing MPN modified scaffolds mostly use simple dipping methods to prepare coatings, which makes it difficult to achieve long-term release of MPN and lack of intelligent response to the infected microenvironment (acidic) and the repair stage (neutral). SUMMARY

[0004] The present application aims to at least partially solve one of the technical problems in the related art.

[0005] To this end, one object of the present application is to propose a pH-responsive metal-polyphenol modified 3D-printed bone repair scaffold and a preparation method thereof. The metal-polyphenol network dynamically releases antibacterial components according to the pH change of the infected microenvironment, and can release antibacterial components (Ag + ) to kill bacteria in the acidic environment of the infected site, and release bone regeneration-promoting components (Zn 2+ ) and anti-inflammatory polyphenols after the infection is alleviated, to synergistically regulate immune cells and accelerate the bone repair process, and the drug release during the degradation of the scaffold matches the bone regeneration process.

[0006] To achieve the above object, the first aspect of the present application provides a preparation method of a pH-responsive metal-polyphenol modified 3D printed bone repair scaffold, which comprises the following steps: S1, dissolving PLGA in dichloromethane, adding β-TCP nanoparticles, and stirring to form a uniform PLGA / β-TCP suspension; S2, dissolving tannic acid in a Tris-HCl buffer solution with a pH of 8.5 to obtain a TA solution, mixing the TA solution with an equal volume of a ZnCl2 solution, centrifuging and washing three times, and freeze-drying to obtain Zn-TA-MPN nanoparticles; S3, dispersing the Zn-TA-MPN nanoparticles in dichloromethane, mixing with the PLGA / β-TCP suspension, and forming a bio-printing ink containing Zn-TA-MPN nanoparticles; S4, preparing a scaffold by using the bio-printing ink through a 3D printing technology, obtaining a Zn-TA / PLGA / β-TCP scaffold after freeze-drying, then immersing the scaffold in a TA solution, washing with PBS, and then immersing the scaffold in an AgNO3 solution to form an Ag-TA-MPN coating on the surface of the scaffold, thereby obtaining a Zn-TA / PLGA / β-TCP@Ag-TA scaffold; and S5, vacuum drying the Zn-TA / PLGA / β-TCP@Ag-TA scaffold to remove the solvent, and then sequentially performing ethanol sterilization, ultraviolet sterilization, and PBS washing after oven drying.

[0007] In addition, the pH-responsive metal-polyphenol modified 3D printed bone repair scaffold and the preparation method thereof according to the present application can have the following additional technical features:

[0008] In an embodiment of the present application, the mass ratio of PLGA to β-TCP in step S1 is 70:30, and the stirring time of the suspension is 12 h.

[0009] In an embodiment of the present application, the pore size of the Zn-TA / PLGA / β-TCP scaffold is 500 μm, and the porosity is 60-70%.

[0010] In an embodiment of the present application, the concentration of the TA solution in step S2 is 2.5 mg / mL, and the concentration of the ZnCl2 solution is 1 mg / mL.

[0011] In an embodiment of the present application, in the bio-printing ink, the mass fraction of Zn-TA-MPN nanoparticles is 5 wt%.

[0012] In an embodiment of the present application, in step S4, the time for immersing the scaffold in the TA solution is 12 h, and the time for immersing the scaffold in the AgNO3 solution is 12 h.

[0013] In an embodiment of the present application, in step S5, the drying temperature of the oven is 37℃, and the drying time of the oven is 7 days.

[0014] In another aspect, the second aspect of the embodiments of the present application proposes a pH-responsive metal-polyphenol modified 3D-printed bone repair scaffold, which is prepared by the preparation method in any one of the above technical solutions.

[0015] Compared with the prior art, the present application at least includes the following beneficial effects:

[0016] 1. By 3D printing technology, the fine control of the shape and internal pore structure can be realized. The 3D-printed 500 μm pore size and 60-70% porosity biomimetic porous structure not only promotes cell migration and vascular ingrowth, but also provides spatial support for uniform loading of MPN nanoparticles, enhancing the stability of drug release. The degradation period of the scaffold matches the bone regeneration process, avoiding structural collapse or residue.

[0017] 2. By in-situ loading of Zn-TA-MPN particles and surface Ag-TA-MPN coating modification through 3D printing, the non-uniformity of traditional impregnation method is broken through, realizing the precise distribution of nanoparticles inside and outside the scaffold, and ensuring the synergistic effect of antibacterial and osteogenic functions.

[0018] 3. By using the pH responsiveness of MPN, in the infected micro-acidic environment, Ag + is rapidly released from the Ag-TA-MPN coating and efficiently kills bacteria; after the infection is relieved, in the neutral environment, Zn 2+ is released from the Zn-TA-MPN nanoparticles, significantly improving the osteogenic performance, while tannic acid (TA) plays an anti-inflammatory role by scavenging free radicals and regulating macrophage polarization, solving the problems of single function and uncontrollable release of traditional scaffolds.

[0019] 4. The coordination bond dissociation characteristics of MPN ensure that Ag + is only released during the infection period, avoiding non-specific toxicity; the slow release of Zn 2+ is realized through the stable chelation of TA, continuously promoting osteogenesis and neutralizing the acidic products generated by PLGA degradation, reducing the inflammatory response. Compared with existing antibacterial peptide immobilization technologies, the present application has dual safety of broad-spectrum antibacterial and controllable osteogenesis through dynamic coordination of metal ions and polyphenols.

[0020] Additional aspects and advantages of the present application will be partially given in the following description, partially will become obvious from the following description, or will be understood by the practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0021] The above and / or additional aspects and advantages of the present application will become apparent and more readily appreciated from the following description, taken in conjunction with the following drawings, in which:

[0022] Figure 1Preparation method flow chart of Zn-TA / PLGA / β-TCP@Ag-TA scaffold of the embodiment of the present application;

[0023] Figure 2 Effect diagram of PLGA / β-TCP scaffold and Zn-TA / PLGA / β-TCP@Ag-TA scaffold of the embodiment of the present application;

[0024] Figure 3 SEM diagram of Ag-TA-MPN coating of the embodiment of the present application;

[0025] Figure 4 Column diagram of antibacterial effect of different material scaffolds on Staphylococcus aureus of the embodiment of the present application;

[0026] Figure 5 Antibacterial effect of Zn-TA / PLGA / β-TCP@Ag-TA scaffold on Staphylococcus aureus shown by plate colony counting of the embodiment of the present application;

[0027] Figure 6 Column diagram of antibacterial effect of different material scaffolds on Escherichia coli of the embodiment of the present application;

[0028] Figure 7 Antibacterial effect of Zn-TA / PLGA / β-TCP@Ag-TA scaffold on Escherichia coli shown by plate colony counting of the embodiment of the present application;

[0029] Figure 8 Overall work flow chart of the embodiment of the present application. DETAILED DESCRIPTION

[0030] The embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference signs represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the accompanying drawings are exemplary and are intended to explain the present application, and cannot be understood as a limitation of the present application.

[0031] The pH-responsive metal-polyphenol modified 3D printing bone repair scaffold and the preparation method thereof of the embodiments of the present application are described below in conjunction with the accompanying drawings.

[0032] Bone tissue engineering technology as a frontier of bone regeneration method is concerned, bone tissue engineering mainly involves three factors: scaffold, bioactive factors and cells with osteogenic potential. Bone tissue engineering scaffold is generally prepared using bioactive materials, which can mimic the composition, structure and mechanical properties of human tissue and other biological functions, suitable for the growth and reproduction of seed cells, and is one of the key factors affecting the successful reconstruction of bone tissue. However, there are still some problems in the clinical application of bone tissue engineering scaffold, and the scaffold has a high risk of infection in the early stage of implantation, especially in some exposed or easily infected conditions. For example, periodontitis, large area of maxillofacial bone defect caused by trauma and other factors, there are many bacteria in the oral environment, so patients need to use a large amount of antibiotics after implanting the scaffold. The regeneration and repair of bone tissue is a complex process, and the presence of infection makes it more difficult to repair bone. At present, the treatment of infected bone defects mainly depends on traditional surgery and the use of broad-spectrum antibiotics, and the treatment cycle is long, and patients often have to bear the side effects of antibiotics and the risk of secondary operation of bone grafting, which brings additional economic burden to the patient's family and society. Therefore, it is urgent to develop a treatment strategy to solve the limitations of current treatment methods.

[0033] In the treatment of bone defects, three-dimensional porous scaffold is concerned in the field of bone tissue engineering. 3D printing, also known as additive manufacturing, is based on the principle of layer manufacturing, and the material is overlapped layer by layer. According to the computer-aided design model, through the use of solid modeling precise accumulation of materials, it can quickly manufacture components with any complex shape. The performance of the material such as pore size or porosity affects the biological behavior of cells. Compared with traditional scaffold preparation methods, which can only control the overall characteristics of the scaffold and cannot accurately control the internal topology of the scaffold, 3D printing technology is widely used in the preparation of bone repair materials due to its advantages of individual customization, rapid prototyping, precise control of internal and external structure of the scaffold. Biological 3D printing can use cells, proteins and biomaterials to construct three-dimensional structures, which is expected to become a powerful tool in the field of bone tissue engineering. 3D printed scaffold not only has a three-dimensional shape, but also has an internal network structure similar to the extracellular matrix, which is similar to human bone and more conducive to bone tissue reconstruction, and has important significance for individualized treatment of bone defects.

[0034] Poly(lactic-co-glycolic acid) (PLGA) is a polymer formed by the dehydration condensation of lactic acid and glycolic acid molecules, which has been approved by the Food and Drug Administration for clinical use, and has good biocompatibility, adjustable biodegradability and processing performance. The ratio of glycolic acid and lactic acid is different, and the molecular weight of the synthesized PLGA is different, and the degradation time is also different. For different purposes, we can choose the appropriate ratio of PLGA to control the required degradation time. However, the acidic microenvironment formed after the degradation of PLGA can cause inflammation of the surrounding tissue, which is not conducive to tissue regeneration. In addition, the mechanical properties of PLGA are poor. Studies have shown that the combination of PLGA and inorganic components can effectively enhance its mechanical properties. β-tricalcium phosphat (β-TCP) is the most commonly used calcium phosphate material in bone tissue engineering, which has good biocompatibility, bioactivity and biodegradability. β-TCP can enhance the mechanical properties of PLGA, neutralize acidic products, and endow the material with bone conduction properties. At the same time, the complete degradation time of PLGA / β-TCP composite material in the animal body is about 24 weeks, which is close to the natural healing time of bone. It has been proved to be suitable for clinical application, which is conducive to the matching of the degradation rate of the scaffold and the bone regeneration rate in the body. PLGA / β-TCP composite scaffolds have become excellent artificial bone graft materials due to their osteogenic properties and appropriate degradation behavior, but their lack of osteoinduction has hindered their clinical application. There have been many efforts in the modification of PLGA / β-TCP scaffolds, such as loading fibroblast growth factor 2 (FGF-2) to regulate the proliferation and differentiation of osteoblasts, improving osteoinduction; using the scaffold to deliver osteogenic peptide and mesenchymal stem cells to improve bone formation ability. Although the above methods have made some progress, growth factors and osteogenic peptides are expensive and easy to lose activity, and their osteogenic effect is uncertain. Therefore, new scaffold modification methods need to be developed to promote the osteogenic effect of bone repair materials. Moreover, considering the frequent occurrence of clinical infection of bone defects, the antibacterial properties of scaffold materials cannot be ignored.

[0035] Metal phenolic network (MPN) is a new type of organic-inorganic hybrid network system that has been gradually developed in recent years. It is simple to synthesize and has excellent biocompatibility, antibacterial, antioxidant, anti-inflammatory and other functions, and has been widely concerned in the field of anti-microbial infection. MPN is a supramolecular complex structure formed by the coordination of metal ions and phenolic ligands, and has the beneficial properties of universal adhesion, stimulus responsiveness, selective permeability and thermal stability. Due to the abundance of natural polyphenols and the wide selection of metal ions, MPN provides a new method for designing various functional materials. The synthesis of MPN can be directly assembled in one step, using natural polyphenols as organic ligands, which can rapidly react with metal ions. The material can be formed by simply mixing the polyphenol and metal ions at room temperature. The composition of the synthesized material is easy to obtain, safe and low in price. The rapid and simple reaction is the most attractive feature of MPN. Due to its rapid and direct synthesis, MPN has been widely used in coating materials. The raw materials of MPN, polyphenol and metal ions, both have biological activity. For example, tannic acid (TA), the most commonly used polyphenol, is a natural weakly acidic polyphenol. As a reducing agent, it has certain antibacterial and anti-inflammatory effects. Metal ions, as another raw material, can exert different biological effects. For example, Ag ions have been widely studied for their antibacterial effects, while Zn ions can promote osteogenesis and have certain antibacterial effects. Biologically active metal ions such as Fe, Cu and Mg can also exert different biological effects. It is worth noting that the formation of MPN requires certain conditions, the most important of which is the pH value of the reaction. At different pH values, polyphenols and metal ions combine and separate. Different combinations of polyphenols and metals have different pH values, which is another attractive feature of MPN, i.e. pH responsiveness. Generally, MPN is combined under alkaline conditions and dissociated under acidic conditions. This feature has high medical application value. The pH of the infected microenvironment is relatively low compared to the normal internal environment of the human body. In an acidic environment, the polyphenols and metal ions that make up the MPN can be quickly released and exert their corresponding biological activities. Using pH-responsive MPN as a drug carrier can target the release of drugs to the affected area, avoiding the side effects of drugs on normal tissues. Therefore, selecting the appropriate MPN for material modification is expected to develop more ideal bone tissue engineering scaffold materials.

[0036] The pH-responsive metal-polyphenol modified 3D printed bone repair scaffold of the present application can be used for the repair of infected bone defects, and can achieve the synergistic treatment of infection control and bone regeneration by releasing antibacterial and osteogenic components in a pH-responsive manner.

[0037] As shown in Figure 1 and Figure 8 , the preparation method of the pH-responsive metal-polyphenol modified 3D printed bone repair scaffold of the present application can include the following steps:

[0038] S1. Dissolve PLGA in dichloromethane, add β-TCP nanoparticles, and stir to form a uniform PLGA / β-TCP suspension;

[0039] Furthermore, the mass ratio of PLGA to β-TCP is 70:30, and the stirring time of the PLGA / β-TCP suspension is 12 hours.

[0040] S2. Tannic acid (TA) was dissolved in Tris-HCl buffer solution at pH 8.5 to obtain TA solution. The TA solution was mixed with ZnCl2 solution in equal volume, centrifuged and washed 3 times, and then freeze-dried to obtain Zn-TA-MPN nanoparticles.

[0041] Furthermore, the concentration of the TA solution is 2.5 mg / mL, and the concentration of the ZnCl2 solution is 1 mg / mL.

[0042] S3. Disperse Zn-TA-MPN nanoparticles in dichloromethane and mix with PLGA / β-TCP suspension to form a bioprinting ink containing Zn-TA-MPN nanoparticles.

[0043] Furthermore, the Zn-TA-MPN nanoparticles in the bioprinting ink have a mass fraction of 5 wt%.

[0044] S4. The bioprinting ink is prepared into a scaffold using 3D printing technology. After freeze-drying, a Zn-TA / PLGA / β-TCP scaffold is obtained. Then, the Zn-TA / PLGA / β-TCP scaffold is immersed in a TA solution, washed with PBS, and then immersed in an AgNO3 solution to form an Ag-TA-MPN coating on the scaffold surface, thus obtaining a Zn-TA / PLGA / β-TCP@Ag-TA scaffold.

[0045] Furthermore, the Zn-TA / PLGA / β-TCP scaffold has a pore size of 500 μm and a porosity of 60-70%. The scaffold is immersed in TA solution for 12 hours and in AgNO3 solution for 12 hours.

[0046] It is understood that this embodiment uses 3D printing technology to precisely construct a PLGA / β-TCP scaffold with a pore size of 500μm and a porosity of 60-70%. By uniformly loading Zn-TA-MPN nanoparticles and modifying the surface with an Ag-TA-MPN coating, MPN is synergistically distributed inside and on the surface of the scaffold, thus overcoming the functional limitations of traditional preparation methods.

[0047] S5. The Zn-TA / PLGA / β-TCP@Ag-TA scaffold is vacuum dried to remove the solvent, then dried in an oven, and then successively sterilized with ethanol, sterilized with ultraviolet light, and washed with PBS.

[0048] Furthermore, in step S5, the drying temperature of the oven is 37°C, and the drying time of the oven is 7 days.

[0049] It should be noted that the composition and structure of the Zn-TA / PLGA / β-TCP@Ag-TA scaffold prepared by the above method are as follows:

[0050] The substrate is a composite of polylactic-co-glycolic acid copolymer (PLGA) and β-tricalcium phosphate (β-TCP).

[0051] The materials are combined in a mass ratio of 70:30.

[0052] Zinc-tannic acid metal-polyphenol nanoparticles (Zn-TA-MPN) were loaded onto the PLGA / β-TCP scaffold, with a Zn-TA-MPN loading mass fraction of 5 wt%.

[0053] The surface is modified with a silver-tannic acid metal-polyphenol nano-network coating (Ag-TA-MPN); and the scaffold has a pore size of 500±50μm and a porosity of 60-70%.

[0054] refer to Figure 3 , Figure 3 Here is a SEM image of the Ag-TA-MPN coating, where... Figure 3 (Top left) and Figure 3 (Top right) is a top-view electron microscope image of the coating on the surface of the support. Figure 3 (bottom left) and Figure 3 (Bottom right) is a cross-sectional electron microscope image of the stent after liquid nitrogen brittle fracture.

[0055] It should be noted that this stent is pH-responsive, releasing Ag in slightly acidic environments. + Antibacterial, releases TA for anti-inflammatory effects, and continuously releases Zn as the stent degrades in a neutral environment. 2+ It promotes bone regeneration and simultaneously releases TA for anti-inflammatory effects.

[0056] Therefore, this Zn-TA / PLGA / β-TCP@Ag-TA scaffold can achieve:

[0057] 1. Dynamic pH-responsive MPN network achieves multifunctionality: through silver (Ag) + ) and zinc (Zn 2+ Ag ions coordinate with tannic acid (TA) to form a smart nanonetwork. The acidic environment during the infection period triggers Ag... + Rapidly releases bactericidal agents; long-term release of Zn in a neutral environment after infection resolution.2+ Promote bone regeneration, while continuously releasing TA to play an anti-inflammatory role to regulate immunity, achieve antibacterial, anti-inflammatory, and pro-osteogenic multifunctional dynamic switching.

[0058] 2. Synergistic structure and function of the scaffold: combining biomimetic porous structure and pH-responsive release mechanism, optimizing cell migration and bone ingrowth, and matching the degradation rate with the bone regeneration process to achieve dynamic balance of infection control and bone repair.

[0059] 3. Through the deep integration of intelligent MPN network and 3D printing technology, the functional dynamic regulation of bone tissue engineering scaffolds in pathological microenvironment is realized for the first time, and the clinical problem of simultaneous anti-infection and bone regeneration is solved.

[0060] Example:

[0061] a) Solution preparation: dissolve PLGA in dichloromethane, add β-TCP nanoparticles, the mass ratio of PLGA to β-TCP is 70:30, stir for 12 h to form a uniform suspension;

[0062] b) Preparation of Zn-TA-MPN: dissolve tannic acid (TA) in Tris-HCl buffer solution with pH=8.5 to obtain TA solution (2.5 mg / mL), mix with ZnCl2 solution (1 mg / mL) in equal volume, centrifuge and wash 3 times, freeze-dry to obtain Zn-TA-MPN nanoparticles;

[0063] c) Ink preparation: disperse Zn-TA-MPN nanoparticles in dichloromethane, mix with PLGA / β-TCP suspension to form a bio-printing ink containing 5 wt% Zn-TA-MPN nanoparticles;

[0064] d) Scaffold modification: prepare the scaffold by 3D printing technology using the bio-ink obtained in step (c), control the pore size to be 500 μm and the porosity to be 60-70%, freeze-dry to obtain Zn-TA / PLGA / β-TCP scaffold; then immerse in TA solution for 12 h, wash with PBS, and then immerse in AgNO3 solution for 12 h, form Ag-TA-MPN coating on the surface of the scaffold, obtain Zn-TA / PLGA / β-TCP@Ag-TA scaffold, as shown in Figure 2 (right);

[0065] e) Post-processing: remove the solvent by vacuum drying, dry in a 37°C oven for 7 days, sterilize with ethanol, sterilize with ultraviolet light, and wash with PBS.

[0066] Comparative Example 1: Preparation of PLGA / β-TCP scaffold

[0067] a) PLGA was dissolved in dichloromethane, β-TCP nanoparticles were added, the mass ratio of PLGA to β-TCP was 70:30, and stirring was carried out for 12 h to form a uniform suspension;

[0068] b) PLGA / β-TCP scaffolds were prepared at room temperature by using 3D printing technology, such as Figure 2 (Left). The pore size was controlled to be 500 μm, and the porosity was 60-70%. Freeze-drying solidification.

[0069] Comparative Example 2: Preparation of Zn-TA / PLGA / β-TCP scaffolds

[0070] a) PLGA was dissolved in dichloromethane, β-TCP nanoparticles were added, the mass ratio of PLGA to β-TCP was 70:30, and stirring was carried out for 12 h to form a uniform suspension

[0071] b) Tannic acid (TA) was dissolved in Tris-HCl buffer solution with pH = 8.5 to obtain a TA solution (2.5 mg / mL), and an equal volume of ZnCl2 solution (1 mg / mL) was mixed, centrifuged and washed 3 times, and freeze-dried to obtain Zn-TA-MPN nanoparticles;

[0072] c) Zn-TA-MPN nanoparticles were dispersed in dichloromethane and mixed with PLGA / β-TCP suspension to form a bio-printing ink containing 5 wt% Zn-TA-MPN nanoparticles;

[0073] d) The bio-ink obtained in step c) was prepared into a scaffold by 3D printing technology, the pore size was controlled to be 500 μm, and the porosity was 60-70%, and freeze-drying was carried out to obtain a Zn-TA / PLGA / β-TCP scaffold.

[0074] In order to reflect the antibacterial performance of the Zn-TA / PLGA / β-TCP@Ag-TA scaffold, three control groups were set up, namely the Control group (blank control group), the PLGA / β-TCP group and the Zn-TA / PLGA / β-TCP group, which were compared with the Zn-TA / PLGA / β-TCP@Ag-TA group, in order to compare the antibacterial effects of different material scaffolds on Staphylococcus aureus and Escherichia coli.

[0075] I. Antibacterial effect on Staphylococcus aureus

[0076] Figure 4 The bar chart of the antibacterial effect of different material scaffolds on Staphylococcus aureus is shown in terms of OD 600 value (reflecting the concentration of bacterial solution, the lower the value, the better the antibacterial effect) as the evaluation index to show the antibacterial effect of different material scaffolds on Staphylococcus aureus.

[0077] Control group (blank control group): OD600 The higher the value, the higher the concentration of the bacterial solution, indicating that Staphylococcus aureus grows normally without antibacterial effect.

[0078] PLGA / β-TCP group: OD 600 The value is similar to the Control group, indicating that the material scaffold has no significant antibacterial effect on Staphylococcus aureus.

[0079] Zn-TA / PLGA / β-TCP group: Compared with the Control group and the PLGA / β-TCP group, the OD 600 value decreased. It should be noted that there was no statistically significant difference between groups.

[0080] Zn-TA / PLGA / β-TCP@Ag-TA group: OD 600 The value is significantly lower than that of other groups, and the difference is extremely significant compared with the Control group, the PLGA / β-TCP group, and the Zn-PLGA / β-TCP group, indicating that the material scaffold has the most outstanding antibacterial effect on Staphylococcus aureus, and the antibacterial effect of the material scaffold is the best.

[0081] Further, through the plate colony counting method, the differences in antibacterial effect of different material scaffolds on Staphylococcus aureus were compared. Referring to Figure 5 , the number of colonies of Staphylococcus aureus on the plates of each group was counted, and the colony count results were quantified to obtain the antibacterial effect of the Zn-TA / PLGA / β-TCP@Ag-TA scaffold and each control group on Staphylococcus aureus.

[0082] Figure 5 From left to right are the Control group, the PLGA / β-TCP group, the Zn-TA / PLGA / β-TCP group, and the Zn-TA / PLGA / β-TCP@Ag-TA group.

[0083] The plate colony counting results show that compared with the control group, the experimental group scaffolds exhibit significant antibacterial effect. Among them, the antibacterial rate of the Zn-TA / PLGA / β-TCP scaffold on Staphylococcus aureus reaches 64.8%, and the antibacterial rate of the Zn-TA / PLGA / β-TCP@Ag-TA scaffold reaches 94.1%.

[0084] II. Antibacterial effect on E. coli

[0085] Figure 6 The bar chart of the antibacterial effect of different material scaffolds on E. coli shows the antibacterial effect of different material scaffolds on E. coli with OD 600 value as the evaluation index.

[0086] Control group (blank control group): OD 600The higher the value, the higher the concentration of the E. coli solution, indicating that the E. coli can grow and reproduce normally without the antibacterial effect.

[0087] PLGA / β-TCP group: OD 600 The value is similar to that of the Control group, indicating that the material scaffold has little obvious antibacterial effect on E. coli.

[0088] Zn-TA / PLGA / β-TCP group: Compared with the Control group and the PLGA / β-TCP group, the OD 600 The value is slightly lower.

[0089] Zn-TA / PLGA / β-TCP@Ag-TA group: OD 600 The value is significantly lower than that of the other groups, and the difference is extremely significant compared with the Control group, the PLGA / β-TCP group, and the Zn-PLGA / β-TCP group, indicating that the material scaffold has the most outstanding antibacterial effect on E. coli and has the best antibacterial performance among the several scaffolds.

[0090] Further, the differences in the antibacterial effects of different material scaffolds on E. coli were compared by plate colony counting method. Figure 7 The number of E. coli colonies on the plates of each group was counted, and the amount of viable bacteria was quantified by colony counting results, so as to obtain the antibacterial effect of the Zn-TA / PLGA / β-TCP@Ag-TA scaffold and each control group on E. coli.

[0091] Figure 7 From left to right, they are the Control group, the PLGA / β-TCP group, the Zn-TA / PLGA / β-TCP group, and the Zn-TA / PLGA / β-TCP@Ag-TA group.

[0092] The plate colony counting results show that the scaffolds in the experimental groups exhibit significant antibacterial effects compared with the control groups. Among them, the antibacterial rate of the Zn-TA / PLGA / β-TCP scaffold on E. coli reaches 52.9%, and the antibacterial rate of the Zn-TA / PLGA / β-TCP@Ag-TA scaffold reaches 87.9%.

[0093] In summary, the pH-responsive metal-polyphenol modified 3D printed bone repair scaffold and the preparation method thereof have the following beneficial effects:

[0094] (1) Controllable structure: Through 3D printing technology, the shape and internal pore structure can be precisely controlled. The 3D printed 500 pm pore size and 60-70% porosity biomimetic porous structure not only promotes cell migration and vascular ingrowth, but also provides spatial support for uniform loading of MPN nanoparticles, enhancing drug release stability. The degradation period of the scaffold matches the bone regeneration process, avoiding structural collapse or residue. (2) High antibacterial effect: The Zn-TA / PLGA / β-TCP@Ag-TA scaffold has the most outstanding antibacterial effect on E. coli among the several scaffolds, and the antibacterial performance is the best. (3) Biocompatibility: The Zn-TA / PLGA / β-TCP@Ag-TA scaffold has good biocompatibility and biodegradability, and can be used for bone repair and regeneration.

[0095] (II) Preparation process is highly efficient and controllable: by in-situ loading of Zn-TA-MPN particles and surface Ag-TA-MPN coating modification through 3D printing, the non-uniformity of traditional immersion method is broken through, the precise distribution of nanoparticles in the interior and exterior of the scaffold is realized, and the synergistic effect of antibacterial-osteogenic function is ensured.

[0096] (III) Antibacterial-osteogenic-anti-inflammatory multifunctional: by the pH responsiveness of MPN, Ag + is rapidly released from the Ag-TA-MPN coating and efficiently kills bacteria; after the infection is relieved, Zn 2+ is released from the Zn-TA-MPN nanoparticles, significantly improving the osteogenic performance, and tannic acid (TA) plays an anti-inflammatory role by scavenging free radicals and regulating macrophage polarization, solving the problems of single function and uncontrollable release of traditional scaffolds.

[0097] (IV) Precise release and biological safety: the coordination bond dissociation characteristics of MPN ensure that Ag + is only released during the infection period, avoiding non-specific toxicity; the slow release of Zn 2+ is realized through the stable chelation of TA, continuously promoting osteogenesis and neutralizing the acidic products generated by PLGA degradation, reducing inflammatory response. Compared with existing antibacterial peptide immobilization technologies, the present application has dual safety of broad-spectrum antibacterial and controllable osteogenesis through dynamic coordination of metal ions and polyphenols.

[0098] In the description of the present specification, the terms "first", "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined with "first", "second" can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise specifically limited.

[0099] In the description of the present specification, the description of the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples without contradiction.

[0100] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and that changes, modifications, substitutions and variations can be made by those skilled in the art without departing from the scope of the present application.

Claims

1. A method for preparing a pH-responsive metal-polyphenol modified 3D-printed bone repair scaffold, characterized in that, The preparation method includes the following steps: S1. Dissolve PLGA in dichloromethane, add β-TCP nanoparticles, and stir to form a uniform PLGA / β-TCP suspension; S2. Dissolve tannic acid in Tris-HCl buffer solution at pH=8.5 to obtain TA solution. Mix the TA solution with ZnCl2 solution in equal volume, centrifuge and wash 3 times, and freeze dry to obtain Zn-TA-MPN nanoparticles. S3. Disperse Zn-TA-MPN nanoparticles in dichloromethane and mix with PLGA / β-TCP suspension to form a bioprinting ink containing Zn-TA-MPN nanoparticles. S4. The bioprinting ink is prepared into a scaffold using 3D printing technology. After freeze-drying, a Zn-TA / PLGA / β-TCP scaffold is obtained. Then, the Zn-TA / PLGA / β-TCP scaffold is immersed in a TA solution, washed with PBS, and then immersed in an AgNO3 solution to form an Ag-TA-MPN coating on the scaffold surface, thus obtaining a Zn-TA / PLGA / β-TCP@Ag-TA scaffold. S5. The Zn-TA / PLGA / β-TCP@Ag-TA scaffold is vacuum dried to remove the solvent, then dried in an oven, and then sterilized with ethanol, sterilized with ultraviolet light, and washed with PBS in sequence.

2. The method for preparing a pH-responsive metal-polyphenol modified 3D-printed bone repair scaffold according to claim 1, characterized in that, In step S1, the mass ratio of PLGA to β-TCP is 70:30, and the stirring time of the PLGA / β-TCP suspension is 12 hours.

3. The method for preparing a pH-responsive metal-polyphenol modified 3D-printed bone repair scaffold according to claim 1, characterized in that, The Zn-TA / PLGA / β-TCP scaffold has a pore size of 500 μm and a porosity of 60-70%.

4. The method for preparing a pH-responsive metal-polyphenol modified 3D-printed bone repair scaffold according to claim 1, characterized in that, In step S2, the concentration of the TA solution is 2.5 mg / mL, and the concentration of the ZnCl2 solution is 1 mg / mL.

5. The method for preparing a pH-responsive metal-polyphenol modified 3D-printed bone repair scaffold according to claim 1, characterized in that, The bioprinting ink contains 5 wt% Zn-TA-MPN nanoparticles.

6. The method for preparing a pH-responsive metal-polyphenol modified 3D-printed bone repair scaffold according to claim 1, characterized in that, In step S4, the stent is immersed in the TA solution for 12 hours and in AgNO3 solution for 12 hours.

7. The method for preparing a pH-responsive metal-polyphenol modified 3D-printed bone repair scaffold according to claim 1, characterized in that, In step S5, the drying temperature of the oven is 37°C and the drying time is 7 days.

8. A pH-responsive metal-polyphenol modified 3D-printed bone repair scaffold, characterized in that, It is prepared by the preparation method according to any one of claims 1-7.

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