Bone repair scaffold of ZIF-8 gene-loaded hydrogel as well as preparation method and application of bone repair scaffold

Abstract

The invention provides a ZIF-8 gene-loaded hydrogel bone repair scaffold as well as a preparation method and application thereof, and belongs to the technical field of biomedical material manufacturing. The bone repair scaffold of the ZIF-8 gene-loaded hydrogel comprises a porous base material, hydrogel and ZIF-8 particles. The porous base material is a bone implant material; the hydrogel is sodium alginate hydrogel, the surface of the porous base material is coated with the hydrogel, and pores of the porous base material are filled with the hydrogel; the ZIF-8 particles are distributed in the hydrogel, the genes are loaded in the ZIF-8 particles, the genes are osteogenesis promoting genes, and the bone repair scaffold has multiple functions of resisting bacteria, promoting osseointegration, promoting osteogenesis and the like, so that the clinical application requirements of the bone repair scaffold are better met.

Description

Technical Field

[0001] The present application relates to the technical field of biomedical material manufacturing, and in particular to a bone repair scaffold of ZIF-8 gene-loaded hydrogel and a preparation method and application thereof. Background Art

[0002] Currently, the development of clinical bone repair materials must meet multi-dimensional functional properties. Specifically, while ensuring basic mechanical compatibility, they must also possess key properties such as antibacterial ability, the ability to promote bone integration, and the ability to promote osteogenesis. However, traditional bone repair materials (such as single metals, ceramics, or polymers) often only meet some of these requirements and struggle to meet multiple functional needs simultaneously.

[0003] Based on this, technicians began to modify bone implant materials to give them richer functions. Common modification methods are usually to prepare a modified coating on the surface of the bone implant material or to graft bioactive factors on the surface of the bone implant material. However, the modification effect of a single modification method is poor. For example: (1) Coating a hydroxyapatite modified coating on the surface of the bone repair material can only improve the ability to promote bone integration; (2) Coating an antibacterial coating (such as silver nanoparticles) on the surface of the bone repair material can only improve the antibacterial performance; (3) Grafting bioactive factors (usually protein biomacromolecules) on the surface of the bone repair material can only improve the bone-promoting performance, and this type of bioactive factor also has disadvantages such as instability, short half-life, and high cost.

[0004] Therefore, how to use multiple modification methods to modify bone repair materials at the same time so that they can take into account multiple functions such as antibacterial, promoting bone integration and promoting osteogenesis is a major problem currently faced. The breakthrough of this technology not only has important scientific and research significance, but also has broad market prospects. Summary of the Invention

[0005] The purpose of this application is to provide a bone repair scaffold of ZIF-8 gene-loaded hydrogel and its preparation method and application. The bone repair scaffold has multiple functions such as good antibacterial properties, promoting bone integration and promoting osteogenesis, so as to better meet the clinical application needs of bone repair scaffolds.

[0006] The embodiment of the present application is implemented as follows:

[0007] First, the present invention provides a bone repair scaffold comprising a ZIF-8 gene-loaded hydrogel, comprising a porous substrate, a hydrogel, and ZIF-8 particles. The porous substrate is a bone implant material; the hydrogel is a sodium alginate hydrogel, coated on the surface of the porous substrate and filling the pores within the porous substrate; and the ZIF-8 particles are distributed within the hydrogel, loaded with a gene that promotes bone growth.

[0008] In the above technical solution, the bone repair scaffold is composed of a porous substrate, a hydrogel and ZIF-8 particles loaded with genes. Specifically, the porous substrate can provide basic mechanical adaptability; ZIF-8 particles can provide antibacterial properties by releasing zinc ions on the one hand, and can serve as a carrier and protective layer of genes on the other hand, so as to smoothly deliver genes to the cell nucleus and then release and exert the gene's ability to promote osteogenesis (wherein, the load corresponds to genes rather than proteins, and gene editing methods are used to make cells express related active factors, which has better economy, controllability and effectiveness than direct protein delivery); hydrogel can improve the surface hydrophilicity of the scaffold, enhance cell adhesion, and thus promote bone integration, and on the other hand, it can also form a two-stage pH-responsive controlled release system with ZIF-8, so as to slowly and continuously release genes in the body and exert the ability to promote osteogenesis. By integrating the above-mentioned multiple functional components together, the bone repair scaffold can have multiple functions such as good antibacterial, bone integration promotion and osteogenesis promotion, so as to better meet the clinical application needs of bone repair scaffolds.

[0009] In some optional embodiments, the particle size of the ZIF-8 particles is 100 to 300 nm.

[0010] In the above technical solution, the particle size of ZIF-8 particles is limited to the above range so that the ZIF-8 particles loaded with genes have an appropriate size, thereby facilitating the safe and smooth delivery of genes into the cell nucleus and enabling the cells to express the corresponding genes, thereby better playing the role of promoting osteogenesis.

[0011] In some optional embodiments, the gene is selected from at least one of pVEGF, pBMP-2 and pTGF-β; or / and, the material of the porous substrate is selected from at least one of metal, ceramic and polymer materials; optionally, the polymer material is selected from at least one of polyetheretherketone, polylactic acid and polycaprolactone.

[0012] The above technical solution offers a wide range of applicable genes and porous substrates, providing a wide range of feasible implementation options, thereby facilitating the promotion and application of the technical solution provided in the embodiments of this application. Furthermore, when the porous substrate is a polymer material, limiting the polymer material to the above range offers the advantages of good biocompatibility, excellent mechanical properties, and ease of processing and molding.

[0013] In the second aspect, an embodiment of the present application provides a method for preparing a bone repair scaffold of a ZIF-8 gene-loaded hydrogel as provided in the first aspect, comprising the following steps: mixing zinc salt, 2-methylimidazole and gene to obtain a first mixed solution, and then placing the first mixed solution at room temperature for reaction. After the reaction is completed, the solution is subjected to solid-liquid separation and redispersion to obtain a dispersion containing ZIF-8 particles loaded with genes; mixing the dispersion and an aqueous sodium alginate solution to obtain a second mixed solution; immersing the porous substrate in the second mixed solution for immersion treatment, and after the immersion treatment is completed, obtaining a cross-linked precursor solution; adding a cross-linking agent to the cross-linked precursor solution to cross-link the sodium alginate in the cross-linked precursor solution to form a sodium alginate hydrogel, thereby obtaining a bone repair scaffold of a ZIF-8 gene-loaded hydrogel.

[0014] In the above technical solution, the first mixed solution is placed at room temperature to react (i.e., zinc salt and 2-methylimidazole react to form a ZIF-8 vector, and the gene is wrapped inside it), which can form ZIF-8 particles loaded with genes, and then undergo solid-liquid separation and redispersion to achieve constant volume dispersion of ZIF-8 particles loaded with genes, thereby facilitating subsequent accurate weighing of genes according to actual needs; the porous substrate is immersed in the second mixed solution for immersion treatment, so that the second mixed solution composed of the dispersion liquid and the sodium alginate aqueous solution fully enters and fills the pores of the porous substrate and covers its surface; after the impregnation is completed, a cross-linking agent is added to cross-link the sodium alginate to form a sodium alginate hydrogel. According to the above process, a bone repair scaffold of ZIF-8 gene-carrying hydrogel can be prepared.

[0015] In some optional embodiments, in the first mixed solution, the molar ratio of zinc element in the zinc salt to 2-methylimidazole is 1:(45-55); or / and, the ratio of the molar amount of zinc element to the mass of the gene is 6.25 μmol:(20-40) μg; optionally, the molar concentration of zinc element in the first mixed solution is 0.02-0.03 mol / L.

[0016] In the above technical solution, the molar ratio of zinc to 2-methylimidazole in the first mixed solution is limited to the above range, so that the formed ZIF-8 particles contain a relatively suitable weight ratio of zinc ions, thereby making the scaffold have relatively suitable antibacterial properties; the molar amount of zinc and the mass ratio of the gene are limited to the above range, so that the formed gene-loaded ZIF-8 particles carry a relatively suitable weight ratio of genes, thereby making the scaffold have relatively suitable osteogenic ability. Furthermore, limiting the molar concentration of zinc in the first mixed solution to the above range helps the raw materials to fully react, and the appropriate concentration also helps to form particles with a relatively suitable particle size; at the same time, the appropriate amount of gene can appropriately reduce the surface potential of ZIF-8 while maintaining positive charge, which is beneficial for gene delivery to the cell nucleus and can reduce cytotoxicity (the cell membrane surface is negatively charged, and the appropriate positive charge can promote the interaction between the nanocarrier and the cell membrane through electrostatic adsorption, enhance endocytosis, and thus improve gene delivery efficiency; at the same time, appropriately reducing the surface potential can reduce the damage caused to the membrane structure during the binding of the positively charged carrier to the cell membrane and maintain relatively suitable electrostatic adsorption capacity).

[0017] In some optional embodiments, in the step of placing the first mixed solution at room temperature for reaction, the reaction time is 15 to 120 minutes.

[0018] In the above technical solution, in the process of preparing ZIF-8 particles loaded with genes, the reaction time is limited to the above range, which helps to form particles with a particle size closer to 200 nm, thereby facilitating the gene-loaded ZIF-8 to enter the cell nucleus through endocytosis.

[0019] In some optional embodiments, in the step of impregnation, the duration of the impregnation treatment is 10 to 30 minutes; or / and, in the step of cross-linking, the duration of the treatment is 5 to 20 minutes.

[0020] In the above technical solution, the duration of the immersion treatment step is limited to the above range, so that the second mixed solution composed of the dispersion liquid and the sodium alginate aqueous solution can more fully enter and fill the pores of the porous substrate and more comprehensively cover its surface; in addition, the duration of the cross-linking step is limited to the above range, which helps the sodium alginate to fully cross-link to form a sodium alginate hydrogel.

[0021] In some optional embodiments, in the second mixed solution, the ratio of the gene concentration to the sodium alginate concentration is 1:(1.5-2.5); optionally, the mass concentration of sodium alginate in the second mixed solution is (1-3) μg / 100 μL.

[0022] In the above technical solution, the ratio of the concentration of the gene and sodium alginate in the second mixed solution is limited to the above range so that the two have an appropriate mass ratio, which helps to fully mix the ZIF-8 particles loaded with the gene and the sodium alginate, so that the sodium alginate hydrogel formed by subsequent cross-linking can be more completely wrapped on the surface of the particles; in addition, the mass concentration of sodium alginate in the second mixed solution is limited to the above range. The appropriate concentration helps the sodium alginate to subsequently cross-link efficiently and thoroughly form a sodium alginate hydrogel.

[0023] In some optional embodiments, in the cross-linking precursor solution, the ratio of the concentration of sodium alginate to the concentration of the cross-linking agent is 5:(1-3); optionally, the gene is pVEGF, and the ratio of the concentration of sodium alginate to the concentration of the cross-linking agent is 5:1; optionally, the gene is pBMP-2 or / and pTGF-β, and the ratio of the concentration of sodium alginate to the concentration of the cross-linking agent is 5:3.

[0024] In the above technical solution, the concentration ratio of sodium alginate and cross-linking agent in the cross-linking precursor solution is limited to the above range, which helps the sodium alginate to cross-link to form a sodium alginate hydrogel, and also enables the sodium alginate hydrogel formed after cross-linking to have a more suitable cross-linking density, thereby providing a more suitable sustained release time (the more cross-linking agent, the greater the cross-linking density, and the longer the sustained release time); further, for different types of genes, the concentration ratio of sodium alginate and cross-linking agent is further limited so as to better match the action time of different types of genes.

[0025] In a third aspect, an embodiment of the present application provides a use of a bone repair scaffold of a ZIF-8 gene-loaded hydrogel as provided in the first aspect in the preparation of a bone implant device. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without creative work.

[0027] Figure 1 A process flow chart of a method for preparing a ZIF-8 gene-loaded hydrogel bone repair scaffold provided in an embodiment of the present application;

[0028] Figure 2 This is a morphology of the gene-loaded ZIF-8 particles prepared in Example 1 of the present application;

[0029] Figure 3: This is a particle size distribution diagram of the gene-loaded ZIF-8 particles prepared in Example 1 of the present application;

[0030] Figure 4 These are bright field images and fluorescence images of the gene-loaded ZIF-8 particles prepared in Example 1 of the present application in water;

[0031] Figure 5 Infrared spectra of a series of substances provided for this application;

[0032] Figure 6 This is a fluorescence image of the bone repair scaffold prepared in Example 1 of the present application;

[0033] Figure 7 This is a morphological diagram of the bone repair scaffold prepared in Example 1 of the present application;

[0034] Figure 8 This is a morphological image of the bone repair scaffold prepared in Example 2 of the present application;

[0035] Figure 9 This is a morphology diagram of the bone repair scaffold prepared in Comparative Example 1 of the present application;

[0036] Figure 10 This is the gene release curve of the bone repair scaffold prepared in Example 1 of the present application;

[0037] Figure 11 This is the gene release curve of the gene-loaded ZIF-8 prepared in Example 1 of the present application;

[0038] Figure 12 This is the gene release curve of the bone repair scaffold prepared in Comparative Example 3 of this application. DETAILED DESCRIPTION

[0039] To make the purpose, technical solutions and advantages of the examples of the present application clearer, the technical solutions in the examples of the present application will be described clearly and completely below. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0040] It should be noted that “and / or” in this application, such as “Feature 1 and / or Feature 2”, refers to three situations: “Feature 1” alone, “Feature 2” alone, or “Feature 1” plus “Feature 2”.

[0041] In addition, in the description of this application, unless otherwise specified, the meaning of "multiple" in "one or more" refers to two or more; the range of "value a to value b" includes the two end values "a" and "b", and the "unit of measurement" in "value a to value b+unit of measurement" represents the "unit of measurement" of both "value a" and "value b".

[0042] The following is a detailed description of a ZIF-8 gene-loaded hydrogel bone repair scaffold, its preparation method, and its application in the embodiments of the present application.

[0043] First, the present invention provides a bone repair scaffold comprising a ZIF-8 gene-loaded hydrogel, comprising a porous substrate, a hydrogel, and ZIF-8 particles. The porous substrate is a bone implant material; the hydrogel is a sodium alginate hydrogel, coated on the surface of the porous substrate and filling the pores within the porous substrate; and the ZIF-8 particles are distributed within the hydrogel, loaded with a gene that promotes bone growth.

[0044] In the present application, the bone repair scaffold is composed of a porous substrate, a hydrogel and a gene-loaded ZIF-8 particle composition, specifically, the porous substrate can provide basic mechanical adaptability; ZIF-8 particles can provide antibacterial properties by releasing zinc ions on the one hand, and can be used as a carrier and protective layer of genes on the other hand, so that the genes are smoothly delivered to the nucleus and then released and the ability of genes to promote osteogenesis is exerted (wherein, the corresponding load is a gene rather than a protein, and gene editing means are used to make cells express related active factors, which has better economy, controllability and effectiveness than directly delivering proteins); hydrogel can improve the surface hydrophilicity of the scaffold on the one hand, enhance cell adhesion, thereby promoting bone integration, and on the other hand, it can also constitute a controlled release system of a two-stage pH response with ZIF-8, so that the genes are slowly and continuously released in vivo and the ability to promote osteogenesis is exerted. By integrating the above-mentioned multiple functional components together, the bone repair scaffold can be made to have multiple functions such as good antibacterial, promoting bone integration and promoting osteogenesis, so as to better meet the clinical application needs of the bone repair scaffold.

[0045] It should be noted that due to the significant differences in the functional components and preparation processes used in different modification methods, coupled with the fact that different functional components generally have different physical and chemical properties, the integration of multiple modification methods can easily affect the physical and chemical properties or structure of some functional components, making it difficult to effectively perform their inherent functions. Therefore, in the process of integrating multiple modification methods, how to ensure that each modification method can achieve a relatively excellent performance improvement effect is a major challenge currently faced, which is one of the reasons why there are currently few patents or literature related to combined modification.

[0046] Specifically in the examples of the present application, the researchers found that, on the one hand, ZIF-8 is used as a vector, which can effectively load genes and provide protection for them, and it also has relatively excellent antibacterial properties and pH response functions. If another vector is replaced, it may be necessary to additionally add components with antibacterial properties and pH response functions (it should be noted that this application does not mean that components with antibacterial properties and pH response functions cannot be added, but that this application may not add components with antibacterial properties and pH response functions), resulting in a more complex design scheme, a long preparation cycle, and high costs. On the other hand, sodium alginate hydrogel is used, which can improve the bone integration ability of the material and can assist ZIF-8 in realizing the effect of secondary slow control, and it does not involve conditions such as high temperature, strong acid, strong alkali and ultraviolet radiation in the whole preparation process, ZIF-8 and gene can be preferably protected, so that ZIF-8 has good structural integrity and gene maintains good activity, if replaced by other hydrogels, due to the conditions such as high temperature, strong acid, strong alkali and ultraviolet radiation that may be involved in the preparation process, ZIF-8 is easily decomposed in advance and causes gene activity to decrease, then it is difficult to obtain the bone repair scaffold with the multiple functions such as good antibacterial, promoting bone integration and promoting osteogenesis. Therefore, the specific collocation system in the embodiment of the present application is that researchers pay a lot of creative labor to finally obtain.

[0047] It should be noted that bone implant materials refer to biomedical materials used to repair or fill bone defects, or provide fixation and support for fracture sites. They need to have good biocompatibility and be easy to process and shape.

[0048] It should be noted that the porosity and pore size of the porous substrate are not limited and can be set according to conventional selection in the art.

[0049] It should be noted that the specific type of bone implant material is not limited and can be selected according to routine selection in the art.

[0050] As an example, the material of the porous substrate (ie, bone repair material) is selected from at least one of metal, ceramic and polymer materials.

[0051] As an example, the metal is selected from at least one of stainless steel, titanium alloy, and cobalt-chromium alloy.

[0052] As an example, the ceramic is at least one selected from zirconia ceramics, alumina ceramics, calcium phosphate ceramics, zirconium silicon phosphate ceramics, and aluminum oxide ceramics.

[0053] As an example, the polymer material is selected from at least one of polyetheretherketone, polylactic acid and polycaprolactone.

[0054] In this embodiment, when the porous substrate is a polymer material, the material of the polymer material is limited to the above range, which has the advantages of good biocompatibility, excellent mechanical properties and easy processing and molding.

[0055] As an example, the particle size of the ZIF-8 particles is 100 to 300 nm, for example but not limited to any one of 100 nm, 150 nm, 180 nm, 200 nm, 250 nm, 280 nm and 300 nm or a range between any two thereof.

[0056] In this embodiment, the particle size of the ZIF-8 particles is limited to the above-mentioned range so that the ZIF-8 particles loaded with genes have an appropriate size (so that they can adapt to the size of the nuclear pores of the cell membrane), thereby facilitating the safe and smooth delivery of genes into the cell nucleus and allowing the cells to express the corresponding genes, thereby better playing the role of promoting bone formation.

[0057] It should be noted that the type of gene is not limited, as long as it is a gene related to osteogenesis, and can be specifically selected and set according to routine selection in the art.

[0058] As an example, the gene is selected from at least one of pVEGF, pBMP-2 and pTGF-β.

[0059] It should be noted that any structural or functional unit not specifically described or limited in the bone repair scaffold may be configured according to conventional selections in the art.

[0060] In the second aspect, an embodiment of the present application provides a method for preparing a bone repair scaffold of a ZIF-8 gene-loaded hydrogel as provided in the first aspect, comprising the following steps: mixing zinc salt, 2-methylimidazole and gene to obtain a first mixed solution, and then placing the first mixed solution at room temperature for reaction. After the reaction is completed, the solution is subjected to solid-liquid separation and redispersion to obtain a dispersion containing ZIF-8 particles loaded with genes; mixing the dispersion and an aqueous sodium alginate solution to obtain a second mixed solution; immersing the porous substrate in the second mixed solution for immersion treatment, and after the immersion treatment is completed, obtaining a cross-linked precursor solution; adding a cross-linking agent to the cross-linked precursor solution to cross-link the sodium alginate in the cross-linked precursor solution to form a sodium alginate hydrogel, thereby obtaining a bone repair scaffold of a ZIF-8 gene-loaded hydrogel.

[0061] In the present application, the first mixed solution is placed at room temperature to react (i.e., zinc salt and 2-methylimidazole react to form a ZIF-8 vector, and the gene is wrapped inside it), and a ZIF-8 particle loaded with a gene can be formed, and then subjected to solid-liquid separation and redispersion treatment, to achieve constant volume dispersion of the ZIF-8 particle loaded with a gene, so as to facilitate subsequent accurate weighing of the gene according to actual needs; the porous substrate is immersed in the second mixed solution and impregnated, so that the second mixed solution composed of a dispersion liquid and an aqueous sodium alginate solution fully enters and fills the pores of the porous substrate and covers its surface; after the impregnation is completed, a cross-linking agent is added to cross-link sodium alginate to form a sodium alginate hydrogel. According to the above process, it is possible to prepare a bone repair scaffold of a ZIF-8 gene-carrying hydrogel.

[0062] As an example, the step of mixing zinc salt, 2-methylimidazole and gene to obtain a first mixed solution includes first mixing 2-methylimidazole and gene to obtain a first mixed solution intermediate; and then mixing zinc salt and the first mixed solution intermediate to obtain a first mixed solution.

[0063] In other possible implementations, the zinc salt, 2-methylimidazole and the gene may be mixed in a one-step mixing manner to obtain a first mixed solution.

[0064] As an example, the step of placing the first mixed solution at room temperature for reaction is carried out under stirring conditions, wherein the stirring speed is 900 to 1500 rpm, for example but not limited to any one of 900 rpm, 1000 rpm, 1100 rpm, 1200 rpm, 1300 rpm, 1400 rpm and 1500 rpm or a range between any two of them.

[0065] It should be noted that the cross-linking agent needs to be added after the impregnation treatment is completed because the cross-linking reaction is very fast. If the cross-linking agent is added before the impregnation treatment is completed, the pores of the porous substrate cannot be fully and effectively filled with sodium alginate hydrogel.

[0066] It should be noted that the cross-linking step in the embodiment of the present application is not carried out by ultraviolet irradiation (ultraviolet irradiation can easily damage genes, resulting in reduced activity), which can better protect the genes, so that the genes in the prepared bone repair scaffold have better biological activity.

[0067] It should be noted that the type of zinc salt is not limited, and for example, it can be at least one of zinc nitrate, zinc chloride, zinc sulfate, and hydrates thereof.

[0068] It should be noted that the type of cross-linking agent is not limited, and for example, it can be at least one of calcium chloride, calcium sulfate, calcium hydroxide, calcium carbonate and calcium nitrate.

[0069] As an example, in the first mixed solution, the molar ratio of zinc element in the zinc salt to 2-methylimidazole is 1:(45-55), for example, but not limited to, a molar ratio of 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54 and 1:55, or a range value between any two of them; or / and, the ratio of the molar amount of zinc element to the mass of the gene is 6.25 μmol:(20-40) μg, for example, but not limited to, a ratio of 6.25 μmol:20 μg, 6.25 μmol:25 μg, 6.25 μmol:30 μg, 6.25 μmol:35 μg and 6.25 μmol:40 μg, or a range value between any two of them.

[0070] In this embodiment, the molar ratio of zinc element and 2-methylimidazole in the first mixed solution is limited to the above range, so that the formed ZIF-8 particles contain zinc ions with a relatively suitable mass ratio, thereby making the scaffold have a relatively suitable antibacterial property; the ratio of the molar amount of zinc element to the mass of gene is limited to the above range, so that the formed ZIF-8 particles loaded with genes carry genes with a relatively suitable mass ratio, thereby making the scaffold have a relatively suitable osteogenic ability; at the same time, the appropriate amount of gene makes the surface potential of ZIF-8 appropriately reduced while maintaining positive charge, which is beneficial for delivering genes into the cell nucleus and reducing cytotoxicity (the cell membrane surface is negatively charged, and moderate positive charge can promote the interaction between nanocarriers and cell membranes through electrostatic adsorption, enhance endocytosis, and thus improve gene delivery efficiency; at the same time, appropriately reducing the surface potential can reduce the damage to the membrane structure caused by the positively charged carrier during the binding process with the cell membrane and maintain a relatively suitable electrostatic adsorption capacity).

[0071] As an example, the molar concentration of zinc element in the first mixed solution is 0.02-0.03 mol / L, for example, but not limited to, the molar concentration is any one of 0.02 mol / L, 0.021 mol / L, 0.022 mol / L, 0.023 mol / L, 0.024 mol / L, 0.025 mol / L, 0.026 mol / L, 0.027 mol / L, 0.028 mol / L, 0.029 mol / L and 0.03 mol / L, or a range between any two of them.

[0072] In this embodiment, limiting the molar concentration of zinc element in the first mixed solution to the above range is conducive to sufficient reaction of the raw materials, and the appropriate concentration also helps to form particles with a more appropriate particle size.

[0073] As an example, in the step of placing the first mixed solution at room temperature for reaction, the reaction time is 15 to 120 minutes, for example, but not limited to, any one of 15 minutes, 30 minutes, 60 minutes, 90 minutes and 120 minutes, or a range between any two of them.

[0074] In this embodiment, during the preparation of gene-loaded ZIF-8 particles, limiting the reaction time to the above range helps to form particles with a particle size closer to 200 nm, thereby facilitating the gene-loaded ZIF-8 to enter the cell nucleus through endocytosis.

[0075] As an example, in the impregnation step, the impregnation time is 10 to 30 minutes, for example but not limited to any one of 10 minutes, 15 minutes, 20 minutes, 25 minutes and 30 minutes, or a range between any two of them; or / and, in the crosslinking step, the treatment time is 5 to 20 minutes, for example but not limited to any one of 5 minutes, 10 minutes, 15 minutes and 20 minutes, or a range between any two of them.

[0076] In this embodiment, the duration of the immersion treatment step is limited to the above range so that the second mixed solution composed of the dispersion and the sodium alginate aqueous solution can more fully enter and fill the pores of the porous substrate and more comprehensively cover its surface; in addition, the duration of the cross-linking step is limited to the above range, which helps the sodium alginate to fully cross-link to form a sodium alginate hydrogel.

[0077] As an example, in the second mixed solution, the ratio of the gene concentration to the sodium alginate concentration is 1:(1.5~2.5), for example, but not limited to, the concentration ratio is any one of 1:1.5, 1:1.75, 1:2, 1:2.25 and 1:2.5 or a range value between any two of them.

[0078] In this embodiment, the concentration ratio of the gene and sodium alginate in the second mixed solution is limited to the above range so that the two have an appropriate mass ratio, which helps to fully mix the gene-loaded ZIF-8 particles and sodium alginate, and thus allows the sodium alginate hydrogel formed by subsequent cross-linking to be more completely wrapped on the particle surface.

[0079] As an example, the mass concentration of sodium alginate in the second mixed solution is (1 to 3) μg / 100 μL, for example, but not limited to, a mass concentration of any one of 1 μg / 100 μL, 1.5 μg / 100 μL, 2 μg / 100 μL, 2.5 μg / 100 μL and 3 μg / 100 μL, or a range between any two of them.

[0080] In this embodiment, the mass concentration of sodium alginate in the second mixed solution is limited to the above range. The appropriate concentration helps the sodium alginate to be subsequently efficiently and thoroughly cross-linked to form a sodium alginate hydrogel.

[0081] As an example, in the cross-linking precursor solution, the ratio of the concentration of sodium alginate to the concentration of the cross-linking agent is 5:(1~3), for example, but not limited to, the concentration ratio is any one of 5:1, 5:1.5, 5:2, 5:2.5 and 5:3 or a range value between any two of them.

[0082] In this embodiment, the concentration ratio of sodium alginate and cross-linking agent in the cross-linking precursor solution is limited to the above range, which helps the sodium alginate to cross-link to form a sodium alginate hydrogel, and also enables the sodium alginate hydrogel formed after cross-linking to have a more suitable cross-linking density, thereby providing a more suitable sustained release time (the more cross-linking agent, the greater the cross-linking density, and the longer the sustained release time; conversely, the less cross-linking agent, the smaller the cross-linking density, and the shorter the sustained release time).

[0083] As an example, if the gene is pVEGF, the ratio of the concentration of sodium alginate to the concentration of the cross-linking agent is 5:1; if the gene is pBMP-2 or / and pTGF-β, the ratio of the concentration of sodium alginate to the concentration of the cross-linking agent is 5:3.

[0084] In this embodiment, the ratio of the concentrations of sodium alginate and the cross-linking agent is further limited for different types of genes so as to better match the duration of action of different types of genes (wherein the duration of action required for pVEGF is shorter, and the duration of action required for pBMP-2 and / or pTGF-β is longer).

[0085] It should be noted that after the cross-linking treatment is completed, a step of washing with water to remove the cross-linking agent is also included.

[0086] It should be noted that any process or step not specifically described or limited in the preparation of the bone repair scaffold may be arranged according to conventional selection in the art.

[0087] As an example, before the step of immersing the porous substrate in the second mixed solution for immersion treatment, the porous substrate is pretreated by sulfonation treatment, anodization treatment or plasma activation treatment.

[0088] In this embodiment, pre-treating the porous substrate can increase its hydrophilicity, cell compatibility and antibacterial properties.

[0089] As an example, the process flow chart of the preparation method of the bone repair scaffold is shown in FIG. Figure 1 .

[0090] In a third aspect, an embodiment of the present application provides a use of a bone repair scaffold of a ZIF-8 gene-loaded hydrogel as provided in the first aspect in the preparation of a bone implant device.

[0091] It should be noted that the type of bone implant device is not limited, and it can be, for example, a joint fixation device or a joint replacement device.

[0092] The features and performance of the present application are further described in detail below with reference to the embodiments.

[0093] Example 1

[0094] The present invention provides a method for preparing a bone repair scaffold, comprising the following steps:

[0095] S1: 2-Methylimidazole (125 μL, 2.5 mol / L) and a gene (20 μg of pVEGF) were first mixed to obtain a first mixed solution intermediate; then zinc nitrate hexahydrate (125 μL, 0.05 mol / L) was mixed with the first mixed solution intermediate to obtain a first mixed solution. The first mixed solution was then placed at room temperature and stirred at 1000 rpm for 30 minutes. After the reaction was completed, solid-liquid separation was performed by centrifugation, and the collected material was dispersed in 50 μL of sterile water to obtain a dispersion containing gene-loaded ZIF-8 particles, wherein the concentration of pVEGF in the dispersion was 0.4 μg / μL.

[0096] S2: 1.25 μL of the above dispersion (containing 0.5 μg of pVEGF) and a sodium alginate aqueous solution (100 μL, concentration of 1 μg / 100 μL) were mixed to obtain a second mixed solution.

[0097] S3 3D printed porous substrate (made of polyetheretherketone, with a volume of 3mm 3 ) Surface modification is performed by a sulfonation process, specifically, the porous substrate is immersed in 98% concentrated sulfuric acid at room temperature for 3 minutes, then the porous substrate is rinsed with deionized water for 1 minute, and then the washed porous substrate is transferred to a reactor containing deionized water and heated at 120° C. for 12 hours to further remove residual sulfuric acid, thereby obtaining an activated porous substrate; then, the activated porous substrate is immersed in a second mixed solution for 15 minutes, and after the immersion treatment is completed, a cross-linked precursor solution is obtained;

[0098] S4 adds a calcium chloride aqueous solution (20 μL, concentration of 1 μg / 100 μL) to the cross-linking precursor solution to cross-link the sodium alginate in the cross-linking precursor solution to form a sodium alginate hydrogel, which is then washed with water to remove the cross-linking agent to obtain a ZIF-8 gene-loaded hydrogel bone repair scaffold.

[0099] Example 2

[0100] This embodiment of the present application provides a method for preparing a bone repair scaffold, which differs from Example 1 only in that the material of the porous substrate is replaced with titanium alloy.

[0101] Comparative Example 1

[0102] The comparative example of the present application provides a method for preparing a bone repair scaffold, comprising the following steps:

[0103] The porous substrate obtained by 3D printing (made of polyetheretherketone, with a volume of 3mm 3 ) The surface of the porous substrate was modified by a sulfonation process. The specific process was as follows: the porous substrate was immersed in 98% concentrated sulfuric acid at room temperature for 3 minutes, and then washed with water to remove the residual sulfuric acid to obtain a bone repair material.

[0104] Comparative Example 2

[0105] This embodiment of the present application provides a method for preparing a bone repair scaffold, which differs from Example 1 only in that pVEGF is not added in step S1.

[0106] Comparative Example 3

[0107] The embodiment of the present application provides a method for preparing a bone repair scaffold, which differs from Example 1 only in that: in step S2, the sodium alginate aqueous solution is replaced with a carboxymethyl chitosan aqueous solution (due to the presence of carboxyl groups, the corresponding solution is acidic).

[0108] Test example

[0109] (1) The gene-loaded ZIF-8 particles prepared in Example 1 were used as samples, and their morphology, particle size distribution, and fluorescence images were tested respectively.

[0110] See Figure 2 、 Figure 3 and Figure 4 , among which, Figure 2 and Figure 3 It can be seen that the particle size of ZIF-8 particles loaded with genes is relatively uniform, and the particle size is mainly around 200 nm. Figure 4 It can be seen (the left side is the bright field image of ZIF-8 particles loaded with genes in water, the right side is the CLSM image of ZIF-8 particles loaded with genes, and the red one is the gene stained with Cy3 fluorescence) that the gene is successfully loaded inside ZIF-8.

[0111] (2) The bone repair scaffold prepared in Example 1 was used as a sample, and its FTIR spectrum and fluorescence spectrum were tested.

[0112] See Figure 5 (Infrared spectra of ZIF-8 alone, sodium alginate hydrogel alone, and bone repair scaffolds) and Figure 6 (CLSM image of bone repair scaffold, red is the gene stained by Cy3 fluorescence), where Figure 5 It shows that the characteristic peaks of ZIF-8 were detected in the bone repair scaffold. Figure 6 It showed that the gene-loaded ZIF-8 particles were distributed in the hydrogel.

[0113] (3) The bone repair scaffolds prepared in Examples 1 to 2 and Comparative Example 1 were used as samples, and their morphologies were tested.

[0114] See Figure 7 、 Figure 8 and Figure 9 It can be seen that the pores of the bone repair scaffolds in Examples 1 and 2 are filled with hydrogel, and the surface is also covered with hydrogel; while the surface of the bone repair scaffold prepared in Comparative Example 1 has many pores but no filler.

[0115] (4) The bone repair scaffolds prepared in Example 1 and Comparative Example 3 were used as samples. At the same time, the gene-loaded ZIF-8 particles obtained in step S1 of Example 1 were used as controls, and their gene release curves were then tested.

[0116] See Figure 10 、 Figure 11 and Figure 12 ,Depend on Figure 10 and Figure 11 By comparison, Figure 10 The sustained release of genes lasted up to 10 days. Figure 11 The sustained release of genes in the solution lasted only a few hours, indicating that the use of sodium alginate hydrogel for coating and combining with ZIF-8 to form a secondary sustained release system can effectively prolong the sustained action time of genes. Figure 10 and Figure 12 By comparison, Figure 10 The sustained release of genes lasted up to 10 days. Figure 12 The amount of gene released is getting smaller and smaller. The reason is that the carboxymethyl chitosan aqueous solution is acidic, which will cause ZIF-8 to degrade in advance and release genes during the preparation process and also cause gene inactivation, so that there are few or even no genes loaded in the bone repair scaffold finally prepared. In addition, the content test process is also accompanied by the continuous reduction of genes (it is necessary to continuously take out the sample solution and replenish water), which leads to the release of genes over time. In other words, in the specific system constructed by the embodiment of the present application, sodium alginate hydrogel is a necessary choice and cannot be replaced at will.

[0117] (5) Performance testing of bone repair scaffolds

[0118] Example 1, Example 2, Comparative Example 1 and Comparative Example 2 were used as samples, and the porous substrate polyetheretherketone (not activated) was used as a control. The water contact angle (i.e., bone integration ability), antibacterial property, and osteogenic ability of each sample were tested (including angiogenesis-promoting ability and alkaline phosphatase activity. Among them, the initial stage of osteogenesis is angiogenesis, which facilitates the transportation of various nutrients for osteogenesis. Therefore, the strong angiogenesis-promoting ability of the bone repair scaffold can indicate its strong osteogenic ability. The enhanced activity of alkaline phosphatase (ALP) indicates the differentiation of pre-osteoblasts into osteoblasts and the beginning of extracellular matrix calcification. Therefore, the higher the ALP activity, the stronger the osteogenic ability). The results are then statistically summarized in Table 1.

[0119] A. The contact angle test steps are as follows:

[0120] The contact angle test is a common method for characterizing the hydrophilic and hydrophobic properties of a material surface based on the Young-Laplace equation for the energy balance analysis of the solid-liquid-gas three-phase interface. The specific steps are as follows: First, fix the sample to be tested horizontally on a temperature-controlled platform, and use a microsyringe to accurately drip a 2μL droplet of deionized water on the surface. Let it stand for 3 minutes to reach the interface equilibrium state, and then use a CCD high-definition camera system equipped with a telephoto lens to capture the droplet contour image. Use VCA Optima image analysis software to automatically identify the three-phase contact point of the droplet, and calculate the contact angle value by ellipse fitting method. According to Young's wetting theory, a contact angle θ>90° indicates that the material surface is hydrophobic; θ<90° indicates hydrophilic properties. By comparing the changes in the contact angle of the material before and after modification, the surface wettability of the bone repair scaffold can be quantitatively evaluated.

[0121] B Antibacterial performance test steps are as follows:

[0122] Count colonies using the plate spreading method: Place the sample to be tested and the LB solid culture medium plate to be spread under ultraviolet light for 15 minutes. Prepare 100 μl of the concentration of 1x10 7 cfu / ml bacterial suspension, inoculate 1mL of the suspension on the surface of the sample to be tested, then co-culture at 37°C for 24 hours, and sonicate for 5 minutes to elute the bacteria adhered to the support into the solution. Take 1mL of the co-culture bacterial solution and use sterile water to dilute the original co-culture bacterial solution in a gradient until 4 dilutions of different concentrations are obtained. Take 100μL of the dilutions of different concentrations and drop them onto the LB solid culture medium plate prepared in advance and sterilized by UV. Use an L-shaped spreading rod to evenly spread the bacterial solution on the plate and transfer it to a bacterial incubator for inverted culture. After culturing for 24 hours, observe and take pictures and use Image J to count the number of colonies.

[0123] The steps for the C angiogenesis test are as follows:

[0124] Use Transwell to co-culture the sample and cells. Use the standard cell passage process to select cells that have been passaged for 3 to 5 generations and are in good condition. Culture the cells until they cover 80% of the area of the culture dish within 24 hours. Next, replace the complete culture medium with DMEM culture medium containing 0.2% FBS and incubate for another 24 hours. At the same time, take out the matrix gel from the -20°C refrigerator and move it to a 4°C refrigerator for thawing. After thawing, use a pre-cooled pipette or gun tip to thoroughly mix the matrix gel. Make sure that the completely thawed matrix gel is placed on ice to prevent it from gelling prematurely. Add 20-30μL of matrix gel to each well, then transfer the 96-well plate covered with matrix gel to a cell culture incubator and incubate at 37°C for 30 minutes to promote the formation of basement membrane gel.

[0125] When the HUVECs cell confluence in the culture dish reached 80%, they were digested, centrifuged, and counted. Single-cell suspension was prepared according to standard procedures, and the cell density was accurately measured with the help of a cell counting plate. The cell density was adjusted to 3×10-5×10 cells / mL, and the culture medium used was ECM culture medium. Quantitative inoculation was performed in a 96-well plate, and 100 μL of cell suspension was added to each well (i.e., each well contained 3×10-5×10 cells). The 96-well plate was then transferred to an incubator at a constant temperature of 37°C, a 5% CO concentration, and a relative humidity of 90% for continued culture.

[0126] Phase contrast microscopy was used to dynamically monitor cell attachment and spreading at 2, 4, 6, and 8 hours after intervention. Three images were acquired for each well using a 10x objective lens for subsequent quantitative analysis. Quantitative analysis was performed using the Angiogenesis Analyzer plugin in Image J.

[0127] D. The steps for alkaline phosphatase activity test are as follows:

[0128] The samples were placed in osteogenic induction medium (containing 10 mM sodium β-glycerophosphate, 50 μg / mL ascorbic acid, and 0.1 μM dexamethasone) and cocultured with cells at 37°C and 5% CO for 7 days. The three groups of cocultured cells were then stained using an ALP staining kit, and ALP activity was quantitatively analyzed using an alkaline protease (AKP) assay kit.

[0129] Table 1

[0130]

[0131] Referring to Table 1, it can be seen from the test results of Examples 1-2, Comparative Examples 1-2 and the control group that the bone repair scaffold constructed according to the specific system in the embodiments of the present application, specifically the bone repair scaffold of ZIF-8 gene-loaded hydrogel, has multiple functions such as better antibacterial, promoting bone integration and promoting osteogenesis, compared with the use of a single modification method.

[0132] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

Claims

1. A ZIF-8 gene-loaded hydrogel bone repair scaffold, characterized in that: include: A porous substrate, wherein the porous substrate is a bone implant material; A hydrogel, wherein the hydrogel is a sodium alginate hydrogel, and the hydrogel is coated on the surface of the porous substrate and filled in the pores of the porous substrate; ZIF-8 particles are distributed in the hydrogel, and the ZIF-8 particles are loaded with genes, wherein the genes are osteogenic genes.

2. The bone repair scaffold of ZIF-8 gene-loaded hydrogel according to claim 1, characterized in that: The particle size of the ZIF-8 particles is 100 to 300 nm.

3. The bone repair scaffold of the ZIF-8 gene-loaded hydrogel according to claim 1, characterized in that: The gene is selected from at least one of pVEGF, pBMP-2 and pTGF-β; or / and the material of the porous substrate is selected from at least one of metal, ceramic and polymer materials; Optionally, the polymer material is selected from at least one of polyetheretherketone, polylactic acid and polycaprolactone.

4. A method for preparing a bone repair scaffold of a ZIF-8 gene-loaded hydrogel according to any one of claims 1 to 3, characterized in that: The following steps are involved: Mixing zinc salt, 2-methylimidazole and the gene to obtain a first mixed solution, then placing the first mixed solution at room temperature to react, and after the reaction is completed, performing solid-liquid separation and redispersion to obtain a dispersion containing the ZIF-8 particles loaded with the gene; mixing the dispersion and a sodium alginate aqueous solution to obtain a second mixed solution; immersing the porous substrate in the second mixed solution for immersion treatment, and obtaining a cross-linking precursor solution after the immersion treatment is completed; A cross-linking agent is added to the cross-linked precursor solution to cross-link the sodium alginate in the cross-linked precursor solution to form the sodium alginate hydrogel, thereby obtaining the bone repair scaffold of the ZIF-8 gene-carrying hydrogel.

5. The method for preparing the bone repair scaffold of the ZIF-8 gene-loaded hydrogel according to claim 4, wherein: In the first mixed solution, the molar ratio of the zinc element in the zinc salt to the 2-methylimidazole is 1:(45-55); or / and the ratio of the molar amount of the zinc element to the mass of the gene is 6.25 μmol:(20-40) μg; Optionally, the molar concentration of the zinc element in the first mixed solution is 0.02 to 0.03 mol / L.

6. The method for preparing the bone repair scaffold of ZIF-8 gene-loaded hydrogel according to claim 5, characterized in that: In the step of placing the first mixed solution at room temperature for reaction, the reaction time is 15 to 120 minutes.

7. The method for preparing the bone repair scaffold of ZIF-8 gene-loaded hydrogel according to claim 4, characterized in that: In the step of immersion treatment, the duration of the immersion treatment is 10 to 30 minutes; Or / and, in the cross-linking step, the treatment time is 5 to 20 minutes.

8. The method for preparing the bone repair scaffold of ZIF-8 gene-loaded hydrogel according to claim 4, characterized in that: In the second mixed solution, the ratio of the concentration of the gene to the concentration of the sodium alginate is 1:(1.5-2.5); Optionally, the mass concentration of the sodium alginate in the second mixed solution is (1-3) μg / 100 μL.

9. The method for preparing the bone repair scaffold of ZIF-8 gene-loaded hydrogel according to claim 7, characterized in that: In the cross-linking precursor solution, the ratio of the concentration of the sodium alginate to the concentration of the cross-linking agent is 5:(1-3); Optionally, the gene is pVEGF, and the ratio of the concentration of sodium alginate to the concentration of the cross-linking agent is 5:1; Optionally, the gene is pBMP-2 or / and pTGF-β, and the ratio of the concentration of sodium alginate to the concentration of the cross-linking agent is 5:

3.

10. Use of the bone repair scaffold of the ZIF-8 gene-loaded hydrogel according to any one of claims 1 to 3 in the preparation of a bone implant device.

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