Cell-loaded bionic bone scaffold with oxygen self-production and bacteriostasis effects as well as preparation method and application of cell-loaded bionic bone scaffold

Abstract

The invention provides a cell-loaded bionic bone scaffold with oxygen self-production and bacteriostasis effects as well as a preparation method and application thereof, and belongs to the technical field of bone tissue engineering. The bionic bone scaffold provided by the invention is formed by stacking and assembling a plurality of cylindrical bone scaffold units, has a natural bone simulated Hauses tube structure, has good biocompatibility and excellent mechanical properties, and can accurately simulate the pore and structural characteristics of a natural bone. The functional GelMA hydrogel filled in the grooves is loaded with different cells, biological factors and antibacterial drugs, so that osteogenesis and vascularization effects at specific positions of the stent are realized, formation of a vascularization network and bone repair by simulating a natural bone structure of a human body are facilitated, and a good antibacterial effect is achieved. According to the invention, the cylindrical bone scaffold unit is loaded with the self-oxygen-producing MOFs particles, and the self-oxygen-producing effect of slowly releasing oxygen can be achieved after the cylindrical bone scaffold unit is implanted in vivo, so that bone marrow mesenchymal stem cell osteogenic differentiation and vascularization network construction are promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of bone tissue engineering, and in particular to a cell-laden bionic bone scaffold with self-oxygenation and antibacterial effects, and a preparation method and application thereof. Background Art

[0002] Bone defects are a common clinical condition, usually caused by various factors such as trauma, tumor resection, and congenital bone dysplasia, which seriously affect patients' daily lives. Although bone tissue has a certain self-repair ability, the body's own repair mechanism can play a role in achieving autologous repair for smaller bone defects. However, large bone defects cannot be repaired through autologous growth due to the relative lack of local osteoblasts, the large distance between the bone ends making it difficult to form callus, and the severe damage to blood vessels in the defect area, which hinders the delivery of nutrients and oxygen.

[0003] In the treatment of large bone defects, traditional treatments, such as autologous bone transplantation and allogeneic bone transplantation, can repair bone defects to a certain extent, but they have many limitations. The development of bone tissue engineering technology has provided a new technical approach for the treatment of large bone defects. By using biomaterials (such as biodegradable polymers and bioceramics), bone scaffolds with specific structures and properties are constructed. Functional cells or drugs are then added to the scaffolds and implanted into the bone defect. During this process, the scaffolds provide space for cells to attach and grow, promoting the formation of new bone tissue and achieving bone regeneration in large bone defects.

[0004] However, current bone scaffolds have many limitations. In terms of microstructure, it is difficult to accurately simulate the microstructure of natural bone, and the gradient design, porosity, pore size and connectivity are not reasonable enough; in terms of macrostructure, it is impossible to accurately simulate the structural composition of natural bone and the biocompatibility is poor. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a cell-loaded bionic bone scaffold with self-oxygenation and antibacterial effects, as well as its preparation method and application. The bionic bone scaffold provided by the present invention has good biocompatibility, excellent mechanical properties, can accurately simulate the pores and structural characteristics of natural bones, and has good osteogenesis and vascularization effects.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a cell-loaded bionic bone scaffold with self-oxygenation and antibacterial effects, comprising a plurality of cylindrical bone scaffold units assembled in layers;

[0008] One side surface of a single cylindrical bone support unit is provided with one or more grooves, and the other side surface is provided with one or more protrusions, and adjacent cylindrical bone support units are fixed by the grooves and protrusions;

[0009] The raw materials for preparing the cylindrical bone scaffold unit include methacrylic anhydride gelatin and amino-modified hydroxyapatite nanopowder;

[0010] The cylindrical bone scaffold unit has a loose porous structure;

[0011] The surface and pores of the cylindrical bone scaffold unit are loaded with self-generating MOFs particles, wherein the self-generating MOFs particles include ZIF-8 and CaO2 encapsulated in the internal pores of the ZIF-8;

[0012] The groove of the cylindrical bone scaffold unit is filled with functional GelMA hydrogel, which contains cells, growth factors and antibacterial drugs. The cells include bone marrow mesenchymal stem cells and / or vascular endothelial cells.

[0013] Preferably, the grooves or protrusions on the surface of the single cylindrical bone scaffold unit are in the shape of rings, and the number thereof is 2 to 5;

[0014] The pore size of the cylindrical bone scaffold unit is 80-120 μm.

[0015] Preferably, the mass ratio of the GelMA to the amino-modified hydroxyapatite nanopowder is (10-12):(1-1.5).

[0016] Preferably, the method for preparing the self-generating oxygen MOFs particles comprises the following steps:

[0017] Calcium chloride, polyvinyl pyrrolidone, hydrogen peroxide, ammonia water and alcohol solvent are mixed to carry out precipitation reaction to obtain CaO2-PVP precipitate;

[0018] The CaO2-PVP precipitate, zinc nitrate and 2-methylimidazole are mixed and subjected to a self-assembly reaction to obtain self-generating MOFs particles.

[0019] Preferably, the concentration of bone marrow mesenchymal stem cells in the functional GelMA hydrogel is 1×10 5 ~3×10 5 cells / mL, and the concentration of endothelial cells was 5×10 5 ~1×10 6 cells / mL, the mass concentration of growth factors is 10-300 ng / mL, and the mass concentration of antibacterial drugs is 5-10 μg / mL.

[0020] The present invention provides a method for preparing the above-mentioned cell-loaded bionic bone scaffold with self-oxygenation and antibacterial effects, comprising the following steps:

[0021] GelMA, amino-modified hydroxyapatite nanopowder, buffer solution and photoinitiator are mixed to obtain a composite ink;

[0022] 3D printing and UV curing the composite ink to obtain a cylindrical bone scaffold unit matrix;

[0023] Immersing the cylindrical bone scaffold unit matrix in a dispersion of self-generating MOFs particles for loading to obtain a cylindrical bone scaffold unit;

[0024] Functional GelMA hydrogel is filled in the groove of the cylindrical bone scaffold unit, and multiple cylindrical bone scaffold units are stacked and assembled to obtain a cell-laden bionic bone scaffold with self-oxygenation and antibacterial effects.

[0025] Preferably, the method for preparing the amino-modified hydroxyapatite nanopowder comprises the following steps:

[0026] The hydroxyapatite nanopowder, KH550 and an alcohol solvent are mixed and subjected to a grafting reaction to obtain amino-modified hydroxyapatite nanopowder.

[0027] Preferably, the 3D printing is extrusion 3D printing, and the parameters of the 3D printing include:

[0028] The nozzle diameter is 0.3mm;

[0029] Extrusion pressure is 0.15~0.18MPa;

[0030] UV light cross-linking is used in the 3D printing process;

[0031] The wavelength of the UV light crosslinking is 365-405 nm, and the light intensity is 15-20 mW / cm 2 ,The irradiation time of each layer of 3D printing material is 15 to 30 seconds.

[0032] Preferably, the concentration of the self-generating MOFs particle dispersion is 20 to 25 mg / mL;

[0033] The load time is 12 to 16 hours.

[0034] The present invention provides the use of the cell-laden bionic bone scaffold with self-oxygenation and antibacterial effects in the preparation of bone and cartilage tissue repair materials.

[0035] The present invention provides a cell-loaded bionic bone scaffold with self-oxygenating and antibacterial effects, comprising a plurality of cylindrical bone scaffold units assembled in a stacked manner; one side surface of a single cylindrical bone scaffold unit is provided with one or more grooves, and the other side surface is provided with one or more protrusions, and adjacent cylindrical bone scaffold units are clamped and fixed by the grooves and protrusions; the raw materials for preparing the cylindrical bone scaffold units include GelMA and amino-modified hydroxyapatite; the cylindrical bone scaffold units have a loose porous structure; the surface and pores of the cylindrical bone scaffold units are loaded with self-oxygenating MOFs particles, the self-oxygenating MOFs particles include ZIF-8 and CaO2 encapsulated in the internal pores of the ZIF-8; the grooves of the cylindrical bone scaffold units are filled with functional GelMA hydrogel, and the functional GelMA hydrogel contains cells, growth factors and antibacterial drugs, and the cells include bone marrow mesenchymal stem cells and / or vascular endothelial cells. The biomimetic bone scaffold provided by the present invention is assembled from multiple cylindrical scaffold units, mimicking the Haversian canal structure of natural bone. It can be easily assembled into any size, allowing for the creation of a scaffold optimally suited to the bone defect site before implantation. The cylindrical scaffold units are composed of methacrylated gelatin (GelMA) and amino-modified hydroxyapatite (nHAP) nanopowder. Methacrylated gelatin has excellent biocompatibility and is non-toxic to living organisms, while hydroxyapatite, a major inorganic component of the extracellular matrix, not only exhibits excellent biocompatibility but also enhances the structural strength of the scaffold.

[0036] In the present invention, the grooves of the cylindrical bone scaffold unit can be used for the protrusion assembly connection of adjacent cylindrical bone scaffold units, and can also be used to fill functional GelMA hydrogel, which is loaded with different cells, biological factors and antibacterial drugs, thereby performing osteogenesis and vascularization effects at specific positions of the scaffold, which is beneficial to form a vascularized network and bone repair by imitating the natural bone structure of the human body, and has good antibacterial efficacy. In the present invention, the cylindrical bone scaffold unit is loaded with self-generating MOFs particles, which can play a self-generating oxygen effect of slowly releasing oxygen after implantation in the body, thereby promoting the osteogenic differentiation of bone marrow mesenchymal stem cells and the construction of a vascularized network. The present invention can load different cells and growth factors according to different parts of the scaffold, and has important practical significance and clinical application value.

[0037] The present invention provides a method for preparing the aforementioned cell-laden biomimetic bone scaffold with self-oxygenating and antibacterial properties. This method utilizes 3D printing technology to print cylindrical bone scaffold units of predetermined dimensions, with precise pore size control, enabling easy simulation of the porosity and structural characteristics of natural bone. Furthermore, the preparation method provided by the present invention is simple to operate and readily adaptable to industrialized mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1Schematic diagram of the composition structure of the bionic bone scaffold unit in the present invention;

[0039] Figure 2 Schematic diagram of the preparation of ZIF-8-CaO2 particles in the present invention;

[0040] Figure 3 Flow chart of the preparation of GelMA in the present invention;

[0041] Figure 4 Flow chart of the preparation of modified HAP nanopowder in the present invention;

[0042] Figure 5 Flow chart of the preparation of GelMA / nHAP composite ink in the present invention;

[0043] Figure 6 This is a flow chart for preparing the cylindrical bone scaffold unit of the present invention;

[0044] Figure 7 This is a schematic diagram of the assembly of the bionic bone scaffold of the present invention;

[0045] Figure 8 This is a diagram showing the mechanism of the bone repair process of the bionic bone scaffold in the present invention. DETAILED DESCRIPTION

[0046] The present invention provides a cell-loaded bionic bone scaffold with self-oxygenation and antibacterial effects, comprising a plurality of cylindrical bone scaffold units assembled in layers;

[0047] One side surface of a single cylindrical bone support unit is provided with one or more grooves, and the other side surface is provided with one or more protrusions, and adjacent cylindrical bone support units are fixed by the grooves and protrusions;

[0048] The raw materials for preparing the cylindrical bone scaffold unit include GelMA and amino-modified hydroxyapatite nanopowder;

[0049] The cylindrical bone scaffold unit has a loose porous structure;

[0050] The surface and pores of the cylindrical bone scaffold unit are loaded with self-generating MOFs particles, wherein the self-generating MOFs particles include ZIF-8 and CaO2 encapsulated in the internal pores of the ZIF-8;

[0051] The groove of the cylindrical bone scaffold unit is filled with functional GelMA hydrogel, which contains cells, growth factors and antibacterial drugs. The cells include bone marrow mesenchymal stem cells and / or vascular endothelial cells.

[0052] In the present invention, the number of the plurality of cylindrical bone scaffold units is determined according to the size of the bone defect. The greater the number of the plurality of cylindrical bone scaffold units, the higher the height of the bone scaffold. As a specific embodiment of the present invention, the number of the plurality of cylindrical bone scaffold units is preferably 5 to 7.

[0053] In the present invention, one or more grooves are provided on one side surface of the single cylindrical bone scaffold unit. In the present invention, the grooves on the surface of the single cylindrical bone scaffold unit are preferably annular in shape, and the number is preferably 2 to 5, more preferably 3. In the present invention, when there are multiple annular grooves, the multiple annular grooves are preferably arranged concentrically from the inside to the outside. As a specific embodiment of the present invention, the height of the single cylindrical bone scaffold unit is 4.0±0.1mm, the diameter is 12.0±0.1mm, the outer diameter of the outer annular groove is 10.8±0.1mm, and the inner diameter is 7.6±0.1mm. The outer diameter of the middle annular groove is 6.0±0.1mm, and the inner diameter is 2.8±0.1mm. The outer diameter of the inner annular groove is 1.6±0.1mm. The groove dimensions are all 1.6±0.1mm in width and 2.5±0.1mm in depth.

[0054] In the present invention, the other side surface of the single cylindrical bone scaffold unit is provided with one or more protrusions, and the position and number of the protrusions match the position and number of the adjacent cylindrical bone scaffold units. In the present invention, the depth of the groove is preferably greater than the height of the protrusion to facilitate the filling of the functional GelMA hydrogel. As a specific embodiment of the present invention, the protrusions are 1.6±0.1mm in width and 1.0±0.1mm in height.

[0055] In the present invention, the cylindrical bone scaffold unit has a loose porous structure, the pore size is preferably 80-120 μm, more preferably 90-100 μm, and the porosity is preferably 85-90%.

[0056] In the present invention, the cylindrical bone scaffold unit is prepared from raw materials comprising methacrylated gelatin (GelMA) and amino-modified hydroxyapatite nanopowder (nHAP). The particle size of the amino-modified hydroxyapatite nanopowder is preferably 50 to 200 nm, more preferably 100 nm. The mass ratio of GelMA to amino-modified hydroxyapatite nanopowder is preferably (10 to 12):(1 to 1.5), more preferably 10:1.

[0057] In the present invention, the method for preparing the amino-modified hydroxyapatite nanopowder preferably comprises the following steps:

[0058] The hydroxyapatite nanopowder, KH550 and an alcohol solvent are mixed and subjected to a grafting reaction to obtain amino-modified hydroxyapatite nanopowder.

[0059] In the present invention, the particle size of the hydroxyapatite nanopowder is preferably 50 to 200 nm. Prior to mixing, the hydroxyapatite nanopowder is preferably subjected to an acid wash treatment. The acidic agent used in the acid wash treatment is preferably a 0.1 to 0.15 M HCl solution, and the acid wash time is preferably 1.5 to 2 hours. After the acid wash treatment, the acid-washed hydroxyapatite nanopowder is preferably centrifuged, washed with water, and dried.

[0060] In the present invention, the alcohol solvent is preferably an ethanol solution with a volume concentration of 50%. The ratio of the hydroxyapatite nanopowder, KH550, and alcohol solvent is preferably 5.0-7.5 g: 1-1.5 mL: 10-15 mL, more preferably 6.0-7.0 g: 1.2-1.4 mL: 12-14 mL. In the present invention, the KH550 and alcohol solvent are preferably premixed before adding the hydroxyapatite nanopowder to the premixed solution. In the present invention, the premixing is preferably performed by ultrasonic mixing.

[0061] In the present invention, the grafting reaction is preferably carried out under stirring and in the dark. The temperature of the grafting reaction is preferably 65-70°C, more preferably 66-68°C, and the time is preferably 5-6 hours. After the grafting reaction is completed, the grafting reaction solution is preferably centrifuged, washed, and dried, and the resulting amino-modified hydroxyapatite nanopowder is stored in the dark.

[0062] In the present invention, the surface and pores of the cylindrical bone scaffold units are loaded with self-oxygenating MOF particles, which include ZIF-8 and CaO2 encapsulated in the internal pores of the ZIF-8. In the present invention, the particle size of the self-oxygenating MOF particles is preferably 180 to 220 nm, more preferably 200 nm. By limiting the particle size of the self-oxygenating MOF particles to ensure that the self-oxygenating MOF particles can be relatively evenly deposited on the surface and pores of the cylindrical bone scaffold units, the present invention achieves a controlled release of oxygen, thereby promoting the osteogenic differentiation of bone marrow mesenchymal stem cells and the construction of a vascularized network.

[0063] In the present invention, the loading amount of the self-generating MOFs particles in the cylindrical bone scaffold unit is preferably 0.5 to 2 mg / cm 2 , more preferably 1 to 1.5 mg / cm 2 .

[0064] In the present invention, the method for preparing the self-generating oxygen MOFs particles comprises the following steps:

[0065] Calcium chloride, polyvinyl pyrrolidone, hydrogen peroxide, ammonia water and alcohol solvent are mixed to carry out precipitation reaction to obtain CaO2-PVP precipitate;

[0066] The CaO2-PVP precipitate, zinc nitrate and 2-methylimidazole are mixed and subjected to a self-assembly reaction to obtain self-generating MOFs particles.

[0067] In the present invention, the mass ratio of calcium chloride (CaCl2·2H2O) to polyvinyl pyrrolidone (PVP) is preferably 1:(1-1.5), more preferably 1:(1.2-1.4), and the dosage ratio of calcium chloride to hydrogen peroxide and ammonia water is preferably 0.6-1g:480-700μL:1-1.5mL, more preferably 0.6-0.8g:500-600μL:1.2-1.4mL. In the present invention, the alcohol solvent is preferably methanol. In the present invention, the temperature of the precipitation reaction is preferably 0-4°C, and the time is preferably 1-1.5h. In the present invention, during the precipitation reaction, CaCl2 reacts with H2O2 and NH3·H2O to generate CaO2. By introducing PVP, the present invention can react with Ca 2+ Forming coordination to prevent CaO2 from agglomerating, while the pyrrolidone ring (C--O) of PVP and Zn in ZIF-8 2+ The affinity between CaO2 and ZIF-8 is enhanced by the weak coordination interaction between them.

[0068] In the present invention, the mass ratio of zinc nitrate (Zn(NO3)2·6H2O) to 2-methylimidazole is preferably (3.5-4):(1-1.3); the mass ratio of CaO2-PVP precipitate to zinc nitrate is preferably (20-25):(1-1.5). In the present invention, the temperature of the self-assembly reaction is preferably room temperature, and the time is preferably 20-24 hours, more preferably 22-24 hours. After the self-assembly reaction, the present invention preferably centrifuges, washes, and dries the resulting self-assembly reaction solution.

[0069] In the present invention, the groove of the cylindrical bone scaffold unit is filled with a functional GelMA hydrogel, and the functional GelMA hydrogel contains cells, growth factors and antibacterial drugs. In the present invention, the cells include bone marrow mesenchymal stem cells and / or vascular endothelial cells. In the present invention, the growth factors preferably include bone morphogenetic protein and / or vascular endothelial growth factor. Specifically, when the cells are bone marrow mesenchymal stem cells, the growth factor is preferably bone morphogenetic protein, and when the cells are vascular endothelial cells, the growth factor is preferably vascular endothelial growth factor. In the present invention, the antibacterial drugs preferably include gentamicin and / or vancomycin.

[0070] In the present invention, the bone marrow mesenchymal stem cells differentiate and proliferate into osteoblasts under the stimulation of bone morphogenetic protein, thereby better promoting bone regeneration at the bone defect site; the vascular endothelial cells migrate and proliferate under the action of vascular endothelial growth factor, thereby promoting the formation of a vascularized network.

[0071] In the present invention, the concentration of bone marrow mesenchymal stem cells in the functional GelMA hydrogel is preferably 1×10 5 ~3×10 5 cells / mL, more preferably 2×10 5 ~3×10 5 cells / mL; the concentration of the vascular endothelial cells is preferably 5×10 5 ~1×10 6 cells / mL, more preferably 5×10 5 ~7×10 5 cells / mL; the mass concentration of the growth factor is preferably 10-300 ng / mL, and the mass concentration of the antibacterial drug is preferably 5-10 μg / mL, more preferably 6-8 μg / mL. In the present invention, when the growth factor includes bone morphogenetic protein, the mass concentration of the bone morphogenetic protein is preferably 50-300 ng / mL, more preferably 100-200 ng / mL; when the growth factor includes vascular endothelial growth factor, the mass concentration of the vascular endothelial growth factor is preferably 10-50 ng / mL, more preferably 20-40 ng / mL.

[0072] In the present invention, the method for preparing the cell-laden bionic bone scaffold with self-oxygenation and antibacterial effects comprises the following steps:

[0073] GelMA, amino-modified hydroxyapatite nanopowder, buffer solution and photoinitiator are mixed to obtain a composite ink;

[0074] 3D printing and UV curing the composite ink to obtain a cylindrical bone scaffold unit matrix;

[0075] Immersing the cylindrical bone scaffold unit matrix in a dispersion of self-generating MOFs particles for loading to obtain a cylindrical bone scaffold unit;

[0076] Functional GelMA hydrogel is filled in the groove of the cylindrical bone scaffold unit, and multiple cylindrical bone scaffold units are stacked and assembled to obtain a cell-laden bionic bone scaffold with self-oxygenation and antibacterial effects.

[0077] The present invention mixes GelMA, amino-modified hydroxyapatite nanopowder, a buffer solution and a photoinitiator to obtain a composite ink. In the present invention, the photoinitiator preferably includes LAP and / or I2959. In the present invention, the mass ratio of the GelMA to the amino-modified hydroxyapatite nanopowder is preferably (10-12):(1-1.5), more preferably 10:1; the mass ratio of the amino-modified hydroxyapatite nanopowder to the photoinitiator is preferably 2-3:1-1.5, more preferably 2:1; the buffer solution is preferably PBS buffer solution, and the amount ratio of the GelMA to the buffer solution is preferably 1-1.5 g:10-15 mL, more preferably 1 g:10 mL. In the present invention, the mixing method is preferably: GelMA, amino-modified hydroxyapatite nanopowder and buffer solution are first mixed, and the photoinitiator is added under light-proof conditions for second mixing.

[0078] After obtaining the composite ink, the present invention performs 3D printing and UV curing on the composite ink to obtain a cylindrical bone scaffold unit matrix. Before the 3D printing, the present invention constructs a cylindrical bone scaffold unit matrix model using computer-aided design software.

[0079] In the present invention, the 3D printing is preferably extrusion 3D printing, and the parameters of the 3D printing preferably include:

[0080] The nozzle diameter is preferably 0.3 mm;

[0081] The extrusion pressure is preferably 0.15 to 0.18 MPa, more preferably 0.15 to 0.16 MPa;

[0082] UV light cross-linking is used in the 3D printing process;

[0083] The wavelength of the UV light crosslinking is preferably 365-405 nm, more preferably 385 nm; the light intensity is preferably 15-20 mW / cm 2 , more preferably 15 to 18 mW / cm 2 The irradiation time of each layer of 3D printing material is preferably 15 to 30 seconds, more preferably 15 to 20 seconds.

[0084] After the 3D printing, the printed material is subjected to UV curing in the present invention. The wavelength of the UV curing is preferably 365 to 405 nm, more preferably 385 nm, and the curing time is preferably 1 to 2 minutes.

[0085] After 3D printing and UV curing, the cylindrical bone scaffold unit matrix is ​​preferably washed and dried. In the present invention, the washing preferably includes soaking in ethanol and ultrasonic cleaning in a PBS solution. The ethanol soaking time is preferably 4 hours, and the ultrasonic cleaning time is preferably 5 minutes. The washing removes uncrosslinked ink. In the present invention, the drying is preferably freeze-drying.

[0086] After obtaining the cylindrical bone scaffold unit matrix, the present invention immerses the cylindrical bone scaffold unit matrix in a dispersion of self-oxygenating MOF particles for loading to obtain the cylindrical bone scaffold unit. In the present invention, the solvent for the self-oxygenating MOF particle dispersion is preferably ethanol; the concentration of the self-oxygenating MOF particle dispersion is preferably 20-25 mg / mL, more preferably 22-24 mg / mL; the loading temperature is preferably room temperature, and the loading time is preferably 12-16 hours, more preferably 13-15 hours. After loading, the present invention preferably washes and dries the obtained cylindrical bone scaffold unit, preferably rinsing with PBS, and preferably drying under vacuum.

[0087] After obtaining the cylindrical bone scaffold unit, the present invention fills the grooves of the cylindrical bone scaffold unit with functional GelMA hydrogel, and stacks and assembles multiple cylindrical bone scaffold units to obtain a cell-laden biomimetic bone scaffold with self-oxygenation and antibacterial properties. The present invention preferably uses a syringe to fill the functional GelMA hydrogel. The present invention stacks and assembles multiple cylindrical bone scaffold units using the grooves and protrusions of adjacent cylindrical bone scaffold units.

[0088] The present invention provides the use of the above-mentioned cell-loaded bionic bone scaffold with self-oxygenation and antibacterial effects in the preparation of osteochondral tissue repair materials. In the present invention, the osteochondral tissue repair material is preferably an osteochondral tissue repair material for large bone defects.

[0089] The cell-laden bionic bone scaffold with self-oxygenation and antibacterial effects, its preparation method and application provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0090] Example 1

[0091] A cell-loaded bionic bone scaffold with a Haversian tube structure, self-oxygenating and antibacterial properties, is composed of multi-layer cylindrical bone scaffold units, self-oxygenating MOFs particles attached to the surface and pores of the bone scaffold units, and functional GelMA hydrogel filling the grooves of the cylindrical bone scaffold units. The functional GelMA hydrogel is composed of a hydrogel solution, bone marrow mesenchymal stem cells, vascular endothelial cells, growth factors (bone morphogenetic protein and / or vascular endothelial growth factor), and an antibacterial drug (gentamicin). The schematic diagram of the composition structure of the bionic bone scaffold is shown in the figure below. Figure 1 Specifically, the outer annular groove of the cylindrical bone scaffold unit is filled with bone marrow mesenchymal stem cells (2×10 5 cells / mL), bone morphogenetic protein (159 ng / mL) and gentamicin (8 μg / mL); the middle annular groove was filled with bone marrow mesenchymal stem cells (2×10 5 cells / mL), bone morphogenetic protein (159 ng / mL) and gentamicin (8 μg / mL); the inner annular groove was filled with vascular endothelial cells (5×10 5 cells / mL), vascular endothelial growth factor (30 ng / mL), and gentamicin (8 μg / mL).

[0092] The preparation method of the cell-loaded bionic bone scaffold with self-oxygenation and antibacterial effects comprises the following steps:

[0093] (1) Preparation of self-generating MOFs particles ZIF-8-CaO2, the preparation flow chart is as follows Figure 2 As shown. At room temperature, 0.6 g of CaCl₂·2H₂O and 0.6 g of polyvinylpyrrolidone (PVP) were dissolved in 30 mL of anhydrous methanol and stirred at room temperature for 3 hours with a magnetic stirrer. Subsequently, 480 μL of a mixed solution of H₂O₂ and 1 mL of aqueous ammonia was added. The reaction was stirred at 0°C for 1 hour with a magnetic stirrer. The resulting solution was centrifuged, and the resulting precipitate was collected, washed three times with methanol, and dispersed in 1 mL of methanol for later use. The resulting sample was named CaO₂-PVP. 37.2 mg, 0.13 mmol of Zn(NO₃)₂·6H₂O was added to 1 mL of the CaO₂-PVP dispersion and fully dissolved. The solution was then added to 5 mL of a methanol solution containing 10.3 mg, 0.13 mmol of 2-methylimidazole. The mixed solution was allowed to stand at room temperature for 24 hours. The resulting precipitate was washed three times with methanol and dried in vacuo at 60°C overnight. The resulting sample was named ZIF-8-CaO₂.

[0094] (2) Preparation of methacrylic anhydride gelatin GelMA, the preparation flow chart is as follows Figure 320 g of gelatin was dissolved in 200 mL of PBS at 60°C and stirred for 2 hours using a magnetic stirrer at 700 rpm. At 50°C, 16 mL of 94% pure methacrylic anhydride was added to the gelatin-containing PBS solution using a micropump at a rate of 0.5 mL / min. The mixture was then heated in a 50°C oil bath and magnetically stirred at 700 rpm for 4 hours. After this, 300 mL of 50°C PBS was added and magnetic stirring continued for 30 minutes to terminate the reaction. The prepared mixed solution was divided into dialysis bags and dialyzed against deionized water in a water bath at 40°C for 7 days using a 13,000 molecular weight dialysis bag to remove any unreacted small molecules. The dialyzed liquid was poured into a centrifuge tube and centrifuged at 3500g for 3 minutes. The supernatant was collected to obtain the GelMA solution. The obtained GelMA solution was frozen in a -80°C refrigerator for 12 hours and then freeze-dried in a freeze dryer for 72 hours to obtain freeze-dried GelMA.

[0095] (3) Preparation of amino-modified HAP nanopowder. The preparation flow chart is as follows: Figure 4 First, 5.0 g of hydroxyapatite nanopowder (50-200 nm) was added to 50 mL of 0.1 M HCl and stirred for 2 hours. After centrifugation at 8000 rpm for 10 minutes, it was washed with deionized water until neutral. It was vacuum-dried at 60 ° C for 12 hours to remove impurities to obtain pretreated HAP nanopowder. Then, 1 mL of 50% ethanol solution was added to 10 mL of ethanol in a fume hood. The pretreated HAP nanopowder was dispersed in the hydrolyzate and subjected to 200W ultrasonic treatment for 30 minutes to achieve uniform dispersion. The mixture was then stirred in a 70°C water bath for 6 hours in the dark, and the container was wrapped with aluminum foil to complete the surface amino grafting of the HAP nanopowder. After the reaction, the precipitate was collected by centrifugation at 8000 rpm for 10 minutes, washed three times with anhydrous ethanol, and ultrasonically removed unreacted KH550. Finally, the modified HAP nanopowder was vacuum-dried at 60°C for 12 hours, passed through a 200-mesh sieve, and sealed in a dark container for future use.

[0096] (4) Preparation of GelMA / nHAP composite ink, the preparation flow chart is as follows Figure 5As shown. Weigh 3g of freeze-dried GelMA and add it to 27mL of 4℃ pre-cooled PBS solution. Stir magnetically on ice for 3 hours until completely dissolved and the solution is clear and free of bubbles. Weigh 0.3g of amino-modified hydroxyapatite nanopowder (nHAP) and add it to 3mL of PBS solution. Ultrasonicate on ice for 30 minutes to form a homogeneous suspension. Slowly pour the HaP nanopowder suspension into the GelMA solution and stir magnetically on ice for 90 minutes to ensure that the amino-modified hydroxyapatite nanopowder (nHAP) is evenly dispersed. Add 0.15g of photoinitiator LAP under light-proof conditions and stir magnetically for 90 minutes until completely dissolved. Transfer the mixture to a centrifuge tube and centrifuge at 2000rpm at 4℃ for 5 minutes to obtain a printable GelMA / nHAP composite ink.

[0097] (5) Prepare cylindrical bone scaffold units. The preparation flow chart is as follows: Figure 6 As shown. First, a porous cylindrical bone scaffold unit model was constructed using computer-aided design software. The cylindrical bone scaffold unit has a height of 4.0±0.1mm and a diameter of 12.0±0.1mm. The outer annular groove has an outer diameter of 10.8±0.1mm and an inner diameter of 7.6±0.1mm. The middle annular groove has an outer diameter of 6.0±0.1mm and an inner diameter of 2.8±0.1mm. The outer diameter of the inner annular groove is 1.6±0.1mm. The groove dimensions are all 1.6±0.1mm in width and 2.5±0.1mm in depth. The protrusion dimensions are all 1.6±0.1mm in width and 1.0±0.1mm in height. The total height of the bone scaffold is designed according to the size of the defect and customized stacking assembly is performed.

[0098] An extrusion-type bio-3D printer was used for printing, with a nozzle diameter of 0.3 mm and an extrusion pressure of 0.15 MPa. UV light was used for cross-linking during the printing process, with a light intensity of 15 mW / cm 2 Each layer was irradiated for 15 seconds. After printing, the entire cylindrical scaffold unit was cured with UV light for 1-2 minutes. After printing and curing, the printed cylindrical scaffold unit was immersed in anhydrous ethanol solution at room temperature for 4 hours, then ultrasonically cleaned in a 40°C PBS solution for 5 minutes to remove any uncrosslinked ink. After ultrasonic cleaning, the cylindrical scaffold unit was frozen at -80°C for 12 hours and then freeze-dried in a freeze dryer for 24 hours. The volume shrinkage of the cylindrical scaffold unit after freeze-drying was pre-designed to ensure that the final size of the freeze-dried cylindrical scaffold unit remained within the desired preset dimensions. The freeze-dried cylindrical scaffold unit was then immersed in a dispersion of 2g ZIF-8-CaO2 in 100mL of anhydrous ethanol at room temperature for 12 hours. The scaffold unit was then rinsed with PBS and vacuum-dried for 12 hours to obtain the final cylindrical scaffold unit.

[0099] (6) Assemble the cylindrical bone scaffold unit. The assembly diagram is as follows: Figure 7 As shown. The annular groove of the bone scaffold unit is filled with GelMA hydrogel containing cells, bioactive factors and antibacterial drugs, and then the cylindrical bone scaffold units are assembled layer by layer according to the structure of the bone scaffold to form a bionic bone scaffold. The mechanism of the bone repair process of the bionic bone scaffold is shown in the figure. Figure 8 As shown in the figure, vascular endothelial growth factor can promote the migration and proliferation of endothelial cells, form a vascular network, and provide nutrition for bone marrow mesenchymal stem cells; CaO2 loaded in ZIF-8 can supply oxygen; ZIF-8 can not only achieve slow release of oxygen, but also degrade to provide Zn for the bone microenvironment. 2+ ; Bone morphogenetic protein induces osteogenic differentiation of bone marrow mesenchymal stem cells; gentamicin exerts an antibacterial effect; the various components build a complex biological regulatory network through synergistic regulatory effects such as nutrient supply, ion supply, oxygen supply, differentiation induction and antibacterial effect, promoting the repair of bone defects.

[0100] Comparative Example 1

[0101] The difference between this embodiment and embodiment 1 is that:

[0102] No self-oxygenating MOFs particles were deposited on the surface of the circular bone scaffold units;

[0103] Other steps are the same as in Example 1.

[0104] Comparative Example 2

[0105] The difference between this embodiment and embodiment 1 is that:

[0106] Gentamycin was not added to the GelMA hydrogel composition;

[0107] Other steps are the same as in Example 1.

[0108] Performance Testing

[0109] In order to study the oxygen release effect of self-generated MOFs particles on the bone scaffold, the effects of the degradation products of ZIF-8 and the addition of antibacterial substances on the various properties of the bionic bone scaffold, the following performance tests were performed on the three bionic bone scaffolds prepared in Example 1, Comparative Example 1 and Comparative Example 2:

[0110] (1) The compressive strength of the three bionic bone scaffolds prepared in Example 1 and Comparative Examples 1-2 was tested using a universal mechanical testing machine.

[0111] (2) The three bionic bone scaffolds prepared in Example 1 and Comparative Examples 1 to 2 were subjected to CCK-8 experiments on vascular endothelial cells (HUVECs) and bone marrow mesenchymal stem cells (BMSCs), and the cell proliferation rate on the third day compared with that on the first day was calculated. The specific experimental process was as follows: cell culture medium was added to a culture dish containing the bone scaffold units in Example 1 and Comparative Examples 1 to 3 (no corresponding cells were added to the functionalized hydrogel at this time), and the scaffold was soaked in the cell culture medium for 24 hours; cells were cultured in the scaffold impregnation solution until the cell confluence reached 70% to 80%, and the cells were resuspended to prepare a cell density of 5×10 4 cells / mL of cell suspension; then, the cell suspension was inoculated into a 96-well plate at a volume of 100 μL per well, with three replicates for each sample; the plate was incubated in a 37°C, 5% CO2 incubator for 4 hours to allow the cells to adhere to the wall. After the cells adhered, 10 μL of CCK-8 solution was accurately added to each well and the plate was gently shaken to mix; the plate was returned to the incubator and incubated for another 4 hours; finally, the absorbance (OD value) of each well was measured at a wavelength of 450 nm using a microplate reader, and cell viability was analyzed by data comparison; the absorbance values ​​of the CCK-8 reagent in different experimental groups were compared to obtain the cell proliferation rate. The proliferation rate was calculated as follows: Cell proliferation rate = (absorbance value of experimental group - absorbance value of blank control) / (absorbance value of control group - absorbance value of blank control) × 100%; where the experimental group refers to the cell group cultured with different scaffold impregnation solutions, the control group refers to the cell group cultured with no scaffold impregnation solution, and the blank control refers to the well group containing no cells but only culture medium and CCK-8 reagent.

[0112] (3) The three bionic bone scaffolds prepared in Example 1 and Comparative Examples 1-2 were subjected to an in vitro angiogenesis experiment. The results of the angiogenesis network were quantitatively analyzed using ImageJ software to evaluate the tubular formation ability of the three bionic bone scaffolds. The specific experimental process was as follows: 24 hours before the experiment, Matrigel was placed in a 4°C refrigerator to slowly melt to avoid repeated freezing and thawing. At the same time, consumables such as 12-well plates and gun tips were pre-cooled for standby use. Matrigel matrix glue was added to the 12-well plate with a pre-cooled gun tip at 500 μL / well, and then the bionic bone scaffold unit (the functionalized hydrogel at this time had been added with the corresponding The scaffolds were placed on Matrigel with the grooves facing up. Gently press to embed the bottom third of the scaffold into the Matrigel. The 12-well plate was then placed in a cell culture incubator for 60 minutes to solidify. The scaffolds were then tightly bonded to the Matrigel, and the pores of the scaffolds were connected to the Matrigel. The Matrigel solidified to form a gel layer. Tube formation induction medium was then added. The culture plates were incubated in a 37°C, 5% CO2 incubator for 48 hours, avoiding vibration. Following incubation, the cells were fixed with 4% paraformaldehyde for 15 minutes, washed three times with PBS, and then incubated in the dark for 30 minutes with Calcein AM / PI double staining. Five randomly selected fields of view were then observed under a fluorescence microscope. ImageJ software was used to quantify the vascularization network and evaluate the angiogenic capacity of the three biomimetic bone scaffolds.

[0113] (4) Determine the ALP activity of the three bionic bone scaffolds prepared in Example 1 and Comparative Examples 1-2, and evaluate the osteogenic differentiation of the three bionic bone scaffolds; the specific experimental process is carried out in accordance with the instructions of the ALP activity detection kit, specifically: place the three bionic bone scaffold units (loaded with cell-functionalized hydrogel) prepared in Example 1 and Comparative Examples 1-2 in a 12-well plate, and culture them in an incubator for 14 days, during which the osteogenic induction culture medium is regularly replaced; then discard the culture medium, gently rinse the scaffold surface with PBS three times to remove unattached cells; add cell lysis solution at a ratio of 100 μL lysis solution per well, and pipette several times to allow the lysis solution and cells to fully contact, and all operations in the lysis step are carried out on ice; after sufficient lysis, centrifuge at 10,000 g for 5 minutes, and take the supernatant for later use; the supernatants of different scaffolds are added to 96-well plates respectively. Remove the chromogenic substrate solution and standard working solution from the kit and return to room temperature; take a tube of chromogenic substrate, dissolve it in 2.5 mL of detection buffer, dissolve and mix thoroughly, and place on ice; take 10 μL of p-nitrophenol solution (10 mM) and dilute it to 0.2 mL with detection buffer, with a final concentration of 0.5 mM; use Western and IP cell lysis buffer (without inhibitors) to lyse the cells on the relevant scaffolds, perform appropriate homogenization, and then centrifuge to obtain the supernatant; use a 96-well plate to set up blank control wells, standard wells, and sample wells, and add detection buffer, chromogenic substrate, sample, and standard working solution according to the amount specified in the instructions; then gently blow and mix with a pipette tip, or use a shaker to mix; incubate at 37°C for 5 to 10 minutes; add 100 μL of reaction stop solution to each well to terminate the reaction. At this point, the standards or wells with alkaline phosphatase activity will show different shades of yellow; the absorbance is measured at 405 nm; the absorbance value is recorded at a wavelength of 405 nm using a microplate reader, thereby accurately quantifying the ALP activity in BMSCs cultured within the specified time interval.

[0114] (5) The oxygen release time and Ca release time of the three bionic bone scaffolds prepared in Example 1 and Comparative Examples 1-2 were measured. 2+ Release rate determination; The specific experimental process is as follows: the stents of Example 1 and Comparative Examples 1 and 2 are immersed in PBS at 37°C, and the dissolved O2 is measured every two days. Then, after each measurement, the O2 is removed until no dissolved O2 is detected, and the number of days of oxygen release is measured. The stents of Example 1 and Comparative Examples 1 and 2 are immersed in PBS at 37°C, and the dissolved Ca2+ is measured every two days. 2+ content, until dissolved Ca 2+ The content no longer changes, so the Ca 2+ Release rate.

[0115] (6) An inhibition zone experiment was conducted on the three bionic bone scaffolds prepared in Example 1 and Comparative Examples 1-2, and the diameters of the inhibition zones of the three scaffolds were measured. The specific experimental process is as follows: streak the frozen Staphylococcus aureus strain onto the plate culture medium and culture it at 37°C for 24 hours; pick a single colony and inoculate it into 100 mL of liquid culture medium, culture it in a shaker at 37°C and 200 r / min overnight, and set the bacterial solution aside; first pour about 20 mL of plate culture medium into the sterilized plate, let it stand horizontally to solidify, inoculate 0.1 mL of bacterial solution, spread it evenly and set it aside; place the brackets of Example 1 and Comparative Examples 1 and 2 on the agar plate coated with the bacterial solution, and place multiple brackets on each plate to ensure that there is sufficient spacing between the brackets to prevent overlapping of the inhibition zones; turn the inoculated plate upside down and place it in an incubator at 37°C for incubation for 16-24 hours; after the incubation is completed, observe the inhibition zone on the agar plate. The inhibition zone refers to the transparent area around the tablet where no bacteria grows; use a ruler or caliper to measure the diameter of the inhibition zone and record it in millimeters.

[0116] The above experimental test results are shown in Table 1.

[0117] Table 1 Performance test data of bionic bone scaffolds prepared in Example 1 and Comparative Examples 1-2

[0118] Example 1 Comparative Example 1 Comparative Example 2 Compressive strength (MPa) 5.2 4.8 5.1 HUVECs proliferation rate (%) 95 72 87 BMSCs proliferation rate (%) 124 80 118 Number of tube formations 21 14 20 <![CDATA[Total branch length (×10 3 )]]> 4.7 2.3 4.3 ALP activity (U / mg) 45 22 43 <![CDATA[Ca 2+ Release rate (μg / d)]]> 120 0 119 <![CDATA[O2 release duration (days)]]> 5～7 0 5～7 Diameter of inhibition zone (mm) 12.5 10.2 4.1

[0119] As shown in Table 1, in terms of compressive strength, the biomimetic bone scaffold prepared in Example 1 exhibits significantly improved mechanical properties due to the addition of modified HAP nanoparticles in the GelMA / nHAP composite ink. Furthermore, the pore-filling effect of ZIF-8-CaO2 particles further optimizes structural stability, resulting in high compressive strength. The bone scaffold in Comparative Example 1 lacks MOFs deposition, resulting in increased porosity and decreased compressive strength. While the bone scaffold in Comparative Example 2 lacks gentamicin, the reinforcing effect of pore-filling by nHAP and ZIF-8-CaO2 particles is retained, resulting in a higher compressive strength than that of Comparative Example 1. Compared to the biomimetic bone scaffold prepared in Example 1, the biomimetic bone scaffolds in Comparative Examples 1 and 2 exhibit lower compressive strength.

[0120] Cell proliferation and angiogenesis: The ZIF-8-CaO2 on the bionic bone scaffold prepared in Example 1 continuously releases oxygen, alleviates the hypoxic microenvironment, significantly promotes HUVECs proliferation and angiogenesis, and at the same time enhances branch extension through high expression of VEGF, and BMSCs proliferation is good because the aerobic environment promotes metabolic cell proliferation and angiogenesis; the bionic bone scaffold in Comparative Example 1 lacks ZIF-8-CaO, and the hypoxic environment inhibits HUVECs proliferation and angiogenesis, and BMSCs proliferation is limited; the bionic bone scaffold in Comparative Example 2 lacks gentamicin, and the cell proliferation and angiogenesis are similar to those in Example 1.

[0121] Osteogenic differentiation: High Ca content of the scaffold in Example 1 2+ The environment can activate the BMP-2 / Smad pathway and promote osteogenic differentiation, and the aerobic environment is also conducive to cell osteogenic differentiation; the hypoxic environment of the bone scaffold in Comparative Example 1 inhibits the HIF-1α pathway, resulting in a decrease in the osteogenic differentiation ability of osteoblasts; the osteogenic differentiation effect of the bone scaffold in Comparative Example 2 is similar to that in Example 1, and the addition of antibacterial drugs does not interfere with the osteogenic signal.

[0122] Antibacterial effect: Zn generated by degradation of ZIF-8 in the scaffold in Example 1 2+ It works synergistically with gentamicin, covering both Gram-positive and Gram-negative bacteria, and has a good antibacterial effect. In Comparative Example 1, gentamicin was added to the scaffold, and there was no Zn produced by ZIF-8 degradation. 2+ The antibacterial effect is general. Comparative Example 2 does not contain gentamicin and only relies on Zn produced by ZIF-8 degradation. 2+ It interferes with bacterial metabolism and has a poor antibacterial effect.

[0123] It can be seen that the method for preparing a bionic bone scaffold in Example 1 of the present invention solves the problems of traditional scaffolds such as hypoxia inhibition, low osteogenesis efficiency, bacterial contamination and insufficient mechanical adaptability through the multifunctional synergistic effect of self-oxygen production, drug controlled release and mechanical enhancement, and provides an efficient solution for the repair of complex bone defects.

[0124] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A cell-loaded bionic bone scaffold with self-oxygenation and antibacterial effects, characterized in that: It includes a plurality of cylindrical bone scaffold units assembled in layers; One side surface of a single cylindrical bone support unit is provided with one or more grooves, and the other side surface is provided with one or more protrusions, and adjacent cylindrical bone support units are fixed by the grooves and protrusions; The raw materials for preparing the cylindrical bone scaffold unit include methacrylic anhydride gelatin and amino-modified hydroxyapatite nanopowder; The cylindrical bone scaffold unit has a loose porous structure; The surface and pores of the cylindrical bone scaffold unit are loaded with self-generating MOFs particles, wherein the self-generating MOFs particles include ZIF-8 and CaO2 encapsulated in the internal pores of the ZIF-8; The groove of the cylindrical bone scaffold unit is filled with functional GelMA hydrogel, which contains cells, growth factors and antibacterial drugs. The cells include bone marrow mesenchymal stem cells and / or vascular endothelial cells.

2. The cell-laden bionic bone scaffold with self-oxygenation and antibacterial effects according to claim 1, characterized in that: The grooves or protrusions on the surface of the single cylindrical bone scaffold unit are in the shape of rings, and the number thereof is 2 to 5; The pore size of the cylindrical bone scaffold unit is 80-120 μm.

3. The cell-laden bionic bone scaffold with self-oxygenation and antibacterial effects according to claim 1 or 2, characterized in that: The mass ratio of the GelMA to the amino-modified hydroxyapatite nanopowder is (10-12):(1-1.5).

4. The cell-laden bionic bone scaffold with self-oxygenation and antibacterial effects according to claim 1, characterized in that: The method for preparing the self-generating oxygen MOFs particles comprises the following steps: Calcium chloride, polyvinyl pyrrolidone, hydrogen peroxide, ammonia water and alcohol solvent are mixed to carry out precipitation reaction to obtain CaO2-PVP precipitate; The CaO2-PVP precipitate, zinc nitrate and 2-methylimidazole are mixed and subjected to a self-assembly reaction to obtain self-generating MOFs particles.

5. The cell-laden bionic bone scaffold with self-oxygenation and antibacterial effects according to claim 1, characterized in that: The concentration of bone marrow mesenchymal stem cells in the functional GelMA hydrogel is 1×10 5 ~3×10 5 cells / mL, and the concentration of endothelial cells was 5×10 5 ~1×10 6 cells / mL, the mass concentration of growth factors is 10-300 ng / mL, and the mass concentration of antibacterial drugs is 5-10 μg / mL.

6. The method for preparing the cell-laden bionic bone scaffold with self-oxygenation and antibacterial effects according to any one of claims 1 to 5, characterized in that: The following steps are involved: GelMA, amino-modified hydroxyapatite nanopowder, buffer solution and photoinitiator are mixed to obtain a composite ink; 3D printing and UV curing the composite ink to obtain a cylindrical bone scaffold unit matrix; Immersing the cylindrical bone scaffold unit matrix in a dispersion of self-generating MOFs particles for loading to obtain a cylindrical bone scaffold unit; Functional GelMA hydrogel is filled in the groove of the cylindrical bone scaffold unit, and multiple cylindrical bone scaffold units are stacked and assembled to obtain a cell-laden bionic bone scaffold with self-oxygenation and antibacterial effects.

7. The preparation method according to claim 6, characterized in that The method for preparing the amino-modified hydroxyapatite nanopowder comprises the following steps: The hydroxyapatite nanopowder, KH550 and an alcohol solvent are mixed and subjected to a grafting reaction to obtain amino-modified hydroxyapatite nanopowder.

8. The preparation method according to claim 6, characterized in that The 3D printing is extrusion 3D printing, and the parameters of the 3D printing include: The nozzle diameter is 0.3mm; Extrusion pressure is 0.15~0.18MPa; UV light cross-linking is used in the 3D printing process; The wavelength of the UV light crosslinking is 365-405 nm, and the light intensity is 15-20 mW / cm 2 ,The irradiation time of each layer of 3D printing material is 15 to 30 seconds.

9. The preparation method according to claim 6, characterized in that The concentration of the self-generating MOFs particle dispersion is 20-25 mg / mL; The load time is 12 to 16 hours.

10. Use of the cell-loaded bionic bone scaffold with self-oxygenation and antibacterial effects according to any one of claims 1 to 5 or the cell-loaded bionic bone scaffold with self-oxygenation and antibacterial effects prepared by the preparation method according to any one of claims 6 to 9 in the preparation of osteochondral tissue repair materials.

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