Three-dimensional bioactive scaffold with ordered micro-nanowires modified on surface as well as preparation method and application of three-dimensional bioactive scaffold

Abstract

The invention relates to a three-dimensional bioactive scaffold with ordered micro-nanowires modified on the surface as well as a preparation method and application of the three-dimensional bioactive scaffold, and belongs to the field of biological materials. The three-dimensional bioactive scaffold comprises a bioactive scaffold base body and a topological structure layer modified on the surface of the bioactive scaffold base body, and the topological structure layer is composed of micro-nanowires which are orderly and directionally arranged in the fixed direction. The ordered micro-nano topological structure is successfully integrated into the three-dimensional tissue engineering scaffold, effective physical intervention can be performed on various cells, and meanwhile, the three-dimensional tissue engineering scaffold can be used as a flexible tissue engineering platform for regeneration of complex tissues. Besides, the 3D printing technology and the surface brushing technology are combined, the bioactive scaffold with the ordered micro-nanowires on the surface is constructed, and the method has important significance on development of tissue engineering scaffolds.

Description

Technical Field

[0001] The invention relates to a three-dimensional bioactive scaffold with surface modified ordered micro-nano wires and a preparation method and application thereof, belonging to the field of biomaterials. Background Art

[0002] Human bones have a highly ordered, complex hierarchical structure, which is assembled step by step from collagen fibers mineralized from hydroxyapatite. Bone tissue also contains a variety of bone-resident cells, and the spatial distribution and mutual crosstalk of these cells are crucial to the remodeling of bone tissue. In addition, the directional and ordered topological structure and mechanical signals in bone tissue also have an important influence on the behavior and fate of bone-resident cells. Based on this, it is of great significance to construct a three-dimensional tissue engineering scaffold with an ordered topological structure.

[0003] Traditional tissue engineering scaffolds cannot effectively expand ordered micro-nano structures to three-dimensional scales. At present, although photolithography, laser engraving, and ion etching technologies can achieve the construction of ordered micro-nano structures, these technologies have strict preparation conditions and can only act on flat and smooth surfaces. They are also expensive and cannot be applied to three-dimensional tissue engineering scaffolds. Therefore, how to expand ordered micro-nano topological structures into three-dimensional tissue engineering scaffolds remains a major challenge. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a three-dimensional bioactive scaffold with ordered micro-nanowires modified on the surface, and a preparation method and application thereof. The present invention successfully integrates the ordered micro-nanotopological structure into the three-dimensional tissue engineering scaffold, which can effectively physically intervene in a variety of cells, and can also be used as a flexible tissue engineering platform for the regeneration of complex tissues. In addition, the present invention combines 3D printing technology with surface brushing technology to construct a bioactive scaffold with ordered micro-nanowires on the surface, which is of great significance to the development of tissue engineering scaffolds.

[0005] In a first aspect, the present invention provides a three-dimensional bioactive scaffold with surface modified ordered micro-nanowires. The three-dimensional bioactive scaffold comprises a bioactive scaffold matrix and a topological structure layer modified on the surface of the bioactive scaffold matrix, wherein the topological structure layer is composed of micro-nanowires orderly and directional arranged along a fixed direction.

[0006] Preferably, the micro-nanowires are hydroxyapatite micro whiskers or hydroxyapatite nanowires; preferably, the diameter of the hydroxyapatite whiskers is 0.5-5 μm and the length is 20-80 μm; preferably, the diameter of the hydroxyapatite nanowires is 10-200 nm and the length is 20-2000 μm.

[0007] Preferably, the topological structure layer has a thickness of 1-15 μm.

[0008] Preferably, the material of the bioactive scaffold matrix is ​​one or more of bioceramics, biocompatible metals and polymers; preferably, the material of the bioactive scaffold matrix is ​​β-phase tricalcium phosphate.

[0009] Preferably, the three-dimensional bioactive scaffold is an integrally formed three-dimensional bioactive scaffold or a three-dimensional bioactive scaffold formed by independently forming bioactive scaffold modules with ordered micro-nanowires modified on the surface by means of the chimeric assembly of the modules themselves.

[0010] In the second aspect, the present invention provides a method for preparing a three-dimensional bioactive scaffold with surface modified ordered micro-nanowires. The preparation method comprises: preparing a scaffold embryo by photocuring 3D printing; crosslinking the scaffold embryo; subjecting the crosslinked scaffold embryo to a first calcination to obtain an unmodified bioactive scaffold; applying a micro / nanowire coating slurry to the surface of the scaffold at a uniform speed along a fixed direction, and after the slurry is dried, subjecting the scaffold to a second calcination to obtain the three-dimensional bioactive scaffold with surface modified ordered micro-nanowires.

[0011] Preferably, the temperature of the first calcination is 1050-1200° C., the time is 2-5 hours, and the atmosphere is air.

[0012] Preferably, the second calcination is carried out at a temperature of 600-900° C., for a time of 2-5 hours, in an air atmosphere.

[0013] Preferably, the micro / nanowire coating slurry is a mixture of a biocompatible organic solution and micro / nanowires or their original slurry; preferably, the microwire coating slurry is a mixture of a biocompatible organic solution and microwire whiskers, wherein the mass volume ratio of microwire whiskers to biocompatible organic solution is 2-6g:100mL; or, the nanowire coating slurry is a mixture of a biocompatible polymer solution and a nanowire original slurry in a volume ratio of 1-3:1-3; more preferably, the mass concentration of the biocompatible polymer solution is 1-5%, and the mass content of nanowires in the nanowire original slurry is 2-6%; further preferably, the biocompatible polymer is one or more of sodium alginate, chitosan, and carboxymethyl cellulose.

[0014] In a third aspect, the present invention provides an application of the three-dimensional bioactive scaffold with surface modified ordered micro-nanowires in the preparation of bone tissue engineering products. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1Hydroxyapatite micro whiskers and nanowires synthesized by hydrothermal method. (a) SEM image of hydroxyapatite micro whiskers. (b) Diameter distribution of hydroxyapatite micro whiskers. (c) SEM image of hydroxyapatite nanowires. (d) Diameter distribution of hydroxyapatite nanowires.

[0016] Figure 2 Schematic diagram of the process of preparing three-dimensional bioactive scaffold modules with surface modified ordered micro-nanowires by brush coating. (a) Schematic diagram of the auxiliary template. (b) Schematic diagram of the brush coating process. (c) Photos of the auxiliary template and scaffold template. (d) Photo of the brush coating process.

[0017] Figure 3 The surface morphology scanning electron microscope images of bioactive scaffolds prepared with different brushing process parameters. (ac) SEM images of the scaffold surface obtained by brushing the hydroxyapatite micro whisker coating slurry once at 80℃, 100℃ and 200℃ respectively. (df) SEM images of the scaffold surface obtained by brushing the hydroxyapatite micro whisker coating slurry four times at 80℃, 100℃ and 200℃ respectively. (gi) SEM images of the scaffold surface obtained by brushing the hydroxyapatite nanowire coating slurry once at 80℃, 100℃ and 200℃ respectively. (jl) SEM images of the scaffold surface obtained by brushing the hydroxyapatite nanowire coating slurry once at 80℃, 100℃ and 200℃ respectively.

[0018] Figure 4 Optical photographs and morphological characterizations of bioactive scaffolds with different surface topologies. (ae) Optical photographs of different bioactive scaffolds. (fj) Scanning electron microscope images of the surfaces of different bioactive scaffolds. (ko) Scanning electron microscope images of cross-sections of different bioactive scaffolds. Including: bioactive scaffolds with unmodified surfaces (Rough), bioactive scaffolds with disordered hydroxyapatite whiskers modified on the surface (HA-WR), bioactive scaffolds with ordered hydroxyapatite whiskers modified on the surface (HA-WA), bioactive scaffolds with disordered hydroxyapatite nanowires modified on the surface (HA-NR), and bioactive scaffolds with ordered hydroxyapatite nanowires modified on the surface (HA-NA).

[0019] Figure 5In vitro cell biological effects of bioactive scaffolds with different surface topologies. (a) Confocal images of bone marrow mesenchymal stem cells adhering to the surface of bioactive scaffolds with different surface topologies. (b) Analysis of the tropism of bone marrow mesenchymal stem cells adhering to bioactive scaffolds with different surface topologies. (c) Proliferation activity of bone marrow mesenchymal stem cells on bioactive scaffolds with different surface topologies. (d) Expression of osteogenic-related genes of bone marrow mesenchymal stem cells after 3 days of culture on bioactive scaffolds with different surface topologies. (e) Confocal images of human umbilical vein endothelial cells adhering to the surface of bioactive scaffolds with different surface topologies. (f) Analysis of the tropism of human umbilical vein endothelial cells adhering to bioactive scaffolds with different surface topologies. (g) Proliferation activity of human umbilical vein endothelial cells on bioactive scaffolds with different surface topologies. (h) Expression of angiogenic-related genes of human umbilical vein endothelial cells after 3 days of culture on bioactive scaffolds with different surface topologies.

[0020] Figure 6 Characterization of the cell biological properties of the bone-vascular co-culture system constructed by assembling the bioactive scaffolds loaded with bone marrow mesenchymal stem cells and human umbilical vein endothelial cells. Unmodified bioactive scaffolds (Rough) and bioactive scaffolds modified with ordered hydroxyapatite nanowires were selected as independent scaffolds to assemble and construct the co-culture system. The upper two layers of scaffolds loaded human umbilical vein endothelial cells, and the lower two layers of scaffolds loaded bone marrow mesenchymal stem cells. (a) Osteogenesis-related gene expression of bone marrow mesenchymal stem cells on the scaffolds after 3 days of incubation. (b) Angiogenesis gene expression of human umbilical vein endothelial cells on the scaffolds after 3 days of incubation. (c) Immunofluorescence image of OPN osteogenic protein expression of bone marrow mesenchymal stem cells on the scaffolds after 3 days of incubation. (d) Immunofluorescence image of CD31 protein expression of human umbilical vein endothelial cells on the scaffolds after 3 days of incubation.

[0021] Figure 7 Bone regeneration of three-dimensional bioactive scaffolds with different surface topologies in the rabbit lateral femoral condyle defect model. (a) Micro-CT scanning reconstruction photo. (b) H&E staining of the longitudinal section of the defect. (c) Masson staining of the longitudinal section of the defect.

[0022] In the above figures, Blank refers to the blank group, Rough refers to the bioactive scaffold with unmodified surface, HA-WR refers to the bioactive scaffold with disordered hydroxyapatite whiskers modified on the surface, HA-WA refers to the bioactive scaffold with ordered hydroxyapatite whiskers modified on the surface, HA-NR refers to the bioactive scaffold with disordered hydroxyapatite nanowires modified on the surface, and HA-NA refers to the bioactive scaffold with ordered hydroxyapatite nanowires modified on the surface. DETAILED DESCRIPTION

[0023] The present invention is further described by the following embodiments. It should be understood that the following embodiments are only used to illustrate the present invention, but not to limit the present invention. In the absence of special instructions, each percentage content refers to the percentage content by mass.

[0024] The three-dimensional bioactive scaffold of the present invention comprises a bioactive scaffold matrix and a topological structure layer modified on the surface of the bioactive scaffold matrix, wherein the topological structure layer is composed of micro-nano wires arranged in an orderly and directional manner along a fixed direction. In other words, the surface of the bioactive scaffold has an ordered and directional micron or nanowire topological structure, and is distributed in a three-dimensional scale. The three-dimensional bioactive scaffold with surface modified ordered micro-nano wires of the present invention has an ordered and directional micro-nano topological structure on its surface that can regulate various cell behaviors and fates, and can also be used as a flexible tissue engineering platform to achieve orderly spatial distribution of various cells.

[0025] In some embodiments, the surface ordered directional topological structure is composed of one-dimensional micron or nanometer-scale fibers; preferably, the surface ordered directional topological structure is composed of hydroxyapatite micron whiskers or hydroxyapatite nanowires. For example, in a specific embodiment, a one-dimensional fiber (hydroxyapatite micron whisker or hydroxyapatite nanowire) is selected as the modified material. The diameter of the hydroxyapatite whisker can be 1 μm and the length can be 20-80 μm. The diameter of the hydroxyapatite nanowire can be 120 nm and the length can be 20-2000 μm.

[0026] The source of hydroxyapatite micro whiskers or hydroxyapatite nanowires is not limited. They can be purchased through commercial channels or synthesized by home-made methods. Hydroxyapatite micro whiskers and hydroxyapatite nanowires can be synthesized by hydrothermal method. The raw materials for synthesizing hydroxyapatite micro whiskers by hydrothermal method may include: calcium nitrate tetrahydrate (Ca(NO 3 ) 2 ·4H 2 O), diammonium hydrogen phosphate ((NH 4 ) 2 HPO 4 ), acetamide (CH 3 CONH 2 ), nitric acid (HNO 3 ) and ammonia (NH 3 ·H 2 O). The raw materials for synthesizing hydroxyapatite nanowires by hydrothermal method may include: oleic acid (C 18 H 34 O 2 ), methanol (CH 3 OH), sodium hydroxide (NaOH), calcium chloride (CaCl 2 ) and sodium dihydrogen phosphate (Na2 HPO 4 ).

[0027] In some embodiments, the thickness of the surface ordered directional topological structure is between 1-15 μm. For the surface topological structure modified by hydroxyapatite micro whiskers, the thickness may be between 5-15 μm. For the surface topological structure modified by hydroxyapatite nanowires, the thickness may be between 1-5 μm. Preferably, the thickness of the surface topological structure modified by hydroxyapatite micro whiskers is 15 μm, and the thickness of the surface topological structure modified by hydroxyapatite nanowires is 5 μm.

[0028] The material of the bioactive scaffold matrix is ​​not limited. Preferably, the material of the bioactive scaffold matrix includes but is not limited to bioceramics, biocompatible metals and polymers, etc. More preferably, the material of the bioactive scaffold matrix is ​​β-phase tricalcium phosphate.

[0029] The shape of the bioactive stent is also not limited. Preferably, the overall structure of the bioactive stent includes but is not limited to circular, square, etc. More preferably, the bioactive stent is a circular stent.

[0030] The three-dimensional bioactive scaffold can be an integrally formed three-dimensional bioactive scaffold, or a three-dimensional bioactive scaffold formed by independently surface-modified ordered micro-nanowire bioactive scaffold modules by means of the module's own structure. In some embodiments, the bioactive scaffold can be assembled into a three-dimensional scaffold from bottom to top. For example, the bioactive scaffold modules all have structures such as large pillars, small pillars, key-shaped grooves and holes for the interlocking of independent modules during assembly.

[0031] In summary, the three-dimensional bioactive scaffold with surface modified ordered micro-nanowires provided by the present invention has oriented and ordered one-dimensional fibers at the micrometer or nanometer scale on its surface. This oriented and ordered physical topological structure can effectively regulate a variety of cell behaviors and fates, and the surface ordered micro-nanostructure is distributed in the three-dimensional scaffold.

[0032] The present invention also provides a method for preparing the three-dimensional bioactive scaffold with surface modified ordered micro-nanowires. The preparation method is exemplarily described below.

[0033] Bioactive scaffold embryos are prepared by photocuring 3D printing. Bioactive scaffold embryos can be printed by photocuring 3D printer. The composition of photocuring printing slurry is a conventional technical means in the field, and is not the innovation of the present invention. Those skilled in the art can adjust the composition of photocuring printing slurry as needed. For example, the mass ratio of raw material powder: photosensitive resin in the ratio of photocuring printing slurry can be 1: (1.22-2.2). Raw material powders include but are not limited to bioceramics, biocompatible metals and polymers. In a specific embodiment, β-TCP (β-tricalcium phosphate) is used as raw material powder. Photosensitive resins commonly used in the field can be used. The photosensitive resin can be a water-washable photosensitive resin, a standard resin, a flexible resin, etc. In the embodiment, a water-washable photosensitive resin is specifically used. Pour the printing slurry into the printing tank. Print the blank according to the pre-set printing file. The printing parameters of the photocuring printer can be set as needed. For example, the model layer thickness is 50 μm; the exposure time of each layer is 6s; the light power is 10%. After the printing process is completed, the uncured photosensitive resin remaining on the bracket module can be removed by cleaning. All the bracket modules can be collected and soaked in deionized water, and then cleaned in an ultrasonic cleaner for at least 5 minutes to remove the uncured photosensitive resin remaining on the bracket module.

[0034] The scaffold embryo is cross-linked. The scaffold embryo can be placed under 450nm blue light to allow it to be deeply cross-linked for more than 1 hour.

[0035] The cross-linked scaffold blank is subjected to high temperature calcination (first calcination) to obtain a bioactive scaffold. The parameters for the blank calcination are: heating rate is 1-3°C / min; holding temperature is 1050-1200°C; holding time is 2-5 hours; cooling method is furnace cooling; calcination atmosphere is air atmosphere. For example, the parameters for the blank calcination are: heating rate is 2°C / min; holding temperature is 1150°C; holding time is 3 hours; cooling method is furnace cooling.

[0036] The micro-nano wires or their original paste are mixed with a biocompatible organic solution to prepare a slurry. In a specific embodiment, the synthesized hydroxyapatite micro whiskers or hydroxyapatite nanowires original paste is mixed with a sodium alginate solution to prepare a slurry.

[0037] As one of the examples, a hydroxyapatite micro whisker coating slurry is prepared: a biocompatible organic solution with a mass concentration of 1-5% is prepared. Biocompatible organics include but are not limited to sodium alginate, chitosan, carboxymethyl cellulose, etc. The solvent of the prepared biocompatible organic solution includes but is not limited to water, etc. Hydroxyapatite micro whiskers are added to the biocompatible organic solution, and stirred with an electric stirrer for 1 hour to obtain a coating slurry of hydroxyapatite micro whiskers (HA-W) with a uniform texture. Among them, the mass volume ratio of hydroxyapatite micro whiskers to the biocompatible organic solution is 2-6g:100mL. In some embodiments, a sodium alginate aqueous solution with a mass concentration of 2% is prepared, and hydroxyapatite micro whiskers accounting for 4% are added to the sodium alginate aqueous solution.

[0038] As a second example, a hydroxyapatite nanowire coating slurry is prepared: a biocompatible organic solution with a mass concentration of 1-5% is prepared. Biocompatible organics include but are not limited to sodium alginate, chitosan, carboxymethyl cellulose, etc. The solvent of the prepared biocompatible organic solution includes but is not limited to water, etc. A certain volume of hydroxyapatite nanowire slurry (hydroxyapatite nanowire stock) is mixed with the biocompatible organic solution. The volume ratio of the hydroxyapatite nanowire slurry to the biocompatible organic solution can be 1-3:3.

[0039] In some embodiments, a sodium alginate solution with a mass concentration of 1-5% is prepared to synthesize a hydroxyapatite nanowire stock paste with a mass content of 2-6% of hydroxyapatite nanowires, and 10-30 ml of the hydroxyapatite nanowire stock paste is mixed with 30 ml of the sodium alginate solution to obtain a hydroxyapatite nanowire coating slurry. Preferably, a sodium alginate aqueous solution with a mass concentration of 2% is prepared to synthesize a hydroxyapatite nanowire stock paste with a mass content of 4% of hydroxyapatite nanowires, and 20 ml of the hydroxyapatite nanowire stock paste is mixed with 30 ml of the sodium alginate solution to obtain a hydroxyapatite nanowire coating slurry.

[0040] A bioactive scaffold with an ordered topological structure on the surface is prepared by brushing on the surface of the scaffold. Specifically, the scaffold is placed in a prefabricated mold, a certain amount of one-dimensional fiber slurry is dipped with a wool brush, and brushed on the surface of the scaffold in a certain direction and speed. The heating temperature of the prefabricated mold can be controlled during the brushing process. For example, the heating temperature of the prefabricated mold is controlled to be 80-200°C. Due to the shear force in the brushing process, the anisotropic one-dimensional fibers are oriented and parallel to the brushing direction. After the slurry is dried, the bioactive scaffold with an ordered topological structure on the surface is further sintered to enhance the bonding force between the modified layer and the substrate.

[0041] During the specific operation, the bracket module and the supporting auxiliary mold are placed on a heating table together, and preheated at 100°C for 10 minutes. Next, a wool brush is used to dip the prepared slurry containing micro-nano wires, and the slurry is brushed on the surface of the bracket from a fixed direction at a uniform speed. After the heating table bakes the slurry on the surface of the module, all the modified brackets are collected with tweezers. For example, a wool brush is used to dip the hydroxyapatite micro whisker coating slurry, and the slurry is brushed on the surface of the bracket module from a fixed direction at a uniform speed. The number of brushing times is 1-4 times. Preferably, the number of brushing times is 4 times.

[0042] The modified bracket module is placed in a crucible and calcined (second calcination) to remove organic components in the slurry. The parameters for the calcination of the modified bracket module are: the heating rate is 1-2°C / min; the insulation temperature is 600-900°C; the insulation time is 2-5 hours; the cooling method is furnace cooling; and the calcination atmosphere is air atmosphere. For example, the parameters for the calcination of the modified bracket module are: the heating rate is 2°C / min; the insulation temperature is 800°C; the insulation time is 3 hours; and the cooling method is furnace cooling.

[0043] In addition, the three-dimensional bioactive scaffold with surface modified ordered micro-nano structure prepared by the present invention can be constructed by combining 3D printing and brushing strategies to construct a single module scaffold with surface ordered micro-nano structure, and then further assembling the scaffold layer by layer.

[0044] The three-dimensional scaffold of the present invention is made by photocuring 3D printing, and has a precise structure, morphology and macroporous structure. At the same time, the ordered micro-nano structure can be expanded to the controllable 3D printed scaffold surface by combining the brushing method and the assembly strategy. The silver nanowire-mineralized collagen co-assembled bionic scaffold prepared by the prior art is disordered inside, and is only an orderly assembly of nanohydroxyapatite in the confined space of collagen fibers, rather than a directional and orderly arrangement of fibers on a macroscopic scale. In other words, the silver nanowire-mineralized collagen co-assembled bionic scaffold is disordered on the micron scale, and the ordered arrangement only occurs inside the fibrous collagen during the assembly process, which is a nanoscale confined scale. Therefore, the scaffold of the present invention is a three-dimensional bioceramic scaffold with an ordered micro-nano structure on the surface prepared by integrating the 3D printed scaffold, the brushing method and the assembly strategy, which realizes the three-dimensional expansion of the ordered micro-nano structure. This method has stronger expansibility and practicality.

[0045] The present invention also provides the application of the above-mentioned three-dimensional bioactive scaffold with surface modified ordered micro-nanowires in bone tissue engineering. One of the biological applications is that the scaffold of the present invention can be used as a universal co-culture platform to explore the mutual crosstalk between multiple cells. The second biological application is that the three-dimensional scaffold described in the present invention can be used for implantation treatment of hard tissue defects such as bones and teeth.

[0046] The present invention also provides a co-culture system constructed using the surface structured three-dimensional bioactive scaffold. For example, a three-dimensional bioactive scaffold with surface modified ordered micro-nanowires is selected as an independent scaffold assembly to construct a co-culture system. The upper two-layer scaffold module carries human umbilical vein endothelial cells, and the lower two-layer scaffold module carries bone marrow mesenchymal stem cells, and then the co-culture system is formed by bottom-up composition.

[0047] In summary, the bioactive scaffold of the present invention has an ordered and oriented micrometer or nanometer wire topology on its surface, which can effectively physically intervene in different types of cells and mediate cell behavior. This three-dimensional bioactive scaffold with ordered micro-nanowires on its surface is of great significance to the development of tissue engineering scaffolds.

[0048] The following examples are further listed to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific values ​​​​exemplified below.

[0049] The bioactive scaffold embryo was printed using a photocuring 3D printer. The mass ratio of the raw material powder to the photosensitive resin in the printing slurry was 1:1.7. The printing slurry was poured into the printing tank and the blank was printed according to the pre-set printing file. The printing parameters of the photocuring printer were set as follows: the model layer thickness was 50 μm; the exposure time of each layer was 6 s; the light power was 10%. After the printing program was completed, all the scaffold modules were collected and soaked in deionized water. Then the scaffold was cleaned in an ultrasonic cleaner for at least 5 minutes to remove the uncured photosensitive resin remaining on the scaffold module. Next, the blank of the scaffold module was placed under 450 nm blue light to make it deeply cross-linked for more than 1 hour. The blanks of all scaffold modules were calcined at high temperature to obtain bioactive scaffolds. The parameters for the calcination of the blanks were: the heating rate was 2 ° C / min; the holding temperature was 1150 ° C; the holding time was 3 hours; and the cooling method was furnace cooling.

[0050] Hydrothermal method was used to synthesize hydroxyapatite micro whiskers. 7.93 g of Ca(NO 3 ) 2 ·4H 2 O, 2.64 g (NH 4 ) 2 HPO 4 , 11.81 g CH 3 CONH 2 and 1.33 mL of HNO3 Add to 400 mL of deionized water and hydrate with HNO 3 and NH 3 ·H 2 The pH of the mixture was adjusted to 3. The solution was mixed evenly using a magnetic stirrer, and then transferred to a 500 mL hydrothermal autoclave for reaction at 180°C for 8 hours. After the hydrothermal autoclave was cooled to room temperature naturally, the obtained product was filtered and washed three times with deionized water and then dried overnight.

[0051] Hydrothermal method was used to synthesize hydroxyapatite nanowires. 210 mL of oleic acid (C 18 H 34 O 2 ) and 120 mL of methanol (CH 3 OH) was added to 270 mL of deionized water and stirred with an electric stirrer for 30 minutes to uniformly mix the solution. Next, sodium hydroxide (NaOH) aqueous solution (21 g / 300 mL) was added to the solution and stirred for 30 minutes. Calcium chloride (CaCl 2 ) aqueous solution (6.6 g / 240 mL) was added to the solution and stirred for 30 minutes. 2 HPO 4 ) aqueous solution (18.72 g / 360 mL) was added to the solution and stirred for 30 minutes. Finally, the solution was poured into four 500 mL hydrothermal kettles respectively and reacted at 200 ° C for 24 hours. After the hydrothermal kettle was naturally cooled to room temperature, hydroxyapatite nanowire slurry was obtained.

[0052] Example 1 Preparation of surface modified ordered microwire three-dimensional bioactive scaffold

[0053] (1) Using photocuring to print a bioactive ceramic module, and sintering the scaffold according to the above example. The obtained scaffold is recorded as Rough, that is, a bioactive scaffold with unmodified surface.

[0054] (2) Hydroxyapatite micro whiskers were hydrothermally synthesized according to the above exemplary method.

[0055] (3) Add 2 g of hydroxyapatite micro whiskers into 50 ml of 2% sodium alginate aqueous solution and stir thoroughly for 1 hour.

[0056] (4) The sintered bioceramic scaffold is clamped in the auxiliary mold and placed on a heating table, and preheated at 100°C for 10 minutes. Next, a wool brush is dipped into the coating slurry containing hydroxyapatite micro whiskers, and the slurry is brushed on the surface of the scaffold module at a uniform speed in the horizontal direction. After the heating table bakes the slurry on the surface of the module, all the modified modules are collected with tweezers.

[0057] (5) The modified module is placed in a crucible and calcined to remove the organic components in the slurry to obtain an assembleable / disassembled bioactive scaffold with surface modified ordered microwires. The parameters for calcining the modified scaffold module are: heating rate is 2°C / min; holding temperature is 800°C; holding time is 3 hours; cooling method is furnace cooling. The obtained scaffold is denoted as HA-WA, i.e., a bioactive scaffold with surface modified ordered hydroxyapatite whiskers.

[0058] Example 2 Preparation of three-dimensional bioactive scaffolds with surface modified ordered nanowires

[0059] The preparation process of the three-dimensional bioactive scaffold with surface modified ordered nanowires in this Example 2 is referred to Example 1, except that: in step (3), a sodium alginate aqueous solution with a mass concentration of 2% is prepared, a hydroxyapatite nanowire stock slurry with a hydroxyapatite nanowire mass content of 4% is taken, and 20 ml of the hydroxyapatite nanowire stock slurry is mixed with 30 ml of the sodium alginate solution to obtain a hydroxyapatite nanowire coating slurry. In step (4), the slurry dipped with a wool brush is the hydroxyapatite nanowire coating slurry. The obtained scaffold is recorded as HA-NA, that is, a bioactive scaffold with surface modified ordered hydroxyapatite nanowires.

[0060] Example 3 Construction of a bone-vascular co-culture model using a three-dimensional bioactive scaffold modified with ordered nanowires

[0061] The HA-NA scaffold modules were placed in a 24-well plate, and 1 mL of HUVECs cell suspension (2 x 10 5 cells·mL -1 ) were inoculated onto the module to form a module loaded with HUVECs; 1 mL of RBMSCs cell suspension (1x10 5 cells·mL -1 ) were seeded onto the module to form a module loaded with RBMSCs. Then, after the cells were completely adhered to the module, two layers of modules loaded with HUVECs and two layers of modules loaded with RBMSCs were assembled into a HA-NA bone-vascular co-culture model.

[0062] Comparative Example 1 Preparation of three-dimensional bioactive scaffolds with surface modified disordered microwires

[0063] The preparation process of the assembleable / disassembled bioactive scaffold with disordered micron-wires on the surface in this comparative example 1 is similar to that in Example 1, except that in step (4), 50 μL of the coating slurry containing hydroxyapatite micron whiskers is drawn with a pipette and evenly dripped on the surface of the scaffold module. The obtained scaffold is recorded as HA-WR, i.e., a bioactive scaffold with disordered hydroxyapatite whiskers on the surface.

[0064] Comparative Example 2 Preparation of three-dimensional bioactive scaffolds with surface modified disordered nanowires

[0065] The preparation process of the three-dimensional bioactive scaffold with disordered microwires on the surface in this comparative example 2 refers to Example 1, except that in step (4), 50 μL of the coating slurry containing hydroxyapatite nanowires is sucked with a pipette and evenly dripped on the surface of the scaffold module.

[0066] Comparative Example 3 Construction of a bone-vascular co-culture model using a three-dimensional bioactive scaffold without surface modification

[0067] The preparation process of constructing a bone-vascular co-culture model using a three-dimensional bioactive scaffold without surface modification in this comparative example 3 refers to Example 3, except that the scaffold used is a three-dimensional bioactive scaffold without surface modification.

[0068] Morphological characterization of hydroxyapatite micro-nanowires:

[0069] As can be seen from the figure, hydroxyapatite whiskers and hydroxyapatite nanowires present a typical one-dimensional structure. The peak diameter of hydroxyapatite whiskers is about 1 μm; the peak diameter of hydroxyapatite nanowires is about 120 nm (see Figure 1 ).

[0070] The process flow of brushing three-dimensional bioactive scaffolds with surface modified ordered micro-nanowires:

[0071] As shown in the figure, the bioactive scaffold is embedded in the auxiliary mold and placed on a heating plate. The corresponding slurry is applied with a wool brush at a fixed direction and at a constant speed. Due to the shearing effect of the brush during the application process, the one-dimensional material in the slurry can be oriented in an orderly manner along a fixed direction (see Figure 2 ).

[0072] Optimization of brush coating process:

[0073] The brushing process is regulated, and the surface microstructure of the stent is regulated by regulating the heating temperature (80-120°C) and the number of brushing times (1-4 times) during the brushing process. As can be seen from the figure, the increase in heating temperature has little effect on the orderliness of the surface micro-nanostructure, but with the increase in the number of brushing times, the orderliness of the surface micro-nanostructure increases significantly. (See Figure 3 ).

[0074] Surface and cross-sectional morphology of bioactive scaffolds with different surface modified micro-nanowires:

[0075] HA-WA and HA-NA groups are bioactive scaffolds with surface-modified ordered hydroxyapatite whiskers and hydroxyapatite nanowires, respectively, and their surfaces have obvious orientation. As controls, Rough, HA-WR, and HA-NR groups are bioactive scaffolds with disordered surfaces (see Figure 4 ).

[0076] In vitro osteogenic and angiogenic properties of three-dimensional bioactive scaffolds modified with ordered micro-nanowires:

[0077] Bone marrow mesenchymal stem cells and human umbilical vein endothelial cells were cultured on bioactive scaffolds with different surface topologies. Compared with bioactive scaffolds with disordered surfaces, bioactive scaffolds with ordered micro-nano structures on the surface can well guide the directional growth of cells. It also shows that compared with bioactive scaffolds with ordered microwire structures on the surface, bioactive scaffolds with ordered nanowire structures on the surface can further enhance the proliferation activity of cells and promote the expression of osteogenic-related genes OPN, ALP, BMP-2 and angiogenic-related genes VEGF-165, HIF-α, bFGF (see Figure 5 ). Therefore, next we used the bioactive scaffolds with surface modified ordered nanowire structures to construct the bone-vascular co-culture model and test the in vivo osteogenic bioactivity.

[0078] Construction of bone-vascular co-culture model:

[0079] Based on the three-dimensional performance of biological activity, a bone-blood vessel co-culture system was constructed. It consists of four layers of separate scaffolds, the upper two layers carry human umbilical vein endothelial cells, and the lower two layers carry bone marrow mesenchymal stem cells. As can be seen from the figure, in the co-culture system, compared with the unmodified micro-nanowire bioactive scaffold, the surface-modified ordered micro-nanowire bioactive scaffold can better promote the expression of bone marrow mesenchymal stem cell osteogenesis and human umbilical vein endothelial cell angiogenesis-related genes, and at the same time can enhance the expression of bone marrow mesenchymal stem cell osteogenesis-related protein OPN and human umbilical vein endothelial cell CD31 protein (see Figure 6 ).

[0080] In vivo osteogenic bioactivity of bioactive scaffolds modified with ordered nanowires on the surface:

[0081] The bioactive scaffold with surface modified with ordered nanowires was spliced ​​into four layers to form a three-dimensional scaffold (here it refers to a separate four-layer scaffold without cells) and implanted into the defect of the lateral femoral condyle of rabbits, and the sample was collected after 8 weeks. Micro-CT results showed that compared with the blank group (the defect was created in the lateral femoral condyle but no scaffold was implanted), the three-dimensional bioactive scaffold with unmodified micro-nanowires on the surface and the three-dimensional bioactive scaffold with disordered nanowires on the surface, the bioactive scaffold with surface modified with ordered nanowires induced more new bone tissue, and histological staining also showed the same results. Immunofluorescence staining of tissues showed that the bone tissue induced by the bioactive scaffold with surface modified with ordered nanowires contained more OPN and CD31 proteins. The above results all indicate that the bioactive scaffold with surface modified with ordered nanowires has better bone formation ability (see Figure 7 ).

[0082] In summary, the present invention combines 3D printing technology and brushing method to construct a bioactive scaffold with ordered micro-nanowires on the surface, which can be assembled into a three-dimensional scaffold from bottom to top. At the same time, the ordered surface topological structure can effectively intervene in the cell behavior and fate of various cells. Therefore, the scaffold expands the ordered micro-nanotopological structure to a three-dimensional scale, truly simulates the bone microenvironment, and is of great significance to the development of tissue engineering scaffolds.

Claims

1. A three-dimensional bioactive scaffold with surface modified ordered micro-nanowires, characterized in that: The three-dimensional bioactive scaffold comprises a bioactive scaffold matrix and a topological structure layer modified on the surface of the bioactive scaffold matrix, wherein the topological structure layer is composed of micro-nano wires arranged in an orderly and directional manner along a fixed direction.

2. The three-dimensional bioactive scaffold according to claim 1, characterized in that: The micro-nano wires are hydroxyapatite micro whiskers or hydroxyapatite nanowires; preferably, the diameter of the hydroxyapatite whiskers is 0.5-5 μm and the length is 20-80 μm; preferably, the diameter of the hydroxyapatite nanowires is 10-200 nm and the length is 20-2000 μm.

3. The three-dimensional bioactive scaffold according to claim 1 or 2, characterized in that: The thickness of the topological structure layer is 1-15 μm.

4. The three-dimensional bioactive scaffold according to any one of claims 1 to 3, characterized in that: The material of the bioactive scaffold matrix is ​​one or more of bioceramics, biocompatible metals and polymers; preferably, the material of the bioactive scaffold matrix is ​​β-phase tricalcium phosphate.

5. The three-dimensional bioactive scaffold according to any one of claims 1 to 4, characterized in that: The three-dimensional bioactive scaffold is an integrally formed three-dimensional bioactive scaffold or a three-dimensional bioactive scaffold formed by independently forming a bioactive scaffold module with ordered micro-nanowires modified on the surface by means of the module's own structure.

6. The method for preparing a three-dimensional bioactive scaffold with surface modified ordered micro-nanowires according to any one of claims 1 to 5, characterized in that: The preparation method comprises: preparing a scaffold embryo by photocuring 3D printing; cross-linking the scaffold embryo; subjecting the cross-linked scaffold embryo to a first calcination to obtain an unmodified bioactive scaffold; coating a micro / nanowire coating slurry on the surface of the scaffold at a uniform speed along a fixed direction, and after the slurry is dried, subjecting the scaffold to a second calcination to obtain a three-dimensional bioactive scaffold with surface modified ordered micro / nanowires.

7. The preparation method according to claim 6, characterized in that: The first calcination is carried out at a temperature of 1050-1200° C., for a time of 2-5 hours, in an air atmosphere.

8. The preparation method according to claim 6 or 7, characterized in that: The second calcination is carried out at a temperature of 600-900° C., for a time of 2-5 hours, in an air atmosphere.

9. The preparation method according to any one of claims 6 to 8, characterized in that: The micro / nanowire coating slurry is a mixture of a biocompatible organic solution and micro / nanowires or their original slurry; preferably, the microwire coating slurry is a mixture of a biocompatible organic solution and microwire whiskers, wherein the mass volume ratio of the microwire whiskers to the biocompatible organic solution is 2-6g:100mL; or, the nanowire coating slurry is a mixture of a biocompatible polymer solution and a nanowire original slurry in a volume ratio of 1-3:1-3; more preferably, the mass concentration of the biocompatible polymer solution is 1-5%, and the mass content of nanowires in the nanowire original slurry is 2-6%; further preferably, the biocompatible polymer is one or more of sodium alginate, chitosan, and carboxymethyl cellulose.

10. Use of the three-dimensional bioactive scaffold with surface modified ordered micro-nanowires according to any one of claims 1 to 5 in the preparation of bone tissue engineering products.

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