Gradient scaffold material based on particle gel as well as preparation method and application of gradient scaffold material

Abstract

The invention relates to a gradient scaffold material based on particle gel as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) constructing a shaping framework; (2) respectively preparing a plurality of hydrogel particles with different properties; and (3) respectively injecting the hydrogel particles with different properties into the shaping frame, and carrying out in-situ crosslinking on the hydrogel particles to prepare the gradient scaffold material. The scaffold material prepared by the method has the characteristic that the composition, the structure and the mechanical property are continuously adjustable in space, and the scaffold also has good biocompatibility and a penetrating porous structure, so that inward migration, proliferation and directional differentiation of cells are facilitated, and the scaffold material has good application prospects. The method has important application in the fields of bone-cartilage, tendon-bone, skin and other gradual change tissue engineering interface repair and the like.

Description

Technical Field

[0001] The present invention relates to the field of tissue engineering scaffolds, and particularly to a gradient scaffold material based on particulate gels, its preparation method and applications. Background Art

[0002] This section aims to provide background or context for the embodiments of the present invention recited in the claims. The description herein is not admitted to be prior art merely by virtue of its inclusion in this section.

[0003] Gradient materials are composite materials designed with a continuous or gradual change in composition, structure, or physical properties (such as hardness, thermal conductivity, elastic modulus, etc.) in space. Different from traditional materials, gradient materials no longer rely solely on a single uniform property, but by designing the gradual change of each layer or structure inside the material, the overall performance of the material can better match different usage requirements. Especially in the field of biomedical applications, traditional materials often suffer from performance mismatches due to interface mutations or the combination of different physical properties, which can affect the overall effect and even lead to failure. Gradient materials can provide a more uniform performance transition inside and outside the material by eliminating these mutations, thereby improving their biocompatibility and physicochemical properties. Among them, hydrogel gradient materials are an important type of gradient materials. They have excellent biocompatibility, physicochemical adjustability, and intelligent responsiveness, and can effectively promote cell growth, migration, and tissue reconstruction, thus receiving extensive attention in tissue engineering and wound repair.

[0004] Common preparation methods for hydrogel gradient materials include: (1) Layer-by-layer assembly method: preparing hydrogel layers with different compositions or properties layer by layer to form a stepped gradient. (2) Diffusion control method: using molecular diffusion or reaction diffusion mechanisms to form a continuous gradient during the gelation process. (3) Light-controlled polymerization: controlling the area and intensity of the photopolymerization reaction through a mask or digital light processing (DLP) technology to form a gradient crosslinked structure. (4) Microfluidic technology: precisely controlling the mixing ratio of different solutions using a microfluidic chip to generate gradient hydrogels. (5) 3D printing: achieving the precise construction of complex gradient structures through multi-material printing or gradient ink design. The hydrogel gradient materials prepared by these methods have problems such as poor control accuracy, unstable gradient structure, complex processes, no large pore porosity, and mismatches between the degradation rate and tissue repair rate. Summary of the Invention

[0005] The object of the present invention is to provide a gradient scaffold material based on particulate gels and its preparation method, so as to provide a new solution for the repair of gradually changing interface defects in tissue engineering.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides a method for preparing a gradient scaffold material based on particulate hydrogels, comprising the following steps:

[0008] (1) Construct a shaping framework;

[0009] (2) Prepare a plurality of hydrogel particles with different properties respectively;

[0010] (3) Inject the plurality of hydrogel particles with different properties into the shaping framework respectively, and in-situ crosslink the hydrogel particles to obtain the gradient scaffold material.

[0011] According to some specific embodiments, in step (2), the hydrogel particles are formed by crosslinking a dispersion liquid containing gelatin monomers, polymer monomers, and optionally inorganic nanoparticles.

[0012] Further, the gelatin monomers include one or more of gelatin, methacrylated gelatin (Gel-MA), and thiolated gelatin (SH-Gel).

[0013] Further, the polymer monomers include one or more of thiolated hyaluronic acid (HA-SH), methacrylated hyaluronic acid (HA-MA), thiolated sodium alginate (Alg-SH), methacrylated sodium alginate (Alg-MA), and polyethylene glycol diacrylate (PEGDA).

[0014] Further, the inorganic nanoparticles include one or several of hydroxyapatite, β-tricalcium phosphate, calcium carbonate, bioactive glass, and silicon-based nanoparticles.

[0015] According to some further embodiments, the concentration of the gelatin monomer in the dispersion is 2% to 5%, such as 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%; the concentration of the polymer monomer in the dispersion is 2% to 10%, such as 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10%; the concentration of the inorganic nanoparticles in the dispersion is 0% to 5%, such as 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%.

[0016] Further, by preparing dispersions with different concentrations of gelatin monomers and / or different concentrations of polymer monomers and / or different concentrations of inorganic nanoparticles, the various hydrogel particles with different properties are prepared. For example, the concentrations of gelatin monomers and polymer monomers can be fixed, and by preparing dispersions with different concentrations of inorganic nanoparticles and crosslinking, hydrogel particles with different properties are obtained. Generally, the modulus of the hydrogel particles increases with the increase in the concentration of inorganic nanoparticles. Alternatively, the concentration of inorganic nanoparticles can be fixed, and by preparing dispersions with different concentrations of gelatin monomers and / or different concentrations of polymer monomers and crosslinking, hydrogel particles with different properties are obtained. Generally, the modulus of the hydrogel particles increases with the increase in the concentration of gelatin monomers and / or polymer monomers.

[0017] In step (3), the order of injecting hydrogel particles with different properties is determined according to the gradient properties required for the application scenario of the material. For example, hydrogel particles with different moduli can be injected layer by layer from the bottom to the top of the self-shaped frame in the order of decreasing modulus.

[0018] Furthermore, the dispersion also contains a photoinitiator. The photoinitiator is one or more of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, 2,4,6-trimethylbenzoyldiphenylphosphine oxide, 2-methyl-1-[4-(methylthio)phenyl]-2-morpholinopropanone, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

[0019] Further, one of the crosslinking in step (2) and the in-situ crosslinking in step (3) is carried out under enzyme catalysis, and the other is carried out under light irradiation. By using two different methods for crosslinking, it is convenient to control the two crosslinking processes. Preferably, the crosslinking in step (2) is carried out under enzyme catalysis, and the in-situ crosslinking in step (3) is carried out under light irradiation, which is more convenient for operation.

[0020] Still further, steps (2) and (3) specifically include the following steps:

[0021] (2.1) Prepare a dispersion and an enzyme solution;

[0022] (2.2) Inject the dispersion, the enzyme solution, and the oil phase containing a surfactant into the corresponding channels of the microfluidic chip respectively to prepare water-in-oil droplets;

[0023] (2.3) Incubate the droplets to cause crosslinking of the droplets under the catalysis of the enzyme, and wash away the oil phase containing the surfactant to obtain the hydrogel particles;

[0024] (3) Inject the hydrogel particles into the shaping framework and then irradiate with light to cause in-situ crosslinking of the hydrogel microspheres.

[0025] Furthermore, the enzyme is transglutaminase.

[0026] Furthermore, the light is blue light or ultraviolet light.

[0027] According to some specific embodiments, the particle size of the hydrogel particles is 10 - 300 μm, such as 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280 μm, 290 μm, 300 μm. By controlling the particle size of the hydrogel particles, the pore size of the gradient scaffold material can be regulated.

[0028] According to some specific embodiments, the hydrogel particles are spherical. In this case, the particle size of the hydrogel particles is their diameter.

[0029] According to some specific embodiments, the gradient scaffold material is a porous structure.

[0030] According to some specific embodiments, the hydrogel particles with different properties are hydrogel particles with different moduli.

[0031] According to some specific embodiments, in step (1), the shaping framework is prepared by electrospinning or extrusion-based 3D printing.

[0032] Among them, the extrusion-based 3D printing method is specifically as follows: After putting the polymer required for printing into a steel feed cylinder, set the printing temperature (10 °C above the melting point), printing voltage (4 - 7 kV), printing air pressure (10 - 30 kPa), and printing speed (1000 - 2000 mm / min) on the 3D printer software. Furthermore, set the height of the shaping framework (50 - 200 layers), the spacing of the fiber grid of the shaping framework (0.2 - 5 mm), and the length and width of the shaping framework. The fiber diameter is determined by various factors. It has a positive correlation with the air pressure and a negative correlation with factors such as temperature, voltage, and printing speed. The fiber diameter can be controlled within the range of 10 - 100 μm by adjusting the parameters.

[0033] According to some specific embodiments, in step (2), the hydrogel particles are prepared by microfluidics, membrane emulsification, or suspension polymerization.

[0034] According to some specific embodiments, in step (3), the various hydrogel particles with different properties are respectively injected into the shaping framework by direct pouring or extrusion 3D printing method.

[0035] Further, the specific method of injecting the various hydrogel particles with different properties into the shaping framework by extrusion 3D printing method is as follows: Hydrogel particles with different mechanical strengths are respectively loaded into a feeding cylinder with a robotic arm. Taking the shaping framework as a template, programmed codes are set on 3D printing software, and by controlling different robotic arms to extrude in the specified shaping framework grid, the filling of hydrogel particles is completed, and a particulate gel gradient scaffold material is formed after in-situ crosslinking. Since the shaping framework grid is used as a template, this scaffold can achieve modulus gradient changes both in the horizontal direction and in the vertical direction.

[0036] According to some specific embodiments, the main components of the shaping framework are one or more of polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), and polyhydroxyalkanoates (PHA).

[0037] The second aspect of the present invention provides a gradient scaffold material prepared by the preparation method as described above.

[0038] The third aspect of the present invention provides an application of the gradient scaffold material prepared by the preparation method as described above or the gradient scaffold material as described above in tissue engineering interface repair materials.

[0039] Further, the tissue engineering interface repair materials include, but are not limited to, repair materials for bone-cartilage, tendon-bone, and skin.

[0040] Due to the application of the above technical solutions, the present invention has the following advantages compared with the prior art:

[0041] The preparation method of the present invention is simple, easy to control, and has high preparation precision. The scaffold material prepared by this method has the characteristics that its composition, structure, and mechanical properties can be continuously adjusted in space. Moreover, the scaffold also has good biocompatibility and a through-porous structure, which is convenient for the inward migration, proliferation, and directional differentiation of cells, and has important applications in the fields of repair of gradient tissue engineering interfaces such as bone-cartilage, tendon-bone, and skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The accompanying drawings here are incorporated into the specification and form a part of this specification, which show embodiments consistent with this application and, together with the specification, are used to explain the principles of this application. To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, for those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings. These accompanying drawings and written descriptions are not intended to limit the scope of the concept of this application in any way, but to illustrate the concept of this application to those skilled in the art by referring to specific embodiments.

[0043] Figure 1 The fiber-based shaping frame prepared in an embodiment of this application;

[0044] Figure 2 The schematic diagram of preparing hydrogel microspheres by droplet microfluidics technology in an embodiment of this application;

[0045] Figure 3 The schematic diagram of the principle of droplet enzyme crosslinking and photo-crosslinking of hydrogel microspheres in an embodiment of this application;

[0046] Figure 4 The optical microscope images of nano-hydroxyapatite hydrogel microspheres with contents of 0%, 1%, 3%, and 5% in an embodiment of this application;

[0047] Figure 5 The comparison result diagram of the storage modulus of nano-hydroxyapatite hydrogel microspheres with contents of 0%, 1%, 3%, and 5% before and after photo-crosslinking in an embodiment of this application;

[0048] Figure 6 The comparison result diagram of the storage modulus of particulate gels before and after adding the shaping frame in an embodiment of this application;

[0049] Figure 7 The equipment schematic diagram of the 3D printer used in an embodiment of this application;

[0050] Figure 8 The schematic diagram of the gradient scaffold prepared in an embodiment of this application;

[0051] Figure 9 The optical microscope images of the nano-hydroxyapatite hydrogel microspheres with contents of 0%, 1%, 3%, and 5% after being filled into the shaping frame and secondarily crosslinked in an embodiment of this application;

[0052] Figure 10 The SEM images of the nano-hydroxyapatite hydrogel microspheres with contents of 0%, 1%, 3%, and 5% after being filled into the shaping frame and secondarily crosslinked in an embodiment of this application;

[0053] Figure 11 Confocal microscopy image after secondary crosslinking of the shaping frame filled with nano-hydroxyapatite hydrogel microspheres with a content of 0% in an embodiment of the present application;

[0054] Figure 12 Optical microscopy image at the interface of hydrogel microspheres with different moduli in an embodiment of the present application;

[0055] Figure 13 Graph of storage modulus results of gelatin with different concentrations over time;

[0056] Figure 14 Graph of storage modulus results of Gel-MA with different concentrations over time. Detailed implementation manners

[0057] To address the deficiencies of the prior art, the present invention has developed a new method for preparing gradient scaffold materials. It prepares hydrogel microspheres through the first crosslinking step, then injects hydrogel microspheres with different properties such as different moduli into the shaping frame in a predetermined order, and then crosslinks the hydrogel microspheres in situ, thereby preparing a gradient scaffold with the characteristics of continuously adjustable composition, structure, and mechanical properties in space. Moreover, this scaffold also has good biocompatibility and a penetrating porous structure.

[0058] Compared with the existing technical solutions, the present invention has the following effects: (1) The gradient scaffold material based on particulate gels of the present invention can be combined with strategies such as additive manufacturing, and customized structures and sizes can be obtained, with flexible design and application. (2) Compared with ordinary hydrogels, this gradient scaffold has a penetrating and uniform porous structure, facilitating the inward migration, adhesion, and proliferation of cells. During this process, the scaffold material still maintains its integrity, avoiding structural distortion or collapse. (4) The mechanically properties that can be adjusted highly in space can very conveniently obtain a scaffold "micro"-environment with different mechanical properties by filling microspheres with different moduli, which is beneficial for guiding host or exogenous stem cells to differentiate in different directions, better integrating with complex tissues or organs, and realizing tissue regeneration; (3) The gradient scaffold material based on particulate gels has good biocompatibility, and the preparation process is mild, and it can simultaneously achieve in-situ encapsulation of cells, achieving purposes such as cell therapy or tissue regeneration.

[0059] The present invention will be further described below in conjunction with embodiments. However, the present invention is not limited to the following embodiments. The implementation conditions adopted in the embodiments can be further adjusted according to different specific usage requirements, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in each implementation manner of the present invention can be combined with each other as long as they do not conflict with each other.

[0060] Unless otherwise specified in this text, the preparation methods and detection methods involved in the following examples or comparative examples refer to the prior art. Unless otherwise specified in the following examples, all raw materials are commercially available or prepared by conventional methods in the art. The percentages of the components in the dispersion of this application are mass-volume percentages (w / v), with the unit of g / mL. For example, if the dispersion contains 10% (w / v) methacrylated gelatin, that is, 0.1 g of methacrylated gelatin is contained in 1 mL of the dispersion.

[0061] Example 1

[0062] (1) Printing of the fiber-based shaping frame

[0063] Weigh 800 mg of polycaprolactone and 200 mg of poly(lactic-co-glycolic acid) copolymer into a centrifuge tube, vortex for 10 min to uniformly mix the two. After loading into a steel syringe, set the syringe temperature to 85 °C and the needle temperature to 95 °C on the software supporting the printer, and heat for half an hour to completely melt it. First, adjust the distance between the printing needle and the printing platform to 2.5 mm, and keep this height unchanged to ensure the temperature of the printing voltage. Select a 35G printing needle, set the printing speed to 1500 mm / min, the length and width of the fiber-based shaping frame are 20 mm × 20 mm respectively, the spacing in the length and width directions of the grid in the fiber-based shaping frame is 1 mm × 1 mm respectively, the printing air pressure is 20 kPa, and the printing voltage is 4.5 kV. After setting, start printing to complete the printing of the fiber-based shaping frame. The shaping frame is as Figure 1 shown.

[0064] (2) Preparation of hydrogel microspheres with controllable modulus

[0065] Prepare a solution containing 10% (w / v) methacrylated gelatin (Gel-MA), 5% (w / v) gelatin, and 0.5% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphate in PBS in a centrifuge tube. After passing through a 0.22 μm filter head, add 5% (w / v) nano-hydroxyapatite to this solution and ultrasonically treat for 2 h to disperse the nano-hydroxyapatite and uniformly disperse it in the solution to obtain a dispersion (Gel@HAP). Dissolve transglutaminase (20 U / mL) in PBS in a centrifuge tube, vortex to dissolve, and pass through a 0.22 μm aqueous filter head to obtain a transglutaminase solution (TG). Select a polydimethylsiloxane chip with a 150×150×75 μm channel, and connect the dispersion, the transglutaminase solution, and the surfactant-containing oil phase (Oil) to the corresponding channels of the chip to prepare water-in-oil droplets. The schematic diagram is as Figure 2As shown. After collection with centrifuge tubes, crosslinking was carried out overnight at 37 °C. After crosslinking, the surfactant on the surface of the hydrogel microspheres was washed away, and then the oil was washed away with PBS. The prepared hydrogel microspheres had a diameter of 130 μm. Since there were still unreacted chemical bonds on the surface and inside of the microspheres, the microspheres could complete photocrosslinking to form a whole. The specific crosslinking schematic diagram is as shown in Figure 3 As shown, where glutamine and lysine on gelatin crosslinked to form hydrogel microspheres under the action of transglutaminase, and the acrylic functional groups on the surface and inside of the hydrogel microspheres crosslinked in situ under light to form a porous structure.

[0066] Modulus-controllable hydrogel microspheres were achieved by changing the concentration of nano-hydroxyapatite. The nano-hydroxyapatite contents in the prepared hydrogel microspheres with different moduli were 0% (w / v), 1% (w / v), 3% (w / v), and 5% (w / v) respectively, and the preparation method was the same as that of 5% (w / v) nano-hydroxyapatite above. The photos of the modulus-controllable hydrogel microspheres under an optical microscope are as shown in Figure 4 As shown, where the upper left figure is the hydrogel microspheres containing 0% (w / v) nano-hydroxyapatite, the upper right figure is the hydrogel microspheres containing 1% (w / v) nano-hydroxyapatite, the lower left figure is the hydrogel microspheres containing 3% (w / v) nano-hydroxyapatite, and the lower right figure is the hydrogel microspheres containing 5% (w / v) nano-hydroxyapatite.

[0067] (3) Rheological testing of modulus-controllable hydrogel microspheres

[0068] After selecting a modulus of hydrogel microspheres and blending them with PBS, they were centrifuged at 7500 rpm for 5 min to discard the supernatant. 200 μL was taken and piled up into a cylindrical sample with a diameter of 8 mm and a height of 3 mm, and then placed on a HAAKE rheometer for testing. The strain was set from 0.1% to 1000% and the angular frequency was set at 10 rad / s to test the stress-strain curve.

[0069] After the above-mentioned cylindrical sample was crosslinked with blue light for 5 min, in-situ crosslinking occurred between the hydrogel microspheres, and the stress-strain curve was tested according to the above parameters.

[0070] The rheological tests of the gels with 0%, 1%, 3%, and 5% nano-hydroxyapatite particle contents were carried out in the same way. After data processing, the changes in modulus were compared, as shown in Figure 5 As shown, where the first crosslinking is the result of testing after droplet enzyme crosslinking, and the second crosslinking is the result of testing after in-situ crosslinking of the hydrogel microspheres.

[0071] (4) Injecting and shaping the modulus-controllable hydrogel into a frame

[0072] After being treated by the same centrifugation method as in step (3), the hydrogel microspheres with 0% HAP were filled into a cylindrical shaping frame template with a diameter of 8 mm and a height of 3 mm, and in-situ crosslinked by blue light. The storage modulus was measured by the method in step (3), and the modulus change was compared. As Figure 6 shown, where MAP is the test result after in-situ crosslinking of the hydrogel microspheres without being filled into the shaping frame, and MAP / P is the test result after in-situ crosslinking of the hydrogel microspheres filled into the shaping frame. Combining Figure 5 and Figure 6 it can be seen that the modulus of the hydrogel microspheres after in-situ crosslinking increases, and as the content of nano-hydroxyapatite gradually increases, the modulus also increases, thus providing hydrogel microspheres with different moduli for the preparation of gradient scaffold materials. Further, after the hydrogel microspheres are combined with the shaping frame into an integral body, the modulus is further increased, so that the gradient scaffold materials can be applied to the repair of gradient tissue engineering interfaces such as bone-cartilage, tendon-bone, skin, etc.

[0073] After being treated by the same centrifugation method as in step (3), the hydrogel microspheres were loaded into a printing syringe to remove the air bubbles between the hydrogel microspheres. The 3D printer has its own specified spatial coordinates (represented by XYZ) at a fixed position, and there are also fixed codes (i.e., E) for opening and closing the air pump switch. Based on this, the movement of the 3D printer at any position, the extrusion of printing ink, and the filament breakage can be controlled at will. Edit the spatial coordinates of the printing needle head, 1.5 mm above the shaping frame grid prepared in step (1). At this time, turn on the pneumatic switch and move downward to the bottom of the grid at a speed of 50 mm / min, then turn off the pneumatic switch and move upward 1.5 mm at a speed of 250 mm / min. According to this principle, the hydrogel microspheres are filled into specific grids, and the feeding cylinder is switched to extrude hydrogel microspheres with another modulus by the same principle. Connect the codes together, so that the 3D printer can be used to fill hydrogel microspheres with different moduli efficiently and stably. After the code is edited, select a 20G printing needle head and set the printing air pressure to 130 kPa to uniformly extrude the hydrogel microspheres into the shaping frame grid. After all the filling is completed, in-situ crosslinking is completed after irradiating with blue light for 5 min to combine the microspheres and the shaping frame into an integral body. The equipment schematic diagram of the 3D printer is as Figure 7 shown, and the schematic diagram of the gradient scaffold is as Figure 8 shown.

[0074] The stacking mode of hydrogel microspheres with different moduli in the shaping frame is as Figure 9 shown, where the hydrogel microspheres containing 0%, 1%, 3%, and 5% nano-hydroxyapatite are corresponding from left to right.

[0075] The SEM images of hydrogel microspheres with different moduli after in-situ crosslinking are as Figure 10As shown, it can be seen from the figure that after in-situ crosslinking, hydrogel microspheres with different moduli can all form a porous structure with uniform macropores, and the pore size of the porous structure has little relationship with the content of nano-hydroxyapatite, mainly depending on the diameter of the hydrogel microspheres. The confocal microscopy image of the hydrogel microspheres containing 0% nano-hydroxyapatite after in-situ crosslinking is as Figure 11 shown, from Figure 11 it can be seen that compared with SEM, confocal microscopy was taken under the condition that the microspheres were not freeze-dried, which can better reflect the real situation of the microsphere stacking, and it can be seen that there is a continuous porous structure between the hydrogel microspheres, providing more adhesion sites for subsequent cell migration and proliferation.

[0076] The optical microscope photos at the interface of hydrogel microspheres with different moduli are as Figure 12 shown, from Figure 12 it can be seen that the gradient scaffold structure of this application has high precision and stable gradient structure.

[0077] Example 2

[0078] Dissolve different masses of gelatin and transglutaminase in PBS to prepare solutions of 1% gelatin, 3% gelatin and 5% gelatin respectively, wherein the concentration of transglutaminase is 20 U / mL. Incubate and crosslink at 37 °C, and respectively test the storage modulus of each hydrogel with the change of incubation time according to the method in step (3) of Example 1. The test results are as Figure 13 shown, from Figure 13 it can be seen that the moduli of the hydrogels prepared from the solutions with 3% and 5% gelatin content are relatively high. Among them, the modulus of the hydrogel with 5% gelatin content is about 1000 Pa, which is most suitable for making microspheres.

[0079] Dissolve different masses of Gel-MA and lithium phenyl(2,4,6-trimethylbenzoyl)phosphate in PBS to prepare solutions of 4% Gel-MA, 7% Gel-MA and 10% Gel-MA respectively, wherein the content of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate is 0.5%. Crosslinking is completed after blue light irradiation, and respectively test the storage modulus of each hydrogel with the change of light irradiation time according to the method in step (3) of Example 1. The test results are as Figure 14 shown, from Figure 14 it can be seen that the moduli of the hydrogels prepared from the solutions with 7% and 10% Gel-MA content are relatively high. Among them, the modulus of the hydrogel with 10% Gel-MA content is about 11000 Pa, which is most suitable for in-situ crosslinking between microspheres.

[0080] Figure 13 and Figure 14It is reflected that as the gelation time of the hydrogel progresses, the modulus gradually increases and reaches stability, completing the gelation, which indirectly reflects that other low concentrations are not suitable for droplet crosslinking and in-situ crosslinking between microspheres.

[0081] In addition, from Figure 13 and Figure 14 it can be seen that the moduli of hydrogels with different concentrations of gelatin and Gel-MA are different. Therefore, hydrogel microspheres with different moduli used for preparing gradient scaffold materials can also have equal concentrations of inorganic nanoparticles while the concentrations of gelatin or Gel-MA vary.

[0082] Through further research, when preparing the dispersion for preparing hydrogel microspheres, the preferred concentration range of Gel-MA is 5% - 10%, and the preferred concentration range of gelatin is 2% - 5%. If the concentration is too low, the crosslinking degree of the hydrogel is low and the system is unstable; if the concentration is too high, the viscosity of the solution is too large, resulting in non-uniform particle sizes of the microspheres. Within this concentration range, the highest concentration of inorganic nanoparticles does not exceed 5%. If the concentration is too high, it is not conducive to the dispersion of inorganic nanoparticles and leads to too high viscosity of the dispersion.

[0083] Example 3

[0084] It is basically the same as Example 1, with the only difference being the preparation of the dispersion in step (2).

[0085] The preparation method of the dispersion in this example is as follows:

[0086] Prepare a solution containing 10% (w / v) polyethylene glycol diacrylate (PEGDA), 5% (w / v) gelatin, and 0.5% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphate in a centrifuge tube with PBS. After passing through a 0.22 μm filter head, add 5% (w / v) nano-hydroxyapatite to this solution and ultrasonically treat for 2 h to disperse the nano-hydroxyapatite and uniformly disperse it in the solution to obtain the dispersion.

[0087] The gradient scaffold material prepared in this example has similar properties to the gradient scaffold material prepared in Example 1, both having a porous structure with uniform macropores.

[0088] Through further research, when preparing the dispersion for preparing hydrogel microspheres, the preferred concentration range of PEGDA is 3% - 10%, and the preferred concentration range of gelatin is 2% - 5%. If the concentration is too low, the crosslinking degree of the hydrogel is low and the system is unstable; if the concentration is too high, the viscosity of the solution is too large, resulting in non-uniform particle sizes of the microspheres. Within this concentration range, the highest concentration of inorganic nanoparticles does not exceed 5%. If the concentration is too high, it is not conducive to the dispersion of inorganic nanoparticles and leads to too high viscosity of the dispersion.

[0089] Example 4

[0090] It is basically the same as Example 1, except for the preparation of the dispersion in step (2).

[0091] The preparation method of the dispersion in this example is as follows:

[0092] Prepare a solution containing 2% (w / v) methacrylated hyaluronic acid (HA-MA), 5% (w / v) gelatin, and 0.5% (w / v) lithium phenyl(2,4,6-trimethylbenzoyl)phosphate in a centrifuge tube with PBS. After passing through a 0.22 μm filter head, add 5% (w / v) nano-hydroxyapatite to this solution and ultrasonically treat it for 2 h to disperse the nano-hydroxyapatite and uniformly disperse it in the solution to obtain the dispersion.

[0093] The gradient scaffold material prepared in this example has similar properties to the gradient scaffold material prepared in Example 1, and both have a porous structure with uniform macropores.

[0094] After further research, when preparing the dispersion for preparing hydrogel microspheres, the preferred concentration range of HA-MA is 2% - 4%, and the concentration range of gelatin is 2% - 5%. If the concentration is too low, the crosslinking degree of the hydrogel is low and the system is unstable; if the concentration is too high, the viscosity of the solution is too large, resulting in non-uniform particle sizes of the microspheres. Within this concentration range, the concentration of inorganic nanoparticles does not exceed 5% at most. If the concentration is too high, it is not conducive to the dispersion of inorganic nanoparticles and results in too high viscosity of the dispersion.

[0095] The above has described the present invention in detail. The purpose is to enable those skilled in this field to understand the content of the present invention and implement it, and it cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for preparing a gradient scaffold material based on particle gel, characterized in that: The steps include: (1) Constructing a shaping framework; (2) preparing a variety of hydrogel particles with different properties; (3) Injecting the plurality of hydrogel particles with different properties into the shaping frame respectively, and cross-linking the hydrogel particles in situ to prepare the gradient scaffold material.

2. The method for preparing a gradient scaffold material based on particle gel according to claim 1, characterized in that: In step (2), the hydrogel particles are formed by cross-linking a dispersion containing gelatin monomers, polymer monomers and optionally inorganic nanoparticles.

3. The method for preparing a gradient scaffold material based on particle gel according to claim 2, characterized in that: The gelatin monomer includes one or more of gelatin, methacrylated gelatin, and thiolated gelatin; and / or, The polymer monomer includes one or more of thiolated hyaluronic acid, methacrylated hyaluronic acid, thiolated sodium alginate, methacrylated sodium alginate, and polyethylene glycol diacrylate; and / or, The inorganic nanoparticles include one or more of hydroxyapatite, β-tricalcium phosphate, calcium carbonate, bioactive glass, and silicon-based nanoparticles.

4. The method for preparing a gradient scaffold material based on particle gel according to claim 2, characterized in that: The concentration of the gelatin monomer in the dispersion is 2% to 5%, the concentration of the high molecular monomer in the dispersion is 2% to 10%, and the concentration of the inorganic nanoparticles in the dispersion is 0% to 5%.

5. The method for preparing a gradient scaffold material based on particle gel according to claim 4, characterized in that: The plurality of hydrogel particles with different properties are prepared by preparing dispersions with different gelatin monomer concentrations and / or different high molecular monomer concentrations and / or different inorganic nanoparticle concentrations.

6. The method for preparing a gradient scaffold material based on particle gel according to claim 2, characterized in that: The dispersion also contains a photoinitiator.

7. The method for preparing a gradient scaffold material based on particle gel according to claim 2 or 6, characterized in that: One of the cross-linking in step (2) and the in-situ cross-linking in step (3) is performed under enzyme catalysis, and the other is performed under light irradiation.

8. The method for preparing a gradient scaffold material based on particle gel according to claim 7, characterized in that: Step (2) and step (3) specifically include the following steps: (2.1) preparing dispersion and enzyme solution; (2.2) injecting the dispersion, the enzyme solution and the oil phase containing the surfactant into corresponding channels of the microfluidic chip respectively to prepare water-in-oil droplets; (2.3) incubating the droplets so that the droplets are cross-linked under the catalysis of the enzyme, and washing away the oil phase containing the surfactant to obtain the hydrogel particles; (3) injecting the hydrogel particles into the shaping frame and then irradiating the frame with light to cause in situ cross-linking of the hydrogel microspheres.

9. The method for preparing a gradient scaffold material based on particle gel according to claim 7, characterized in that: The enzyme is transglutaminase, and / or The photoinitiator capable of initiating a crosslinking reaction under light irradiation is one or more of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide, 2,4,6-trimethylbenzoyl diphenyl phosphine oxide, 2-methyl-1-[4-(methylthio)phenyl]-2-morpholinyl-1-propanone, and phenyl-2,4,6-trimethylbenzoyl phosphite lithium, and / or, The light is blue light or ultraviolet light.

10. The method for preparing a gradient scaffold material based on particle gel according to claim 1, characterized in that: The particle size of the hydrogel particles is 10 to 300 μm; and / or the hydrogel particles are spherical; and / or the gradient scaffold material is a porous structure.

11. The method for preparing a gradient scaffold material based on particle gel according to claim 1, characterized in that: The hydrogel particles with different properties are hydrogel particles with different moduli.

12. The method for preparing a gradient scaffold material based on particle gel according to claim 1, characterized in that: In step (1), the shaping frame is prepared by electrospinning or extrusion 3D printing; and / or, In step (2), the hydrogel particles are prepared by microfluidics, membrane emulsification or suspension polymerization; and / or, In step (3), the plurality of hydrogel particles with different properties are respectively injected into the shaping frame by direct casting or extrusion 3D printing.

13. The method for preparing a gradient scaffold material based on particle gel according to claim 1, characterized in that: The main component of the shaping frame is one or more of polycaprolactone, polylactic acid-glycolic acid copolymer, polylactic acid, and polyhydroxyalkanoate.

14. A gradient scaffold material obtained by the preparation method according to any one of claims 1 to 13.

15. Use of the gradient scaffold material prepared by the preparation method according to any one of claims 1 to 13 or the gradient scaffold material according to claim 14 in tissue engineering interface repair materials.

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