阅读说明：本技术 高综合性能光固化生物3d打印复合水凝胶及其制备方法和应用 (High-comprehensive-performance photocuring biological 3D printing composite hydrogel and preparation method and application thereof ) 是由 黄传真 李淑颖 刘含莲 姚鹏 朱洪涛 邹斌 王军 于 2021-09-06 设计创作，主要内容包括：本申请提供一种高综合性能光固化生物3D打印复合水凝胶及其制备方法和应用。所述高综合性能光固化生物3D打印复合水凝胶为以甲基丙烯酰化明胶、甲基丙烯酰化透明质酸、甲基丙烯酰化丝素蛋白为单体共价交联固化形成的聚合物凝胶,具有生物相容性高、成形性好、机械强度可调、快速凝胶化的优势,可用于制备脊髓支架,可在支架上接种神经细胞,或着进行载神经细胞打印,用于神经损伤的修复、神经药物的筛选等。本发明材料还可用于周围神经组织支架的打印,或者多孔复杂组织结构的打印。(The application provides a high-comprehensive-performance photocuring biological 3D printing composite hydrogel and a preparation method and application thereof. The high comprehensive performance photocuring biological 3D printing composite hydrogel is a polymer gel formed by covalent crosslinking and curing monomers of methacryloylated gelatin, methacryloylated hyaluronic acid and methacryloylated silk fibroin, has the advantages of high biocompatibility, good formability, adjustable mechanical strength and rapid gelation, can be used for preparing a spinal cord stent, can be used for inoculating nerve cells on the stent or printing nerve-carrying cells, and is used for repairing nerve injury, screening nerve drugs and the like. The material of the invention can also be used for printing the peripheral nerve tissue scaffold or porous complex tissue structure.)

1. A high comprehensive performance photocuring biological 3D printing composite hydrogel is a polymer gel formed by covalent crosslinking and curing monomers of methacryloylated gelatin, methacryloylated hyaluronic acid and methacryloylated silk fibroin.

2. A method of making the high overall performance photocurable bio-3D printed composite hydrogel of claim 1, comprising: respectively adding the methacryloylated gelatin, the methacryloylated hyaluronic acid and the methacryloylated silk fibroin into the photoinitiator standard solution to be dissolved in the dark to obtain a composite solution, adding the light blocking agent to be stirred to be completely dissolved, and crosslinking and curing under the light to form gel.

3. The method of claim 2, wherein the photoinitiator is lithium phenyl-2, 4, 6-trimethylbenzoylphosphite;

preferably, the light-blocking agent is lemon yellow.

4. The method according to claim 1 or 2, characterized in that the method comprises: adding methacrylated gelatin into a photoinitiator standard solution, heating in a water bath at 55-60 ℃ in the dark until the methacrylated gelatin is completely dissolved, then adding methacrylated hyaluronic acid, heating in a water bath at 55-60 ℃ in the dark until the methacrylated hyaluronic acid is completely dissolved, then adding methacrylated silk fibroin to dissolve at room temperature to obtain a mixed solution of the methacrylated gelatin, the methacrylated hyaluronic acid and the methacrylated silk fibroin, adding a light-blocking agent, stirring until the mixed solution is completely dissolved, and crosslinking and curing to form gel under the irradiation of ultraviolet light to obtain the compound.

5. The method according to claim 1 or 2, wherein the mass-to-volume concentration ratio of the methacrylated gelatin, the methacrylated hyaluronic acid and the methacrylated silk fibroin in the mixed solution of the methacrylated gelatin, the methacrylated hyaluronic acid and the methacrylated silk fibroin is 8:2: 1-5.

6. A neural tissue scaffold or porous scaffold starting from the high comprehensive performance photocurable bio-3D printed composite hydrogel of claim 1.

7. The neural tissue scaffold according to claim 1, which is a spinal cord scaffold, a peripheral neural tissue scaffold.

8. The nerve tissue scaffold or the porous scaffold according to claim 6 or 7, which is prepared by 3D printing from the high comprehensive performance photocuring biological 3D printing composite hydrogel according to claim 1.

9. A method of preparing a neural tissue scaffold or porous scaffold using 3D printing techniques, comprising: carrying out photocuring 3D printing by using the high-comprehensive-performance photocuring biological 3D printing composite hydrogel as a raw material according to claim 1 or carrying nerve cells on the high-comprehensive-performance photocuring biological 3D printing composite hydrogel according to claim 1, wherein the photocuring 3D printing parameters are as follows: the temperature of a deposition platform of the photocuring 3D printer is 27 ℃, the temperature of a trough is 29 ℃, the layer height is 10 mu m, and the light intensity is 10mw/cm²The number of the base layer layers is 5, the exposure time of the base layer is 5s, the exposure time of the lamella layer is 10-12s, the Z-axis speed is 25mm/min, the stripping distance is 6mm, the stripping speed is 2mm/min, and the stripping recovery speed is 200 mm/min.

10. Use of the high-integration-performance photocuring biological 3D-printed composite hydrogel of claim 1 or the neural tissue scaffold or porous scaffold of any one of claims 6 to 8 in the preparation of a material for repairing nerve damage or in nerve drug screening.

Technical Field

The application relates to medical modified polymer hydrogel, in particular to high-comprehensive-performance photocuring biological 3D printing composite hydrogel and a preparation method and application thereof.

Background

The information disclosed in this background of the invention is intended to enhance an understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information has become known as prior art to a person skilled in the art.

The spinal cord, the spinal cord located in the spinal canal and connecting the nerves around the brain and the nerve system, is responsible for the conduction of nerves, and once the spinal cord is damaged, paralysis of limbs and loss of sensation can occur, and other nerve function injuries can also occur. Spinal Cord Injury (SCI) has a high disability rate and is difficult to recover, and is a serious clinical disease, especially a series of secondary reactions after primary injury make the disease more complicated. Injury from secondary reactions can spread from the initial area of injury to other areas, forming astrocytic scars, hindering axonal regeneration, and also leading to the formation of cystic spaces. Therefore, spinal cord injury has been a difficult problem in the medical field.

With the progress of scientific technology, the biological 3D printing technology provides a brand-new clinical medical strategy for tissue and organ repair, and is applied to the medical field, such as tissue and organ substitute construction, organ transplantation and regeneration, drug screening models, anatomical models and the like. Biological 3D printing can realize printing of complex tissue structures, cell-carrying printing, controllable and accurate deposition of different cell numbers and cell densities, easily obtains the physical and chemical property gradient of biological materials, and has a plurality of advantages in tissue organ repair.

Hydrogel capable of wrapping living cells is particularly important for biological 3D printing, and the proper physical and chemical properties of hydrogel materials are the premise of cell adhesion, migration and proliferation. Therefore, the hydrogel material needs to have good biocompatibility, not only can provide a specific microenvironment for cell growth, but also is the basis for cell functional behavior induction; meanwhile, the ink has good printability, degradation rate matched with the regeneration speed of tissue cells, swelling rate suitable for cell growth and good mechanical property. At present, the preparation of a hydrogel material with good comprehensive performance is still a difficulty of biological 3D printing.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides the photocuring biological 3D printing composite hydrogel with high comprehensive performance, which has the advantages of high biocompatibility, good formability, adjustable mechanical strength and rapid gelation, and can be used for printing a spinal cord stent. The spinal cord stent printed in a 3D mode can be used for inoculating nerve cells or printing nerve cells, and is used for repairing nerve injury, screening nerve drugs and the like. The composite hydrogel material can also be used for printing a peripheral nerve tissue scaffold or printing a porous complex tissue structure.

Specifically, the present invention provides the following technical features, and one or a combination of the following technical features constitutes the technical solution of the present invention.

In a first aspect of the invention, the invention provides a high-comprehensive-performance photocuring biological 3D printing composite hydrogel which is a polymer gel formed by covalent crosslinking and curing monomers of methacryloylated gelatin (GelMA), methacryloylated hyaluronic acid (HAMA) and methacryloylated silk fibroin (SilMA).

The gelatin contains a plurality of bioactive sites, particularly integrin binding sequence RGD peptide, and has good biodegradability and cell adhesion regulation. Gelatin is a temperature sensitive material, a hydrogel network is formed through a physical crosslinking mechanism, but physical crosslinking is greatly influenced by the external environment and has poor stability.

Hyaluronic acid, due to its high molecular weight and ability to absorb a large amount of water, helps to maintain the mechanical integrity, homeostasis, viscoelasticity and lubricity of the tissue, and is an important constituent of the neurocytoplasmic matrix. At the same time, it can bind with cell surface specific receptor (such as CD44) to regulate cell adhesion, migration, proliferation and differentiation. Hyaluronic acid-based biomaterials do not induce allergy and inflammation. But the formability and mechanical strength of hyaluronic acid biological 3D printing are relatively poor.

The silk fibroin is a natural high molecular fiber polymer extracted from silk, contains 18 amino acids, has cell binding sites on a silk fibroin molecular chain, has good bioactivity compared with a synthetic high molecular material, and is beneficial to the adhesion of nerve tissue cells; in addition, the methacrylated silk fibroin can be quickly dissolved in water after being modified, and has a higher gelation speed. Thus, silk fibroin has been used in the biomedical field.

In the implementation mode of the invention, the materials are selected, and on the basis of the materials, double bonds are introduced on the basis of the structure of the materials and are combined for use, so that on one hand, the photosensitivity of the materials is increased, on the other hand, the modified materials can form covalent bonds among molecules in a monomer form to generate a polymerization network, and the gel material with excellent comprehensive performances such as biocompatibility, moldability, mechanical property and the like and high crosslinking speed is obtained

The material is the combination of methacrylated gelatin, methacrylated hyaluronic acid and methacrylated silk fibroin, the materials are important components of a nerve cell cytoplasm matrix, the adhesion, proliferation and differentiation of nerve stem cells are facilitated, the material can be rapidly solidified under ultraviolet light after being modified, the formability is good, particularly, the compression modulus of the scaffold material can be adjusted by adjusting the content of the methacrylated silk fibroin, and the elastic modulus of the scaffold is in the modulus range of natural nervous tissues.

In a second aspect of the present invention, the present invention provides a method of preparing the comprehensive performance photocuring bio-3D printing composite hydrogel described in the first aspect above, comprising: respectively adding the methacryloylated gelatin, the methacryloylated hyaluronic acid and the methacryloylated silk fibroin into the photoinitiator standard solution to be dissolved in the dark to obtain a composite solution, adding the light blocking agent to be stirred to be completely dissolved, and crosslinking and curing under the light to form gel.

In an embodiment of the invention, the photoinitiator is lithium phenyl-2, 4, 6-trimethylbenzoylphosphite (LAP); the light-resisting agent is lemon yellow.

In some embodiments of the invention, the method comprises: adding methacrylated gelatin into a photoinitiator standard solution, heating in a water bath at 55-60 ℃ in the dark until the methacrylated gelatin is completely dissolved, then adding methacrylated hyaluronic acid, heating in a water bath at 55-60 ℃ in the dark until the methacrylated hyaluronic acid is completely dissolved, then adding methacrylated silk fibroin to dissolve at room temperature to obtain a mixed solution of the methacrylated gelatin, the methacrylated hyaluronic acid and the methacrylated silk fibroin, adding a light-blocking agent, stirring until the mixed solution is completely dissolved, and crosslinking and curing to form gel under the irradiation of ultraviolet light to obtain the compound.

In some embodiments of the present invention, the mass to volume concentration ratio of the methacrylated gelatin, the methacrylated hyaluronic acid, and the methacrylated silk fibroin in the mixed solution of the methacrylated gelatin, the methacrylated hyaluronic acid, and the methacrylated silk fibroin is 8:2: 1-5. In a further embodiment, the volume concentration ratio is 8:2: 3-5.

In an embodiment of the present invention, the inventors also provide a method for producing methacrylated gelatin and methacrylated hyaluronic acid, which comprises introducing a carbon-carbon double bond into a molecular chain of gelatin or hyaluronic acid.

Specifically, in some embodiments herein, the process for preparing methacrylated gelatin comprises: soaking gelatin, stirring under water bath heating condition (maintaining 55 deg.C) to dissolve completely, adding methacrylic anhydride, mixing, reacting, adjusting pH to about 8, dialyzing, centrifuging after reaction, freeze drying (at least 2 hr in ultra-low temperature refrigerator) directly without thawing, and freeze drying to completely dehydrate (about 3-4 days) to obtain the final product. In an embodiment of the invention, the dialysis is performed using a dialysis bag with a cut-off molecular weight of 12000.

In some embodiments of the invention, the method of preparing an acylated hyaluronic acid comprises: soaking hyaluronic acid, heating in water bath (50 ℃) to be completely dissolved, then dropwise adding dimethyl formamide, fully reacting, then adding methacrylic anhydride, simultaneously adjusting the pH value of a hydrogel solution to be 8-9, fully reacting overnight, then adding reverse osmosis water to dilute the solution, transferring the hydrogel solution into a dialysis bag to be dialyzed after the sodium chloride is fully dissolved, freezing the dialyzed solution (at least 2 hours in an ultra-low temperature refrigerator), directly freeze-drying without unfreezing after freezing, and freeze-drying until the solution is completely dehydrated (about 3-4 days), thus obtaining the hyaluronic acid gel. In an embodiment of the invention, the dialysis is performed using a dialysis bag with a cut-off molecular weight of 12000.

In the embodiment of the invention, carbon-carbon double bonds are introduced into the molecular chain of the gelatin or the hyaluronic acid, and the modified gelatin or the modified hyaluronic acid has photosensitivity and can be crosslinked and cured into the gelatin by ultraviolet light under the action of an initiator.

By adopting the method, under the irradiation of ultraviolet light with the wavelength of 405nm, the initiator absorbs light energy to generate a polymerization reaction active center, the active center and GelMA monomers, HAMA monomers and SilMA monomers generate free radical reaction, the monomer free radicals and the monomers generate new free radicals through addition, so that the extended chain free radicals are repeatedly generated, the extended chain free radicals lose activity to generate polymer molecules, namely covalent bonds are formed among GelMA, HAMA and SilMA macromolecules to generate a polymerization network, and the GelMA, HAMA and SilMA molecules form hydrogel with good biocompatibility, high forming precision, high strength and high crosslinking speed, and can be used for photocuring biological 3D printing.

In a third aspect of the invention, the invention provides a neural tissue scaffold or porous scaffold, which uses the high comprehensive performance photo-curing biological 3D printing composite hydrogel as described in the first aspect as a raw material. In some embodiments of the invention, the neural tissue scaffold is a spinal cord scaffold, a peripheral neural tissue scaffold.

In some embodiments of the present invention, the nerve tissue scaffold or the porous scaffold is prepared by 3D printing using the high comprehensive performance photo-curable biological 3D printing composite hydrogel described in the first aspect as a raw material.

In a fourth aspect of the present invention, the present invention provides a method for preparing a neural tissue scaffold or a porous scaffold using a 3D printing technique, comprising: carrying out photocuring 3D printing by taking the high comprehensive performance photocuring biological 3D printing composite hydrogel as a raw material or carrying out photocuring 3D printing by taking the high comprehensive performance photocuring biological 3D printing composite hydrogel carrying nerve cells as a raw material, wherein the photocuring 3D printing parameters are as follows: the temperature of a deposition platform of the photocuring 3D printer is 27 ℃, the temperature of a trough is 29 ℃, the layer height is 10 mu m, and the light intensity is 10mw/cm²The number of the base layer layers is 5, the exposure time of the base layer is 5s, the exposure time of the lamella layer is 10-12s, the Z-axis speed is 25mm/min, the stripping distance is 6mm, the stripping speed is 2mm/min, and the stripping recovery speed is 200 mm/min.

In a fifth aspect of the invention, the invention provides the use of the high comprehensive performance photocuring biological 3D printing composite hydrogel described in the first aspect or the neural tissue scaffold or porous scaffold described in the third aspect in the preparation of a material for repairing nerve damage or in nerve drug screening.

Through one or more technical means, the following beneficial effects can be achieved:

(1) GelMA, HAMA and SilMA materials are added, all photosensitive materials can be added, the gel time can be controlled by photocuring and printing a complex organization structure with high precision, good formability and good stability, and adjusting the illumination intensity and the exposure time.

(2) The addition of lemon yellow light-blocking agent is helpful for printing micro-pore diameter.

(3) The GelMA is added to provide a cell adhesion site, so that the cell compatibility and the cell reactivity are excellent; the addition of HAMA improves the water absorption of the hydrogel, and helps to maintain the mechanical integrity, homeostasis, viscoelasticity and lubricity of the tissue; after the SilMA is added, the mechanical strength of the hydrogel is improved, the gelation time is shortened, and the biocompatibility is improved. The composite hydrogel has high biocompatibility, high mechanical strength, rapid gelation and high comprehensive performance with good formability, and provides a novel photocuring material for preparing spinal cord stents.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application. Embodiments of the present application are described in detail below with reference to the attached drawing figures, wherein:

fig. 1 is a photograph of a hydrogel photocured 3D printed spinal cord scaffold and porous scaffold prepared in examples 1-4 of the present invention.

FIG. 2 is a microscopic topography of the hydrogel prepared in examples 1-4 of the present invention under a scanning electron microscope.

FIG. 3 is a graph of the rheological properties of hydrogels prepared in examples 1-4 of the present invention.

Detailed Description

The present application is further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or starting materials used in the present application can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present application can be used in the conventional manner in the art or in the product specification. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present application. The preferred embodiments and materials described herein are intended to be exemplary only.

As some embodiments of the invention, the high-comprehensive-performance photocuring biological 3D printing composite hydrogel material is prepared from GelMA 8% (w/v), HAMA 2% (w/v), SilMA 1% -5% (w/v), LAP 0.25% (w/v) and lemon yellow 0.05% (w/v).

Example 1

(1) Preparation of methacrylic acylated gelatin: 200g of gelatin was soaked in 200ml of deionized water and stirred in a magnetic stirrer for 90min to promote dissolution of the gelatin, while stirring the mixture was heated in a water bath to 55 deg.C (and held at this temperature) until the gelatin was completely dissolved and the solution became clear. After the gelatin is dissolved, 12g of methacrylic anhydride is added dropwise while stirring, the mixture is fully mixed and reacted for 4 hours, and the pH value of the hydrogel solution is adjusted to be about 8 by using 0.5M sodium hydroxide solution in the reaction process. After the reaction is finished, the solution is transferred to a dialysis bag with the molecular weight cutoff of 12000 to be dialyzed in a large amount of deionized water for 4-5 days, and the deionized water is replaced every day. Centrifuging at 5710r/min for 5min in a centrifuge after dialysis, transferring the centrifuged solution into an ultra-low temperature refrigerator for freezing for 2h, transferring the sample into a freeze dryer without thawing after freezing, freeze-drying until the sample is completely dehydrated (about 3-4 days), and finally storing the freeze-dried gel in a brown bottle in a sealed and refrigerated manner for later use.

(2) Preparation of methacrylic acylated hyaluronic acid: 2g of hyaluronic acid was soaked in 150ml of reverse osmosis water, heated to 50 ℃ in a water bath and stirred in a magnetic stirrer until the hyaluronic acid was completely dissolved. Subsequently, 100ml of dimethylformamide is added dropwise, the reaction is carried out for 60min fully, 2.2ml of methacrylic anhydride is added dropwise, the pH value of the hydrogel solution is kept between 8 and 9 by using 0.5M NaOH, 1000ml of reverse osmosis water diluted hydrogel solution is added after the reaction is carried out for overnight fully, 29g of sodium chloride is added, the hydrogel solution is transferred to a dialysis bag with the molecular weight cutoff of 12000 for dialysis for 4 days after the sodium chloride is fully dissolved, the dialyzed solution is equally divided and placed in an ultra-low temperature refrigerator for freeze-drying for 2h, the sample is transferred to a freeze-drying machine without thawing after freezing, freeze-drying is carried out until the gel is completely dehydrated (about 3 to 4 days), and finally the freeze-dried gel is placed in a brown bottle for sealed refrigeration and storage for use.

(3) Preparation of methacrylated gelatin (GelMA), methacrylated hyaluronic acid (HAMA) complex solution: measuring 20ml of PBS solution, adding 0.05g of photoinitiator phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite, heating and dissolving for 15min in water bath at 40-50 ℃, and stirring and shaking for several times during the heating and dissolving process to obtain 0.25% (w/v) initiator standard solution; putting 1.6g GelMA into an initiator standard solution, heating in a water bath at 55-60 ℃ in a dark place until GelMA is completely dissolved, and stirring and shaking the mixture to obtain 8% (w/v) GelMA solution; 0.4g of HAMA is put into 8 percent (w/v) GelMA solution, and is heated in a water bath with the temperature of 55-60 ℃ in a dark place until the HAMA is completely dissolved, and the mixture is stirred and shaken during the heating process to obtain 8 percent (w/v) GelMA and 2 percent (w/v) HAMA mixed solution; 0.005g of light-blocking agent lemon yellow is taken and put into a mixed solution of 8% (w/v) GelMA and 2% (w/v) HAMA, and the mixture is stirred until the mixture is completely dissolved. (Note: lemon yellow is added to the composite hydrogel solution only during photocuring 3D printing (to facilitate printing of micro-pore structures), and lemon yellow is not added to the composite hydrogel solution during hydrogel performance testing.)

(4) Photocuring 3D printing of composite hydrogel: the temperature of a deposition platform of the photocuring 3D printer is 27 ℃, the temperature of a trough is 29 ℃, the layer height is 10 mu m, the light intensity is 10mw/cm2, the number of base layer is 5, the exposure time of the base layer is 5s, the exposure time of a lamella layer is 10-12s, the Z-axis speed is 25mm/min, the stripping distance is 6mm, the stripping speed is 2mm/min, and the stripping recovery speed is 200 mm/min.

Example 2

(1) Preparation of methacrylic acylated gelatin: the same procedure as in step (1) of example 1.

(2) Preparation of methacrylic acylated hyaluronic acid: the same procedure as in step (2) of example 1.

(3) Preparation of methacrylated gelatin (GelMA), methacrylated hyaluronic acid (HAMA), methacrylated silk fibroin (SilMA, ex EFL) complex solutions: measuring 20ml of PBS solution, adding 0.05g of photoinitiator phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite, heating and dissolving for 15min in water bath at 40-50 ℃, and stirring and shaking for several times during the heating and dissolving process to obtain 0.25% (w/v) initiator standard solution; putting 1.6g GelMA into an initiator standard solution, heating in a water bath at 55-60 ℃ in a dark place until GelMA is completely dissolved, and stirring and shaking the mixture to obtain 8% (w/v) GelMA solution; 0.4g of HAMA is put into 8 percent (w/v) GelMA solution, and is heated in a water bath at 55-60 ℃ in a dark place until the HAMA is completely dissolved, and the mixture is stirred and vibrated during the heating process to obtain 8 percent (w/v) GelMA and 2 percent (w/v) HAMA mixed solution; taking 0.2g of SilMA0, putting the SilMA0.2g into 8% (w/v) GelMA and 2% (w/v) HAMA solution, and dissolving at room temperature (avoiding intense ultrasound, high temperature and strong shearing) to obtain a mixed solution of 8% (w/v) GelMA, 2% (w/v) HAMA and 1% SilMA. 0.005g of lemon yellow is taken and put into a mixed solution of 8% (w/v) GelMA, 2% (w/v) HAMA and 1% SilMA, and stirred until the lemon yellow is completely dissolved. (Note: lemon yellow is added to the composite hydrogel solution only during photocuring 3D printing (to facilitate printing of micro-pore structures), and lemon yellow is not added to the composite hydrogel solution during hydrogel performance testing.)

(4) Photocuring 3D printing of composite hydrogel: the temperature of a deposition platform of the photocuring 3D printer is 27 ℃, the temperature of a trough is 29 ℃, the layer height is 10 mu m, the light intensity is 10mw/cm2, the number of base layer is 5, the exposure time of the base layer is 5s, the exposure time of a lamella layer is 7-12s, the Z speed is 25mm/min, the stripping distance is 6mm, the stripping speed is 2mm/min, and the stripping recovery speed is 200 mm/min.

Example 3

(1) Preparation of methacrylic acylated gelatin: the same procedure as in step (1) of example 1.

(2) Preparation of methacrylic acylated hyaluronic acid: the same procedure as in step (2) of example 1.

(3) Preparation of methacrylated gelatin (GelMA), methacrylated hyaluronic acid (HAMA), methacrylated silk fibroin (SilMA, ex EFL) complex solutions: measuring 20ml of PBS solution, adding 0.05g of photoinitiator phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite, heating and dissolving for 15min in water bath at 40-50 ℃, and stirring and shaking for several times during the heating and dissolving process to obtain 0.25% (w/v) initiator standard solution; putting 1.6g GelMA into an initiator standard solution, heating in a water bath at 55-60 ℃ in a dark place until GelMA is completely dissolved, and stirring and shaking the mixture to obtain 8% (w/v) GelMA solution; 0.4g of HAMA is put into 8 percent (w/v) GelMA solution, and is heated in a water bath at 55-60 ℃ in a dark place until the HAMA is completely dissolved, and the mixture is stirred and vibrated during the heating process to obtain 8 percent (w/v) GelMA and 2 percent (w/v) HAMA mixed solution; taking 0.6g of SilMA0, putting the SilMA0 into 8% (w/v) GelMA and 2% (w/v) HAMA solution, and dissolving at room temperature (avoiding violent ultrasound, high temperature and strong shearing) to obtain a mixed solution of 8% (w/v) GelMA, 2% (w/v) HAMA and 3% SilMA. 0.005g of lemon yellow is taken and put into a mixed solution of 8% (w/v) GelMA, 2% (w/v) HAMA and 3% SilMA, and stirred until the lemon yellow is completely dissolved. (Note: lemon yellow is added to the composite hydrogel solution only during photocuring 3D printing (to facilitate printing of micro-pore structures), and lemon yellow is not added to the composite hydrogel solution during hydrogel performance testing.)

(4) Photocuring 3D printing of composite hydrogel: the temperature of a deposition platform of the photocuring 3D printer is 27 ℃, the temperature of a trough is 29 ℃, the layer height is 10 mu m, the light intensity is 10mw/cm2, the number of base layer is 5, the exposure time of the base layer is 5s, the exposure time of a lamella layer is 7-12s, the Z speed is 25mm/min, the stripping distance is 6mm, the stripping speed is 2mm/min, and the stripping recovery speed is 200 mm/min.

Example 4

(1) Preparation of methacrylic acylated gelatin: the same procedure as in step (1) of example 1.

(2) Preparation of methacrylic acylated hyaluronic acid: the same procedure as in step (2) of example 1.

(3) Preparation of methacrylated gelatin (GelMA), methacrylated hyaluronic acid (HAMA), methacrylated silk fibroin (SilMA, ex EFL) complex solutions: measuring 20ml of PBS solution, adding 0.05g of photoinitiator phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite, heating and dissolving for 15min in water bath at 40-50 ℃, and stirring and shaking for several times during the heating and dissolving process to obtain 0.25% (w/v) initiator standard solution; putting 1.6g GelMA into an initiator standard solution, heating in a water bath at 55-60 ℃ in a dark place until GelMA is completely dissolved, and stirring and shaking the mixture to obtain 8% (w/v) GelMA solution; 0.4g of HAMA is put into 8 percent (w/v) GelMA solution, and is heated in a water bath at 55-60 ℃ in a dark place until the HAMA is completely dissolved, and the mixture is stirred and vibrated during the heating process to obtain 8 percent (w/v) GelMA and 2 percent (w/v) HAMA mixed solution; taking SilMA1g, putting into 8% (w/v) GelMA and 2% (w/v) HAMA solution, and dissolving at room temperature (avoiding intense ultrasound, high temperature and strong shearing) to obtain 8% (w/v) GelMA, 2% (w/v) HAMA and 3% SilMA mixed solution. 0.005g of lemon yellow is taken and put into a mixed solution of 8% (w/v) GelMA, 2% (w/v) HAMA and 5% SilMA, and stirred until the lemon yellow is completely dissolved. (Note: lemon yellow is added to the composite hydrogel solution only during photocuring 3D printing (to facilitate printing of micro-pore structures), and lemon yellow is not added to the composite hydrogel solution during hydrogel performance testing.)

(4) Photocuring 3D printing of composite hydrogel: the temperature of a deposition platform of the photocuring 3D printer is 27 ℃, the temperature of a trough is 29 ℃, the layer height is 10 mu m, the light intensity is 10mw/cm2, the number of base layer is 5, the exposure time of the base layer is 5s, the exposure time of a lamella layer is 7-12s, the Z speed is 25mm/min, the stripping distance is 6mm, the stripping speed is 2mm/min, and the stripping recovery speed is 200 mm/min.

The following tests illustrate the advantages of the hydrogel materials of the examples in tissue engineering scaffold or cell-loaded printing tissue applications.

1. And (3) forming property test: example 1, example 2, example 3, example 4 hydrogel material 3D printing spinal cord scaffolds and porous scaffolds as shown in (a), (b), (c), (D) of fig. 1, the exposure time of the sheet layer of example 1 was 10-12s, the printing effect of the porous scaffold was slightly poor, and the porous scaffold was prone to adhesion when removed from the printer deposition station. Examples 2, 3 and 4 have better formability of the printed spinal cord scaffold and the porous scaffold compared with example 1, and the pores are not easy to collapse, in examples 2, 3 and 4, the gelation time is obviously reduced along with the increase of the content of SilMA, and the printing and forming can be carried out within 7-12s of the exposure time of a lamella, which is important for cell-carrying printing.

2. Microscopic morphology of hydrogel: the microscopic morphologies of the hydrogel materials of example 1, example 2, example 3 and example 4 after being solidified, freeze-dried and gold-sprayed are shown in (a), (b), (c) and (d) of fig. 2. Fig. 2 shows that the micro-morphologies of the hydrogels of the embodiments 1, 2, 3, and 4 are porous networks, and the pore sizes of the hydrogel networks are uniform after the addition of the SilMA, and the pores of the hydrogel networks are more dense with the increase of the content of the SilMA, so that other nutrients can easily enter and exit the hydrogel in the process of culturing cells, and the metabolism of the cells is facilitated.

3. Rheological property test: real-time synchronization of rheological testing and UV curing reactions was achieved using an Anton Paar MCr 302 rheometer, with the UV source radiation intensity and wavelength range adjustable by radiometers and filters, and 60% for the experimental light sources of examples 1-4. To evaluate the gelation time of the hydrogels of examples 1-4, tests were carried out at 25 ℃ using a 50mm parallel plate system, the gap distance was fixed at 50um, 0.5ml of homogeneous solution was loaded on the fixture, time scanning was carried out at a frequency of 1Hz with a strain of 1%, the uv lamp was turned on at 50s and irradiation was continued for 300 s. As can be seen from the rheological data in fig. 3, the curing of example 1 starts within 2-3s, the curing of examples 2, 3 and 4 starts rapidly within 2s, the curing degree of examples 1-4 reaches 65% within 35s, the curing is complete within 50s, and the rapid gelation is cell-loaded photocuring 3D printing, so that the time is saved, and the cell viability is improved. In examples 1 to 4, as the amount of SilMA was increased, the storage modulus of the hydrogel was increased, and the print formability was greatly improved.

4. And (3) testing mechanical properties: the compression modulus of several hydrogels of examples 1-4 was tested with a pre-load of 1N and a compression rate of 1mm/min, and the parameters obtained are shown in Table 1.

TABLE 1 hydrogel Material compression modulus test results

As can be seen from Table 1, after the SilMA is added into the hydrogel component, the compressive modulus of the hydrogel is increased, and the compressive modulus is increased along with the increase of the SilMA content, when the SilMA content is increased to 5%, the compressive modulus of the hydrogel can reach 8623.7Pa, compared with example 1, the compressive modulus of the hydrogel is increased by 1 time, and the modulus of the hydrogel can be adjusted by changing the content of the silk fibroin methacrylated, so that the spinal cord scaffold with certain mechanical properties is obtained.

5. CCK-8 measures cell proliferation: the hydrogel blocks of the cylinders obtained after curing in examples 1-4 were pressed by 1.25cm²Soaking in culture medium at a leaching medium ratio of/ml (surface area of leaching medium to volume of culture medium) for 24 hr to obtain leaching solution, and sterilizing with filter for use. Taking mouse fibroblast L929, adjusting the concentration of the cell suspension to 3 x 10⁷And (2) inoculating the prepared cell suspension into a 96-well plate, culturing at 100 uL/well in a 5% CO 2 incubator at 37 ℃ for 24h to ensure that the cells are attached to the wall completely, removing the culture solution, adding 100uL of leaching liquor into an experimental group, and adding 100uL of fresh culture medium into a control group. At the time points of 1 day, 3 days, 5 days and 7 days respectively, the culture solution is absorbed and discarded, 100ul of leaching liquor is added into the experimental group, 100ul of fresh culture medium is added into the control group, 10ul of CCK-8 solution is added, after 1 hour of culture, the experimental group is vibrated for 10s in an enzyme labeling instrument, the light absorption value (OD) is measured under the wavelength of 450nm, 5 holes are parallelly measured in each group, and the average value is taken.

TABLE 2 light absorption test results for different materials

Relative increase in cell% (% Absorbance of Experimental group/Absorbance of control group) (% Absorbance of Experimental group/100%

TABLE 3 relative cell proliferation Rate

As shown in Table 2, in examples 1 to 4, the absorbance (OD) at a wavelength of 450nm increased with the number of days of culture. As shown in Table 3, the cell viability was higher than 80% for examples 1-4, according to GB/T16886.5-2017/ISO 10993-5: 2009, the materials of the examples of the invention are not potentially cytotoxic. Examples 2, 3 and 4 showed significantly improved cell viability compared to example 1, i.e., the cell viability was significantly improved after methacrylated silk fibroin was added in example 1, and the cell viability was the highest in example 2, in which the content of methacrylated silk fibroin was 1% (w/v), which promoted cell proliferation compared to the control group. These results indicate that these composite hydrogels have good cell compatibility, non-toxicity of spinal cord scaffolds and porous scaffolds.

The invention prints a spinal cord stent, can inoculate nerve cells on the stent or print nerve cells, and is used for repairing nerve injury, screening nerve drugs and the like. The material of the invention can also be used for printing the peripheral nerve tissue scaffold or porous complex tissue structure.

Although the present application has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described in the foregoing embodiments, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.