High-strength gelatin-based hydrogel suitable for volume additive manufacturing, preparation method of high-strength gelatin-based hydrogel and model printing method

Abstract

The invention discloses a high-strength gelatin-based hydrogel suitable for volume additive manufacturing. The high-strength gelatin-based hydrogel is NAA-GelMA hydrogel with a cross-linked structure, wherein the NAA-GelMA hydrogel is obtained through a reaction of N-acryloyl glycinamide and methacrylic anhydride gelatin. The invention further discloses a preparation method of the high-strength gelatin-based hydrogel suitable for volume additive manufacturing and a method for printing a model by using the high-strength gelatin-based hydrogel suitable for volume additive manufacturing. The strength of the hydrogel is improved through the reaction of the N-acryloylglycinamide and the methacrylic anhydride gelatin, and the mechanical strength of a printed object is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of bio-inks, and in particular to a high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing, a preparation method thereof, and a model printing method. Background Art

[0002] Commonly used 3D printing technologies include fused deposition modeling (FDM) and direct light projection (DLP). The typical fused deposition modeling (FDM) method uses melt extrusion to create three-dimensional objects; direct light projection (DLP) uses a projector to solidify photosensitive polymer liquid layer by layer. With the development of 3D printing technology, volumetric additive manufacturing (VAM) has emerged. By projecting light onto photopolymer resin at one time, the resin is solidified and formed, achieving high resolution and high-speed molding processes. Compared with the cumbersome printing methods of FDM and DLP, which print and stack layers, volumetric additive manufacturing (VAM) greatly reduces printing time with its advanced printing method.

[0003] Hydrogels are polymers with a three-dimensional network structure that have hydrophilic groups and are swollen but insoluble in water. They can contain more than 90% water. Currently, natural products such as hyaluronic acid, chitosan, and gelatin are widely used in the production of hydrogels, resulting in good biocompatibility. Due to their excellent hydrophilic properties and biocompatibility, hydrogels are considered to be ideal materials for use as extracellular matrix (ECM) mimics. However, traditional hydrogels have the disadvantage of weak mechanical properties, which has also restricted the development of hydrogels in in vivo experiments and clinical applications. In order to improve the mechanical properties of hydrogels, scientists have conducted many studies, some of which have introduced nano-ions into hydrogels, some have constructed double-cross-linked networks, and some have added materials that can generate hydrogen bonds or van der Waals forces into hydrogels. Through these methods, the mechanical properties of hydrogels have been greatly improved.

[0004] The methacrylated gelatin (GelMA) hydrogel currently used in volumetric additive manufacturing has the disadvantage of weak mechanical strength. Hollow tube models such as tracheal channels produced by the hydrogels will collapse, making them difficult to use in in vivo experiments. There is an urgent need for a high-strength hydrogel that can be used in volumetric additive manufacturing. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the purpose of this application is to provide a high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing, which improves the strength of the hydrogel by reacting N-acryloylglycineamide with methacrylic anhydride-treated gelatin, thereby improving the mechanical strength of the printed object.

[0006] To solve the above problems, the technical solutions adopted in this application are as follows:

[0007] The embodiment of the present application provides a high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing, which is a NAGA-GelMA hydrogel with a cross-linked structure obtained by reacting N-acryloylglycinamide (NAGA) with methacrylic anhydride gelatin (GelMA).

[0008] As a further preferred scheme, the high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing described in the embodiment of the present application is a hydrogel obtained by mixing N-acryloyl glycinamide, methacrylic anhydride gelatin and a visible light initiator, wherein the mass ratio of N-acryloyl glycinamide and methacrylic anhydride gelatin is (6-10):1.

[0009] As a further preferred solution, the visible light photoinitiator described in the embodiment of the present application is a composite photoinitiator composed of a Ru compound and SPS, and the mass concentration of Ru in the composite photoinitiator is 0.018%-0.054%.

[0010] As a further preferred solution, the methacrylic anhydride gelatin described in the examples of the present application is prepared by the following method:

[0011] Prepare gelatin solution: Add gelatin to PBS buffer and dissolve until the liquid is clear and transparent to obtain gelatin solution;

[0012] Reaction: adding methacrylic anhydride (MA) to the gelatin solution to react and obtain a reactant;

[0013] Dialysis: Add the above reactants into a dialysis bag and place it in a warm water bath for dialysis to obtain a dialysate, which is then filtered to obtain a clear solution;

[0014] Adjust the pH value: Use sodium bicarbonate solution to adjust the pH of the obtained liquid to 5.6-6.0;

[0015] Freezing: The obtained liquid is divided into portions and placed in a refrigerator for freezing. The frozen liquid is placed in a freeze dryer for freeze drying to obtain methacrylic anhydride gelatin.

[0016] The present invention also provides a method for preparing a high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing, comprising:

[0017] Prepare gelatin solution: add gelatin into PBS (phosphate) buffer and dissolve until the liquid is clear and transparent to obtain gelatin solution;

[0018] Reaction: adding methacrylic anhydride (MA) to the gelatin solution to react and obtain a reactant;

[0019] Dialysis: Add the above reactants into a dialysis bag and place it in a warm water bath for dialysis to obtain a dialysate, which is then filtered to obtain a clear solution;

[0020] Adjust the pH value: Use sodium bicarbonate solution to adjust the pH of the obtained liquid to 5.6-6.0;

[0021] Freezing: The obtained liquid is divided into portions and then frozen, and the frozen liquid is placed in a freeze dryer for freeze drying to obtain methacrylic anhydride gelatin;

[0022] Prepare N-acryloyl glycinamide solution: dissolve N-acryloyl glycinamide in PBS buffer to obtain N-acryloyl glycinamide solution;

[0023] Preparation of hydrogel: The methacrylic anhydride-treated gelatin and N-acryloylglycinamide solution were mixed, a visible light photoinitiator was added, and the mixture was mixed evenly to obtain NAGA-GelMA hydrogel.

[0024] As a further preferred solution, the concentration of the gelatin in the PBS buffer solution described in the examples of the present application is 0.5-2 g / 10 mL, and the volume ratio of the methacrylic anhydride to the gelatin solution is (0.8-2.5):25.

[0025] As a further preferred embodiment, the freezing process described in the embodiment of the present application is carried out at a temperature of -80°C or lower than -80°C, and the freezing time is not less than 4 hours; the freeze-drying temperature is -20°C or lower than -20°C.

[0026] The present invention also provides a method for printing a model using the high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing described in the present invention, comprising:

[0027] Hydrogel pretreatment: NAGA-GelMA hydrogel was dispensed into printing bottles and then placed in an ice box for low-temperature solidification;

[0028] Parameter setting and printing: obtain the required model file through slicing software, set the volumetric additive manufacturing machine parameters, place the printing bottle in the card slot of the volumetric bioprinter, and print;

[0029] Demolding: Place the printed bottle in a water bath to thaw, then wash out the printed model to obtain the product.

[0030] As a further preferred solution, in the hydrogel pretreatment described in the examples of the present application, the temperature of the ice box is -4°C to 4°C, and the coagulation time is 30-45 minutes.

[0031] As a further preferred solution, the printing parameters described in the embodiment of the present application include printing size: 2-10 mm, printing light intensity: 1300-4000 Lux, and printing time: 50-120 s.

[0032] Compared to existing technologies, the present invention offers significant advantages: the high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing (VAM) described herein utilizes N-acryloylglycinamide and methacrylic anhydride-treated gelatin as raw materials. NAGA enhances the hydrogel's mechanical strength through hydrogen bonding, and when mixed with GelMA, the resulting hydrogel improves GelMA's inherent mechanical strength. This composite material can be used to print complex models using a visible light photoinitiator and can be applied to VAM, improving the mechanical strength of printed objects and meeting the strength requirements of organ transplants.

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0035] Figure 1 Schematic diagram of a printing model for a high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing as described in an embodiment of the present application.

[0036] Figure 2 The compressive stress-pressure curves of NAGA-GelMA hydrogels with different concentrations.

[0037] Figure 3 Box plot of the tensile strength of NAGA-GelMA hydrogels with different concentrations.

[0038] Figure 4 SEM images of NAGA-GelMA hydrogels with different concentrations.

[0039] Figure 5 This is a rendering of a model printed using the gelatin-based hydrogel described in the examples of this application.

[0040] Figure 6 This is the transmittance result diagram described in Comparative Example 2 of this application. DETAILED DESCRIPTION

[0041] To make the purpose, technical solutions, and advantages of the embodiments of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings in the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0042] The term "comprising" and other equivalent descriptions in the description and claims of this application are intended to cover non-exclusive inclusions, including both the contents clearly described in the description and claims and the steps or units that are not described in the description and claims but are inherent in the product, method or structure.

[0043] The embodiment of the present application provides a high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing. It is a NAGA-GelMA hydrogel with a cross-linked structure obtained by reacting N-acryloyl glycinamide (NAGA) with methacrylic anhydride gelatin (GelMA). The high-strength gelatin-based hydrogel described in the present application has a compressive strength range of 0.6-5.2 MPa and a tensile strength range of 0.05-1.7 MPa. Specifically, it is a hydrogel obtained by mixing N-acryloyl glycinamide, methacrylic anhydride gelatin and a visible light photoinitiator. Methacrylic anhydride gelatin (GelMA) has good compatibility, but weak mechanical strength, and the printed model often collapses easily due to insufficient support strength. N-acryloyl glycinamide NAGA has two amide groups on the molecule, which easily forms hydrogen bonds between molecules. There are multiple hydrogen bonds between and within its molecules. After polymerization with methacrylic anhydride gelatin (GelMA), the two interact synergistically to form a high-strength hydrogel, solving the strength problem of the printed model. The mechanical properties of the bio-ink described in the embodiments of this application are influenced by the mass ratio of N-acryloylglycinamide to methacrylic anhydride gelatin. By adjusting the ratio, the mechanical strength of the printed model can be controlled. Therefore, to improve the mechanical strength of the printed model, in some embodiments, the mass ratio of N-acryloylglycinamide to methacrylic anhydride gelatin is (6-10):1. Preferably, in other embodiments, the mass ratio of N-acryloylglycinamide to methacrylic anhydride gelatin is (8-10):1.

[0044] The high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing described in this application is a photocurable hydrogel having a photosensitive group. A cross-linked polymer is formed by photocuring during the printing process. In order to control the printing time and the mold forming time, an initiator needs to be added. In this application, a photoinitiator can be used. Preferably, the photoinitiator described in the embodiment of the present application is a composite photoinitiator composed of a Ru compound and SPS (sodium persulfate), which can initiate the reaction under visible light. In some embodiments, the Ru compound is tris(2,2′-bipyridine) ruthenium (II) chloride) hexahydrate, and the mass concentration of Ru in the composite photoinitiator is 0.018%-0.054%.

[0045] The methacrylic anhydride gelatin used in the embodiments of the present application can be a methacrylic anhydride gelatin product disclosed in the prior art. In some preferred embodiments, the methacrylic anhydride gelatin used is prepared by the following method:

[0046] Prepare gelatin solution: add gelatin into PBS buffer and dissolve until the liquid is clear and transparent to obtain gelatin solution.

[0047] Reaction: Add methacrylic anhydride (MA) to the above gelatin solution to react and obtain the reactant

[0048] Dialysis: Add the above reactants into a dialysis bag and place it in a warm water bath for dialysis to obtain a dialysate, which is then filtered to obtain a clear solution;

[0049] Adjust the pH value: Use sodium bicarbonate solution to adjust the pH of the obtained liquid to 5.6-6.0;

[0050] Freezing: The obtained liquid is divided into portions and placed in a refrigerator for freezing. The frozen liquid is placed in a freeze dryer for freeze drying to obtain methacrylic anhydride gelatin.

[0051] The methacrylic anhydride gelatin prepared above is more compatible with volumetric additive manufacturing technology and has a higher cell survival rate.

[0052] The present invention also provides a method for preparing a high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing, comprising:

[0053] Prepare gelatin solution: add gelatin into PBS (phosphate) buffer and dissolve until the liquid is clear and transparent to obtain gelatin solution;

[0054] Reaction: adding methacrylic anhydride (MA) to the gelatin solution to react and obtain a reactant;

[0055] Dialysis: Add the above reactants into a dialysis bag and place it in a warm water bath for dialysis to obtain a dialysate, which is then filtered to obtain a clear solution;

[0056] Adjust the pH value: Use sodium bicarbonate solution to adjust the pH of the obtained liquid to 5.6-6.0;

[0057] Freezing: The obtained liquid is divided into portions and then frozen, and the frozen liquid is placed in a freeze dryer for freeze drying to obtain methacrylic anhydride gelatin;

[0058] Prepare N-acryloyl glycinamide solution: dissolve N-acryloyl glycinamide in PBS buffer to obtain N-acryloyl glycinamide solution;

[0059] Preparation of bio-ink: The methacrylic anhydride-treated gelatin and N-acryloylglycinamide solution were mixed, a photoinitiator was added, and the mixture was mixed evenly to obtain NAGA-GelMA bio-ink.

[0060] As a further preferred solution, the concentration of the gelatin described in the examples of the present application in the PBS buffer solution is 0.5-2 g / 10 mL, and the volume ratio of the methacrylic anhydride (MA) to the gelatin solution is (0.8-2.5):25.

[0061] As a further preferred embodiment, the freezing process described in the embodiment of the present application is carried out at a temperature of -80°C or lower than -80°C, and the freezing time is not less than 4 hours; the freeze-drying temperature is -20°C or lower than -20°C.

[0062] like Figure 1 The present invention also provides a method for printing a model using the high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing described in the present invention, comprising:

[0063] Bio-ink pretreatment: The bio-ink NAGA-GelMA hydrogel was dispensed into printing bottles and then placed in an ice box for low-temperature solidification;

[0064] Parameter setting and printing: obtain the required model file through slicing software, set the volumetric additive manufacturing machine parameters, place the printing bottle in the card slot of the volumetric bioprinter, and print;

[0065] Demolding: Place the printed bottle in a water bath to thaw, then wash out the printed model to obtain the product.

[0066] As a further preferred solution, in the bio-ink pretreatment described in the embodiment of the present application, the temperature of the ice box is -4°C to 4°C, and the solidification time is 30-45 minutes (the bio-ink can only form a gel after being frozen for a sufficient time to ensure that the model does not shift during the printing process, which would affect the printing effect).

[0067] As a further preferred solution, the printer used in this application example is a VBP-T200 volumetric bioprinter produced by GreenKey Biotech. The printing parameters set for printing the mold include a print size of 5-10 mm, a print light intensity of 1300-4000 Lux, and a print time of 50-120 seconds. (Printing parameters affect the time required for model formation, size, and accuracy.)

[0068] The following are specific embodiments of this application.

[0069] Example 1

[0070] This embodiment provides a methacrylic anhydride gelatin, which is prepared by the following steps:

[0071] Prepare 0.1M phosphate buffer (PBS) with deionized water and preheat in a 50°C water bath. Then add gelatin at a ratio of 1g / 10mL to PBS (phosphate buffer) and dissolve until the liquid is clear and transparent to obtain a gelatin solution.

[0072] Add MA to the gelatin solution at a ratio of 40 mL / 500 mL. Use a pipette to add MA slowly. After addition, allow the mixture to react in the dark for 2 h. After the reaction is complete, obtain the reaction solution.

[0073] The reaction solution was added to a dialysis bag and placed in a 40°C water bath for 7 days. After dialysis, the liquid was filtered three times using three layers of qualitative filter paper to obtain a clear solution.

[0074] The pH of the clear solution was adjusted to 5.9 with 0.1 M sodium bicarbonate solution, and the resulting liquid was divided and placed in a -80°C refrigerator for more than 4 hours.

[0075] The frozen liquid was placed in a -20°C freeze dryer and freeze-dried for 3 days to obtain methacrylic anhydride gelatin (GelMA).

[0076] Example 2

[0077] This embodiment provides a high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing, and the preparation method is as follows:

[0078] Using an analytical balance, 0.6 g of N-acryloylglycinamide (NAGA) and 0.1 g of methacrylic anhydride-treated gelatin (GelMA) described in Example 1 were weighed into a 15 mL centrifuge tube. 2 mL of PBS solution was then injected into the 15 mL centrifuge tube, shaken, and heated in a 50°C water bath for 10 min to obtain a clear, transparent, viscous NAGA (30%)-GelMA (5%) mixed solution.

[0079] Take 2 mL of the mixed solution obtained above and place it in a 15 mL centrifuge tube. Add 25.2 μL of Ru (Ruthenium (II) hexahydrate) and shake carefully upside down. Then add 25.2 μL of SPS (sodium persulfate) and shake carefully upside down. Dispense into printing bottles (2 mL) to obtain NAGA (30%)-GelMA (5%) hydrogel.

[0080] A printing vial containing NAGA (30%)-GelMA (5%) hydrogel was placed in a -4°C freezer for 30 minutes. The vial was then removed and placed in the slot of a volumetric bioprinter. Using the printing software, a bone screw model was selected, with the following parameters set: a print size of 8 mm, a light intensity of 1300 Lux, and a print time of 60 seconds. After printing, the vial was heated in a water bath to melt the uncrosslinked portion into a liquid that was washed away, leaving the crosslinked portion.

[0081] Example 3

[0082] This embodiment provides a high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing, and the preparation method is as follows:

[0083] Use an analytical balance to weigh 0.8 g of N-acryloylglycinamide (NAGA) and 0.1 g of methacrylic anhydride gelatin (GelMA) into a 15 mL centrifuge tube. Then inject 2 mL of PBS solution into the 15 mL centrifuge tube, shake well, and heat in a 50°C water bath for 10 min to obtain a clear, transparent, viscous NAGA (40%)-GelMA (5%) mixed solution.

[0084] Take 2 mL of the mixed solution obtained above and place it in a 15 mL centrifuge tube. Add 25.2 μL of Ru (Ruthenium (II) hexahydrate) and shake carefully upside down. Then add 25.2 μL of SPS (sodium persulfate) and shake carefully upside down. Dispense into printing bottles (2 mL) to obtain NAGA (40%)-GelMA (5%) hydrogel.

[0085] The printing bottle containing NAGA (40%)-GelMA (5%) hydrogel was placed in a -4°C refrigerator for 30 minutes. The printing bottle was removed and placed in the card slot of the VBP-T200 volumetric bioprinter. Using the printing software, the bone screw model was selected and the parameters were set to a print size of 8mm-10mm, a printing light intensity of 1300Lux, and a printing time of 60s. After printing, the printing bottle was heated in a water bath to melt the unphotocrosslinked part into liquid and wash it away, leaving the cross-linked part.

[0086] Example 4

[0087] This embodiment provides a high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing, and the preparation method is as follows:

[0088] Use an analytical balance to weigh 1.0 g of N-acryloylglycinamide (NAGA) and 0.1 g of methacrylic anhydride gelatin (GelMA) into a 15 mL centrifuge tube. Then inject 2 mL of PBS solution into the 15 mL centrifuge tube, shake well, and heat in a 50°C water bath for 10 min to obtain a clear, transparent, viscous NAGA (50%)-GelMA (5%) mixed solution.

[0089] Transfer 2 mL of the mixed solution obtained above to a 15 mL centrifuge tube, add 25.2 μL of Ru (Ruthenium (II) hexahydrate, terpyridine ruthenium chloride hexahydrate), and carefully shake the mixture upside down. Then, add 25.2 μL of SPS (sodium persulfate), carefully shake the mixture upside down, and dispense into a 2 mL printing bottle to obtain a NAGA (30%)-GelMA (5%) hydrogel.

[0090] A printing vial containing NAGA (50%)-GelMA (5%) hydrogel was placed in a -4°C freezer for 40 minutes. The vial was then removed and placed in the slot of a volumetric bioprinter. Using the printing software, a bone screw model was selected, with the following parameters set: a print size of 8 mm, a light intensity of 1300 Lux, and a print time of 80 seconds. After printing, the vial was heated in a water bath to melt the uncrosslinked portion into a liquid that was washed away, leaving the crosslinked portion.

[0091] Example 5

[0092] This embodiment provides a high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing, and the preparation method is as follows:

[0093] Use an analytical balance to weigh 0.6 g of N-acryloylglycinamide (NAGA) and 0.1 g of methacrylic anhydride gelatin (GelMA) into a 15 mL centrifuge tube. Then inject 2 mL of PBS solution into the 15 mL centrifuge tube, shake well, and heat in a 50°C water bath for 10 min to obtain a clear, transparent, viscous NAGA (30%)-GelMA (5%) mixed solution.

[0094] Take 2 mL of the mixed solution obtained above and place it in a 15 mL centrifuge tube. Add 25.2 μL of Ru (Ruthenium (II) hexahydrate) and shake carefully upside down. Then add 25.2 μL of SPS (sodium persulfate) and shake carefully upside down. Dispense into printing bottles (2 mL) to obtain NAGA (30%)-GelMA (5%) hydrogel.

[0095] A printing vial containing NAGA (30%)-GelMA (5%) hydrogel was placed in a -4°C freezer for 30 minutes. The vial was then removed and placed in the slot of a VBP-T200 volumetric bioprinter. Using the printing software, a hollow vessel wall model was selected, with the following parameters set: a print size of 5 (10 mm), a print intensity of 4000 Lux, and a print time of 50 seconds. After printing, the vial was heated in a water bath to melt the uncrosslinked portions into a liquid that was washed away, leaving the crosslinked portions.

[0096] Example 6

[0097] This embodiment provides a high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing, and the preparation method is as follows:

[0098] Use an analytical balance to weigh 0.8 g of N-acryloylglycinamide (NAGA) and 0.1 g of methacrylic anhydride gelatin (GelMA) into a 15 mL centrifuge tube. Then inject 2 mL of PBS solution into the 15 mL centrifuge tube, shake well, and heat in a 50°C water bath for 10 min to obtain a clear, transparent, viscous NAGA (40%)-GelMA (5%) mixed solution.

[0099] Take 2 mL of the mixed solution obtained above and place it in a 15 mL centrifuge tube. Add 25.2 μL of Ru (Ruthenium (II) hexahydrate) and shake carefully upside down. Then add 25.2 μL of SPS (sodium persulfate) and shake carefully upside down. Dispense into printing bottles (2 mL) to obtain NAGA (30%)-GelMA (5%) hydrogel.

[0100] A printing vial containing NAGA (40%)-GelMA (5%) hydrogel was placed in a -4°C freezer for 30 minutes. The vial was then removed and placed in the slot of a VBP-T200 volumetric bioprinter. Using the printing software, a DNA double helix model was selected, with the following parameters set: a print size of 8 mm, a light intensity of 1500 Lux, and a print time of 60 seconds. After printing, the vial was heated in a water bath to melt the uncrosslinked portions into a liquid that was washed away, leaving the crosslinked portions.

[0101] Example 7

[0102] This embodiment provides a high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing, and the preparation method is as follows:

[0103] Use an analytical balance to weigh 1.0 g of N-acryloylglycinamide (NAGA) and 0.1 g of methacrylic anhydride gelatin (GelMA) into a 15 mL centrifuge tube. Then inject 2 mL of PBS solution into the 15 mL centrifuge tube, shake well, and heat in a 50°C water bath for 10 min to obtain a clear, transparent, viscous NAGA (50%)-GelMA (5%) mixed solution.

[0104] Take 2 mL of the mixed solution obtained above and place it in a 15 mL centrifuge tube. Add 25.2 μL of Ru (Ruthenium (II) hexahydrate) and carefully shake the tube upside down to mix. Then add 25.2 μL of SPS (sodium persulfate) and carefully shake the tube upside down to mix. Dispense the solution into 2 mL printing bottles to obtain a NAGA (30%)-GelMA (5%) hydrogel.

[0105] A printing vial containing NAGA (50%)-GelMA (5%) hydrogel was placed in a -4°C freezer for 30 minutes. The vial was then removed and placed in the slot of a VBP-T200 volumetric bioprinter. Using the printing software, a Y-shaped hollow blood vessel model was selected, with the following parameters set: a print size of 8 mm, a light intensity of 1600 Lux, and a print time of 60 seconds. After printing, the vial was heated in a water bath to melt the uncrosslinked portions into a liquid that was washed away, leaving the crosslinked portions.

[0106] Comparative Example 1

[0107] Provided is a pure GelMA bio-ink, the preparation method of which comprises the following steps:

[0108] Add 1 g of methacryloylated gelatin (GelMA) to 20 mL of PBS buffer and incubate at 50°C in a water bath for 2 h to obtain a methacryloylated gelatin solution. Transfer 6 mL of this solution to a 15 mL centrifuge tube and, protected from light, add 75.6 μL of Ru and 75.6 μL of SPS, sequentially. Shake well to obtain the pure GelMA bioink for the control group.

[0109] Mechanical properties testing

[0110] Uniaxial compression testing: Uniaxial compression testing was performed using a UTM4304X electronic universal testing machine (Sansi Zongheng). Cylindrical models were prepared using the NAGA-GelMA hydrogels obtained in Examples 2-4 (print size: 4 mm, printing intensity: 1300 Lux, printing time: 60 s), corresponding to Sample Groups 1, 2, and 3. A cylindrical model was prepared using 5% GelMA hydrogel (print size: 4 mm, printing intensity: 1300 Lux, printing time: 60 s) as Control 1. At least three groups of samples (three cylindrical compression models each) were prepared for each concentration.

[0111] See also Figure 2 Uniaxial compression test: For sample group 1 (30% NAGA + 5% GelMA), the cylindrical model had a diameter of 7 mm and a height of 9 mm. The compression strain rate was set at 5 mm / min, and the compressive strength of sample group 1 was measured to be 0.61 ± 0.05 MPa. For sample group 2 (40% NAGA + 5% GelMA), the cylindrical model had a diameter of 7 mm and a height of 10 mm. The compression strain rate was set at 5 mm / min, and the compressive strength of sample group 2 was measured to be 1.07 ± 0.21 MPa. For sample group 3 (50% NAGA + 5% GelMA), the cylindrical model had a diameter of 7 mm and a height of 10 mm. The compression strain rate was set at 5 mm / min, and the compressive strength of sample group 3 was measured to be 5.23 ± 0.76 MPa. For the control group (5% GelMA), the cylindrical model had a diameter of 10 mm and a height of 8 mm. The compression strain rate was set at 5 mm / min, and the compressive strength of sample group 4 was measured to be 0.09 ± 0.01 MPa. The compressive strength of sample 1 was 7 times that of the pure GelMA group, the compressive strength of sample 2 was 10 times that of the pure GelMA group, and the compressive strength of sample 3 was as high as 50 times that of the pure GelMA group.

[0112] Uniaxial tensile testing: Uniaxial tensile testing was performed using a UTM4304X electronic universal testing machine (Sansi Zongheng). Dumbbell-shaped models were prepared using the NAGA-GelMA hydrogels obtained in Examples 2-4 (preparation process: liquid bio-ink was injected into the mold and irradiated with strong light for 2 minutes to ensure complete consumption of the photoinitiator in the bio-ink). These models correspond to Sample Groups 1, 2, and 3. A dumbbell-shaped model was prepared using 5% GelMA hydrogel as Control 1. At least three groups of samples (three dumbbell-shaped models each) were prepared for each concentration.

[0113] See also Figure 3. The dumbbell-shaped model of sample group 1 (30% NAGA + 5% GelMA) has a thickness of 1.0 mm, a gauge length of 26 mm, a sample width of 4.0 mm, and a tensile strain rate of 8 mm / min. The tensile strength of sample group 1 was measured to be 0.71±0.08 MPa. The dumbbell-shaped model of sample group 2 (40% NAGA + 5% GelMA) has a thickness of 1.0 mm, a gauge length of 26 mm, a sample width of 4.0 mm, and a compressive strain rate of 8 mm / min. The tensile strength of sample group 2 was measured to be 1.09±0.11 MPa. The dumbbell-shaped model of sample group 3 (50% NAGA + 5% GelMA) has a thickness of 1.0 mm, a gauge length of 26 mm, a sample width of 4.0 mm, and a tensile strain rate of 8 mm / min. The tensile strength of sample group 3 was measured to be 1.68±0.23 MPa. The dumbbell-shaped model of the control group (5% GelMA) was 1.0 mm thick, with a gauge length of 26 mm and a sample width of 4.0 mm. The tensile strain rate was set to 8 mm / min, and the tensile strength of the sample group 4 was measured to be 0.05±0.01 MPa.

[0114] SEM analysis

[0115] The printed samples were frozen (sample group 1: 30% NAGA + 5% GelMA, sample group 2: 40% NAGA + 5% GelMA, sample group 3: 50% NAGA + 5% GelMA, control group: 5% GelMA). After freezing for two hours, they were placed in a freeze dryer and freeze-dried for one day. After freeze-drying, the samples were sprayed with gold and observed and photographed using a scanning electron microscope.

[0116] See also Figure 4 Scanning electron microscopy images showed that the pore size of freeze-dried sections of sample group 1 (30% NAGA + 5% GelMA) was smaller than that of the control group, and the pore walls were thicker than those of the control group; the pore size of freeze-dried sections of sample group 2 (40% NAGA + 5% GelMA) was smaller than that of sample group 1, and the pore walls were thicker; and the pore size of freeze-dried sections of sample group 3 (50% NAGA + 5% GelMA) was significantly reduced, and the pore walls were very thick.

[0117] Further adoption Figure 5 It can be seen that the effect of the product printed using the gelatin-based hydrogel described in this application.

[0118] Comparative Example 2

[0119] A NAGA (30%)-GelMA (5%) printing ink containing 0.018% LAP (a common photoinitiator) was placed on a volumetric bioprinter for printing. The printing parameters used were: a print size of 4 (print size 1 corresponds to 2 mm, 2 corresponds to 4 mm, and so on), a printing light intensity of 1300 Lux, and a printing time of 60 seconds (consistent with the parameters used with the Ru / SPS composite photoinitiator). The results are shown in Table 1.

[0120] Table 1 Comparison of printability of Ru / SPS composite photoinitiator and LAP photoinitiator

[0121] NAGA (30%)-GelMA (5%) printing ink Visible light (523nm light source) Ru / SPS composite photoinitiator √ LAP photoinitiator ×

[0122] √ means the printing can be completed before the ink melts (within 120 seconds)

[0123] × indicates that the printing cannot be completed before the ink melts (within 120 seconds)

[0124] Light transmittance experiment

[0125] The printing light intensity was set to 1300 Lux, and NAGA (30%)-GelMA (5%) printing inks containing different mass fractions of Ru (0%, 0.0045%, 0.009%, 0.018%, 0.036%, 0.054%) / SPS (the SPS concentration was proportional to the Ru) were placed on the working volume bioprinter in sequence. The initial light transmittance of the printing ink was measured with an illuminometer, and the transmittance was calculated using a formula (a transmittance greater than 37% meets the printing conditions of the volume bioprinter). Formula: Transmittance = I1 / I0, (I1 is the light intensity of the experimental group, and I0 is the light intensity of the blank control group). The measurement results are shown in Figure 2. Figure 6 shown.

[0126] Printability test

[0127] The printing parameters were set as a printing size of 8 mm, a printing light intensity of 1300 Lux, and a printing time of 60 s. NAGA (30%)-GelMA (5%) printing inks containing different concentrations of Ru (0.0045%, 0.009%, 0.018%, 0.036%, 0.054%) / SPS (the SPS concentration varied in proportion to Ru) were placed on the volumetric bioprinter for printing. The printing results are shown in Table 2.

[0128] Table 2 Printing results of NAGA (30%)-GelMA (5%) printing inks containing different concentrations of Ru / SPS

[0129] Mass fraction of Ru (%) 0.0045 0.009 0.018 0.036 0.054 Printing × × √ √ √

[0130] √: Printing can be done before the ink melts (within 120 seconds)

[0131] ×: Unable to print before the ink melts (within 120 seconds)

[0132] The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and replacements made by technicians in this field on the basis of the present invention fall within the scope of protection required by the present invention.

Claims

1. A high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing, characterized in that: It is a NAGA-GelMA hydrogel with a cross-linked structure obtained by the reaction of N-acryloylglycinamide and methacrylic anhydride-treated gelatin.

2. The high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing according to claim 1, characterized in that The hydrogel is obtained by mixing N-acryloyl glycinamide, methacrylic anhydride gelatin and a visible light photoinitiator, wherein the mass ratio of N-acryloyl glycinamide to methacrylic anhydride gelatin is (6-10):

1.

3. The high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing according to claim 1, characterized in that The visible light photoinitiator is a composite photoinitiator composed of a Ru compound and SPS, and the mass concentration of Ru in the composite photoinitiator is 0.018%-0.054%.

4. The high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing according to claim 1, characterized in that The methacrylic anhydride gelatin is prepared by the following method: Prepare gelatin solution: Add gelatin to PBS buffer and dissolve until the liquid is clear and transparent to obtain gelatin solution; Reaction: adding methacrylic anhydride to the above gelatin solution to react and obtain a reactant; Dialysis: Add the above reactants into a dialysis bag and place it in a warm water bath for dialysis to obtain a dialysate, which is then filtered to obtain a clear solution; Adjust the pH value: Use sodium bicarbonate solution to adjust the pH of the resulting liquid to 5.6-6.0; Freezing: The obtained liquid is divided into portions and placed in a refrigerator for freezing. The frozen liquid is placed in a freeze dryer for freeze drying to obtain methacrylic anhydride gelatin.

5. A method for preparing a high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing according to any one of claims 1 to 4, characterized in that: include Prepare gelatin solution: Add gelatin into PBS buffer and dissolve until the liquid is clear and transparent to obtain gelatin solution; Reaction: adding methacrylic anhydride to the above gelatin solution to react and obtain a reactant; Dialysis: Add the above reactants into a dialysis bag and place it in a warm water bath for dialysis to obtain a dialysate, which is then filtered to obtain a clear solution; Adjust the pH value: Use sodium bicarbonate solution to adjust the pH of the obtained liquid to 5.6-6.0; Freezing: The obtained liquid is divided into portions and then frozen, and the frozen liquid is placed in a freeze dryer for freeze drying to obtain methacrylic anhydride gelatin; Prepare N-acryloyl glycinamide solution: dissolve N-acryloyl glycinamide in PBS buffer to obtain N-acryloyl glycinamide solution; Preparation of hydrogel: The methacrylic anhydride-treated gelatin and N-acryloylglycinamide solution were mixed, a visible light photoinitiator was added, and the mixture was mixed evenly to obtain NAGA-GelMA hydrogel.

6. The preparation method according to claim 5, characterized in that The concentration of the gelatin in the PBS buffer solution is 0.5-2 g / 10 mL, and the volume ratio of the methacrylic anhydride to the gelatin solution is (0.8-2.5):

25.

7. The preparation method according to claim 5, characterized in that The freezing process is carried out at a temperature of -80°C or lower than -80°C, and the freezing time is not less than 4 hours; the freeze-drying temperature is -20°C or lower than -20°C.

8. A method for printing a model using the high-strength gelatin-based hydrogel suitable for volumetric additive manufacturing according to any one of claims 1 to 4, characterized in that: include Hydrogel pretreatment: NAGA-GelMA hydrogel was dispensed into printing bottles and then placed on ice for low-temperature solidification; Parameter setting and printing: obtain the required model file through slicing software, set the volumetric additive manufacturing machine parameters, place the printing bottle in the card slot of the volumetric bioprinter, and print; Demolding: Place the printed bottle in a water bath to thaw, then wash out the printed model to obtain the product.

9. The method according to claim 8, characterized in that During the hydrogel pretreatment, the temperature of the ice box is between -4°C and 4°C, and the solidification time is 30-45 minutes.

10. The method according to claim 8, characterized in that The printing parameters include printing size: 5-10 mm, printing light intensity: 1300-4000 Lux, and printing time: 50-120 s.

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