Gelatin-based hydrogel, visual printing bio-ink, preparation method of visual printing bio-ink and method for manufacturing printing model based on volume additive

Abstract

The invention discloses gelatin-based hydrogel, visual printing bio-ink, a preparation method of the visual printing bio-ink and a method for manufacturing a printing model based on volume additive manufacturing. The invention discloses gelatin-based hydrogel. The gelatin-based hydrogel is hydrogel formed by interweaving an N-isopropylacrylamide chain and a methacrylated gelatin chain, wherein the N-isopropylacrylamide chain and the methacrylated gelatin chain are formed by polymerizing N-isopropylacrylamide and methacrylated gelatin. The invention also provides a preparation method of the gelatin-based hydrogel and a method for printing a model by using the gelatin-based hydrogel. According to the gelatin-based hydrogel, the problems of visualization and compatibility of printing ink are solved, and the printing ink which is clear and visible in the forming process during printing is provided for volume additive manufacturing.

Description

Technical Field

[0001] The present invention relates to the field of volumetric additive manufacturing technology, and in particular to a gelatin-based hydrogel, a visual printing bio-ink, a preparation method thereof, and a method for printing a model based on volumetric additive manufacturing of a gelatin-based hydrogel. Background Art

[0002] Researchers published their latest findings in Science, introducing volumetric additive manufacturing (VAM), which improves the layer-by-layer stacking printing of traditional photocuring printing to volumetric molding, greatly improving the printing speed (Brett E. Kelly et al. Volumetric additive manufacturing via tomographic reconstruction. Science 363, 1075-1079 (2019). DOI: 10.1126 / science.aau7114).

[0003] Hydrogel is a highly hydrophilic network structure formed by water-soluble or hydrophilic polymers through certain chemical or physical crosslinking. In the medical field, a variety of hydrogels with high cell compatibility have been used in drug delivery systems, dressings, organoids, microneedles and other fields. Volumetric bioprinting technology is a volumetric additive technology that replaces the above-mentioned photosensitive resin ink with a highly cell-compatible hydrogel and replaces the ultraviolet light source that has a killing effect on cells with visible light.

[0004] Currently, highly active biomaterials commonly used for 3D cell culture include collagen, fibrin, decellularized extracellular matrix (dECM), gelatin, and methacrylic anhydride gelatin (GelMA). Methacrylic anhydride gelatin (GelMA) is a gelatin modified with olefin double bonds. It is obtained by grafting methacrylic anhydride (MA) onto the amino groups of gelatin. Under the action of visible light and an initiator, it undergoes photocrosslinking to form a three-dimensional structure with a certain strength suitable for cell growth and differentiation. GelMA combines the characteristics of natural and synthetic biomaterials, possessing reversible temperature-sensitive crosslinking and irreversible photocrosslinking. Due to its excellent bioactivity and tunable physicochemical properties, GelMA is widely used in fields such as tissue engineering and has gradually become the material of choice for 3D cell culture. However, the molding process of methacrylic anhydride gelatin during volumetric additive manufacturing (VAM) is not visible, making the printing effect difficult to evaluate. This brings many inconveniences to the practical application of methacrylic anhydride gelatin hydrogels in VAM. At the same time, GelMA hydrogel has poor mechanical strength and small compression modulus, which makes it difficult to expand the application scenarios of pure GelMA printing ink.

[0005] Most existing biological 3D printers use a contact, layer-by-layer printing principle, stacking layers of printing into three-dimensional objects. This method results in long printing times, low cell survival rates, and rough printed surfaces.

[0006] Volumetric bioprinters are high-precision, contactless, ultra-fast, sterile, volumetrically molded, and mild-conditioning biological 3D printers designed based on the reverse application of CT imaging technology. They are a revolutionary technology in the field of 3D additive manufacturing. Compared to existing layer-by-layer printing methods, volumetric bioprinters based on VAM (Volumetric additive manufacturing) and the principle of reverse CT have increased printing speed by at least two orders of magnitude, greatly improving cell survival rates. The printed surface is smooth and can more realistically simulate the surface structure of human organs. However, when GelMA hydrogel, the mainstream material for biological 3D printing, is printed in a volumetric bioprinter, the molding process is completely invisible. The printed object must be washed out after printing to evaluate the printing effect, which brings many difficulties to the exploration of parameters when printing new models. Therefore, the development of printing inks with visual effects is an urgent problem to be solved in volumetric additive manufacturing. Summary of the Invention

[0007] In order to overcome the shortcomings of the existing technology, the purpose of this application is to provide a gelatin-based hydrogel to solve the visualization and compatibility problems of printed biological inks, and to provide a printing ink for volumetric additive manufacturing with a clearly visible molding process during printing.

[0008] To solve the above problems, the technical solutions adopted in this application are as follows:

[0009] The embodiment of the present application provides a gelatin-based hydrogel, which is formed by the polymerization of N-isopropylacrylamide and methacrylated gelatin, and is a hydrogel formed by interweaving N-isopropylacrylamide chains and methacrylated gelatin chains.

[0010] As a further preferred solution, the gelatin-based hydrogel described in the embodiment of the present application is prepared from methacrylated gelatin, N-isopropylacrylamide and a photoinitiator, wherein the w / v% of N-isopropylacrylamide is 7.5% to 0.2%.

[0011] As a further preferred solution, the w / v% of N-isopropylacrylamide described in the examples of the present application is 3.3% to 2.5%.

[0012] As a further preferred solution, the method for preparing methacrylated gelatin used in the examples of the present application is as follows:

[0013] Dissolution: Add gelatin to preheated PBS solution and dissolve until the liquid is clear and transparent;

[0014] Adding methacrylamide: adding methacrylamide to the above liquid, reacting in the dark for 1 to 3 hours to obtain a reaction solution;

[0015] Dialysis: Add the above reaction solution into a dialysis bag and place it in a water bath for dialysis. The dialyzed liquid is filtered to obtain a clear solution;

[0016] Freezing: Adjust the pH value of the above clear solution to 5.5-6.5 and freeze in the refrigerator;

[0017] Drying: The frozen liquid is placed in a freeze dryer and freeze-dried to obtain methacrylated gelatin.

[0018] The present invention also provides a method for preparing a gelatin-based hydrogel, comprising:

[0019] Prepare N-isopropylacrylamide solution: add N-isopropylacrylamide into PBS buffer and dissolve at room temperature to obtain N-isopropylacrylamide solution;

[0020] Prepare methacrylated gelatin solution: add methacrylated gelatin into PBS buffer and dissolve in a warm water bath to obtain methacrylated gelatin solution;

[0021] Mixing: After mixing the above-mentioned N-isopropylacrylamide solution and methacrylated gelatin solution in a predetermined ratio, add a photoinitiator under light-proof conditions, and shake evenly to obtain a visual printing ink.

[0022] As a further preferred solution, in the preparation method described in the examples of the present application, during the preparation of the methacrylated gelatin solution, the temperature of the water bath is 45-55°C.

[0023] As a further preferred solution, in the preparation method described in the examples of the present application, the w / v% of the N-isopropylacrylamide is 7.5% to 0.5%.

[0024] The embodiment of the present application also provides a method for printing a model using the visual printing bio-ink, so that the microneedle model that originally could not stand upright after being removed from the water surface can stand upright after processing.

[0025] Specifically, the method described in the embodiment of the present application includes

[0026] Printing ink pretreatment: put the visual printing bio-ink into the printing bottle and put it into the refrigerator to freeze until the liquid solidifies;

[0027] Printing: Place the frozen visual printing bio-ink into the volumetric bio-printer, select the printing model and set the printing parameters before 3D printing:

[0028] Model processing: After printing is completed, melt the uncrosslinked ink in a warm water bath, wash out the melted ink with ultrapure water, then add anhydrous ethanol to the printing bottle, put the printing bottle into the refrigerator for freezing, and then take it out to obtain the printed model.

[0029] As a further preferred solution, the printing parameters described in the embodiment of the present application include printing light intensity, printing time, and green light intensity.

[0030] As a further preferred solution, in the model treatment process described in the embodiment of the present application, the water temperature is 35 to 40°C, the freezing treatment temperature is -15 to -20°C, and the freezing time is 20 to 40 minutes.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The gelatin-based hydrogel described in the present application is formed by adjusting the polymerization of methacrylated gelatin and N-isopropylacrylamide to form a transparent hydrogel with a double-penetrating network structure interwoven by N-isopropylacrylamide chains and methacrylated gelatin chains. The molding process is clearly visible during printing.

[0033] 2. The gelatin-based hydrogel described in this application achieves a good balance between cell compatibility and printability by adjusting the concentrations of methacrylated gelatin and N-isopropylacrylamide. It also has high cell compatibility under the premise of visualization, simplifies the process of volumetric bioprinter experiments, and optimizes its display effect.

[0034] 3. Furthermore, the gelatin-based hydrogel described in this application is suitable for the emerging technology of volumetric additive manufacturing and has high cell compatibility while having a fast printing speed.

[0035] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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.

[0037] Figure 1 Schematic diagram of the formation of a double-penetrating network structure by the gelatin-based hydrogel described in the examples of the present application;

[0038] Figure 2 A diagram showing the visual mechanism of gelatin-based hydrogels;

[0039] Figure 3A diagram showing the printing process of NIPAAM visual ink with different concentrations;

[0040] Figure 4 for Figure 3 The corresponding printed light intensity graph uses the light intensity fitting line to reflect the opacity of the ink after printing. Figure (A) is the light intensity curve of the NIPAAM visualization ink at different concentrations, and Figure (B) is the slope of the light intensity fitting line of the NIPAAM visualization ink at different concentrations.

[0041] Figure 5 The weight bar graphs of GelMA and NIPAAM before and after treatment with different concentrations of visualization inks are shown;

[0042] Figure 6 is based on Figure 5 Calculated ink water loss rate graph;

[0043] Figure 7 Stress / strain diagrams of visualized inks with different concentrations of GelMA and NIPAAM;

[0044] Figure 8 SEM images of visualized inks with different concentrations of GelMA and NIPAAM;

[0045] Figure 9 Visual ink cell live-dead staining images at different concentrations of GelMA and NIPAAM;

[0046] Figure 10 Comparison of the support properties of models printed using gelatin-based hydrogels, where (A) shows the printed model after being removed from the water surface, and (B) shows the printed model after low-temperature treatment in an ethanol liquid environment.

[0047] Figure 11 The following are visualization effects of different models.

[0048] Figure 12 A comparison chart of the visual effects of printing ethylene glycol dimethacrylate (PEGDA), silk fibroin (SF), and methacrylated gelatin (GelMA) mixed with N-isopropylacrylamide. DETAILED DESCRIPTION

[0049] 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.

[0050] 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.

[0051] like Figure 1 As shown, the present invention provides a gelatin-based hydrogel, which is formed by the polymerization of N-isopropylacrylamide (NIPAAM) and methacrylated gelatin (GelMA), and is composed of interwoven N-isopropylacrylamide chains and methacrylated gelatin chains. In this embodiment, the interwoven NIPAAM and GelMA chains form a double-penetrating network structure, which effectively enhances the mechanical properties of the hydrogel.

[0052] As a further preferred embodiment, the gelatin-based hydrogel described in the embodiment of the present application is prepared from methacrylated gelatin, N-isopropylacrylamide, and a photoinitiator. During the preparation process, the concentrations of GelMA and NIPAAM have a significant effect on the visualization of the ink, elastic modulus measurement, and the regularity and uniformity of the ink porosity and pore size. The effect on the visualization of the ink is manifested as follows: as the concentration ratio of NIPAAM in the visualization ink decreases, the absolute value of the slope of the light intensity fitting line also decreases continuously, and the opacity of the reflection decreases continuously; the higher the proportion of NIPAAM, the more obvious the visualization effect. The effect on the elastic modulus measurement of the ink is manifested as follows: the introduction of NIPAAM significantly improves the mechanical properties of the GelMA printing ink, and the compressive stress is improved compared to the pure GelMA printing ink. Moreover, as the concentration of NIPAAM increases, the compression modulus and toughness of the printed bio-ink increase, and the elastic modulus and stress-strain at fracture increase first. The reason may be that a double-penetrating network is formed between the NIPAAM chain and the GelMA chain, which improves the mechanical properties of the hydrogel from a microstructural perspective. The effect of NIPAAM concentration on the porosity, pore uniformity and regularity of the ink is manifested as follows: as the NIPAAM concentration is too low, the pore structure will gradually become irregular and the pores will become smaller, which cannot meet the printing requirements. Therefore, in this application, in order to obtain a good visualization effect, ensure the regularity of the product pore size and porosity, and improve the mechanical properties of the product, in some embodiments of this application, the w / v% of the N-isopropylacrylamide is 7.5% to 0.2%. Preferably, in some embodiments, the w / v% of the N-isopropylacrylamide described in the embodiments of this application is 3.3% to 0.2%; further, the w / v% of the N-isopropylacrylamide described in the embodiments of this application is 3.3% to 2.5%. In specific embodiments, the w / v% of N-isopropylacrylamide is 0.25% or 0.5% or 1% or 3.3%, 3% or 2.5% or 1.7% or 1.25%. Among them, the w / v% of N-isopropylacrylamide with the best porosity and pore size regularity is 2.5%

[0053] In some embodiments of the present application, the photoinitiator used is a composite photoinitiator composed of a ruthenium compound and sodium persulfate. The composite photoinitiator composed of a Ru compound and SPS can covalently cross-link free tyrosine and acryloyl groups to make the gelatin-based hydrogel have a higher cross-linking efficiency and better cell compatibility. In some embodiments, the concentrations of the ruthenium compound and sodium persulfate are 15-25 mol / L and 180-250 mol / L, respectively. Preferably, in some embodiments of the present application, the ruthenium compound is tris(2,2′-bipyridyl)ruthenium(II) chloride hexahydrate. Preferably, the volume ratio of tris(2,2′-bipyridyl)ruthenium(II) chloride hexahydrate to sodium persulfate is 1:1.

[0054] In some embodiments, the methacrylated gelatin used in this application can be purchased from existing products on the market. Preferably, the preparation method of the methacrylated gelatin used in the embodiments of this application is as follows:

[0055] Dissolution: Add gelatin to preheated PBS solution and dissolve until the liquid is clear and transparent;

[0056] Adding methacrylamide: adding methacrylamide to the above liquid, reacting in the dark for 1 to 3 hours to obtain a reaction solution;

[0057] Dialysis: Add the above reaction solution into a dialysis bag and place it in a water bath for dialysis. The dialyzed liquid is filtered to obtain a clear solution;

[0058] Freezing: Adjust the pH value of the above clear solution to 5.5-6.5 and freeze in the refrigerator;

[0059] Drying: The frozen liquid is placed in a freeze dryer and freeze-dried to obtain methacrylated gelatin.

[0060] As a further preferred embodiment, in the preparation method described in the embodiments of the present application, the w / v % of N-isopropylacrylamide is 7.5% to 0.2%. For example, in specific embodiments, the volume ratio of the two can be 2.5%, 1.7%, 1%, 0.5%, or 0.2%. Preferably, in some embodiments, the w / v % of N-isopropylacrylamide is 7.5% to 2.5%. In some embodiments, when preparing methacrylated gelatin, gelatin and PBS are added at a ratio of 1g of gelatin per 10mL of PBS. Before addition, the PBS is preheated in a 50°C water bath. When adding methacrylamide to the gelatin solution, the concentration of methacrylamide to gelatin solution is 2:25. After the gelatin solution is transferred, it is slowly added using a pipette. After addition, the reaction is carried out in the dark for 2 hours. During dialysis, the temperature is 35-50°C, preferably 40°C, and the dialysis time is 5-10 days, preferably 7 days. In some embodiments, a sodium bicarbonate solution with a concentration of 0.1 mol / L is used to adjust the pH value, preferably a pH value of 6.0. The freezing process is carried out in a -80°C refrigerator for more than 4 hours.

[0061] The present invention also provides a method for preparing a gelatin-based hydrogel, comprising:

[0062] Prepare N-isopropylacrylamide solution: add N-isopropylacrylamide into PBS buffer and dissolve at room temperature to obtain N-isopropylacrylamide solution;

[0063] Prepare methacrylated gelatin solution: add methacrylated gelatin into PBS buffer and dissolve in a warm water bath to obtain methacrylated gelatin solution;

[0064] Mixing: After mixing the above-mentioned N-isopropylacrylamide solution and methacrylated gelatin solution in a predetermined ratio, add a photoinitiator under light-proof conditions, and shake evenly to obtain a visual printing ink.

[0065] As a further preferred solution, in the preparation method described in the examples of the present application, during the preparation of the methacrylated gelatin solution, the temperature of the water bath is 45-55° C. Preferably, in some embodiments, the temperature of the water bath is 50° C.

[0066] Figure 2 The visualization mechanism of gum-based visual printing ink is demonstrated.

[0067] The embodiment of the present application also provides a method for printing a small model using the gelatin-based hydrogel, so that the microneedle model that originally could not stand upright after being removed from the water surface can stand upright after processing.

[0068] Specifically, the method described in the embodiment of the present application includes

[0069] Printing ink pretreatment: gelatin-based hydrogel is placed in a printing bottle and then placed in a refrigerator to freeze until the liquid solidifies;

[0070] Printing: Place the frozen printing ink into the volumetric bioprinter, select the printing model and set the printing parameters before 3D printing:

[0071] Model processing: After printing is completed, melt the uncrosslinked ink in a warm water bath and wash out the melted ink with ultrapure water, leaving the printed model in the bottle. Then add anhydrous ethanol to the printed bottle, put the printed bottle into the refrigerator for freezing, and then take it out to obtain the printed model.

[0072] In the above scheme, the model needs to remain upright in a liquid environment for the low-temperature treatment. However, water has a high melting point and cannot meet the low-temperature treatment requirements. Therefore, a liquid with a lower melting point is required to provide the model with a liquid environment. Therefore, in this embodiment, anhydrous ethanol is used to provide the liquid environment. Furthermore, the water absorption of anhydrous ethanol causes models printed with pure GelMA printing ink to lose water and undergo irreversible deformation during the above treatment. However, due to the water absorption property of NIPAAM at low temperatures, the visualization printing ink can also retain its own water in anhydrous ethanol and prevent deformation. In some embodiments, during the model treatment, the water temperature is 35-40°C, the freezing temperature is -15--20°C, and the freezing time is 20-40 minutes. The warm water bath temperature is preferably no more than 50°C, and a 37°C water bath is preferred. The model is placed in a -20°C refrigerator for 30 minutes before removal. The low-temperature treatment further solidifies the water in the GelMA and the model, improving the model's hardness.

[0073] As a further preferred solution, the printing parameters described in the embodiment of the present application include print size, print intensity, print time, green light intensity, etc.

[0074] The following are specific embodiments of the present application. In the following embodiments, unless otherwise specified in the present application, the raw materials and equipment used can be purchased.

[0075] Example 1

[0076] This embodiment provides a gelatin-based hydrogel, which is prepared by the following method:

[0077] Prepare N-isopropylacrylamide solution: add 0.25 g N-isopropylacrylamide (NIPAAM) to 5 ml PBS buffer and dissolve at room temperature to obtain N-isopropylacrylamide solution;

[0078] Preparation of methacryloyl gelatin solution: 1 g of methacryloyl gelatin (GelMA) was added to 20 ml of PBS buffer and incubated in a water bath at 50°C for 2 h to obtain methacryloyl gelatin solution;

[0079] Mixing: After mixing the above-mentioned N-isopropylacrylamide solution and methacrylated gelatin solution in a volume ratio of 1:2, 75.6 ml of Ru (tris(2,2′-bipyridyl)ruthenium(II) chloride) hexahydrate) and 75.6 ml of SPS as photoinitiators were added in sequence under dark conditions. The concentration of Ru was 20 mol / L and the concentration of SPS was 200 mol / L. After shaking evenly, a visual printing ink was obtained.

[0080] Comparative Example 1

[0081] Provided is a pure GelMA printing ink, the preparation method of which comprises the following steps:

[0082] Step 1: Add 1 g of methacryloylated gelatin (GelMA) to 20 ml of PBS buffer and incubate in a water bath at 50°C for 2 h to obtain a methacryloylated gelatin solution;

[0083] Step 2: Take 6 ml of mixed solution A and add 75.6 ml of Ru and 75.6 ml of SPS as photoinitiators in sequence in the dark, shake and mix, the concentration of Ru is 20 mol / L, and the concentration of SPS is 200 mol / L; obtain the visual printing ink.

[0084] Performance Characterization

[0085] To further verify the effect of N-isopropylacrylamide solution and methacrylated gelatin solution concentrations on ink performance, we investigated the effects of different concentrations based on Example 1. Specifically, we examined the visual printing ink's visualization, elastic modulus measurement, SEM analysis, water loss measurement, and cytocompatibility at N-isopropylacrylamide solution concentrations of 4%, 3.75%, 3.3%, 2.5%, 1.7%, 1%, 0.5%, 0.25%, and 0.2%, respectively. Comparisons were made with Comparative Example 1. The details are as follows.

[0086] 1. Visualization level representation

[0087] NIPAAM ink, which is a transparent liquid before printing and an opaque gel after printing, allows the model to scatter some of the printed light into the human eye as it nears completion, making it visible. However, the degree of visualization is currently subjective and can only be observed by the human eye. In this application, we use light intensity changes to characterize visualization. The specific steps are as follows:

[0088] Step 1: Fix the light intensity detector 5 cm behind the center of the printing base;

[0089] Step 2: Place the frozen printing ink prepared in Example 1 on the printing base, ensure that there is no strong light in the surrounding environment, and start printing. The light intensity detector then records the light intensity data.

[0090] Step 3: After printing is complete, import the light intensity meter data into Origin and generate a line graph. Add a linear fit line. Since the model's visualization is directly related to its opacity, and opacity can be characterized by the intensity of light passing through the model, the absolute value of the slope of the linear fit line can be used to intuitively represent the transmittance of the ink at that ratio.

[0091] The results are as follows Figure 3As the w / v% of N-isopropylacrylamide in the ink increases, the visual effect of the print improves accordingly.

[0092] Figure 2 This is a diagram showing the printing process of pure GelMA in comparative example 1; Figure 3 This is a visualization diagram of the ink printing process of different concentrations of GelMA and NIPAAM. Figure 3 It can be seen that the visualization effect of the printing ink begins to decline when the w / v% of NIPAAM is 0.2%. Visualization printing inks with a w / v% of 7.5% or more do not solidify at -4°C and are therefore not suitable for use as printing inks.

[0093] Depend on Figure 4 It can be seen that as the proportion of NIPAAM in the visualization ink decreases, the absolute value of the slope of the light intensity fitting line continues to decrease, and the opacity is reflected to continue to decrease. The higher the proportion of NIPAAM, the more obvious the visualization effect. However, at 0.2% w / v%, the ink still has a relatively clear printing effect.

[0094] The visualization effects of different models are as follows Figure 11 shown.

[0095] 2. Water loss performance measurement

[0096] Step 1: Place the frozen visualization printing inks of different proportions in Example 1 into the volumetric bioprinter, select the cylindrical model (cylinder r4h8), and set the printing parameters as print size: 4.5, print intensity: 1.3, print time: 60, and green light intensity: 55.

[0097] Step 2: After printing, melt the uncrosslinked ink in a 37°C water bath. Rinse the printed part with ultrapure water. Use absorbent paper to blot dry, taking care not to damage the model.

[0098] Step 3: Place the finished product on a high-precision balance and weigh it to obtain the weight of the hydrogel before shrinkage.

[0099] Step 4: Place the sample back into the printed bottle and soak it in a 50°C water bath for 30 minutes to ensure complete shrinkage of the hydrogel. Remove the sample, blot the surface of the finished product with absorbent paper, and weigh it again to obtain the weight of the hydrogel after shrinkage.

[0100] The water loss rates of visualization printing inks with different concentrations of GelMA and NIPAAM can be calculated using the formula: water loss rate (%) = (W2-W1) / W1x100%.

[0101] The water loss rate is Figure 5 and Figure 6 shown.

[0102] Depend on Figure 6 It can be seen that when the w / v% of NIPAAM is 3.3%, the water loss rate of the printing ink is relatively high, reaching 339.344%. At this concentration, the water loss performance of NIPAAM is not affected by GelMA.

[0103] 3. Elastic modulus measurement and characterization

[0104] Step 1: Place different proportions of the frozen visualization printing inks in Example 1 and Comparative Example 1 into the volumetric bioprinter, select the cylindrical model (cylinder r4h8), and set the printing parameters as print size: 4.5, print light intensity: 1.3, print time: 60, and green light intensity: 55.

[0105] Step 2: After printing is completed, melt the uncrosslinked ink in a 37°C water bath and wash out the printed product with ultrapure water.

[0106] Step 3: Take out the printed cylindrical model and measure it using an electronic universal testing machine (Jinan Hengsi Shengda, UTM4304 microcomputer-controlled electronic universal testing machine).

[0107] The compression modulus obtained is Figure 7 shown.

[0108] Depend on Figure 7 The addition of NIPAAM significantly improved the mechanical properties of the GelMA ink, with the compressive stress increasing by 0.6 MPa compared to pure GelMA ink. With increasing NIPAAM concentration, the compression modulus and toughness of the ink increased, while the elastic modulus and stress-strain at fracture increased first. This may be due to the formation of a double-penetrating network between NIPAAM and GelMA chains, which improves the mechanical properties of the hydrogel at a microstructural level.

[0109] 4.SEM analysis

[0110] Step 1: Place the frozen visualization printing inks of different proportions in Example 1 into the volumetric bioprinter, select the cylindrical model (cylinder r4h8), and set the printing parameters as print size: 4.5, print intensity: 1.3, print time: 60, and green light intensity: 55.

[0111] Step 2: After printing is completed, melt the uncrosslinked ink in a 37°C water bath and wash out the printed product with ultrapure water.

[0112] Step 3: Place the washed sample in a -20°C freezer for at least 2 hours to ensure that the water in the sample is completely solidified. The frozen sample is freeze-dried in a freeze dryer (Nanjing Saifei Biotechnology Co., Ltd., Biosafer-10A) for 24 hours to remove the water in the sample.

[0113] Step 4: Take out the freeze-dried samples from step 4, and use liquid nitrogen to break them into thin slices. Use conductive glue to stick the thin slices to the surface of the scanning electron microscope sample stage with the cross section facing up. Then place them in an automatic plasma sputtering instrument (Zhongke Keyi, SBC-12 small ion sputtering instrument) for gold spraying. Use a desktop scanning electron microscope (Phenom Pro G6) to observe the cross-sectional morphology and pore distribution of the samples. The results are as follows: Figure 6 shown.

[0114] Depend on Figure 6 It can be seen that the pore size of the visual printing ink at 3.3% w / v% is regular and the porosity is large. However, as the NIPAAM concentration decreases, the pore structure gradually becomes irregular and the pores become smaller.

[0115] 5. Cytocompatibility

[0116] Step 1: The liquid printing ink obtained in Example 1 and Comparative Example 1 was filtered through a sterile 0.22 μm filter membrane to obtain sterile printing ink, and the cells collected in advance were mixed in a sterile environment. The number of cells in each bottle of ink was at least 1×10 6 / mL (293T), respectively dispensed into printing bottles and frozen at -4℃ for 20min in preparation for printing.

[0117] Step 2: Place the frozen solid printing ink into a volumetric bioprinter (Green Key Biotech, VBP-T200), select the eight cell spheroid model (8balls-new2), and set the printing parameters as: print size: 4 (8 cm), print light intensity: 1.3 (6300 Lux), and print time: 120 (s).

[0118] Step 3: After printing, melt the uncrosslinked ink in a 37°C water bath and aspirate the melted ink in a sterile environment. The aspirated ink can be reused. Subsequently, rinse the printed cell spheroids twice with sterile PBS to ensure that the uncrosslinked ink is fully removed. After washing, aliquot into 96-well plates and culture in a cell culture incubator using DMEM supplemented with 10% FBS.

[0119] Step 3: On days 1, 4, and 7 of culture, remove one cell sphere from each sample for live / dead staining.

[0120] The results are as follows Figure 9 As shown, there is no significant difference in cell live-death staining and cell survival between the printing ink with a ratio of 1:24 and the pure GelMA printing ink of the control example. Among them, the cell survival rate of the visual printing ink with a w / v% of 0.2% can reach over 90%.

[0121] Application Example 1

[0122] This application embodiment provides a method for visualizing the water absorption performance of printing ink below the LCST temperature to process a small model.

[0123] The specific method is as follows:

[0124] Step 1: Place NIPAAM (w / v% 3.3%) visual printing ink into the volumetric bioprinter, select the microneedle model, and set the printing parameters as print size: 4, print intensity: 1.2, print time: 120, and green light intensity: 49.

[0125] Step 2: After printing, melt the uncrosslinked ink in a 37°C water bath. Wash out the melted ink with ultrapure water. Add anhydrous ethanol to the printing bottle and place it in a -20°C refrigerator for 30 minutes before removing. The resulting model is stronger than before treatment and can support small models upright.

[0126] like Figure 10 As shown in the figure, (A) is a microneedle model that cannot stand upright after leaving the water surface.

[0127] In this application, low-temperature treatment can further solidify the water in GelMA and the model, thereby increasing the hardness of the model. However, the model needs to remain upright in a liquid environment for the above-mentioned low-temperature treatment. Because the melting point of water is too high to meet the requirements of low-temperature treatment, a liquid with a lower melting point is needed to provide a liquid environment for the model, so the present invention chooses anhydrous ethanol. Due to the water absorption of anhydrous ethanol, the model printed by pure GelMA printing ink will lose water and undergo irreversible deformation during the above-mentioned treatment. However, due to the water absorption property of NIPAAM at low temperatures, the visualization printing ink can also retain its own water in anhydrous ethanol and will not deform.

[0128] thus, Figure 10 As shown in (B), the treated model can maintain its shape basically unchanged and improve the mechanical strength.

[0129] Comparative Example 2

[0130] Figure 12 Comparison of the visualization effects of printing ethylene glycol dimethacrylate (PEGDA), silk fibroin (SF), and methacrylated gelatin (GelMA) mixed with N-isopropylacrylamide.

[0131] The concentration of PEGDA is 50%, the concentration of SF is 5%, the concentration of GelMA is 5%, and the concentration of NIPAAM is 5%.

[0132] The three materials were mixed with NIPAAM in a ratio of 1:1, so that the NIPAAM concentration in the final ink was 2.5%.

[0133] The GelMA mixed ink was frozen at -4°C for 20 min to solidify.

[0134] The PEGDA mixed ink and SF mixed ink could not solidify at -4°C, so they were printed directly.

[0135] The results are as follows Figure 12 As shown, the PEGDA mixed ink has good light transmittance and visualization effect, but because it cannot solidify, the formed ink will sink during the printing process, resulting in poor printing effect.

[0136] SF mixed ink has poor light transmittance and no visualization effect.

[0137] The GelMA mixed ink has good light transmittance and visualization effect, and has good printing effect, so GelMA and NIPAAM were finally mixed to prepare visualization ink.

[0138] 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 gelatin-based hydrogel, characterized in that It is a hydrogel formed by the polymerization of N-isopropylacrylamide and methacryloyl gelatin, which is interwoven with N-isopropylacrylamide chains and methacryloyl gelatin chains.

2. The gelatin-based hydrogel according to claim 1, characterized in that The invention is prepared from methacrylated gelatin, N-isopropylacrylamide and a photoinitiator, wherein the w / v% of the N-isopropylacrylamide is 7.5% to 0.2%.

3. The gelatin-based hydrogel according to claim 2, characterized in that The w / v% of the N-isopropylacrylamide is 3.3% to 2.5%.

4. The gelatin-based hydrogel according to claim 1, characterized in that The preparation method of the methacrylated gelatin is as follows: Dissolution: Add gelatin to preheated PBS solution and dissolve until the liquid is clear and transparent; Adding methacrylamide: adding methacrylamide to the above liquid, reacting in the dark for 1 to 3 hours to obtain a reaction solution; Dialysis: Add the above reaction solution into a dialysis bag and place it in a water bath for dialysis. The dialyzed liquid is filtered to obtain a clear solution; Freezing: Adjust the pH value of the above clear solution to 5.5-6.5 and freeze in the refrigerator; Drying: The frozen liquid is placed in a freeze dryer and freeze-dried to obtain methacrylated gelatin.

5. A method for preparing a gelatin-based hydrogel based on volumetric additive manufacturing according to any one of claims 1 to 4, characterized in that: include Prepare N-isopropylacrylamide solution: add N-isopropylacrylamide into PBS buffer and dissolve at room temperature to obtain N-isopropylacrylamide solution; Prepare methacrylated gelatin solution: add methacrylated gelatin into PBS buffer and dissolve in a warm water bath to obtain methacrylated gelatin solution; Mixing: After mixing the above-mentioned N-isopropylacrylamide solution and methacrylated gelatin solution in a predetermined ratio, add a photoinitiator under light-proof conditions, and shake evenly to obtain a visual printing ink.

6. The preparation method according to claim 5, characterized in that The w / v% of the N-isopropylacrylamide is 7.5% to 0.2%.

7. The preparation method according to claim 5, characterized in that During the preparation of the methacrylated gelatin solution, the temperature of the water bath is 45-55°C.

8. Use of the gelatin-based hydrogel according to any one of claims 1 to 4 in preparing visual printing bio-ink.

9. A visual printing bio-ink, characterized in that: The method comprises the gelatin-based hydrogel according to any one of claims 1 to 4.

10. The method for visually printing a bio-ink model according to claim 9, wherein: include Printing ink pretreatment: put the visual printing bio-ink into the printing bottle and put it into the refrigerator to freeze until the liquid solidifies; Printing: Place the frozen printing ink into the volumetric bioprinter, select the printing model and set the printing parameters before 3D printing; Model processing: After printing is completed, melt the uncrosslinked ink in a warm water bath, wash out the melted ink with ultrapure water, then add anhydrous ethanol to the printing bottle, put the printing bottle into the refrigerator for freezing, and then take it out to obtain the printed model.

11. The method according to claim 10, characterized in that The printing parameters include printing light intensity, printing time, and green light intensity.

12. The method according to claim 10, characterized in that During the model treatment process, the water temperature is 35 to 40° C., the freezing treatment temperature is -15 to -20° C., and the freezing time is 20 to 40 minutes.

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