Gelatin-based bio-ink, preparation method and application thereof

Abstract

The application provides a gelatin-based bio-ink for digital light processing, which comprises a gelatin-ureidopyrimidinone polymer GelMA-UPy, a photoinitiator, a light absorber and a solvent; wherein the GelMA-UPy is prepared by reacting methylacrylic anhydride gelatin GelMA with 2-(6-isocyanohexylaminocarbonylamino)-6-methyl-4[1H]pyrimidine UPy-NCO; the application introduces the UPy group into the methylacrylic anhydride gelatin in the form of a group, so that the gelatin-based bio-ink has reversible quadruple hydrogen bonds, that is, a dynamic bond; and due to the fact that the UPy is easy to form a dimer, a dynamic network structure is formed at a micro level; the introduction of the dynamic network makes the gelatin-based bio-ink change from the original pure chemical crosslinking structure to a structure in which the chemical crosslinking and the dynamic network coexist, which is of great help to solve the defects of the gelatin-based bio-ink in the application of the digital light processing technology.

Description

Technical Field

[0001] This application relates to the field of bio-ink preparation technology, and in particular to a gelatin-based bio-ink, its preparation method, and its application. Background Technology

[0002] 3D printing technology is a type of additive manufacturing technology that uses computer-aided software to design a 3D model that meets specific structural requirements, and then prints the designed 3D model into shape using 3D printing equipment. Compared with traditional additive manufacturing technologies, 3D printing technology produces models with more precise aperture sizes, higher spatial structure fidelity, and can meet the personalized needs of users, achieving precise model control. Digital Light Processing (DLP) technology uses ultraviolet light to irradiate and cure bio-ink, solidifying the liquid ink layer by layer. Under the detection of a DLP digital controller, the curing process of each layer is repeated cyclically until the final 3D model input into the printer is formed. Compared with fused deposition modeling (FDM), selective laser sintering (SLS), and extrusion printing, digital light processing technology has higher printing resolution, higher printing fidelity compared to the input model, and better biocompatibility when printing with loaded cells.

[0003] Bioprinting technology uses cells, growth factors, and biomaterials as raw materials, combined with 3D printing technology, to create tissue scaffolds, tissue-like and organ-like models with specific 3D structures and functions in vitro using a bioprinter. Bio-inks are a key component of bioprinting. The main role of bio-inks in bioprinting technology is to simulate the natural extracellular matrix (ECM). Ideal bio-inks need to meet three requirements: biocompatibility, mechanical properties, and printability. Gelatin-based bio-inks are currently the most widely used type of bio-ink, and their excellent biocompatibility, ease of availability, and high controllability are the main reasons for their selection.

[0004] Gelatin-based bioinks generally suffer from poor mechanical properties and limited structural strength of printed models. Furthermore, their chemical cross-linking process restricts the long-term survival of cells loaded within the ink, thus limiting their long-term efficacy. To address these issues, scientists have conducted extensive research. One feasible approach is to modify gelatin-based bioinks into dynamically bonded bioinks, which can simultaneously improve both the poor mechanical properties and limited long-term cell survival inherent in gelatin-based bioinks.

[0005] There has been some research on gelatin-based bio-inks for digital light processing. However, common problems include poor mechanical properties of printed models that are prone to collapse, excessively high exposure conditions that can damage cells, and low long-term survival rate of cells loaded in printed models. There is an urgent need to develop a gelatin-based bio-ink for digital light processing that can overcome these defects. Summary of the Invention

[0006] In view of this, the present invention provides a gelatin-based bio-ink, a preparation method thereof, and its application, which combines printing accuracy with mechanical and biological activity.

[0007] To solve the above problems, this application adopts the following technical solution: One objective of this application is to provide a gelatin-based bio-ink for digital light processing, the bio-ink comprising a gelatin-ureidopyrimidinone polymer GelMA-UPy, a photoinitiator, a light absorber, and a solvent; The GelMA-UPy is prepared by reacting methacrylic anhydride gelatin (GelMA) with 2-(6-isocyanohexylaminocarbonylamino)-6-methyl-4[1H]pyrimidine (UPy-NCO).

[0008] In some embodiments, the GelMA is prepared by reacting gelatin with methacrylic anhydride; and the UPy-NCO is prepared by reacting hexamethylene diisocyanate with 2-amino-4-hydroxy-6-methylpyrimidine.

[0009] In some embodiments, the mass ratio of the components in the bio-ink is: GelMA-UPy 10-20 servings Photoinitiator 0.3–0.7 parts Light absorber 0.02–0.08 parts 100 parts solvent.

[0010] In some embodiments, the mass ratio of the components in the bio-ink is: GelMA-UPy : photoinitiator : light absorber : solvent = (10~15):(0.25~0.5):(0.025~0.05):(100~100).

[0011] In some embodiments, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphine; The light absorber is lemon yellow; The solvent is phosphate buffer.

[0012] The second objective of this application is to provide a method for preparing the bio-ink, comprising the step of mixing and dissolving GelMA-UPy, a photoinitiator, a light absorber, and a solvent.

[0013] In some embodiments, the GelMA-UPy is prepared by a method including the following steps: Methacrylic anhydride gelatin (GelMA) was dissolved in dimethyl sulfoxide under a nitrogen atmosphere. 2-(6-isocyanohexylaminocarbonylamino)-6-methyl-4[1H]pyrimidine UPy-NCO was added to the resulting solution to carry out the reaction; After the reaction, the resulting mixed solution was added to acetone to precipitate the precipitate. After solid-liquid separation, washing and drying, GelMA-UPy was obtained.

[0014] In some embodiments, the GelMA is prepared by a method including the following steps: Dissolve the gelatin in phosphate buffer to obtain a gelatin solution; Methacrylic anhydride was added to the gelatin solution to initiate a reaction; After the reaction, the product was precipitated, dissolved, dialyzed, centrifuged, frozen, and lyophilized to obtain GelMA.

[0015] In some embodiments, the UPy-NCO is prepared by a method comprising the following steps: Under a nitrogen atmosphere, hexamethylene diisocyanate was mixed with 2-amino-4-hydroxy-6-methylpyrimidine and stirred to react. After the reaction, n-pentane was added to precipitate the product. After solid-liquid separation, washing, and vacuum drying, UPy-NCO was obtained.

[0016] The third objective of this application is to provide an application of the aforementioned bio-ink in digital light processing bioprinting, comprising the following steps: The bio-ink is loaded into a digital light processing printing device; Set the printing parameters and print the preset 3D model; Evaluate the print quality.

[0017] The present application adopts the above technical solution, and its beneficial effects are as follows: This application provides a gelatin-based bio-ink for digital light processing, comprising a gelatin-ureidopyrimidinone polymer GelMA-UPy, a photoinitiator, a light absorber, and a solvent. The GelMA-UPy is prepared by reacting methacrylic anhydride-modified gelatin (GelMA) with 2-(6-isocyanohexylaminocarbonylamino)-6-methyl-4[1H]pyrimidinylUPy-NCO. This application introduces the UPy group into the methacrylic anhydride-modified gelatin, giving the gelatin-based bio-ink a reversible quadruple hydrogen bond, i.e., a dynamic bond. Furthermore, because UPy readily forms dimers, a dynamic network structure is formed at the microscopic level. The introduction of this dynamic network transforms the gelatin-based bio-ink from a purely chemical cross-linked structure into a structure where chemical cross-linking and a dynamic network coexist. This structure greatly helps to overcome the shortcomings of gelatin-based bio-inks in digital light processing technology applications. In addition, regarding mechanical properties… In terms of improvement, the introduction of dynamic networks allows the material to undergo the breaking and recombination of quadruple hydrogen bonds when subjected to external forces. This process requires a significant amount of energy, effectively improving the material's stiffness and toughness. Regarding reducing the exposure requirements for printing, the presence of dynamic networks results in a more compact spatial structure in the chemically cross-linked portions, with the carbon-carbon double bonds that dominate photocrosslinking being closer together at the microscopic scale. This facilitates free radical reactions and makes photocrosslinking easier to occur. In terms of improving the long-term survival of loaded cells, the deformability of dynamic networks facilitates normal cellular physiological activities such as proliferation, migration, and material exchange, whereas the rigid and non-deformable structure under chemical crosslinking is detrimental to cellular physiological activities. Simultaneously, dynamic networks facilitate the flow of nutrients. These factors make the gelatin-based bio-ink mentioned in this invention more conducive to long-term cell survival than traditional gelatin-based bio-inks. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The compressive stress-strain curves of the methacrylic anhydride gelatin and different GelMA-UPy of the present invention are shown. Figure 2 Viscosity-shear rate curves of the methacrylic anhydride gelatin of the present invention and different GelMA-UPy. Figure 3 The images show the printing effects of the methacrylic anhydride gelatin and different GelMA-UPy models of this invention. Figure 4This is a 3D staining image of methacrylic anhydride gelatin and different GelMA-UPy cell scaffolds co-cultured according to the present invention. Detailed Implementation

[0020] The embodiments of this application are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application. In the description of this application, it should be understood that the terms "upper", "lower", "horizontal", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified. To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments.

[0021] The gelatin-based bio-ink for digital light processing described in this invention has a core biomaterial of gelatin-ureidopyrimidinone polymer (GelMA-UPy). This material is obtained by reacting methacrylic anhydride gelatin (GelMA) with 2-(6-isocyanohexylaminocarbonylamino)-6-methyl-4[1H]pyrimidine (UPy-NCO).

[0022] Specifically, the gelatin-based bio-ink for digital light processing of the present invention involves reacting raw materials in a reaction system at a certain ratio to prepare methacrylic anhydride gelatin (GelMA) and 2(6-isocyanohexylaminocarbonylamino)-6-methyl-4[1H]pyrimidine (UPy-NCO). Then, GelMA and UPy-NCO are reacted under certain conditions to prepare GelMA-UPy. Different GelMA-UPy samples can be prepared by changing the mass ratio of UPy-NCO to GelMA. The two samples, GelMA and GelMA-UPy, are then formulated into bio-ink. For example, to prepare the bio-ink: 0.15g of GelMA-UPy is dissolved in 100ml of PBS solution, and 0.5g of photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphine sulfate (LAP) and 0.05g of light absorber lemon yellow are added. After mixing evenly, the gelatin-based bio-ink for digital light processing of the present invention is obtained. The specific implementation scheme is described in detail below.

[0023] Preparation of methacrylic anhydride-modified gelatin (GelMA): In a preferred embodiment of the present invention, the GelMA is prepared by a method comprising the following steps: (1) Preparation of gelatin solution: Add gelatin to phosphate-buffered saline (PBS) and stir until the liquid is clear and transparent, and the gelatin is completely dissolved to obtain a gelatin solution. Preferably, the concentration of the gelatin solution is 0.04–0.06 g / ml, more preferably 0.05 g / ml. The stirring and dissolving conditions are preferably at 35–45°C for 1.5–2 h, more preferably at 40°C for 1.5 h.

[0024] (2) Reaction: Add methacrylic anhydride (MA) to the above gelatin solution and stir thoroughly to obtain a mixed solution. Preferably, dimethylformamide (DMF) is added as a co-solvent before adding methacrylic anhydride. More preferably, the volume ratio of methacrylic anhydride to phosphate buffer is 1:200 to 1:300, and more preferably 1:250. In one specific embodiment, DMF equivalent to 35-45% of the volume of phosphate buffer is added first, and then DMF equivalent to 5-10% of the volume of phosphate buffer is thoroughly mixed with methacrylic anhydride and added dropwise to the reaction system using a separatory funnel. The reaction conditions are preferably at 35-45°C in the dark for 1.5-2.5 h, and more preferably at 40°C in the dark for 2 h.

[0025] (3) Precipitation and Dissolution: The mixed solution obtained in step (2) is added to anhydrous ethanol to precipitate a precipitate; then the precipitate is added to deionized water and stirred to dissolve, resulting in a precipitate solution. Preferably, the volume of anhydrous ethanol is not less than 3 times the volume of the mixed solution to ensure complete precipitation. The volume of deionized water is preferably 2 to 3 times the volume of the mixed solution to ensure complete dissolution of the precipitate. The stirring and dissolution conditions are preferably 35 to 45°C for 2 to 2.5 hours, more preferably 40°C for 2 hours.

[0026] (4) Dialysis and centrifugation: The precipitate solution obtained in step (3) is added to a dialysis bag and dialyzed in a warm water bath to obtain dialysate; the dialysate is centrifuged to obtain a clear solution. Preferably, the molecular weight cutoff of the dialysis bag is 8000-14000 Da, the dialysis temperature is 35-45℃ (more preferably 40℃), and dialysis is performed in deionized water for 3-5 days (more preferably 4 days). The centrifugation conditions are preferably centrifugation at 3500-4000 rpm / min for 10-15 min.

[0027] (5) Freezing and lyophilization: The clarified solution obtained in step (4) is aliquoted and frozen. The frozen liquid is then placed in a lyophilizer for lyophilization to obtain methacrylic anhydride gelatin. Preferably, the freezing temperature is -20°C or lower, and the freezing time is not less than 48 hours; the lyophilization ambient temperature is not higher than -70°C, and the lyophilization time is 3 to 5 days, preferably 4 days.

[0028] Preparation of (6-isocyanohexylaminocarbonylamino)-6-methyl-4[1H]pyrimidine (UPy-NCO) In a preferred embodiment of the present invention, the UPy-NCO is prepared by a method comprising the following steps: (6) Creating the reaction environment: Connect the relevant instruments, confirm the airtightness of the reaction environment, and purge with nitrogen to create a nitrogen atmosphere. Preferably, the temperature of the reaction environment is below 30°C.

[0029] (7) Reaction: First, add hexamethylene diisocyanate (HDI), wait for a period of time, and then quickly add 2-amino-4-hydroxy-6-methylpyrimidine (AHM). Stir to obtain a mixed solution. Preferably, the waiting time after adding HDI is 3–5 min. The preferred reaction conditions are mechanical stirring at 95–105 °C (more preferably 100 °C) for 15–17 h (more preferably 16 h), during which the nitrogen atmosphere in the reaction environment is checked every 2 h. Preferably, the mass ratio of AHM to HDI is (2.5–3.5):50, more preferably 3:50.

[0030] (8) Precipitation: Add n-pentane to the mixed solution obtained in step (7) to precipitate and obtain a precipitate. Preferably, the volume of n-pentane is 400-600 ml.

[0031] (9) Solid-liquid separation and washing: The mixture obtained in step (8) is subjected to solid-liquid separation to obtain a solid product, which is then washed with a detergent. Preferably, a Buchner funnel is used for vacuum filtration, followed by washing with n-pentane and acetone. More preferably, the mixture is first washed with n-pentane 2 to 4 times (more preferably 3 times), and then washed with acetone 2 to 4 times (more preferably 3 times), with each wash consuming 400 to 600 ml (more preferably 500 ml) of detergent.

[0032] (10) Vacuum drying: The solid product obtained in step (9) is dried to obtain UPy-NCO. Preferably, the solid is pressed into powder and placed in a vacuum drying oven and vacuum dried at 45-55°C (more preferably 50°C) for 1.5-2.5 days (more preferably 2 days).

[0033] Preparation of GelMA-UPy In a preferred embodiment of the present invention, the GelMA-UPy is prepared by a method comprising the following steps: (11) Creating the reaction environment: Connect the relevant instruments, confirm the airtightness of the reaction environment, and purge with nitrogen to create a nitrogen atmosphere. Preferably, the temperature of the reaction environment is 24–26°C.

[0034] (12) Dissolution: First, add an organic solvent, wait for a period of time, then add GelMA, and stir until the GelMA is completely dissolved to obtain a solution. Preferably, the organic solvent is dimethyl sulfoxide (DMSO), which has been dehydrated before use (e.g., by adding molecular sieves to remove water for at least 2 days). The waiting time after adding DMSO is 3-5 minutes. The preferred dissolution conditions are under stirring at 35-45°C (more preferably 40°C) for 0.5-1.5 hours (more preferably 1 hour), during which the nitrogen atmosphere is monitored. Preferably, after the GelMA is completely dissolved, its mass fraction in the solution is 0.05-0.07 g / ml, more preferably 0.06 g / ml.

[0035] (13) Reaction: Adjust the temperature of the reaction system, add UPy-NCO to the solution obtained in step (12), and stir thoroughly to obtain a mixed solution. Preferably, the reaction environment temperature is lowered to 24-26°C (more preferably 25°C). The mass ratio of UPy-NCO to GelMA can be adjusted as needed, for example, it can be 1:40, 3:40, 3:80 or 9:80, to synthesize GelMA-UPy with different UPy grafting rates. The preferred reaction conditions are stirring, reacting at 24-26°C (more preferably 25°C) for 22-26 h (more preferably 24 h), during which the nitrogen atmosphere in the reaction environment is checked every 2 h.

[0036] (14) Precipitation: The mixed solution obtained in step (13) is added to a precipitant to precipitate and obtain a precipitate. Preferably, the precipitant is acetone, and its volume is 500-800 ml.

[0037] (15) Solid-liquid separation and washing: The mixture obtained in step (14) is subjected to solid-liquid separation to obtain a solid product, which is then washed with a detergent. Preferably, a Buchner funnel is used for vacuum filtration, followed by washing with acetone and anhydrous ethanol. More preferably, the mixture is first washed with acetone 2 to 4 times (more preferably 3 times), and then washed with anhydrous ethanol 2 to 4 times (more preferably 3 times), with each wash consuming 400 to 600 ml (more preferably 500 ml) of detergent. In a more preferred embodiment, after each wash, the precipitate is sonicated for 5 to 8 minutes before the next wash. After the final wash, the precipitate is dried to allow the detergent to evaporate naturally until no obvious residue remains.

[0038] (16) Vacuum drying: The solid product obtained in step (15) is dried to obtain GelMA-UPy. Preferably, the solid is pressed into powder and placed in a vacuum drying oven and vacuum dried at 35-45°C (more preferably 40°C) for 22-26 hours (more preferably 24 hours).

[0039] Formulation of bio-ink The gelatin-based bio-ink for digital light processing described in this invention is obtained by uniformly mixing the above-prepared GelMA-UPy, photoinitiator, light absorber, and solvent.

[0040] In a preferred embodiment of the present invention, the bio-ink comprises the following components in parts by weight: GelMA-UPy 10-20 servings Photoinitiator 0.3–0.7 parts Light absorber 0.02–0.08 parts 100 parts solvent.

[0041] In some implementation schemes, the mass ratio of the components is: GelMA-UPy: Photoinitiator: Light absorber: Solvent = (10~15):(0.25~0.5):(0.025~0.05):(100~100).

[0042] In a more preferred embodiment, the mass ratio of the components is: GelMA-UPy : Photoinitiator : Light absorber : Solvent = 15 : 0.5 : 0.05 : 100.

[0043] In a preferred embodiment of the present invention, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP). The light absorber is tartrazine. The solvent is phosphate buffered saline (PBS).

[0044] The preparation method of the bio-ink includes: weighing each component according to the ratio, mixing them, and dissolving them at a suitable temperature until there are no obvious solids in the solution. Preferably, the dissolution temperature is 35-45℃ (more preferably 40℃), the dissolution time is 1-3 hours, and the dissolution status is observed every 30 minutes during the period.

[0045] Digital light processing printing applications The bio-ink described in this invention is suitable for digital light processing (DLP) bioprinting technology. As one focusing direction of this invention, the printing parameters can be set as follows: single-layer printing thickness 50–150 μm (preferably 100 μm), exposure time 5–20 s, light intensity 5–20 mW / cm², printing temperature 35–40℃ (preferably 37℃), and the wavelength of the light source used is 400–410 nm (preferably 405 nm).

[0046] In application, the prepared bio-ink is loaded into a digital light processing printing device, the aforementioned printing parameters are set, a preset 3D model is printed, and the printing effect is evaluated. The beneficial effects of this invention can be evaluated by comparing the molding effect, precision, and structural fidelity of different bio-inks (such as GelMA-UPy inks with different UPy grafting rates, or unmodified GelMA inks) under the same printing parameters and model.

[0047] This invention introduces the UPy group into gelatin-based bioinks, giving them reversible quadruple hydrogen bonds—a dynamic bond. Furthermore, because UPy readily forms dimers, a dynamic network structure is created at the microscopic level. The introduction of this dynamic network transforms the gelatin-based bioink from a purely chemically cross-linked structure into one where chemical cross-linking and a dynamic network coexist. This structure greatly helps to overcome the shortcomings of gelatin-based bioinks in digital light processing technology applications. Regarding the improvement of mechanical properties, the introduction of dynamic networks allows the material to undergo the breaking and recombination of quadruple hydrogen bonds under external forces. This process requires significant energy but effectively improves the material's stiffness and toughness, which is beneficial for printing 3D models with high mechanical requirements. In terms of reducing the exposure conditions required for printing, the presence of dynamic networks results in a more compact spatial structure in the chemically cross-linked portions, with the carbon-carbon double bonds that dominate photocrosslinking being closer together at the microscopic scale. This facilitates free radical reactions and makes photocrosslinking easier to occur. Regarding improving the long-term survival of loaded cells, the deformability of dynamic networks facilitates normal physiological activities such as cell proliferation, migration, and substance exchange, whereas the rigid and non-deformable structure under chemical crosslinking is detrimental to these activities. Furthermore, dynamic networks facilitate the flow of nutrients. These factors make the gelatin-based bio-ink mentioned in this invention more conducive to long-term cell survival than traditional gelatin-based bio-inks. In summary, this bio-ink provides a feasible solution to address the shortcomings of gelatin-based bio-inks in digital light processing, which is beneficial for advancing the clinical application of digital light processing technology.

[0048] To further understand the present invention, the present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0049] Example 1: A methacrylic anhydride-modified gelatin was prepared by the following steps: A phosphate buffer solution was prepared using deionized water. 400 ml of the phosphate buffer solution was added to a three-necked flask, followed by 20 g of gelatin. The mixture was stirred at 40°C for 1.5 h until the liquid became clear and transparent. Then, 160 ml of dimethylformamide was added to the three-necked flask. Next, 30 ml of dimethylformamide and 1.6 ml of methacrylic anhydride were thoroughly mixed and added to a separatory funnel. The mixture was slowly added to the three-necked flask through the separatory funnel. The mixture was protected from light and reacted at 40°C for 2 h to obtain a mixed solution. The mixture was precipitated with 1.5 L of anhydrous ethanol. The collected precipitate was... Squeeze the precipitate dry and place it in a beaker containing 1L of deionized water. Stir and dissolve at 40℃ for 2 hours to obtain a precipitate solution. Prepare a dialysis bag and beaker with a molecular weight cutoff of 8000-14000 Da. Add the above precipitate solution to the dialysis bag, place the dialysis bag in a beaker containing sufficient deionized water, and dialyze at 40℃ for 4 days. Pour out the liquid from the dialysis bag and aliquot it into a centrifuge. Centrifuge at 4000rpm / min for 10 minutes. Take the supernatant and place it into a suitable container. Freeze the aliquoted clear solution at -20℃ for 2 days and freeze-dry at -70℃ for 4 days to obtain methacrylic anhydride gelatin.

[0050] Example 2: A GelMA-UPy was prepared and further processed into a bio-ink through the following steps: Sufficient dimethyl sulfoxide was measured, and an appropriate amount of molecular sieve was added. The mixture was left to dehydrate for at least 2 days. The experimental apparatus was assembled, ensuring a nitrogen atmosphere. 100 ml of dimethyl sulfoxide was added to a three-necked flask, and after 3 minutes, 6 g of methacrylic anhydride-modified gelatin was quickly added. The mixture was stirred at 40°C for 1 hour, with the nitrogen atmosphere monitored, until the methacrylic anhydride-modified gelatin was completely dissolved, resulting in a solution with no obvious solids. The reaction temperature was lowered to 25°C, and 0.45 g of 2-(6-isocyanohexylaminocarbonylamino)-6-methyl-4[1H]pyrimidine was added to the three-necked flask. The mixture was stirred at 25°C. The reaction should be carried out over 24 hours, with the nitrogen atmosphere observed every 2 hours to ensure the entire reaction is conducted under nitrogen. After the reaction, the mixed solution is poured into 700 ml of acetone to precipitate. The precipitate is then washed by vacuum filtration, using acetone and anhydrous ethanol three times each, consuming 500 ml of the corresponding reagent each time. After each wash, the precipitate is sonicated for 5 minutes before the next wash. After the final anhydrous ethanol wash, the precipitate is dried until no obvious traces of anhydrous ethanol are visible inside or outside the precipitate. The solid obtained after vacuum filtration is pressed into powder and placed in a vacuum drying oven for vacuum drying at 40°C for 24 hours to obtain GelMA-UPy.

[0051] Weigh 0.75g of the above-mentioned GelMA-UPy, 0.025g of phenyl-2,4,6-trimethylbenzoyl lithium phosphinate, 0.0025g of lemon yellow and 5ml of phosphate buffer, mix them and place them in a centrifuge tube, place the centrifuge tube in a 40℃ water bath for 2.5h to dissolve, and obtain a solution with no obvious solids, which is the gelatin-based bio-ink mentioned in this invention.

[0052] Comparative Example 1: A bio-ink of pure methacrylic anhydride gelatin is provided, which is prepared by the following steps: Weigh 0.75g of methacrylic anhydride gelatin from Example 1, 0.025g of phenyl-2,4,6-trimethylbenzoyl lithium phosphine, 0.0025g of lemon yellow and 5ml of phosphate buffer, mix them and place them in a centrifuge tube, place the centrifuge tube in a 40℃ water bath for 1h to dissolve, and obtain a solution with no obvious solids, which is the bio-ink of pure methacrylic anhydride gelatin.

[0053] Pure gelatin, methacrylic anhydride gelatin from Example 1, and GelMA-UPy from Examples 2 to 6 were taken, and their MA grafting rate and UPy grafting rate were detected. The results are recorded in Tables 1 and 2, and they were also named according to the grafting rate of the two groups. The grafting rate was detected by the ninhydrin microplate method, and the amino acid content was detected according to the Regan amino acid assay kit.

[0054] The ninhydrin microplate method is a commonly used method for determining amino acid content. Its detection principle is as follows: under weakly acidic conditions, amino acids and ninhydrin react with each other upon heating to generate diketoindamine. This product is blue-violet, and its absorption peak is at a wavelength of 570 nm. Therefore, the content of free amino groups in the solution can be calculated from the absorbance of the solution. Then, based on the absorbance differences of pure gelatin, methacrylic anhydride gelatin, and various groups of GelMA-UPy, combined with their structural differences, the grafting rate of MA and UPy can be inferred.

[0055] The samples obtained in the above embodiments of this application are tested below.

[0056] (1) Mechanical property testing: Samples of the two groups of bio-inks were taken and injected into cylindrical plastic molds with an inner diameter and length of 5 mm. They were then photocrosslinked using a UV crosslinking chamber to obtain photocrosslinked and cured cylinders. The photocured cylinders were placed in an in-situ mechanical testing instrument to perform compressive strength tests, obtaining stress-strain curves, as shown below. Figure 1 As shown.

[0057] Depend on Figure 1It can be seen that the introduction of the UPy group has a significant impact on the compressive strength of gelatin-based bio-inks, with improved compressive strength and toughness of GelMA-UPy. GelMA fractures at approximately 60% strain, while GelMA-UPy fractures at 70% strain, and the stress curve also shows a significant increase. The figure also shows an increase in the fracture strength and Young's modulus of the GelMA-UPy sample.

[0058] (2) Rheological property testing: Samples of the four types of bio-inks were taken, and rheological tests were conducted on each bio-ink using the "viscosity-shear rate" test mode. The parameters were set as follows: temperature 37℃, test gap 0.2mm, rotational testing using a PP25 plate, and logarithmic shear rate variation from 0.1 to 100 s⁻¹. An oscillating scanning mode was also used to test each bio-ink, with a temperature scanning range of 15 to 40℃, and 40 points were measured. This yields... Figure 2 .

[0059] Figure 2 This is represented by the viscosity-shear rate curves of two sets of samples. From Figure 2 As can be seen, the viscosity of GelMA-UPy is significantly improved compared to GelMA, but the difference between GelMA-UPy is not significant. The highest viscosity of GelMA is 484.03 Pa•s, while the highest viscosity of GelMA-UPy is 4161.4 Pa•s. This indicates that the introduction of the UPy group significantly improves the viscosity and shear-thinning properties of gelatin-based bio-inks compared to GelMA.

[0060] (3) Printing of digital light processing models: Two groups of bio-ink samples, GelMA and GelMA-UPy, were used for digital light processing model printing. The printing effect on larger models was explored during the digital light processing model printing process. This involved printing larger models and photographing them for comparison, thereby obtaining... Figure 3 .

[0061] Figure 3 This is a diagram showing the printed models of two sets of samples. From... Figure 3 As can be seen, after introducing the UPy group into GelMA, the gelatin-based bio-ink becomes easier to photocrosslink, and the mechanical properties of the model after photocuring are improved. The model printed with GelMA bio-ink is softer and more easily deformed, while the model printed with GelMA-UPy is not soft and is not easily deformed. Under the same parameters, the model printed with GelMA-UPy has higher fidelity and more distinct edges and corners, while the model printed with GelMA lacks obvious edges and corners. This may be because the GelMA bio-ink is somewhat underexposed under these exposure conditions.

[0062] (4) 3D co-culture of cell-loaded scaffolds: Two groups of bio-ink samples, GelMA and GelMA-UPy, were used for 3D co-culture on cell-loaded scaffolds, with MSCs selected. 50 μL of cell-loaded bio-ink was irradiated with a blue-violet flashlight for 1 min to obtain a gel. The gel was transferred to 24-well plates containing α-MEM complete medium for culture. After 5 days of 3D co-culture, the cell-loaded scaffolds were removed, and the scaffolds were stained for live / dead cell activity and DAPI / F-actin staining. The images were then captured using a confocal microscope. Figure 4 .

[0063] Figure 4 This is a 3D staining image of cell-carrying scaffolds from two sample groups. From Figure 4 As can be seen from the data, after 5 days of culture, the relationship between GelMA and GelMA-UPy is that the introduction of the UPy group increases the survival rate of the loaded cells, and GelMA-UPy bioink is more conducive to the long-term survival of cells; the higher the UPy grafting rate, the more the cells spread out, indicating that in the cell-loaded scaffold formed by GelMA-UPy bioink, cells are more likely to carry out normal physiological activities such as proliferation, migration, and differentiation.

[0064] The above are merely preferred embodiments of this application, and only specifically describe the technical principles of this application. These descriptions are only for explaining the principles of this application and should not be construed as limiting the scope of protection of this application in any way. Based on this explanation, any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application, as well as other specific embodiments of this application that can be conceived by those skilled in the art without creative effort, should be included within the scope of protection of this application.

Claims

1. A gelatin-based bio-ink for digital light processing, characterized in that, The bio-ink comprises a gelatin-ureidopyrimidinone polymer GelMA-UPy, a photoinitiator, a light absorber, and a solvent; The GelMA-UPy is prepared by reacting methacrylic anhydride gelatin (GelMA) with 2-(6-isocyanohexylaminocarbonylamino)-6-methyl-4[1H]pyrimidine (UPy-NCO).

2. The bio-ink according to claim 1, characterized in that, The GelMA is prepared by reacting gelatin with methacrylic anhydride; the UPy-NCO is prepared by reacting hexamethylene diisocyanate with 2-amino-4-hydroxy-6-methylpyrimidine.

3. The bio-ink according to claim 1 or 2, characterized in that, The mass ratio of the components in the bio-ink is as follows: GelMA-UPy 10-20 servings Photoinitiator 0.3–0.7 parts Light absorber 0.02–0.08 parts 100 parts solvent.

4. The bio-ink according to claim 3, characterized in that, The mass ratio of each component in the bio-ink is: GelMA-UPy : photoinitiator : light absorber : solvent = (10~15):(0.25~0.5):(0.025~0.05):(100~100).

5. The bio-ink according to claim 1, characterized in that: The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphine; The light absorber is lemon yellow; The solvent is phosphate buffer.

6. A method for preparing the bio-ink as described in any one of claims 1-5, characterized in that, This includes the step of mixing and dissolving GelMA-UPy, a photoinitiator, a light absorber, and a solvent.

7. The method according to claim 6, characterized in that, The GelMA-UPy is prepared by a method including the following steps: Methacrylic anhydride gelatin (GelMA) was dissolved in dimethyl sulfoxide under a nitrogen atmosphere. 2-(6-isocyanohexylaminocarbonylamino)-6-methyl-4[1H]pyrimidine UPy-NCO was added to the resulting solution to carry out the reaction; After the reaction, the resulting mixed solution was added to acetone to precipitate the precipitate. After solid-liquid separation, washing and drying, GelMA-UPy was obtained.

8. The method according to claim 7, characterized in that, The GelMA is prepared by a method including the following steps: Gelatin was dissolved in phosphate buffer to obtain a gelatin solution; Methacrylic anhydride was added to the gelatin solution to initiate a reaction; After the reaction, the product was precipitated, dissolved, dialyzed, centrifuged, frozen, and lyophilized to obtain GelMA.

9. The method according to claim 7, characterized in that, The UPy-NCO is prepared by a method including the following steps: Under a nitrogen atmosphere, hexamethylene diisocyanate was mixed with 2-amino-4-hydroxy-6-methylpyrimidine and stirred to react. After the reaction, n-pentane was added to precipitate the product. After solid-liquid separation, washing, and vacuum drying, UPy-NCO was obtained.

10. The application of a bio-ink as described in any one of claims 1-5 in digital light processing bioprinting, characterized in that, Includes the following steps: The bio-ink is loaded into a digital light processing printing device; Set the printing parameters and print the preset 3D model; Evaluate the print quality.

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