Photocuring bio-ink as well as preparation method and application thereof

Abstract

The invention relates to photocuring bio-ink as well as a preparation method and application thereof, and belongs to the technical field of biological materials and tissue engineering. The photocuring bio-ink comprises methacrylated fish scale gelatin and a photoinitiator, the methacrylated fish scale gelatin is prepared from fish scale gelatin and methacrylic anhydride through an acylation reaction, and the substitution degree of the methacrylated fish scale gelatin is 30%-55%. The photocuring bio-ink has the advantages of fast photocuring, low immunogenicity, low fat content and excellent biocompatibility, and is especially suitable for 3D biological printing construction of fine tissue engineering scaffolds and organoids such as cartilage, skin and blood vessels.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomaterials and tissue engineering, and particularly relates to a photocurable biomaterial ink as well as a preparation method and application thereof. BACKGROUND

[0002] Photocurable hydrogels, such as methacrylated gelatin (GelMA), have become a key material in the field of 3D bioprinting due to their excellent light-controlled molding ability, good biocompatibility and adjustable physical properties.

[0003] At present, commercial GelMA is mainly derived from mammals (such as pig skin and cow skin), which has the following inherent defects: (1) immunogenicity risk: mammalian gelatin may carry zoonotic pathogens, and its complete antigen epitope (such as α-Gal antigen) may trigger immune rejection, which limits its clinical conversion application; (2) low light crosslinking efficiency: pig skin and other raw materials are naturally rich in adipose tissue, and although a defatting process is performed, a small amount of lipids may still remain in the finished product. These lipids not only may cause oxidation and spoilage, affecting product stability, but more critically, they may severely scatter ultraviolet light / visible light, resulting in low light crosslinking efficiency, manifested as long curing time (usually 1-3 min) and insufficient curing depth, thereby affecting the precision and fidelity of the printed structure, and GelMA prepared from pig skin and cow skin usually requires a high degree of substitution (usually 70%-90%) to achieve the required light curing effect for 3D bioprinting.

[0004] Therefore, how to provide a new type of photocurable biomaterial ink with low immunogenicity, high light crosslinking efficiency and wide source is a technical problem to be solved in the field. SUMMARY

[0005] To solve the above technical problems, the application provides a photocurable biomaterial ink as well as a preparation method and application thereof. The photocurable biomaterial ink takes methacrylated fish scale gelatin as an active component, and its raw material is derived from renewable fishery processing by-products, has the advantages of extremely low fat content, low immunogenicity and extremely short light curing time, and is particularly suitable for high-precision and high-efficiency 3D bioprinting.

[0006] The application provides a photocurable biomaterial ink, which comprises methacrylated fish scale gelatin and a photoinitiator, wherein the methacrylated fish scale gelatin is prepared by acylation reaction of fish scale gelatin and methacrylic anhydride, and the degree of substitution of the methacrylated fish scale gelatin is 30%-55%.

[0007] In some embodiments, the concentration of the methacrylated fish scale gelatin is 2.5%-15% w / v, and the concentration of the photoinitiator is 0.2%-0.5% w / v.

[0008] In some embodiments, when preparing methacrylated fish scale gelatin, the ratio of the volume of added methacrylic anhydride to the mass of fish scale gelatin is (0.3~1.2):1 mL / g.

[0009] In some of these embodiments, the acylation reaction is carried out at a temperature of 4–80 °C and for a time of 1–8 h when preparing methacrylated fish scale gelatin.

[0010] In some embodiments, fish scale gelatin is obtained from the scales of marine fish.

[0011] In some of these embodiments, the marine fish is selected from any one or more of sea bass, cod, and sea bream.

[0012] In some embodiments, the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0013] The present invention also provides a method for preparing photocurable bio-ink according to any of the above technical solutions, comprising the following steps: dissolving methacrylamide fish scale gelatin in cell culture medium or phosphate buffer, adding a photoinitiator, and obtaining photocurable bio-ink by sterile filtration.

[0014] The present invention further provides the application of the photocurable bio-ink described in any of the above technical solutions in the preparation of tissue engineering scaffolds or organ models by 3D bioprinting.

[0015] In some embodiments, when preparing tissue engineering scaffolds or organ models, the photocurable bio-ink is cured under irradiation with a 405nm light source, with a light intensity of 5~30 mW / cm². 2 The irradiation time is 3-5 seconds.

[0016] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. The photocurable bio-ink provided by this invention uses methacrylamide fish scale gelatin as the active component. The methacrylamide fish scale gelatin is prepared by acylation reaction between fish scale gelatin and methacrylic anhydride. Utilizing the low fat content (less than 0.5%) of fish scale gelatin, the obtained photocurable bio-ink solution has extremely high transparency and negligible scattering effect on ultraviolet / blue light. Light energy can penetrate efficiently and initiate cross-linking. At a relatively low substitution degree of 30%~55%, methacrylamide fish scale gelatin can achieve rapid curing within 3~5 seconds in combination with a photoinitiator. The photocuring efficiency far exceeds that of photocurable bio-inks using existing commercial GelMA as the active component, greatly improving printing efficiency and molding accuracy. 2. The photocurable bio-ink provided by the present invention uses methacrylamide fish scale gelatin, which is prepared by acylation reaction between fish scale gelatin and methacrylic anhydride. By utilizing the low fat content of fish scale gelatin, material degradation, discoloration and potential cytotoxicity caused by lipid oxidation are avoided, thereby improving the storage stability and batch consistency of the bio-ink. 3. The photocurable bio-ink provided by the present invention uses methacrylamide fish scale gelatin as the active component. Since the amino acid sequence homology between fish collagen and human collagen is much lower than that of mammals, its immunogenic epitopes (such as Gal-α-1,3-Gal) are not present in fish scale gelatin, making the photocurable bio-ink have extremely low immunogenicity, which can minimize the immune rejection reaction after implantation in vivo and has higher clinical safety. 4. In the photocurable bio-ink provided by the present invention, the methacrylamide fish scale gelatin is prepared by fish scale gelatin and methacrylic anhydride through an acylation reaction. Since the lysine content in fish scale gelatin is 2.4%~2.9%, which is much higher than the 1.4%~1.9% in pigskin gelatin, and lysine is a substituted amino acid for methacrylamide, the increase in its content can significantly improve the modification efficiency and reduce the amount of methacrylic anhydride used as the modifier. 5. The photocurable bio-ink provided by the present invention uses methacrylamide fish scale gelatin as the active component. The methacrylamide fish scale gelatin is made from fish scales, which are waste products of fishery processing. It is low in cost, sustainable in source, and has a wider market and application acceptance. 6. The photocurable bio-ink provided by this invention can be used to rapidly manufacture various high-precision tissue engineering scaffolds or organ models based on extrusion or photocuring 3D bioprinting technology. It is particularly suitable for soft tissue regeneration and organoid culture fields where high transparency and immunocompatibility are required, such as cartilage, skin, blood vessels and cornea. Attached Figure Description

[0017] Figure 1 The ¹H NMR comparison diagrams are of fish scale gelatin and FGelMA in Example 1 of the present invention and PGelMA in Comparative Example 1. Figure 2 The viscosity-shear rate curve of the photocurable bio-ink prepared in Example 1 of this invention is shown. Figure 3 These are photographs of the photocurable bio-inks prepared in Examples 1-3 and Comparative Examples 1-3 after curing. (a) corresponds to Examples 1-3, and (b) corresponds to Comparative Examples 1-3. The percentages marked on the bottles are the corresponding GelMA concentrations. Figure 4 A photograph of a three-dimensional mesh scaffold obtained by printing using the photocurable bio-ink prepared in Example 1 of this invention; Figure 5The results of the cytotoxicity experiment of the hydrogels formed by curing the photocurable bio-inks prepared in Examples 1-3 of the present invention are shown. (a) corresponds to the blank control group, (b) corresponds to Example 1, (c) corresponds to Example 2, and (d) corresponds to Example 3. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] This invention provides a photocurable bio-ink comprising methacrylamide fish scale gelatin and a photoinitiator. The methacrylamide fish scale gelatin is prepared by an acylation reaction between fish scale gelatin and methacrylic anhydride, with a substitution degree of 30%–55%. It should be noted that the preparation method of the methacrylamide fish scale gelatin is as follows: fish scale gelatin is dissolved in deionized water or phosphate buffer, methacrylic anhydride is slowly added dropwise to carry out the acylation reaction, and the reaction product is dialyzed in the dark and lyophilized to obtain methacrylamide fish scale gelatin powder.

[0020] The aforementioned photocurable bio-ink uses methacrylamide fish scale gelatin as the active ingredient. This methacrylamide fish scale gelatin is prepared by an acylation reaction between fish scale gelatin and methacrylic anhydride. Utilizing the low fat content of fish scale gelatin (less than 0.5%), the resulting photocurable bio-ink solution exhibits extremely high transparency and minimal scattering of ultraviolet / blue light, allowing light energy to penetrate efficiently and initiate cross-linking. Even with a relatively low substitution degree of 30%–55%, methacrylamide fish scale gelatin, in conjunction with a photoinitiator, can achieve rapid curing within 3–5 seconds. This curing efficiency far surpasses that of photocurable bio-inks using existing commercially available GelMA as the active ingredient, significantly improving printing efficiency and molding accuracy. Furthermore, the low fat content of the methacrylamide fish scale gelatin in this photocurable bio-ink avoids material degradation, discoloration, and potential cytotoxicity caused by lipid oxidation, thus improving the storage stability and batch consistency of the bio-ink. Furthermore, the aforementioned photocurable bio-ink uses methacrylamide-modified fish scale gelatin as the active component. Since the amino acid sequence homology between fish collagen and human collagen is much lower than that of mammals, and its immunogenic epitopes (such as Gal-α-1,3-Gal) are not present in fish scale gelatin, this photocurable bio-ink exhibits extremely low immunogenicity, minimizing immune rejection reactions after implantation and resulting in higher clinical safety. Moreover, in the aforementioned photocurable bio-ink, the methacrylamide-modified fish scale gelatin is prepared by an acylation reaction between fish scale gelatin and methacrylic anhydride. Because the lysine content in fish scale gelatin is 2.4%–2.9%, significantly higher than the 1.4%–1.9% in porcine skin gelatin, and lysine is a substituted amino acid in methacrylamide, increasing its content can significantly improve modification efficiency and reduce the amount of methacrylic anhydride used as a modifier. In addition, the aforementioned photocurable bio-ink uses methacrylated fish scale gelatin as the active component. The methacrylated fish scale gelatin is made from fish scales, a waste product of fishery processing, which is low in cost, sustainable in source, and has a wider market and application acceptance.

[0021] In a preferred embodiment, the concentration of methacrylamide fish scale gelatin is 2.5% to 15% w / v, and the concentration of photoinitiator is 0.2% to 0.5% w / v. The photocurable bio-ink obtained under these conditions exhibits higher photocrosslinking efficiency.

[0022] In a preferred embodiment, when preparing methacrylamide fish scale gelatin, the ratio of the volume of added methacrylic anhydride to the mass of fish scale gelatin is (0.3~1.2):1 mL / g. Using this condition to prepare methacrylamide fish scale gelatin results in high modification efficiency, balancing the amount of methacrylic anhydride used with the degree of substitution obtained in the methacrylamide fish scale gelatin.

[0023] In a preferred embodiment, the acylation reaction is carried out at a temperature of 4–80°C and for a time of 1–8 h during the preparation of methacrylated fish scale gelatin. Using these conditions improves the efficiency of the acylation reaction.

[0024] In a preferred embodiment, the fish scale gelatin is extracted from the scales of marine fish. Using fish scale gelatin extracted from marine fish scales ensures that the gelatin has an extremely low fat content. It should be noted that the method for obtaining fish scale gelatin from fish scales is a conventional method in the art, specifically involving a multi-step purification process including demineralization, alkali treatment, and hot water extraction of washed marine fish scales to obtain high-purity fish scale gelatin powder.

[0025] In a preferred embodiment, the marine fish is selected from any one or more of sea bass, cod, and sea bream. This preferred embodiment lists preferred marine fish species whose scales have lower fat content, which is beneficial for obtaining fish scale gelatin with lower fat content.

[0026] In a preferred embodiment, the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate. This preferred embodiment lists preferred types of photoinitiators that exhibit superior cell compatibility.

[0027] This invention also provides a method for preparing the above-mentioned photocurable bio-ink, comprising the following steps: dissolving methacrylamide fish scale gelatin in cell culture medium or phosphate buffer, adding a photoinitiator, and obtaining the photocurable bio-ink through sterile filtration. This method for preparing the photocurable bio-ink is simple and suitable for the commercial production of photocurable bio-inks.

[0028] This invention further provides the application of the above-mentioned photocurable bio-ink in the 3D bioprinting of tissue engineering scaffolds or organ models. The photocurable bio-ink provided by this invention can be used to rapidly manufacture various high-precision tissue engineering scaffolds or organ models based on extrusion or photocuring 3D bioprinting technologies. It is particularly suitable for soft tissue regeneration fields and organoid culture fields where high transparency and immunocompatibility are required, such as cartilage, skin, blood vessels, and cornea.

[0029] In a preferred embodiment, when preparing tissue engineering scaffolds or organ models, the photocurable bio-ink is cured under irradiation with a 405nm light source, where the light intensity is 5~30 mW / cm². 2 The irradiation time is 3-5 seconds. Under these conditions, rapid curing of photocurable bio-inks can be achieved, which is beneficial to improving the efficiency and quality of tissue engineering scaffolds or organ models prepared by 3D bioprinting.

[0030] To provide a clearer and more detailed description of the photocurable bio-ink, its preparation method, and its applications provided in the embodiments of the present invention, the following description will be based on specific embodiments.

[0031] Example 1 The preparation method of photocurable bio-ink includes the following steps: (1) Take 10 g of fish scale gelatin extracted from sea bass scales, dissolve it in 90 mL of phosphate buffer (PBS) at 37 ℃, slowly add 5 mL of methacrylic anhydride (MA), and react for 2 h; put the reaction solution into a dialysis bag, dialyze it with deionized water at 37 ℃ in the dark for 5 days, and change the water 3 times a day; freeze dry the solution to obtain white porous sponge-like methacrylamide fish scale gelatin (abbreviated as FGelMA), with a degree of substitution (DS) of 51.54% (for the specific characterization steps of the degree of substitution, please refer to the methacrylamide substitution degree characterization section in the subsequent performance test); (2) FGelMA was dissolved in DMEM medium containing 0.38% (w / v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) to prepare a solution with an FGelMA concentration of 5% (w / v). The solution was shaken at 37°C until completely dissolved and then sterile filtered through a 0.22 μm filter membrane to obtain photocurable bio-ink.

[0032] Example 2 The difference between this embodiment and Embodiment 1 is that the concentration of FGelMA in the photocurable bio-ink is 2.5% (w / v).

[0033] Example 3 The difference between this embodiment and Embodiment 1 is that the concentration of FGelMA in the photocurable bio-ink is 10% (w / v).

[0034] Example 4 The preparation method of photocurable bio-ink includes the following steps: (1) Take 10 g of fish scale gelatin extracted from sea bass scales, dissolve it in 90 mL of PBS at 4 ℃, slowly add 12 mL of MA, and react for 6 h; put the reaction solution into a dialysis bag, dialyze with deionized water at 37 ℃ in the dark for 5 days, changing the water 3 times a day; freeze-dry the solution to obtain FGelMA, with a DS of 55%; (2) Dissolve FGelMA in DMEM medium containing 0.2% (w / v) LAP to prepare a solution with an FGelMA concentration of 2.5% (w / v), shake at 37°C until completely dissolved, and filter aseptically through a 0.22 μm filter membrane to obtain photocurable bio-ink.

[0035] Example 5 The preparation method of photocurable bio-ink includes the following steps: (1) Take 10 g of fish scale gelatin extracted from sea bass scales, dissolve it in 90 mL of PBS at 37 ℃, slowly add 5 mL of MA, and react for 1 h; put the reaction solution into a dialysis bag, dialyze with deionized water at 37 ℃ in the dark for 5 days, changing the water 3 times a day; freeze-dry the solution to obtain FGelMA, with a DS of 40%; (2) Dissolve FGelMA in DMEM medium containing 0.45% (w / v) LAP to prepare a solution with an FGelMA concentration of 10% (w / v), shake at 37°C until completely dissolved, and filter aseptically through a 0.22 μm filter membrane to obtain photocurable bio-ink.

[0036] Example 6 The preparation method of photocurable bio-ink includes the following steps: (1) Take 10 g of fish scale gelatin extracted from sea bass scales, dissolve it in 90 mL of PBS at 80 ℃, slowly add 3 mL of MA, and react for 8 h; put the reaction solution into a dialysis bag, dialyze with deionized water at 37 ℃ in the dark for 5 days, changing the water 3 times a day; freeze-dry the solution to obtain FGelMA, with a DS of 30%; (2) Dissolve FGelMA in PBS containing 0.5% (w / v) LAP to prepare a solution with an FGelMA concentration of 15% (w / v), shake at 37°C until completely dissolved, and filter aseptically through a 0.22 μm filter membrane to obtain photocurable bio-ink.

[0037] Example 7 The preparation method of photocurable bio-ink includes the following steps: (1) Take 10 g of fish scale gelatin extracted from sea bream scales, dissolve it in 90 mL of PBS at 45 ℃, slowly add 5 mL of MA, and react for 2 h; put the reaction solution into a dialysis bag, dialyze with deionized water at 37 ℃ in the dark for 5 days, changing the water 3 times a day; freeze-dry the solution to obtain FGelMA, with a DS of 53.5%; (2) Dissolve FGelMA in DMEM medium containing 0.38% (w / v) LAP to prepare a solution with an FGelMA concentration of 4% (w / v), shake at 37°C until completely dissolved, and filter aseptically through a 0.22 μm filter membrane to obtain photocurable bio-ink.

[0038] Example 8 The preparation method of photocurable bio-ink includes the following steps: (1) Take 10 g of fish scale gelatin extracted from cod scales, dissolve it in 90 mL of PBS at 40 ℃, slowly add 8 mL of MA, and react for 3 h; put the reaction solution into a dialysis bag, dialyze with deionized water at 37 ℃ in the dark for 5 days, changing the water 3 times a day; freeze-dry the solution to obtain FGelMA, with a DS of 51.4%; (2) Dissolve FGelMA in PBS containing 0.42% (w / v) LAP to prepare a solution with an FGelMA concentration of 8% (w / v), shake at 37°C until completely dissolved, and filter aseptically through a 0.22 μm filter membrane to obtain photocurable bio-ink.

[0039] Comparative Example 1 The difference between this comparative example and Example 1 is that: porcine collagen extracted from pigskin is used instead of fish scale gelatin to prepare methacrylamide porcine gelatin (abbreviated as PGelMA); the concentration of PGelMA in the photocurable bio-ink is 5% (w / v).

[0040] Comparative Example 2 The difference between this comparative example and Comparative Example 1 is that the concentration of PGelMA in the photocurable bio-ink is 2.5% (w / v).

[0041] Comparative Example 3 The difference between this comparative example and Comparative Example 1 is that the concentration of PGelMA in the photocurable bio-ink is 10% (w / v).

[0042] Comparative Example 4 The difference between this comparative example and Example 1 is that commercial GelMA (prepared from porcine collagen with a degree of substitution of 60%) was used instead of FGelMA to prepare the photocurable bio-ink.

[0043] Performance testing 1. Characterization of the degree of substitution of methacrylation The ¹H NMR characterization of the fish scale gelatin used in Example 1 and the obtained FGelMA, and the ¹H NMR characterization of the PGelMA obtained in Comparative Example 1, are as follows: Figure 1 As shown in Table 1, the DS values ​​of the FGelMA prepared in Examples 1-6 and the PGelMA prepared in Comparative Examples 1-4 were calculated based on ¹H NMR.

[0044] Depend on Figure 1 As can be seen, compared with the ¹H NMR spectrum of fish scale gelatin, the characteristic peaks at δ=5.3 ppm and δ=5.6 ppm in the ¹H NMR spectrum of FGelMA prove that the methacryloyl group has been successfully grafted onto fish scale gelatin.

[0045] As shown in Table 1, the DS of FGelMA is 51.54%, which is much higher than that of PGelMA (DS is 38.89%). It can be seen that, under the same amount of MA, the FGelMA obtained by fish scale gelatin in Example 1 of the present invention has a higher degree of substitution.

[0046] 2. Rheological property testing of photocurable bio-ink The viscosity-shear rate curve of the photocurable bio-ink prepared in Example 1 was measured using a rotational rheometer, as shown below. Figure 2 As shown. By Figure 2 As can be seen, the photocurable bio-ink prepared in Example 1 of the present invention exhibits typical shear-thinning characteristics. It has high viscosity at low shear rates, which is beneficial for maintaining the structure, while the viscosity drops sharply at high shear rates, which is beneficial for smooth extrusion.

[0047] 3. Photocuring performance test of photocurable bio-ink The photocurable bio-inks prepared in Examples 1-6 and Comparative Examples 1-4 of this invention were injected into molds and placed in a 405nm blue light generator at 30 mW / cm². 2 The curing results after irradiation are shown in Table 1. Among them, photographs of the cured photocurable bioinks prepared in Examples 1-3 and Comparative Examples 1-3 are shown below. Figure 3 As shown.

[0048] Table 1. Test results of photocurability of photocurable bio-inks in Examples 1-6 and Comparative Examples 1-4

[0049] From Table 1 and Figure 3 As can be seen, the photocurable bio-inks prepared in Examples 1-6 of the present invention were all completely cured within 3-5 seconds of irradiation, and the photocuring speed was significantly faster than that of Comparative Examples 1-4. Moreover, the photocurable bio-inks prepared in Examples 1-6 of the present invention remained transparent and did not turn yellow after curing.

[0050] 4. Experiments on 3D bioprinting with photocurable bio-inks Using an extrusion-type bioprinter equipped with a 405 nm photocuring module, a 10×10×2 mm three-dimensional mesh scaffold was printed with the photocurable bio-ink prepared in Example 1 of this invention. The printing parameters were: nozzle diameter 25G, printing pressure 25 kPa, printing speed 8 mm / s. Immediately after each layer was printed, the ink was applied at 405 nm and 30 mW / cm². 2 Photocuring was performed by irradiation for 5 seconds. The resulting 3D mesh support structure is shown in the image. Figure 4 As shown, by Figure 4 As can be seen, the printed support structure is clear, the fibers are continuous, and the pores are uniform, demonstrating excellent molding accuracy and shape fidelity.

[0051] 5. Cytotoxicity test The photocurable bio-inks prepared in Examples 1-3 were cured using a 405nm blue light generator to obtain hydrogels. Resuscitated mouse fibroblasts L929 were seeded onto the surface of the corresponding hydrogels from Examples 1-3, and cell culture medium was added, serving as the experimental group. Mouse fibroblasts L929 were directly seeded onto cell culture medium, serving as the blank control group. Both the experimental and blank control groups were cultured at 37 ℃ and 5% CO2 for 3 days. L929 cells from the experimental and blank control groups were stained with calcein-AM to assess cell number and morphology. The results are as follows: Figure 5 As shown. By Figure 5 As can be seen, the relative cell survival rate of L929 cells on the surface of the hydrogel cured by the photocurable bio-ink prepared in Examples 1-3 of this invention is as high as 95%, and the cells extend well in the three-dimensional network of the hydrogel, indicating that the material has excellent cell compatibility.

Claims

1. A photocurable bio-ink, characterized in that, It includes methacrylated fish scale gelatin and a photoinitiator, wherein the methacrylated fish scale gelatin is prepared by acylation reaction of fish scale gelatin and methacrylic anhydride, and the degree of substitution of the methacrylated fish scale gelatin is 30%~55%.

2. The photocurable bio-ink according to claim 1, characterized in that, The concentration of methacrylamide fish scale gelatin is 2.5% to 15% w / v, and the concentration of photoinitiator is 0.2% to 0.5% w / v.

3. The photocurable bio-ink according to claim 1, characterized in that, When preparing methacrylated fish scale gelatin, the ratio of the volume of added methacrylic anhydride to the mass of fish scale gelatin is (0.3~1.2):1 mL / g.

4. The photocurable bio-ink according to claim 1 or 3, characterized in that, When preparing methacrylated fish scale gelatin, the acylation reaction temperature is 4~80 ℃ and the reaction time is 1~8 h.

5. The photocurable bio-ink according to claim 1, characterized in that, Fish scale gelatin is extracted from the scales of saltwater fish.

6. The photocurable bio-ink according to claim 5, characterized in that, The saltwater fish are selected from any one or more of the following: sea bass, cod, and sea bream.

7. The photocurable bio-ink according to claim 1, characterized in that, The photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

8. The method for preparing the photocurable bio-ink according to any one of claims 1-7, characterized in that, The process includes the following steps: dissolving methacrylamide fish scale gelatin in cell culture medium or phosphate buffer, adding a photoinitiator, and obtaining photocurable bio-ink through sterile filtration.

9. The application of the photocurable bio-ink according to any one of claims 1-7 in the preparation of tissue engineering scaffolds or organ models by 3D bioprinting.

10. The application according to claim 9, characterized in that, When preparing tissue engineering scaffolds or organ models, photocurable bio-inks are cured under irradiation with a 405 nm light source, with a light intensity of 5–30 mW / cm². 2 The irradiation time is 3-5 seconds.

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