CN113150561A - Collagen-based biological ink for 3D biological printing and preparation method and application thereof - Google Patents
Collagen-based biological ink for 3D biological printing and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological materials, and particularly relates to collagen-based biological ink for 3D biological printing, and a preparation method and application thereof. The preparation method of the collagen-based biological ink comprises the following steps: methacrylic anhydride is added into dilute hydrochloric acid solution of collagen for full reaction to obtain methacrylated collagen; and sequentially adding acetic acid, an ultraviolet light initiator LAP aqueous solution and a procyanidine aqueous solution into the methacryloylated collagen, and fully mixing and crosslinking to obtain the collagen-based bio-ink. The collagen-based bio-ink prepared by the preparation method can be quickly gelatinized within 30-50 seconds under the irradiation of ultraviolet light, the formed hydrogel has good mechanical properties and good biocompatibility, and the collagen-based bio-ink can be used for 3D bioprinting.
Description
Technical Field
The invention belongs to the technical field of biological materials, and particularly relates to collagen-based biological ink for 3D biological printing, and a preparation method and application thereof.
Background
The 3D bioprinting technology is driven by three-dimensional modeling programs such as CAD, CAM and the like, takes cells, proteins and biocompatible materials as raw materials, and forms a three-dimensional entity in a layer-by-layer accumulation mode. In recent years, 3D bio-printing technology has rapidly developed. The emergence of 3D biological printing technology opens up a new way for in vitro biomedical engineering, and can be used for constructing tissue regeneration, in vitro biological models, cell diagnosis and the like.
Bio-ink is a raw material for 3D bio-printing, a cell preparation suitable for processing by automated bio-fabrication techniques. 3D bio-printing currently faces a major challenge in preparing suitable bio-inks. The biological ink for 3D printing application needs to meet the following requirements: (1) the material has a stable structure, can be efficiently and effectively crosslinked, and ensures the integrity and stability of the structure after printing; (2) the material must transition from a fluid state to a solid state at the appropriate time; (3) sufficient mechanical properties to support the printing structure; (4) good biocompatibility, can support cell loading and maintain cell activity; (5) the printed shape has high structural resolution so as to better simulate tissues and organs with complex structures. Among them, it is the biggest challenge that bio-ink has both good bioprintability and maintains cell viability.
The most widely studied bio-ink is prepared from gelatin or modified gelatin. Gelatin is a hydrolysate of collagen, which loses the triple helical structure of collagen and also loses its physiological activity. Collagen is a major component of extracellular matrix and is widely present in living organisms. Collagen is formed by winding 3 polypeptide chains in a supercoiled form, and its basic structural unit, the primary structure of procollagen, has (-Gly-X-Y-)nX is typically proline or hydroxyproline and Y is any amino acid. Complex sequence composed of multiple amino acidsThe collagen has natural cell binding sites and is easy to be cross-linked and modified. As a biological material, the collagen has good biocompatibility and various biological activities, and can promote cell proliferation and platelet aggregation. Based on these characteristics, collagen has been widely used in the manufacture of bone graft materials, wound dressings, surgical sutures, drug carriers, artificial blood vessels, heart valves, repair of myocardial tissue, and the like.
Several studies have demonstrated the feasibility of collagen for 3D bioprinting. According to literature reports, temperature changes can drive collagen to self-assemble into triple-helical structures (Osidak E O, Karalkin P A, Osidak MS, et al, Viscol collagen solution as a novel choice for direct 3D bioprinting [ J ]. Journal of Materials Science: Materials in Medicine,2019,30(3): 31); collagen-based bio-inks (Kim Y B, Lee H, Kim G H. Strategy to achieve high strain pore/biocompatable macromolecular cells, using a collagen/genipin-bio and an optimal 3D printing process [ J ]. ACS applied materials & interfaces,2016,8(47): 32230) 32240) can also be formulated with genipin as a cross-linking agent.
However, the temperature, cross-linker driven collagen gelation time is long, and the resolution of the structure after 3D printing is low; pure collagen hydrogel has poor mechanical properties and is difficult to form a 3D structure in a layer-by-layer stacking mode (Osidak E O, Kozhukhov V I, Osidak M S, et al. collagen as Bioink for Bioprinting: A Comprehensive Review [ J ]. International Journal of Bioprinting,2020,6 (3)); this limits its application in the field of 3D bioprinting. Therefore, the collagen-based bio-ink maintains the physiological activity and excellent biocompatibility of collagen, accelerates the gelation time, improves the mechanical strength of gel, and is an important research direction of the collagen-based bio-ink.
Disclosure of Invention
The first purpose of the invention is to provide a preparation method of collagen-based bio-ink for 3D bioprinting, the collagen-based bio-ink prepared by the preparation method can be rapidly gelatinized within 30-50 seconds under the irradiation of ultraviolet light, the formed hydrogel has good mechanical properties and good biocompatibility, and the collagen-based bio-ink can be used for 3D bioprinting.
The second purpose of the invention is to provide the collagen-based bio-ink for 3D bioprinting prepared by the preparation method.
A third object of the present invention is to provide an application of the collagen-based bio-ink for 3D bio-printing in 3D printing.
The primary purpose of the invention is realized by the following technical scheme:
a method of preparing a collagen-based bio-ink for 3D bio-printing, comprising the steps of:
A. preparation of methacrylated collagen freeze-dried sponge: mixing collagen, methacrylic anhydride and hydrochloric acid solution with the molar concentration of 10 mM-30 mM according to the mass-volume ratio of 1 g-10 g: 2.3 mL-10 mL: 400 mL-600 mL; adding a hydrochloric acid solution into collagen to adjust the pH value to 7.5-9, adding methacrylic anhydride, stirring, reacting in a dark place, dialyzing, and freeze-drying to obtain the methacryloylated collagen freeze-dried sponge;
B. preparing the collagen-based bio-ink: and B, mixing the methacrylated collagen freeze-dried sponge obtained in the step A with an acetic acid solution with the mass concentration of 0.2-0.5% according to the mass-volume ratio of 100-200 mg: 5-10 mL; dissolving methacryloylated collagen freeze-dried sponge in an acetic acid solution, sequentially adding an ultraviolet initiator LAP and procyanidine to ensure that the mass concentration of the ultraviolet initiator LAP in the solution is 0.2-1.0% (w/v) and the mass concentration of the procyanidine is 0.002-0.030% (w/v), and uniformly mixing to obtain the collagen-based bio-ink.
Preferably, the molecular weight of the collagen in the step A is 8000-300000 Da, and the collagen has a triple helix structure.
Preferably, in the step A, the stirring temperature is 4-10 ℃, the dark reaction time is 8-10 hours, the dialysis is carried out for 7 days, and the freeze-drying temperature is-40 to-20 ℃ and the time is 48 hours.
Preferably, the pH value of the methacrylated collagen lyophilized sponge synthesized in the step A is 9.
Preferably, the ultraviolet photoinitiator LAP in step B is phenyl (2,4, 6-trimethylbenzoyl) lithium phosphate.
Preferably, the mass concentration of the ultraviolet photoinitiator LAP in the step B is 0.2-0.5% (w/v).
Preferably, the mass concentration of the procyanidine solution in the step B is 0.004-0.008% (w/v).
The second purpose of the invention is realized by the following technical scheme:
a collagen-based bio-ink for 3D bio-printing is prepared by the preparation method.
Preferably, the collagen-based bio-ink for 3D bio-printing is a cell-free bio-ink.
Preferably, the collagen-based bio-ink for 3D bio-printing is a bio-ink containing living cells.
The third purpose of the invention is realized by the following technical scheme:
use of a collagen-based bio-ink for 3D bioprinting to reconstruct biological tissues or organs and in vitro biological models of constructs.
Preferably, the collagen-based bio-ink for 3D bioprinting has a specific application in reconstructing biological tissues or organs and in constructing an external biological model: placing collagen-based bio-ink in a trough of a DLP 3D printer, irradiating by ultraviolet light, crosslinking, curing and molding through photoreaction to prepare hydrogel, and preparing a hydrogel bracket without cells through 3D printing; placing the printed hydrogel scaffold in cell culture medium, and culturing at 37 deg.C with 5% CO2After culturing for 5 minutes in a medium incubator, absorbing the cell culture medium, adding the cell suspension, and continuously placing in the incubator to perform cell culture on the hydrogel surface.
Preferably, the collagen-based bio-ink for 3D bioprinting has a specific application in reconstructing biological tissues or organs and in constructing an external biological model: preparing collagen-based bio-ink and cells into cell-loaded bio-ink, placing the cell-loaded bio-ink in a trough of a DLP 3D printer, irradiating by ultraviolet light, crosslinking and curing by photoreaction to obtain hydrogel, preparing a hydrogel bracket containing cells by 3D printing, placing the hydrogel bracket in a cell culture medium, and performing cell culture at 37 ℃ and 5% CO2Culturing in a medium incubator.
Preferably, the illumination intensity of the ultraviolet irradiation is 10-50 mW/cm2The wavelength is 365-405 nm, and the irradiation time is 30-150 s.
Preferably, the temperature of the hydrogel support generated by 3D printing is 4-25 ℃.
The principle of the invention is as follows:
as shown in fig. 1 and fig. 2, the reaction principle schematic diagram of the methacrylated collagen prepared by the present invention and the principle schematic diagram of the cross-linking reaction of procyanidine and collagen are that the collagen is first methacrylated and modified, and carbon-carbon double bond groups are grafted on free amino groups of the collagen. And then adding an ultraviolet photoinitiator LAP and an aqueous solution of procyanidine into the methacrylated collagen solution. Wherein, the ultraviolet initiator LAP can generate free radicals under ultraviolet light to initiate the fracture and bonding of carbon-carbon double bonds on the methacryloylated collagen, so that photopolymerization reaction is carried out to form the collagen hydrogel. Procyanidins are natural polyphenols with catechol structure, in which phenolic hydroxyl groups can form hydrogen bonds with nitrogen and oxygen atoms in collagen. In the invention, the procyanidine is taken as a protein cross-linking agent, so that the mechanical property of the collagen hydrogel can be obviously improved, and the printability of the collagen is further improved.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the invention aims to provide a preparation method of collagen-based bio-ink. The preparation method takes natural protein-collagen as raw material, and has the advantages of abundant source, low cost and excellent biocompatibility. The modified collagen still maintains the complete triple-helical structure. The collagen is one of the main components of the extracellular matrix, and the biological ink prepared by the invention can simulate the environment of the extracellular matrix and provides a good environment for the in-vitro three-dimensional culture of cells.
(2) The collagen-based bio-ink of the present invention uses procyanidins as a cross-linking agent. Compared with other crosslinking agents, the procyanidine is a natural material, has good biocompatibility, no cytotoxicity at low concentration and high crosslinking efficiency, and is an ideal protein crosslinking agent. The procyanidin can be used as cross-linking agent of collagen, has various physiological activities, can inhibit bacterial growth, has good antioxidant, anticancer activity and anti-inflammatory activity, and can reduce blood sugar and prevent and treat cardiovascular diseases.
(3) The collagen-based bio-ink has certain shear thinning performance. During extrusion, the collagen-based bio-ink is subjected to shear force, hydrogen bonds between collagen and procyanidine are broken, and the collagen-based bio-ink is subjected to shear thinning, so that the collagen-based bio-ink can be smoothly extruded from an extrusion head, and loaded cells are protected from being damaged by the shear stress. After extrusion, the shear stress is removed, and hydrogen bonds between the modified collagen and the procyanidin can be rapidly reformed to be gel.
(4) The collagen-based bio-ink can be quickly gelatinized within 30-50s under the irradiation of ultraviolet light, has good forming performance, and the generated hydrogel has high mechanical strength and supports the printing of models with various structures of different shapes.
(5) The collagen-based bio-ink can control the mechanical strength, the water content and the like of hydrogel by changing the substitution degree of the methacryloylated collagen, the concentrations of the methacryloylated collagen and the procyanidine and the irradiation duration of ultraviolet light, so as to prepare the hydrogel scaffold with controllable performance.
(6) The collagen-based bio-ink can construct hydrogel with various shapes and structures through a 3D bio-printing technology, can be used for application of various tissue engineering and regenerative medical engineering, can prepare scaffolds with any size and shape through the 3D bio-printing technology, has wide application field, and can be used for tissue repair, blood vessel construction, tissue reconstruction, repair fillers, drug carriers, construct external biological models and the like.
Drawings
FIG. 1 is a schematic diagram of the reaction principle of methacrylated collagen prepared by the present invention.
FIG. 2 is a schematic diagram of the cross-linking reaction between procyanidins and collagen in the present invention.
FIG. 3 is a NMR spectrum of methacryloylated collagen synthesized in accordance with the present invention at various pH's. Wherein, Collagen is unmodified Collagen, and ColMAA7.5, ColMAA8 and ColMAA9 represent methacrylated Collagen synthesized under the conditions of pH 7.5, pH 8 and pH 9, respectively. The substitution degree of methacrylated collagen synthesized under the conditions of pH 7.5, pH 8 and pH 9 was increased in order by calculation of peak integration.
FIG. 4 is a rheological analysis of collagen-based bio-inks prepared in examples 1-3 of the present invention, wherein the gray rectangular areas are the time of UV irradiation; in the figure, PAC is procyanidin, and the values are the concentration of procyanidin in collagen-based bio-ink, in μ g/mL.
Fig. 5 is a graph of collagen-based bio-ink of the present invention before and after cross-linking.
Fig. 6 is a scanning electron microscope image of the collagen-based bio-ink crosslinked in example 1.
Detailed Description
The present invention will be described in further detail with reference to examples.
It should be specifically noted that the specific examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. In addition, after reading the disclosure of the present invention, those skilled in the art and related fields can make various changes or modifications to the present invention based on the description herein, and changes, modifications, substitutions, simplifications, or other applications in the field without departing from the spirit and principle of the present invention are included in the protection scope of the present invention.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1: preparation of collagen-based bio-ink
1g of collagen was dissolved in 400mL of a 10mM hydrochloric acid solution at a molar concentration, and the solution was stirred at 4 ℃ for 8 hours to completely dissolve the collagen, thereby preparing a dilute hydrochloric acid solution of collagen. Adjusting the pH of the collagen dilute hydrochloric acid solution to 9 by using a sodium hydroxide solution with the molar concentration of 1M, dropwise adding 2.3mL of methacrylic anhydride into the collagen dilute hydrochloric acid solution while stirring, dropwise adding a sodium hydroxide solution with the molar concentration of 1M while stirring, stabilizing the pH to 9, and then stirring at 4 ℃ and reacting for 8 hours in a dark place. The reacted solution was poured into a 8000-and 14000Da cut-off dialysis bag and dialyzed against 10mM HCl at 4 ℃ for 7 days, and the dialysate was changed twice a day. After dialysis, the solution is frozen at-20 ℃, and then is frozen and dried in a vacuum freeze dryer to obtain the methacryloylated collagen freeze-dried sponge. The modified collagen still has an intact triple helix structure (fig. 6), and the grafting rate is 68.0% by the integration of hydrogen nuclear magnetic resonance spectroscopy data (fig. 3).
Dissolving 100mg of methacryloylated collagen lyophilized sponge in 5mL of 0.2% acetic acid solution, fully dissolving, adjusting the pH to 7 by using 1M sodium hydroxide, and adding a photoinitiator LAP aqueous solution and a procyanidine aqueous solution into the solution to ensure that the concentration of the LAP in the solution is 0.5% (w/v) and the concentration of the procyanidine is 0.002% (w/v). And uniformly mixing to obtain the collagen-based biological ink.
Uniformly placing a proper amount of collagen-based bio-ink in a cylindrical die, and irradiating by ultraviolet light with power of 10W and wavelength of 365nm for 30-50s to form the hydrogel through photocrosslinking and curing.
FIG. 6 shows that: freezing the solidified hydrogel by using liquid nitrogen, and then freeze-drying the hydrogel at the temperature of-40 ℃, wherein the interior of the freeze-dried hydrogel can be found to be a porous structure by scanning electron microscopy.
Example 2: preparation of collagen-based bio-ink
1g of collagen was dissolved in 400mL of a 10mM hydrochloric acid solution at a molar concentration, and the solution was stirred at 4 ℃ for 8 hours to completely dissolve the collagen, thereby preparing a dilute hydrochloric acid solution of collagen. Adjusting the pH of the collagen dilute hydrochloric acid solution to 9 by using a sodium hydroxide solution with the molar concentration of 1M, dropwise adding 2.3mL of methacrylic anhydride into the collagen dilute hydrochloric acid solution while stirring, dropwise adding a sodium hydroxide solution with the molar concentration of 1M while stirring, stabilizing the pH to 9, and then stirring at 4 ℃ and reacting for 8 hours in a dark place. The reacted solution was poured into a 8000-and 14000Da cut-off dialysis bag and dialyzed against 10mM HCl at 4 ℃ for 7 days, and the dialysate was changed twice a day. After dialysis, the solution is frozen at-20 ℃, and then is frozen and dried in a vacuum freeze dryer to obtain the methacryloylated collagen freeze-dried sponge.
Dissolving 100mg of methacrylated collagen lyophilized sponge in 5mL of 0.2% acetic acid solution, fully dissolving, adjusting the pH to 7 by using 1M sodium hydroxide with the molar concentration, and adding an ultraviolet light initiator LAP aqueous solution and a procyanidine aqueous solution into the solution to ensure that the concentration of the LAP in the solution is 0.5% (w/v) and the concentration of the procyanidine is 0.004% (w/v). After being mixed evenly, the mixture is centrifuged to remove air bubbles, thus obtaining the collagen-based bio-ink which is placed in a refrigerator at 4 ℃ for 24 hours.
And (3) putting a proper amount of collagen-based bio-ink into a mould, irradiating by using ultraviolet light with the power of 10W and the wavelength of 365nm, and curing and forming for 30-50s to obtain the hydrogel.
Example 3: preparation of collagen-based bio-ink
The preparation method of the methacrylated collagen lyophilized sponge is the same as that of example 2.
Dissolving 100mg of methacrylated collagen lyophilized sponge in 5mL of 0.2% acetic acid solution, fully dissolving, adjusting the pH to 7 by using 1M sodium hydroxide with a molar concentration, and adding a photoinitiator LAP aqueous solution and a procyanidine aqueous solution into the solution to ensure that the concentration of the LAP in the solution is 0.5% (w/v) and the concentration of the procyanidine is 0.008% (w/v). After being mixed evenly, the mixture is centrifuged to remove air bubbles, thus obtaining the collagen-based bio-ink which is placed in a refrigerator at 4 ℃ for 24 hours.
And (3) putting a proper amount of collagen-based bio-ink into a mould, irradiating by using ultraviolet light with the power of 10W and the wavelength of 365nm, and curing and forming for 30-50s to obtain the hydrogel.
The collagen-based bio-inks of examples 1-3 were used for rheological analysis, and as can be seen in fig. 4, the storage modulus of the gel rapidly increased under uv light irradiation and reached a maximum within 30-50s, and then stabilized at the maximum. The value of the storage modulus is related to the grafting ratio of the methacrylated collagen, the concentration of the methacrylated collagen and the procyanidin.
Example 4: surface cell culture of collagen-based bio-ink hydrogel
The preparation method of the methacrylated collagen lyophilized sponge is the same as that of example 2. Dissolving 100mg of methacryloylated collagen lyophilized sponge in 5mL of 0.2% acetic acid solution, fully dissolving, adjusting the pH to 7 by using 1M sodium hydroxide with a molar concentration, and adding a photoinitiator LAP aqueous solution and a procyanidine aqueous solution into the solution to ensure that the concentration of the LAP in the solution is 0.5% (w/v) and the concentration of the procyanidine is 0.004% (w/v). And after uniform mixing, centrifuging to remove air bubbles to obtain the collagen-based bio-ink.
The collagen-based bio-ink was sterilized through a sterile filter with a pore size of 0.22 μm. And under the aseptic condition, placing the sterilized hydrogel precursor liquid into a trough of a DLP 3D printer, and printing the hydrogel precursor liquid into a three-dimensional bracket through 3D. Placing the three-dimensional scaffold in a 24-well plate, adding cell culture medium, and culturing at 37 deg.C with 5% CO2After culturing for 5 minutes in the middle incubator, absorbing the cell culture medium, adding mouse fibroblast L929 suspension, and continuously placing in the incubator to perform cell culture on the hydrogel surface.
Example 5: printing of cellular collagen-based bio-inks
The preparation method of the methacrylated collagen lyophilized sponge is the same as that of example 2. Dissolving 100mg of methacryloylated collagen lyophilized sponge in 5mL of 0.2% acetic acid solution, fully dissolving, adjusting the pH to 7 by using 1M sodium hydroxide with a molar concentration, and adding a photoinitiator LAP aqueous solution and a procyanidine aqueous solution into the solution to ensure that the concentration of the LAP in the solution is 0.5% (w/v) and the concentration of the procyanidine is 0.004% (w/v). And after uniform mixing, centrifuging to remove air bubbles to obtain the collagen-based bio-ink.
The collagen-based bio-ink was sterilized through a sterile filter with a pore size of 0.22 μm. Under sterile conditions, pre-cultured mouse fibroblasts L929 were resuspended in the hydrogel precursor solution to a final cell concentration of 2 × 105and/mL. And (3) placing the collagen-based biological ink containing the cells in a trough of a DLP 3D printer, and performing 3D biological printing to obtain the three-dimensional scaffold containing the cells. The scaffolds were placed in 24-well plates, cell culture medium was added, and culture was performed at 37 ℃ in a 5% carbon dioxide incubator.
Claims (10)
1. A method of preparing a collagen-based bio-ink for 3D bio-printing, comprising the steps of:
A. preparation of methacrylated collagen freeze-dried sponge: mixing collagen, methacrylic anhydride and hydrochloric acid solution with the molar concentration of 10 mM-30 mM according to the mass-volume ratio of 1 g-10 g: 2.3 mL-10 mL: 400 mL-600 mL; adding a hydrochloric acid solution into collagen to adjust the pH value to 7.5-9, adding methacrylic anhydride, stirring, reacting in a dark place, dialyzing, and freeze-drying to obtain the methacryloylated collagen freeze-dried sponge;
B. preparing the collagen-based bio-ink: and B, mixing the methacrylated collagen freeze-dried sponge obtained in the step A with an acetic acid solution with the mass concentration of 0.2-0.5% according to the mass-volume ratio of 100-200 mg: 5-10 mL; dissolving methacryloylated collagen freeze-dried sponge in an acetic acid solution, sequentially adding an ultraviolet initiator LAP and procyanidine to ensure that the mass concentration of the ultraviolet initiator LAP in the solution is 0.2-1.0% (w/v) and the mass concentration of the procyanidine is 0.002-0.030% (w/v), and uniformly mixing to obtain the collagen-based bio-ink.
2. The method of claim 1, wherein the collagen-based bio-ink used in the step A has a molecular weight of 8000-300000 Da, and the collagen has a triple-helix structure.
3. The preparation method of the collagen-based bio-ink for 3D bio-printing according to claim 1, wherein in the step A, the stirring temperature is 4-10 ℃, the dark reaction time is 8-10 h, the dialysis is performed for 7 days, the freeze-drying temperature is-40 to-20 ℃, and the freeze-drying time is 48 h.
4. The method of claim 1, wherein the ultraviolet photoinitiator LAP in step B is phenyl (2,4, 6-trimethylbenzoyl) lithium phosphate.
5. The method for preparing the collagen-based bio-ink for 3D bio-printing according to claim 1, wherein the mass concentration of the ultraviolet photoinitiator LAP in the step B is 0.2-0.5% (w/v); and the mass concentration of the procyanidine solution in the step B is 0.004-0.008% (w/v).
6. A collagen-based bio-ink for 3D bioprinting according to any one of claims 1 to 5.
7. Use of a collagen-based bio-ink for 3D bioprinting according to claim 6 for reconstructing biological tissues or organs and in vitro biological models of constructs.
8. Use of a collagen-based bio-ink for 3D bioprinting according to claim 7 for the reconstruction of biological tissues or organs and in vitro biological models, characterized by the specific application: placing collagen-based bio-ink in a trough of a DLP 3D printer, irradiating by ultraviolet light, crosslinking, curing and molding through photoreaction to prepare hydrogel, and preparing a hydrogel bracket without cells through 3D printing; placing the printed hydrogel scaffold in cell culture medium, and culturing at 37 deg.C with 5% CO2After culturing for 5 minutes in a medium incubator, absorbing the cell culture medium, adding cell suspension, and continuously placing in the incubator to perform cell culture on the surface of the hydrogel;
or preparing collagen-based bio-ink and cells into cell-loaded bio-ink, placing in a trough of a DLP 3D printer, irradiating with ultraviolet light, crosslinking and curing by photoreaction to obtain hydrogel, 3D printing to obtain hydrogel scaffold containing cells, placing in cell culture medium, and culturing at 37 deg.C and 5% CO2Culturing in a medium incubator.
9. Use of a collagen-based bio-ink for 3D bioprinting according to claim 8 for the reconstruction of biological tissues or organs and in vitro biological models, characterized in that said uv irradiation has an intensity of 10 to 50mW/cm2The wavelength is 365-405 nm, and the irradiation time is 30-150 s.
10. The application of the collagen-based bio-ink for 3D bioprinting in reconstructing biological tissues or organs and in vitro biological models of constructed bodies according to claim 8, wherein the photoreaction crosslinking curing molding is carried out to obtain hydrogel, and the temperature for 3D printing to generate the hydrogel scaffold is 4-25 ℃.
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