Novel photosensitive collagen bio-ink for 3D printing as well as preparation method and application of novel photosensitive collagen bio-ink

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to novel photosensitive collagen bio-ink for 3D printing, a preparation method and application. Comprising the following components: 0.5%-2.0% m / v of allyl succinylated collagen, 0.25%-0.5% m / v of phenyl-2, 4, 6-trimethylbenzoyl lithium phosphate and 0-0.05% m / v of an ultraviolet absorbent, the hydrogel can be completely dissolved in a solution with physiological pH at a high concentration, and a stable and uniform solution is formed; the hydrogel with good mechanical property, fidelity and degradation resistance can be prepared; proliferation, adhesion, migration and differentiation of cells can be promoted, and good biocompatibility and biological activity are achieved; the method is widely applied to the field of tissue engineering such as preparation of skin repair materials and osteochondral repair materials.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a novel photosensitive collagen bioink for 3D printing, a preparation method and applications thereof. Background Art

[0002] 3D printing is an additive manufacturing technology that fabricates scaffolds and complex structures by precisely depositing materials. 3D printing has advantages such as high speed, high efficiency, high resolution, strong personalized manufacturing and bionic ability, and has attracted much attention in the fields of regenerative medicine and tissue engineering. In theory, 3D printing technology can print any designed model; in practice, due to the property limitations of 3D printing bioinks, many designed models still cannot achieve high-precision printing or forming. Therefore, the key to the application of 3D printing technology is to prepare bioinks with good properties, such as printability, high mechanical strength, anti-degradability and good biocompatibility, etc.

[0003] Collagen is an important functional protein in the human body and one of the most widely used biomaterials, and is the main component of the extracellular matrix. Collagen can bind to various cell surface receptors and participate in regulating cell adhesion, proliferation and migration; collagen has good biocompatibility and low immunogenicity, and is widely used in the fields of tissue engineering, etc. However, collagen has poor solubility under physiological pH and low temperature conditions, and usually requires an acidic or alkaline environment to be fully dissolved, which may cause potential stimulation and damage to cells and tissues.

[0004] The development of photosensitive collagen has broadened the application of collagen in bioprinting. Methacrylated collagen (CMA) is currently the main photosensitive collagen bioink. However, its uniformity and stability are poor, and it is difficult to meet the requirements of high-precision 3D printing. Moreover, during the printing process, problems such as structural collapse and nozzle clogging often occur, affecting the printing quality and efficiency. In addition, the mechanical properties of the 3D-printed collagen bio-scaffold are weak and it is difficult to provide sufficient structural support, which limits its application in loading cells and tissue repair. Therefore, those skilled in the art usually choose to add polymer materials to CMA or add cross-linking agents before / after printing to improve its mechanical strength. However, the potential toxicity problems of polymer materials and chemical cross-linking agents are another technical problem that those skilled in the art cannot solve. Summary of the Invention

[0005] To address the above technical problems, the present invention modifies collagen with allyl succinic anhydride to obtain an allyl succinylated collagen (CAA) bioink. The collagen bioink can be completely dissolved in a solution at physiological pH and forms a stable and uniform solution. The collagen bioink can be used for DLP and extrusion-based 3D printing. The collagen bioink can be used to prepare hydrogels with good mechanical properties, fidelity, and anti-degradation ability through 3D printing. The hydrogel has good biocompatibility and bioactivity and can significantly promote cell proliferation, adhesion, differentiation, and migration. The allyl succinylated collagen bioink is widely used in the fields of tissue engineering such as skin repair materials and osteochondral repair materials.

[0006] Specifically, it includes the following content:

[0007] The primary objective of the present invention is to provide a novel photosensitive collagen bioink for 3D printing. The bioink includes the following components: 0.5% - 2.0% m / v allyl succinylated collagen, 0.25% - 0.5% m / v lithium phenyl-2,4,6-trimethylbenzoylphosphate, and 0 - 0.05% m / v ultraviolet absorber.

[0008] Preferably, the allyl succinylated collagen is prepared by the following method: Dissolve collagen in a 0.1 - 0.5M acetic acid solution, adjust the pH to 3 - 9; dropwise add allyl succinic anhydride, react at low temperature for 12 - 72h, and maintain the solution pH at 7 - 9; dialyze and freeze-dry to obtain allyl succinylated collagen containing photosensitive groups.

[0009] Preferably, the collagen is bovine collagen.

[0010] Preferably, the bovine collagen is type I bovine collagen, and / or type II bovine collagen, and / or type III bovine collagen.

[0011] Preferably, the ultraviolet absorber is one or several of tartrazine and brilliant blue.

[0012] The second objective of the present invention is to provide a preparation method of the bioink, including the following steps:

[0013] (1) Dissolve the freeze-dried collagen sponge in an acetic acid solution to prepare a collagen solution, and stir slowly to dissolve.

[0014] (2) Adjust the pH of the collagen solution obtained in step (1) to 7 - 9, dropwise add allyl succinic anhydride, and maintain the solution pH at 7 - 8; react in an ice bath, dialyze, and freeze-dry to obtain allyl succinylated collagen.

[0015] (3) Dissolve the allyl succinylated collagen obtained in step (2) directly in water, sodium chloride solution, PBS or cell culture medium, and slowly stir until completely dissolved;

[0016] (4) Take 0.5% - 2.0% m / v of the allyl succinylated collagen solution obtained in step (3), add 0.25% - 0.5% m / v of lithium phenyl-2,4,6-trimethylbenzoyl phosphate and 0 - 0.05% m / v of ultraviolet absorber, mix evenly, and centrifuge to remove air bubbles to obtain a novel photosensitive collagen bioink.

[0017] The third object of the present invention is to provide the application of the bioink in the preparation of one or more of tissue engineering materials, hemostatic materials, wound dressings, and drug delivery materials.

[0018] Preferably, the tissue engineering materials include one or more of artificial skin, artificial blood vessels, cartilage and bone repair materials, and corneal transplantation materials.

[0019] The beneficial effects of the present invention are: (1) The present invention provides an allyl succinylated collagen (CAA) bioink, which can be completely dissolved at a high concentration in a solution with physiological pH, and forms a stable and uniform solution, and can be used for DLP and extrusion 3D printing; (2) The bioink of the present invention can prepare hydrogels with good mechanical properties, fidelity and anti-degradation ability through 3D printing; (3) The bioink 3D printed hydrogel of the present invention can promote cell proliferation, adhesion and migration, and has good biocompatibility and bioactivity; the novel collagen bioink is widely used in the tissue engineering fields of skin repair materials and osteochondral repair materials. Description of the Drawings

[0020] Figure 1 Preparation and characterization of allyl succinylated collagen (CAA).

[0021] Note: (A) Nuclear magnetic resonance spectroscopy (H-NMR) diagram; (B) Fourier transform infrared spectroscopy (FT-IR) diagram; (C) SDS-PAGE gel electrophoresis diagram; (D) Circular dichroism (CD) diagram; (E) Thermal transition curve; (F) First derivative of the thermal transition curve (d[θ] / dT).

[0022] Figure 2 Characterization of CAA bioink.

[0023] Note: (A) Photocuring performance; (B) Zeta potential; (C) Solubility; (D) Injectability; (E) Transmittance; (F) DLS. Figure 3 Characterization of the properties of collagen hydrogel.

[0024] Note: (A) Contact angle; (B) Stress-strain curve; (C) Toughness of hydrogel; (D) Swelling property of hydrogel; (E) Degradation resistance of hydrogel; (F) SEM image of hydrogel.

[0025] Figure 4 3D bioprinting of CAA bioink.

[0026] Note: (A - B): DLP 3D printing of CAA bioink (A) Rotating channel model; (B) Ear model; (C - E): DIW 3D printing of CAA bioink (D) Grid model; (E) Meniscus model.

[0027] Figure 5 Good biocompatibility and bioactivity of CAA bioink.

[0028] Note: (A) Cellular immunofluorescence staining; (B) Cell proliferation; (C) Live / dead cell staining images of BMSCs incubated on CAA scaffolds for 1, 3, and 7 days; (D) 3D image of cell 3D imaging of the distribution of BMSCs on CAA scaffolds;

[0029] Figure 6 CAA bioink promotes cell migration and differentiation.

[0030] (A) Migration map of BMSCs on CAA material; (B) Cell migration rate; (C) Sox9 gene expression map of BMSCs promoted by CAA scaffold; (D) Col2 a1 gene expression map of BMSCs promoted by CAA scaffold.

[0031] Figure 7 Cell-laden printing of CAA bioink.

[0032] Note: (A) Pentagram printed with CAA bioink loaded with BMSCs; (B) Spade printed with CAA bioink loaded with BMSCs; (C) Grid printed with CAA bioink loaded with BMSCs. Specific embodiments

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the specification. Obviously, the described embodiments are only a part of the present invention, rather than all of the invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0034] It should be noted that in the following embodiments, unless otherwise specified, the methods used are all conventional methods; the reagents used are all conventional reagents and can be purchased from the market.

[0035] In the following examples, the collagen used was extracted in our laboratory. For the extraction method, refer to the invention patent CN113520900B. However, the collagen described in the present invention is not limited to the above-mentioned collagen.

[0036] Example 1 Preparation and Characterization of Collagen Allyl Succinylated (CAA)

[0037] A 0.3% m / v collagen solution was prepared in 0.5M acetic acid solution. After complete dissolution, sodium hydroxide solution was added to adjust the pH to 7 - 9. Allyl succinic anhydride (AA), which is ten times the equivalent of collagen lysine, was added dropwise while maintaining the solution pH at 7 - 8. After reacting in an ice bath for 24 h, dialysis was carried out in ultrapure water and then freeze-dried to prepare collagen allyl succinylated (CAA).

[0038] CAA and Col were respectively dissolved in deuterated DMSO for 1H-NMR spectroscopic analysis ( Figure 1 A). 1 The 1H-NMR spectrum showed that compared with Col, CAA exhibited characteristic resonance peaks of methylene and methyl at δ = 5.5 and 5.8 ppm, δ = 1.85 ppm. In addition, a carboxyl signal was present at δ = 2.5 ppm in the 1H-NMR spectrum of CAA, indicating an increase in the number of carboxyl groups in collagen. The results showed that allyl succinic anhydride was successfully modified onto collagen to form CAA.

[0039] COL and CAA after freeze-drying were co-ground with potassium bromide, and a thin slice was prepared by the tablet pressing method for infrared spectrum determination ( Figure 1 B). In the FT-IR spectrum, the peak of amide A band shifted from 2924 cm-1 to 2979 cm-1 red shift. The FTIR spectra of both collagen allyl succinylated (CAA) and collagen (Col) showed characteristic peaks at 1654, 1547, and 1240 cm -1 corresponding to amide I, II, and III respectively. A unique band at 1055 cm -1 in the CAA molecule was attributed to the in-plane bending vibration of C = CH, confirming the incorporation of double bonds. The results showed the successful modification of allyl succinic anhydride on collagen.

[0040] The purity of CAA was verified by SDS-PAGE gel electrophoresis ( Figure 1 C). The SDS-PAGE results showed that for Col, α1 and α2 bands appeared between ~130 kDa, and a β-dimer band appeared at 180 kDa; while in the SDS-PAGE image of CAA, the α1 and α2 bands were between 130 - 180 kDa, and the β-dimer band was significantly higher than 180 kDa. This indicated that the molecular weight of collagen increased after AA modification, proving the successful modification of AA on the collagen molecule.

[0041] The thermal stability of allyl succinylated collagen and collagen was characterized by circular dichroism. The CD spectra of CAA and Col showed a positive peak at 220 nm and a negative peak at 198 nm, indicating the presence of the typical triple-helix structure of collagen( Figure 1 D). The thermal denaturation curves showed that both Col and CAA maintained a stable triple-helix structure, indicating that the successful modification of AA did not affect the triple-helix structure of collagen( Figure 1 E). At the same time, the first derivative curves were obtained by measuring and differentiating COL and CAA( Figure 1 F), and the results showed that the thermal denaturation temperature of CAA decreased, but it still had a relatively high thermal denaturation temperature. These results indicate that CAA has a typical triple-helix structure and high thermal stability.

[0042] The photocuring performance of CAA bioink was investigated by the vial inversion experiment( Figure 2 A). A 10 mg / mL CAA solution was added to a vial, and after standing, it was placed at an angle of 45°. The results showed that the CAA bioink exhibited good fluidity. Then, the CAA ink was irradiated with 405 nm blue light for 30 s, and the vial was inverted and placed at an angle of 45°. The CAA ink in the vial was in a solid state, indicating that the CAA bioink formed a hydrogel with good support performance under blue light irradiation.

[0043] The charge properties of CAA bioink were investigated by Zeta potential measurement( Figure 2 B). CAA and Col solutions with a concentration of 0.1 mg / mL and different pH values were prepared. The results of the Zeta potential curves showed that the zero potential point of CAA appeared between pH 4 - 4.5, and the isoelectric point of Col was between pH 5.5 - 6. This may be due to the introduction of acidic groups (carboxyl groups) in AA molecules onto collagen molecules, increasing the overall acidity of collagen and thus lowering its isoelectric point. A lower isoelectric point means that CAA has better water solubility and stability in neutral and alkaline environments. In addition, CAA is negatively charged at physiological pH (about 7.4), which may help it interact with positively charged biomolecules (such as proteins, cell membranes) and improve its performance in biomedical applications.

[0044] The effect of allyl succinic anhydride-modified collagen on its solubility at physiological pH was studied by solubility tests Figure 2C). 50 mg of freeze-dried collagen sponge and CAA sponge were respectively dissolved in solutions with pH values of 6, 7, and 8, stirred overnight, centrifuged at 4 °C and 10,000 rpm for 30 min, the supernatant was taken, freeze-dried and reweighed. The results showed that when the pH was 6, the solubilities of Col and CAA were 4.14 mg / mL and 24.7 mg / mL respectively; when the pH was 7, the solubilities of Col and CAA were 4.61 mg / mL and 27.4 mg / mL respectively. When the pH was 8, the solubilities of Col and CAA were 4.80 mg / mL and 28 mg / mL respectively. The results indicated that in solutions with different pH values, the solubility of CAA was significantly higher than that of collagen. This was because the modification of AA introduced new carboxyl groups to collagen, greatly increasing the hydrophilicity of collagen, resulting in excellent water solubility of CAA, which could be directly dissolved in a neutral environment and had a solubility as high as 28 mg / mL. In contrast, due to the easy self-assembly of collagen in a neutral environment, its solubility was less than 5 mg / mL. This showed that AA modification had great advantages in improving the solubility of collagen.

[0045] To study the uniformity of CAA and CMA inks, we conducted extrusion force tests on them. CAA ink and CMA ink were respectively loaded into 1 mL sterile syringes, and the syringes were fixed on the specimen platform of the extrusion force tester and extruded at a speed of 30 mm / min. The results were as Figure 2 shown in Fig. D. When the CAA and CMA bioinks were 0.5%, the extrusion force required for CAA was lower than that for CMA, and the force was evenly applied within 40 s, while CMA required an uneven and significantly variable extrusion force. For 1.0% CMA, a stronger extrusion force was required and needle bursting occurred, while for CAA at high concentrations of 1.0% and 1.5%, the required extrusion force was still very small and the extrusion force was uniform with no significant change. These results indicated that due to its significantly enhanced water solubility, CAA had good uniformity and excellent injectability under neutral conditions and was suitable for different 3D printing technologies.

[0046] Furthermore, the uniformity of CAA and CMA inks was studied by transmittance tests, and the results were as Figure 2 shown in Fig. E. In the wavelength range of 400 - 800 nm, at 0.5% and 1.0% conditions, the transmittance of CAA was significantly higher than that of CMA. In addition, at 600 nm, the transmittance of 1.5% CAA was about 95%, indicating that the particle distribution in the CAA solution was more uniform and there were no obvious microparticles or aggregates. These results showed that CAA had better solubility and uniformity under neutral conditions.

[0047] In addition, we further studied the hydrated particle size (R h ) of CMA and CAA bioinks by DLS. The results showed that the R of CMA bioinkh was approximately 4005 nm, while the R of the CAA bioink h was approximately 698 nm, indicating that the particles formed by CAA in neutral solution were smaller. These results consistently showed that the CAA bioink had good stability and uniformity in neutral solution.

[0048] Preparation and Characterization of CAA Hydrogel in Example 2

[0049] LAP, UV, and 10 mg / mL of CAA ink were put into a mold for curing. The Col hydrogel was prepared using glutaraldehyde as a crosslinking agent. The contact angles of CAA and Col hydrogels with water were measured using a droplet shape analyzer. The hydrogel was placed on the test bench and 5 μL of deionized water was added dropwise. The contact angle values of CAA and Col hydrogels were obtained by measurement. The results showed that the contact angle of the CAA hydrogel with deionized water was 23°, while the contact angle of the Col hydrogel with deionized water was 73.9° ( Figure 3 A). These results further indicated that AA modification significantly improved the hydrophilicity of collagen, and the CAA hydrogel was more hydrophilic. The microstructure of the CAA hydrogel was investigated using a scanning electron microscope (SEM) ( Figure 3 F). The SEM results showed that the CAA hydrogel had a uniform network structure.

[0050] The compressive capacity of the scaffold materials was evaluated by uniaxial compression testing. The stress-strain curves showed that the ultimate fracture strength (UFS) of CMA-5 was 17.15 kPa at a strain of 36.07%; the UFS of CAA-5 was 22.54 kPa at a strain of 43.87%. The UFS of CAA-10 was 81.51 kPa at a strain of 54.84%; the UFS of CAA-15 was 147.41 kPa at a strain of 56.33% ( Figure 3 B). The fracture energies of CMA, CAA-5, CAA-10, and CAA-15 were 100.78 kJ / m 3 , 153.25 kJ / m 3 , 418.86 kJ / m 3 and 800.79 kJ / m 3 ( Figure 3 C). These results showed that the CAA hydrogel had good mechanical properties.

[0051] Through the swelling ratio (SR) test, we evaluated the stability of CAA-5, CAA-10, and CAA-15 scaffolds in deionized water, PBS, and DMEM solutions ( Figure 3D). In deionized water, the SR values of CAA-5, CAA-10, and CAA-15 scaffolds were 123.94%, 113.43%, and 110.27% respectively. As the CAA concentration gradually increased, the SR gradually decreased. The SR values of CAA-5, CAA-10, and CAA-15 scaffolds in PBS solution were 78.62%, 83.67%, and 88.97% respectively. The SR values of CAA-5, CAA-10, and CAA-15 scaffolds in DMEM solution were 78.53%, 84.9%, and 88.32% respectively. As the CAA concentration gradually increased, the SR gradually increased. The results showed that CAA had different swelling properties in different solutions.

[0052] Immerse the CAA-5, CAA-10, and CAA-15 scaffolds in a collagenase solution containing 5 U / mL to investigate the ability of the scaffold materials to resist collagenase degradation ( Figure 3 E). After 12 h, the degradation rates of CAA-5, CAA-10, and CAA-15 were 62.75%, 40.58%, and 29.82% respectively. After 24 h, the degradation rates of CAA-5, CAA-10, and CAA-15 were 91.18%, 73.91%, and 66.67% respectively. After 48 h, the degradation rates of CAA-5, CAA-10, and CAA-15 were 97.06%, 95.17%, and 92.54% respectively. The results showed that the high-concentration CAA hydrogel exhibited excellent resistance to collagenase-mediated degradation.

[0053] Example 3. Preparation and Screening of CAA Bioink

[0054] Add the freeze-dried CAA sponge into water, PBS, or cell culture medium, dissolve it at low temperature, add a blocking reagent to stabilize the pH, and then add components such as a photoinitiator, mix evenly, and centrifuge to remove air bubbles to prepare CAA bioink. The inventor made various bioink formulations, and Table 1 lists 8 bioink formulations.

[0055] Table 1 Formulations of Different Bioinks

[0056]

[0057]

[0058] CAA undergoes crosslinking under the action of a photoinitiator and ultraviolet light to form a gel. Bioinks 1-3 were used to screen the photoinitiator. The experiment showed that Bioink 1 containing LAP was cured under 365 nm light irradiation for 20 s, while Bioinks 2 and 3 required several minutes and had a low gel strength and could not be used for 3D printing. LAP was selected as the photoinitiator.

[0059] Bioinks 4 - 8 contain different concentrations of CAA and are suitable for different printing requirements. Low - concentration bioinks 4 - 6 have good fluidity and are suitable for DLP 3D printing, while high - concentration bioink 8 is suitable for extrusion printing. The concentrations of LAP and ultraviolet absorber can be adjusted according to requirements to optimize printing time, biocompatibility, and precision. Multiple bioink formulations can be prepared according to needs.

[0060] Example 4: 3D Bioprinting of CAA Bioink

[0061] CAA bioink exhibits excellent solubility, fluidity, and uniformity under neutral conditions and has a relatively high gelation modulus, making it very suitable for DLP 3D printing. We selected bioink 6 for DLP 3D printing ( Figure 4 A). The 3D - printed rotating channel model accurately reproduced the details of the CAD model, and the internal pipes were clearly visible. In addition, the printed ear model conformed to the CAD model, accurately reproducing the details of the ear and having good structural integrity and support ( Figure 4 B). The results show that CAA bioink is suitable for DLP 3D printing, and the printed models can reproduce the details of the CAD model with high fidelity.

[0062] Furthermore, 25 mg / mL CAA bioink was used for DIW printing of grid and meniscus models ( Figure 4 C and D). The printing results showed that these models could accurately reproduce the design details and had high shape fidelity. The Pr values of two holes in the calculated grid were 1.06 and 0.93 respectively, indicating that CAA bioink has high precision in DIW 3D printing. In summary, CAA bioink is suitable for DLP and DIW 3D printing and exhibits excellent precision and versatility.

[0063] Example 5: Biocompatibility and Bioactivity of CAA Bioink

[0064] Using fluorescence confocal microscopy, we observed the adhesion and spreading characteristics of BMSCs on Col, CMA, and CAA hydrogel substrates ( Figure 5 A). Phalloidin - tetramethylrhodamine isothiocyanate and 33258 dye were used to stain the actin stress fibers and nuclei of cells respectively. The fluorescence images showed that BMSCs on Col, CMA, and CAA hydrogel substrates were all in the shape of slender spindles. The results of cell adhesion and spreading indicated that the CAA hydrogel significantly promoted cell adhesion.

[0065] The growth of BMSCs in the CAA hydrogel was measured by CCK - 8 to evaluate the biocompatibility of the collagen ink. Figure 5B). The results showed that CAA could promote the proliferation of BMSCs. The fluorescence images of live / dead cell staining showed that with the prolongation of the incubation time, the number of BMSCs encapsulated in the CAA hydrogel gradually increased, indicating that the CAA hydrogel had a significant ability to promote cell proliferation. Figure 5 C). The 3D imaging of live / dead cell staining of BMSCs incubated on the CAA scaffold for 5 days showed that BMSCs were evenly distributed in the hydrogel and the cell growth state was good. Figure 5 D). These results indicated that the CAA hydrogel had excellent biological activity and significantly promoted cell proliferation.

[0066] The ability of the collagen bioink to promote cell migration was evaluated by the cell scratch assay. The microscope images showed that after 0 h of scratching, obvious scratched areas were visible in the blank group, CMA group, and CAA group. After 24 h of cell culture, only a small number of cells migrated in the blank group, while in the CMA and CAA hydrogel groups, a large number of cells migrated towards the scratched area. Among them, in the CAA hydrogel group, the closure effect of the scratched area was the best. Figure 6 A). The cell migration rate of the CAA group was 67.95%, which was significantly higher than that of the blank group (29.50%) and the CMA group (53.30%) respectively. Figure 6 B). These results indicated that the CAA hydrogel could significantly promote cell migration.

[0067] The ability of the collagen hydrogel to promote the differentiation of BMSCs was investigated by detecting the expression of collagen type II alpha 1 chain (Col2 a1) and SRY-box transcription factor 9 (Sox9). Figure 6 C, D). At 12 days of incubation, the expression level of Sox9 was upregulated by 449.51-fold and 52.15-fold in the CAA group and Col group respectively; the expression of the Col2a1 gene was upregulated by 82.70-fold and 13.33-fold in the CAA group and Col group respectively. These results indicated that the CAA hydrogel had excellent biological activity and significantly promoted the differentiation of BMSCs.

[0068] In addition, we further investigated the biological activity of the CAA hydrogel by cell-laden DLP 3D printing. The CAA and BMSCs were mixed to prepare a bioink, and DLP bioprinted pentagram, spade, and grid patterns, and the results were as Figure 7 shown. After 24 h of culture, the fluorescence images of live / dead cell staining showed that BMSCs were evenly distributed in the CAA hydrogel. These results indicated that the CAA bioink-loaded cell DLP bioprinting material had high cell viability and printing accuracy and had broad application prospects in the field of tissue repair.

[0069] In summary, the present invention provides an allyl succinylated collagen (CAA) bioink. The bioink can be completely dissolved in a solution with physiological pH, and forms a stable and homogeneous solution, and can be used for DLP and extrusion 3D printing; SEM results show that the CAA hydrogel has a uniform network structure; the bioink of the present invention can prepare hydrogels with good mechanical properties, fidelity and anti-degradation ability through 3D printing; the 3D printed hydrogel of the bioink of the present invention can promote cell proliferation, adhesion, differentiation and migration, and has good biocompatibility and bioactivity; the novel collagen bioink has wide applications in the fields of tissue engineering such as skin repair materials and osteochondral repair materials.

Claims

1. A novel photosensitive collagen bio-ink for 3D printing, characterized in that: The biological ink comprises the following components: 0.5% to 2.0% m / v allyl succinylated collagen, 0.25% to 0.5% m / v phenyl-2,4,6-trimethylbenzoyl lithium phosphate and 0 to 0.05% m / v ultraviolet absorber.

2. The bio-ink according to claim 1, characterized in that: The allyl succinylated collagen is prepared by the following method: dissolving collagen in a 0.1-0.5M acetic acid solution and adjusting the pH to 3-9; adding allyl succinic anhydride dropwise, reacting at low temperature for 12-72 hours, and maintaining the solution pH at 7-9; dialyzing, and freeze-drying to obtain allyl succinylated collagen containing a photosensitive group.

3. The bio-ink according to claim 1, characterized in that: The collagen is bovine collagen.

4. The bio-ink according to claim 3, characterized in that: The bovine collagen is type I bovine collagen, and / or type II bovine collagen, and / or type II bovine collagen.

5. The bio-ink according to claim 1, characterized in that: The ultraviolet absorber is one or more of lemon yellow and brilliant blue.

6. The method for preparing the biological ink according to any one of claims 1 to 5, characterized in that: The steps include: (1) dissolving freeze-dried collagen sponge in acetic acid solution to prepare collagen solution, and slowly stirring to dissolve; (2) adjusting the pH of the collagen solution obtained in step (1) to 7-9, adding allyl succinic anhydride dropwise, and maintaining the pH of the solution at 7-8; reacting in an ice bath, dialyzing, and freeze-drying to obtain allyl succinylated collagen; (3) directly dissolving the allyl succinylated collagen obtained in step (2) in water, sodium chloride solution, PBS or cell culture medium, and slowly stirring until completely dissolved; (4) Take 0.5% to 2.0% m / v of the allyl succinylated collagen solution obtained in step (3), add 0.25% to 0.5% m / v of phenyl-2,4,6-trimethylbenzoyl lithium phosphate and 0 to 0.05% m / v of an ultraviolet absorber, mix well, and centrifuge to remove bubbles to obtain a novel photosensitive collagen bio-ink.

7. Use of the bio-ink as described in any one of claims 1 to 5 in the preparation of one or more of tissue engineering materials, hemostatic materials, wound dressings, and drug delivery materials.

8. The use according to claim 7, characterized in that The tissue engineering material includes one or more of artificial skin, artificial blood vessels, cartilage and bone repair materials and corneal transplantation materials.

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