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

Abstract

The invention relates to bionic bio-ink as well as a preparation method and application thereof, and relates to the technical field of 3D bio-printing. The preparation method of the bionic bio-ink comprises the following steps: sequentially adding methylacryloylated recombinant human collagen, o-nitrobenzyl modified methylacryloylated hyaluronic acid, elastin-like polypeptide and phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonate into a solvent, stirring and uniformly mixing to obtain an ACEM hydrogel precursor solution, and carrying out ultrasonic treatment on the ACEM hydrogel precursor solution to obtain the bionic bio-ink. Uniformly mixing the ACEM hydrogel precursor solution with a photoresist and seed cells to obtain the bionic biological ink. The sequence of the elastin-like polypeptide is as shown in SEQ ID NO. 1. The biological ink is better in mechanical strength, retains the advantages and functions of natural protein, can be quickly photo-cured into a high-strength and dual-network hydrogel structure, and has excellent printability, biocompatibility and cartilage tissue regeneration promoting capacity.

Description

Technical Field

[0001] The present invention relates to the field of 3D bioprinting technology, and particularly relates to a bionic bioink, a preparation method thereof and an application thereof. Background Art

[0002] The auricle is one of the most complex body surface organs, and its repair and reconstruction are extremely challenging. At the same time, the number of patients with congenital microtia is large. In clinical practice, autologous costal cartilage is often collected for auricle reconstruction to restore the appearance of the auricle, but the elasticity of the auricle cannot be reproduced, and at the same time, risks such as subsequent trauma, pain, and thoracic deformity will be faced. Tissue engineering combined with 3D bioprinting technology can accurately repair and regenerate tissues and organs, which will bring good news to countless patients with microtia.

[0003] Ideally, the bioink used to construct tissue-engineered auricles should be rich in elastin, type II collagen, and proteoglycans like natural extracellular matrix. The application of decellularized matrix brings a glimmer of hope for bionic natural auricle matrix. However, the decellularization process may cause differences between batches and damage the structure, mechanical properties, and biological activity of the matrix. In addition, immunogenic substances such as DNA or animal-derived viruses may still remain after decellularization, posing potential risks of rejection or pathogenicity.

[0004] The current progress of genetic engineering and DNA recombination technology can assemble specific protein domains intercepted into efficient protein materials, thereby obtaining recombinant proteins derived from natural auricle matrix. Their complexity and functionality are no less than natural proteins and the components are completely controllable, combining the best characteristics of biological materials and artificial materials. Recombinant polypeptides and proteins have the advantages of good biocompatibility, low immunogenicity, adjustable biochemical and mechanical properties, high batch consistency, easy purification, and large-scale production, which have attracted wide attention in the field of 3D bioprinting and are also one of the excellent choices for bionic auricular cartilage matrix components.

[0005] Therefore, there is an urgent need to develop a bioink with the following characteristics in the field of auricular cartilage tissue regeneration: (1) no risk of heterologous viruses, high biocompatibility, low immunogenicity, and mild in vivo inflammatory response to ensure the safety of the material; (2) capable of bionically simulating the specific microenvironment of auricular cartilage to promote the function of chondrocytes and the deposition of extracellular matrix; (3) having rapid photocuring and excellent rheological properties to meet the high-efficiency forming requirements of three-dimensional bioprinting; (4) good mechanical properties to accurately maintain the three-dimensional shape of the auricle; (5) moderate degradation rate to match the regeneration rate of cartilage tissue. Summary of the Invention

[0006] The object of the present invention is to provide a bionic bioink and its preparation method and application. The preparation method of the present invention is simple, and the obtained bionic bioink has good mechanical strength, retains the advantages and functions of natural proteins, can be rapidly photocured into a high-strength, double-network hydrogel structure, and has excellent printability, biocompatibility and the ability to promote cartilage tissue regeneration.

[0007] The technical solution of the present invention to solve the above technical problems is as follows:

[0008] The first object of the present invention is to provide a preparation method of a bionic bioink, comprising the following steps:

[0009] Methacrylated recombinant human collagen, o-nitrobenzyl-modified methacrylated hyaluronic acid, elastin-like polypeptide, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate are sequentially added to a solvent, stirred and mixed evenly to obtain an ACEM hydrogel precursor solution. The ACEM hydrogel precursor solution is mixed with a photoresist and seed cells to obtain the bionic bioink;

[0010] The sequence of the elastin-like polypeptide is shown in SEQ ID NO.1.

[0011] VPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVGVPGVGVPGVGVPGKGVPGVGVPGVG(SEQ ID NO:1).

[0012] Preferably, in the bionic bioink, the final concentration of the photoresist is 0.05% m / v to 0.1% m / v; the final concentration of the seed cells is 1×10

[0013] ,

[0012] , , 6 , , 8 , ,

[0015] , ,

[0014] / mL to 1×10 8 / mL.

[0013] Furthermore, the solvent includes but is not limited to PBS and FBS.

[0014] The beneficial effects of the present invention are: (1) The bionic bioink of the present invention can effectively restore the main components of the auricular cartilage matrix, and is significantly superior to the decellularized cartilage matrix in terms of the preservation of the natural protein structure, the carrying of xenogenic viruses or immunogens, and the stability between batches.

[0015] (2) Natural elastin and tropoelastin are difficult to be used as biomaterials because they are not easily separated and obtained and are insoluble in water. Therefore, elastin from animal sources has been widely used as a substitute. However, the isolation of elastin from animals often requires feeding animals a copper-deficient diet, which is not only inefficient and raises ethical issues, but also carries the risk of carrying xenoviruses and triggering immune rejection. The elastin-like polypeptide (ELP) recombinantly synthesized based on genetic engineering technology in this application not only avoids the above problems, but also inherits the biocompatibility of natural elastin due to sequence conservation. At the same time, it also has good water solubility, biodegradability and low toxicity, and is very suitable for mimicking the matrix components of elastic tissues.

[0016] (3) The ability of a single-component bioink to promote cartilage regeneration is limited, while the multi-component bioink synthesized in the present invention can better promote cartilage regeneration in vivo and in vitro.

[0017] (4) Generally, the material can be dissolved in PBS. After adding cells, FBS (fetal bovine serum) can be used to provide nutrition for the cells.

[0018] (5) The preparation method of the present invention overcomes the disadvantages of poor printability, too high or too low viscosity of the bioink synthesized by the prior art. Adding too much raw material will cause the solute to be insoluble, or the final solution to be too viscous, and too little will cause the solution to be too thin, resulting in a poor final shape after 3D printing. The synthesized HAMA-NB itself is a yellow material, and the solution after proper dissolution according to the ratio of the present invention is also of a suitable yellow color. Therefore, during the printing process, part of the light scattering problem of the ultraviolet light emitted by the DLP printer for photocuring is solved. Subsequently, the step of adding a water-soluble photoresist can cooperate to overcome the shortcoming of light path scattering, ensure the accuracy of the printed scaffold, physically increase the strength of the printed scaffold, and ensure the successful construction of the final large-sized tissue engineering auricle.

[0019] On the basis of the above technical solutions, the present invention can be further improved as follows.

[0020] Further, the mass ratio of the methacrylated recombinant human collagen, the o-nitrobenzyl-modified methacrylated hyaluronic acid, the elastin-like polypeptide, and the lithium phenyl-2,4,6-trimethylbenzoylphosphinate is 500-1000:50-100:50-100:25-50; the dosage ratio of the methacrylated recombinant human collagen to the solvent is 500-1000 mg:50-100 mL.

[0021] The beneficial effect of adopting the above further scheme is that the recombinant human collagen modified with methacrylic anhydride has fast photocuring performance and excellent printability.

[0022] Further, the seed cells include at least one of auricular chondrocytes, articular chondrocytes, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, embryonic stem cells, and induced pluripotent stem cells.

[0023] The beneficial effect of adopting the above further solution is that tissue engineering biological scaffolds of various tissue types can be constructed. For example, when a variety of seed cells are mixed and applied, diversified comprehensive bionic tissue engineering constructs can be constructed.

[0024] Further, the preparation method of the methacrylated recombinant human collagen includes the following steps:

[0025] Weigh the recombinant humanized collagen lyophilized powder and dissolve it in PBS to obtain a recombinant humanized collagen solution; add the methacrylic anhydride solution to the recombinant humanized collagen solution, centrifuge after the reaction to obtain the supernatant; take the supernatant and dilute it with water, then adjust the pH, dialyze, and lyophilize in sequence to obtain methacrylated recombinant human collagen.

[0026] Further, the temperature of the reaction is room temperature, and the time is 0.5 h to 2 h.

[0027] The beneficial effect of adopting the above further solution is that collagen plays a key role in promoting cell growth, adhesion, and arrangement during tissue regeneration. However, bio-printing based on collagen often has deficiencies such as poor resolution, low printing fidelity, and weak mechanical strength. The methacrylated recombinant humanized collagen prepared in the present invention can avoid the above problems and has good biocompatibility, excellent printability, and sufficient mechanical strength.

[0028] Further, the preparation method of o-nitrobenzyl-modified methacrylated hyaluronic acid includes the following steps:

[0029] (1) Take hyaluronic acid and PBS and add them to water in sequence to dissolve, then add the methacrylic anhydride solution to react, then adjust the pH to weakly alkaline, react again at low temperature, then dialyze and lyophilize in sequence to obtain methacrylated hyaluronic acid;

[0030] (2) Dissolve the methacrylated hyaluronic acid in water to obtain a methacrylated hyaluronic acid solution; dissolve 1-hydroxybenzotriazole, (2-aminoethyl)-4-[4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy]-butyramide, and (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) in dimethyl sulfoxide to obtain a mixed solution; mix the mixed solution with the methacrylated hyaluronic acid solution, then react in the dark, perform dialysis after the reaction ends to obtain a dialysate, adjust the pH of the dialysate to 3-4, and add sodium chloride to the dialysate, and lyophilize after dialysis ends to obtain o-nitrobenzyl-modified methacrylated hyaluronic acid.

[0031] Further, the reaction at low temperature is specifically: reacting at 3°C to 5°C for 22h to 26h.

[0032] Further, the dosage ratio of the recombinant humanized collagen solution to the methacrylic anhydride solution is 1g to 10g: 0.5mL to 5mL; wherein the concentration of the recombinant humanized collagen solution is 0.01g / mL to 0.05g / mL; the volume concentration of the methacrylic anhydride solution is 0.05% to 0.01%.

[0033] Further, the dosage ratio of the hyaluronic acid, PBS, and methacrylic anhydride solution in step (1) is 0.1g to 2g: 100mL to 200mL: 0.4mL to 8mL.

[0034] In step (2), the mass ratio of 1-hydroxybenzotriazole, (2-aminoethyl)-4-[4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy]-butyramide, and (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) is 1 to 2: 0.1 to 2: 1 to 2; the total concentration of the mixed solution is 30g / L to 40g / L; the dosage ratio of the dialysate to the sodium chloride is 4L to 5L: 30g to 200g.

[0035] The second object of the present invention is to provide a bionic bioink.

[0036] The beneficial effects of the present invention are: The present invention successfully prepares a bioink synthesized from recombinant proteins and polypeptide derivatives with better mechanical strength, retaining the advantages and functions of natural proteins, and obtained based on genetic engineering technology. After being modified with methacrylic acid groups and o-nitrobenzyl groups, it can be rapidly photocured into a high-strength, double-network hydrogel structure, and has excellent printability, biocompatibility, and the ability to promote cartilage tissue regeneration.

[0037] The third object of the present invention is to provide an application of the bionic bioink, and use the bionic bioink to construct a human auricle tissue substitute.

[0038] The beneficial effects of the present invention are as follows: By using the bioink of the present invention, adding seed cells and combining with 3D bioprinting technology, an engineered auricle with precise morphology and excellent biological properties is constructed, meeting the actual needs of ear reconstruction.

[0039] Furthermore, the construction of the human auricle tissue substitute includes the following steps:

[0040] Mix the seed cells with the above-mentioned bionic bioink to obtain a cell printing solution; apply laser scanning and construct a three-dimensional digital model of the human ear morphology through computer-aided design; based on the three-dimensional digital model of the human ear morphology, use a 3D bioprinter to evenly extrude the bionic bioink and obtain a human auricle tissue substitute by photocuring.

[0041] Preferably, the construction of the three-dimensional digital model of the human auricle can use one or more of CT, laser scanning, MRI, etc.

[0042] Preferably, the three-dimensional model cutting software can use one or more of Mimics Medical, CAD, Creo, PotatoOSP, Zbrush.

[0043] Preferably, the 3D bioprinter is one or more of a projection stereolithography printer, a technique-based printing combined with suspension printing, and a dynamic interface printing.

[0044] The beneficial effects of adopting the above further scheme are as follows: By using the bionic bioink of the present invention to prepare a human auricle tissue substitute, it has good mechanical strength, retains the advantages and functions of natural proteins, can be quickly photocured into a high-strength, double-network hydrogel structure, and has excellent printability, biocompatibility and the ability to promote cartilage tissue regeneration. Description of the Drawings

[0045] Figure 1 Appearance of the materials in Example 1; among them, (A) is RHCMA; (B) is HAMA; (C) is HAM A-NB; (D) is ELP;

[0046] Figure 2 Modification of the materials and principle of photocrosslinking reaction of ACEM hydrogel; among them, (A) is the 1 1H NMR liquid nuclear magnetic resonance hydrogen spectrum of the ACEM hydrogel precursor solution in Example 5, Comparative Example 1, and Comparative Example 2; (B) is the principle of photocrosslinking preparation of ACEM hydrogel;

[0047] Figure 3Construction of the ACEM hydrogel in Example 4; among which (A) shows the precursor solution of the ACEM hydrogel in Comparative Example 1, the appearance of the ACEM hydrogel, and the corresponding SEM detection results; (B) shows the precursor solution of the ACEM hydrogel in Comparative Example 2, the appearance of the ACEM hydrogel, and the corresponding SEM detection results; (C) shows the precursor solution of the ACEM hydrogel in Example 5, the appearance of the ACE M hydrogel, and the corresponding SEM detection results; (D) shows the pore sizes of the ACEM hydrogels in Example 5, Comparative Example 1, and Comparative Example 2; (E) shows the porosities of the ACEM hydrogels in Example 5, Comparative Example 1, and Comparative Example 2; (F) shows the swelling ratios of the ACEM hydrogels in Example 5, Comparative Example 1, and Comparative Example 2. (*p<0.05, **p<0.01, ***p<0.001);

[0048] Figure 4 Physical and chemical properties of the ACEM hydrogels in Example 5, Comparative Example 1, and Comparative Example 2; among which (A) shows the change curves of the ultraviolet curing storage modulus (G') and loss modulus (G") with time; (B) shows the change curve of the ultraviolet curing loss factor tan(δ) with time; (C) shows the compressive Young's modulus; (D) shows the compressive stress-strain curve;

[0049] Figure 5 Detection pictures of the printability of the ACEM bioink 3D printing constructs; among which (A) shows the side view, front view, material tank, and printing platform of a DLP printer (BP8601 Pro, EFL) (from left to right); (B) shows the general appearance of the ACEM hydrogels in Example 5, Comparative Example 1, and Comparative Example 2; (C) shows the optimal printing light intensity and exposure time of the ACEM hydrogels in Example 5, Comparative Example 1, and Comparative Example 2; (D) shows the pore size morphology display of the incompletely crosslinked, optimally crosslinked, and overcrosslinked hydrogels with a model pore size parameter of 700μm;

[0050] Figure 6 Gross appearance of the ACEM bioink 3D printing constructs immediately after printing and 6 weeks after implantation in nude mice.

[0051] Figure 7 In vivo cartilage regeneration of the ACEM bioink 3D printing constructs. (A) shows the low-magnification H&E staining whole field, high-magnification H&E, SO / FG, Masson, EVG, and CoL II staining results of the regenerated cartilage tissue in the R-Cross group, (B) shows those in the RH-Cross group, and (C) shows those in the RHE-Cross group 6 weeks after in vivo implantation;

[0052] Figure 8For the wet weight and biochemical quantitative analysis of the regenerated tissue. Among them, (A) is the wet weight of the regenerated cartilage tissue before implantation and the wet weight after 6 weeks of implantation; (B) is the DNA content of the regenerated cartilage tissue after 6 weeks of implantation, (C) is the GAG content of the regenerated cartilage tissue after 6 weeks of implantation, (D) is the total collagen content of the regenerated cartilage tissue after 6 weeks of implantation, (E) is the elastin content of the regenerated cartilage tissue after 6 weeks of implantation, and (F) is the CoL II content of the regenerated cartilage tissue after 6 weeks of implantation. (*p<0.05, **p<0.01, ***p<0.001)

[0053] Figure 9 It is a tissue-engineered auricle constructed using the ACEM bioink of the RHE group;

[0054] Figure 10 It is the implantation in nude mice of the tissue-engineered auricle constructed using the ACEM bioink of the RHE group. Specific implementation manner

[0055] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention. For those not specifying specific techniques or conditions in the examples, follow the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be purchased through regular channels.

[0056] Example 1: Isolation and culture of rabbit auricular chondrocytes

[0057] (1) After anesthesia, skin preparation, disinfection, and ligation of blood vessels, an adequate amount of auricular cartilage tissue of Japanese white rabbits was excised. The perichondrium was carefully dissected, and the rabbit ear cartilage was soaked in sterile PBS and washed repeatedly 3 times. Subsequently, it was cut into cartilage blocks the size of millet grains with autoclaved ophthalmic scissors and forceps, and washed 3 times again with sterile PBS. Then, it was transferred to a 0.25% trypsin solution (Gibco, USA) equal to the total volume of the cartilage blocks and digested with shaking in a constant temperature shaker at 37°C and 120 rpm for 30 min. An appropriate amount of type II collagenase (Sigma, USA) was weighed to prepare a 40 mL 0.2% collagenase solution, which was added to the cartilage blocks after centrifugation to remove the supernatant, and continued to be digested with shaking in a constant temperature shaker with the same parameters overnight.

[0058] (2) When the cartilage blocks showed a viscous and slightly transparent appearance, they were filtered through a cell sieve, the filtrate was collected and centrifuged at 1200 rpm for 10 min. After removing the supernatant, it was resuspended with complete medium, counted and the cell viability was detected by a cell counter Countess II (Countstar, USA). Approximately 1×10 6Cells were used to prepare a complete medium containing 10% fetal bovine serum (Gibico, USA) and cultured in a constant temperature incubator at 37 °C, 5% CO 2 2, and saturated humidity. The culture medium was changed completely every two days. When the cells were cultured to 90% confluence, the culture medium was washed with PBS, digested with 0.25% trypsin, and then passaged to obtain the second generation of rabbit auricular chondrocytes.

[0059] Example 2: Preparation of biomimetic bioink I

[0060] Preparation of methacrylated recombinant human collagen (RHCMA):

[0061] Weigh the freeze-dried powder of recombinant humanized collagen (RHC), dissolve it in 0.03% PBS solution at room temperature, and use a magnetic stirrer with a magnetic stir bar to fully dissolve it to obtain an RHC solution with a concentration of 0.01 - 0.05 g / mL. Use a micro-injection pump to slowly inject a methacrylic anhydride solution with a concentration of 0.05% - 0.01% (purity 97%, Sines Co., 760-93-0) into the 5 mL RHC solution that is being stirred (the dosage ratio of the RHC solution to the methacrylic anhydride solution is 1 - 10 g: 0.5 - 5 mL); after slow stirring at room temperature for 1 h, a mixed solution is obtained. Transfer the mixed solution to a 50 mL centrifuge tube, centrifuge at 4000 g for 9 min, and take the supernatant and dilute it with pure water by 3 times. Subsequently, add baking soda to adjust the pH to approximately 7, transfer it to a dialysis bag, and dialyze at 37 °C for 4 days, changing the water one to two times a day. After dialysis, it is freeze-dried to obtain methacrylated recombinant human collagen (RHCM A);

[0062] Preparation of o-nitrobenzyl-modified methacrylated hyaluronic acid (HAMA-NB):

[0063] (1) Dissolve 1 g of hyaluronic acid in 100 mL of water, then add 200 mg of 1xPBS powder (labshark Co.) and stir until dissolved. Add 3.8 mL of methacrylic anhydride solution (HAMA solution, purity 97%, Sines Co., 760-93-0) to make its volume ratio to the total solution 0.05% - 0.01%. At this time, the pH is approximately 3.5. After reacting for 75 min, titrate the reaction system with sodium hydroxide solution to a pH of approximately 8, react at 4 °C for 24 h, then dialyze for 3 days, freeze at -80 °C for 1 h, freeze at -40 °C to -50 °C for 4 h, and evacuate for 24 h to obtain methacrylated hyaluronic acid;

[0064] (2) Dissolve 1 g of freeze-dried methacrylated hyaluronic acid in 100 mL of water to obtain an aqueous solution of methacrylated hyaluronic acid. Dissolve 1-hydroxybenzotriazole, 0.1 - 2 g of (2-aminoethyl)-4-[4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy]-butyramide, and 1.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in 1.5 g of dimethyl sulfoxide. After thorough mixing, mix it with the aqueous solution of methacrylated hyaluronic acid and adjust the pH to between 4.5. Place it in the dark at room temperature for a reaction of 48 h. After the reaction is completed, start dialysis. Add dilute hydrochloric acid to the dialysis solution to adjust the pH to between 3.5, and add 115 g of sodium chloride. After dialysis is completed, freeze-dry to obtain o-nitrobenzyl-modified methacrylated hyaluronic acid (HAMA-NB);

[0065] Preparation of elastin-like polypeptide (ELP):

[0066] (1) The elastin-like sequence is SEQ ID NO.1: [(VPGVG) 2 VPGKG(VPGVG) 2 25 Insert the base fragment encoding this gene into the PJ54 plasmid and transform it into Escherichia coli competent cells (Shanghai Beyotime Biotechnology Co., Ltd.) for induced expression, and continuously culture at 37 °C for 36 h. Subsequently, use a centrifuge tube to collect the culture solution, and after high-speed centrifugation, ultrasonically lyse the collected bacterial cells to obtain the supernatant of the bacterial cells. Mix the supernatant of the bacterial cells with polyethyleneimine (Sigma-Aldrich, molecular weight about 750,000) to form a mixed solution with a polyethyleneimine concentration of 0.4% - 0.7% m / v. Centrifuge the mixed solution at 20,000 g for 15 minutes, and then remove the precipitate to obtain the supernatant of the mixed solution;

[0067] (2) Centrifuge the supernatant of the mixed solution at 4 °C and 20,000 g, and then pour out the supernatant from the precipitated cell debris. Subsequently, incubate the solution at 37 °C for 1 h, and then centrifuge at the same temperature and 20,000 g for 1 h. Take this as 1 cycle and repeat this cycle 4 - 6 times. Each time, adjust the pH of the solution to about 7.4 with NaCl or ammonium sulfate;

[0068] (3) After the cycle is completed, dialyze the obtained supernatant in 4 L of water for 21 h, changing the water every 3 - 4 h. After the finally obtained dialysis solution is freeze-dried, the final product elastin-like polypeptide (ELP) is obtained;

[0069] Preparation of ACEM hydrogel precursor solution:

[0070] ​After sterilizing 10% m / v RHCMA, 1% m / v HAMA-NB, 1% m / v ELP, and 0.25% m / v lithium phenyl-2,4,6-trimethylbenzoylphosphinate, continuously stir overnight at room temperature and thoroughly mix them in a complete medium of 10% v / v PBS to prepare an ACEM hydrogel precursor solution.

[0071] Preparation of biomimetic bioink:

[0072] Twenty-four hours before bioprinting, add a water-soluble photoresist to the ACEM hydrogel precursor solution until the final concentration reaches 0.05% m / v. After pasteurization, mix it with passage 2 rabbit auricular chondrocytes to prepare a biomimetic bioink with a final cell concentration of 1 - 10×10 7 / mL.

[0073] Example 3: Preparation of biomimetic bioink II

[0074] Preparation of methacrylated recombinant human collagen (RHCMA):

[0075] Weigh the freeze-dried powder of recombinant humanized collagen (RHC), dissolve it in 0.01% PBS solution at room temperature, and use a magnetic stirrer with a magnetic bar to fully dissolve it to obtain an RHC solution with a concentration of 0.01 - 0.05 g / mL. Slowly inject a 0.05% - 0.01% methacrylic anhydride solution (the purity of methacrylic anhydride is 97%, Xinsi Company, 760 - 93 - 0) into 0.1 mL of the stirring RHC solution (the dosage ratio of the RHC solution to the methacrylic anhydride solution is 1 - 10 g: 0.5 - 5 mL) using a micro-injection pump. After slow stirring at room temperature for 1 h, obtain a mixed solution. Transfer the mixed solution to a 50 mL centrifuge tube, centrifuge at 4000 g for 8 min, and take the supernatant and dilute it with pure water by 1-fold. Subsequently, add baking soda to adjust the pH to approximately 7, transfer it to a dialysis bag, and dialyze at 37°C for 4 days, changing the water one to two times a day. After dialysis, lyophilize to obtain methacrylated recombinant human collagen (RHCMA);

[0076] Preparation of o-nitrobenzyl-modified methacrylated hyaluronic acid (HAMA-NB):

[0077] (1) Dissolve 1 g of hyaluronic acid in 100 mL of water. Subsequently, add 200 mg of 1xPBS powder (Labshark) and stir until dissolved. Add 3.8 mL of methacrylic anhydride solution (purity 97%, Siyansi Co., 760 - 93 - 0) so that its volume ratio in the total solution is 0.05% - 0.01%. At this time, the pH is approximately 3.5. After reacting for 75 min, titrate the reaction system with sodium hydroxide solution to a pH of approximately 8, react at 4 °C for 24 h, then dialyze for 3 days, freeze - dry at - 80 °C for 1 h, freeze at - 40 °C to - 50 °C for 4 h, and evacuate for 24 h to obtain methacrylated hyaluronic acid;

[0078] (2) Dissolve 1 g of freeze - dried methacrylated hyaluronic acid in 100 mL of water to obtain an aqueous solution of methacrylated hyaluronic acid. Dissolve 1 - hydroxybenzotriazole, 0.1 g of (2 - aminoethyl)-4 - [4 - (hydroxymethyl)-2 - methoxy - 5 - nitrophenoxy]-butyramide, and 1 g of 1 - ethyl-(3 - dimethylaminopropyl)carbodiimide hydrochloride in 1 - 2 g of dimethyl sulfoxide. After thorough mixing, mix with the aqueous solution of methacrylated hyaluronic acid and adjust the pH to between 4. Place it in the dark at room temperature and react for 48 h. After the reaction, start dialysis. Add dilute hydrochloric acid to the dialysis solution to adjust the pH to between 3, and add 30 g of sodium chloride. After dialysis, freeze - dry to obtain o - nitrobenzyl - modified methacrylated hyaluronic acid (HAMA - NB);

[0079] Preparation of elastin - like polypeptide (ELP):

[0080] (1) The elastin - like sequence is SEQ ID NO.1: [(VPGVG) 2 VPGKG(VPGVG) 2 25 , Insert the base fragment encoding this gene into the PJ54 plasmid and transform it into Escherichia coli competent cells (Shanghai Beyotime Biotechnology Co., Ltd.) for induced expression, and continuously culture at 37 °C for 36 h. Subsequently, use a centrifuge tube to collect the culture solution, and after high - speed centrifugation, ultrasonically lyse the collected bacteria. Mix the supernatant with polyethyleneimine (purchased from Sigma - Aldrich, molecular weight approximately 750000) to form a mixed solution with a concentration of 0.4% - 0.7%. Centrifuge the mixed solution at 20000 g for 15 minutes, and then remove the precipitate to obtain the supernatant of the mixed solution;

[0081] ​(2) Centrifuge the supernatant of the mixed solution at 4°C and 20,000 g. Then pour out the supernatant from the precipitated cell debris. Subsequently, incubate the solution at 37°C for 1 hour and then centrifuge it at the same temperature and 20,000 g for 1 hour. Take this as one cycle and repeat this cycle 4 - 6 times. Each time, adjust the pH of the solution to about 7.4 with NaCl or ammonium sulfate.

[0082] (3) After the cycle is completed, dialyze the obtained supernatant in 4 L of water for 21 h, changing the water every 3 - 4 h. After freeze-drying the finally obtained dialysis solution, the final product, elastin-like polypeptide (ELP), is obtained.

[0083] Preparation of ACEM hydrogel precursor solution I:

[0084] Sterilize 10% m / v RHCMA, 1% m / v HAMA-NB, 1% m / v ELP, and 0.25% m / v lithium phenyl-2,4,6-trimethylbenzoylphosphonate, and then continuously stir and thoroughly mix them overnight at room temperature in a complete medium of 10% v / v PBS to prepare an ACEM hydrogel precursor solution.

[0085] Preparation of biomimetic bioink:

[0086] Twenty-four hours before bioprinting, add a water-soluble photoresist to the ACEM hydrogel precursor solution until the final concentration reaches 0.05% m / v. After pasteurization, mix it with the second-generation rabbit auricular chondrocytes to prepare a biomimetic bioink with a final cell concentration of 1 - 10×10 7 cells / mL.

[0087] Example 4: Preparation of biomimetic bioink III

[0088] Preparation of methacrylated recombinant human collagen (RHCMA):

[0089] Weigh the recombinant humanized collagen (RHC) lyophilized powder, dissolve it in 0.04% PBS solution at room temperature, use a magnetic bar with a magnetic stirrer to fully dissolve to obtain a RHC solution with a concentration of 0.01-0.05 g / mL, use a microinjection pump to slowly inject a 0.05%-0.01% methacrylic anhydride solution (purity of 97%, Xiens Company, 760-93-0) into the stirring 10 mL RHC solution (the amount ratio of RHC solution to methacrylic anhydride solution is 1-10 g: 0.5-5 mL); after slowly stirring at room temperature for 1 hour, a mixed solution is obtained, which is transferred to a 50 mL centrifuge tube, centrifuged at 4000g for 10 minutes, and the supernatant is diluted to 5 times with pure water. Subsequently, baking soda is added to adjust the pH to about 7, and the solution is transferred to a dialysis bag for dialysis at 37°C for 5 days, with water changed once or twice a day. Methacryloylated recombinant human collagen (RHCMA) can be obtained by lyophilization after dialysis;

[0090] Preparation of o-nitrobenzyl-modified methacryloylated hyaluronic acid (HAMA-NB):

[0091] (1) Dissolve 1g of hyaluronic acid (HA) in 100mL of water, then add 200mg of 1xPBS powder (Labshark) and stir until dissolved. Add 3.8mL of methacrylic anhydride solution (purity 97%, Hiens, 760-93-0) until its volume ratio of the total solution is 0.05% to 0.01%. At this time, the pH is about 3.5. After 75 minutes of reaction, the reaction system is titrated to a pH of about 8 with sodium hydroxide solution, react at 4℃ for 24h, then dialyze for 3 days, freeze at -80℃ for 1h, freeze at -40℃ to -50℃ for 4h, and vacuum for 24h to obtain methacryloyl hyaluronic acid (HAMA).

[0092] (2) Take 2g of freeze-dried methacryloyl hyaluronic acid and dissolve it in 100mL of water to obtain a methacryloyl hyaluronic acid aqueous solution. Use 2g of dimethyl sulfoxide to dissolve 1-hydroxybenzotriazole, 0.1-2g of (2-aminoethyl)-4-[4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy]-butyramide and 2g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride. After fully mixing, mix with the methacryloyl hyaluronic acid aqueous solution and adjust the pH to between 4 and 5. Place it at room temperature and avoid light to react for 48 hours. After the reaction is completed, start dialysis, add dilute hydrochloric acid to the dialysate to adjust the pH to between 4, and add 200g of sodium chloride. After dialysis, freeze-dry to obtain o-nitrobenzyl-modified methacryloyl hyaluronic acid (HAMA-NB);

[0093] Preparation of Elastin-like Polypeptide (ELP):

[0094] The elastin-like sequence is SEQ ID NO.1: [(VPGVG) 2 VPGKG(VPGVG) 2 25 , insert the base fragment encoding this gene into the PJ54 plasmid and transform it into Escherichia coli competent cells (Shanghai Beyotime Biotechnology Co., Ltd.) for induced expression, and continuously culture at 37 °C for 36 h. Subsequently, collect the culture solution using a centrifuge tube, and after high-speed centrifugation, ultrasonically lyse the collected bacterial cells. Mix the supernatant with polyethyleneimine (purchased from Sigma-Aldrich, with a molecular weight of approximately 750,000) to form a mixed solution with a concentration of 0.4% - 0.7% m / v. Centrifuge the mixed solution at 20,000 g for 15 minutes, and then remove the precipitate to obtain the supernatant of the mixture;

[0095] (2) Centrifuge the supernatant of the mixture at 4 °C and 20,000 g, and then pour out the supernatant from the precipitated cell debris. Subsequently, incubate the solution at 37 °C for 1 hour, and then centrifuge at the same temperature and 20,000 g for 1 hour. Take this as 1 cycle and repeat this cycle 4 - 6 times. Each time, adjust the pH of the solution to about 7.4 with NaCl or ammonium sulfate;

[0096] (3) After the cycle is completed, dialyze the obtained supernatant in 4 L of water for 21 h, changing the water every 3 - 4 h. After freeze-drying the final dialysis solution, the final product elastin-like polypeptide (ELP) is obtained.

[0097] Preparation of ACEM hydrogel precursor solution:

[0098] Sterilize 10% m / v RHCMA, 1% m / v HAMA-NB, 1% m / v ELP, and 0.25% m / v lithium phenyl-2,4,6-trimethylbenzoylphosphonate, and then continuously stir and mix thoroughly overnight at room temperature in a complete medium of 10% v / v PBS to prepare an ACEM hydrogel precursor solution.

[0099] Preparation of biomimetic bioink:

[0100] 24 h before bioprinting, add a water-soluble photoresist to the ACEM hydrogel precursor solution to a final concentration of 0.05% m / v. After pasteurization, mix it with the second-generation rabbit auricular chondrocytes to prepare a biomimetic bioink with a final cell concentration of 1 - 10×10 7 cells / mL.

[0101] Example 5: Preparation of Biomimetic Bioink IV

[0102] ​This example is the same as Example 1, except that the dosage of each raw material added to the prepared ACEM hydrogel precursor solution is different, and the total cell concentration of the bionic bioink is 1×10 6 cells / mL.

[0103] After sterilizing 10% m / v RHCMA, 1% m / v HAMA-NB, 1% m / v ELP, and 0.25% m / v lithium phenyl-2,4,6-trimethylbenzoylphosphonate, continuously stir overnight at room temperature and mix well in a complete medium of 10% v / v FBS to prepare an ACEM hydrogel precursor solution.

[0104] Example 6: Preparation of Bionic Bioink V

[0105] This example is the same as Example 5, except that the final cell concentration of the prepared bionic bioink is 20×10 6 cells / mL.

[0106] Comparative Example 1: Preparation of Bionic Bioink

[0107] This comparative example is the same as Example 5, except that the dosage of each raw material added to the prepared ACEM hydrogel precursor solution is different.

[0108] After sterilizing 10% m / v RHCMA and 0.25% m / v lithium phenyl-2,4,6-trimethylbenzoylphosphonate, continuously stir overnight at room temperature and mix well in a complete medium of 10% v / v FBS to prepare an ACEM hydrogel precursor solution.

[0109] Comparative Example 2: Preparation of Bionic Bioink

[0110] This comparative example is the same as Example 5, except that the dosage of each raw material added to the prepared ACEM hydrogel precursor solution is different.

[0111] After sterilizing 10% m / v RHCMA, 1% m / v HAMA-NB, and 0.25% m / v lithium phenyl-2,4,6-trimethylbenzoylphosphonate, continuously stir overnight at room temperature and mix well in a complete medium of 10% v / v FBS to prepare an ACEM hydrogel precursor solution.

[0112] Comparative Example 3: Preparation of Bionic Bioink

[0113] This comparative example is the same as Comparative Example 1, except that the final cell concentration of the prepared bionic bioink is 20×10 6 cells / mL.

[0114] Comparative Example 4: Preparation of Bionic Bioink

[0115] This comparative example is the same as Comparative Example 2, except that the final cell concentration of the prepared bionic bioink is 20×10 6 cells / mL.

[0116] Example 7: Preparation of Bionic Auricle

[0117] The three-dimensional scanning of the real human auricle morphology was carried out by laser scanning and processed by a CAD computer-aided design system. Subsequently, the three-dimensional model of the mirrored human auricle was obtained by Boolean operation;

[0118] The temporal bone part of the three-dimensional model of the human auricle was removed by Mimics Medical 21.0 software, only the auricle part was retained, and the digital model of the auricle in STL format was exported. The auricle digital model was divided into corresponding slices with a height of 50 - 200 μm as a unit, imported into PotatoOSP-Client V1.2.6 software, sliced at 50 - 200 μm per layer, and uploaded to a 3D bio-printer (BP8601 Pro, EFL) for printing. 1 - 5 mL of the bionic bioink prepared in Example 5 was added to the 3D printer as the printing solution, and after adjusting to an appropriate light intensity (10 - 20 mW / cm 2 ), exposure time (5 - 20 s), base layer exposure time (5 - 20 s) and base layer number (2 - 10 layers), printing was carried out to obtain the tissue-engineered auricle, as Figure 9 shown.

[0119] The tissue-engineered auricle was immersed in complete medium and pre-cultured in an incubator for 24 h to ensure that the construct was free of contamination and to remove as much as possible the excess bioink adhering to the pore structure of the construct. After the nude mice were anesthetized routinely and carefully disinfected, an incision was designed in the middle of the back, and the construct was implanted on both sides of its back, as Figure 10 shown.

[0120] Test Example 1: Performance Test of ACEM Hydrogel Precursor Solution

[0121] 1. Liquid Nuclear Magnetic Resonance Hydrogen Spectrum Test ( 1 H NMR)

[0122] 50 mg of the freeze-dried samples of RHC and RHCMA, HA and HAMA, HAMA and HAMA-NB in Example 1 were respectively taken out and dissolved in 500 uL of PBS. The grafting of MA and NB groups was detected by a Bruker AVAME III 600M nuclear magnetic resonance spectrometer to determine whether RHCMA, HAMA and HAMA-NB were successfully constructed. The test results are as Figure 2 (A) shown.

[0123] From Figure 2(A) It can be obtained that RHCMA, HAMA, and HAMA-NB in Example 1 have been successfully constructed.

[0124] 2. Performance testing of the ACEM hydrogel precursor solution

[0125] Before detection, the ACEM hydrogel precursor solution was first prepared into a hydrogel: the ACEM hydrogel precursor solutions prepared in Example 5, Comparative Example 1, and Comparative Example 2 were respectively poured into a silicone mold, and a blue light flashlight (wavelength: 405 nm; light intensity: 20 mW / cm 2 ; vertical distance 10 cm; exposure time: 20 s) was used to induce the ACEM hydrogel precursor solution to solidify into a hydrogel, and its principle is as Figure 2 (B) shown: HAMA was modified to nitrobenzyl-modified methacrylated hyaluronic acid (HAMA-NB) through nitrobenzyl (NB). Under light illumination, the aldehyde group photogenerated by the NB group can rapidly anchor with amino-containing ELP, enabling ELP and HAMA to be connected by an imine bond. At the same time, HAMA-NB and RHCMA can also be tightly cross-linked through free radical polymerization under light illumination. Subsequently, the performance testing of the hydrogel was carried out.

[0126] (1) Scanning electron microscope detection (SEM)

[0127] The hydrogels of each group were rinsed three times with PBS and then placed in an electron microscopy solution, fixed in the dark at 4 °C in a cold storage for 48 h, then frozen by liquid nitrogen and freeze-dried. After the freeze-dried samples were broken and sputter-coated with gold, the microporous characteristics of the hydrogels of each group were observed under a scanning electron microscope (Leica, Germany). The electron microscopy photos were analyzed using ImageJ software, as Figure 3 (A), Figure 3 (B), Figure 3 (C), Figure 3 (D), Figure 3 (E) shown.

[0128] From Figure 3 (A), Figure 3 (B), Figure 3 (C), Figure 3 (D), Figure 3 (E), it can be obtained that:

[0129] After 20 s of photo-crosslinking in the mold, the hydrogels of the three groups can all be solidified. The SEM observation results of the solidified samples show that the hydrogels of the R group, RH group, and RHE group are all loose and porous structures, and the pore size of the R group is significantly larger than that of the other two groups ( Figure 3D, **p < 0.01, ***p < 0.001), which means that the R group is more suitable for cells to grow in, but the mechanical strength may be relatively weak. The pore sizes of the RH group and the RHE group after adding HAMA-NB both decreased significantly compared to the R group, meaning that the RH group and the RHE group slightly reduced the liquid exchange and proliferation space required for cell survival, but the mechanical strength of the hydrogel may be improved. Compared with the RH group, the RHE group after adding ELP had no significant differences in pore size and porosity ( Figure 3 D, E, p > 0.05), but the porosity of the RHE group increased and was able to approach that of the R group ( Figure 3 E, p > 0.05).

[0130] (2) Swelling ratio test

[0131] Accurately weigh the initial weight of the hydrogels in each group above and record it as W 0 , soak the hydrogels in each group above in PBS for 24 h, dry the moisture and then accurately weigh and record it as W swelling . The test was repeated four times, and the swelling ratio was calculated according to the data obtained from the test. The swelling ratio was the percentage of W swelling to W 0 , and the results are shown in Figure 3 (F).

[0132] From Figure 3 (F), it can be obtained that:

[0133] The swelling ratio detection of the three groups of materials showed that although the three groups of hydrogels all swelled to varying degrees compared with the initial state, the swelling ratio of the R group was significantly higher than that of the other two groups (*p < 0.05), while there was no significant difference between the RH group and the RHE group. This may be because the R group has a larger porosity and pore size, resulting in more water absorption.

[0134] (3) Rheological detection

[0135] Using an Anton Paar MCR 302 optical rheometer, equipped with a PP25 test rotor, supplemented with a P-PTD200 / GL and Omicure S200 measurement system, the storage modulus (G') and loss modulus (G") of the hydrogel were detected by oscillatory time testing, and the loss factor tan(δ) was calculated, tan(δ) = G" / G', and the results are shown in Figure 4 (A), Figure 4 (B).

[0136] From Figure 4 (A), Figure 4 (B), it can be obtained that: as the sample cures, tan(δ) will gradually decrease. The point where tan(δ) = 1 is the phase transition point.

[0137] (4) Mechanical testing

[0138] Use a clean Instron universal testing machine (Instron, USA) to press the upper and lower platens, turn on the humidifier, and conduct the test at room temperature with an indoor humidity of 60%. After drying the surface moisture of each group of hydrogels with a gauze, place them in the center of the platen, move the upper platen to touch the upper boundary of the sample, and measure the sample height with a vernier caliper. After resetting the gauge length, perform vertical compression at 5 mm / min until the sample undergoes maximum deformation. Record the force and displacement data, analyze the compressive stress-strain curve of the sample, and obtain the compressive Young's modulus of the sample. Measure 4 parallel samples for each group, as Figure 4 (C) shows.

[0139] As Figure 4 (C) shows: After adding HAMA-NB, the compressive Young's modulus of the material increased significantly (****p < 0.0001, Figure 4 C, D). Compared with the single-component R group, adding HAMA-NB and ELP can increase its compressive Young's modulus by more than 3 times. There was no statistically significant difference in the compressive Young's modulus between the RH group and the RHE group, indicating that although an additional crosslinking network between NB groups and ELP was added in the RHE group, the effect was relatively weak and did not significantly improve the mechanical properties of the RHE group (p > 0.05).

[0140] Experimental Example 2: Performance testing of 3D printed constructs

[0141] 1. Confirmation of printing parameters

[0142] Use the ACEM hydrogel precursor solutions of Example 5, Comparative Example 1, and Comparative Example 3 as printing inks. Construct an orthogonal grid (Cross) model with a diameter of 1 cm, a thickness of 2 mm, a truss width of 1 mm, and a truss spacing and edge thickness of 0.7 mm through Creo5.0 software. Import the constructed model into the PotatoOSP-Client V1.2.6 software in STL format and slice it at 50 μm per layer. Add sufficient printing ink to the trough of the DLP printer (BP8601 Pro, EFL) with a pipette (the side view, front view, trough, and printing platform of the DLP printer (BP8601 Pro, EFL) are as Figure 5 shown in A), debug the printing parameters of each group of inks by changing the light intensity and exposure time, and observe whether the truss spacing is the set size under a stereomicroscope to determine the optimal printing parameters of the material. The basic printing parameters are shown in Table 1.1.

[0143] Table 1.1 Basic parameters of DLP printing ACEM photosensitive hydrogels

[0144]

[0145]

[0146] From Figure 5 it can be obtained that the optimal printing parameters for group R are light intensity of 20 mW / cm 2 , and exposure time of 10 s; both group RH and group RHE are light intensity of 10 mW / cm 2 , and exposure time of 6 s.

[0147] 2. 3D printing of ACEM bioink

[0148] Twenty-four hours before bioprinting, a water-soluble photoresist was added to the ACEM hydrogel precursor solutions of Examples 5-6 and Comparative Examples 1-4 until the final concentration reached 0.05% m / v. After being treated by pasteurization, it was mixed with second-generation rabbit ear chondrocytes to prepare ACEM bioink. ACEM bioinks with final cell concentrations of 1×10 6 cells / mL and 20×10 6 cells / mL were used for in vitro and in vivo experiments, respectively. The feeding trough was irradiated in the built-in ultraviolet disinfection mode of the DLP printer for 30 min in advance, and the temperature of the feeding trough was set at 37 °C. The Cross model was imported into the DLP printer (BP8601 Pro, EFL), and sliced according to the preset height. After printing, bioprinted constructs of group R-Cross (Comparative Example 1, Comparative Example 3), group RH-Cross (Comparative Example 2, Comparative Example 4), and group RHE-Cross (Example 5, Example 6) were obtained. The printed constructs were added with complete medium and placed in an incubator for culture.

[0149] (1) In vivo implantation and regeneration of 3D printed constructs

[0150] The bioprinted constructs of group R-Cross, group RH-Cross, and group RHE-Cross were immersed in complete medium and pre-cultured in an incubator for 24 h to ensure that the constructs were pollution-free and to remove as much as possible the excess bioink adhering to the pore structure of the constructs. After the nude mice were anesthetized routinely and carefully disinfected, an incision was designed in the middle of the back, and the constructs were implanted on both sides of their backs. The formation of cartilage in the nude mice was observed, and the regenerated tissue was obtained after harvesting at 6 w, as Figure 6 shown.

[0151] From Figure 6It can be obtained that the three groups of constructs all showed a bright yellow appearance immediately after printing, with a soft texture, rich in moisture, and having a regular pore structure. After being implanted in vivo for 6 weeks, they all showed a porcelain-white appearance, indicating the possible formation of new cartilage tissue. However, although the R-Cross group could maintain the appearance of a circular structure, its height decreased significantly; although the RH-Cross group did not change significantly in height, curling deformation occurred on the surface of the construct; the general appearance of the RHE-Cross group was well maintained in terms of shape and height.

[0152] (2) Histological and immunohistochemical detection of regenerated tissue

[0153] The regenerated tissue formed in Example (1) of this experiment was taken out from the nude mice, observed and weighed, and then immediately immersed and fixed in 4% tissue cell fixative for 48 h. After conventional dehydration, paraffin embedding, and 4-μm sectioning, the structure and matrix deposition status of the regenerated tissue were observed by hematoxylin-eosin (H&E) staining, safranin O-fast green (SO / FG) staining, Masson trichrome staining, elastic-van Gieson (EVG) staining, and immunohistochemical staining of type II collagen (Col II) (Invitrogen, USA). Except for special instructions, all the above reagents were sourced from Solarbio. The specific process is as follows:

[0154] ① H&E staining: Bake the slides at 65 °C for 2 h, dewax to water. Stain the nuclei with hematoxylin for 10 min, differentiate with 0.5% hydrochloric acid alcohol for 30 s, blue return for 1 min, stain with eosin for 2 min, then dehydrate with gradient alcohol, clear with xylene, and seal with neutral resin. Observe and collect under the microscope.

[0155] ② SO / FG staining: Bake the slides at 65 °C for 2 h, dewax to water, stain with Weigert stain for 5 min, wash with water, differentiate with acidic differentiating solution for 15 s, stain with fast green stain for 5 min, wash with weak acid solution for 10 s, stain with Safranin-O stain for 5 min, dehydrate with 95% ethanol, clear with xylene, and seal with neutral resin. Observe under the microscope.

[0156] ③ Masson staining: Bake the slides at 65 °C for 2 h, dewax to water. Stain with mordant solution in an incubator at 60 °C for 1 h, stain with celestine blue dropwise for 2 min, wash with water twice, stain with Mayer hematoxylin stain dropwise for 3 min, wash with distilled water twice, differentiate with acidic ethanol for 10 s, stain with ponceau fuchsin for 10 min, stain with phosphomolybdic acid for 10 min, stain with aniline blue for 5 min, stain with 0.3% weak acid for 2 min, dehydrate, clear, and seal. Observe the staining results under the microscope.

[0157] ④ EVG staining: Bake the slides at 65 °C for 2 h, dewax to water, immerse in Verhoeff stain for 30 min, differentiate with Verhoeff differentiating solution until elastic fibers are clear, treat with 95% ethanol for 5 min, counterstain for 2 min, dehydrate, clear, mount, and observe under a microscope.

[0158] ⑤ CoL II staining: Bake the slides at 65 °C for 2 h, dewax to water, incubate with 3% hydrogen peroxide at room temperature for 10 min, incubate with complex enzyme original digestion solution at 37 °C for 30 min, incubate with avidin C at 37 °C for 10 min, develop color with DAB chromogenic solution for 5 min, counterstain with hematoxylin for 2 min, quickly differentiate with 0.5% hydrochloric acid alcohol, return to blue with running water for 30 min, dehydrate, clear, mount, and observe under a microscope.

[0159] The above test results are as Figure 7 shown.

[0160] It can be Figure 7 seen that the chondrogenesis in the RHE-Cross group and the R-Cross group was significantly better than that in the RH-Cross group. At the same time, in the RHE-Cross group, not only typical cartilage lacunae and cartilage-specific ECM deposition were observed, but also "cartilage island"-like structures similar to the formation of chondrocyte clusters in natural cartilage appeared. "Cartilage island"-like structures were not observed in the other two groups, which means that the materials in the RHE-Cross group have better cartilage regeneration ability in in vivo experiments.

[0161] (5) Biochemical quantification of regenerated tissue

[0162] Perform DNA quantification detection, glycosaminoglycan quantification detection, total collagen quantification, and biochemical quantification analysis on the regenerated tissue. The specific test procedures are as follows:

[0163] ① DNA quantification detection

[0164] The regenerated tissue was snap-frozen in liquid nitrogen after being taken out of the body. Before testing, an appropriate amount of tissue was cut and accurately weighed, and then the DNA content in the regenerated tissue was measured using a DNA quantification detection kit. The DNA content in each group of samples was measured using the Quant-iT™ PicoGreen dsDNA Assay kit (Invitrogen, USA). The detection steps and calculation methods were as shown in the kit instructions to reflect cell proliferation: Each group of samples was accurately weighed and the weight was recorded. The Shanghai Jingxin grinder was pre-cooled, and the PicoGreen reagent was placed at room temperature in advance. The TE stock solution was diluted to a TE working solution with sterile water. The PicoGreen working solution was prepared with the TE working solution, and then the proteinase K working solution was prepared. 500 μL of the proteinase K working solution was added to each grinding tube. The samples were rinsed 3 times with pre-cooled PBS and then transferred to the grinding tubes containing the proteinase K working solution. An ophthalmic scissors was inserted under the liquid surface in the grinding tube to cut the samples into pieces, 3 grinding beads were added, and the samples were ground in a pre-cooled grinder at 60 Hz for 3 minutes, and then placed in a 56 °C oven for digestion for 10 - 12 h, with several oscillations in the middle. At the end of digestion, the liquid in the grinding tube should be basically clear, without debris or only a little flocculent substance. The centrifuge was pre-cooled in advance. After taking the grinding tubes out of the oven, they were directly placed in the pre-cooled centrifuge and centrifuged at 12,000 rpm for 10 min. The supernatant was aspirated, and the sample dilution range was determined by preliminary experiments. 100 μL of the standard product and the diluted test sample were respectively added to a 96-well plate, and then 100 μL of the PicoGreen working solution was added to each well. After incubating in the dark at room temperature for 5 min, the fluorescence value could be detected with a microplate reader (PerkinElmer EnSpire, Singapore) at 480 - 520 nm and compared with the standard curve. Each group of samples was repeated 4 times, and finally the DNA content of the samples was calculated corresponding to the standard curve.

[0165] ② Quantitative detection of glycosaminoglycan (GAG)

[0166] The total content of tissue glycosaminoglycan (GAG) was quantitatively detected using an Alcian blue colorimetric assay kit (JemeGen, China) to measure the GAG content in the regenerated tissue. After the regenerated tissue was excised, it was immediately frozen in liquid nitrogen. Before testing, an appropriate amount of tissue was cut and precisely weighed as a sample. The sample was washed 3 times with pre-cooled PBS, then minced and placed in a cryogenic grinder for 1 minute of oscillatory grinding at 60 Hz. 500 μL of extraction solution was added, vortexed for 1 minute, and then extracted at 4 °C for 24 hours, with vortexing 2 times during the extraction process. Finally, the supernatant was obtained by centrifugation at 16,000 g for 10 minutes. The dilution range was determined by preliminary experiments, and after dilution by the corresponding multiple, it was reserved for testing. Prepare 5 EP tubes and add 50 μL of Reagent A to each to start serial dilution to prepare 5 standards: Add 50 μL of Reagent G standard solution to tube 1 and mix well; Take out 50 μL from tube 1 and add it to tube 2 and mix well; Take out 50 μL from tube 2 and add it to tube 3 and mix well; Take out 50 μL from tube 3 and add it to tube 4 and mix well. Take out 50 μL from tube 4 and place it in another EP tube for standby. Place all the remaining tubes 1 - 5 on ice for use as the following standards. Subsequently, prepare the GENMED staining working solution and keep it in the dark for standby. Add 50 μL of Reagent B uniformly to the standards and the samples to be tested and mix well. Then add 50 μL of Reagent C and mix well. Incubate at room temperature for 15 minutes. Add 750 μL of the GENMED staining working solution, vortex for 15 s, incubate in the dark at room temperature for 15 minutes, and then centrifuge at 16,000 rpm for 15 minutes. Retain the blue precipitate and carefully discard the supernatant until no water droplets remain. Add 1 mL of Reagent E, vortex for 15 s (ensure thorough resuspension of the particles), centrifuge again at 16,000 g for 15 minutes, retain the blue precipitate, and carefully discard the supernatant until no water droplets remain. Add 1 mL of Reagent F, vortex for 15 s, and incubate in the dark at room temperature for 15 minutes to ensure complete dissolution of the precipitate. Transfer to a 96-well plate, set 3 replicates for each sample, measure the absorbance at 600 nm using an enzyme-linked immunosorbent assay reader, and obtain the corresponding GAG content of the sample according to the standard curve.

[0167] ③ Total collagen quantification

[0168] The Hydroxyproline (HYP) quantification kit (Nanjing Jiancheng, China) was used, and the HYP content in the regenerated tissue was measured according to the kit instructions. Subsequently, the total collagen content in the regenerated tissue was calculated from its content. The specific detection process is as follows:

[0169] After sampling, rinse the regenerated tissues in the R-Cross group, RH-Cross group, and RHE-Cross group thoroughly, quick-freeze them in liquid nitrogen. Before testing, cut an appropriate amount of tissue as the test sample, accurately weigh it, wash the sample 3 times with pre-cooled PBS, then put the sample into a 15 mL tube, add 500 μL of hydrolysis solution, and hydrolyze at 95 °C for 20 min (mix once at the 10th minute. After hydrolysis, the liquid is basically clear and no tissue can be seen). Add 10 μL of indicator to each tube, shake well, add "pH-adjusting solution A", mix for 20 s until the liquid turns red. Dropwise add "pH-adjusting solution B", mix well after each drop until the red color of the liquid disappears. At this time, the pH value is about 6.0 - 6.8. Add double-distilled water to 10 mL and mix well. Take 4 mL of each tube sample, add 30 mg of activated carbon and mix well, and centrifuge at 3500 rpm for 10 min (the supernatant is clear and colorless after centrifugation).

[0170] Take 1 mL of the test sample and put it into a new 15 mL tube. Prepare blank tube samples, standard tube samples, and determination tube samples according to the instructions. Add each detection reagent in sequence and mix well. Incubate in a water bath at 60 °C for 15 min. After cooling for 10 min, centrifuge at 3500 rpm for 10 min. Add 100 μL of the test sample to each well of a 96-well plate. Set three sub-wells for each sample. Use an enzyme-linked immunosorbent assay reader to measure the absorbance at 550 nm, and calculate the hydroxyproline content in the sample according to the calculation formula in the instructions to estimate the total collagen content.

[0171] ④ CoL II and elastin quantification

[0172] Use enzyme-linked immunosorbent assay to detect the contents of CoL II and elastin in the regenerated tissues of the R-Cross group, RH-Cross group, and RHE-Cross group. Specifically, use CoL II quantitative detection kits and elastin quantitative detection kits (Jianglai Biotech, China), and determine the HYP content in the regenerated tissues through the kit instructions. Subsequently, calculate the total collagen content in the regenerated tissues from its content. The specific detection process is as follows:

[0173] After washing the regenerated tissues of the R-Cross group, RH-Cross group, and RHE-Cross group with pre-cooled PBS, dry them to remove moisture, accurately weigh them as test samples, cut them into pieces and place them in a grinding tube containing RIPA lysis buffer, and grind them at 30 Hz for 6 min in a low-temperature grinding instrument. After sufficient lysis, centrifuge the test samples of each group at 4°C and 12,000 g for 10 min. Then, dilute the supernatant by the corresponding multiple. Take 50 μL of the test sample and each concentration standard product and place them in a microplate coated with the corresponding antibody. Set 3 replicate wells for each sample, and set a blank well at the same time. Then add the secondary antibody labeled with HRP in the amount specified in the instruction manual. After incubation and washing, color with TMB. The color depth is positively correlated with the content of CoL II and elastin in the sample. Measure the OD value of each well at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader, plot the standard curve and calculate.

[0174] Statistical analysis: Enter all quantitative data into GraphPAD Prism 8.0 software for analysis and plotting. After performing a normality test, the comparison between two groups of samples uses Student's t-test, and for three or more groups of samples, one-way analysis of variance (One-Way ANOVA) or two-way analysis of variance (Two-Way ANOVA) is used according to the situation to evaluate the significant differences between the values of each group. p < 0.05 represents a statistically significant difference. Measurement test data are expressed as mean ± standard deviation. The test results are as Figure 8 shown.

[0175] It can be Figure 8 seen that there were no significant differences in the wet weights of the three constructs before implantation ( Figure 8 A, p > 0.05). However, after the in vivo culture ended, the wet weights of each group increased, but the wet weight of the RH-Cross group was lower and significantly lower than that of the R-Cross group ( Figure 8 A, *p < 0.05), which may mean that less new cartilage tissue was formed in the RH-Cross group. The subsequent detection results of DNA content, GAG content, collagen content, elastin content, and CoL II content also confirmed this conjecture. After the in vivo culture ended, the contents of each detection index in the RH-Cross group were significantly lower than those of the other two groups ( Figure 8 B-F, *p < 0.05, **p < 0.01, ***p < 0.001), indicating that in terms of both in vitro and in vivo experiments, the ability of the RH-Cross group to promote chondrocyte growth and cartilage tissue formation was lower. Compared with the R-Cross group, the RHE-Cross group had no significant differences except for containing more elastin ( Figure 8 E, *p < 0.05).

[0176] The above results indicate that among the three groups of ACEM bioinks, the RHE group has the best comprehensive ability, with rapid photocuring ability to form hydrogels, good printability and mechanical strength. It can be precisely constructed into a biological scaffold through DLP printing and exhibits good biocompatibility in in vitro and in vivo experiments, which is beneficial to the proliferation of chondrocytes and the secretion of matrix, promoting cartilage tissue regeneration and laying a solid foundation for the successful construction of tissue-engineered auricles.

[0177] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing a biomimetic biological ink, characterized in that: The preparation method comprises the following steps: Adding methacryloyl recombinant human collagen, o-nitrobenzyl-modified methacryloyl hyaluronic acid, elastin-like polypeptide, and lithium phenyl-2,4,6-trimethylbenzoylphosphonate to a solvent in sequence, stirring and mixing to obtain an ACEM hydrogel precursor solution, and mixing the ACEM hydrogel precursor solution with a photoresist and seed cells to obtain a biomimetic bio-ink; The sequence of the elastin-like polypeptide is shown in SEQ ID NO.

1.

2. The method for preparing a biomimetic biological ink according to claim 1, characterized in that: The mass ratio of the methacryloyl recombinant human collagen, the o-nitrobenzyl-modified methacryloyl hyaluronic acid, the elastin-like polypeptide, and the phenyl-2,4,6-trimethylbenzoylphosphonic acid lithium is 500-1000:50-100:50-100:25-50; the dosage ratio of the methacryloyl recombinant human collagen to the solvent is 100 mg-1000 mg:1 mL-10 mL.

3. The method for preparing a biomimetic biological ink according to claim 1, characterized in that: The seed cells include at least one of auricular chondrocytes, articular chondrocytes, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, embryonic stem cells, and induced pluripotent stem cells.

4. The method for preparing a biomimetic biological ink according to claim 1, characterized in that: The method for preparing the methacrylylated recombinant human collagen comprises the following steps: Weigh the recombinant humanized collagen lyophilized powder and dissolve it in PBS to prepare a recombinant humanized collagen solution; add the methacrylic anhydride solution to the recombinant humanized collagen solution, centrifuge after reaction, and obtain a supernatant; take the supernatant and dilute it with water, then adjust the pH, dialyze, and lyophilize in sequence to obtain methacrylylated recombinant human collagen.

5. The method for preparing a biomimetic biological ink according to claim 1, characterized in that: The preparation method of the o-nitrobenzyl-modified methacrylylated hyaluronic acid comprises the following steps: (1) dissolving hyaluronic acid and PBS in water, then adding methacrylic anhydride solution to react, then adjusting the pH to weak alkalinity, reacting again at low temperature, then dialyzing and freeze-drying in sequence to obtain methacrylylated hyaluronic acid; (2) dissolving the methacryloyl hyaluronic acid in water to obtain a methacryloyl hyaluronic acid solution; adding 1-hydroxybenzotriazole, (2-aminoethyl)-4-[4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy]-butyramide, and (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride to dimethyl sulfoxide for mixed dissolution to obtain a mixed solution; mixing the mixed solution with the methacryloyl hyaluronic acid solution, and then reacting in the dark, dialyzing after the reaction to obtain a dialysate, adjusting the pH of the dialysate to 3-4, and adding sodium chloride to the dialysate, and freeze-drying after the dialysis to obtain o-nitrobenzyl-modified methacryloyl hyaluronic acid.

6. The method for preparing a biomimetic biological ink according to claim 4, characterized in that: The dosage ratio of the recombinant humanized collagen solution and the methacrylic anhydride solution is 1g-10g:0.5mL-5mL; the concentration of the recombinant humanized collagen solution is 0.01g / mL-0.05g / mL; and the volume concentration of the methacrylic anhydride solution is 0.05%-0.01%.

7. The method for preparing a biomimetic biological ink according to claim 5, characterized in that: The dosage ratio of hyaluronic acid, PBS and methacrylic anhydride solution in step (1) is 0.1 g to 2 g: 100 mL to 200 mL: 0.4 mL to 8 mL; In step (2), the mass ratio of 1-hydroxybenzotriazole, (2-aminoethyl)-4-[4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy]-butyramide, and (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1-2:0.1-2:1-2; the total concentration of the mixed solution is 30 g / L-40 g / L; and the usage ratio of the dialysate to the sodium chloride is 4 L-5 L:30 g-200 g.

8. A biomimetic biological ink, characterized in that: Prepared by the preparation method according to any one of claims 1 to 7.

9. An application of a biomimetic biological ink, characterized in that: The bionic biological ink described in claim 8 is used to construct a human auricle tissue substitute.

10. The use of a biomimetic biological ink according to claim 9, characterized in that: The construction of a human auricle tissue substitute includes the following steps: Laser scanning and computer-aided design are used to construct a three-dimensional digital model of the human ear morphology; based on the three-dimensional digital model of the human ear morphology, a 3D bioprinter is used to uniformly extrude the biomimetic bio-ink, and photocuring is performed to obtain a human auricle tissue substitute.

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