A solvent-free citric acid-based bio-ink, a preparation method thereof, a 3D printing method and application thereof

Abstract

The application belongs to the field of 3D printing biotechnology, and particularly relates to a kind of solvent-free citric acid-based bio-ink and its preparation method, 3D printing method and application. Including the following steps: citric acid and carboxyl, hydroxyl, amino or anhydride group-containing compound are prepared by polycondensation, ester exchange or substitution reaction to obtain citric acid-based prepolymer;The citric acid-based prepolymer is compounded with thickening agent to prepare citric acid-based bio-ink suitable for extrusion 3D printing. The application provides an efficient and accurate method for expanding the application of citric acid-based biomaterials in tissue engineering based on the extrusion 3D printing citric acid-based bio-ink. By adjusting the reaction conditions of citric acid-based prepolymer and the compounding ratio of thickening agent, the rheological curing characteristics of citric acid-based bio-ink are optimized.

Description

Technical Field

[0001] This invention belongs to the field of 3D printing biotechnology, specifically relating to a solvent-free citric acid-based bio-ink, its preparation method, 3D printing method, and application. Background Technology

[0002] Additive manufacturing technology has made rapid progress in the field of tissue engineering, particularly in the construction of personalized medical devices, biodegradable scaffolds, and patient-customized prostheses. Some biodegradable polymers, such as polylactic acid (PLA) and its copolymer PLGA, have become thermosetting biodegradable synthetic polymers authorized by the U.S. Food and Drug Administration (FDA). These polymers can be used in melt extrusion 3D printing (FDM) to meet the customized needs of patients, precisely matching the morphology of tissue defects, improving the fit between the scaffold and the host tissue, and contributing to enhanced local tissue regeneration and functional recovery. However, these materials typically suffer from long degradation cycles, mechanical properties that are difficult to match with tissue regeneration rates, and a general tendency to be biologically inert, preventing them from actively participating in local metabolic regulation or promoting tissue function recovery. This limits their applicability in the construction of high-functionality tissue scaffolds and related applications for functional recovery.

[0003] In recent years, novel thermosetting elastomer biomaterials based on citric acid have demonstrated significant application potential in areas such as tissue mechanical property regulation and organoid culture. Among them, the composite product of poly(octyl glycol) citrate and hydroxyapatite became one of the first FDA-approved biodegradable thermosetting polymers. Citric acid monomers contain three carboxyl groups and one hydroxyl group, and can react with polyols via ester bonds. By controlling the reaction ratio of citric acid to polyols, the catalytic type, and subsequent cross-linking conditions, a three-dimensional polymer network with controllable chain length can be formed. These materials are prepared through the condensation or cross-linking reaction of citric acid and polyols, featuring a simple synthesis process that can be achieved under mild, solvent-free, or low-temperature conditions. Furthermore, the degradation product citric acid is an important intermediate in energy metabolism in cellular mitochondria. Exogenous citric acid intake is absorbed by cells, participates in the tricarboxylic acid cycle, and generates ATP, better supporting tissue repair and regeneration. This unique metabolic regulatory function allows citric acid-based biomaterials not only to serve as physical scaffolds but also to promote cell function and tissue regeneration at the biochemical level, providing new insights for the construction of functional tissue engineering.

[0004] However, the mismatch between the rapid deposition window (seconds) and the long cross-linking time (days), along with the lack of rheological properties such as shear thinning and thixotropy, makes it difficult to directly use FDM for citrate-based biomaterials. Currently, the vast majority of citrate-based material products can only be prepared using mold-making processes, which severely restricts the promotion and application of citrate-based materials in 3D printing and broader biomanufacturing fields. Although a few teams have attempted to improve the printability of citrate-based materials using organic solvents, these solvents are highly volatile, resulting in a very short printing window, and the inks must be prepared and used immediately, which greatly limits the promotion of citrate-based materials in the 3D printing field. Therefore, developing a long-lasting, stable citrate-based bio-ink and a 3D method with a long printing window is of great significance for improving the metabolic regulation, biodegradability, and tissue regeneration compatibility of personalized medical implants. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to design and provide a technical solution for a solvent-free citric acid-based bio-ink, its preparation method, 3D printing method, and application.

[0006] The present invention is specifically implemented using the following technical solutions:

[0007] The first aspect of this invention provides a method for preparing a solvent-free citric acid-based bio-ink, comprising the following steps:

[0008] S.1 Citric acid-based prepolymers are prepared by reacting citric acid with compounds containing carboxyl, hydroxyl, amino, or anhydride groups through polycondensation, transesterification, or substitution reactions.

[0009] S.2 The citric acid-based prepolymer is compounded with a thickener to prepare a citric acid-based bio-ink suitable for extrusion 3D printing.

[0010] Furthermore, the compounds containing carboxyl, hydroxyl, amino, or anhydride groups mentioned in step S.1 include ethylene glycol, propylene glycol, hexanediol, polyethylene glycol, glycerol, pentaerythritol, xylitol, octanediol, serine, cysteine, glutamine, folic acid, tartaric acid, adipic acid, succinic acid, fumaric acid, acetic anhydride, or maleic anhydride.

[0011] Further, the molar ratio of citric acid to the compound containing carboxyl, hydroxyl, amino, or anhydride groups in step S.1 is 0.5 to 4; the reaction conditions are: reaction temperature of 60 to 160°C, reaction time of 0.5 to 12 h, and rotation speed of 100 to 1000 rpm.

[0012] Furthermore, the thickener in step S.2 includes citric acid powder, bentonite, montmorillonite, fumed silica, xanthan gum, hydroxymethyl cellulose, carrageenan, starch, gelatin, polyvinyl alcohol, or agar.

[0013] Furthermore, the citric acid-based powder is obtained through the following steps: taking citric acid-based prepolymer and uniformly mixing it with sodium chloride, potassium chloride, sodium bicarbonate, zinc chloride, or copper sulfate at a mass ratio of 0.5 to 4, and thermally crosslinking it to obtain a citric acid-based scaffold; soaking the citric acid-based scaffold in deionized water, physiological saline, or PBS buffer, lyophilizing it, and then grinding it to a particle size of 50 to 400 μm to obtain the citric acid-based powder.

[0014] Furthermore, the mass ratio of citric acid-based prepolymer to thickener in step S.2 is 0.5~4, and the viscosity of the compounded bio-ink is 50~5000 mPa·s.

[0015] Furthermore, the thermal crosslinking conditions are as follows: thermal crosslinking temperature is 60~160℃, and crosslinking time is 1~4 days.

[0016] A second aspect of the present invention provides a solvent-free citrate-based bio-ink prepared by the method described above.

[0017] The third aspect of the present invention provides a 3D printing method for bio-ink, which uses an extrusion 3D printer to print the solvent-free citric acid-based bio-ink into shape, and prepares a scaffold of citric acid-based biomaterial by means of heating crosslinking, photocrosslinking or chemical crosslinking.

[0018] Furthermore, the printing temperature and platform temperature of the extrusion 3D printing are both 4~37℃, and the extrusion speed is 0.1~2 cm. 3 The printing speed is 1~10 cm / s, and the printing layer thickness is 0.1~0.5 mm; the heating crosslinking temperature is 60~160℃, and the crosslinking time is 1~4 days.

[0019] The fourth aspect of the present invention provides a citrate-based biomaterial scaffold prepared by the method described above.

[0020] The fifth aspect of this invention provides the application of the aforementioned citrate-based biomaterial scaffold in the fields of mechanical property regulation, cell culture, or skin damage repair.

[0021] The present invention has the following beneficial effects:

[0022] (1) This invention is the first to develop a citric acid-based bio-ink based on extrusion 3D printing, providing an efficient and precise preparation method for expanding the application of citric acid-based biomaterials in tissue engineering. Based on solvent-free citric acid, by controlling the reaction conditions of citric acid-based prepolymer and the compounding ratio of thickener, the rheological properties and curing characteristics of citric acid-based bio-ink are optimized, effectively overcoming the problem of mismatch between printing time and curing speed of thermosetting materials in extrusion 3D printing, and significantly improving the printing accuracy and molding stability of complex structure scaffolds.

[0023] (2) The citrate-based biomaterial scaffold prepared by the present invention has adjustable mechanical strength and degradation rate. It can achieve drug delivery or tissue regeneration by adding functional components. It is widely applicable to the repair of tissues such as bone, cartilage, and skin, as well as the preparation of personalized implants.

[0024] (3) This invention utilizes the acceleration of the addition reaction between isocyanate and active groups such as hydroxyl and amino groups in the prepolymer molecule to accelerate the polymer reaction rate, thereby achieving the purpose of significantly shortening the post-printing processing time.

[0025] (4) The present invention uses Poisson's ratio pattern design to regulate the contraction stress of the scaffold, which is used to match the mechanical environment of human tissue, induce the qualitative behavior of cells and improve the stability after implantation into the recipient.

[0026] (5) The citric acid-based prepolymer prepared in this invention can be 3D printed to form a three-dimensional scaffold with suitable mechanical properties, pore structure and biocompatibility. It can be used to construct a biomimetic microenvironment to support cell adhesion, proliferation and differentiation. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the synthesis of citric acid-based prepolymers;

[0028] Figure 2 The mass spectrum of the citric acid-based prepolymer matrix is ​​obtained through laser-assisted refractive index mass spectra.

[0029] Figure 3 Images of citrate ester powder are shown, where (a) is an actual image of citrate ester powder and (b) is a scanning electron microscope image of citrate ester powder.

[0030] Figure 4 This is a picture of a pure citrate ester ink.

[0031] Figure 5 The rheological test results for pure citrate ester ink are shown in (a) and (b) respectively.

[0032] Figure 6 Images of the pure citrate scaffold are shown, where (a) is an actual image of the pure citrate scaffold and (b) is a scanning electron microscope image of the pure citrate scaffold.

[0033] Figure 7 Image of citrate ester bio-ink;

[0034] Figure 8 The images show the extrusion effect of citrate ester bio-ink, where (a) shows the extrusion effect of different ratios and (b) shows the printability evaluation image.

[0035] Figure 9 The rheological tests of citrate ester bio-ink are shown in (a) for viscosity test results and (b) for cyclic strain test results.

[0036] Figure 10 This is a diagram illustrating the printing process of citrate ester bio-ink.

[0037] Figure 11 The images show the physical object and electron microscope image of the three-dimensional cross-linked citric acid-based material scaffold, where (a) is the physical object of the citric acid-based material scaffold and (b) is its scanning electron microscope image.

[0038] Figure 12 The image shows the nuclear magnetic resonance (1H NMR) spectrum of photogel citrate, where (a) is the 1H NMR peak of citrate prepolymer, (b) is the 1H NMR of glycidyl methacrylate, and (c) is the 1H NMR peak of citrate-glycidyl methacrylate.

[0039] Figure 13 The images show the physical object and scanning electron microscope (SEM) image of the porous citrate ester three-dimensional scaffold, where (a) is the physical object of the porous citrate-based material scaffold and (b) is the SEM image.

[0040] Figure 14 The compressive modulus diagram of the citrate ester three-dimensional scaffold;

[0041] Figure 15 The diagram shows the mechanical control of the citrate ester three-dimensional scaffold, where (a) is a citrate ester three-dimensional scaffold with different Poisson's ratio patterns, and (b) is a diagram of the shrinkage stress of different Poisson's ratio patterns.

[0042] Figure 16 The images show rabbit chondrocytes cultured on a citrate 3D scaffold. The left image is a bright-field immunofluorescence image of rabbit chondrocytes on the 3D scaffold, and the right image is an immunofluorescence image of phalloidin on rabbit chondrocytes on the 3D scaffold.

[0043] Figure 17 The diagram shows the angiogenesis status of human umbilical vein endothelial cells, where (a) is a fluorescence image of angiogenesis in human umbilical vein endothelial cells cultured in blank and citrate scaffold extract, (b) is a diagram showing the number of branch nodes in angiogenesis, and (c) is a diagram showing the total length of the vessels.

[0044] Figure 18This image shows the effect of a citrate ester three-dimensional scaffold in skin damage repair. Detailed Implementation

[0045] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0046] Example 1: Preparation of Citric Acid-Based Prepolymers by Reaction of Citric Acid with Polyols Citric acid was reacted with polyols to prepare citric acid-based prepolymers, the structural formula of which is as follows: Figure 1 As shown.

[0047] Add 19.2 g of citric acid and 14.6 g of octanediol to a 200 mL volumetric flask, set the oil bath temperature to 160℃, and the reaction speed to 100 rpm for 10 min until the citric acid and octanediol are completely dissolved.

[0048] The oil bath temperature was set to 140℃, the rotation speed was set to 600 rpm, nitrogen gas was introduced, and the reaction was carried out for 60 min to obtain the citrate prepolymer.

[0049] Turn off the oil bath, add 20 mL of hexaoxide, set the stirring speed to 100 rpm, and stir for 5 min to dissolve the citrate prepolymer in the hexaoxide.

[0050] Pour the citrate prepolymer solution into 2 L of deionized water, wait for the prepolymer to precipitate, remove the supernatant, repeat 4 times to remove unreacted citric acid and octanediol monomer.

[0051] The citrate prepolymer was transferred to a wide-mouth bottle and freeze-dried for 3 days to obtain a purified citrate prepolymer. Figure 2 ).

[0052] Example 2: Preparation of pure citric acid-based bio-ink by compounding citric acid-based prepolymer with citric acid-based materials

[0053] Referring to Example 1, a citrate prepolymer was synthesized by mixing 5 g of citrate prepolymer with 10 g of sodium chloride and then adding the mixture into a polytetrafluoroethylene dish. The mixture was heated at 80°C for two days to obtain a cross-linked citrate scaffold.

[0054] The cross-linked citrate scaffold was soaked in PBS for 5 days to remove sodium chloride, and then transferred to a freeze dryer for freeze drying to obtain the freeze-dried citrate scaffold.

[0055] The freeze-dried citrate scaffold was ground to obtain citrate powder (e.g. Figure 3 ).

[0056] 1 g of citrate prepolymer was mixed evenly with 0.7 g of citrate particles to obtain pure citrate bio-ink.

[0057] Figure 4 , Figure 5 The results showed that pure citrate bio-ink has shear-thinning properties and is suitable for extrusion 3D printing; Figure 5 Part (b) is a cyclic strain scanning structure, which is tested cyclically from 1% to 100% strain. The results show that the pure citrate bio-ink has self-healing properties.

[0058] Example 3: Pure citrate ester bio-ink for 3D printing

[0059] Pure citrate bio-ink was prepared according to Example 2. The pure citrate bio-ink was then printed using a BioMaker bio-3D printer. The ink-containing cartridge was transferred into the printer, with the cartridge temperature controlled at 25°C, the platform temperature at 10°C, and the base speed controlled at 0.1 cm. 3 The printing speed is 2 cm / s, and the printing layer thickness is 0.22 mm.

[0060] The printed citrate ester scaffold was transferred to 80 °C and heated for two days to obtain a three-dimensional cross-linked pure citrate-based material scaffold, as shown in Figure 6.

[0061] Example 4: Preparation of citric acid-based bio-ink by compounding citric acid-based prepolymer with thickener

[0062] 1 g of citrate prepolymer was uniformly mixed with 0 mg, 150 mg, 250 mg, and 350 mg of fumed silica to obtain citrate bio-ink. The citrate bio-ink was transferred to a 5 mL syringe and centrifuged at 7000 rpm for 5 min to obtain de-aerated citrate bio-ink. Figure 7 This is a picture of a citrate ester bio-ink.

[0063] Figure 8Part (a) shows the extrusion results of 1 g of citrate prepolymer uniformly mixed with 0 mg, 150 mg, 250 mg, and 350 mg of fumed silica. At a mixing ratio of 0 mg, the citrate ink collapses; at a mixing ratio of 150 mg, the citrate ink fails to maintain its morphology well; at a mixing ratio of 250 mg, the citrate bio-ink maintains the stability of the printed structure; and at a mixing ratio of 350 mg, the citrate ink is too viscous. Figure 8 Part (b) is the printability evaluation of inks mixed in different proportions. The printability of the citrate ink mixed with 250 mg of fumed silica is 1.02, which is closest to 1, while the printability of 150 mg and 350 mg is 0.88 and 1.08, respectively, which are relatively poor.

[0064] Figure 9 The results showed that citrate ester bio-ink has shear-thinning properties and is suitable for extrusion 3D printing; Figure 9 Part (b) is a cyclic strain scanning structure, which is tested under cyclic strain from 1% to 200%. The results show that the citrate bio-ink has good self-healing properties.

[0065] Example 5: Citrate ester bio-ink for 3D printing

[0066] Refer to Example 3 for printing citrate bio-ink. Figure 10 This is a diagram of the citrate ester bio-ink printing process; the printed citrate ester scaffold is transferred to 80℃ and heated for two days to form a three-dimensional cross-linked citrate-based material scaffold.

[0067] Figure 11 Part (a) indicates that, using the BioMaker bio-3D printer, citrate ester bio-ink can be printed into structures such as noses, bones, and blood vessels; Figure 11 Part (b) indicates that the citrate ester ink, after crosslinking, has a substantial filamentous structure.

[0068] Example 6: Preparation and Printing Method of Photogel Citrate Bio-ink

[0069] Referring to Example 1, a citrate prepolymer was synthesized by dissolving the citrate prepolymer in 50% w / v hexaoxide. 5 g of the citrate solution, 5 g of glycidyl methacrylate, and 0.05 g of lithium phenyl-2,4,6-trimethylbenzoylphosphonate were added to a 200 mL flask and reacted overnight at 80 °C, 300 rpm, under nitrogen protection to obtain a photocrosslinkable citrate prepolymer.

[0070] Pour the photocrosslinked citrate prepolymer solution into 2 L of deionized water, wait for the prepolymer to precipitate, remove the supernatant, repeat 4 times to remove unreacted glycidyl methacrylate and lithium phenyl-2,4,6-trimethylbenzoylphosphonate.

[0071] The citrate prepolymer was transferred to a wide-mouth bottle and freeze-dried for 3 days to obtain a purified photocrosslinked citrate prepolymer. Figure 12 The image shows the 1H NMR peaks of photogel citrate, where... Figure 12 Part (a) represents the 1H NMR peak of the citrate prepolymer. Figure 12 Part (b) is the 1H NMR of glycidyl methacrylate. Figure 12 Part (c) is the 1H NMR peak of citrate-glycidyl methacrylate. The purified prepolymer was characterized by 1H NMR. The results showed that the integral ratio of the characteristic peaks of citric acid and 1,8-octanediol was basically consistent with the initial reaction feed ratio. The results of citrate-glycidyl methacrylate indicate the presence of the characteristic peak of glycidyl methacrylate, and the integral ratio of the characteristic peaks is basically consistent with the initial feed ratio.

[0072] Referring to Example 4, a photo-crosslinked citrate prepolymer was compounded with fumed silica to obtain a photogel citrate bio-ink.

[0073] Referring to Example 3, a photocrosslinked citric acid ester prepolymer was printed. During the printing process, a 405 nm excitation light source was applied to obtain a three-dimensional photocrosslinked citric acid-based material scaffold.

[0074] Example 7: Preparation of porous citrate ester scaffold. The citrate ester prepolymer was synthesized according to Example 1.

[0075] Sodium chloride was ground into powder and filtered through a 300-mesh filter to obtain sodium chloride particles with a diameter of less than 50 mm.

[0076] 1g of citrate prepolymer was uniformly mixed with 3g of sodium chloride particles to obtain citrate bio-ink.

[0077] Referring to Example 3, after printing citrate ester bio-ink and obtaining a three-dimensional cross-linked citrate-based material scaffold, the scaffold was transferred to deionized water, soaked for 5 days, and then freeze-dried to obtain a porous citrate ester three-dimensional scaffold.

[0078] Figure 13 Part (a) indicates that porous citrate 3D scaffolds can be printed using a BioMaker bio-3D printer to create porous ear, nose, and bone morphologies from citrate bio-ink. Figure 13 Part (b) indicates that the porous citrate 3D scaffold exhibits a porous structure left by sodium chloride occupancy.

[0079] Example 8: Preparation and Printing Method of Rapidly Crosslinked Citric Acid-Based Bio-Ink

[0080] Citric acid ester prepolymer was synthesized according to Example 1.

[0081] Referring to Example 4, 1g of citrate prepolymer, 50mg of isocyanate, and 250mg of fumed silica were uniformly mixed to obtain a citrate bio-ink that can be rapidly crosslinked.

[0082] Referring to Example 3, a citrate ester scaffold was printed. The printed three-dimensional scaffold was left at room temperature overnight to obtain a cross-linked citrate ester three-dimensional scaffold.

[0083] Figure 14 The diagram shows a comparison of the compressive modulus of the three-dimensional scaffolds obtained in Examples 3, 5, 6, 7, and 8. The compressive modulus of the pure citrate ester three-dimensional scaffold is 120 kPa, the compressive modulus of the citrate ester three-dimensional scaffold is 140 kPa, the compressive modulus of the photocrosslinked citrate ester three-dimensional scaffold is 210 kPa, the compressive modulus of the porous citrate ester three-dimensional scaffold is 50 kPa, and the compressive modulus of the rapidly crosslinked citrate ester three-dimensional scaffold is 610 kPa.

[0084] Example 9: Citrate ester three-dimensional scaffold for mechanical property regulation

[0085] Citric acid ester bio-ink was obtained with reference to Example 4.

[0086] Using the BioMaker 3D printer, a citrate ester three-dimensional scaffold with a length of 1cm, a width of 1.2cm, a single layer height of 0.2mm, and a total height of 4 layers was printed. The scaffolds had four Poisson ratio structures: honeycomb square, honeycomb rectangle, honeycomb hexagon, and concave hexagon.

[0087] The printed scaffold was transferred to 80°C and heated for 2 days to obtain a cross-linked citrate ester three-dimensional scaffold, as shown below. Figure 15 Part (a).

[0088] Finite element analysis of the three-dimensional citrate scaffold yielded injury stress ranges of 4505-236394 Pa, 10044-206938 Pa, 11682-277316 Pa, and 231-1003551 Pa. Figure 15 Part (b).

[0089] Example 10: Citrate ester three-dimensional scaffold for cell culture

[0090] A three-dimensional scaffold made of citrate was obtained with reference to Example 5.

[0091] The citrate ester three-dimensional scaffold was immersed in deionized water for 5 days to equilibrate the pH.

[0092] The citrate 3D scaffold was sterilized with ethylene oxide and then soaked in DMEM culture medium overnight.

[0093] The citrate 3D scaffold was washed three times with PBS. (The last part, "1x10", appears to be a typo and should be left as is.) 6 Rabbit chondrocytes were seeded on the surface of a scaffold and placed in an incubator for 20 minutes. The scaffold was then flipped over and placed in the incubator for another 20 minutes. DMEM culture medium was then added to culture the cells.

[0094] Figure 16 This is a diagram of rabbit chondrocytes cultured on a citrate ester scaffold, in which... Figure 16 The left image is a bright-field immunofluorescence image of rabbit chondrocytes on a three-dimensional scaffold. The cells have completely covered the scaffold surface. Figure 16 The right image shows an immunofluorescence image of phalloidin on rabbit chondrocytes on a three-dimensional scaffold.

[0095] A pH-balanced citrate ester three-dimensional scaffold was obtained with reference to Example 10.

[0096] The citrate 3D scaffold was sterilized with ethylene oxide and then immersed in ECM culture medium at a concentration of 200 mg / mL for 24 h.

[0097] The obtained extract was diluted to a 20% concentration using ECM culture medium and added to 2x10⁻¹⁰ mL of water. 6 Human umbilical vein endothelial cells were cultured overnight.

[0098] Human umbilical vein endothelial cells were digested and placed into a matrix gel-coated culture plate at a density of 1.5 x 10⁻⁶. 5 Cells were cultured at high density for 6 hours.

[0099] Figure 17 The image shows the angiogenesis status of human umbilical vein endothelial cells. Human umbilical vein endothelial cells cultured with citrate scaffold extract have strong angiogenesis ability, forming 176 branch nodes and a total vessel length of 43.5 micrometers, which is higher than the 114 and 5 micrometers of the control group.

[0100] Example 11: Citrate ester three-dimensional scaffold for skin damage repair

[0101] A porous citrate ester three-dimensional scaffold was obtained with reference to Example 5.

[0102] The porous citrate 3D scaffold was immersed in deionized water for 5 days to equilibrate the pH.

[0103] The porous citrate 3D scaffold was sterilized with ethylene oxide.

[0104] A full-scale skin injury model was created in SD rats. A sterile porous citrate ester three-dimensional scaffold was filled into the skin wound, the skin wound was fixed with a fixation ring, and gauze was applied.

[0105] Figure 18 The image shows the effect of the citrate ester three-dimensional scaffold on skin damage repair. Compared with the control group, the mice containing the citrate ester three-dimensional scaffold have higher skin repair efficiency.

[0106] The above-described embodiments are a detailed description of the preparation method and printing application of citric acid-based bio-ink, intended to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be easily made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the interpretation of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A method for preparing a solvent-free citric acid-based bio-ink, characterized in that, Includes the following steps: S.1 Citric acid-based prepolymers are prepared by polycondensation reaction of citric acid and hydroxyl-containing compounds; The hydroxyl-containing compounds include ethylene glycol, propylene glycol, glycerol, xylitol, or octanediol; S.2 The citric acid-based prepolymer is compounded with a thickener to prepare a citric acid-based bio-ink suitable for extrusion 3D printing; The thickener includes citric acid powder, bentonite, montmorillonite, fumed silica, xanthan gum, hydroxymethyl cellulose, carrageenan, starch, gelatin, polyvinyl alcohol, or agar. The citric acid-based powder is obtained through the following steps: citric acid-based prepolymer is uniformly mixed with sodium chloride, potassium chloride, sodium bicarbonate, zinc chloride, or copper sulfate at a mass ratio of 0.5-4, and thermally crosslinked to obtain a citric acid-based scaffold; the citric acid-based scaffold is soaked in deionized water, physiological saline, or PBS buffer, freeze-dried, and then ground to a particle size of 50-400 μm to obtain the citric acid-based powder.

2. The method for preparing a solvent-free citric acid-based bio-ink as described in claim 1, characterized in that, The molar ratio of citric acid to the hydroxyl-containing compound in step S.1 is 0.5 to 4; the reaction conditions are: reaction temperature of 60 to 160°C, reaction time of 0.5 to 12 h, and rotation speed of 100 to 1000 rpm.

3. The method for preparing a solvent-free citric acid-based bio-ink as described in claim 1, characterized in that, The mass ratio of citric acid-based prepolymer to thickener mentioned in step S.2 is 0.5~4, and the viscosity of the compounded bio-ink is 50~5000 mPa·s.

4. The method for preparing a solvent-free citric acid-based bio-ink as described in claim 3, characterized in that, The thermal crosslinking conditions are as follows: thermal crosslinking temperature is 60~160℃, and crosslinking time is 1~4 days.

5. A solvent-free citric acid-based bio-ink prepared by any of the preparation methods described in claims 1-4.

6. A 3D printing method based on the bio-ink of claim 5, characterized in that, The solvent-free citric acid-based bio-ink was printed using an extrusion 3D printer, and a scaffold of citric acid-based biomaterial was prepared by thermal crosslinking, photocrosslinking, or chemical crosslinking.

7. The 3D printing method as described in claim 6, characterized in that, The extrusion 3D printing process involves a printing temperature of 4~37℃, a platform temperature of 4~37℃, and an extrusion speed of 0.1~2 cm. 3 The printing speed is 1~10 cm / s, and the printing layer thickness is 0.1~0.5 mm; the heating crosslinking temperature is 60~160℃, and the crosslinking time is 1~4 days.

8. A citrate-based biomaterial scaffold obtained by printing according to claim 6 or 7.

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