3D printing method for forming directional filament structures in situ

Abstract

A three-dimensional (3D) printing method for forming directional filament structures in situ, comprising: preparing a reversible temperature-sensitive material A into microparticles / microgels, and mixing the microparticles / microgels as a discrete phase with a continuous phase material B solution to obtain a multiphase printing ink; loading the multiphase printing ink into an extrusion printer, controlling the temperature so that the discrete phase is thermally softened into a sol state, simultaneously performing extrusion printing to extrude the ink from a cartridge to obtain discrete phase-continuous phase composite directional filament structures, and on the basis of a designed 3D model, implementing 3D printed structure forming; and after printing is finished, performing optional and independent post-crosslinking treatment on the continuous phase and the discrete phase in a printed structure to obtain different 3D structures having directional characteristics. After the multiphase ink undergoes the printing process that couples phase change and shape change, manufacture of personalized complex 3D structures having directional microfiber characteristics can be achieved.

Description

A 3D printing method for in-situ formation of oriented filament structures Technical Field

[0001] The present invention belongs to the fields of advanced manufacturing and biomedicine, and specifically relates to a 3D printing method for in-situ forming of oriented filament structures, and more specifically focuses on a 3D printing method that can produce oriented microstructures. Background Art

[0002] Directed structures are widely present in nature, which endow structures with specific properties and functions. For example, plants have many directional vertical channels that are used to transport ions, water and other components to upper tissues to achieve nutrient delivery and meet the needs of plant photosynthesis [Zhu M, Song J, Li T, Gong A, Wang Y, Dai J, Yao Y, Luo W, Henderson D and Hu L 2016 Highly Anisotropic, Highly Transparent Wood CompositesAdvanced Materials285181-7]. Animals also have tissues with directional structures, such as muscles, cartilage, tendons, nerves, blood vessels and parts of the intestine [Khuu N, Kheiri S and Kumacheva E 2021 Structurally anisotropic hydrogels for tissue engineeringTrends in Chemistry 31002-26,Xing J, Liu N, Xu N, Chen W and Xing D 2022 Engineering Complex Anisotropic Scaffolds beyond Simply Uniaxial Alignment for Tissue EngineeringAdvanced Functional Materials 322110676].Compared with non-directional structures, directional structures not only play an important role in life systems, but their anisotropic characteristics in mechanical, electrical, thermal, optical and other aspects, and the unique properties they bring, are also widely used in engineering applications. For example, the mechanical enhancement properties brought by directional structures are used in the manufacture of load-bearing steel bars [Chen J, Liu X, Tian Y, Zhu W, Yan C, Shi Y, Kong LB, Qi HJ and Zhou K 2022 3D-Printed Anisotropic Polymer Materials for Functional ApplicationsAdvanced Materials 342102877], directional porous structures are used in the development of supercapacitors [Wang F, Chen L, He S, Zhang Q, Liu K, Han X, Duan G and Jiang S 2022 Design of wood-derived anisotropic structural carbon electrode for high-performance supercapacitorWood Science and Technology 561191-203], and equipment [Wang Z, Luan C, Liao G, Yao X and Fu J 2019 Mechanical and self-monitoring behaviors of 3D printing smart continuous carbon fiber-thermoplastic lattice truss sandwich structure Composites Part B: Engineering 176107215] or the design and manufacture of wearable sensors [ Chen C, Wang Y, Wu Q, Wan Z, Li D and Jin Y 2020 Highly strong and flexible composite hydrogel reinforced by aligned wood cellulose skeleton via alkali treatment for muscle-like sensors Chemical Engineering Journal 400125876]. Therefore, the construction of oriented structures is of great significance.

[0003] 3D printing is based on the concept of free-form fabrication and can produce personalized, complex three-dimensional structures. Based on the characteristics of the molding technology, current 3D printing technologies generally include powder bed melting, vat photopolymerization, spraying, extrusion, and sheet lamination [Ligon SC, Liska R, Stampfl J, Gurr M and Muelhaupt R 2017 Polymers for 3D Printing and Customized Additive ManufacturingChemical Reviews 11710212-90]. Extrusion methods include melt extrusion and solution extrusion. These printing methods can print a variety of materials and have a wide range of applications. Melt extrusion printing materials include polylactic acid, acrylonitrile butadiene styrene, polyurethane, polyether ether ketone, etc. [Daminabo SC, Goel S, Grammatikos SA, Nezhad HY and Thakur VK 2020 Fused deposition modeling-based additive manufacturing (3D printing): techniques for polymer material systemsMaterials Today Chemistry 16100248]. Solution extrusion supports the printing of materials with high water content, such as hydrogels and living cells [Zhang YS, Haghiashtiani G, Hubscher T, Kelly DJ, Lee JM, Lutolf M, McAlpine MC, Yeong WY, Zenobi-Wong M and Malda J 2021 3D extrusion bioprintingNature Reviews Methods Primers 1]. Therefore, 3D printing is used to manufacture directional structures of different materials.

[0004] Conventional extrusion printing methods can extrude material filaments with unit sizes generally exceeding 100 microns, which limits the minimum features of the directional structures that can be constructed [Ligon SC, Liska R, Stampfl J, Gurr M and Muelhaupt R 2017 Polymers for 3D Printing and Customized Additive ManufacturingChemical Reviews11710212-90]. Traditional extrusion printing processes can reduce the size of material filament units under the action of an electric field: electrospinning technology can produce micron-sized filaments and obtain a certain degree of oriented structure, but the directional controllability is limited, it is difficult to achieve precise deposition of filaments, and it is difficult to form thick structures [Sun B, Long YZ, Zhang HD, Li MM, Duvail JL, Jiang XY and Yin HL 2014 Advances in three-dimensional nanofibrous macrostructures via electrospinning Prog. Polym. Sci. 39862-90]; near-field direct writing technology can control the arrangement of micron-sized filaments and form good directionality, but it is also limited by the manufacturing speed and it is difficult to form thick structures [Kade JC and Dalton PD 2021 Polymers for Melt Electrowriting Advanced Healthcare Materials 102001232].

[0005] Existing technologies preload fine fibers within the extruded material and orient the fine fibers through extrusion shearing, but short fibers are generally used and the end-to-end alignment of the fibers is uncontrollable, making it difficult to achieve a directional continuous fiber distribution with a large aspect ratio. For example, after using electrospinning technology to make a fiber membrane of norbornene-modified hyaluronic acid, it can be chopped to obtain short fibers less than 20 microns in length. These short fibers can be mixed with bio-ink and rearranged along the extrusion direction to form a directional arrangement after extrusion printing, but the continuity of the short fibers remains to be solved [Prendergast ME, Davidson MD and Burdick JA 2021 A biofabrication method to align cells within bioprinted photocrosslinkable and cell-degradable hydrogel constructs via embedded fibers BIOFABRICATION 13]. There is also a technology that extrudes a two-phase immiscible system material. Under the action of extrusion shear, the two materials form a layered directional fluid, thereby obtaining a structure with a certain directional effect, but the size uniformity of the internal directional through-pore structure is poor. For example, a mixed material system of methacrylated gelatin and polyethylene oxide can be used to obtain layered oriented fibers through coaxial extrusion and photocuring crosslinking. However, there is a clear phenomenon of interconnectedness between the layers within the oriented fibers [Shao L, Hou R, Zhu Y and Yao Y 2021 Pre-shear bioprinting of highly oriented porous hydrogel microfibers to construct anisotropic tissues Biomater. Sci. 96763-71]. Another method is to use a Kenics static mixer to achieve the layering of two different materials, resulting in a fiber structure with an oriented effect.Specifically, two materials are injected into a mixer at the same time. During the flow and mixing process, the materials will produce stratification. The delaminated fibers are cross-linked and fixed to obtain oriented fibers. However, the internal oriented structure is lamellar, and it is difficult to obtain filamentous substructures [Bolívar-Monsalve EJ, Ceballos-González CF, Borrayo-Montaño KI, Quevedo-Moreno DA, Yee-de León JF, Khademhosseini A, Weiss PS, Alvarez MM and Trujillo-de Santiago G 2021 Continuous chaotic bioprinting of skeletal muscle-like constructsBioprinting21e00125, Bolívar-Monsalve EJ, Ceballos-González CF, Chávez-Madero C, de la Cruz-Rivas BG, Velásquez Marín S, Mora-Godínez S, Reyes-Cortés LM, Khademhosseini A, Weiss PS, Samandari M, Tamayol A, Alvarez MM and Trujillo-de Santiago G 2022 One-Step Bioprinting of Multi-Channel Hydrogel Filaments Using Chaotic Advection: Fabrication of Pre-Vascularized Muscle-Like Tissues Advanced Healthcare Materials 112200448]. In summary, it remains challenging to construct continuous micron-oriented structures within 3D printed models. SUMMARY OF THE INVENTION

[0006] The present invention prepares a multiphase printing ink by preparing a reversible temperature-sensitive material A into microparticles / microgels, mixing the microparticles / microgels as a discrete phase with a solution of a continuous phase material B; the multiphase printing ink is loaded into an extrusion printer, and the temperature is controlled so that the discrete phase microparticles / microgels are thermally softened into a sol state; at the same time, extrusion printing is performed to extrude the ink from a silo; during the extrusion flow, the discrete phase is sheared and deformed to form fine filaments, thereby obtaining a discrete-continuous phase composite oriented filament structure; based on a designed three-dimensional model, 3D printing structure formation is implemented; after printing, the continuous phase and the discrete phase in the printed structure are optionally and independently post-processed and cross-linked to obtain different three-dimensional structures with directional characteristics. Technical issues

[0007] Existing 3D printing has difficulty in producing continuous micron-oriented structures within its printed structures. Technical Solutions

[0008] The purpose of the present invention is to provide a new 3D printing method that can couple the phase change and deformation of multiphase ink to form a directional filament microstructure in situ, so as to solve the problem that 3D printing is difficult to produce continuous micron directional structures within its printed structure.

[0009] The 3D printing method for in-situ formation of an oriented filament structure provided by the present invention comprises the following steps:

[0010] 1) The reversible thermosensitive material A is made into microparticles / microgels, which are used as the discrete phase and mixed with the continuous phase material B solution to obtain a multiphase printing ink;

[0011] 2) Loading the multiphase printing ink into an extrusion printer, controlling the temperature to thermally soften the discrete phase microparticles / microgels into a sol state, and simultaneously performing extrusion printing to squeeze the ink from the hopper. During the extrusion flow, the discrete phase is sheared and deformed to form fine filaments, resulting in a discrete-continuous phase composite oriented filament structure. Based on the designed 3D model, 3D printing is then performed to form the structure.

[0012] 3) After printing, the continuous and discrete phases in the printed structure are optionally and independently post-processed and cross-linked to obtain different three-dimensional structures with directional characteristics.

[0013] In step 1) of the above method, the reversible temperature-sensitive material A may be at least one of the following 1)-3): 1) gelatin or modified gelatin or a mixture thereof; 2) gelatin or modified gelatin or a mixture thereof + a blended phase, wherein the blended phase may be one or more of hyaluronic acid, silk fibroin, heparin, polyethylene glycol, dextran, chondroitin sulfate, sodium alginate, chitosan, and fibrinogen; 3) poly(N-isopropylacrylamide) and its modified products;

[0014] The reversible temperature-sensitive material A may further contain a hydrogel material that can form a stable structure through chemical or physical crosslinking. The hydrogel material that can form a stable structure through chemical or physical crosslinking may serve as the main component or additive of the discrete phase microparticles / microgels.

[0015] Furthermore, the hydrogel material capable of forming a stable structure through chemical and physical cross-linking can specifically be methacrylated gelatin;

[0016] The method for preparing the reversible temperature-sensitive material A into microparticles / microgels is not limited and can be selected from any one of droplet microfluidics, oil-water emulsion method, complex coacervation method, electrospray method, mechanical crushing method, etc.;

[0017] First, the reversible temperature-sensitive material A is prepared into a reversible temperature-sensitive material A solution, and then prepared into microparticles / microgels by any of the above methods;

[0018] In the reversible temperature-sensitive material A solution, the mass concentration of the reversible temperature-sensitive material A may be 2-20%, preferably 10%;

[0019] The reversible temperature-sensitive material A solution can be prepared using 0.9% sodium chloride solution, phosphate buffered saline solution or ultrapure water as a solvent.

[0020] The microparticles / microgels may be in the form of spheres (diameter between 10-5000 microns), polyhedrons, and other irregular three-dimensional shapes (volume between 1000 cubic microns and 125 cubic millimeters); specifically, they may be spheres with a diameter ranging from 100-250 microns.

[0021] The continuous phase material B maintains extrudability within the operating temperature range and has poor miscibility with the reversible temperature-sensitive material A;

[0022] Specifically, the continuous phase material B may be selected from: 1) Pluronic or modified Pluronic or a mixture thereof; 2) Pluronic or modified Pluronic or a mixture thereof + a blended phase, wherein the blended phase may be one or more of hyaluronic acid, silk fibroin, heparin, polyethylene glycol, dextran, chondroitin sulfate, sodium alginate, chitosan, fibrinogen, etc.; 3) polyethylene glycol or modified polyethylene glycol or a mixture thereof;

[0023] The continuous phase material B may also contain a photosensitive crosslinking component to facilitate subsequent post-processing crosslinking of the continuous phase;

[0024] Preferably, the continuous phase material B contains photosensitive modified Pluronic,

[0025] The continuous phase material B is Pluronic or modified Pluronic or a mixture thereof, and the mass concentration of Pluronic or modified Pluronic or a mixture thereof in the solution of the continuous phase material B is 10-50%, preferably 30%;

[0026] The continuous phase material B is polyethylene glycol or modified polyethylene glycol or a mixture thereof, and the mass concentration of the polyethylene glycol or modified polyethylene glycol or a mixture thereof in the solution of the continuous phase material B is 5-50%;

[0027] The continuous phase material B solution further contains a photoinitiator, and the mass concentration of the photoinitiator can be 0.05-0.5%;

[0028] The photoinitiator can be specifically phenyl (2,4,6-trimethylbenzoyl) lithium phosphate,

[0029] The continuous phase material B solution can use 0.9% sodium chloride solution, phosphate buffered saline solution or ultrapure water as a solvent.

[0030] The mixed mass volume ratio of the discrete phase and the continuous phase material B solution can range from 1 g: 2 ml to 1 g: 128 ml, preferably 1 g: 8 ml;

[0031] The temperature control is to control the ink reservoir or nozzle to be above the drawing temperature, which refers to the temperature at which the discrete phase microparticles / microgels can be thermally softened into a sol state, and the drawing temperature is the sol temperature / melting temperature of material A or above;

[0032] For reversible temperature-sensitive materials with different components or different concentrations, the drawing temperature is different;

[0033] The reversible temperature-sensitive material A is mainly gelatin or modified gelatin, and the drawing temperature is 32° or above;

[0034] The reversible temperature-sensitive material A is poly(N-isopropylacrylamide) and its modified products, and the drawing temperature is 32° C. or above;

[0035] The nozzle diameter is 0.2-2 mm, preferably 0.26 mm, and the printing speed is controlled at 5-20 mm / s, preferably 10 mm / s;

[0036] Step 3) involves post-crosslinking the continuous phase in the printed structure to crosslink the hydrogel material contained in the reversible thermosensitive material A microparticles / microgel, which can form a stable structure through chemical and physical crosslinking, to obtain a hydrogel structure with oriented microfilaments inside.

[0037] Alternatively, the continuous phase in the printed structure is post-crosslinked, and the hydrogel structure after the post-crosslinking treatment is immersed in a temperature above the dissolution temperature of the reversible thermosensitive material A, so that the reversible thermosensitive material A sol is dissolved and directional through-holes are formed inside the hydrogel structure, thereby obtaining a directional through-hole hydrogel structure;

[0038] Alternatively, the continuous phase in the printed structure and the hydrogel material contained in the reversible thermosensitive material A microparticles / microgel, which can form a stable structure through chemical or physical crosslinking, are selectively crosslinked (specific operation: selectively irradiate the printed structure through a mask to remove the uncured continuous phase), to obtain a single fiber or an aligned fiber scaffold;

[0039] The continuous phase in the printed structure is post-crosslinked by irradiating light. The wavelength of the light source may be 200-410 nm, specifically 405 nm, and the irradiation time may be 10-300 seconds, preferably 180 seconds.

[0040] The three-dimensional structure with directional characteristics prepared by the above method (hydrogel structure with directional fine filaments inside, directional through-hole hydrogel structure, single fiber or directional fiber scaffold) also falls within the scope of protection of the present invention. Beneficial effects

[0041] The preparation method of the present invention has the following advantages over the prior art:

[0042] 1. The multiphase ink of the present invention can obtain complex 3D structures after a printing process that couples phase change and deformation, enabling the fabrication of three-dimensional solids with personalized oriented fine fibers or fine fiber bundles.

[0043] 2. The fabrication of three-dimensional oriented micronized hydrogel structures, oriented channel structures, single fibers or fiber bundles can be achieved through selective cross-linking;

[0044] 3. It can be combined with biological 3D printing to realize the preparation of cell-carrying three-dimensional structures with directional characteristics while carrying cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG1 is a flow chart of 3D printing for in-situ formation of oriented filament structures according to the present invention.

[0046] FIG2 is a schematic diagram of the technical solution of the 3D printing method for in-situ forming of an oriented filament structure according to the present invention.

[0047] FIG3 is a schematic diagram of the manufacture of the oriented microfilament / through-pore hydrogel structure in Example 1 of the present invention.

[0048] FIG4 shows the effect of printing temperature on the multiphase system ink extrusion printing process in Example 1 of the present invention.

[0049] FIG5 is a three-dimensional hydrogel model with an oriented filament substructure prepared in Example 1 of the present invention.

[0050] FIG6 is a diagram showing the diameter distribution of microwires within the oriented structure prepared in Example 1 of the present invention.

[0051] FIG7 shows the cell survival statistics in the oriented cell hydrogel structure prepared in Example 2 of the present invention.

[0052] FIG8 is a staining image of a muscle fiber with a large aspect ratio of the oriented cell hydrogel structure prepared in Example 2 of the present invention.

[0053] FIG9 is a schematic diagram of the technology for preparing a single fiber according to Example 3 of the present invention.

[0054] FIG10 is a technical schematic diagram of preparing an oriented fiber scaffold according to Example 3 of the present invention.

[0055] FIG11 is an oriented fiber scaffold after the continuous phase is removed in Example 3 of the present invention.

[0056] FIG12 shows the cell extension induced by the oriented fiber scaffold in Example 3 of the present invention.

[0057] FIG13 is a schematic diagram of the oriented fiber pore scaffold prepared in Example 4 of the present invention.

[0058] Figure 14 shows the oriented hydrogel silk structure based on modular discrete phase multiphase ink.

[0059] Figure 15 shows the fabrication of directional heterostructures based on modular discrete phase multiphase inks.

[0060] FIG16 shows the fabrication of a directional hydrogel silk structure based on physically cross-linked materials in the discrete phase.

[0061] FIG17 shows the fabrication of a directional hydrogel silk structure based on a continuous phase material with physical cross-linking properties. Best Mode for Carrying Out the Invention

[0062] The present invention adopts a multiphase system ink. During the extrusion printing process, the in-situ shear effect is utilized to shear the blocky discrete phases in the printing ink into fibers with a large aspect ratio, construct fine oriented fibers within the extruded material filament units, and achieve a graded oriented effect (Figures 1 and 2).

[0063] Basic operation process: Prepare microparticles A as the discrete phase and mix them with the continuous phase solution B to prepare a multiphase printing ink (S10). The ink is loaded into an extrusion printer, and the solid A is adjusted to a fluid state (solid-liquid transition) by temperature control, and extrusion printing is performed simultaneously. Phase A is sheared and deformed during flow to form fine filaments. The final extruded material filaments contain highly oriented fine filaments of the discrete phase of material A and a continuous phase of material B. Then, under computer control, based on the designed three-dimensional model, 3D printing structure formation is implemented (S20). After printing is completed, the continuous phase and discrete phase in the printed structure are optionally and independently post-processed and cross-linked. (S30) (Figure 1)

[0064] In the following examples, methacrylated gelatin was prepared by the following method:

[0065] 1. Add 1 g of gelatin solid to 10 ml of ultrapure water and stir at 50°C until completely dissolved to obtain a gelatin solution.

[0066] 2. While stirring at 50°C, add 0.6 ml of methacrylic anhydride solution dropwise to the gelatin solution in step 1 and continue stirring for 3 hours.

[0067] 3. After the reaction is complete, transfer the solution from step 2 to a 50 ml centrifuge tube to remove unreacted methacrylic acid. Centrifuge at 2000 rpm at room temperature for 3 minutes. Pour the supernatant into a glass beaker and dilute it with 30 ml of ultrapure water.

[0068] 4. Transfer the solution from step 3 to a 8000-14000 Da dialysis bag and dialyze at 40°C for 7 days;

[0069] 5. Adjust the pH of the solution from step 4 to 7.4 with 1 M NaOH solution;

[0070] 6. After freeze-drying, the methacrylated gelatin material was obtained.

[0071] Fluorescent gelatin is prepared by the following method:

[0072] 1. Adjust the pH of the phosphate buffered saline to 8.1 by adding 1 M NaOH solution.

[0073] 2. Prepare a gelatin solution by dissolving 1 g of gelatin in 5 ml of the alkaline phosphate buffered saline solution described in step 1 and stirring at 50°C.

[0074] 3. Add 30 mg of fluorescein material (fluorescein isothiocyanate) to the alkaline phosphate buffered saline solution (5 ml) in step 1 and stir to dissolve at 50°C;

[0075] 4. Add the fluorescein solution from step 3 dropwise to the gelatin solution from step 2 and stir in a dark environment at 50°C for 3 hours.

[0076] 5. Pause the reaction with 40 ml of PBS, transfer the reaction liquid to a dialysis bag, and dialyze at 40°C for 7 days;

[0077] 6. After freeze-drying, fluorescent gelatin solid material is obtained. Modes for Carrying Out the Invention

[0078] Example 1: Fabrication of oriented microfilament / through-pore hydrogel structure

[0079] The schematic diagram is shown in Figure 3.

[0080] 1. Preparation of fluorescent gelatin thermosensitive spheres. The preparation method for gelatin spheres is not limited and can include droplet microfluidics, oil-water emulsion, complex coacervation, electrospraying, mechanical disruption, and other methods. In this example, droplet microfluidics was used to prepare the thermosensitive gelatin spheres. A 10% fluorescent gelatin solution was prepared using 0.9% sodium chloride solution as the solvent, and the gelatin solid was dissolved at 60°C. By adjusting the flow rates of the homemade droplet microfluidics device to 8 mL / hour for oil and 1 mL / hour for gelatin solution, gelatin spheres with a diameter of approximately 200 μm were consistently obtained. The spheres were concentrated by centrifugation, and a cleaning solution (0.9% sodium chloride solution) was added to remove the oily components on the surface of the spheres. Through repeated concentration and cleaning steps, the oil content was reduced to a level that would not affect subsequent steps.

[0081] 2. Prepare a photosensitive modified Pluronic continuous phase solution by using 0.9% sodium chloride solution as solvent to prepare a 30% mass fraction of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate containing 0.15% of the photoinitiator. Dissolve the photosensitive modified Pluronic solid at 0-4°C to obtain a photosensitive modified Pluronic continuous phase solution of corresponding concentration.

[0082] 3. Preparation and printing of a multiphase system ink. The gelatin thermosensitive gel spheres were mixed with a photosensitive modified Pluronic solution at a mass-to-volume ratio of 1g:8ml. After stirring thoroughly, the mixture was loaded onto the extrusion printer nozzle for printing. The nozzle and receiving plate temperatures were controlled at 32°C. The nozzle diameter was 0.26mm, and the printing speed was controlled at 10 mm / s. After printing, the hydrogel structure was irradiated with a 405nm wavelength light source for 180 seconds to crosslink the photosensitive modified Pluronic.

[0083] 4. The scaffold was then gently washed three times with 4°C PBS to remove excess crosslinker. The printed hydrogel structure was recorded under a confocal microscope. The shape of the thermosensitive gelatin spheres within the hydrogel was related to the printing temperature (Figure 4). At a suitable temperature (denoted as the drawing temperature), the thermosensitive gelatin microspheres stably transformed into oriented filaments parallel to the extrusion direction. In conjunction with the movement of the 3D printer, the hydrogel filaments with an aligned fiber structure were deposited to form a three-dimensional structure (Figure 5). The majority of the internal oriented filaments had diameters less than 10 microns (Figure 6).

[0084] 5. This embodiment can control the material composition of the thermosensitive gel microspheres, and can optionally sacrifice or retain the oriented structure. In the sacrifice scenario, gelatin is used as the thermosensitive gel microspheres. The oriented hydrogel structure obtained by printing above the drawing temperature is then immersed in PBS at 37°C. The oriented gelatin filaments dissolve and flow out, forming oriented through-holes within the hydrogel structure. The dissolution rate of the gelatin is positively correlated with the volume and temperature of the immersion liquid. In the retention scenario, a hydrogel material that can form a stable structure through chemical or physical cross-linking is used as the main component or additive of the thermosensitive gel microspheres. After printing above the drawing temperature, cross-linking is performed to obtain a hydrogel structure with oriented microfilaments inside. In this embodiment, methacrylated gelatin is used as the material for the thermosensitive gel microspheres. The oriented microfilaments obtained can still be well maintained after 10 days at 37°C.

[0085] Example 2: Fabrication of Oriented Cell Hydrogel Structures

[0086] 1. As in steps 1 and 2 of Example 1, prepare a 10% by mass gelatin solution containing gelatin thermosensitive gel spheres with a diameter of approximately 200 μm; and a solution containing 5% by mass of photosensitive modified Pluronic and 25% by mass of Pluronic (non-photosensitive Pluronic), containing 0.15% of a photoinitiator (lithium phenyl (2,4,6-trimethylbenzoyl) phosphate).

[0087] 2. Culture mouse myoblast cell line (abbreviated as "C2C12") in DMEM high-glucose medium containing 10% fetal bovine serum. Change the medium every 2 days. Once the cells are in the logarithmic growth phase, digest them with 0.25% trypsin and collect them by centrifugation.

[0088] 3. Preparation of Multiphase Ink: Add the gelatin thermosensitive gel balls and the Pluronic solution to a sterile centrifuge tube at a mass-to-volume ratio of 1 g:8 ml. Mix thoroughly and set aside.

[0089] 4. Prepare cell bio-ink. Resuspend C2C12 cells in the multiphase system ink obtained in step 3 to obtain 5×10 6 / mL of bio-ink was mixed evenly and loaded into a pre-sterilized extrusion printer.

[0090] 5. Printing of the cell hydrogel structure. The nozzle diameter was selected to be 0.26 mm, the printing speed was controlled at 5 mm / s, and the printing temperature was 32°C. After printing, the hydrogel structure was irradiated with a 405 nm wavelength light source for 30 seconds to crosslink the photosensitive modified Pluronic.

[0091] 6. Gently wash the cell hydrogel structure three times with 4°C PBS for 10 minutes each time to remove excess crosslinker and uncrosslinked Pluronic. Transfer the printed cell structure to a culture dish / well plate and culture in DMEM high-glucose medium supplemented with 10% fetal bovine serum. Replace the culture medium in the hydrogel scaffold with fresh medium every 48 hours.

[0092] 7. Regularly check the cell status within the construct and assess the growth of the C2C12 cells. Cell survival remained remarkably good over seven days (Figure 7). After seven days of culture, the cells began to fuse, forming muscle fibers with a large aspect ratio (Figure 8).

[0093] Example 3: Fabrication of a partially cross-linked oriented hydrogel silk structure

[0094] 1. As in steps 1 and 2 of Example 1, prepare a 10% by mass methacrylated gelatin solution containing methacrylated gelatin thermosensitive gel spheres with a diameter of approximately 200 μm; and a 5% by mass solution of photosensitive modified Pluronic and 25% by mass Pluronic (non-photosensitive Pluronic) containing 0.15% of a photoinitiator (lithium phenyl (2,4,6-trimethylbenzoyl) phosphate).

[0095] 2. Preparation and printing of multiphase system ink. Methacrylated gelatin thermosensitive gel balls were mixed with Pluronic solution in a mass-volume ratio of 1 g:8 ml. After stirring the two evenly, they were loaded onto the extrusion printer nozzle for printing. The temperatures of the nozzle and the receiving base were controlled at 32°C and 32°C, respectively. The nozzle diameter was selected to be 0.26 mm, the printing speed was controlled at 10 mm / s, and the printing temperature was greater than or equal to the drawing temperature. After printing, the structure was selectively irradiated with a 405 nm wavelength light source through a mask. The irradiation time range was 20 seconds, which was used to selectively cross-link the photosensitive modified Pluronic to obtain single fibers (Figure 9) or oriented fiber scaffolds (Figure 10).

[0096] 3. Gently wash the hydrogel structure by immersing it in 4°C PBS solution three times for 10 minutes each time to remove excess cross-linker and uncross-linked Pluronic.

[0097] 4. After washing, the hydrogel structure was re-immersed in 4°C PBS containing 0.15% photoinitiator and irradiated a second time with a 405 nm wavelength light source to achieve secondary crosslinking of the methacrylated gelatin filaments. The resulting aligned fiber scaffold, after removal of the uncrosslinked continuous phase, is shown in Figure 11.

[0098] 5. The oriented fiber scaffold structure of this embodiment can induce cell extension. As in step 2 of Example 2, prepare 5×10 6 A C2C12 cell suspension of 100 μg / mL was added dropwise to the aligned fiber scaffold structure prepared in this example for culture. Every 48 hours, the culture medium in which the scaffold was immersed was replaced with fresh culture medium.

[0099] 6. Regularly check the status of cells in the structure and test and evaluate the growth status of C2C12. Cells are able to attach and grow on the oriented fiber scaffold structure (Figure 12).

[0100] Example 4: Fabrication of Oriented Hydrogel Silk Structure with Partial Crosslinking of Continuous and Discrete Phases

[0101] 1. As in steps 1 and 2 of Example 1, prepare a 10% by mass gelatin solution containing gelatin thermosensitive gel spheres with a diameter of approximately 200 μm; and a solution containing 5% by mass of photosensitive modified Pluronic and 25% by mass of Pluronic (non-photosensitive Pluronic), containing 0.15% of a photoinitiator (lithium phenyl (2,4,6-trimethylbenzoyl) phosphate).

[0102] 2. Preparation and printing of multiphase system ink. The gelatin thermosensitive gel spheres were mixed with the Pluronic solution in a mass-to-volume ratio of 1 g:8 ml. After stirring the two together, they were loaded onto the extrusion printer nozzle for printing. The temperature of the nozzle and the receiving base were controlled at 32°C. The nozzle diameter was selected to be 0.26 mm, the printing speed was controlled at 10 mm / s, and the printing temperature was greater than or equal to the drawing temperature. After printing, the hydrogel structure was selectively irradiated with a 405 nm wavelength light source through a mask (Figure 13). The illumination time range was 20 seconds for optional cross-linking of the photosensitive modified Pluronic.

[0103] 3. Gently wash the hydrogel structure with 4°C PBS solution three times for 10 minutes each time to remove excess crosslinker and uncrosslinked Pluronics. Soaking the hydrogel structure at above 37°C will cause the gelatin filaments to dissolve and flow out, forming oriented through-holes within the hydrogel structure, resulting in an oriented fiber pore scaffold.

[0104] Example 5: Fabrication of composite oriented hydrogel microfilament-oriented through-hole structure

[0105] 1. As in steps 1 and 2 of Example 1, a 10% by mass red fluorescent gelatin solution and a green fluorescent methacrylated gelatin solution were prepared, respectively, and red gelatin thermosensitive gelatin spheres and green methacrylated gelatin thermosensitive gelatin spheres with a diameter of approximately 200 μm were prepared, respectively; a solution of 5% by mass photosensitive modified Pluronic and 25% by mass Pluronic (non-photosensitive Pluronic) containing 0.15% of a photoinitiator (lithium phenyl (2,4,6-trimethylbenzoyl) phosphate) was prepared.

[0106] 2. Preparation and Printing of Multiphase Ink. Red gelatin thermosensitive gelatin spheres and green methacrylated gelatin thermosensitive gelatin spheres were premixed. The mass ratio of the mixture depended on the application scenario; in this example, a mass ratio of 1 g:1 g was used for verification. After obtaining the two-color gelatin thermosensitive gelatin spheres, they were mixed with the pluronic solution at a mass-to-volume ratio of 1 g:8 ml. After mixing thoroughly, the mixture was loaded onto the extrusion printer nozzle for printing. The nozzle and receiving plate temperatures were controlled at 32°C. The nozzle diameter was 0.26 mm, the printing speed was 10 mm / s, and the printing temperature was greater than or equal to the drawing temperature. After printing, the hydrogel structure was irradiated with a 405 nm wavelength light source for 180 seconds to crosslink the photosensitive modified pluronic and oriented methacrylated gelatin fibers (Figure 14).

[0107] 3. Gently wash the hydrogel structure by soaking it in 4°C PBS solution three times for 10 minutes each time to remove excess crosslinker and uncrosslinked Pluronics. Soaking the hydrogel structure at temperatures above 37°C will cause the red gelatin oriented fibers to dissolve and flow out, forming oriented through-holes within the hydrogel structure. The green methacrylated gelatin oriented fibers will remain insoluble and form an oriented fiber structure within the hydrogel structure, resulting in a composite scaffold with both oriented fiber pores and oriented fibers.

[0108] 4. In this embodiment, a modular discrete phase ink composition can be achieved by combining microparticles / microgels prepared from different materials. Through the printing process in step 2, a composite oriented hydrogel structure composed of multi-material hydrogel fibers is achieved, where the crosslinking method of the hydrogel fibers is determined by the material of the discrete phase microparticles / microgel.

[0109] Example 6: Fabrication of composite hydrogel heterogeneous-oriented structures

[0110] 1. As in step 1 of Example 1, a 10% mass fraction of red fluorescent gelatin solution and a green fluorescent methacrylated gelatin solution were prepared, and red gelatin thermosensitive gel balls and green methacrylated gelatin thermosensitive gel balls with a diameter of approximately 200 μm were prepared, respectively. The green methacrylated gelatin thermosensitive gel balls were pre-immersed in a solution containing 0.15% of a photoinitiator (phenyl (2,4,6-trimethylbenzoyl) lithium phosphate) and irradiated with a 405 nm wavelength light source for 180 seconds to crosslink the green methacrylated gelatin thermosensitive gel balls.

[0111] 2. As in step 2 of Example 1, a solution of 5% by weight of photosensitive modified Pluronic and 25% by weight of Pluronic (non-photosensitive Pluronic) was prepared, containing 0.15% of a photoinitiator (lithium phenyl (2,4,6-trimethylbenzoyl) phosphate).

[0112] 3. Preparation and Printing of Multiphase Ink. Pre-mix red thermosensitive gelatin spheres and green methacrylated gelatin spheres. The mass ratio depends on the application scenario; in this example, a mass ratio of 1 g:1 g is used for verification. After obtaining the two-color gel spheres, they are mixed with the Pluronic solution at a mass-to-volume ratio of 1 g:8 ml. After mixing the two solutions thoroughly, the mixture is loaded onto the extrusion printer nozzle for printing. The nozzle and receiving plate temperatures are each controlled at 32°C. The nozzle diameter is selected to be 0.26 mm, the printing speed is controlled at 10 mm / s, and the printing temperature is greater than or equal to the drawing temperature. After printing, the hydrogel structure is irradiated with a 405 nm wavelength light source for 180 seconds to crosslink the photosensitive modified Pluronic (Figure 15).

[0113] 4. Gently wash the hydrogel structure by soaking it in 4°C PBS three times for 10 minutes each time to remove excess crosslinker and uncrosslinked Pluronics. Soaking the hydrogel structure at temperatures above 37°C will cause the red gelatin oriented fibers to dissolve and flow out, forming oriented through-holes within the hydrogel structure. The green methacrylated gelatin microspheres will remain insoluble and form microspheres within the hydrogel structure, resulting in a composite scaffold with both oriented fiber pores and microspheres.

[0114] 5. In this embodiment, by combining microparticles / microgels with and without temperature-sensitive properties as modular discrete phase ink components, the printing process in step 2 can be used to manufacture a composite hydrogel heterogeneous directional structure composed of multiple materials, whose internal structure is a composite structure of a directional structure and a microparticle / microgel shape.

[0115] Example 7: Fabrication of oriented hydrogel microfilament structures based on physically cross-linked materials in the discrete phase

[0116] 1. As in steps 1 and 2 of Example 1, a mixed solution of 10% gelatin and 0.5% alginate was prepared, and gelatin-alginate thermosensitive gel spheres with a diameter of approximately 200 μm were prepared; a solution of 5% photosensitive modified Pluronic and 25% Pluronic (non-photosensitive Pluronic) by mass was prepared, and the solution contained 0.15% photoinitiator (lithium phenyl (2,4,6-trimethylbenzoyl) phosphate).

[0117] 2. Preparation and printing of multiphase system ink. The gelatin-alginate thermosensitive gel spheres were mixed with the Pluronic solution in a mass-volume ratio of 1 g:8 ml. After stirring the two evenly, they were loaded onto the extrusion printer nozzle for printing. The temperature of the nozzle and the receiving base were controlled at 32°C. The nozzle diameter was selected to be 0.26 mm, the printing speed was controlled at 10 mm / s, and the printing temperature was greater than or equal to the drawing temperature. After printing, the hydrogel structure was selectively irradiated with a 405 nm wavelength light source through a mask (Figure 16). The illumination time range was 20 seconds for optional cross-linking of the photosensitive modified Pluronic.

[0118] 3. Soak the hydrogel structure in a deionized water solution containing 3% calcium chloride for 3 minutes to cross-link the oriented alginate hydrogel fibers; gently soak and wash the hydrogel structure in 4°C PBS solution three times for 10 minutes each time to remove excess cross-linker and uncross-linked Pluronic, thereby realizing the fabrication of oriented hydrogel silk structures based on physically cross-linked materials.

[0119] Example 8: Fabrication of oriented hydrogel microfilament / through-pore structures based on physically cross-linked materials in the continuous phase

[0120] 1. As in step 1 of Example 1, prepare a 10% gelatin solution and gelatin thermosensitive gel balls with a diameter of approximately 200 μm;

[0121] 2. Prepare a composite continuous phase solution of alginic acid and Pluronic. Use 0.9% sodium chloride solution as solvent to prepare a 1% mass fraction alginate solution. Dissolve the alginate solid at 40-60°C. Then add Pluronic solid to prepare a 30% mass fraction Pluronic solution. Dissolve it at 0-4°C to obtain a composite continuous phase solution of alginic acid and Pluronic of corresponding concentration.

[0122] 3. Preparation and printing of multiphase system ink. Mix the gelatin thermosensitive gel balls with the composite continuous phase solution of step 2 in a mass volume ratio of 1 g: 8 ml. After stirring the two evenly, load them onto the extrusion printer nozzle for printing. The temperature of the nozzle and the receiving base plate are controlled at 32°C. The nozzle diameter is selected to be 0.26 mm, the printing speed is controlled at 10 mm / s, and the printing temperature is greater than or equal to the drawing temperature. After printing, the printed structure is soaked in a 3% calcium chloride solution at 37°C (Figure 17) for 3 minutes for optional cross-linking of alginate.

[0123] 4. Gently soak and wash the hydrogel structure three times with 4℃ PBS solution for 10 minutes each time to remove excess cross-linker and uncross-linked Pluronic. Soak the hydrogel structure above 37℃, and the gelatin oriented fibers will dissolve and flow out, forming oriented through-holes inside the hydrogel structure, thus realizing the fabrication of oriented hydrogel fiber structures based on physically cross-linked materials.

[0124] 5. As in step 5 of Example 1, this embodiment controls the material composition of the thermosensitive gel microspheres, allowing for the optional sacrifice or retention of the oriented structure. In the sacrifice scenario, gelatin is used as the thermosensitive gel microsphere. In the retention scenario, a hydrogel material that can form a stable structure through chemical or physical crosslinking is used as the main component or additive of the thermosensitive gel microspheres. After printing above the drawing temperature, crosslinking is performed to obtain a hydrogel structure with oriented microfilaments within. In this embodiment, methacrylated gelatin is used as the thermosensitive gel microsphere material, and the oriented microfilaments obtained are still well maintained after 10 days at 37°C. Industrial Applicability

[0125] The multiphase ink in the present invention can obtain a complex 3D structure after a printing process that couples phase change and deformation, thereby realizing the three-dimensional solid manufacturing with personalized oriented fine fibers or fine fiber bundle structures; selective cross-linking can be used to realize the manufacture of three-dimensional oriented micron hydrogel structures, oriented channel structures, single fibers or fiber bundles; and it can be combined with biological 3D printing to realize the preparation of cell-carrying three-dimensional structures with oriented characteristics under the condition of carrying cells.

Claims

1. A 3D printing method for in-situ forming a directional filament structure, comprising the following steps: 1) Making the reversible thermosensitive material A into microparticles / microgels, and mixing them with the continuous phase material B solution as the discrete phase to obtain a multiphase printing ink; 2) Loading the multiphase printing ink into an extrusion printer, controlling the temperature to thermally soften the discrete phase microparticles / microgels into a sol state, and at the same time performing extrusion printing to extrude the ink from the hopper. During the extrusion flow process, the discrete phase is sheared and deformed to form fine filaments, obtaining a directional filament structure of discrete-continuous phase composite, and implementing 3D printing structure forming based on the designed three-dimensional model; 3) After printing, perform optional and independent post-treatment crosslinking on the continuous phase and the discrete phase in the printed structure to obtain different three-dimensional structures with directional characteristics.

2. The method according to claim 1, wherein: In step 1), the reversible thermosensitive material A is at least one of the following 1)-3): 1) gelatin or modified gelatin or a mixture thereof; 2) gelatin or modified gelatin or a mixture thereof + a blend phase, wherein the blend phase is one or more of hyaluronic acid, silk fibroin, heparin, polyethylene glycol, dextran, chondroitin sulfate, sodium alginate, chitosan, fibrinogen; 3) poly(N-isopropylacrylamide) and its modified products.

3. The method according to claim 1, wherein: In step 1), the reversible thermosensitive material A further contains a hydrogel material that can form a stable structure through chemical or physical crosslinking, and the hydrogel material that can form a stable structure through chemical or physical crosslinking is used as the main component or additive of the discrete phase microparticles / microgels.

4. The method according to claim 1, wherein: First, prepare the reversible thermosensitive material A into a reversible thermosensitive material A solution, and then make microparticles / microgels by any one of the following methods: droplet microfluidics, oil-in-water emulsion method, complex coacervation method, electrospray method, mechanical fragmentation method; Among them, in the reversible thermosensitive material A solution, the mass concentration of the reversible thermosensitive material A is 2-20%; Use 0.9% sodium chloride solution, phosphate buffer solution or ultrapure water as the solvent to prepare the reversible thermosensitive material A solution; The morphology of the microparticles / microgels is spherical, polyhedral and other irregular three-dimensional shapes.

5. The method according to claim 1, wherein: The discrete phase microparticles / microgels can be modularly combined as needed as components of the multiphase ink, such as at least one of 1)-4): 1) a combination of microparticles / microgels with different sizes; 2) a combination of microparticles / microgels with different shapes; 3) a combination of microparticles / microgels with different material concentrations / compositions with thermosensitive properties; 4) a combination of microparticles / microgels with thermosensitive properties and microparticles / microgels without thermosensitive properties.

6. The method according to claim 1, wherein: The continuous phase material B maintains extrudability within the operating temperature range and has poor miscibility with the reversible thermosensitive material A.

7. The method according to claim 5, It is characterized in that: The continuous phase material B is selected from at least one of the following 1)-3): 1) Pluronic or modified Pluronic or a mixture thereof; 2) Pluronic or modified Pluronic or a mixture thereof + a blending phase, where the blending phase is one or more of hyaluronic acid, silk fibroin, heparin, polyethylene glycol, dextran, chondroitin sulfate, sodium alginate, chitosan, fibrinogen; 3) polyethylene glycol or modified polyethylene glycol or a mixture thereof.

8. The method according to claim 5, It is characterized in that: The continuous phase material B is Pluronic or modified Pluronic or a mixture thereof, and in the solution of the continuous phase material B, the mass concentration of Pluronic or modified Pluronic or a mixture thereof is 10-50%; The continuous phase material B is polyethylene glycol or modified polyethylene glycol or a mixture thereof, and in the solution of the continuous phase material B, the mass concentration of polyethylene glycol or modified polyethylene glycol or a mixture thereof is 5-50%; The solution of the continuous phase material B further contains a photoinitiator, and the mass concentration of the photoinitiator is 0.05-0.5%.

9. The method according to claim 1, It is characterized in that: The mixing mass-volume ratio range of the discrete phase and the solution of the continuous phase material B is 1 g:2 ml to 1 g:128 ml; The temperature control is to control the ink reservoir or nozzle above the drawing temperature, and the drawing temperature is the sol temperature / melting temperature of the material A and above; The diameter of the nozzle is 0.2-2 mm, and the printing speed is controlled at 5-20 mm / s.

10. The method according to claim 1, It is characterized in that: The operation of step 3) is: performing post-crosslinking treatment on the continuous phase in the printed structure, crosslinking the hydrogel material contained in the reversible thermosensitive material A microparticles / microgels that can form a stable structure through chemical or physical crosslinking to obtain a hydrogel structure with oriented filaments inside; Or performing post-crosslinking treatment on the continuous phase in the printed structure, soaking the hydrogel structure after the post-crosslinking treatment of the continuous phase above the dissolution temperature of the reversible thermosensitive material A, so that the reversible thermosensitive material A sol dissolves out, and forming oriented through-holes inside the hydrogel structure to obtain an oriented through-hole hydrogel structure; Or performing optional crosslinking on the continuous phase in the printed structure and the hydrogel material contained in the reversible thermosensitive material A microparticles / microgels that can form a stable structure through chemical or physical crosslinking to obtain a single fiber or an oriented fiber scaffold.

11. A three-dimensional structure with micron-scale oriented features prepared by the method according to any one of claims 1-9, It is characterized in that: The three-dimensional structure with micron-scale oriented features is generated in the printing process of synchronous coupling of multiphase ink phase change and deformation.

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