A 3D printing method for in-situ formation of directional filament structures

Abstract

The application discloses a kind of 3D printing methods of in situ forming directional filament structure. Including: reversible temperature-sensitive characteristic material A is made into microparticle / microgel, it is mixed as discrete phase with continuous phase material B solution, and multiple-phase printing ink is obtained;Multiple-phase printing ink is loaded into extrusion type printer, temperature is controlled to make discrete phase heat-induced softening into sol state, while extrusion printing is carried out to extrude ink from bin, and directional filament structure of discrete-continuous phase composite is obtained, based on the three-dimensional model designed, 3D printing structure forming is implemented;After printing, the continuous phase and discrete phase in the structure printed are selectively and independently post-processed crosslinking, and different three-dimensional structures with directional characteristics are obtained. In the application, after the printing process of phase change and deformation coupling of multiple-phase ink, personalized complex 3D structure manufacturing with directional micron fiber characteristic structure can be realized.

Description

Technical Field

[0001] This invention belongs to the fields of advanced manufacturing and biomedicine, specifically relating to a 3D printing method for forming oriented filament structures in situ, and more specifically focusing on a 3D printing method that can produce oriented microstructures. Background Technology

[0002] Oriented structures are widespread in nature, giving structures unique properties and functions. For example, plants contain many oriented vertical channels that transport ions, water, and other components to upper tissues to deliver nutrients and meet the needs of 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 Composites Advanced Materials 285181-7]; animals also have tissues with oriented 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 engineering Trends 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 Engineering Advanced Functional Materials]. 322110676).Compared to non-oriented structures, oriented structures not only play an important role in living systems, but their unique properties, exhibiting anisotropy in mechanical, electrical, thermal, and optical aspects, are also widely used in engineering applications. For example, the mechanical reinforcement properties of oriented 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 Applications Advanced Materials 342102877], oriented porous structures are used in the research and 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 supercapacitor Wood Science and Technology 561191-203], and in equipment [Wang Z, Luan C, Liao G, Yao X and Fu J 2019 Mechanical and self-monitoring behaviors of 3Dprinting smart continuous carbon fiber-thermoplastic lattice truss sandwich structure Composites Part B: Engineering 176107215] or 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] design and fabrication, etc. Therefore, the construction of oriented structures is of great significance.

[0003] 3D printing, based on the concept of freeform molding, can create personalized and complex three-dimensional structures. From the perspective of molding technology characteristics, current 3D printing technologies can be broadly categorized into powder bed melting, reduction photopolymerization, jetting, extrusion, and sheet lamination [Ligon SC, Liska R, Stampfl J, Gurr M and Muelhaupt R 2017 Polymers for 3D Printing and Customized Additive Manufacturing Chemical Reviews 11710212-90]. Extrusion includes melt extrusion or solution extrusion. This type of printing method can print different materials, with a wide range of material applications. Melt extrusion printing materials include polylactic acid, acrylonitrile butadiene styrene, polyurethane, and polyetheretherketone (PEEK) [Daminabo SC, Goel S, Grammatikos SA, Nezhad HY and Thakur V K2020 Fused deposition modeling-based additive manufacturing (3D printing): techniques for polymer material systems Materials Today Chemistry 16100248]. Solution extrusion, on the other hand, supports the printing of materials with high water content, such as hydrogels and living cells [Zhang YS, Haghhiashtiani G, Hubscher T, Kelly DJ, Lee JM, Lutolf M, McAlpine MC, Yeong WY, Zenobi-Wong M and Malda J 20213 Deextrusion bioprinting Nature Reviews Methods Primers 1]. Therefore, 3D printing is applied to fabricate oriented structures of different materials.

[0004] Traditional extrusion printing methods can typically extrude material filaments with a unit size of 100 micrometers or larger, which limits the minimum feature size of the oriented structures that can be constructed [Ligon SC, Liska R, Stampfl J, Gurr M and Muelhaupt R 2017 Polymers for 3D Printing and Customized Additive Manufacturing Chemical Reviews 117 10 212-90]. Traditional extrusion printing processes can reduce the size of material filament units under the influence of an electric field: electrospinning technology can produce micron-sized filaments and obtain a certain degree of oriented structure, but the orientation 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 orientation, but it is also limited by the manufacturing speed and it is difficult to form thick structures [Kade J C and Dalton PD 2021 Polymers for Melt Electrowriting Advanced HealthcareMaterials 102001232].

[0005] Existing technologies preload fine fibers into extruded materials and orient them through extrusion shearing. However, these typically use short fibers, and the alignment of the fiber ends is uncontrollable, making it difficult to achieve a high aspect ratio and continuous oriented fiber distribution. For example, using electrospinning to produce fiber membranes from norbornene-modified hyaluronic acid, and then cutting them into short fibers less than 20 micrometers in length, these short fibers can be mixed with bio-ink and then extruded and printed to rearrange themselves along the extrusion direction to form an oriented arrangement. However, the continuity of the short fibers needs to be addressed [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]. Other technologies extrude immiscible two-phase systems, where the two materials form a layered oriented fluid under extrusion shearing, resulting in a structure with a certain degree of orientation. However, the uniformity of the internal oriented through-hole structure is poor. For example, a layered oriented fiber can be obtained by using a mixture of methacrylamide gelatin and polyethylene oxide through coaxial extrusion and photocuring crosslinking. However, there is a relatively obvious interconnection between the layers within the oriented fiber [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 uses a Kenics static mixer to achieve layering of two different materials, resulting in a oriented fiber structure. Specifically, both materials are simultaneously injected into the mixer. During the flow mixing process, the materials undergo layering. Crosslinking and fixing the layered fibers yields oriented fibers, but the internal oriented structure is lamellar, making it difficult to obtain a filamentous substructure [Bolívar-Monsalve EJ, Ceballos-González CF, Borrayo- KI, Quevedo-Moreno DA, Yee-de León JF, Khademhosseini A, WeissP S, Alvarez MM and Trujillo-de Santiago G 2021Continuous chaotic bioprinting of skeletal muscle-like constructs Bioprinting21 e00125, 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 2022One-Step Bioprinting ofMulti-Channel Hydrogel Filaments Using Chaotic Advection: Fabrication of Pre-Vascularized [Muscle-Like Tissues, Advanced Healthcare Materials, 112200448]. In summary, constructing continuous micron-oriented structures within 3D printed models remains challenging. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a novel 3D printing method that can couple the phase change and deformation of multiphase inks to form oriented filament microstructures in situ, thereby solving the problem that 3D printing is unable to generate continuous micron-oriented structures within its printed structure.

[0007] The 3D printing method for in-situ formation of oriented filament structures provided by this invention includes the following steps:

[0008] 1) The reversible temperature-sensitive material A is made into microparticles / microgels, which are then mixed with the solution of the continuous phase material B as the discrete phase to obtain multiphase printing ink;

[0009] 2) Load multiphase printing ink into an extrusion printer, control the temperature to thermally soften discrete phase microparticles / microgels into a sol state, and simultaneously perform extrusion printing to extrude 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 three-dimensional model, implement 3D printing to form the structure.

[0010] 3) After printing, the continuous and discrete phases in the printed structure are selectively and independently crosslinked to obtain different three-dimensional structures with directional characteristics.

[0011] In step 1) of the above method, the reversible temperature-sensitive material A can 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 blend phase, wherein the blend phase can 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;

[0012] The reversible temperature-sensitive material A may also contain a hydrogel material that can form a stable structure through chemical and physical cross-linking. The hydrogel material that can form a stable structure through chemical and physical cross-linking may be used as the main component or additive of the discrete phase microparticles / microgels.

[0013] Furthermore, the hydrogel material that can form a stable structure through chemical and physical cross-linking can specifically be methacrylated gelatin;

[0014] The method for preparing reversible temperature-sensitive material A into microparticles / microgels is not limited and can be selected from any of the following: droplet microfluidics, oil-water emulsion method, composite coagulation method, electro-injection method, mechanical fragmentation method, etc.

[0015] First, prepare a solution of reversible temperature-sensitive material A, and then prepare microparticles / microgels using any of the methods described above.

[0016] In the solution of the reversible temperature-sensitive material A, the mass concentration of the reversible temperature-sensitive material A can be 2-20%, preferably 10%;

[0017] The reversible temperature-sensitive material A solution can be prepared using 0.9% sodium chloride solution, phosphate buffer solution, or ultrapure water as solvents.

[0018] The microparticles / microgels can be in the form of spheres (diameter between 10-5000 micrometers), polyhedra, or other irregular three-dimensional shapes (volume between 1000 cubic micrometers and 125 cubic millimeters); specifically, they can be spheres with a diameter range of 100-250 micrometers.

[0019] The continuous phase material B maintains extrudability within the operating temperature range and has poor miscibility with the reversible temperature-sensitive material A.

[0020] Specifically, the continuous phase material B can be selected from: 1) Pluronic or modified Pluronic or a mixture thereof; 2) Pluronic or modified Pluronic or a mixture thereof + a blend phase, wherein the blend phase can 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;

[0021] The continuous phase material B may also contain photosensitive crosslinking components to facilitate subsequent post-processing crosslinking of the continuous phase;

[0022] Preferably, the continuous phase material B contains photosensitive modified Prönnicke.

[0023] The continuous phase material B is Prädchenol or modified Prädchenol or a mixture thereof, and the mass concentration of Prädchenol or modified Prädchenol or a mixture thereof in the solution of the continuous phase material B is 10-50%, preferably 30%.

[0024] The continuous phase material B is polyethylene glycol or modified polyethylene glycol or a mixture thereof, and the mass concentration of polyethylene glycol or modified polyethylene glycol or a mixture thereof in the solution of the continuous phase material B is 5-50%.

[0025] The continuous phase material B solution also contains a photoinitiator, the mass concentration of which can be 0.05-0.5%;

[0026] The photoinitiator may specifically be lithium phenyl (2,4,6-trimethylbenzoyl)phosphate.

[0027] The continuous phase material B solution can be prepared using 0.9% sodium chloride solution, phosphate buffer solution, or ultrapure water as a solvent.

[0028] The mixing mass-volume ratio of the discrete phase and the continuous phase material B solution can range from 1g:2ml to 1g:128ml, preferably 1g:8ml;

[0029] The temperature control refers to controlling the ink tank or nozzle above the drawing temperature. The drawing temperature refers to the temperature at which discrete phase microparticles / microgels can be thermally softened into a sol state. The drawing temperature is the sol temperature / melting temperature of material A and above.

[0030] The drawing temperature varies for reversible temperature-sensitive materials with different compositions or concentrations.

[0031] The reversible temperature-sensitive material A is mainly gelatin or modified gelatin, and the drawing temperature is 32°C or above;

[0032] The reversible temperature-sensitive material A is poly(N-isopropylacrylamide) and its modified products, and the drawing temperature is 32°C or higher;

[0033] The nozzle diameter is 0.2-2mm, preferably 0.26mm, and the printing speed is controlled at 5-20mm / s, preferably 10mm / s;

[0034] Step 3) involves performing a post-crosslinking treatment on the continuous phase in the printed structure, crosslinking the hydrogel material contained in the reversible temperature-sensitive material A microparticles / microgels that can form a stable structure through chemical and physical crosslinking, to obtain a hydrogel structure with oriented fine filaments inside.

[0035] Alternatively, the continuous phase in the printed structure can be post-crosslinked, and the hydrogel structure after the continuous phase post-crosslinking treatment can be immersed above the dissolution temperature of the reversible temperature-sensitive material A, so that the sol of the reversible temperature-sensitive material A dissolves and forms directional through-pores inside the hydrogel structure, thus obtaining a directional through-pore hydrogel structure.

[0036] Alternatively, selective crosslinking can be performed on the continuous phase in the printed structure and the hydrogel material contained in the reversible thermosensitive material A microparticles / microgels, which can form a stable structure through chemical and physical crosslinking (specific operation: selectively irradiate the printed structure through a mask to remove the uncured continuous phase) to obtain a single fiber or oriented fiber scaffold.

[0037] In this process, the continuous phase of the printed structure is post-crosslinked by illuminating it with light. The wavelength of the light source can be 200-410nm, specifically 405nm, and the duration can be 10-300 seconds, preferably 180 seconds.

[0038] The three-dimensional structures with directional features obtained by the above method (hydrogel structures with directional fine filaments inside, directional through-pore hydrogel structures, single fiber or directional fiber scaffolds) are also within the scope of protection of this invention.

[0039] The preparation method of the present invention has the following advantages over the prior art:

[0040] 1. In this invention, multiphase ink can obtain complex 3D structures after a printing process that couples phase change and deformation, thereby realizing the manufacturing of three-dimensional solids with personalized oriented fine fiber or fine fiber bundle structures.

[0041] 2. Three-dimensional oriented micron hydrogel structures, oriented channel structures, single fibers or fiber bundles can be fabricated through selective crosslinking;

[0042] 3. It can be combined with bio-3D printing to achieve the preparation of cell-borne three-dimensional structures with directional features in the presence of cells. Attached Figure Description

[0043] Figure 1 This is a flowchart illustrating the 3D printing process for in-situ formation of oriented filament structures according to the present invention.

[0044] Figure 2 This is a schematic diagram of the technical solution for the 3D printing method of forming oriented filament structures in situ according to the present invention.

[0045] Figure 3 This is a schematic diagram illustrating the fabrication of the directional microfilament / through-pore hydrogel structure in Embodiment 1 of the present invention.

[0046] Figure 4 This illustrates the effect of printing temperature on the multiphase ink extrusion printing process in Embodiment 1 of the present invention.

[0047] Figure 5 This is a three-dimensional hydrogel model with a directional filament substructure prepared in Example 1 of the present invention.

[0048] Figure 6 This is a diagram showing the diameter distribution of the microfilaments inside the oriented structure prepared in Example 1 of the present invention.

[0049] Figure 7 This is a statistical analysis of cell survival in the directional cell hydrogel structure prepared in Example 2 of the present invention.

[0050] Figure 8 This is a staining image of a high aspect ratio myofiber of a directional cell hydrogel structure prepared in Example 2 of the present invention.

[0051] Figure 9 This is a schematic diagram illustrating the technique for preparing a single fiber in Example 3 of the present invention.

[0052] Figure 10 This is a schematic diagram illustrating the technology for preparing a directional fiber scaffold in Embodiment 3 of the present invention.

[0053] Figure 11 This is the directional fiber scaffold after continuous phase removal in Embodiment 3 of the present invention.

[0054] Figure 12 This is an example of directional fiber scaffold induction of cell extension in Embodiment 3 of the present invention.

[0055] Figure 13 This is a schematic diagram of the directional fiber optic scaffold obtained in Embodiment 4 of the present invention.

[0056] Figure 14 This is a directional hydrogel filament structure based on modular discrete phase multiphase ink.

[0057] Figure 15To fabricate directional heterostructures based on modular discrete phase multiphase inks.

[0058] Figure 16 This describes the fabrication of oriented hydrogel filament structures based on the physical cross-linking properties of discrete phase materials.

[0059] Figure 17 Fabrication of oriented hydrogel filament structures based on physical cross-linking properties of continuous phase materials. Detailed Implementation

[0060] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0062] This invention employs a multiphase ink system. During the extrusion printing process, it utilizes in-situ shearing effects to shear the blocky discrete phases within the printing ink into fibers with a large aspect ratio. This constructs finely oriented fibers within the extruded material filament units, achieving a graded orientation effect. Figure 1 , Figure 2 ).

[0063] Basic operating procedure: Microparticles A are prepared as a discrete phase and mixed with a continuous phase solution B to prepare a multiphase printing ink (S10). This ink is loaded into an extrusion printer, and the solid phase A is adjusted to a fluid state (solid-liquid transition) by temperature control, while extrusion printing is performed. During the flow, the A phase undergoes shear deformation to form fine filaments. The extruded filaments ultimately contain highly oriented discrete phase fine filaments of material A and a continuous phase of material B. Then, under computer control, based on a pre-designed 3D model, 3D printing of the structure is carried out (S20). After printing, the continuous and discrete phases in the printed structure undergo optional and independent post-processing crosslinking (S30). Figure 1 )

[0064] The methacrylamide gelatin in the following examples was prepared by a method comprising the following steps:

[0065] 1. Add 1g of solid gelatin to 10ml of ultrapure water and stir at 50℃ 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 from 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 for 3 minutes at room temperature, then transfer the supernatant to a glass beaker and add 30 mL of ultrapure water to dilute the supernatant.

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

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

[0070] 6. After freeze-drying, methacrylamide gelatin material is obtained.

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

[0072] 1. Adjust the pH of the phosphate buffer solution to 8.1 by adding 1 mole of NaOH solution for later use;

[0073] 2. Dissolve 1g of gelatin in 5ml of the alkaline phosphate buffer solution described in step 1, and stir at 50°C to prepare a gelatin solution;

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

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

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

[0077] 6. After freeze-drying, fluorescent gelatin solid material is obtained.

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

[0079] Its schematic diagram is as follows Figure 3 As shown.

[0080] 1. Preparation of fluorescent gelatin thermosensitive gel spheres. The preparation method of the gelatin spheres is not limited, and can be droplet microfluidics, oil-water emulsion method, composite coagulation method, electrospraying method, mechanical fragmentation method, etc. In this embodiment, droplet microfluidics is used to prepare gelatin thermosensitive gel spheres. A 10% (w / w) fluorescent gelatin solution is prepared using 0.9% sodium chloride solution as solvent, and the gelatin solid is dissolved at 60°C. By adjusting the flow rate of the self-made droplet microfluidic device, the oil flow rate is 8 mL / h and the gelatin solution flow rate is 1 mL / h, and gel spheres with a diameter of approximately 200 micrometers are stably obtained. The microspheres are enriched by centrifugation, and a washing solution (0.9% sodium chloride solution) is added to remove the oil phase components on the surface of the gel spheres. Through repeated enrichment-washing steps, the oil phase content is reduced to a level that does not affect subsequent steps.

[0081] 2. Prepare a photosensitive modified Prönnick continuous phase solution. Use 0.9% sodium chloride solution as solvent to prepare a 30% (w / w) lithium phenyl (2,4,6-trimethylbenzoyl)phosphate photoinitiator containing 0.15% lithium phenylphosphate. Dissolve the photosensitive modified Prönnick solid at 0-4℃ to obtain a photosensitive modified Prönnick continuous phase solution of the corresponding concentration.

[0082] 3. Preparation and Printing of Multiphase Ink System. Gelatin thermosensitive gel spheres were mixed with a photosensitive modified Prönnicke solution at a mass-to-volume ratio of 1g:8ml. After thorough mixing, the mixture was loaded onto the printhead of an extrusion printer for printing. The temperatures of the nozzle and receiving plate were controlled at 32℃. The nozzle diameter was 0.26mm, and the printing speed was controlled at 10mm / s. After printing, the hydrogel structure was irradiated with a 405nm wavelength light source for 180 seconds to crosslink the photosensitive modified Prönnicke.

[0083] 4. Then, gently wash the scaffold three times with 4°C PBS solution to remove excess cross-linking agent. Record the printed hydrogel structure under a confocal microscope; the shape of the internal gelatin thermosensitive spheres is related to the printing temperature. Figure 4 At a suitable temperature (denoted as the drawing temperature), gelatin thermosensitive gel microspheres can stably transform into oriented filaments parallel to the extrusion direction. Combined with the movement of a 3D printer, these oriented fiber-structured hydrogel filaments can be stacked and deposited to form a three-dimensional structure. Figure 5 The internal oriented filaments are mostly less than 10 micrometers in diameter. Figure 6 ).

[0084] 5. This embodiment allows control over the material composition of the temperature-sensitive gel microspheres, with the option to sacrifice or retain the oriented structure. In the sacrifice scenario, gelatin is used as the temperature-sensitive gel sphere. The oriented hydrogel structure obtained is printed above the drawing temperature. The hydrogel structure is then immersed in PBS at 37°C, causing the gelatin oriented filaments to dissolve and flow out, forming oriented through-pores 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 and physical cross-linking is used as the main component or additive of the temperature-sensitive gel microspheres. After printing above the drawing temperature, cross-linking is performed to obtain a hydrogel structure with internal oriented fine filaments. In this embodiment, methacrylamide gelatin is used as the temperature-sensitive gel microsphere material, and the obtained oriented fine filaments are well maintained after 10 days at 37°C.

[0085] Example 2: Fabrication of a Directed Cell Hydrogel Structure

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

[0087] 2. The mouse myoblast cell line (referred to as "C2C12") was cultured in DMEM high glucose medium containing 10% fetal bovine serum. The medium was changed every 2 days. After the cells were in the logarithmic growth phase, they were digested with 0.25% trypsin, centrifuged and collected for later use.

[0088] 3. Preparation of multiphase system ink. Add gelatin thermosensitive gel beads and Pluronic solution to a sterile centrifuge tube at a mass-to-volume ratio of 1g:8ml, stir well, and set aside.

[0089] 4. Preparation of cell bio-ink. Resuspend C2C12 cells in the multiphase ink system obtained in step 3 to obtain 5 × 10⁶ cells / day. 6 / mL of bio-ink is mixed evenly and then loaded into a pre-sterilized extrusion printer.

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

[0091] 6. The cell hydrogel structure was then gently washed three times with 4°C PBS solution for 10 minutes each time to remove excess cross-linking agent and uncross-linked Prönnicke. The printed cell structure was then transferred to culture dishes / well plates and cultured in DMEM high-glucose medium containing 10% fetal bovine serum. The medium soaking the hydrogel scaffold was replaced with fresh medium every 48 hours.

[0092] 7. Regularly check the cell status within the structure and assess the growth status of C2C12. Its cell viability remains relatively good for seven days. Figure 7 After seven days of culture, the cells fused, forming muscle fibers with a high aspect ratio. Figure 8 ).

[0093] Example 3: Fabrication of a directional hydrogel filament structure with partial cross-linking of a continuous phase

[0094] 1. As in steps 1 and 2 of Example 1, prepare a 10% (w / w) methacrylamide gelatin solution and methacrylamide gelatin thermosensitive gel spheres with a diameter of approximately 200 micrometers; and a solution containing 5% (w / w) photosensitive modified Pluronic and 25% Pluronic (non-photosensitive Pluronic), wherein the solution contains 0.15% (lithium phenyl(2,4,6-trimethylbenzoyl)phosphate).

[0095] 2. Preparation and Printing of Multiphase Ink System. Methacrylamide gelatin thermosensitive gel spheres were mixed with Prönnicke solution at a mass-to-volume ratio of 1g:8ml. After thorough mixing, the mixture was loaded onto the printhead of an extrusion printer for printing. The nozzle and receiving plate temperatures were controlled at 32℃ and 32℃, respectively. The nozzle diameter was 0.26mm, the printing speed was controlled at 10mm / s, and the printing temperature was greater than or equal to the drawing temperature. After printing, the structure was selectively irradiated through a mask with a 405nm wavelength light source for 20 seconds to selectively crosslink photosensitive modified Prönnicke, obtaining a single fiber (…). Figure 9 or for directional fiber optic scaffolds ( Figure 10 ).

[0096] 3. Gently wash the hydrogel structure three times with PBS solution at 4°C for 10 minutes each time to remove excess cross-linking agent and uncross-linked Prönnicke.

[0097] 4. After cleaning, the hydrogel structure was re-immersed in PBS containing 0.15% photoinitiation at 4°C, and then irradiated a second time with a 405nm wavelength light source to achieve secondary crosslinking of the methacrylamide gelatin filament structure. The final oriented fiber scaffold after removal of the uncrosslinked continuous phase is shown below. Figure 11 As shown.

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

[0099] 6. Regularly check the cell status within the structure and assess the growth status of C2C12 cells. Cells are able to attach to and extend on the oriented fiber scaffold structure. Figure 12 ).

[0100] Example 4: Fabrication of directional hydrogel filament structures with partial crosslinking of continuous and discrete phases

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

[0102] 2. Preparation and Printing of Multiphase Ink System. Gelatin thermosensitive gel spheres were mixed with Prönkel solution at a mass-to-volume ratio of 1g:8ml. After thorough mixing, the mixture was loaded onto the nozzle of an extrusion printer for printing. The temperatures of the nozzle and receiving plate were controlled at 32℃. The nozzle diameter was 0.26mm, the printing speed was controlled at 10mm / s, and the printing temperature was greater than or equal to the drawing temperature. After printing, the hydrogel structure was selectively irradiated through a mask using a 405nm wavelength light source. Figure 13 (), with an illumination time range of 20 seconds, for optional cross-linked photosensitive modified Prönnicke.

[0103] 3. Gently wash the hydrogel structure three times with 4°C PBS solution for 10 minutes each time to remove excess crosslinking agent and uncrosslinked Prönnicke. Immerse the hydrogel structure at above 37°C; the gelatin oriented fibers will dissolve and flow out, forming oriented pores inside the hydrogel structure, thus obtaining an oriented fiber pore scaffold.

[0104] Example 5: Fabrication of Composite Directed Hydrogel Microfilament-Directed Through-Pore Structure

[0105] 1. As in steps 1 and 2 of Example 1, prepare a 10% red fluorescent gelatin solution and a green fluorescent methacrylated gelatin solution, respectively, and prepare red gelatin thermosensitive gel spheres and green methacrylated gelatin thermosensitive gel spheres with a diameter of approximately 200 micrometers, respectively; and prepare solutions with a mass fraction of 5% photosensitive modified Pluronic and 25% Pluronic (non-photosensitive Pluronic), which contain 0.15% photoinitiator (lithium phenyl(2,4,6-trimethylbenzoyl)phosphate).

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

[0107] 3. Gently wash the hydrogel structure three times with 4°C PBS solution for 10 minutes each time to remove excess crosslinking agent and uncrosslinked Prönnicke. Immerse the hydrogel structure at above 37°C. The red gelatin oriented filaments will dissolve and flow out, forming oriented pores inside the hydrogel structure. The green methacrylamide gelatin oriented filaments will not dissolve and will form oriented fiber structures inside the hydrogel structure, finally yielding a composite scaffold with both oriented fiber pores and oriented fibers.

[0108] 4. In this embodiment, 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 can be realized, wherein the crosslinking method of the hydrogel fibers is determined by the materials of the discrete phase microparticles / microgels.

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

[0110] 1. As in step 1 of Example 1, prepare a red fluorescent gelatin solution and a green fluorescent methacrylated gelatin solution with a mass fraction of 10%, and prepare red gelatin thermosensitive gel balls and green methacrylated gelatin thermosensitive gel balls with a diameter of about 200 micrometers, respectively. The green methacrylated gelatin thermosensitive gel balls are pre-soaked in a solution containing 0.15% photoinitiator (lithium phenyl (2,4,6-trimethylbenzoyl)phosphate), and the solution is irradiated with a 405 nm wavelength light source for 180 seconds for crosslinking of the green methacrylated gelatin thermosensitive gel balls.

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

[0112] 3. Preparation and Printing of Multiphase Ink System. Red gelatin thermosensitive gel spheres and green methacrylamide gel spheres were pre-mixed. The mixing ratio depended on the application scenario; in this example, a ratio of 1g:1g was used for verification. After obtaining the two-color gel spheres, they were mixed with Pluronic solution at a mixing volume ratio of 1g:8ml. After thorough mixing, the mixture was loaded onto the nozzle of an extrusion printer for printing. The temperatures of the nozzle and receiving plate were controlled at 32℃. The nozzle diameter was 0.26mm, the printing speed was controlled at 10mm / s, and the printing temperature was greater than or equal to the drawing temperature. After printing, the hydrogel structure was irradiated with a 405nm wavelength light source for 180 seconds to crosslink the photosensitive modified Pluronic solution. Figure 15 ).

[0113] 4. Gently wash the hydrogel structure three times with PBS solution at 4°C for 10 minutes each time to remove excess crosslinking agent and uncrosslinked Prönnicke. Immerse the hydrogel structure at above 37°C. The red gelatin oriented filaments will dissolve and flow out, forming oriented pores inside the hydrogel structure. The green methacrylamide gelatin microspheres will not dissolve and will form microsphere structures inside the hydrogel structure, finally yielding a composite scaffold with both oriented fiber pores and microspheres.

[0114] 5. In this embodiment, microparticles / microgels with and without temperature-sensitive properties can be combined as modular discrete phase ink components. Through the printing process in step 2, a composite hydrogel heterogeneous oriented structure with multiple material compositions can be manufactured. Its internal structure is a composite structure of oriented structure and microparticle / microgel shape.

[0115] Example 7: Fabrication of directional hydrogel microfilament structures based on discrete phase materials with physical cross-linking properties

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

[0117] 2. Preparation and printing of multiphase system ink. Gelatin-algin thermosensitive gel spheres were mixed with Prönkel solution at a mass-to-volume ratio of 1g:8ml. After thorough mixing, the mixture was loaded onto the nozzle of an extrusion printer for printing. The temperatures of the nozzle and receiving plate were controlled at 32℃. The nozzle diameter was 0.26mm, the printing speed was controlled at 10mm / s, and the printing temperature was greater than or equal to the filament drawing temperature. After printing, the hydrogel structure was selectively irradiated through a mask using a 405nm wavelength light source. Figure 16 (), with an illumination time range of 20 seconds, for optional cross-linked photosensitive modified Prönnicke.

[0118] 3. Soak the hydrogel structure in a deionized aqueous solution containing 3% calcium chloride for 3 minutes to crosslink the oriented alginate hydrogel fibers; gently soak and wash the hydrogel structure three times with PBS solution at 4°C for 10 minutes each time to remove excess crosslinking agent and uncrosslinked Prönnicke, thereby realizing the fabrication of oriented hydrogel fiber structures based on materials with physical crosslinking properties.

[0119] Example 8: Fabrication of directional hydrogel microfilaments / through-pore structures based on continuous phase materials with physical cross-linking properties

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

[0121] 2. To prepare a composite continuous phase solution of alginate and Prönnicke, a 1% (w / w) alginate solution was prepared by dissolving the alginate solid in 0.9% sodium chloride solution as a solvent. After dissolving the alginate solid at 40-60℃, a 30% (w / w) Prönnicke solution was added and dissolved at 0-4℃ to obtain a composite continuous phase solution of alginate and Prönnicke of the corresponding concentration.

[0122] 3. Preparation and Printing of Multiphase Ink System. Gelatin thermosensitive gel beads were mixed with the composite continuous phase solution from step 2 at a mass-to-volume ratio of 1g:8ml. After thorough mixing, the mixture was loaded onto the nozzle of an extrusion printer for printing. The temperatures of the nozzle and receiving plate were controlled at 32℃. The nozzle diameter was 0.26mm, the printing speed was controlled at 10mm / s, and the printing temperature was greater than or equal to the drawing temperature. After printing, the printed structure was immersed in a 3% calcium chloride solution at 37℃. Figure 17 (The soaking time is 3 minutes, and it is used for optional cross-linked alginate.)

[0123] 4. Gently soak and wash the hydrogel structure three times with PBS solution at 4℃ for 10 minutes each time to remove excess crosslinking agent and uncrosslinked Prönnicke. Soak the hydrogel structure at 37℃ or higher. The gelatin oriented filaments will dissolve and flow out, forming oriented through-pores inside the hydrogel structure, thus realizing the fabrication of oriented hydrogel filament structures based on materials with physical crosslinking properties.

[0124] 5. As in step 5 of Example 1, this example allows control over the material composition of the temperature-sensitive gel microspheres, with the option to sacrifice or retain the oriented structure. In the scenario of sacrifice, gelatin is used as the temperature-sensitive gel microsphere. In the scenario of retention, a hydrogel material that can form a stable structure through chemical and physical cross-linking is used as the main component or additive of the temperature-sensitive gel microsphere. After printing above the drawing temperature, cross-linking is performed to obtain a hydrogel structure with internally oriented fine filaments. In this example, methacrylamide gelatin is used as the temperature-sensitive gel microsphere material, and the obtained oriented fine filaments can still be well maintained after 10 days at 37°C.

[0125] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A 3D printing method for in-situ forming of oriented filament structures, comprising the following steps: 1) Reversible thermosensitive material A is made into microparticles / microgels, which are then mixed with a solution of continuous phase material B as a discrete phase to obtain multiphase printing ink; 2) Load multiphase printing ink into an extrusion printer, control the temperature to thermally soften discrete phase microparticles / microgels into a sol state, and simultaneously perform extrusion printing to extrude 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, implement 3D printing to form the structure. 3) After printing, the continuous and discrete phases in the printed structure are selectively and independently crosslinked to obtain different three-dimensional structures with directional characteristics. The continuous phase material B maintains extrudability within the operating temperature range and has poor miscibility with the reversible temperature-sensitive material A.

2. The method according to claim 1, characterized in that: In step 1), the reversible temperature-sensitive material A is at least one of the following 1)-3): 1) gelatin or modified gelatin or a mixture thereof; 2) any one of gelatin, modified gelatin, or a mixture of gelatin and modified gelatin and a blended phase, wherein the blended phase is 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.

3. The method according to claim 1, characterized in that: In step 1), the reversible temperature-sensitive material A also contains a hydrogel material that can form a stable structure through chemical and physical cross-linking. The hydrogel material that can form a stable structure through chemical and physical cross-linking serves as the main component or additive of the discrete phase microparticles / microgels.

4. The method according to claim 1, characterized in that: First, prepare a solution of reversible temperature-sensitive material A, and then prepare microparticles / microgels using any of the following methods: droplet microfluidics, oil-water emulsion method, composite coagulation method, electro-injection method, and mechanical fragmentation method. The reversible temperature-sensitive material A solution has a mass concentration of 2-20%. The reversible temperature-sensitive material A solution was prepared using 0.9% sodium chloride solution, phosphate buffer solution, or ultrapure water as solvents. The microparticles / microgels are in the form of spheres, polyhedra, and other irregular three-dimensional shapes.

5. The method according to claim 1, characterized in that: Discrete phase microparticles / microgels are modularly combined as components of multiphase inks as needed, such as at least one of 1)-4): 1) combinations of microparticles / microgels with different sizes; 2) combinations of microparticles / microgels with different shapes; 3) combinations of microparticles / microgels with different material concentrations / compositions that have temperature-sensitive properties; 4) combinations of microparticles / microgels that have temperature-sensitive properties and microparticles / microgels that do not have temperature-sensitive properties.

6. The method according to claim 1, characterized in that: The continuous phase material B is selected from at least one of the following 1)-3): 1) Prönkel or modified Prönkel or a mixture thereof; 2) a mixture of at least one of Prönkel, modified Prönkel, Prönkel and a mixture of modified Prönkel with 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, and fibrinogen; 3) polyethylene glycol or modified polyethylene glycol or a mixture thereof.

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

8. The method according to claim 1, characterized in that: The mixing mass-volume ratio of the discrete phase and the continuous phase material B solution ranges from 1 g: 2 ml to 1 g: 128 ml. The temperature control is to keep the ink tank or nozzle above the drawing temperature, where the drawing temperature is the sol temperature / melting temperature of material A and above. The nozzle diameter is 0.2-2mm, and the printing speed is controlled between 5-20mm / s.

9. The method according to claim 1, characterized in that: Step 3) involves performing a post-crosslinking treatment on the continuous phase in the printed structure, crosslinking the hydrogel material contained in the reversible temperature-sensitive material A microparticles / microgels that can form a stable structure through chemical and physical crosslinking, to obtain a hydrogel structure with oriented filaments inside. Alternatively, the continuous phase in the printed structure can be post-crosslinked, and the hydrogel structure after the continuous phase post-crosslinking treatment can be immersed above the dissolution temperature of the reversible temperature-sensitive material A, so that the sol of the reversible temperature-sensitive material A dissolves and forms directional through-pores inside the hydrogel structure, thus obtaining a directional through-pore hydrogel structure. Alternatively, the hydrogel material contained in the continuous phase and reversible thermosensitive material A microparticles / microgels in the printed structure can be selectively cross-linked to form a stable structure through chemical and physical cross-linking, to obtain a single fiber or oriented fiber scaffold.

10. A three-dimensional structure with micron-level directional features prepared by the method according to any one of claims 1-9, characterized in that: The three-dimensional structure with micron-level directional features is generated during the printing process in which the phase change and deformation of multiphase ink are synchronously coupled.

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