A non-yield stress fluid based suspension printing method

Abstract

The application provides a suspension printing method based on a non-yield stress fluid. The method comprises the following steps: using a yield stress fluid material as a first support bath; using a non-yield stress fluid material as a second support bath; wrapping the second support bath in the first support bath; suspension printing a biological ink in the second support bath, and then constructing a three-dimensional structure in the non-yield stress fluid material; cross-linking the non-yield stress fluid material, removing the first support bath, removing or cross-linking the biological ink, and obtaining a three-dimensional structure based on the non-yield stress fluid. The yield stress fluid material wrapped outside the non-yield stress fluid material is used as the first support bath, which can provide omnidirectional space restraint and elastic mechanical restraint, and the suspension printing of the three-dimensional structure in the non-yield stress fluid material is realized.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering and biomanufacturing technology, specifically relating to a suspension printing method based on non-yield stress fluid. Background Technology

[0002] Suspension 3D printing, by freely depositing bio-ink in a support bath, enables the three-dimensional shaping of low-viscosity soft hydrogel bio-inks with unrestricted forming direction, and has been widely used in tissue engineering, pathological model research, drug screening, and other fields. While suspension 3D printing has achieved numerous technological and application breakthroughs, it still faces a significant challenge: the range of support bath materials is limited. Most high-performance bio-gels, such as extracellular matrix-derived or extracellular matrix-derived hydrogels, cannot support suspension printing.

[0003] In suspension printing, yield-stress fluids are the most commonly used support bath materials, possessing excellent rheological properties, including a suitable storage modulus, a certain yield stress, and good self-healing properties. However, most high-performance hydrogel materials (such as extracellular matrix-derived hydrogels) typically exhibit a solid-like or liquid-like state, making them unsuitable for suspension printing. Liquid-like support bath materials, with insufficient storage modulus, are prone to flow, causing deposited ink to float or break, leading to printing failure. Solid-like support bath materials cannot self-heal promptly after the nozzle passes through, resulting in vertical cracks and ink escaping, causing printing failure. Therefore, directly constructing three-dimensional vascular channels or heterogeneous structures in these hydrogel materials via suspension printing is not feasible, significantly limiting the range of materials for constructing engineered tissues and organs, as well as their functionalization and vascularization.

[0004] Rheological modification is one approach to constructing fine three-dimensional structures in non-yield stress fluid materials. A common method is to prepare non-yield stress fluid hydrogels into particulate microgels, thereby giving them yield stress fluid properties. Skylar-Scott et al. prepared organ building blocks based on extracellular matrix and induced pluripotent stem cells (iPSCs), exhibiting the rheological properties of yield stress fluids, which can support omnidirectional printing of sacrificial gelatin ink (MarkSkylar-Scott, Sebastien Uzel, Lucy Nam, et al. Science Advances, 2019, 5(9): eaaw2459.). Similarly, Yongcong Fang et al. developed a microgel biphasic bioink based on methacrylamide gelatin (GelMA) with good shear thinning and self-healing properties, which can be used to construct vascular channels through suspension printing (Yongcong Fang, Yihan Guo, Bingyan Wu, et al. Advanced Materials, 2023, 35, 220582.). However, the complicated process of preparing particulate microgels is very time-consuming and may affect the activity of the encapsulated cells.

[0005] Unlike rheological modification, external force assistance offers an alternative approach. Xie Ruoxiao et al. developed a novel magnetically assisted manufacturing strategy, where pre-prepared two-dimensional hydrogel scaffolds were transformed into three-dimensional structures under the influence of gravity and magnetism, successfully fabricating permeable three-dimensional vascular channels in various soft matrix materials such as GelMA, collagen, and fibrin. However, unlike suspension printing, this magnetically controlled method cannot achieve precise shaping of complex structures (Ruoxiao Xie, Yuanxiong Cao, Rujie Sun, et al. Science Advances, 2024, 10, eadl 1549.). Therefore, there is an urgent need to develop new suspension printing strategies to further broaden the range of materials that can be selected for the support bath, enabling the construction of three-dimensional structures in various hydrogel materials, especially extracellular matrix-derived or extracellular matrix-derived hydrogels. Summary of the Invention

[0006] To address the technical challenge of levitation printing to construct three-dimensional structures within non-yield stress fluid materials, this invention aims to provide a levitation printing method based on non-yield stress fluids. This invention utilizes a yield stress fluid material encased within the non-yield stress fluid material as a first support bath, providing omnidirectional spatial and elastic mechanical constraints, thus enabling levitation printing to construct three-dimensional structures within the non-yield stress fluid material.

[0007] To achieve the above objectives, the present invention provides a suspension printing method based on non-yield stress fluid, which includes the following steps:

[0008] S1. A yield stress fluid material is used as the first support bath;

[0009] S2. A non-yielding stress fluid material is used as the second support bath;

[0010] S3. Wrap the second support bath in the first support bath;

[0011] S4. Suspend and print bio-ink in the second support bath to construct a three-dimensional structure in the non-yielding stress fluid material;

[0012] S5. Crosslink the non-yield stress fluid material, remove the first support bath, remove or crosslink the bio-ink to obtain a three-dimensional structure based on non-yield stress fluid.

[0013] In the above-described suspension printing method based on non-yield stress fluid, preferably, in step S1, the yield stress of the yield stress fluid material is 5 to 100 Pa.

[0014] In the above-described suspension printing method based on non-yield stress fluid, preferably, in step S1, the yield stress fluid material includes a support bath material with self-healing properties. More preferably, the support bath material with self-healing properties includes a microparticle-based hydrogel material, a continuous phase-based hydrogel material, an inorganic colloid, or an organic-inorganic composite colloid. Further preferably, the microparticle-based hydrogel material is a particulate hydrogel material; the continuous phase-based hydrogel material is a hydrogel material containing reversible dynamic bonds, preferably including one or more of host-guest hydrogel materials, metal ion coordination bond hydrogel materials, and interpenetrating polymer network hydrogel materials; the inorganic colloid or organic-inorganic composite colloid includes a colloidal material containing one or more of nano-clay, mineral oil-fumed silica, polydimethylsiloxane (PDMS)-fumed silica, etc.

[0015] In the above-described suspension printing method based on non-yield stress fluid, preferably, in step S1, the raw materials of the microparticle-based hydrogel material and the continuous phase-based hydrogel material may respectively include natural polymers and / or synthetic polymers, more preferably including one or a combination of several of the following: sodium alginate, gelatin, carrageenan, gellan gum, agarose, carbomer, and acrylamide dimethyl taurate ammonium / VP copolymer (Aristoflex AVC).

[0016] In the above-mentioned suspension printing method based on non-yield stress fluid, preferably, in step S1, the microparticle-based hydrogel material can be prepared by at least one of the following methods: electrospraying, mechanical crushing, photolithography, composite agglomeration, or microfluidic technology.

[0017] In the above-described suspension printing method based on non-yield stress fluid, preferably, in step S1, the mass-volume concentration of the material providing the hydrogel or colloidal three-dimensional network structure in the yield stress fluid material is 1 to 100 mg / mL, more preferably 1 to 10 mg / mL.

[0018] In the above-described suspension printing method based on non-yield stress fluid, preferably, in step S2, the non-yield stress fluid material is a solid-like or liquid-like state.

[0019] In the above-described suspension printing method based on non-yield stress fluid, preferably, in step S2, the yield stress of the non-yield stress fluid material is less than the yield stress of the yield stress fluid material. More preferably, the yield stress of the non-yield stress fluid material is 4% to 80% of the yield stress of the yield stress fluid material.

[0020] In the above-described suspension printing method based on non-yield stress fluid, preferably, in step S2, the non-yield stress fluid material includes natural polymeric hydrogel materials and / or synthetic polymeric hydrogel materials, more preferably including extracellular matrix-derived and / or extracellular matrix-derived hydrogel materials. More preferably, the non-yield stress fluid material includes a hydrogel material containing one or more of gelatin, collagen, hyaluronic acid, and their methacrylamide products.

[0021] In the above-described suspension printing method based on non-yield stress fluid, preferably, in step S2, the mass-volume concentration of the material providing the hydrogel three-dimensional network structure in the non-yield stress fluid material is 1-100 mg / mL, more preferably 2-25 mg / mL.

[0022] In the above-described suspension printing method based on non-yield stress fluid, preferably, in step S2, the non-yield stress fluid material contains cells with a cell density of 1×10⁻⁶. 6 / mL~5×10 7 / mL.

[0023] In the above-described suspension printing method based on non-yield stress fluid, preferably, in step S3, the second support bath is wrapped in the first support bath by suspension printing or by direct injection.

[0024] In the above-described suspension printing method based on non-yield stress fluid, preferably, in step S3, the second support bath is completely enclosed in the first support bath.

[0025] In the above-described suspension printing method based on non-yield stress fluid, preferably, in step S4, the bio-ink includes natural polymer hydrogel materials and / or synthetic polymer hydrogel materials, etc.

[0026] In the above-described suspension printing method based on non-yield stress fluid, preferably, in step S4, the bio-ink contains cells with a cell density of 10-1. 6 / mL~5×10 7 / mL.

[0027] In the above-described suspension printing method based on non-yield stress fluid, preferably, in step S4, the mass-volume concentration of the material providing the hydrogel three-dimensional network structure in the bio-ink is 1-100 mg / mL, more preferably 4-30 mg / mL.

[0028] In the above-described suspension printing method based on non-yield stress fluid, preferably, in step S5, the crosslinking of the non-yield stress fluid material includes one or a combination of several of the following: temperature change, ion reaction, ultraviolet light irradiation, and pH change.

[0029] In the above-described suspension printing method based on non-yield stress fluid, preferably, in step S5, the first support bath is removed by one or a combination of temperature change, ion reaction and mechanical cleaning.

[0030] In the above-described suspension printing method based on non-yield stress fluid, preferably, in step S5, the bio-ink is cross-linked or removed by one or a combination of temperature change, ion reaction, ultraviolet light irradiation and pH change.

[0031] The technical solution of the present invention has at least the following beneficial effects:

[0032] This invention utilizes a yield-stress fluid material as the first support bath and a non-yield-stress fluid material as the second support bath, encapsulating the second support bath within the first. The yield-stress fluid material surrounding the non-yield-stress fluid material provides omnidirectional spatial and elastic mechanical confinement, and allows control over the yield stress relationship between the two materials. This enables the suspension printing of complex three-dimensional fine structures within solid-like or liquid-like non-yield-stress fluid hydrogel materials (such as extracellular matrix-derived and / or extracellular matrix-derived hydrogels), overcoming the limitation of support bath materials in existing suspension printing technologies. This invention facilitates the direct construction of vascular networks or heterogeneous structures in tissues and organs from various hydrogel materials (such as extracellular matrix-derived and / or extracellular matrix-derived hydrogels), providing new ideas for the construction of functionalized and vascularized engineered tissues and organs. It has significant scientific and application value in tissue and organ repair, drug development, and pathological research. Attached Figure Description

[0033] Figure 1 A schematic diagram of a suspension printing method based on non-yield stress fluid according to a specific embodiment of the present invention is shown.

[0034] Figure 2 A schematic diagram of direct suspension printing in a non-yield stress fluid material is shown.

[0035] Figure 3 The simulation results show the process of suspending and printing in a solid-like non-yielding stress fluid-mass hydrogel material.

[0036] Figure 4 The simulation diagram shows the velocity distribution during suspension printing in a liquid-like non-yielding stress fluid-like blocky hydrogel material.

[0037] Figure 5 The shear stress-shear rate curves of yield stress fluid materials and non-yield stress fluid materials are shown, as well as optical images of yield stress fluid materials with different yield stresses, in which cylindrical non-yield stress fluid materials are wrapped, and then suspended to print bio-ink.

[0038] Figure 6 The image shows a comparison of the gelatin bio-inks printed by direct suspension in solid-like and liquid-like GelMA in Example 1 and the gelatin bio-inks printed by suspension in solid-like and liquid-like GelMA in yield stress fluid materials.

[0039] Figure 7 The illustration shows the printing of gelatin bio-ink suspended in quasi-solid and quasi-liquid GelMA, which was placed directly in a yield stress fluid material as a first support bath, in Example 2.

[0040] Figure 8 The rheological properties of the HAMA hydrogel in Example 3 are shown, including the construction of a red HAMA helical structure by suspension printing in a green HAMA hydrogel cylinder, and the fidelity of the suspension-printed helical structure.

[0041] Figure 9 The image shows the printing of red gelatin bio-ink suspended in a green single-opening GelMA hydrogel chamber in Example 4.

[0042] Figure 10 The image shows a single-opening oral cavity of HAMA hydrogel with a complex heterostructure obtained by suspension printing in Example 5.

[0043] Figure 11 The high-cell-density GelMA hydrogel myocardial compartment with channels, which was suspended and printed in Example 6, is shown. Detailed Implementation

[0044] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0045] According to specific embodiments of the present invention, such as Figure 1 As shown, this invention provides a suspension printing method based on non-yield stress fluid, which includes the following steps:

[0046] S1. A yield stress fluid material is used as the first support bath;

[0047] S2. A non-yielding stress fluid material is used as the second support bath;

[0048] S3. Wrap the second support bath inside the first support bath;

[0049] S4. Suspend and print bio-ink in the second support bath to construct a three-dimensional structure in a non-yield stress fluid material;

[0050] S5. Crosslink the non-yield stress fluid material, remove the first support bath, and remove or crosslink the bio-ink to obtain a three-dimensional structure based on the non-yield stress fluid.

[0051] In some embodiments, in step S2, the non-yielding stress fluid material is a solid-like or liquid-like state.

[0052] Figure 2This diagram illustrates direct suspension printing in non-yield stress fluid materials. It can be seen that liquid-like non-yield stress fluid hydrogel materials have insufficient storage modulus and are prone to flow, leading to ink floating or breaking and printing failure. Solid-like non-yield stress fluid hydrogel materials cannot self-heal promptly after the nozzle passes through, resulting in vertical cracks and ink escaping, causing printing failure. Figure 3 The simulation results show the suspension printing in a solid-like non-yield stress fluid-mass hydrogel material, with a nozzle movement speed of 5 mm / s; Figure 3 In (a), direct suspension printing is shown, and obvious cracks can be seen. Figure 3 (b) shows the suspension printing under the condition of non-yield stress fluid material being wrapped in yield stress fluid material in the present invention. It can be seen that the cracks are generated briefly and then heal quickly. Figure 4 The simulation diagram of velocity distribution during suspension printing in a liquid-like non-yielding stress fluid-like blocky hydrogel material is shown, with a nozzle movement speed of 5 mm / s. Figure 4 In the middle (a), direct hover printing is used. Figure 4 (b) shows the suspension printing under the condition of non-yield stress fluid material being wrapped in yield stress fluid material in the present invention. It can be seen that the flow rate of the latter is significantly lower than that of the former.

[0053] In this invention, a yield stress fluid material, encased within a non-yield stress fluid material, serves as the first support bath. This first support bath provides omnidirectional spatial and elastic mechanical confinement, enabling the construction of three-dimensional structures through levitation printing within the non-yield stress fluid material. For the quasi-solid-state non-yield stress fluid material, the elastic confinement of the external first support bath is crucial for ensuring crack healing and preventing internal deformation. During bio-ink printing, as the levitation printing nozzle cuts through the quasi-solid-state non-yield stress fluid material, localized deformation (e.g., cracking) occurs on both sides of the nozzle, causing localized elastic deformation in the external yield stress fluid material domain. After the nozzle passes, the localized elastic deformation of the yield stress fluid material domain recovers, promoting crack healing. For the liquid-like non-yield stress fluid material, the omnidirectional confinement significantly reduces the influence area during nozzle movement, thereby minimizing the impact on the deposited filaments. Therefore, this invention overcomes the limitation of support bath materials in existing levitation printing technologies, enabling the construction of three-dimensional structures within non-yield stress fluid materials.

[0054] In some embodiments, in step S1, the yield stress of the yield stress fluid material is 5–100 Pa, preferably 30–90 Pa. This invention uses a material with yield stress fluid properties and a high yield stress as the first support bath.

[0055] In some embodiments, in step S2, the yield stress of the non-yield stress fluid material is less than the yield stress of the yield stress fluid material. Preferably, the yield stress of the non-yield stress fluid material is 4 to 80% of the yield stress of the yield stress fluid material, more preferably 4 to 70%.

[0056] Figure 5 Shear stress-shear rate curves of yield stress fluid materials and non-yield stress fluid materials are shown, as well as optical images of yield stress fluid materials with different yield stresses, in which cylindrical non-yield stress fluid materials are wrapped, and then suspended for printing bio-ink. Figure 5 In the diagram, the yield stress fluid materials with different yield stresses are acrylamide dimethyl taurate ammonium / VP copolymer (Aristoflex AVC) hydrogels with mass-volume concentrations of 0.3 mg / mL, 0.6 mg / mL, and 1.0 mg / mL, respectively. The non-yield stress fluid material is methacrylamide gelatin (GelMA) hydrogel with a mass-volume concentration of 5 mg / mL, and the bio-ink is a gelatin hydrogel. It can be seen that the yield stress of the yield stress fluid material is greater than that of the non-yield stress fluid material. Therefore, the structure of the non-yield stress fluid material does not rotate during levitation printing, and vice versa.

[0057] This invention enables the suspension printing of complex three-dimensional fine structures in solid-like or liquid-like non-yield stress fluid hydrogel materials (such as extracellular matrix-derived and / or extracellular matrix-derived hydrogel materials) by providing all-round spatial and elastic mechanical constraints through a non-yield stress fluid material and a yield stress fluid material outside the non-yield stress fluid material, as well as controlling the relationship between the yield stress magnitudes of the yield stress fluid material and the non-yield stress fluid material.

[0058] In some embodiments, in step S1, the yield stress fluid material includes a support bath material with self-healing properties. Preferably, the support bath material with self-healing properties includes a microparticle-based hydrogel material, a continuous phase-based hydrogel material, an inorganic colloid, or an organic-inorganic composite colloid. More preferably, the microparticle-based hydrogel material is a particulate hydrogel material; the continuous phase-based hydrogel material is a hydrogel material containing reversible dynamic bonds, preferably including one or more of host-guest hydrogel materials, metal ion coordination bond hydrogel materials, and interpenetrating polymer network hydrogel materials; the inorganic colloid or organic-inorganic composite colloid includes a colloidal material containing one or more of nano-clay, mineral oil-fumed silica, polydimethylsiloxane (PDMS)-fumed silica, etc.

[0059] In some embodiments, in step S1, the raw materials for the microparticle-based hydrogel material and the continuous phase-based hydrogel material may respectively include natural polymers and / or synthetic polymers, preferably including one or a combination of several of sodium alginate, gelatin, carrageenan, gellan gum, agarose, carbomer, and acrylamide dimethyl taurate ammonium / VP copolymer (Aristoflex AVC).

[0060] In some embodiments, in step S1, the microparticle-based hydrogel material can be prepared by at least one of the following methods: electrospraying, mechanical fragmentation, photolithography, composite agglomeration, or microfluidics.

[0061] In this invention, the aforementioned hydrogel materials with self-healing properties are all existing hydrogel materials or hydrogel materials that can be prepared by methods in the prior art.

[0062] In some embodiments, in step S1, the mass-volume concentration of the material providing the hydrogel or colloidal three-dimensional network structure in the yield stress fluid material is 1 to 100 mg / mL, preferably 1 to 10 mg / mL.

[0063] In this invention, it should be noted that the material providing the three-dimensional network structure of the hydrogel or colloidal material refers to the substance that forms the three-dimensional network structure in the hydrogel or colloidal material (such as the polymer or nano-clay mentioned above).

[0064] In some embodiments, in step S2, the non-yield stress fluid material comprises natural polymeric hydrogel materials and / or synthetic polymeric hydrogel materials, preferably including extracellular matrix-derived and / or extracellular matrix-derived hydrogel materials. More preferably, the non-yield stress fluid material comprises a hydrogel material containing one or more of gelatin, collagen, hyaluronic acid and their methacrylamide products.

[0065] In some embodiments, in step S2, the mass-volume concentration of the material providing the hydrogel three-dimensional network structure in the non-yielding stress fluid material is 1 to 100 mg / mL, preferably 2 to 25 mg / mL.

[0066] In some embodiments, in step S2, the non-yielding stress fluid material contains cells with a cell density of 1×10⁻⁶. 6 / mL~5×10 7 / mL, preferably 1×10 7 / mL~5×10 7 / mL.

[0067] In some embodiments, in step S3, enclosing the second support bath within the first support bath is achieved either by suspension printing or by direct injection. Specifically, suspension printing can be used to suspend and print the non-yield stress fluid material within the first support bath to form the second support bath; alternatively, direct injection can be used to place the non-yield stress fluid material directly into the first support bath to form the second support bath. The second support bath can have various shapes; for example, suspension printing allows for the construction of various desired three-dimensional shapes, while direct injection allows the non-yield stress fluid material to be shaped into various desired forms before injection.

[0068] In some embodiments, in step S3, the second support bath is completely enclosed within the first support bath.

[0069] In some embodiments, the three-dimensional structure constructed in step S4 includes a three-dimensional network structure.

[0070] In some embodiments, in step S4, the bio-ink comprises natural polymeric hydrogel materials and / or synthetic polymeric hydrogel materials. The raw materials for the natural polymeric hydrogel materials include, but are not limited to, sodium alginate, gelatin, collagen, chitosan, silk fibroin, hyaluronic acid, and their methacrylamide products, or combinations thereof. The raw materials for the synthetic polymeric hydrogel materials include, but are not limited to, polyethylene glycol, polyacrylamide, polyethylene glycol diacrylate, polyethylene oxide, polyacrylamide, polyacrylic acid, polyphosphazene, poly(N-isopropylacrylamide), and their methacrylamide products, or combinations thereof.

[0071] In some embodiments, in step S4, the bio-ink contains cells at a cell density of 10-1. 6 / mL~5×10 7 / mL, preferably 1×10 7 / mL~5×10 7 / mL.

[0072] In some embodiments, in step S4, the mass-volume concentration of the material providing the hydrogel three-dimensional network structure in the bio-ink is 1–100 mg / mL, preferably 4–30 mg / mL.

[0073] In some embodiments, in step S5, the crosslinking of the non-yielding stress fluid material includes one or a combination of several of the following: temperature change, ion reaction, ultraviolet light irradiation, and pH change.

[0074] In some embodiments, in step S5, the first support bath is removed by one or a combination of temperature change, ion reaction and mechanical cleaning.

[0075] In some embodiments, in step S5, the bio-ink is crosslinked or removed by one or a combination of temperature changes, ionic reactions, ultraviolet light irradiation, and pH changes.

[0076] The technical solutions of the present invention are specifically illustrated by the following embodiments, but the present invention is not limited to these embodiments. Of course, various modifications can be made within the scope of the key points of the present invention.

[0077] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0078] In the following embodiments, the yield stress of the hydrogel material was obtained by testing using the following method: The yield stress of the first and second support baths was measured using a rheometer (MCR301, Anton Paar). The rheometer probe was a 25mm diameter conical plate with a gap size of 800μm. The stress was measured over a period of 0–1000 s⁻¹. -1 Shear stress was measured at a gradually varying shear rate, and the results were fitted with Hershel-Bulkly to obtain the yield stress.

[0079] Example 1

[0080] Preparation of the first support bath: AVC powder was added to deionized water (DI water) and stirred overnight to obtain an AVC hydrogel with a mass-volume concentration of 1.0 mg / mL. Before coating the second support bath, the AVC hydrogel was centrifuged at 1200g for 3 minutes to remove air bubbles. The yield stress of the AVC hydrogel was 83.12 Pa.

[0081] Preparation of the second support bath: Commercially purchased GelMA (60% substitution, Qingyuan Taishuo Company) was used to prepare a GelMA precursor solution with a mass-volume concentration of 5% (deionized water as solvent). Then, a photoinitiator (LAP) was added, wherein the concentration of the photoinitiator in the continuous phase precursor solution was 0.25 mg / mL, to obtain a GelMA block hydrogel.

[0082] Preparation of bio-ink: Gelatin (Sigma-Aldrich) was dissolved in deionized water to prepare a gelatin hydrogel with a mass-volume concentration of 7.5 mg / mL, and then stained with red dye (Thermo Fisher Scientific).

[0083] GelMA block hydrogels were incubated at 16℃ and 22℃ respectively to achieve a near-solid or near-liquid state. The yield stress of the near-solid GelMA hydrogel was 56.12 Pa, and the yield stress of the near-liquid GelMA hydrogel was 4.47 Pa.

[0084] In contrast, red gelatin bio-ink was directly suspended and printed in it; for liquid-like GelMA block hydrogel, the deposited filaments floated significantly due to its inability to provide effective support, resulting in low print fidelity; for solid-like GelMA block hydrogel, non-healing cracks appeared after the needle passed through, causing the deposited ink to escape and resulting in printing failure.

[0085] The 5 mg / mL GelMA block hydrogel was stained with a green dye (Thermo Fisher Scientific).

[0086] The second support bath was completely enclosed in the first support bath: green GelMA hydrogel was suspended and printed in AVC hydrogel to form a cylinder, and kept at 16℃ / 22℃ respectively.

[0087] Bio-ink was suspended and printed in a second support bath: Red gelatin bio-ink was printed between the inner and outer walls of a GelMA hydrogel cylinder at 16℃ and 22℃ respectively, and both were well formed, thus constructing a three-dimensional structure in a non-yielding stress fluid material.

[0088] Figure 6 This example shows a comparison of printing gelatin bio-ink directly suspended in solid-like and liquid-like GelMA and printing gelatin bio-ink suspended in solid-like and liquid-like GelMA within a yield stress fluid material. Figure 6 (a) shows the direct suspension printing of gelatin bio-ink in solid-like and liquid-like GelMA, which shows the phenomenon of filament floating and cracks. Figure 6 In (b), gelatin bio-ink is suspended and printed in a semi-solid and semi-liquid three-dimensional cylinder with GelMA as the second support bath, using yield stress fluid material as the first support bath. Good forming can be seen.

[0089] The above experiments demonstrate that, under the condition that the external yield stress fluid material serves as the first support bath, it is possible to perform levitation printing on non-yield stress fluid materials that have been obtained through levitation printing to construct three-dimensional structures.

[0090] Then, the GelMA hydrogel was crosslinked, and the AVC hydrogel was removed, and the gelatin bio-ink was removed or crosslinked to obtain a three-dimensional structure based on non-yield stress fluid.

[0091] Example 2

[0092] Preparation of the first support bath: AVC powder was added to deionized water (DI water) and stirred overnight to obtain an AVC hydrogel with a mass-volume concentration of 1.0 mg / mL. Before coating the second support bath, the AVC hydrogel was centrifuged at 1200g for 3 minutes to remove air bubbles. The yield stress of the AVC hydrogel was the same as in Example 1.

[0093] Preparation of the second support bath: A GelMA precursor solution (deionized water) with a mass-volume concentration of 5 mg / mL was prepared using commercially available GelMA (60% substitution, Qingyuan Taishuo Company). A photoinitiator (LAP) was then added, with a concentration of 0.25 mg / mL in the continuous phase precursor solution, resulting in a blocky GelMA hydrogel. This hydrogel was then stained with a red dye (Thermo Fisher Scientific). The yield stress of the solid-like GelMA hydrogel and the yield stress of the liquid-like GelMA hydrogel were the same as in Example 1.

[0094] Preparation of bio-ink: Gelatin (Sigma-Aldrich) was dissolved in deionized water to prepare a gelatin hydrogel with a mass-volume concentration of 7.5 mg / mL, and then stained with blue dye (Thermo Fisher Scientific).

[0095] Completely enclose the second support bath in the first support bath: place the red GelMA block hydrogel directly in the AVC hydrogel and keep it at 16℃ / 22℃ respectively to make it in a near-solid or near-liquid state.

[0096] Bio-ink was suspended and printed in a second support bath: Blue gelatin bio-ink was suspended and printed in GelMA hydrogel at 16℃ and 22℃ respectively, and it was found that it formed well, thus constructing a three-dimensional structure in a non-yield stress fluid material.

[0097] Figure 7 This embodiment illustrates the suspension printing of gelatin bio-ink in quasi-solid and quasi-liquid GelMA, which is placed directly in a yield stress fluid material as a first support bath.

[0098] The above experiments demonstrate that, under the condition that an external yield stress fluid material serves as the first support bath, it is possible to perform suspension printing in directly placed solid-like or liquid-like non-yield stress fluid materials to construct three-dimensional structures.

[0099] Then, the GelMA hydrogel was crosslinked, and the AVC hydrogel was removed, and the gelatin bio-ink was removed or crosslinked to obtain a three-dimensional structure based on non-yield stress fluid.

[0100] Example 3

[0101] Preparation of the first support bath: AVC powder was added to deionized water (DI water) and stirred overnight to obtain an AVC hydrogel with a mass-volume concentration of 1.0 mg / mL. Before coating the second support bath, the AVC hydrogel was centrifuged at 1200g for 3 minutes to remove air bubbles. The yield stress of the AVC hydrogel was the same as in Example 1.

[0102] Preparation of the second support bath and bio-ink: A commercially available HAMA (methacryloyl hyaluronic acid, molecular weight 400K, Qingyuan Taishuo Company) precursor solution with a mass-volume concentration of 3 mg / mL was prepared (using deionized water as solvent). The solution was then fluorescently stained with a red or green fluorescent dye (Thermo Fisher Scientific), and a photoinitiator (LAP) was added. The concentration of the photoinitiator in the continuous phase precursor solution was 0.25 mg / mL, resulting in a bulk hydrogel. The green HAMA hydrogel served as the second support bath, and the red HAMA hydrogel served as the bio-ink. The yield stress of the HAMA hydrogel was 3.34 Pa.

[0103] Rheological characterization of HAMA hydrogel and strain scanning showed that the loss modulus of HAMA hydrogel was consistently higher than the storage modulus, proving that it was in a liquid-like state and could not be used as a support bath for suspension printing.

[0104] The second support bath is completely enclosed in the first support bath: green HAMA hydrogel is suspended and printed in AVC hydrogel to form a cylinder with an outer diameter of 16 mm, a height of 12 mm, and a wall thickness of 2 mm.

[0105] Bio-ink was suspended and printed in a second support bath: Red HAMA hydrogel was suspended and printed between the inner and outer walls of a HAMA hydrogel cylinder, creating a red spiral structure.

[0106] The above-mentioned whole was photocrosslinked by ultraviolet irradiation with a light intensity of 60 mW / cm. 2 The time was 100s, and the AVC hydrogel was removed (e.g., by dissolving the AVC hydrogel with a 1.0 mg / mL calcium chloride solution to remove the first support bath) to obtain a three-dimensional structure based on non-yield stress fluid.

[0107] Figure 8 The rheological properties of the HAMA hydrogel in this embodiment are shown. Figure 8 In (a) of the diagram, a red HAMA spiral structure is constructed by suspension printing within a green HAMA hydrogel cylinder. Figure 8 (b) in the middle, and the fidelity of the suspended printed spiral ( Figure 8(c) shows that this embodiment constructs a HAMA cylindrical model with a helical structure. Furthermore, comparing the pitch and linewidth of the suspended helical structure with the designed structure demonstrates good print fidelity.

[0108] The above results demonstrate that the suspension printing strategy based on non-yield stress fluid materials of the present invention can effectively construct three-dimensional structures in liquid-like soft hydrogel materials.

[0109] Example 4

[0110] Preparation of the first support bath: AVC powder was added to deionized water (DI water) and stirred overnight to obtain an AVC hydrogel with a mass-volume concentration of 1.0 mg / mL. Before coating the second support bath, the AVC hydrogel was centrifuged at 1200g for 3 minutes to remove air bubbles. The yield stress of the AVC hydrogel was the same as in Example 1.

[0111] Preparation of the second support bath: A GelMA precursor solution with a mass-volume concentration of 5 mg / mL was prepared using commercially available GelMA (60% substitution, Qingyuan Taishuo Company) (solvent: deionized water). The solution was stained with a green fluorescent dye (ThermoFisher Scientific), and then a photoinitiator (LAP) was added. The concentration of the photoinitiator in the continuous phase precursor solution was 0.25 mg / mL, resulting in a bulk hydrogel. At 19 °C, the yield stress of the GelMA hydrogel was 14.21 Pa.

[0112] Preparation of bio-ink: Gelatin (Sigma-Aldrich) was dissolved in deionized water to prepare a gelatin hydrogel with a mass-volume concentration of 7.5 mg / mL, and then stained with a red fluorescent dye (Thermo Fisher Scientific).

[0113] The second support bath is completely enclosed in the first support bath: at 19°C, green GelMA hydrogel is suspended and printed in AVC hydrogel to form a single-open oral chamber with an outer diameter of 12 mm, a height of 16 mm, and a wall thickness of 2 mm.

[0114] Bio-ink was suspended and printed in a second support bath: Red gelatin bio-ink was suspended and printed between the inner and outer walls of a single-open oral chamber of GelMA hydrogel at 22–24°C, constructing a complex red network structure.

[0115] The above-mentioned whole was photocrosslinked by ultraviolet irradiation with a light intensity of 60 mW / cm. 2 The time was 150s, and the AVC hydrogel was removed to obtain a three-dimensional structure based on non-yield stress fluid.

[0116] Figure 9 This embodiment demonstrates the suspension and printing of red gelatin bio-ink within a green single-opening GelMA hydrogel chamber. Figure 9 (a) in the diagram is the design drawing of the suspended printing structure. Figure 9 (b) shows the structure of a single-open oral cavity in GelMA hydrogel with a complex network structure, embossed during printing. This embodiment successfully constructed a model of a single-open oral cavity in GelMA hydrogel with a complex network structure. Furthermore, the printed structure exhibits good print fidelity compared to the designed structure.

[0117] The above results demonstrate that the suspension printing strategy based on non-yield stress fluid materials of the present invention can effectively construct three-dimensional structures in extracellular matrix-derived hydrogel materials.

[0118] Example 5

[0119] Preparation of the first support bath: AVC powder was added to deionized water (DI water) and stirred overnight to obtain an AVC hydrogel with a mass-volume concentration of 1.0 mg / mL. Before coating the second support bath, the AVC hydrogel was centrifuged at 1200g for 3 minutes to remove air bubbles. The yield stress of the AVC hydrogel was the same as in Example 1.

[0120] Preparation of the second support bath: A commercially available HAMA (methacrylamide hyaluronic acid, molecular weight 400K, Qingyuan Taishuo Company) precursor solution with a mass-volume concentration of 3 mg / mL was prepared (solvent: deionized water). A photoinitiator (LAP) was added, wherein the concentration of the photoinitiator in the continuous phase precursor solution was 0.25 mg / mL, to obtain a bulk hydrogel. The yield stress of the HAMA hydrogel was the same as in Example 3.

[0121] Preparation of bio-ink: A GelMA precursor solution with a mass-volume concentration of 5 mg / mL was prepared using GelMA (60% substitution degree, Qingyuan Taishuo Company) (solvent was deionized water), and fluorescently stained with red fluorescent dye (Thermo Fisher Scientific Company). Then, a photoinitiator (LAP) was added, wherein the concentration of the photoinitiator in the continuous phase precursor solution was 0.25 mg / mL, to obtain GelMA hydrogel bio-ink.

[0122] The second support bath is completely enclosed within the first support bath: A single-opening chamber with an outer diameter of 10 mm, a height of 20 mm, and a wall thickness of 2 mm is suspended and printed in AVC hydrogel using HAMA hydrogel at room temperature. The printing speed is 3 mm / s, and the extrusion speed is 0.4 mm / s. 3 / s.

[0123] Bio-ink was suspended and printed in a second support bath: Red GelMA hydrogel bio-ink was suspended and printed between the inner and outer walls of the single-open oral chamber of the HAMA hydrogel, constructing a complex red network structure.

[0124] The above-mentioned whole was photocrosslinked by ultraviolet irradiation with a light intensity of 60 mW / cm. 2 The time is 150 seconds.

[0125] After crosslinking is complete, 1.0 mg / mL calcium chloride solution is added to dissolve the AVC hydrogel, thereby removing the first support bath and taking out the HAMA hydrogel containing the complex network structure into a single oral cavity.

[0126] Figure 10 This embodiment shows a single-open oral cavity with a complex heterostructure obtained by suspension printing of HAMA hydrogel. Figure 10 (a) shows the design diagram of the heterogeneous structure. Figure 10 (b) in the diagram represents the levitation printing process. Figure 10 In Figure (c), the single-open oral cavity containing a complex heterogeneous structure, is eluted from the first support bath of the AVC hydrogel. This embodiment realizes the construction of a single-open oral cavity model of HAMA hydrogel containing a complex heterogeneous network. Furthermore, the printed structure exhibits good print fidelity compared to the designed structure.

[0127] The above results demonstrate that the suspension printing strategy based on non-yield stress fluid materials of the present invention can effectively construct complex heterogeneous structures in extracellular matrix-derived hydrogel materials.

[0128] Example 6

[0129] Preparation of the first support bath: A microparticle-based gelatin hydrogel material was prepared as the first support bath using a composite coagulation method. Specifically, a mixture containing 9.6 mg / mL type A gelatin (Sigma-Aldrich), 2 mg / mL Pluronic F-127 (Sigma-Aldrich), and 2.4 mg / mL gum arabic (Sigma-Aldrich) was boiled in deionized water. Then, an equal volume of ethanol was added, and the beaker was sealed with a sealing film to minimize evaporation. The mixture was cooled to room temperature and stirred overnight at 500 rpm. The resulting solution containing microgels was centrifuged at 500 g for 3 minutes, and the supernatant was discarded. The resulting gelatin microgels were washed three times with culture medium (DMEM, Thermo Fisher) to remove any residual ethanol and Pluronic F-127. Before use, the resulting granular gelatin was centrifuged at 1000 g for 3 minutes to obtain the first support bath, in which the gelatin mass-volume concentration was 6.4 mg / mL, and the yield stress of the first support bath was 44.85 Pa.

[0130] Preparation of the second support bath: GelMA (60% substitution, Qingyuan Taishuo Company) was dissolved in DMEM medium (Thermo Fisher Scientific) to prepare a GelMA precursor solution with a mass-volume concentration of 5 mg / mL. A photoinitiator (LAP) was then added, with a concentration of 0.25 mg / mL in the continuous phase precursor solution. Human induced pluripotent stem cells were cultured in vitro and induced to differentiate into cardiomyocytes. These cardiomyocytes were then thoroughly mixed with the 5 mg / mL GelMA precursor solution to obtain a cardiomyocyte density of 5 × 10⁶ cells / mL. 7 / mL of GelMA block hydrogel. The yield stress of the GelMA hydrogel at 19℃ is 14.21Pa.

[0131] Preparation of bio-ink: Gelatin (Sigma-Aldrich) was dissolved in deionized water to prepare a gelatin bio-ink with a mass-volume concentration of 7.5%.

[0132] The second support bath was completely enclosed within the first support bath: GelMA hydrogel containing cardiomyocytes was suspended and printed in the granular gelatin first support bath at 19°C, constructing a single-opening chamber with an outer diameter of 8 mm, a height of 10 mm, and a wall thickness of 2 mm. The printing temperature was 22°C, the printing speed was 3 mm / s, and the extrusion speed was 0.4 mm / s. 3 / s.

[0133] Bio-ink was suspended and printed in a second support bath: 7.5 mg / mL gelatin bio-ink was printed in the intermediate layer between the inner and outer walls of a single-open oral chamber of GelMA hydrogel at 22–24 °C to construct a hierarchical vascular network structure. The printing temperature was 21 °C, the printing speed was 3 mm / s, and the extrusion speed was 0.3 mm / s. 3 / s.

[0134] After printing, photocrosslinking is performed with a light intensity of 30 mW / cm². 2 The time is 40 seconds.

[0135] After cross-linking, the cells were incubated in an incubator (37°C and 5% CO2) for 30 minutes to dissolve the first support bath and sacrifice the gelatin bio-ink, thereby constructing a myocardial compartment containing a hollow channel.

[0136] Endothelial cells were seeded into the channels via perfusion implantation to form vascularized channels. Furthermore, the vascularized channels of the myocardial chambers were continuously perfused with culture medium in vitro to provide the necessary oxygen and nutrients to the cardiomyocytes. After two weeks of culture, the myocardial chambers exhibited overall beating, thus obtaining large-scale, functionally mature vascularized myocardial chambers. Immunofluorescence staining confirmed sarcomere maturation.

[0137] Figure 11 This embodiment shows a channel-containing, high-cell-density GelMA hydrogel myocardial compartment that has been suspended and printed. Figure 11 (a) in the diagram is a design diagram of the myocardial chambers. Figure 11 (b) in the figure represents a myocardial chamber containing channels obtained through suspension printing. Figure 11 (c) in the figure represents the synchronous myocardial contraction curve. Figure 11 In the diagram, (d) represents staining for myocardial-specific proteins. This embodiment demonstrates the construction of a myocardial compartment using a soft hydrogel material containing vascular channels.

Claims

1. A suspension printing method based on non-yield stress fluid, comprising the following steps: S1. A yield stress fluid material is used as the first support bath; S2. A non-yielding stress fluid material is used as the second support bath; S3. Wrap the second support bath in the first support bath; S4. Suspend and print bio-ink in the second support bath to construct a three-dimensional structure in the non-yielding stress fluid material; S5. Crosslink the non-yield stress fluid material, remove the first support bath, remove or crosslink the bio-ink to obtain a three-dimensional structure based on non-yield stress fluid.

2. The suspension printing method based on non-yield stress fluid according to claim 1, wherein, In step S1, the yield stress of the yield stress fluid material is 5~100 Pa.

3. The suspension printing method based on non-yield stress fluid according to claim 1, wherein, In step S1, the yield stress fluid material includes a support bath material with self-healing properties.

4. The suspension printing method based on non-yield stress fluid according to claim 3, wherein, In step S1, the support bath material with self-healing properties includes microparticle-based hydrogel materials, continuous phase-based hydrogel materials, inorganic colloids, or organic-inorganic composite colloids.

5. The suspension printing method based on non-yield stress fluid according to claim 4, wherein, In step S1, the microparticle-based hydrogel material is a particulate hydrogel material; the continuous phase-based hydrogel material is a hydrogel material containing reversible dynamic bonds; the inorganic colloid or organic-inorganic composite colloid includes a colloidal material containing one or more of the following: nano-clay, mineral oil-fumed silica, and polydimethylsiloxane-fumed silica.

6. The suspension printing method based on non-yield stress fluid according to claim 5, wherein, In step S1, the continuous phase-based hydrogel material further includes one or a combination of several of host-guest hydrogel materials, metal ion coordination bond hydrogel materials, and interpenetrating polymer network hydrogel materials.

7. The suspension printing method based on non-yield stress fluid according to claim 4, wherein, In step S1, the raw materials for the microparticle-based hydrogel material and the continuous phase-based hydrogel material respectively include natural polymers and / or synthetic polymers.

8. The suspension printing method based on non-yield stress fluid according to claim 7, wherein, In step S1, the raw materials for the microparticle-based hydrogel material and the continuous phase-based hydrogel material respectively include one or a combination of several of sodium alginate, gelatin, carrageenan, gellan gum, agarose, carbomer, and acrylamide dimethyl taurate ammonium / VP copolymer.

9. The suspension printing method based on non-yield stress fluid according to claim 4, wherein, In step S1, the microparticle-based hydrogel material is prepared by at least one of the following methods: electrospraying, mechanical fragmentation, photolithography, composite agglomeration, or microfluidics.

10. The suspension printing method based on non-yield stress fluid according to claim 4, wherein, In step S1, the mass-volume concentration of the material providing the hydrogel or colloidal three-dimensional network structure in the yield stress fluid material is 1~100 mg / mL.

11. The suspension printing method based on non-yield stress fluid according to claim 10, wherein, In step S1, the mass-volume concentration of the material providing the hydrogel or colloidal three-dimensional network structure in the yield stress fluid material is 1~10 mg / mL.

12. The suspension printing method based on non-yield stress fluid according to claim 1, wherein, In step S2, the non-yield stress fluid material is in a solid-like or liquid-like state.

13. The suspension printing method based on non-yield stress fluid according to claim 1, wherein, In step S2, the yield stress of the non-yield stress fluid material is less than the yield stress of the yield stress fluid material.

14. The suspension printing method based on non-yield stress fluid according to claim 13, wherein, In step S2, the yield stress of the non-yield stress fluid material is 4 to 80% of the yield stress of the yield stress fluid material.

15. The suspension printing method based on non-yield stress fluid according to claim 1, wherein, In step S2, the non-yielding stress fluid material includes natural polymer hydrogel materials and / or synthetic polymer hydrogel materials.

16. The suspension printing method based on non-yield stress fluid according to claim 15, wherein, In step S2, the non-yielding stress fluid material includes hydrogel materials derived from and / or derived from extracellular matrix.

17. The suspension printing method based on non-yield stress fluid according to claim 15, wherein, In step S2, the non-yielding stress fluid material includes a hydrogel material containing one or more of gelatin, collagen, hyaluronic acid and their methacrylamide products.

18. The suspension printing method based on non-yield stress fluid according to claim 15, wherein, In step S2, the mass-volume concentration of the material providing the hydrogel three-dimensional network structure in the non-yielding stress fluid material is 1~100 mg / mL.

19. The suspension printing method based on non-yield stress fluid according to claim 18, wherein, In step S2, the mass-volume concentration of the material providing the hydrogel three-dimensional network structure in the non-yielding stress fluid material is 2~25 mg / mL.

20. The suspension printing method based on non-yield stress fluid according to claim 1, wherein, In step S2, the non-yielding stress fluid material contains cells with a cell density of 1×10⁻⁶. 6 / mL~5×10 7 / mL.

21. The suspension printing method based on non-yield stress fluid according to claim 1, wherein, In step S3, the second support bath is wrapped in the first support bath by either suspension printing or direct injection.

22. The suspension printing method based on non-yield stress fluid according to claim 1, wherein, In step S3, the second support bath is completely enclosed within the first support bath.

23. The suspension printing method based on non-yield stress fluid according to claim 1, wherein, In step S4, the bio-ink comprises natural polymer hydrogel materials and / or synthetic polymer hydrogel materials.

24. The suspension printing method based on non-yield stress fluid according to claim 1, wherein, In step S4, the bio-ink contains cells at a density of 1×10⁻⁶ cells. 6 / mL~5×10 7 / mL.

25. The suspension printing method based on non-yield stress fluid according to claim 23, wherein, In step S4, the mass-volume concentration of the material providing the hydrogel three-dimensional network structure in the bio-ink is 1~100 mg / mL.

26. The suspension printing method based on non-yield stress fluid according to claim 25, wherein, In step S4, the mass-volume concentration of the material providing the hydrogel three-dimensional network structure in the bio-ink is 4~30 mg / mL.

27. The suspension printing method based on non-yield stress fluid according to claim 1, wherein, In step S5, the crosslinking of the non-yielding stress fluid material includes one or a combination of temperature change, ionic reaction, ultraviolet light irradiation, and pH change.

28. The suspension printing method based on non-yield stress fluid according to claim 1, wherein, In step S5, the first support bath is removed by one or a combination of temperature change, ion reaction and mechanical cleaning.

29. The suspension printing method based on non-yield stress fluid according to claim 1, wherein, In step S5, the bio-ink is cross-linked or removed by one or a combination of temperature change, ionic reaction, ultraviolet light irradiation and pH change.

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