Multimodal method and system for printing a three-dimensional construct or a portion thereof

Abstract

The invention provides a multi-modal method and system for printing a three-dimensional (3D) construct or a portion thereof, the method comprising: (i) supporting a flowable precursor with a flowable support at a location that allows the flowable precursor to be cross-linked by radiation of a radiation source; (ii) depositing one or more materials on or within the flowable precursor; and (iii) cross-linking at least a portion of the flowable precursor by radiation of a radiation source to form the 3D construct or a portion thereof, wherein at least a portion of the flowable precursor is in contact with the one or more materials in the flowable precursor.

Description

Technical Field

[0001] This application broadly relates to multimodal methods and systems for printing three-dimensional (3D) structures or portions thereof. Background Technology

[0002] Multimodal 3D printing is a field of great interest because most manufactured products are made from multiple materials. Multimodal printing can be used for bioprinting to combine soft and hard materials, for example, to create structures such as bone / ligament interfaces, and to create bioprinted structures containing blood vessels. Multimodal 3D printing can also be combined with other material deposition methods, such as inkjet printing, to allow for the establishment of concentration gradients of materials such as biopharmaceuticals within bioprinted structures.

[0003] However, conventional 3D printers may not be suitable for multimodal printing because they typically have two solid surfaces: a print platform and a patterning screen / projection window. In use, the print platform is usually close to the patterning screen and gradually rises as the material crosslinks. This configuration leaves no space between the print platform and the patterning screen for introducing a second morphology (e.g., an extruder and / or printhead) for depositing other materials.

[0004] Furthermore, most bioprinters employ a syringe extrusion method, where a rasterized syringe is used to deposit hydrogel bio-ink that may contain cells to create each layer, progressing incrementally to the next. In such systems, a trade-off is made between resolution, speed, and cell compatibility, with one of these always being sacrificed. For example, inventors previously developed a 3D printer capable of printing living cells at speeds up to 200 mm / hr, at least an order of magnitude faster than previous systems. However, the absence of a vascular system in the printed structure limited its thickness to a few hundred micrometers before diffusion limitations affected cell viability.

[0005] Therefore, there is a need for multimodal methods and systems for printing 3D structures or parts thereof, which seek to solve or at least improve one of the aforementioned problems. Summary of the Invention

[0006] On one hand, this application provides a multimodal method for printing a three-dimensional (3D) structure or a portion thereof, the method comprising: (i) supporting a flowable precursor with a flowable support at a location where the flowable precursor is allowed to crosslink by radiation from a radiation source; (ii) depositing one or more materials onto or within the flowable precursor; and (iii) crosslinking at least a portion of the flowable precursor by radiation from a radiation source to form the 3D structure or a portion thereof, wherein at least a portion of the flowable precursor is in contact with one or more materials in the flowable precursor.

[0007] In one embodiment, the method further includes: (iv) supporting the flowable precursor at a new location that allows the flowable precursor to be crosslinked by radiation from the radiation source; (v) optionally further depositing one or more materials on or within the flowable precursor; (vi) further crosslinking at least a portion of the flowable precursor by radiation from the radiation source to form the 3D structure or a portion thereof, wherein at least a portion of the flowable precursor is optionally in contact with one or more materials at the new location; and (vii) optionally repeating steps (iv) to (vi) until the desired 3D structure is formed.

[0008] In one embodiment, depositing one or more materials includes extruding sacrificial materials to form an arrangement having one or more sacrificial structures in the 3D construction.

[0009] In one embodiment, the method further includes removing the sacrificial material from an arrangement having one or more sacrificial structures to form an arrangement having one or more channels in the 3D construction.

[0010] In one embodiment, the one or more channels include an open end that facilitates fluid communication between the internal environment of the 3D fabrication and the external environment of the 3D fabrication.

[0011] In one embodiment, the sacrificial material is in a semi-solid or gel state when it is extruded to form an arrangement having one or more sacrificial structures.

[0012] In one embodiment, removing the sacrificial material includes liquefying the sacrificial material to facilitate its removal from the 3D structure or a portion thereof.

[0013] In one embodiment, liquefying the sacrificial material includes cooling the sacrificial material to a temperature that would cause the sacrificial material to liquefy.

[0014] In one embodiment, depositing one or more materials includes advancing droplets containing one or more materials onto the flowable precursor.

[0015] In one embodiment, advancing a droplet comprising one or more materials on the flowable precursor includes advancing the droplet at a variable rate such that a spatial concentration gradient of one or more materials is formed in the 3D structure.

[0016] In one embodiment, the cross-linking step by radiation from the radiation source is performed in an upward direction, such that the 3D structure or a portion thereof is formed layer by layer in an upward direction.

[0017] In one embodiment, the crosslinking step by radiation from the radiation source is performed continuously.

[0018] In one embodiment, depositing one or more materials onto or within the flowable precursor includes depositing the one or more materials in a top-down direction toward the top surface of the flowable precursor.

[0019] In one embodiment, the flowable support and the flowable precursor are immiscible.

[0020] In one embodiment, the flowable precursor is biocompatible with the one or more materials.

[0021] In one embodiment, the density of the 3D construct is higher than the density of the flowable precursor and the flowable support.

[0022] In one embodiment, the flowable precursor and / or one or more materials comprise cells.

[0023] On one hand, a multimodal system is provided for performing the multimodal method disclosed herein, the system comprising: a tank configured to include a flowable support and a flowable precursor disposed above the flowable support; a radiation source configured to radiate the flowable precursor; and one or more dispensing outlets disposed between the tank and the radiation source and configured to deposit the one or more materials onto or within the flowable precursor; wherein the radiation source is configured to crosslink at least a portion of the flowable precursor by radiation to form the 3D structure or a portion thereof, wherein at least a portion of the flowable precursor is in contact with one or more materials in the flowable precursor.

[0024] In one embodiment, the one or more dispensing outlets include extruder nozzles for extruding sacrificial material to form a template for arranging one or more channels in a 3D structure.

[0025] In one embodiment, the one or more dispensing outlets include inkjet printheads for propelling droplets containing one or more materials onto the flowable precursor.

[0026] definition

[0027] The term “biocompatible” as used in this article should be interpreted broadly as the ability of a substance to perform its intended function without inducing a significant inflammatory response, immunogenicity, or cytotoxicity in natural cells, tissues, or organs.

[0028] Unless otherwise specified, the term “cell” as used herein refers to a single cell, cell line, primary culture, or culture derived from such cells.

[0029] The term “substrate” as used in this article should be interpreted broadly to refer to any supporting structure.

[0030] The term "layer," when used to describe a first material, should be interpreted broadly as referring to a first depth of the first material, which can be distinguished from a second depth of the second material. The first material of the layer can exist in the form of a continuous film, a discontinuous structure, or a mixture of both. The layer can also have a substantially uniform depth throughout its length or a varying depth. Therefore, when the layer is formed from individual structures, the dimensions of each individual structure can be different. The first material and the second material can be the same or different, and the first depth and the second depth can be the same or different.

[0031] The term "continuous" in the context of describing the printing of 3D structures refers broadly to the way in which a 3D structure is formed without any noticeable pauses, breaks, or breaks between continuous parts or segments of the formed 3D structure.

[0032] When used in this article to describe objects, the term "substantially transparent to light" should be interpreted broadly as meaning that 50% or more of incident light perpendicular to the object's surface can be transmitted through it. In some examples, substantially transparent objects allow 60% or more, 65% or more, 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more of incident light perpendicular to the object's surface to be transmitted. In one example, substantially transparent objects allow more than 70% of incident light perpendicular to the object's surface to be transmitted.

[0033] The term “micro” as used in this article should be interpreted broadly to include sizes ranging from about 1 micrometer to about 1,000 micrometers.

[0034] The term “nano” as used in this article should be interpreted broadly to include sizes smaller than approximately 1000 nm.

[0035] As used herein, the term "particle" broadly refers to a discrete entity or discrete body. The particles described herein may include organic particles, inorganic particles, or biological particles. The particles described herein may also be large particles formed from aggregates of multiple sub-particles or fragments of small objects. The particles of this application may be spherical, substantially spherical, or non-spherical, such as irregularly shaped particles or elliptical particles. When used to refer to a particle, the term "size" broadly refers to the maximum size of the particle. For example, when the particle is substantially spherical, the term "size" may refer to the diameter of the particle; or when the particle is substantially non-spherical, the term "size" may refer to the maximum length of the particle.

[0036] Unless otherwise stated, the terms “coupled” or “connected” as used in this specification are intended to cover direct connection or connection via one or more intermediate means.

[0037] The term "association" used in this article when referring to two elements refers to a broad relationship between the two elements. This relationship includes, but is not limited to, physical, chemical, or biological relationships. For example, when element A is associated with element B, element A and element B may be directly or indirectly connected to each other, or element A may contain element B, and vice versa.

[0038] When referring to two elements, the term “adjacent” as used herein means that one element is very close to another element and may be elements that are in contact with each other, but may further include elements separated by one or more other elements disposed between them.

[0039] The term “and / or”, such as “X and / or Y”, should be understood as “X and Y” or “X or Y”, and should be regarded as providing explicit support for both or one of these meanings.

[0040] Furthermore, throughout this description, the word “substantially” should be understood to include, but is not limited to, “completely” or “entirely” whenever used. Additionally, terms such as “comprising” or “comprise” are non-limiting descriptive language, as they broadly encompass elements / components listed after them, in addition to other components not explicitly enumerated. For example, when “comprising” is used, referring to “one” feature should also mean referring to “at least one” of that feature. In the appropriate context, terms such as “consisting” or “consist” can be considered a subset of terms such as “comprising” or “comprise”. Therefore, in the embodiments disclosed herein that use terms such as “comprising” or “comprise”, it should be understood that these embodiments provide teachings on the use of terms such as “consisting” or “consist” for the respective embodiments. Furthermore, whenever used, terms such as “approximately” or “about” generally imply a reasonable deviation, such as a deviation of + / -5% from the published value, or a deviation of 4% from the published value, or a deviation of 3% from the published value, or a deviation of 2% from the published value, or a deviation of 1% from the published value.

[0041] Furthermore, in this description, certain values ​​may be disclosed within a range. The values ​​shown at the endpoints of the range are intended to illustrate preferred ranges. Whenever a range is described, it is intended to indicate that the range covers and teaches all possible subranges and individual values ​​within that range. In other words, the endpoints of a range should not be interpreted as inflexible limitations. For example, the description of a range of 1% to 5% is intended to specifically disclose subranges such as 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3%, etc., and individually, values ​​within that range, such as 1%, 2%, 3%, 4%, and 5%. It should be understood that individual values ​​within this range also include integers, fractions, and decimals. Furthermore, whenever a range is described, it is also intended to indicate that the range covers and teaches values ​​with up to two additional decimal places or significant figures (where appropriate) from the endpoints of the indicated values. For example, the description of the 1% to 5% range is intended to specifically disclose ranges of 1.00% to 5.00% and 1.0% to 5.0% and all intermediate values ​​spanning these ranges (e.g., 1.01%, 1.02%... 4.98%, 4.99%, 5.00% and 1.1%, 1.2%... 4.8%, 4.9%, 5.0%, etc.). The above specific disclosure is intended to apply to any depth / width of the range.

[0042] Additionally, when describing some embodiments, this disclosure may disclose methods and / or processes as a specific sequence of steps. However, unless otherwise required, it should be understood that the method or process should not be limited to the specific order of steps disclosed. Other sequences of steps are also possible. The specific order of steps disclosed herein should not be construed as an undue limitation. Unless otherwise required, the methods and / or processes disclosed herein should not be limited to steps performed in the order written. The order of steps may be changed and still remain within the scope of this disclosure.

[0043] Furthermore, it should be understood that while this disclosure provides embodiments having one or more of the features / characteristics discussed herein, one or more of these features / characteristics may also be omitted in other alternative embodiments, and this disclosure provides support for such omissions and these related alternative embodiments. Detailed Implementation

[0044] The following discloses a multimodal method for printing 3D structures or portions thereof, and non-limiting embodiments of a multimodal system for performing the multimodal method for printing 3D structures or portions thereof.

[0045] In various embodiments, a multimodal method is provided for printing a three-dimensional (3D) structure or a portion thereof, the method comprising: (i) supporting a flowable precursor with a flowable support at a location where the flowable precursor is allowed to crosslink by radiation from a radiation source; (ii) depositing one or more materials onto or within the flowable precursor; and (iii) crosslinking at least a portion of the flowable precursor by radiation from a radiation source to form the 3D structure or a portion thereof, wherein at least a portion of the flowable precursor is in contact with one or more materials in the flowable precursor.

[0046] In various embodiments, one or more modalities different from the modality used to crosslink the flowable precursor are used to deposit the one or more materials. In various embodiments, the step of crosslinking the flowable precursor by radiation from a radiation source is performed via a first printing modality. The first printing modality may be, for example, a fluid-supported liquid interface polymerization (FLIP) printer according to various embodiments disclosed herein. In various embodiments, the step of depositing one or more materials on or within the flowable precursor is performed using a second printing modality or another printing modality (e.g., inkjet printing or extrusion printing different from the first printing modality (e.g., fluid-supported liquid interface polymerization)). In various embodiments where more than two printing modalities are used in the method, the second printing modality and another printing modality (e.g., a third printing modality) may be the same as or different from each other, but both the second and third printing modalities are different from the first printing modality.

[0047] In various embodiments, the one or more materials may be the same as or different from the crosslinked flowable precursor. In various embodiments, the one or more materials may differ from the crosslinked flowable precursor in terms of physical / mechanical characteristics / properties (e.g., shape, size, mass, volume, density, melting point, ductility, stiffness, etc.) and / or chemical characteristics / properties (e.g., chemical composition, pH value, reactivity, reaction rate, ionic strength, bond strength, etc.). In various embodiments, if multiple different materials are deposited on or within the flowable precursor, the modes used for depositing the different materials may differ from each other. In various embodiments, one or more materials may be incorporated into a 3D fabrication, such as the final structure of the 3D fabrication.

[0048] In various embodiments, the method further includes: (iv) supporting the flowable precursor at a new location that allows the flowable precursor to crosslink under radiation from the radiation source; (v) optionally further depositing one or more materials onto or within the flowable precursor; (vi) further crosslinking at least a portion of the flowable precursor under radiation from the radiation source to form the 3D structure or a portion thereof, wherein at least a portion of the flowable precursor is optionally in contact with one or more materials at the new location; and (vii) optionally repeating steps (iv) to (vi) until the desired 3D structure is formed. In various embodiments, step (v) of further depositing or within the flowable precursor the one or more materials is optional for some iterations of steps (iv) to (vi), such that the one or more materials are present only in selected portions or regions of the 3D structure.

[0049] Advantageously, the multimodal approach can be used to print 3D structures comprising more than one material, i.e., multimaterial printing. Various embodiments of this method can be used in bioprinting applications to print living 3D structures (i.e., structures containing cells), such as bone / ligament interfaces and bioprinted structures containing blood vessels, and structures with complex geometries associated with anatomical structures.

[0050] In various embodiments, the flowable precursor supported by a flowable support includes a top surface that is connected to or exposed to a fluid medium. In various embodiments, the fluid medium can be air, or an inert gas or liquid (e.g., mineral oil or silicone oil), which, for example, does not react with the precursor and / or does not react in the presence of radiation (e.g., cross-linking). In various embodiments, the fluid medium is also a fluid medium that allows radiation to pass through. In various embodiments, the method does not have a screen or panel (e.g., a glass screen) in contact with the top surface of the flowable precursor. In various embodiments, the pattern is projected directly onto a thin layer (e.g., about 3 mm thick) of the fluid-supported precursor (e.g., a hydrogel precursor), which serves as a floating liquid projection screen. This advantageously avoids unintentional adhesion to screens or panels (e.g., glass screens), thus preventing disruption to typical resin-based 3D printers and enabling rapid continuous printing.

[0051] In various embodiments, the top surface of the flowable precursor provides a relatively unobstructed free surface at the precursor-fluid medium interface. Advantageously, this configuration of the first printing modality offers great flexibility in introducing / incorporating other different printing modalities due to the space utilization provided by the free surface. The free surface of the flowable precursor can advantageously allow or facilitate the addition of one or more materials to the flowable precursor. For example, in a 3D printer apparatus (e.g., a fluid-supported liquid interface polymerization (FLIP) printer) for performing the methods disclosed herein, the free surface can advantageously allow other printing modalities (e.g., extrusion or piezoelectric deposition of one or more different materials) to be combined or integrated into the 3D printer setup. Some advantages of using different modalities may include increased throughput (e.g., certain other modalities can print certain more intricate and complex structures within the 3D construct faster); easy introduction of different types of materials into the 3D construct; and allowing intermittent cooling / non-operation / charging while still allowing seamless and continuous printing, thereby increasing part life and efficiency, when different modalities are interchanged during operation.

[0052] In various embodiments, the flowable precursor supported by the flowable support includes a bottom surface connected to or exposed to the flowable support. In other words, the flowable precursor is disposed (e.g., floating) on ​​the flowable support. In various embodiments, the flowable precursor may be directly connected to the flowable support or indirectly connected to the flowable support via one or more intervening intermediate layers disposed between the flowable precursor and the flowable support (e.g., another flowable liquid or separating substrate / solid). In various embodiments, the flowable precursor and the flowable support are immiscible and form separate, distinct phases. Advantageously, the flowable support provides the necessary buoyancy to support the flowable precursor, allowing it to be appropriately presented / supported toward the radiation source for crosslinking. In various embodiments, the buoyancy of the flowable support is sufficient to support the flowable precursor but insufficient to allow the 3D fabrication to float in or on the flowable support. Therefore, in various embodiments, the 3D fabrication is still supported by the printing platform plate. In various embodiments, the density of the flowable support is higher than the density of the flowable precursor, and the density of the fluid medium is lower than the density of the flowable precursor. In various embodiments, the density of the 3D construct is higher than the densities of the flowable precursor, the flowable support, and the fluid medium.

[0053] In various embodiments, a flowable precursor is disposed between a flowable support and a fluid medium (e.g., air), for example, the flowable precursor is connected to both the flowable support and the fluid medium. In various embodiments, the flowable precursor (the entire flowable precursor) can be directly connected to the flowable support and the fluid medium, i.e., without any other intermediate layer; in other words, the flowable precursor can be in direct contact with the fluid medium and the flowable support, for example, the top surface of the flowable precursor forms an interface with the fluid medium, and the bottom surface of the flowable precursor forms an interface with the flowable support. In various embodiments, the flowable precursor, the flowable support, and the fluid medium are connected in the following configuration: flowable support - flowable precursor - fluid medium. In other words, the flowable precursor can be sandwiched between the flowable support and the fluid medium without an intermediate solid screen / panel (e.g., a glass screen).

[0054] In various embodiments, the step of supporting the flowable precursor in a new location includes adjusting / changing the position / level of the flowable precursor. In various embodiments, the step of adjusting / changing the position / level of the flowable precursor includes adjusting / changing the position or level of the flowable support. For example, adjusting the position of the top surface of the flowable precursor to be crosslinked includes adjusting the position of the upper interface of the flowable support (e.g., the top surface of the flowable support connected to another medium, such as the precursor). The adjustment step may then further include adjusting the position of the intervening intermediate layer. In various embodiments, adjusting the position of the upper interface of the flowable support results in a simultaneous change or adjustment of the position / level of any intermediate layer and / or the position of the top surface of the flowable precursor. In various embodiments, as the position / level of the flowable precursor is raised, a pattern corresponding to the cross-section of the 3D structure to be printed is sequentially transferred onto the flowable precursor via a radiation source, causing the flowable precursor to crosslink according to the pattern.

[0055] In various embodiments, the step of adjusting / changing the position / level of the flowable support includes raising the level of the flowable support (e.g., by increasing the volume of the flowable support and / or by moving the level of a base (such base may be a solid substrate or an intermediate liquid layer), wherein the flowable support rests upward on the base without changing the volume of the flowable support). In various embodiments, the step of increasing the volume of the flowable support may include maintaining laminar flow of the flowable support into a container / tank (e.g., having a Reynolds number of about 3) that contains the flowable precursor to minimize or prevent disruption to the crosslinking process of the flowable precursor. In various embodiments, the flowable precursor is attached to the flowable support. Therefore, the adjustment step may additionally / alternatively include adjusting the position of the interface between the flowable precursor and the flowable support.

[0056] In various embodiments, the step of supporting the flowable precursor at a new location includes adjusting / changing the position / level of the flowable precursor relative to the 3D structure or a portion thereof. In various embodiments, the step of adjusting / changing the relative position / level of the flowable precursor may include adjusting / changing the position / level of the 3D structure or a portion thereof. For example, adjusting / changing the position / level of the flowable precursor relative to the 3D structure or a portion thereof may include maintaining a constant volume of the flowable support and changing the position of the 3D structure or a portion thereof relative to the flowable precursor. This can be achieved by moving the print bed, which supports the 3D structure or a portion thereof, in a downward direction, such that the relative positions of the flowable precursor and the 3D structure or a portion thereof change.

[0057] In various embodiments, the flowable support can be a liquid immiscible with the precursor and can be selected based on the flowable precursor. For example, for aqueous precursors, the flowable support can be a perfluorinated oil, such as FC-40, HFE7500, etc., or an immiscible aqueous solution (i.e., forming an aqueous two-phase system). For common resins (if used as flowable precursors), the supporting fluid (or flowable support) can be water. In various embodiments, one criterion for selecting the flowable support is that the density of the flowable support is greater than that of the flowable precursor.

[0058] In various embodiments, the flowable precursor comprises at least one polymerizable monomer / crosslinkable polymer and at least one photoinitiator. In various embodiments, the flowable precursor is a hydrogel precursor capable of crosslinking to form a hydrogel. The hydrogel can be formed using monomers (e.g., acrylic acid, methacrylic acid) or macromonomers (e.g., methacrylate gelatin, PEG-diacrylate). Non-limiting examples of polymers or copolymers suitable for forming hydrogels include polyacrylates, polymethacrylates, polyacrylamide, polymethacrylamide, polyvinylpyrrolidone, and copolymers thereof. Other examples include polyethers, polyurethanes, and poly(ethylene glycol) functionalized with crosslinking groups or used in combination with compatible crosslinking agents. In various embodiments, the polymer chains can be modified with reactive groups such as acrylates or methacrylates so that the polymer chains are photocrosslinkable. In various embodiments, the hydrogel may comprise macromolecules, including but not limited to modified polycaprolactone, gelatin, methacrylate gelatin, alginate, methacrylate alginate, modified chitosan, methacrylate chitosan, ethylene glycol chitosan, ethylene glycol chitosan methacrylate, modified hyaluronic acid (HA), HA methacrylate, and other non-crosslinked natural or synthetic polymer chains. In various embodiments, the macromolecules may be modified to make them crosslinkable. For example, acrylates, methacrylates, or other types of conjugated chemicals may be used to form covalent crosslinks. In various embodiments, the flowable precursor may include methacrylate gelatin (GelMA) and / or methacrylate alginate (ALMA).

[0059] In various embodiments, the photoinitiator may be a free radical photoinitiator that generates free radicals upon exposure to radiation (e.g., ultraviolet or visible radiation) and thereby initiates the polymerization reaction. Non-limiting examples of suitable photoinitiators include benzophenones (aromatic ketones), such as benzophenone and methyl benzophenone; acylphosphine oxide photoinitiators such as diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide; and benzoin and benzoin alkyl ethers, such as benzoin, benzoin methyl ether, benzoin isobutyl ether, etc.

[0060] In various embodiments, the starting material for the flowable precursor may include monomers (e.g., acrylic acid) or macromonomers (e.g., poly(ethylene glycol) diacrylate (PEGDA), said macromonomer being a polymer chain modified with two acrylate groups). The macromonomers may have different chain lengths (e.g., about 100 Da to about 10,000 kDa, about 200 Da to about 9,500 kDa, about 300 Da to about 9,000 kDa, about 400 Da to about 8,500 kDa, about 500 Da to about 8,000 kDa).

[0061] In various embodiments, depending on the type of material used to manufacture the 3D structure, the concentration of the monomer or macromonomer ranges from about 1% to about 100% of the total weight of the flowable precursor. For 3D hydrogel structures, the concentration of the monomer or macromonomer can be selected from the following numerical groups at the start and end points: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, and 50% of the total weight of the flowable precursor. For 3D plastic resin structures, the concentration of the monomer or macromonomer can be selected from the following numerical groups at the start and end points: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100% of the total weight of the flowable precursor. In various embodiments, the concentration of the photoinitiator is from about 0.05% to about 10% of the total weight of the flowable precursor. The concentration range of the photoinitiator can be, for example, from about 0.05% to about 10%, from about 0.065% to about 9%, from about 0.08% to about 8%, from about 0.095% to about 7%, from about 0.11% to about 6%, and from about 0.125% to about 5%.

[0062] In various embodiments, one or more materials in step (ii) differ from the flowable precursor. One or more materials may differ from the flowable precursor in terms of physical and / or chemical properties / properties. In various embodiments, the one or more materials may be deposited using different modalities, such as inkjet printing or extrusion printing.

[0063] In various embodiments, the step of depositing one or more materials includes extruding sacrificial material to form an arrangement having one or more sacrificial structures in a 3D fabrication. In various embodiments, the sacrificial material can be extruded from a dispensing outlet (e.g., an extruder nozzle positioned between a radiation source and a flowable precursor). An arrangement having one or more sacrificial structures can include an arrangement having one or more sacrificial fibers that act as a mold / template for an arrangement of one or more channels in the 3D fabrication. The sacrificial fibers can be straight or curved. The one or more sacrificial fibers can be arranged in a regular or irregular pattern. One or more sacrificial structures can be shaped and sized according to a predetermined design and can be used as molds / templates for various structures in 3D fabrications (e.g., overhanging structures, freestanding hollow structures, complex geometries associated with anatomical structures). The sacrificial structure itself can also be three-dimensional and span different planes of the 3D fabrication.

[0064] In various embodiments, the method further includes removing sacrificial material from an arrangement having one or more sacrificial structures to form an arrangement in the 3D construction having one or more channels. In various embodiments, the one or more channels include open ends that facilitate communication between the internal environment and the external environment of the 3D construction. In various embodiments, the method further includes removing sacrificial material from an arrangement having one or more sacrificial structures to form various structures in the 3D construction, such as overhanging structures, freestanding hollow structures, and complex geometries associated with anatomical structures.

[0065] In various embodiments, the sacrificial material is in a semi-solid or gel state when it is extruded to form the sacrificial structure. Advantageously, the gel state helps the sacrificial structure maintain its shape and form in the liquid-flowable precursor, so that the sacrificial material does not mix with the flowable precursor and substantially does not lose its shape and form during the formation of the 3D structure.

[0066] In various embodiments, the step of removing the sacrificial material may include liquefying the sacrificial material to facilitate its removal from the 3D structure or a portion thereof. In various embodiments, the step of liquefying the sacrificial material depends on the type of sacrificial material used. For example, the sacrificial material may be liquefied using physical methods (e.g., by changing the temperature of the sacrificial material), chemical methods (e.g., by modifying / altering the chemical composition of the sacrificial material), or a combination thereof.

[0067] In various embodiments, liquefying the sacrificial material may include cooling the sacrificial material to a temperature that liquefies it. Cooling the sacrificial material may include immersing the 3D structure or a portion thereof in a water bath at a temperature that liquefies the sacrificial material. In various embodiments, the sacrificial material may be a thermosensitive material that changes its physical and / or chemical properties in response to temperature changes. The temperature at which the physical and / or chemical properties change depends on the type of sacrificial material used.

[0068] In one example, the sacrificial material may include a fadeable ink (e.g., poloxamer 407, also known by the trade name Pluronic F127). Depending on the concentration, F127 is configured to switch between liquid and semi-solid or gel states. For example, at a 20% w / w concentration, F127 material is liquid below 20°C, gel-like between about 20°C and about 70°C, and liquefies again above 70°C. The sacrificial ink is configured to remain in a gel state at the printing temperature and melt at elevated or lower temperatures. In other words, other materials, such as agarose, can be used if the printing application allows temperatures to rise to 40°C to 50°C. For bioprinting applications, temperatures above 40°C are unsuitable for living cells because the high temperatures can affect cell viability and may even lead to cell death. Therefore, a sacrificial material that makes the printing temperature suitable for cells should be selected. For other printing applications that do not involve living cells, the choice of sacrificial material may have greater flexibility, as cell viability may not be a consideration in such applications.

[0069] In various embodiments, liquefying the sacrificial material may include altering / modifying the chemical composition of the sacrificial material to liquefy it. For example, altering the chemical composition of the sacrificial material may include removing a reversible crosslinking agent used to maintain the sacrificial material in a solid or semi-solid form. In various embodiments, liquefying the sacrificial material may include dissolving the sacrificial material. In various embodiments, the sacrificial material may comprise alginate. In the presence of a crosslinking solution containing polyvalent cations (e.g., calcium), alginate molecules form a gel. In various embodiments, the gel-state alginate fibers may be extruded to form an arrangement having one or more sacrificial structures in a 3D construction. In various embodiments, liquefying a sacrificial material made of alginate may include immersing the 3D construction in a buffer solution containing a chelating agent (e.g., citrate, EDTA, EGTA), which removes cations and causes the alginate fibers to dissolve.

[0070] In various embodiments, the step of removing the sacrificial material is primarily accomplished by melting the sacrificial material (e.g., sacrificial ink) under suitable temperature or chemical conditions. In various embodiments, the step of removing the sacrificial material may also include actively removing or diluting the liquefied sacrificial material from the 3D structure, for example, by rinsing / washing with water, a suitable buffer solution (e.g., a buffer containing a chelating agent), or cell culture medium to remove the liquefied sacrificial material from the 3D structure. In various embodiments, the step of removing the sacrificial material may also include passively removing or diluting the liquefied sacrificial material from the 3D structure, for example, by immersing or soaking the 3D structure in water, a suitable buffer solution (e.g., a buffer containing a chelating agent), or cell culture medium for a period of time to allow the liquefied sacrificial material to flow out of the 3D structure. In various embodiments, the temperature of the fluid (e.g., culture medium) is used to induce a phase change in the sacrificial material (e.g., sacrificial ink). In various embodiments, the step of removing the sacrificial material may include both active and passive removal of the liquefied sacrificial material from the 3D structure.

[0071] In various embodiments, 3D constructs comprising an arrangement of one or more channels can be applied to bioprinting 3D structures for culturing cells. The presence of channels in the 3D construct can advantageously address or improve the fundamental challenge in bioprinting of providing nutrients beyond diffusion limitations to cells in thicker structures. The size, density, and arrangement of the channels can be optimized to ensure adequate nutrient supply to cells distributed throughout the 3D construct.

[0072] In various embodiments, the step of depositing one or more materials includes advancing droplets containing one or more materials onto a flowable precursor. In various embodiments, one or more materials can be advanced from a droplet dispensing outlet (e.g., an inkjet printhead, an acoustophoresis printhead). The droplet dispensing outlet is located between the radiation source and the flowable precursor.

[0073] In various embodiments, the step of advancing a droplet comprising one or more materials onto a flowable precursor includes advancing the droplet at a substantially uniform rate such that the one or more materials are substantially uniformly dispersed in the 3D fabrication. The one or more materials may be substantially uniformly dispersed in a selected region of the 3D fabrication or throughout the entire 3D fabrication.

[0074] In various embodiments, the step of advancing a droplet comprising one or more materials onto a flowable precursor includes advancing the droplet at a variable rate such that a spatial concentration gradient of one or more materials is formed in the 3D fabrication, such as a spatial patterning of molecules in the 3D fabrication. One or more materials may be dispersed in a selected region of the 3D fabrication or throughout the entire 3D fabrication with a spatial concentration gradient. A “spatial concentration gradient” is a concentration gradient in which the concentration can vary with one or more spatial dimensions.

[0075] Non-limiting examples of one or more materials contained in the droplet may include biological response modifiers, cytokines, growth factors, antigens, drugs, hormones, etc. In one example, the method can be used to print a controlled spatial gradient of angiogenic factors to facilitate desired vascularization outcomes within 3D constructs for tissue engineering applications. In another example, the method can be used to print a 3D construct having a first region and a second region, the first region containing a first growth factor for promoting a first type of cell (e.g., a growth factor for promoting chondrocyte growth in the first region), and the second region containing a second growth factor for promoting a second type of cell (e.g., a growth factor for promoting osteoblast growth in the second region).

[0076] In various embodiments, the step of depositing one or more materials may simply include extruding sacrificial material to form an arrangement having one or more sacrificial structures in a 3D fabrication. In various embodiments, the step of depositing one or more materials may simply include advancing droplets comprising one or more materials onto a flowable precursor. In various embodiments, the step of depositing one or more materials may include extruding sacrificial material to form an arrangement having one or more sacrificial structures in a 3D fabrication and advancing droplets comprising one or more materials onto a flowable precursor.

[0077] In various embodiments, crosslinking of the flowable precursor occurs at, near, adjacent to, or close to the interface between the flowable precursor and the fluid medium. Advantageously, in various embodiments, only a small amount or a thin layer of the flowable precursor is required at any given time during crosslinking. In various embodiments, a minimum thickness can reduce the amount of expensive precursor used and can also help improve resolution by avoiding excessive photocrosslinking. This reduces waste of valuable resources while allowing for better control over the formation of structures at high resolution and speed. However, the amount of precursor material should be sufficient to print the desired structure (e.g., 10 mL of precursor is insufficient to print a 20 mL structure). The actual amount of flowable precursor required also depends on, for example, the properties of the precursor (e.g., viscosity, surface tension, etc.), but in various embodiments, as an example, a layer of flowable precursor approximately 3 mm thick can be used or is sufficient for 3D printing structures. Ideally, the amount of flowable precursor should be sufficient to maintain a continuous layer of the precursor. Low surface tension (e.g., by using surfactants) can help the precursor spread on the flowable support, but this may not be desirable if it is toxic to cells. In various embodiments, the thickness / height of the flowable precursor layer ranges from about 0.5 mm to about 5 mm. The flowable precursor layer can have a start and end point selected from the following numerical groups: 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, and 5 mm.

[0078] In various embodiments, the step of crosslinking at least a portion of the precursor by radiation includes irradiating at least a portion of the precursor to be crosslinked with electromagnetic waves from an electromagnetic source (i.e., a radiation source). In various embodiments, the flowable precursor is capable of crosslinking upon application of electromagnetic radiation. Electromagnetic radiation may include, but is not limited to, infrared waves, ultraviolet waves, or electromagnetic waves with visible spectral wavelengths. In various embodiments, the flowable support cannot be crosslinked by applying the same electromagnetic radiation as crosslinking the flowable precursor. Similarly, in various embodiments, the fluid medium connected to the top of the flowable precursor layer cannot be crosslinked by applying the same electromagnetic radiation as crosslinking the flowable precursor.

[0079] In various embodiments, electromagnetic radiation exists as a projection from an electromagnetic source (e.g., an illumination or radiation source) onto a flowable precursor. In various embodiments, the projection onto the flowable precursor corresponds to the cross-sectional area of ​​the 3D structure to be formed by cross-linking the precursor. In various embodiments, the projection (corresponding to the cross-sectional area of ​​the 3D structure to be formed) changes continuously as the position / level of the flowable precursor, which is in contact with or connected to a portion of the 3D structure to be formed, changes and / or when cross-linking occurs.

[0080] In various embodiments, the flowable precursor is radiated in a direction substantially perpendicular to the surface of the flowable precursor (e.g., the plane of the flowable precursor-fluid medium interface). In various embodiments, the crosslinking step by radiation from the radiation source is performed in a bottom-up direction, such that the 3D structure or portions thereof are formed layer by layer in a bottom-up direction. In various embodiments, the step of depositing one or more materials on or within the flowable precursor includes depositing one or more materials toward the top surface of the flowable precursor in a top-down direction.

[0081] In various embodiments, the steps of radiation-crosslinking at least a portion of the precursor and depositing one or more materials onto or within the flowable precursor occur simultaneously. For example, when crosslinking occurs in the flowable precursor, a sacrificial material can be extruded to form an arrangement with one or more sacrificial structures in a 3D construction. Although extrusion of the sacrificial material may require temporarily positioning the extruder nozzle between the projector and the flowable precursor, the extrusion of the sacrificial material may occur faster than the crosslinking of the flowable precursor. Therefore, the extrusion of the sacrificial material does not significantly affect the crosslinking process. In alternative embodiments, the steps of radiation-crosslinking at least a portion of the precursor and depositing one or more materials onto or within the flowable precursor are performed sequentially. For example, one or more materials may first be deposited onto or within the flowable precursor, followed by crosslinking of the flowable precursor via a radiation source.

[0082] In various embodiments, the rate at which the steps of adjusting / changing the position / level of the flowable precursor are performed is substantially matched or substantially consistent with (or substantially proportional to) the crosslinking rate of the flowable precursor. For example, the rate at which the position of the top surface of the flowable precursor is adjusted is substantially matched with the crosslinking rate of the flowable precursor. In various embodiments, the rate at which the volume of the flowable support is increased and / or the rate at which the position / level of the flowable precursor is adjusted / changed is matched or consistent with (or substantially proportional to) the crosslinking rate of the flowable precursor. In various embodiments, the method is a continuous printing method and / or a rapid printing method. In various embodiments, the method is a continuous printing method, for example, even using different methods to deposit one or more materials. In various embodiments, the crosslinking step by irradiation from an irradiation source is performed continuously without interruption. In various embodiments, continuous printing advantageously enables the printing of 3D structures substantially free of delamination artifacts, which can occur due to non-continuous printing processes.

[0083] In various embodiments, the method may further include removing a flowable support from the 3D construct. During printing, the printed 3D construct, or a portion thereof, is placed on a printing platform and immersed in a flowable support (i.e., support fluid). After printing, the support fluid is removed from the printed 3D construct, for example by rinsing with another fluid, such as water or a suitable buffer solution.

[0084] In various embodiments, the printing speed in the method is from about 0.05 mm / min (or about 3 mm / h) to about 5 mm / min (or about 300 mm / h) along a vertical axis perpendicular to the surface plane of the flowable precursor. For ease of understanding, the X-axis and Y-axis are defined as parallel to the surface plane of the flowable precursor, and the Z-axis is defined as perpendicular to the surface plane of the flowable precursor. The printing speed can be selected from the following number groups for the start and end points: 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, and 5 mm / min. In various embodiments, the printing speed may depend on factors such as the formulation of the flowable precursor, the reaction rate of the flowable precursor, and the behavior of the flowable precursor during rapid crosslinking (e.g., whether the structure warps). Depending on the formulation of the flowable precursor, 3D structures (e.g., soft hydrogel structures with complex geometries) can be printed in the range of minutes to hours using the methods disclosed herein, depending on the materials used and the size of the structure. For example, for a 3 cm high structure, the printing time for a fast-curing material can be 5 to 10 minutes, while the printing time for a very slow-curing material can be several hours. In one embodiment, the method is capable of printing 3D hydrogel structures at a speed of 3.3 mm / min, which is faster than many DLP (Digital Light Processing) printers, whose printing speeds are typically around 0.1 mm / min to 1 mm / min.

[0085] In various embodiments, the 3D structure may include dimensions on the order of centimeters. In various embodiments, channels formed within the 3D structure may include dimensions on the order of millimeters or sub-millimeters. In various embodiments, the 3D structure may be a 3D hydrogel structure with a stiffness ranging from about 7 kPa to about 4 MPa. The stiffness of the 3D hydrogel structure can be selected from the following numerical ranges at the start and end points: 7, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, and 4000 kPa. In various embodiments, the stiffness of the 3D hydrogel structure can be greater than 4 MPa. In various embodiments, the 3D structure can be a 3D plastic resin structure with a stiffness greater than about 0.1 GPa. In various embodiments, depending on the material and structure to be printed, the stiffness of the 3D structure does not exceed about 1 kPa.

[0086] In various embodiments, the method can be used for bioprinting 3D constructs containing cells. In various embodiments, the flowable precursor and one or more materials are biocompatible. In various embodiments, the flowable precursor and / or one or more materials contain cells. In various embodiments, the method can advantageously enable rapid printing of soft materials without subjecting them to high shear forces, extreme temperatures, or other harsh conditions, and can be compatible with cell printing. Because printing conditions typically differ from the final incubation conditions of the printed structure (in terms of osmotic concentration, temperature, pH, etc.), the printing speed ensures that cells can quickly return to optimal culture conditions. In various embodiments, speed, ease of use, and versatility allow the method to be used in a variety of manufacturing and biomedical applications.

[0087] In various embodiments, a system is provided for performing the methods disclosed herein, the system comprising: a tank configured to include a flowable support and a flowable precursor disposed above the flowable support; a radiation source configured to radiate the flowable precursor; and one or more dispensing outlets disposed between the tank and the radiation source and configured to deposit one or more materials onto or within the flowable precursor; wherein the radiation source is configured to crosslink at least a portion of the flowable precursor by radiation to form the 3D structure or a portion thereof, wherein at least a portion of the flowable precursor is in contact with one or more materials in the flowable precursor.

[0088] In various embodiments, based on a first printing modality, the components of the system (i.e., the canister, the flowable support, the radiation source) cooperate to crosslink the flowable precursor. The first printing modality can be, for example, a fluid-supported liquid interfacial polymerization (FLIP) printer according to various embodiments disclosed herein. In various embodiments, one or more dispensing outlets are based on a second printing modality or another printing modality, such as inkjet printing or extrusion printing, which differs from the first printing modality (e.g., fluid-supported liquid interfacial polymerization).

[0089] In various embodiments, the first printing mode of the system provides a relatively unobstructed free surface at the top surface of the flowable precursor. Due to the space utilization provided by the free surface, the free surface of the flowable precursor can advantageously offer great flexibility in introducing / incorporating other different modes, thereby allowing or facilitating the addition of one or more materials to or within the flowable precursor. Advantageously, various embodiments of the system overcome the limitations of conventional 3D printers, where there is a lack of space between the printing platform and the patterning screen to introduce / incorporate other different printing modes. Some advantages of using different modes may include increased throughput (e.g., certain other modes can print certain more intricate and complex structures within the 3D construct faster); easy introduction of different types of materials into the 3D construct; and allowing intermittent cooling / non-running / charging of modes while still allowing seamless and continuous printing, thus increasing part lifespan and efficiency, as different modes are interchanged during operation.

[0090] In various embodiments, one or more dispensing outlets include extruder nozzles for extruding sacrificial material to form a template for arranging one or more channels in a 3D construction. In various embodiments, one or more dispensing outlets include propulsion nozzles (e.g., inkjet nozzles, sonication nozzles) for propelling droplets containing one or more materials over a flowable precursor. In various embodiments, one or more dispensing outlets (e.g., extruder nozzles, inkjet nozzles, sonication nozzles) are coupled to a respective reservoir containing one or more materials (e.g., sacrificial material, additives).

[0091] In various embodiments, the propulsion nozzle can be configured to propel droplets comprising one or more materials onto a flowable precursor at a substantially uniform rate, such that the one or more materials are substantially uniformly dispersed in the 3D fabrication. The propulsion nozzle can be configured to disperse one or more materials substantially uniformly in a selected region or throughout the entire 3D fabrication. In various embodiments, the propulsion nozzle can be configured to propel droplets comprising one or more materials onto a flowable precursor at a variable rate, such that a spatial concentration gradient of one or more materials is formed in the 3D fabrication, for example, a molecular spatial pattern in the 3D fabrication. The propulsion nozzle can be configured to disperse one or more materials with a spatial concentration gradient in a selected region or throughout the entire 3D fabrication.

[0092] In various embodiments, one or more dispensing outlets (e.g., extruder nozzles, inkjet nozzles, sonication nozzles) are configured to be movable. The one or more dispensing outlets may be configured to move along one or more axes. For example, the one or more dispensing outlets may be configured to move in a horizontal direction substantially parallel to the surface of the flowable precursor. The one or more dispensing outlets may be configured to move in a vertical direction substantially perpendicular to the surface of the flowable precursor. The mobility of the one or more dispensing outlets can advantageously facilitate the deposition of one or more materials onto or within the flowable precursor.

[0093] In various embodiments, the system may include a combination of an extruder nozzle and a propulsion nozzle, the extruder nozzle being used to extrude sacrificial material to form an arrangement having one or more sacrificial structures in a 3D construct, and the propulsion nozzle being used to propel droplets containing one or more materials over a flowable precursor. Advantageously, various embodiments of the system can combine extrusion-based printing methods with continuous 3D printing methods such as DLP (Digital Light Processing) to create multimodal printers that can be used to create bioprinted structures with channels that can provide the nutrients needed to maintain cell viability. In addition to fiber extruders that create sacrificial structures for channels, various embodiments of the system can also be used in conjunction with propulsion nozzles such as inkjet nozzles and / or sonication nozzles to establish concentration gradients of additives (e.g., cytokines, growth factors, etc.) to produce heterogeneous matrices.

[0094] In various embodiments, the container / tank includes an inlet (i.e., at least one inlet) for allowing flowable supports to flow in at a predetermined rate. The system may also include actuators (e.g., pumps, lifting arms) for facilitating the flow of flowable supports through the container / tank's inlets. In various embodiments, the container / tank may include one or more inlets, and in such embodiments, actuators may accordingly facilitate the flow of flowable supports through one or more inlets of the container / tank.

[0095] In various embodiments, the system further includes a processing module configured to adjust the position of the top surface of the flowable precursor at a rate substantially matching the rate at which the flowable precursor crosslinks through the radiation source. In various embodiments, the processing module may be configured to control, for example, the rate at which the flowable support material flows into the container / tank, the radiation intensity of the radiation source, and the position of the radiation source, synchronizing these parameters, which can affect the rate of position adjustment of the top surface of the flowable precursor and / or the rate at which the flowable precursor crosslinks through the radiation source. The processing module may also control the type and amount of one or more materials to be deposited on or within the flowable precursor, as well as the deposition time and rate. The processing module may also control and determine the type or shape of the projection to be radiated onto the flowable precursor as the relative horizontality of the flowable precursor (e.g., relative to a portion of an already formed 3D structure) changes or varies along a vertical axis perpendicular to the top surface of the flowable precursor.

[0096] In various embodiments, the system further includes a reservoir for accommodating a volume of flowable support outside the container / tank. In various embodiments, the reservoir is in fluid communication with the container / tank (e.g., via an inlet). In various embodiments, a passive pumping system (e.g., a gravity pumping system) is used to transfer the flowable support from the reservoir to the container / tank by raising the position of the reservoir relative to the container / tank (e.g., by using a lifting arm). Advantageously, this ensures that the flowable support enters the container / tank in a laminar flow to minimize or prevent disruption of the crosslinking process of the flowable precursor.

[0097] In various embodiments, the system further includes a print bed / print platform plate disposed on or near the bottom of the container / tank. The print bed can support the 3D structure or a portion thereof. In various embodiments, the position of the print platform plate can also be adjusted along a vertical axis substantially perpendicular to the top surface of the flowable precursor (e.g., via a processing module), such that the position of the 3D structure or a portion thereof relative to the flowable precursor is altered.

[0098] In various embodiments, the radiation source is positioned above the tank (e.g., a top-down projection system), below the tank / object (e.g., a bottom-up projection system), next to the tank / object (e.g., a side projection system), and / or around the tank / object (e.g., a circumferential projection system such as a radial / circular projection system).

[0099] In various embodiments, the container / tank is substantially transparent to radiation from the radiation source. For example, the container / tank allows electromagnetic waves emitted from the radiation source to be transmitted to the flowable precursor. In various embodiments, such a container / tank may be particularly useful when using projection systems other than top-down projection systems, i.e., when using bottom-up projection systems, side projection systems, and / or circumferential projection systems. In various embodiments, the flowable precursor, flowable support, and 3D structure, or portions thereof, are disposed within the tank.

[0100] In various embodiments, a multimodal 3D printer is provided that can be used to fabricate cell-loaded hydrogel structures and utilizes a free surface with a flowable precursor. In various embodiments, the multimodal 3D printer is combined with other printing modalities, such as extrusion or piezoelectric deposition of different materials. In various embodiments, the multimodal 3D printer uses specific inks to create cell-loaded structures (e.g., methacrylate gelatin, alginate, etc.) and uses easily fading inks that liquefy at low temperatures and are compatible with cells (e.g., Pluronic F127) to create channels within the cell-loaded structures as a means of nutrient supply. In various embodiments, the multimodal 3D printer combines free surface printing with inkjet deposition of molecules (e.g., biomaterials) to create a concentration gradient of said molecules within the hydrogel structure.

[0101] Brief description of the attached diagram

[0102] Figure 1 This is a schematic diagram showing a side view of a system (e.g., a fluid-supported liquid interface polymerization (FLIP) printer) for printing 3D structures or portions thereof according to various embodiments disclosed herein.

[0103] Figure 2 This is a schematic diagram showing a side view of a conventional 3D printing system.

[0104] Figures 3A to 3D These are a series of schematic diagrams illustrating methods for printing 3D structures or portions thereof using a FLIP printer according to various embodiments disclosed herein.

[0105] Figure 4A , 4B 4C are screenshots from a video demonstrating the process of printing 3D constructs according to various embodiments disclosed herein. Figure 4D It is a cross-sectional image of the 3D structure, showing the interference-free channels within the 3D structure.

[0106] Figure 5A and 5B These are micrographs showing cross-sections of hydrogel structures according to various embodiments disclosed herein. Scale bar = 200 micrometers.

[0107] Figures 6A to 6C This is a micrograph showing a cross-section of another hydrogel structure according to various embodiments disclosed herein. Scale bar = 200 micrometers.

[0108] Figure 7 This is the phase diagram of the Pluronic F127.

[0109] Detailed description of the attached figures

[0110] Exemplary embodiments of this disclosure will be better understood and readily apparent to those skilled in the art through the following discussion and, where applicable, in conjunction with the accompanying drawings. It should be understood that other modifications relating to structural changes may be made without departing from the scope of the invention. Exemplary embodiments are not necessarily mutually exclusive, as some exemplary embodiments may be combined with one or more embodiments to form new exemplary embodiments. Exemplary embodiments should not be construed as limiting the scope of this disclosure.

[0111] The following example illustrates a strategy for printing 3D constructs.

[0112] Systems for printing 3D structures

[0113] Figure 1 This is a schematic side view of a system 100 (e.g., a fluid-supported liquid interface polymerization (FLIP) printer) for printing 3D structures 102 or portions thereof according to various embodiments disclosed herein. System 100 includes a tank 104 and a flowable precursor 108, the tank 104 being configured to contain a flowable support 106 (e.g., a fluid support), the flowable precursor 108 (e.g., a hydrogel precursor) being disposed above the flowable support 106. System 100 further includes a radiation source 110 (e.g., a patterning light source or projector) configured to radiate the flowable precursor 108. System 100 also includes a dispensing outlet 112 disposed between the tank 104 and the radiation source 110 and configured to deposit material 114 onto or within the flowable precursor 108. System 100 may include more than one dispensing outlet 112 configured to deposit one or more materials 114 onto or within a flowable precursor 108. Radiation source 110 is configured to crosslink at least a portion of the flowable precursor 108 in contact with the materials 114 by radiation to form a 3D structure 102 or a portion thereof. The formed 3D structure 102 or a portion thereof is placed on a printing platform plate 116.

[0114] In such Figure 1In the system 100 shown, a pattern from a radiation source 110 is directly projected onto a flowable precursor 108, and photocrosslinking occurs on the surface of the flowable precursor 108, the pattern floating on top of a flowable support 106. This configuration frees up the top surface of the flowable precursor 108 and provides a free surface that allows a dispensing outlet 112 (e.g., an extruder) to be introduced into the gap between the radiation source 110 and the flowable precursor 108 and to deposit a different material (e.g., a second material different from the flowable precursor 108) into the flowable precursor 108 and onto the newly printed material of the 3D structure 102 or a portion thereof. This configuration addresses a problem encountered in conventional 3D printers, where there is a lack of space to introduce additional printing modes different from the FLIP printing mode.

[0115] For example, Figure 2 This is a schematic side view of a conventional 3D printing system 200, highlighting the problems encountered by conventional 3D printers. The 3D printing system 200 includes a printing platform 202, a patterning screen 204 (e.g., an oxygen-permeable window), and an imaging unit 206 configured to project a patterned light image onto a mirror 208, which in turn projects the patterned light image onto the patterning screen 204. The conventional 3D printing system 200 includes two solid surfaces: the printing platform 202 and the patterning screen 204. In use, the printing platform 202 is positioned close to the patterning screen 204 and gradually rises as the liquid resin 210 crosslinks to form a 3D structure 212 or a portion thereof suspended from the underside of the printing platform 202. In the conventional 3D printing system 200, a dead zone 214 is formed between the printing platform 202 and the patterning screen 204 when the 3D structure 212 or a portion thereof is formed. No space is left between the printing platform 202 and the pattern screen 204 to introduce other printing modes for depositing other materials.

[0116] Methods of printing 3D structures

[0117] Figures 3A to 3D These are a series of schematic diagrams illustrating methods for printing 3D structures or portions thereof using a FLIP printer according to various embodiments disclosed herein.

[0118] like Figure 3A As shown, the flowable precursor 308 (comparison) Figure 1 108 in the middle) is composed of a movable support 306 (in contrast) Figure 1 106) is supported in a way that allows the flowable precursor 308 to pass through a radiation source (not shown, for comparison). Figure 1 The cross-linking site is located at the radiation cross-linking point of 110 in the middle. The flowable precursor 308 is positioned such that it contacts the already printed cylindrical structure 302 (e.g., hydrogel structure) (contrast). Figure 1 The top surface of part of 102). Part of the cylindrical structure 302 is set on the printing platform plate 316 (compare). Figure 1 116 in the middle).

[0119] In FLIP, photocrosslinking occurs in the flowable precursor 308. Since the space above the flowable precursor 308 is relatively unobstructed, this provides a free surface, allowing the use of extruders 312 based on different printing modalities (for comparison) while printing is still in progress. Figure 1 (112) Deposit easily faded ink fibers (such as Pluronic F127 fibers) onto newly printed material. For example... Figure 3A As shown, when the easily fading ink is extruded from the extruder 312 and horizontally translated through the flowable precursor 308, as indicated by the boxed arrow 318, fiber 314 (contrast) Figure 1 114) is formed on the top surface of the cylindrical structure 302. During printing, easily faded ink is extruded onto the free surface of the flowable precursor 308 to create a mold / template for nutrient supply channels. As the cylindrical structure 302 continues to print, the position / level of the flowable precursor 308 rises upward by increasing the volume of the flowable support 306, as indicated by box arrow 320.

[0120] like Figure 3B As shown, the flowable precursor 308 continues to flow through the fiber, embedding the fiber into the cylindrical structure 302 during crosslinking. The cylindrical structure 302 is then immersed in an ice bath at 4°C to liquefy the fiber 314. Figure 3C A schematic perspective view of the cylindrical structure 302 is shown, wherein a channel 322 is formed at the location where the fiber 314 is initially embedded. Figure 3D As shown, the cylindrical structure 302 with channel 322 is then immersed in culture medium 324, such that the culture medium fills the channel 322.

[0121] Figure 4A , 4B 4C are screenshots from a video demonstrating the process of printing 3D constructs according to various embodiments disclosed herein. Figure 4D It is a cross-sectional image of the 3D structure, showing the interference-free channels within the 3D structure.

[0122] During FLIP printing, a free surface is created at the precursor-air interface. This allows for the direct deposition of materials, such as Pluronic F127 fibers, using an extruder. Figure 4A As shown, GelMA (methacrylate gelatin) cylinder 402 (comparison) Figure 1 102) is derived from its precursor 408 (contrast) Figure 1108) were formed. Pluronic F127 gel fibers 414 (contrast) Figure 1 114) is extruded from syringe 412 during GelMA formation (contrast) Figure 1 (112) was incorporated. For example... Figure 4B As shown, when the supporting fluid (not visible in the figure, in contrast) Figure 1 As 106) flows in, the precursor 408, floating on top of the supporting fluid, is lifted upwards and flows over the deposited fibers 414. Figure 4C As shown, fiber 414 is encapsulated in gel as photocrosslinking continues. Although the projector (not visible in the image, for comparison) Figure 1 The position of syringe 412 between 110 and precursor 408 may obstruct the projected pattern, but Pluronic F127 extrusion occurs within seconds, much faster than photocrosslinking, and therefore will not significantly affect the crosslinking process. Figure 4D As shown, after photocrosslinking is completed, the entire cylinder is immersed in a cold bath / ice bath, whereby F127 is liquefied and removed, forming channels within the GelMA cylinder. Figure 7 As shown in the phase transition diagram, F127 is a thermosensitive material whose physical state changes with temperature. For example, at a concentration of 20% w / w, F127 is a liquid below 20°C, a gel between about 20°C and about 70°C, and liquefies again above 70°C. The sacrificial ink remains in a gel state at printing temperatures and melts as the temperature rises or falls.

[0123] Bioprinting of cells in hydrogels containing nutrient supply channels

[0124] The ability to print soft hydrogel structures has clear applications in 3D bioprinting. However, two further goals must be achieved to successfully print living structures.

[0125] First, the 3D printing process itself should not be cytotoxic. To demonstrate the bioprinting potential of the FLIP printer disclosed herein, fibroblasts expressing green fluorescent protein (GFP) were suspended in gelatin methacrylate (GelMA) and printed onto a gelatin methacrylate (GelMA) matrix. The hydrogel construct was cultured in cell culture medium under static conditions (i.e., no active flow of the medium). After 7 days, the hydrogel construct was cut in half and stained with propidium iodide (PI) to identify dead cells.

[0126] Due to the mild printing conditions (no high shear force, high temperature, or excessive radiation) and rapid printing, the cells remained viable for up to a week after printing. However, as... Figure 5AAs shown, without printed channels, GFP-expressing cells are only visible within a few hundred micrometers of the surface 500 facing the medium (culture medium). Beyond a few hundred micrometers from the surface, limited nutrient diffusion leads to a sharp decline in cell viability, and almost no live cells are visible in the core region 502 of this gel. Figure 5B This is a micrograph of the core region / necrotic core 502, showing only dead, PI-stained cells.

[0127] This highlights the second condition required for bioprinting: the printed structure must provide sufficient nutrients to support cellular metabolism, for example, through one or more channels within the printed structure. While channels can be printed directly, the tendency for excessive cross-linking often results in very slow printing speeds (due to the use of excessive inhibitory dyes to control the process) or partially blocked channels. The method disclosed in this application utilizes an easily fading ink strategy to directly deposit F127 fibers onto partially printed structures during the printing process (see [link to application]). Figures 3A to 3D This is possible because FLIP uses a contactless strategy, allowing fibers to be deposited onto a free surface using an extruder, while photocrosslinking occurs uninterruptedly. After printing, the construct is immersed in a cold medium, and the fibers are subsequently liquefied to form channels through which cells are supported (see...). Figure 3C (322 in the text). The advantage of these channels is evident around the channel lumen, where GFP-expressing cells can be clearly seen, even though they are located near the necrotic core of the gel.

[0128] like Figures 6A to 6C As shown, mouse fibroblasts expressing green fluorescent protein (GFP) were directly printed into a gelatin methacrylate (GelMA) matrix containing printing channels 600 at a similar location to the necrotic core 502 in Figure 5A. The GFP-expressing cells were clearly visible around the lumen of the channels (see Figure 5A). Figure 6A Although cell viability decreases at a distance of 100-200 micrometers from the lumen (see...) Figure 6B ). Figure 6C yes Figure 6A and 6B The overlapping images show a gradual transition to lower vitality.

[0129] If culture medium exchange is achieved through active flow instead of passive diffusion as in this experiment, cell viability may be prolonged. Proper channel design that allows the culture medium to flow actively through the channels, rather than through passive diffusion as in this experiment, would further enhance cell viability.

[0130] This experiment demonstrates the feasibility of using the methods and systems disclosed in this application for rapid and versatile bioprinting, as it enables the rapid printing of cell-loaded GelMA (printing speeds exceeding 70 mm / hr) and facilitates the integration of arbitrarily designed channel networks to support cell viability.

[0131] discuss

[0132] Embodiments of the FLIP method and system disclosed in this application provide a continuous 3D printing platform that uses a fluid support strategy to print soft structures. Embodiments of the FLIP method and system disclosed in this application can expand the role of 3D printing in biomedical applications, such as tissue bioprinting. To this end, FLIP attempts to address several technical challenges associated with bioprinting, including the low mechanical stiffness of hydrogels; complex geometries related to anatomical structures; the low printing speeds of most existing bioprinters; and the need for mild, cell-compatible printing conditions.

[0133] One of the main components of bioinks is often hydrogels because they are compatible with cell physiology. While hydrogels are softer than traditional engineering materials such as metals, plastics, and even many elastomers, it's worth noting that even hydrogels made from materials with the same surface (such as PEG-diacrylate) can have significantly different mechanical properties depending on the formulation. Although megapascal hydrogels can be printed using various printers (including FLIP) for bioprinting purposes, these materials typically have a Young's modulus of several hundred kilopascals or less. This necessitates the use of supports for overhanging structures, a need exacerbated by the thinness of many anatomical structures (such as blood vessels) and the potential for inclusion of voids (such as ventricles).

[0134] To address this issue, possible support strategies include printing with cryogenic supports, printing directly into gels that melt at high temperatures, and printing into particulate gels. While these strategies have their advantages, they all employ syringe extrusion, which has its limitations. Furthermore, the required low temperatures in cryogenic support are incompatible with cells, making direct cell printing challenging. In embodiments of the methods disclosed in this application, FLIPs are employed. FLIPs use an inert, stable fluid to provide buoyancy, supporting complex structures from collapse during printing. The use of a perfluorinated support fluid allows it to be immiscible with aqueous precursors and also minimizes interactions with the organic components of the precursors, such as monomers, crosslinking agents, and initiators. Therefore, the support fluid in the embodiments of the methods disclosed in this application does not cause any swelling of the hydrogel structure and can be easily removed by rinsing under running water. In addition to printing overhanging structures, these examples also demonstrate that FLIP printers can be used to estimate the conditions required for successfully printing submillimeter-scale freestanding hollow structures, which is difficult for many resin-based 3D printers.

[0135] It is worth emphasizing that the high printing speed is not merely a matter of convenience; rather, because the printing conditions typically differ from the final incubation conditions of the printed structure (in terms of osmotic concentration, temperature, pH, etc.), the speed ensures that cells can quickly return to optimal culture conditions. FLIP enables the rapid printing of soft materials without subjecting them to high shear forces, extreme temperatures, or other harsh conditions, and has proven compatible with cell printing (see Figures 5 and 6). By introducing channels into the printed structure, embodiments of the methods and systems disclosed in this application also demonstrate their ability to address fundamental challenges in bioprinting, providing nutrients to cells in thicker structures beyond diffusion limitations. Due to the versatility of embodiments of the systems and methods disclosed in this application, the size, density, and arrangement of the channels can be optimized to ensure adequate supply of cells distributed throughout the structure. In addition to fiber extruders, embodiments of the methods disclosed in this application can also be used in conjunction with sonication nozzles, which can establish concentration gradients of cytokines, growth factors, or other additives to generate a heterogeneous matrix. The speed, ease of use, and versatility currently available for FLIP enable its widespread application in a variety of manufacturing and biomedical applications.

[0136] Those skilled in the art will understand that other variations and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of this disclosure, which is broadly described herein. For example, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included, etc., among different exemplary embodiments in the description herein. Therefore, these embodiments are to be considered illustrative rather than restrictive in all respects.

Claims

1. A multimodal method for printing 3D structures or portions thereof, the method comprising: (i) Support the flowable precursor with a flowable support at a location where the flowable precursor can be crosslinked by radiation from a radiation source; (ii) Depositing one or more materials onto or into the flowable precursor; as well as (iii) Crosslinking at least a portion of the flowable precursor by radiation from a radiation source to form the 3D structure or a portion thereof, wherein at least a portion of the flowable precursor is in contact with one or more materials in the flowable precursor. The flowable precursor comprises at least one polymerizable monomer / crosslinkable polymer and at least one photoinitiator; The step of crosslinking at least a portion of the flowable precursor by radiation from the radiation source is performed by a first printing mode, and the step of depositing the one or more materials on or within the flowable precursor is performed by a second printing mode or another printing mode different from the first printing mode.

2. The method according to claim 1, further comprising: (iv) Support the flowable precursor at a new location that allows the flowable precursor to crosslink through radiation from the radiation source; (v) Optionally, one or more materials may be further deposited on or within the flowable precursor; (vi) Further crosslinking at least a portion of the flowable precursor by radiation from the radiation source to form the 3D structure or a portion thereof, wherein at least a portion of the flowable precursor may optionally contact one or more materials at the new location; as well as (vii) Optionally repeat steps (iv) to (vi) until the desired 3D structure is formed.

3. The method according to claim 1, wherein, Depositing the one or more materials includes extruding sacrificial materials to form an arrangement having one or more sacrificial structures in the 3D construction.

4. The method of claim 3, further comprising removing the sacrificial material from the arrangement having one or more sacrificial structures to form an arrangement having one or more channels in the 3D construction.

5. The method according to claim 4, wherein, The one or more channels include an open end that facilitates fluid communication between the internal environment of the 3D structure and the external environment of the 3D structure.

6. The method according to any one of claims 3 to 5, wherein, The sacrificial material is in a semi-solid or gel state when it is extruded to form the arrangement having one or more sacrificial structures.

7. The method according to any one of claims 4 to 5, wherein, Removing the sacrificial material includes liquefying the sacrificial material to facilitate its removal from the 3D structure or a portion thereof.

8. The method according to claim 7, wherein, Liquefying the sacrificial material includes cooling the sacrificial material to a temperature that would cause it to liquefy.

9. The method according to claim 1, wherein, Depositing the one or more materials involves advancing droplets containing the one or more materials onto the flowable precursor.

10. The method according to claim 9, wherein, Propelling a droplet containing one or more of the materials on the flowable precursor includes propelling the droplet at a variable rate, such that a spatial concentration gradient of the one or more materials is formed in the 3D structure.

11. The method according to claim 1, wherein, The cross-linking step, which is carried out by radiation from the radiation source, is performed in the bottom-up direction, so that the 3D structure or a portion thereof is formed layer by layer in the bottom-up direction.

12. The method according to claim 1, wherein, The cross-linking process, which is carried out through radiation from the radiation source, is performed continuously.

13. The method according to claim 1, wherein, Depositing one or more materials onto or within the flowable precursor includes depositing one or more materials in a top-to-bottom direction toward the top surface of the flowable precursor.

14. The method according to claim 1, wherein, The flowable support and the flowable precursor are immiscible.

15. The method according to claim 1, wherein, The flowable precursor is biocompatible with the one or more materials.

16. The method according to claim 1, wherein, The density of the 3D structure is higher than that of the flowable precursor and the flowable support.

17. The method according to claim 1, wherein, The flowable precursor and / or one or more materials include cells.

18. A multimodal system for performing the multimodal method of claim 1, the system comprising: A can, the can being configured to include a flowable support and a flowable precursor disposed above the flowable support; A radiation source configured to radiate the flowable precursor; One or more dispensing outlets are disposed between the tank and the radiation source and are configured to deposit one or more materials onto or into the flowable precursor; The radiation source is configured to crosslink at least a portion of the flowable precursor by radiation to form the 3D structure or a portion thereof, wherein at least a portion of the flowable precursor is in contact with one or more materials in the flowable precursor.

19. The system according to claim 18, wherein, The one or more dispensing outlets include extruder nozzles for extruding sacrificial material to form a template for arranging one or more channels in the 3D structure.

20. The system according to claim 18 or 19, wherein, The one or more dispensing outlets include inkjet printheads for propelling droplets containing the one or more materials onto the flowable precursor.

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